Stocker and Dehner's Pediatric Pathology

EDITORS
J. Thomas Stocker MD
Professor of Pathology, Pediatrics and Emerging Infectious Disease
Department of Pathology
Uniformed Services University of the Health Sciences
Bethesda, Maryland
Louis P. Dehner MD
Professor
Division of Anatomic and Molecular Pathology
Department of Pathology and Immunology
Washington University in St. Louis
Attending Surgical Pathologist
Lauren V. Ackerman Laboratory of Surgical Pathology
Barnes-Jewish and St. Louis Childrens Hospitals at the Washington University Medical Center
St. Louis, Missouri
Aliya N. Husain MD
Professor
Department of Pathology
University of Chicago
Chicago, Illinois
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Contributors
Hikmat A. Al-Ahmadie MD
Assistant Attending, Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York
Kevin E. Bove MD
Professor, Department of Pathology and Laboratory Medicine, University of Cincinnati, College of Medicine
Staff Pathologist, Division of Pathology and Laboratory Medicine, Cincinnati Children's Hospital Medical
Center, Cincinnati, Ohio
David S. Brink MD
Associate Professor, Departments of Pathology and Pediatrics, St. Louis University School of Medicine
Associate Pathologist, Department of Pathology, SSM
Cardinal Glennon Children's Medical Center, St. Louis, Missouri
John J. Buchino MD
Emeritus Professor, Department of Pathology, Department of Pediatrics, University of Louisville
Emeritus Chief, Department of Pathology, Kosair
Children's Hospital, Louisville, Kentucky
J. Douglas Cameron MD
Professor of Ophthalmology, Departments of Ophthalmology and Pathology, Mayo Clinic, Rochester,
Minnesota
Ellen Chung
Assistant Professor, Department of Radiology and Radiological Sciences and Department of Pediatrics,
Uniformed Services University of the Heath Sciences
Integrated Chief, Diagnostic Radiology Service, Department of Radiology, Walter Reed National Military
Medical Center, Bethesda, Maryland
Kim A. Collins MD
Professor, Department of Pathology and Laboratory Medicine, Medical University of South Carolina,
Charleston, South Carolina
Richard M. Conran MD, PhD, JD

Professor of Pathology and Emerging Infectious Diseases, Department of Pathology, Uniformed Services
University of the Health Sciences, Bethesda, Maryland
Tracey S. Corey
Clinical Professor, Division of Forensic Pathology, University of Louisville School of Medicine Chief Medical
Examiner, Kentucky Medical Examiner Program, Commonwealth of Kentucky, Louisville, Kentucky
Robert F. Debski MD
Assistant Professor, Department of Pathology, Department of Pediatrics, University of Louisville
Chief, Department of Pathology, Kosair Children's Hospital, Louisville, Kentucky
Louis P. Dehner MD
Professor, Division of Anatomic and Molecular Pathology, Department of Pathology and Immunology,
Washington University in St. Louis Attending Surgical Pathologist, Lauren V Ackerman Laboratory of Surgical
Pathology, Barnes-Jewish and St. Louis Children's Hospitals at the Washington University Medical Center, St.
Louis, Missouri
Christopher Dunham MD
Clinical Fellow, Department of Pathology and Immunology, Washington University Clinical Fellow, Department
of Pathology and Immunology, Barnes-Jewish Hospital, St. Louis, Missouri
Michael K. Fritsch MD, PhD

Associate Professor, Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison,
Wisconsin
Dorothy K. Grange MD
Professor of Pediatrics, Department of Pediatrics, Division of Genetics and Genomic Medicine, Washington
University School of Medicine
Professor of Pediatrics, Department of Pediatrics, St. Louis Children's Hospital, St. Louis, Missouri
John Hart MD
John Hart MD
Professor, Department of Pathology, University of Chicago, Chicago, Illinois
Anjum Hassan MD
Assistant Professor, Division of Anatomic and Molecular Pathology, Washington University School of Medicine
Assistant Director FISH Laboratory, Department of Pathology, Washington University Medical Center, Barnes
Jewish Hospital, St. Louis, Missouri
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M. John Hicks MD, DDS, MS, PhD
Professor of Pathology, Department of Pathology and Immunology, Baylor College of Medicine
Attending Pathologist, Department of Pathology, Texas Children's Hospital, Houston, Texas
D. Ashley Hill MD
Associate Professor, Department of Pathology and Pediatrics, The George Washington University
Chief, Department of Pathology, Children's National Medical Center, Washington, DC
Aliya N. Husain MD
Jason A. Jarzembowski MD, PhD

Assistant Professor, Department of Pathology, Medical College of Wisconsin Program Director, Perinatal
Pathology, Department of Pathology, Children's Hospital of Wisconsin, Milwaukee, Wisconsin
Raj P. Kapur MD, PhD

Professor, Department of Pathology, University of Washington Staff Pathologist, Department of Laboratories,
Seattle Children's Hospital, Seattle, Washington DC
Jochen K. M. Lennerz MD, PhD

Division of Anatomic and Molecular Pathology, Department of Pathology and Immunology, Washington
University School of Medicine, St. Louis, Missouri
Haresh Mani MD
Assistant Professor, Department of Pathology, Northwestern University's Feinberg School of Medicine
Pathologist-in-Chief, Children's Memorial Hospital, Chicago, Illinois
Thomas L. McCurley MD
Associate Professor, Department of Pathology, Vanderbilt University Director, Department of Immunopathology
Laboratory, Nashville, Tennessee
Deborah E. McFadden MD
Clinical Professor, Department of Pathology and Laboratory Medicine, University of British Columbia
Head and Medical Director, Department of Pathology and Laboratory Medicine, BC Children's Hospital and BC
Women's Hospital and Health Centre, Vancouver, British Columbia
Gary W. Mierau PhD
Electron Microscopist, Department of Pathology and Laboratory Medicine, The Children's Hospital, Aurora,
Colorado
Lili Miles MD
Associate Professor, Department of Pathology and Laboratory Medicine, University of Cincinnati, College of
Medicine
Staff Pathologist, Division of Pathology and Laboratory Medicine, Cincinnati Children's Hospital Medical
Center, Cincinnati, Ohio
Jeffrey Mueller MD
Assistant Professor, Department of Pathology, University of Chicago, Chicago, Illinois
Rish K. Pai MD, PhD

Associate Staff, Department of Anatomic Pathology, Cleveland Clinic Foundation, Cleveland, Ohio
Kathleen Patterson MD
Associate Professor, Department of Pathology, University of Washington Associate Pathologist, Department of
Pathology, Seattle Children's Hospital, Seattle, Washington DC
Elizabeth J. Perlman MD
Pathologist-in-chief, Children's Memorial Hospital
Professor of Pathology, Northewestern University's Feinberg School of Medicine, Chicago, Illinois
Arie Perry MD
Professor of Pathology and Neurological Surgery, Director of Neuropathology and Vice Chair of Pathology,
Department of Pathology, Division of Neuropathology, University of California, San Francisco (UCSF), San
Francisco, California
Theodore J. Pysher MD
Professor of Pathology, Department of Pathology, University of Utah School of Medicine
Division Chief of Pediatric Pathology and Director of Laboratories, Primary Children's Medical Center, Salt
Lake City, Utah
Vijaya B. Reddy MD
Professor, Department of Pathology, Rush Medical College Senior Attending, Department of Pathology, Rush
University Medical Center, Chicago, Illinois
Raymond W. Redline MD
Professor, Pathology and Reproductive Biology, Case Western Reserve University School of Medicine,
Cleveland, OH Co-director, Pediatric Pathology, Pathology, University Hospitals Case Medical Center,
Cleveland, OH
Andrea M. Sheehan MD
Assistant Professor, Department of Pathology and Immunology, Department of Pediatrics, Section of
Hematology and Oncology, Baylor College of Medicine
Director of Hematology and Flow Cytometry, Department of Hematopathology, Department of Pathology, Texas
Children's Hospital, Houston, Texas
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Hiroyuki Shimada MD, PhD
Professor, Department of Pathology, University of Southern California Keck School of Medicine
Pathologist, Department of Pathology and Laboratory Medicine, Children's Hospital Los Angeles, Los Angeles,
California
Joseph R. Siebert PhD

Professor of Pathology, Adjunct Professor of Pediatrics, Departments of Pathology, Pediatrics, University of
Washington Director of Autopsy Services, Department of Laboratories, Seattle Children's Hospital, Seattle,
Washington
Professor of Pathology, Pediatrics and Emerging Infectious Disease, Department of Pathology, Uniformed
Services University of the Health Sciences, Bethesda, Maryland
Mariko Suchi MD, PhD

Assistant Professor, Department of Pathology, Medical College of Wisconsin Department of Pathology and
Laboratory Medicine, Children's Hospital of Wisconsin, Milwaukee, Wisconsin
Jerome B. Taxy MD
Carole A. Vogler MD
Professor and Chair, Department of Pathology, Saint Louis University School of Medicine Pathologist,
Department of Pediatric Pathology, SSM Cardinal Glennon Children's Medical Center, St. Louis, Missouri
Christopher R. Weber MD, PhD

Instructor, Department of Pathology, The University of Chicago, Chicago, Illinois
Rebecca Wilcox MD
Assistant Professor, School of Medicine, The University of Vermont Assistant Professor, Department of
Pathology, Fletcher Allen Hospital, Burlington, Vermont
Mary M. Zutter MD
Professor of Pathology and Cancer Biology, Department of Pathology, Vanderbilt University Director of
Hematopathology, Scientific Director, HTAP Shared Resource, Department of Pathology, Vanderbilt University
Medical Center, Nashville, Tennessee
Dedication
To my children and grandchildren—Louis, Jr., Carl, Christopher, Elizabeth, Rebecca, Rachael, Jennifer, and
Charlie.
Louis P. Dehner
To my mother, Khadija Omar, who has inspired me throughout my life and in memory of my father, Zahid Omar,
who saw only the beginnings of his children's lives.
Aliya N. Husain
To my wife, Pat, the Center of my life, at 44 years together and counting: to our children; Rick, his wife Cathy
and sons Jack and Joseph; David, his wife Carol and daughter Sydney; and Meg. How full they make our lives!
J. Thomas Stocker
Preface
There is no other way to put it, a book, especially one that is now in its third edition, has long since become an
enduring burden. Almost 10 years have elapsed since the last edition and no decade passes without advances
being made and old “truths” being dismissed as folly. We have attempted to capture within the front and back
covers of this book a reasonable approximation of the latest truths as they relate to our understanding of those
unique disorders of maldevelopmental, infectious, and neoplastic nature that are found predominantly in children
and in the period of 9 months preceding childhood.
The codex of pediatric pathology is still largely based upon morphologic features that are apparent in their gross
manifestations as in the case of congenital heart disease and skeletal dysplasias or on visualizing microscopic
features. Immunohistochemistry has become as routine today as the trichrome and Gomori methenamine-silver
stains. However, we have now moved into the era of application of molecular diagnostic methodologies that have
admitted us into an unseen world in a sense. The pace of discoveries has created a world where yesterday
becomes a part of the deep past as we catapult through today. In the microcosm of pediatric pathology, the
current endeavor is our attempt to capture what is reasonably stable as the foundation of morphologic pediatric
pathology. In the yesterdays, we may have speculated about the pathogenesis or puzzled over the morphologic
findings, but today, we have the ability to test those speculations and begin the process of diagnostic discovery
and investigation. As we have all experienced, this process is oftentimes one door leading to another. An attempt
has been made in this edition to not only lay out the foundation of pediatric pathology but also convey the idea
that another door awaits to be opened. It is to our younger colleagues in pediatric pathology that we address this
latter challenge.
For the majority of the chapters, about 200 of the most important references are in the printed book; the rest are
available as eReferences as indicated in the text. Similarly, some additional photomicrographs are available as
eFigures.
We have gathered a distinguished group of contributors to this tome whose collective efforts are every bit as
worthy as the three names that appear on the front cover. It is very difficult to find individuals as knowledgeable
as they are who choose to devote their precious time to the arduous task of writing a chapter that conforms to
the goals of this volume as discussed in the preceding paragraph. These authors met and exceeded those
aspirations. Some authors questioned whether the third edition would ever become a reality with legitimate
cause since we all recognize the prolonged gestation of this enterprise. One of us (LPD) had to be coaxed and
even harangued into the yoke but here we are through the monumental efforts of another one of us (ANH). We
are grateful to our authors who are also our colleagues, both past and present. Your patience and perseverance
are appreciated well beyond this acknowledgment.
Louis P. Dehner MD
Aliya N. Husain MD
Preface to the First Edition
Several years ago, the editors of this volume were lamenting the fact that a third edition of Kissane's Pathology
of Infancy and Childhood was unlikely because John Kissane had committed himself to another formidable
publishing enterprise. Our British colleagues in pediatric pathology have authored two fine references (Keeling's
Fetal and Neonatal Pathology and Berry's Paediatric Pathology), but it was our opinion that a comprehensive
volume on all major aspects of the pathologic anatomy of chlldhood disorders ranging from chromosomal
syndromes and neoplasms to forensic pathology was needed. At this juncture in our deliberations, we were
confronted with the daunting nature of the potential task at hand given the required range of expertise necessary
to cover all of these areas. Our attention turned to the reservoir of such abilities that exists in an organization of
which we are privileged to be members, the Society for Pediatric Pathology, formerly known as the Pediatric
Pathology Club. Many of the contributors to this volume are friends and colleagues whom we met through the
Society for Pediatric Pathology.
It was Albert Einstein who acknowledged the fact that we all stand on the shoulders of giants, and certainly
pediatric pathology has evolved to its present state through the seminal contributions of the “first” generation of
North American pediatric pathologists. Some of these include Maude Abbott, Dorothy Andersen, James B. Arey,
J. Bruce Beckwith, Jay Bernstein, William A. Blanc, Robert P. Bolande, John Craig, John R. Esterly, Sidney
Farber, George Fetterman, Enid Gilbert-Barness, M. Daria Haust, John M. Kissane, Benjamin
H. Landing, A. James McAdams, Harry B. Neustein, William A. Newton, Ella Oppenheimer, Eugene V. Perrin,
Edith Potter, Harvey S. Rosenberg, Marie Valdes-Dapena, Gordon Vawter, and F.W. Wigglesworth, to mention
only a few. Virtually all of us in the second and now third generation of pediatric pathologists can call one of
these extraordinary individuals our mentor.
Because we the editors are also the co-authors of several chapters in this textbook, we appreciate firsthand the
many hours that our contributors have invested in the completion of their manuscripts. The time, experience, and
patience necessary to compile information into the concise prose required by a textbook of this type are greatly
appreciated. This textbook belongs to all of these authors collectively, despite the connotations of the cover and
title page.
We also thank our colleagues and friends at our respective institutions for their understanding and support
during the prolonged gestation and difficult delivery of this textbook. At the Armed Forces Institute of Pathology,
these include Robert McMeekin, MD, Robert F. Karnei, MD, Vernon Armbrustmacher, MD, Nancy Roberts,
Luther Duckett, Lisa Penalver, and Venetia Valiga. At the University of Minnesota, Ellis S. Benson, MD, Dale
Snover, MD, and Diane Perez, and at the Washington University Medical Center, Emil Unanue, MD, Mark R.
Wick, MD; Eleanor Grob, and Patricia Dixon are recognized for their special support.
Louis P. Dehner MD
Acknowledgments
Dr. Stocker would like to acknowledge the authors of prior editions who are no longer with us: Drs. Laurence E.
Becker and Patricia A. O'Shea.
Dr. Dehner would like to acknowledge Jeannie Doerr, Margaret Chesney, and Walter Clermont whose tireless
efforts and encouragement brought him back from the lip of the abyss.
Dr. Husain would like to acknowledge the strong support and advice given by Drs. Thomas Krausz and Vinay
Kumar and the excellent secretarial service provided by Dorothy Peoples and Margaret Rietman.
Chapter 1A
The Pediatric Autopsy
J. Thomas Stocker
As described by the Autopsy Committee of the College of American Pathologists, the autopsy is “a medical-
surgical procedure by a physician for the welfare of the living through the study of those patients for whom all our
current knowledge and technology were inadequate”(1). The use of the autopsy in medicine as a tool of
discovery and education has declined frighteningly in the past 25 years, with some newer hospitals not even
including an autopsy suite in their design. In many hospitals, including university hospitals, the autopsy incidence
(autopsies compared to number of deaths) has dropped well below 20%, often reaching as low as 2% to 5%.
Pediatric hospitals have historically had a higher incidence, often as high as 75% or more, but in recent years
this incidence has declined as well. In a survey in 2005 (by the author) of 15 children's hospitals, the autopsy
rate for in-hospital deaths varied from 15% to 48% with an average of 32%, and that figure represented a 5% to
10% drop from the rate in 2000 at these same children's hospitals.
Many excellent textbooks and protocols have been written describing methods for performing an autopsy. The
following is a technique the author has developed over the past 40 years, often incorporating many techniques
from these textbooks and colleagues' experience. This type of autopsy has proven useful to the author, but is by
no means the only procedure that might be used.
THE STANDARD PEDIATRIC AUTOPSY

Autopsy Permit
The first step with any autopsy is examination of the autopsy permit for its completeness. This includes a
determination of the nature of the death of the patient and whether it may be under medical examiner jurisdiction
(i.e., a coroner's case). It is imperative that the pathologist performing the autopsy be intimately familiar with the
criteria for medical examiner jurisdiction in the community in which the patient died. The College of American
Pathology maintains a state-by-state file of these criteria (www.CAP.org).
Following examination of the autopsy permit, the clinical chart of the patient should be reviewed and a call placed
(if possible) to the attending physician or other members of the medical team responsible for the patient. In
addition to a review of the clinical or hospital course of the patient, the medical team should be asked what
questions they might have that the autopsy should address.
A variety of forms and protocols are available for the pediatric autopsy and one of these might be used (see
Appendix) or one designed specifically to address the types of patients in a particular hospital.
Instrumentation
The instruments used in performing the pediatric autopsy are often quite different than those used in adult
autopsies, both in type and the size (Figure 1A-1). Pediatric autopsies, particularly those done on fetuses and
neonates, require smaller and more delicate instruments than the “full-sized” instruments used on larger children
or adults (Table 1A-1). In morgues where adult autopsies are also done, it is often wise to keep the instruments
used for the pediatric autopsy in a separate area, even under lock and key, if necessary, to assure they are not
used (and abused) doing autopsies on adults.
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FIGURE 1A-1 ▪ Top: Small instruments such as these scissors, that are proportional to the size of the infant are
vital to the performance of the autopsy. Bottom: Curved and tapered ends are helpful in dissecting and holding
tissues.
External Examination
The external examination of the body is one of the most important aspects of the pediatric autopsy for it offers
information about the general health of the infant/child, evidence of therapy, and portends what might be
expected when the body is opened. And since, with the exception of skin sections taken for microscopic
examination, the “shell” of the patient will be documented and recorded only as measurements, photographs and
descriptive phrases, accuracy and completeness of the examination are all the more important.
Table 1A-1 ▪ INSTRUMENTS USED IN PERFORMING THE PEDIATRIC AUTOPSY
Scissors
a. Thin, small, with tapered points and curved tip, used more for dissection than cutting. Limit their
use to soft tissues, and organs, not for bone, cartilage, or dense tissues.
b. Medium sized, straight or curved for opening bowel.
c. Large or heavier ones for opening calvarium and vertebral column.
Forceps
a. Small and medium sized, but WITHOUT teeth (which only tears tissue)
Hemostats
a. Small and medium sized
b. Straight and curved
Scalpels
a. No. 10 size curved for most routine work
b. No. 1 size with pointed tip for delicate cutting
c. Double edged, rectangular for sectioning organs such as spleen, lung, liver, kidneys
Knives
a. Straight, of various sizes for sectioning larger organs such as liver, brain, or organs of larger
children
Balances for weighing
a. Standard hanging balance for weighing neonates or small children
b. Electronic balance (accurate to 0.1 g) for weighing organs
Probes of various diameters to establish patency of various openings including nares, ears, ureters,
urethra, biliary tract, heart valves, patency of foramen ovale, and ductus arteriousus.
General measurements include body weight, body length (crown-heel and crown-rump [in neonates]) (Figure 1A-
2), arm span from the tip of the fingers of one hand to the tip of the fingers on the other hand (which in most
cases approximates the crown-heel length) (eFigure 1A-1), and head (occiput to frontal) (Figure 1A-3), chest (at
level of nipples) (eFigure 1A-2), and abdominal (at level of umbilicus) circumference (eFigure 1A-3). This
information can be recorded in the autopsy description or on drawings included in the final autopsy report.
External markings such as needle marks, IV tubes, chest tubes, incisions, abrasions, etc. also need to be
recorded and can be illustrated on standard drawings (Figure 1A-4).
FACE, EYES, EARS, MOUTH (EXTERNAL AND INTERNAL)

Examination of the face begins with an overall view to determine symmetry and gross abnormalities. As facial
abnormalities often predict brain abnormalities, special attention should be paid to midfacial development
(hypertelorism/hypotelorism, nasal bridge deformities) and hair patterns. The hair growth pattern usually consists
of one or two whorls in the upper occipital/parietal area. More than two whorls or actual defects in the scalp
(eFigure 1A-4) are associated with underlying CNS abnormalities. The anterior and posterior fontanelles should
be examined for their size (maximum length and width), shape, and “fullness” (i.e., bulging, depressed) (Figure
1A-5). The neck should be
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flexed and extended as far as possible to determine its range of motion (eFigure 1A-5A,B).
FIGURE 1A-2 ▪ Fullbody view showing crown-hell measurement.

FIGURE 1A-3 ▪ Head circumference is measured in the frontal-occipital plane.
FIGURE 1A-4 ▪ Head examination includes palpation and measurement of the anterior fontanel.
The eyes are examined for both size and location. The measurement of each palpebral fissure should, in a
normal infant, equal the intercanthal distance (eFigure 1A-6, Figure 1A-6) effectively dividing the face at the level
of the eyes into three equal expanses. If the fissure length exceeds the intercanthal distance, the eyes are closer
together than normal (hypotelorism), and conversely, if the intercanthal distance exceeds the palpebral fissure
length, the eyes are too far apart (hypertelorism).
Examination of the eye itself includes the diameter of each pupil and comparison with each other to determine if
the eyes are of equal size (eFigure 1A-7). If one is smaller than the other, microphthalmia may be present. If
there is a question, the eyeballs may be removed from within the cranium after the brain is removed (see CNS
examination). The pupils are also examined for their symmetry and completeness (vs. aniridia) and their color
(which may be difficult to determine in a premature infant).
The nose examination includes its position and shape (e.g., upturned, flat) with evaluation of cartilage
development. A curved probe can be used to determine the patency of the choanae (posterior nasal apertures)
(Figure 1A-7). The lip beneath the nose (the prolabium) should be observed and determined if longer that usual
(associated with the fetal alcohol syndrome).
Examination of the ears begins with determining their position on the side of the head relative to the level of the
palpebral fissures. In near-term and term infants, the tip of the ears should be above the level of the palpebral
fissures (Figure1A-8) or they are considered to be “lowset.” As the ears develop in utero, they “move” upward as
the lower face and jaw develop and expand, reaching and then rising above the palpebral fissure level in late
third trimester.
The ears are also examined for patency of the external auditory canal (via small caliber probe) by pulling down
on the earlobe as the probe is inserted (eFigure 1A-8). The external ear should be evaluated for its shape and
completeness and for the presence of cartilage.
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FIGURE 1A-5 ▪ Check the mobility of the neck by flexing (A) and extending (B) it.
The mouth should be inspected both externally and internally. A finger can be inserted into the mouth to examine
the alveolar ridges of the jaw for the presence (or absence) of teeth and for determination of the shape and
completeness of the palate (eFigure 1A-9). The tongue can be palpated as well, but may also be removed intact
after the thoracic organs have been removed (see below).
ARMS, HANDS, FINGERS

The upper extremities are examined for symmetry, mobility, and the presence of skin lesions. The axilla should
be palpated for the presence of lymph nodes or other masses. The positioning and mobility of the fingers should
be noted along with their length. Some chromosomal anomalies (e.g., trisomies 13 and 18) may produce an
overlapping of the little finger over the fourth finger and the index finger over the third finger (eFigure 1A-10).
Children with Down syndrome (trisomy 21) often display short metacarpals and phalanges, and hypoplasia of the
midphalanx of the fifth finger (eFigure 1A-11). Nails should be examined for the presence of hypoplasia or
dysplasia. Radiographs of the extremities may be helpful in identifying skeletal abnormalities (e.g., radial
hypoplasia of the VATER association) or recent or old fractures. In fact, in cases of suspected nonaccidental
trauma (NAT), a full skeletal survey would be important.
FIGURE 1A-6 ▪ Measuring the intercanthal distance.
The palms of the hands should be examined for aberrant patterning, most notably for the presence of a Simian
crease or a malpositioned axial triradius, common findings in Down syndrome but also seen in a wide variety of
other syndromes.
FIGURE 1A-7 ▪ Examination of the nose includes checking for the patency of each nares into the upper pharynx
with a probe or curved suturing needle.
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FIGURE 1A-8 ▪ Probing the external auditory canal.
CHEST: FRONT AND BACK

Examination of the chest begins with the determination of its symmetry, position of the nipples, and length and
positioning of the sternum (e.g., pectus excavatum or carinatum). The junction of the neck with the chest should
be examined to note the shape and the length of the neck (short neck or webbed skin). The clavicles should be
palpated for degree of development (e.g., hypoplasia) and the presence of fractures. Breast development should
be determined using a system such as the Tanner stage I to V system (2).
FIGURE 1A-9 ▪ Top: A needle is inserted between the upper ribs at an angle parallel to the sternum. Bottom: If
air or fluid is present in the thorax it can be withdrawn and measured (or cultured, if appropriate cleansing is
performed).
Turning the body over or rolling it on its side allows examination of the back for symmetry (e.g., scoliosis,
lordosis) and the presence of lesions. Particularly important in infants is the presence of spinal and vertebral
column defects indicative of meningomyelocele and spina bifida, remembering that one form, spinal bifida
occulta, may not be visible as a skin defect.
At this point, prior to the opening of the chest and abdomen, aspiration of the thorax for air, blood and/or fluid can
be performed. In young children, in particular, the presence of air or fluid in each hemithorax can be determined
as well as its amount. With the body in the supine position, a 12- to 14-gauge needle on a 5- to 25-mL syringe
(depending on the size of the child), can be inserted parallel to the autopsy table at the rib-sternal junction
between the 4th and 5th or 5th and 6th ribs, being careful to avoid the heart (Figure 1A-9). When inserted
through the parietal pleura, aspiration of air or fluid within the free space of the hemithorax can be attempted. If
nothing is present as the syringe plunger is pulled back, the plunger when released will move back toward the
needle. If air or fluid is present, it will be withdrawn as the plunger is pulled back until it can no longer be done. At
this point, the amount of fluid/air in the syringe can be measured and, if a sterile draw has been done, the fluid
may be sent for culture. If the plunger is pulled back to its maximum length, the needle and/or syringe can be
removed, the amount of air/fluid measured and expelled from the syringe, and then reinserted into the same
needle hole for aspiration of as much air/fluid as is left (repeating as many times as needed). The same
procedure can then be performed on the other hemithorax. This allows for an accurate measurement of the
amount of pneumothorax, hemithorax, or transudate/exudate present on each side.
ABDOMEN: FRONT AND BACK INCLUDING ANUS/VAGINA, URETHRA

The shape of the abdomen should be evaluated looking for distension (e.g., ascites or abdominal air),
depression (e.g., secondary to dehydration), and wall thickness (edema, muscular atrophy, etc.). Sterile
aspiration of abdominal fluid may be performed for culture prior to incising the abdominal wall. Signs of
premortem medical intervention such as needle marks or incisions should be recorded. In newborn infants, the
umbilicus should be examined for evidence of inflammation or necrosis and, if present, the stump of the umbilical
cord may be examined for the presence of two umbilical arteries and one umbilical vein. Discoloration of the
abdominal wall may indicate underlying hemorrhage, infection, or gastrointestinal necrosis as in neonatal
necrotizing enterocolitis (eFigure 1A-12).
The external genitalia can be examined for anatomic development and, in infant boys, the presence or absence
of testes in the scrotum should be noted along with the size and development of the penis. A staging system can
be used to describe pubic hair growth in male and females. In females,
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the patency of the vaginal opening may be determined with a probe (eFigure 1A-13). Similarly, the anus should
be probed for patency in neonates, recognizing that anal atresia may be higher that the anal opening (Figure 1A-
10). The presence of meconium is a clear sign of anal patency.
FIGURE 1A-10 ▪ The anus (and penis of vagina) should be probed for patency.
FIGURE 1A-11 ▪ Examination of the foot includes its overall development and configuration (arched versus
“rocker-bottom”) (top) as well as it length (bottom) which correlates with gestational age.
LEGS, FEET, TOES

The lower extremities should be examined for symmetry and length (i.e., in proportion to trunk and arm length). In
infants, the hips can be rotated to determine laxity. Feet should be examined for the presence of an arch to the
sole (Figure 1A-11), versus a “rocker-bottom” configuration as may be seen with certain trisomies (eFigure 1A-
14). Five toes should be present on each foot and the spaces between toes should be of equal depth.
Special Techniques and Studies

Photography. Photography is an integral and highly important part of any autopsy, particularly images of
abnormalities or gross pathology for which microscopic sections may be inadequate documentation. And with the
availability of digital technology, many photographs can be taken with the “excess or unnecessary” images easily
removed at no cost. While fixed photography equipment is useful, a handheld camera is more easily utilized and
encourages the taking of images throughout the performance of the autopsy.
Basic images should include the external surface of the body (front and back) and anterior and lateral views of
the face. Incisions and other surface marks on the face trunk and extremities may be photographed and
documented particularly in cases of suspected NAT. A ruler placed at the edge of the picture helps define the
dimensions of a lesion. Images of internal organs are taken as needed to document anatomic abnormalities or
specific pathologic changes (e.g., necrosis, hemorrhage).
Radiography. Imaging via x-rays, MRI, or CT can be important in diagnosing and documenting skeletal
abnormalities from chondrodyplasias to fractures. They may also be helpful in recording the presence of such
things as pneumothorax, pneumopericardium, and pneumoperitonium, along with documenting the extent of
tumor involvement in metastatic diseases.
LABORATORY TECHNIQUES
Cultures. “Standard” cultures (aerobic and anaerobic bacterial) of blood, lung, and CSF may be taken, or
appropriate cultures (fungal and/or viral) might be determined as the clinical history suggests or as the autopsy
progresses and signs of infection are noted (e.g., cloudy abdominal fluid in peritonitis or aspirated fluid from an
unsuspected cyst or abscess).
Unusual studies are those that extend beyond the limits of the standard autopsy as described in the autopsy
permit, for example, examination of the organs of the chest and abdomen, and the brain. Removal of the eyes or
long bones of the arms and legs might be needed to diagnose diseases of the eyes or to define a particular type
of musculoskeletal dysplasia. (Special permission for these procedures may be needed.)
Cytogenetics. Tissue for cell culture should be taken as soon as possible after death (and having the autopsy
permit signed and witnessed). Chromosome studies should be
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considered in embryos and fetuses when the maternal history or the appearance of the embryo/fetus suggests
the presence of chromosomal abnormalities. “Large” chromosomal abnormalities such as trisomies or deletion of
a major portion of a long or a short arm are often associated with significant external abnormalities such as
midline defects (facial dysmorphia), hand or feet changes (syndactyly, “rocker-bottom” feet), and scalp defects
(see Chapters 3 and 4).
The source of the tissue is dependent on the time after death in which the sample is obtained (see below).
Internal Examination
Chest and Abdomen
Unless the clinical history or appearance of the body suggests otherwise (e.g., a large gastroschisis or previous
thoracic or abdominal surgery with sutured incisions still present), the opening of the body is most commonly
done via a “Y”-shaped incision (Figure 1A-12) or some variation (e.g., “U”-shaped over chest with extension to
the symphysis pubis). In either case, the incision begins in the anterior axillary line at the level of the clavicle and
extends to the xyphoid just below the sternum then up to the opposite anterior axillary line. In a neonate or infant,
the chest incision can be positioned through or adjacent to the nipples to allow sampling of breast tissue while
obtaining a section of skin. Subcutaneous tissue may also be measured (thickness) (eFigure 1A-15) and
observed to determine the state of nutrition of the infant or the state of hydration. Edema can often be noted in
the subcutaneous tissue of the chest. From the point below the xyphoid, the incision is extended toward the
symphysis pubis on either side of the umbilicus.
FIGURE 1A-12 ▪ A “standard” Y-shaped incision is used to gain access to the thorax and abdomen. Note the
gastronomy site in the right upper quadrant.
FIGURE 1A-13 ▪ With the abdomen open, but before removal of the chest plate, the abdominal organs can be
examined. Here the size of the liver is determined by measuring its extension below the lower sternal border, in
this measurment in the right midclavicular line.
The skin of the chest and abdomen is reflected to either side after dissecting it free from the sternum and
thoracic cage (eFigure 1A-16). The abdominal skin is freed along the lower rib margin.
The following measurements may be made prior to removing the chest plate (Figure 1A-13). The size of the liver
is judged by measuring the distance that it extends below the rib margin (assuming no diaphragmatic hernia is
present). Measurements are made in the anterior axillary lines, the midclavicular line, and the midline (eFigures
1A-17 and 1A-18). If the liver does not extend to the left anterior axillary line, the distance it does extend to the
left can be recorded by measuring the distance from the midline to where it disappears beneath the rib margin
(eFigure 1A-19). Other measurements include
1. 1. The distance the spleen tip extends below (or above) the rib margin.
2. 2. The distance the gallbladder extends above or below the margin of the liver—done primarily to see that a
gallbladder is present.
3. 3. The distance the urinary bladder extends above the symphysis pubis.
4. 4. The root and the radius of the mesentery. The root is determined by moving the bowel toward the upper
right quadrant and measuring the length of its attachment to the vertebral column (eFigure 1A-20). The radius
is determined by placing one end of a ruler on the vertebral column where the mesentery attaches and pulling
up a segment of small bowel and measuring the distance from the vertebral column to where it attaches at the
mesenteric border of the bowel (eFigure 1A-21).
5. 5. The amount the diaphragm leaflets are pushed up into the thorax by the abdominal organs. This is done by
placing a finger beneath the rib margin in the right and left midclavicular line and feeling how high the leaflets
extend (Figure 1A-14). This is determined by noting where one can feel one's finger in relationship to a rib or
intercostal space (e.g., 5th intercostal space or 6th rib) (eFigure 1A-22). This measurement is significant for
determining whether the diaphragm leaflets are intact, and whether air or fluid (e.g., blood, pus) in the thorax
have forced the leaflets down.
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FIGURE 1A-14 ▪ The height of the diaphragm is measured by inserting a finger up under the lower sternal
border and palpating its upward extension to the highest rib or intercostal space.
THYMUS
The thymus in infants is often quite large and may obstruct the view of the pericardium and great vessels of the
heart. It is usually helpful to dissect the thymus free from the other chest organs and weigh it before proceeding
to the examination of the heart and lungs. Care must be taken to include the portion of the thymus that extends
“outside” the chest into the cervical tissues of the neck.
BLOOD CULTURE
When a blood-borne infection is suspected, a blood culture may be obtained prior to dissecting the
cardiovascular system. An easy approach is to open the pericardial sac and, after measuring any fluid that may
be present, use a needle and syringe (after searing the surface of the right atrium with a heated spatula)
(eFigure 1A-23A,B) to withdraw blood for culture (Figure 1A-15). Caution: if a cardiac anomaly is suspected, one
may choose to sterilize and withdraw blood from the inferior vena cava just prior to its entering the heart, thus
avoiding damage to the atrium by the heated spatula.
FIGURE 1A-15 ▪ A blood culture can be taken from the inferior vena cava or the right atrium after searing the
appropriate area with a heated spatula.
LUNG CULTURE
Lung tissue for culture may easily be obtained from the right or left lower lobe by immobilizing it with a forceps or
hemostat, searing the surface with a heated spatula, and, using a sterile scalpel, excising a piece of lung tissue
(eFigure 1A-24A to C).
TISSUE FOR CYTOGENETICS

Before taking tissue for examination and culture, contact the laboratory performing the analysis and obtain
appropriate media (such as RPMI) for transportation. Tissues that may be used for cell culture include the
amniotic membranes of the placenta, spleen, and blood for lymphocytes and skin, fascia, pericardium, pleura,
and retroperitoneal tissue for fibroblasts. The sooner this tissue can be placed in the appropriate media, the
better chance for successful growth, but fibroblasts from fascia and skin may often still be successfully harvested
24 to 48 hours after death.
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FIGURE 1A-16 ▪ The cardiac-thoracic ratio is determined by measuring the width of the heart (top) at its widest
point and the width of the thoracic cavity (bottom) at its widest internal point.
CARDIAC/THORACIC RATIO
Prior to removal of the organs of the chest, the width of the heart at its widest point should be measured and
compared to the width of the thorax at the same point (Figure 1A-16). The ratio is helpful in detecting cardiac
anomalies since a ratio of greater than 0.5 is often associated with many of these anomalies. With this initial
suspicion, a “nonstandard” approach to the heart's dissection may be employed (see later).
REMOVING THE ORGANS

Organ-by-organ versus Rokitansky. Removal and examination of the chest and abdominal organs can be done
one organ at a time or by removing the neck, chest, and abdominal organs as one unit (Rokitansky technique).
The latter technique is best performed by beginning in the area of the neck and working caudally. The neck
organs are dissected by working around the larynx, esophagus, and descending aorta, freeing them with blunt
dissection from the soft tissues laterally and behind. Anteriorly, the left brachial artery, the left carotid artery, and
the right brachiocephalic artery may be tied off and transected to allow access to them by the mortician (eFigure
1A-25). When dissection has extended behind and laterally above the larynx, the region above the epiglottis can
be transected allowing the complete larynx with attached esophagus to be pulled inferiorly (eFigure 1A-26A,B).
Following this, the left lung can be pulled aside to allow an incision to be made along the spinal column just
behind the esophagus and aorta. When this procedure is repeated on the right side, the neck organs along with
the heart/lung/esophagus can be pulled forward (eFigure 1A-27). The abdominal organs are mobilized by cutting
the diaphragm (a good time to take a section for microscopic examination) along the contour of the body wall and
dissecting inferiorly along the spinal canal, freeing up the spleen and kidney on the left and the liver and kidney
on the right. Care must be taken to avoid cutting across the ureters as they pass along the sides of the spinal
column before entering the bladder. At this point, the urethra and rectum (and vagina in a female) must be
transected (eFigure 1A-28A,B) and freed from the soft tissue of the pelvis allowing the entire neck, chest, and
abdominal block to be removed intact.
Note: In a premature infant, it is often prudent to remove the ovaries or testes (if undescended) prior to
performing the Rokitansky technique in order to not “lose” them during the dissection (eFigure 1A-29).
It may also be helpful in some cases to perform a “modified” Rokitansky technique and remove one or more of
the organs before removing the entire block (e.g., remove the spleens in cases of polysplenia, or the bowel in
cases of intestinal atresia or duplication).
TESTES/OVARIES
As noted above, it is often easier to remove the ovaries (and testes if undescended) shortly after opening the
abdomen. The small size of an infant's ovaries may make locating them difficult. By finding the uterus and
fallopian tubes behind the urinary bladder, one can locate the ovaries adjacent to the tubes. They can then be
removed, weighed, and often submitted in toto for microscopic examination. Larger ovaries from older infants and
young girls may be hemisected. These often contain small fluid-filled cysts.
Testes that are present in the scrotum may be removed by pressure on the scrotum in the direction of the
inguinal canal, then inserting a forceps into the canal from the open abdomen, pulling on the vas deferens, and
extracting both the vas deferens and testis. This can then be examined for the presence of a vascular
malformation or a hydrocele before dissecting the testis free from the vas deferens and attached soft tissue,
weighing it and submitting a section for microscopic examination.
EXAMINATION OF THE BODY CAVITY

Following removal of the chest and abdominal organs, the body cavity can be examined for abnormalities of the
ribs and spinal cord (Figure 1A-17). Vertebral bodies may be noted to be irregular, for example, butterfly
vertebrae in the VATER association (Vertebral or Vascular anomaly, Anal atresia, TracheoEsophageal fistula,
Renal or Radial abnormality), or out of alignment, for example, scoliosis or lordosis (eFigure 1A-30). Special
attention should be given to the area in which the ribs abut the spinal column as this is a region in which rib
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fractures, both old and recent, may be observed. A section of rib including the costo-chondral junction may be
taken for microscopic examination of bone and bone marrow.
FIGURE 1A-17 ▪ With all organs removed the ribs and vertebral column can be examined for developmental
abnormalities and the psoas muscle can be sampled for histological sections.
VERTEBRAL COLUMN/SPINAL CORD

CSF culture. A culture of the cerebral spinal fluid is relatively easy in an infant and a young child. Once all the
abdominal organs have been removed, the intervertebral disc region of one of the lumbar discs can be seared
with a hot spatula (eFigure 1A-31), and a long needle (in an infant) or a spinal tap needle may be inserted
through the disc into the spinal canal (Figure 1A-18). Aspiration not only supplies fluid for culture but can also be
used for documenting hemorrhage (eFigure 1A-32). In a small infant, the head may need to be elevated to
provide enough fluid in the spinal canal for aspiration.
Section of psoas muscle. The psoas muscles provide an easily accessible source of skeletal muscle and often
include ganglion cells from the paraspinal ganglia (eFigure 1A-33).
Removing the vertebral column. While older children may require spinal cord removal similar to that of an adult,
infants, particularly neonates, have vertebral columns easily removed from an abdominal approach to provide
ready access to the spinal cord. This is accomplished by making an incision through two of the lowest
intervertebral discs and then bending the pelvis backward to allow a pair of round-ended scissors into the
vertebral canal to transect the pedicles on both sides of the vertebral columns (eFigure. 1A-34A to D). As one
moves caudally, the vertebral column can be lifted to allow the thoracic and cervical vertebral to be freed up by
cutting their pedicles. To remove the vertebral column completely, the highest cervical intervertebral disc
accessible can be transected. Upon removal of the vertebral column, vertebral body anomalies may again be
noted and a vertebral body may be taken for microscopic examination following fixation and decalcification.
FIGURE 1A-18 ▪ CSF can be obtained for culture and other purposes by inserting a long needle through the
intervertebral disc of a lumbar vertebra after sterilizing the area.
Removing the spinal cord. With the vertebral column removed, the spinal cord in its dura can be dissected free
by cutting across the spinal nerves exiting through the dura (Figure 1A-19). The spinal cord may be dissected at
this time with cross sections taken from the upper, mid, and lower levels or may be placed in fixative with the
brain for dissection after fixation (eFigure 1A-35A,B).
SEPARATION AND EXAMINATION OF HEART/LUNG

Prior to separating the heart lung block from the abdominal organs, assuming the Rokitansky technique was
used to remove the organs, an examination of the esophagus should be performed. This is done by placing the
block, so its posterior surface is exposed. The entrance to the esophagus
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behind the larynx can then be entered with a pair of scissors and an incision made from the opening to the point
the esophagus passed through the diaphragm. This posterior exposure allows examination of the internal
surface of the esophagus with particular attention paid to the portion of the anterior wall of the esophagus lying
adjacent to the trachea. Esophageal atresia will be easily discovered if present, and the presence of
tracheoesophageal fistula, particularly of the “H” type (see Chapter 12), can be established prior to the
esophagus being separated from the trachea. Following this examination, the upper portion of the esophagus is
separated from the larynx and the mediastinal tissue and left intact for examination with the remainder of the
gastrointestinal tract.
FIGURE 1A-19 ▪ Following removal of the vertebral column (left) the spinal cord along with it dura can be
removed from the spinal canal (right).
Section thoracic/abdominal aorta and inferior vena cava. With the esophagus separated from the thoracic
organs, the descending aorta can be examined for abnormalities (Figure 1A-20) and, if none are present,
transected beyond the arch and freed from the mediastinal tissues to be left with the abdominal organs. This
leaves the chest and abdominal organs attached by only the inferior vena cava, which, when transected as near
to the diaphragm/liver as possible, separates the two blocks.
Examination of the chest block. If the clinical history suggests a cardiac malformation, if the cardiac/thoracic ratio
is greater than 0.5, or if external examination of the heart is noticeably abnormal, consideration should be given
to a “fixed inflation” of the heart (see below) prior to opening the atria and ventricles. If no abnormality is
suspected, the heart may be opened in a standard fashion.
Standard examination of the heart. The heart should be separated from the lungs following identification and
transection of the pulmonary arteries and veins, noting their anatomic relationships (i.e., origin and position). The
heart may then be weighed and examined by opening the chambers along the line of blood flow. This is most
easily accomplished by opening the right atrium between the inferior vena cava and the atrial appendage. This
incision leaves intact the sinoatrial node that is located in the anterior wall of the right atrium just below the
entrance of the superior vena cava. A pair of scissors can be used to cut through the lateral wall of the right
atrium, through the tricuspid valve and along the lateral portion of the right ventricular wall. With the right side of
the heart thus opened, the atrium can be examined for completeness of the foramen ovale and the entrance of
the coronary sinus. The tricuspid can be measured (circumference) and the leaflets inspected, and the right
ventricle can be measured for the thickness of the free wall.
FIGURE 1A-20 ▪ The thoracic aorta and the esophagus can be opened while the abdominal and thoracic organ
block is still intact. Here the aorta is opened along the posterior aspect of the organ block.
The next incision, most easily accomplished with a pair of blunt-nosed scissors, extends up the anterior wall of
the right ventricle adjacent to the septum and along the outflow tract into and then through the pulmonary valve.
This allows examination of the septum for ventricular septal defects and for measurement of the pulmonary valve
circumference and presence of three cusps. With the pulmonary valve opened, the right and left pulmonary
artery branches can be identified as can the ductus arteriosus (for patency, circumference, and length).
The left side of the heart is examined by cutting between the openings of the pulmonary veins and then down the
lateral wall of the left atrium, through the mitral valve and along the wall of the left ventricle. The mitral valve
circumference can be measured and the leaflets observed for orientation and completeness. The left ventricular
wall thickness is determined and the septum is examined for defects. The systemic outflow tract is then opened
with an incision through the anterior wall of the left ventricle adjacent to the septum and behind the mitral leaflet
into the aorta. When opening the aorta, care must be
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taken to move the opened pulmonary trunk aside and make an incision through the aortic valve. The opened
valve circumference may be measured and the three cusps observed, noting the position of the origin of the
coronary arteries above and behind two of the cusps (the right and left coronary sinuses). Finally, the arch of the
aorta is examined for anomalies (e.g., coarctation, patent ductus, etc.). If myocardial infarction is suspected, the
right and left ventricles may be “bread-loafed” remembering that the papillary muscles are often affected first in
infants with myocardial damage.
Fixed inflation and dissection of the heart—Figures EP1-48 The study of an organ by removing, inflating, and
fixing prior to its dissection is useful primarily for examining (and retaining for teaching) the heart but could also
be used for other “hollow” organs such as the small or large bowel and the urinary or gallbladder.
The technique for the heart involves separating the heart from the lungs by tying off (as far from the heart as
possible) all the vessels including the pulmonary arteries, pulmonary veins, superior and inferior vena cavas, and
arteries of the aortic arch, then attaching the heart via canullas to the superior vena cava and a pulmonary vein,
and inflating it under mild pressure (e.g., 20-cm water) with a mixture in four parts to one of 100% alcohol and
37% formalin. Following approximately 24 hours in this fixative, the heart is “opened” by transecting each of the
vessels that have been tied off and opening a series of “windows” in the atria, ventricles, and pulmonary trunk
and aorta above the valves.
The windows begin with a square or rectangular opening in the right atrium, and after examination of the interior
of the atrium and the tricuspid valve, continue with a triangular opening in the right ventricle dictated by any
anomalies that may be observed, for example, an incomplete tricuspid valve or a high ventricular septal defect.
After observing the anatomy of the right ventricle, its outflow tract can be examined from the window into the right
ventricle as well as from a rectangular window opening in the pulmonary trunk just above the pulmonary valve.
The left side of the heart is approached with a window in the left atrium made from incisions connecting the
openings for the pulmonary veins, or, to spare the veins, a window just “inside” the locations of the pulmonary
veins. With the atrium open, the upper aspect of the mitral valve can be observed and, if normal, the left ventricle
can be opened with an incision from the apex of the ventricle, parallel to the ventricular septum, and upward
through the anterior and posterior wall, creating a hingelike opening through which the interior of the ventricle
and the lower portion of the mitral valve can be examined. The outflow tract through the aorta can also be
observed from the ventricular side and, with a rectangular window cut into the aorta above the aortic valve, from
the aortic side.
The incisions involved in creating the windows also allow access to atrial and ventricular myocardial tissue, as
well as aortic and pulmonary artery wall for microscopic sections.
Following dissection, the heart can be processed through various concentrations of alcohol and then xylene as is
performed with other tissues submitted for processing for microscopic sections. The processing may take a day
or more in each solution, and then when the xylene has cleared the heart, it is placed in a paraffin bath under a
slight vacuum, which will help speed the impregnation of the tissue. When removing the heart from the heated
paraffin, it should be rotated in all planes to clear the paraffin from the heart chambers and vessel openings.
Doing this over Bunsen burner with a low flame allows the excess paraffin to exit more rapidly. The heart can
then be cooled slowly and retained for future study or teaching purposes.
Examination of the thymus. This includes weighing and describing it and submitting a representative section for
microscopic examination.
Examination/removal of thyroid and parathyroids. The thyroid is usually readily visible adjacent to the lower
larynx and can be dissected free intact. The weight should be taken and a representative section submitted for
microscopic examination. The parathyroid glands may only rarely be visible in an infant, and to ensure that they
are available for microscopic examination (if clinical history warrants), the entire thyroid gland and adjacent soft
tissue may need to be submitted.
Removal/examination of the tongue. While not necessary or feasible in most cases, removal of the tongue not
only allows more extensive examination of the mouth and nasopharynx but also provides another specimen of
skeletal muscle for microscopic examination. Once the larynx has been removed (or in continuity with the
removal of the chest organs), the tongue may be freed from the mandible by cutting with a scalpel (or preferably
a pair of scissors) along the inner edge of the mandible. Care must be taken to not cut the lips or outside of the
mouth (Figure 1A-21). A safe way to
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avoid this possibility is to use a pair of scissors (rather than a scalpel) and only opening the blades after inserting
the pair of scissors inside the mouth.
FIGURE 1A-21 ▪ The tongue is removed by lifting it with a forceps toward the top of the mouth (top) and then
incision and separating the base and lateral surfaces of the tongue from the floor of the mouth (bottom).
EXAMINATION OF THE RESPIRATORY SYSTEM

Following removal of the heart from the heart/lung block as described above, the respiratory system can be
examined. The pulmonary arteries and veins should be identified and examined for the presence of clots
(emboli). If present, the arteries or veins should be opened along their length into the lung to determine the
extent of the vascular obstruction.
The larynx (with thyroid and parathyroids removed) should be separated from the trachea and then examined for
patency from above and below. It may then be hemisected from anterior to posterior, allowing a view of the vocal
cords and laryngeal mucosa.
The trachea should also be probed for patency and for the size of the lumen throughout. Externally, the cartilage
plates along its circumference should be examined for the presence of complete rings. The trachea may then be
resected at the carina leaving as much as possible of the right and left main stem bronchi.
Lung examination begins by weighing the right and the left lungs separately and noting the lobation of the lobes
(two on the left and three on the right) and their color and consistency (eFigure 1A-36). At this point, it is often
helpful to inflate one of the lungs with formalin by inserting a syringe in the mainstem bronchus and slowly
injecting 10 to 50 mL of formalin depending on the size of the lungs. A hemostat may then be used to close off
the bronchus and the lung placed in formalin for an hour or two (or overnight if possible) before dissecting. The
other lung is examined by gently probing the bronchi and vessels and then sectioning the lung perpendicular to
the hilum. This allows examination of the parenchyma for lesions (cysts, abscesses, areas of consolidation and
hemorrhage). Sections should be taken from obvious areas of pathology as well as from pleural and hilar
regions. While a section may be taken from each of the five lobes, in small lungs a slice of the entire lung may fit
into one cassette.
EXAMINATION OF THE ABDOMINAL ORGANS

In females, separate the uterus and fallopian tubes from the abdominal block (ovaries already removed and
weighed).
Spleen. The spleen may have been removed earlier (see above), but if not, should now be dissected from the
abdominal block with special attention paid to the areas adjacent to the spleen and liver for smaller “accessory”
spleens. If none are present, the spleen can be weighed, sectioned and a sample taken for microscopic
examination (eFigure 1A-37).
Liver. From the anterior portion of the abdominal block, the diaphragm can be removed and the liver examined.
The biliary tract is difficult to dissect in a small infant, but its patency can be demonstrated by making an incision
in the duodenum in the region of the ampulla of Vater. The gallbladder can then be compressed against the liver,
and if the biliary tree is patent, bile can be expressed through the ampulla (Figure 1A-22). Following this, the liver
can be removed from the block, weighed, and sectioned at 1.0-cm intervals with representative tissue taken for
microscopic examination.
Adrenals and kidneys. From the rear of the abdominal block, the aorta can be opened to observe the origin and
patency of the celiac axis, mesenteric arteries, and renal/adrenal arteries as wall as the iliac arteries. The renal
veins can also be observed entering the inferior vena cava and their patency observed. The adrenals can be
dissected from the kidneys, weighed, and sectioned with a cross section taken from each adrenal for microscopic
examination.
The kidneys, ureters, and bladder can be dissected en bloc either with or without the renal arteries and section
of the aorta (eFigure 1A-38). After identifying the origin, course, and entrance into the bladder of each ureter,
each kidney can be removed, weighed, and examined by clearing off the soft tissue from the capsule (without
stripping the capsule) and bisecting the kidney. The cortex and medullary thicknesses are measured and
examined for lesions before sections are taken for microscopic examination. The renal pelvis should be opened
and the entrance to the ureters examined, followed by opening the entire length of the ureters into the bladder.
The bladder itself should be opened and the mucosa examined before a section is taken for microscopic
examination. The urethra can be probed for patency and when opened in a male the prostate can be examined
and a section submitted for microscopic examination.
Note: In cases of suspected urethral stricture or atresia, it may be helpful to remove the urethra along with the
external genitalia, most noticeably the penis in male infants.
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This is accomplished by separating the symphysis pubis and dissecting the penis in continuity with the bladder. It
may also be helpful to fix the penis and distal portion of the bladder and then cross section the specimen and
submit it in its entirety.
FIGURE 1A-22 ▪ The biliary tree can be examined for patency by first opening the duodenum in the region of the
ampulla of Vater then compressing the gallbladder (note thumb over gallbladder) and observing the flow of bile
from the ampulla (at tip of scissors).
Removing, measuring, and sectioning the bowel . The bowel may be removed prior to removing the
chest/abdomen block or when dissecting the abdominal organs. In either event, the bowel is best separated from
the other organs by beginning in the area of the sigmoid/rectum and working toward the stomach, using a pair of
curved scissors to cut along the mesenteric attachment as close to the bowel wall as possible, being careful to
identify (and not cut across) the appendix when working near the cecum. In a small infant, the bowel may be
wrapped around one's fingers as one progresses from the sigmoid to the duodenum. The bowel may be
transected at the duodenum at the point it passes beneath the inferior duodenal fold. The entire bowel can then
be laid out on a cutting board for measuring the length and width of the small intestine, colon, and appendix. If
lesions are identified along the length of the bowel, they may be cross-sectioned and examined, or the entire
length of the bowel may be opened for inspection before sectioning (eFigure 1A-39).
The most proximal part of the gastrointestinal tract (esophagus, stomach, and upper duodenum) along with the
pancreas is then (if not previously done) separated from the diaphragm and liver. The incision in the previously
opened esophagus (see under “Separation and Examination of Heart/Lung”) can be extended through the
gastroesophageal junction, along the edge of the stomach and through the pylorus into the duodenum. Gastric
contents can be observed and a portion saved for further analysis if appropriate. Beyond the pylorus, the
ampulla of Vater is again identified (see liver above) and its relationship to the pancreas observed. The pancreas
can then be dissected from its attachment to the duodenum, weighed, and sections taken from the head and tail
for microscopic examination. With the entire gastrointestinal tract now opened, portions along its length (2 × 1
cm) may be taken (esophagus ×1, esophageal-gastric junction ×1, stomach ×2, small bowel ×3, appendix ×1 and
colon ×2) and placed on paper (Figure 1A-23) for fixation and sectioning at a later time (overnight is best, but
only 1 to 3 hours in 37% formalin is usually sufficient).
Note: In situations in which the bowel is extremely fragile, particularly in cases of necrotizing enterocolitis, it may
be best to leave the small bowel and colon intact with the mesentery, and fix the entire specimen in formalin prior
to dissection.
Central Nervous System

Examination of the scalp. The scalp should be examined for abnormalities in the pattern of the growth of the
hair, looking for two or more swirls of growth; the more swirls or defects in hair growth, the more likely there will
be abnormalities in the structure of the brain. The anterior and the posterior fontanels should be palpated in
infants to check for fullness or depression.
FIGURE 1A-23 ▪ Technique for fixing thin-walled tissue on paper towels before sectioning.
Opening the scalp. An intermastoid, suboccipital incision (Figure 1A-24) allows reflection of the scalp anteriorly
to the level of the eyebrows and posteriorly to below the posterior fontanel (eFigure 1A-40A,B). In young infants,
pushing a finger between the scalp and the calvarium and rolling the skin forward may accomplish this. In older
children, dissection of the tissue between the scalp and calvarium may require a pair of scissors or a scalpel.
Measuring the calvarium. With the fontanels exposed, they may again be palpated and measured (length and
width) (eFigure 1A-41). The calvarium can also be examined for developmental defects, fractures, or
hemorrhage.
FIGURE 1A-24 ▪ The scalp is opened with an incision between the ears at the level below (posterior to) the
crown of the head, that is, an intermastoid suboccipital incision.
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Opening the calvarium. In infants whose calvarium has not completely ossified, the calvarium may be opened
with a scalpel and a pair of scissors along the unfused sutures. Examination of the saggital sinus may be done
by cutting with a pair of scissors through the parietal bone from the anterior fontanel to the posterior fontanel
about one centimeter to each side of the saggital suture. Lifting the edge of the strip left in the middle allows a
view of the intact sinus (eFigure 1A-42A to C).
Extending the incisions parallel to the saggital suture to the anterior and the posterior portions of the calvarium
and then laterally from both ends of the incision into the parietal bone (on both the right and the left sides) until
they are 1 to 4 cm apart (depending on the size of the head), allow both parietal/frontal bones to be reflected
laterally (Figure 1A-25, eFigure 1A-43). By cutting across the anterior extension of the saggital suture and
reflecting it posteriorly, the brain is exposed. The calvarium of older infants and children is removed as one
would for an adult.
Removing the brain. The brain of a small infant is removed from anterior to posterior by placing one's hand
behind the head (with the reflected saggital suture between the middle and the ring fingers and tilting the head
backward (Figure 1A-26)). As the brain falls away from the base of the skull, the cranial nerves, pituitary stalk,
and tentorium can be cut across as they come into view. Eventually, one can see into the spinal canal and insert
a pair of scissors to cut across the spinal cord well below the brainstem. At this point, the brain should easily be
“delivered” into the hand held beneath the head.
Examination of the external brain. Following removal, the brain should be weighed and the external features
examined including the basic development of the cerebral cortex related to the infant's gestation age (see
Addendum), The vessels at the base of the brain may also be examined, but further manipulation of the brain
should be put off until it can be made more firm by fixing in formalin (10% to 37%) for 1 to 2 weeks. Placing the
container with the brain near a source of low heat (e.g., a radiator or a heat vent) may hasten the fixation.
FIGURE 1A-25 ▪ The calvarium can be opened by cutting lateral to each side of the saggital suture and then
along the sutures between the frontalparietal and parietal-occipital bones. After examining the saggital vein the
suture line is reflected posteriorly (bottom) and the parietal bones are reflected laterally to expose the brain.
FIGURE 1A-26 ▪ Remove the brain by tipping the head posteriorly and transecting the cranial nerves, tentorium,
and spinal cord.
Sectioning the brain and spinal cord. The spinal cord, if removed via the abdominal approach, can be fixed
along with the brain. Examination consists of opening the dura along its length and then sectioning the cord at
0.5- to 1.0-cm intervals saving two or more sections for microscopic examination.
The brain after fixation should be examined for gross abnormalities (e.g., area of hemorrhage or necrosis,
developmental anomalies such as holoprosencephaly) and a unique approach to dissection determined by the
abnormalities. In most instances, however, major anomalies are not seen and a more “standard” approach may
be taken. This consists first of examining the vessels at the base of the brain after gently removing the meninges.
The circle of Willis should be identified and any variations recorded. The cerebellum and brainstem can be
removed from the rest of the brain by making a transverse section in the region of the cerebral peduncles
(eFigure 1A-44A,B). In a small infant's brain, this cerebellar/brainstem block may be cut transversely at 0.5- to
1.0-cm intervals to view the cerebellar folia and dentate nucleus along with the lower brainstem (eFigure 1A-45A
to C). In larger brains, the brainstem might be separated from the cerebellum prior to sectioning.
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If significant hemorrhage is present in the cerebral hemispheres, the meningeal arteries may be followed into the
cerebrum to search for a site of an aneurysm or rupture. After the exterior of the cerebral hemispheres has been
examined, the brain is placed “base up” and transverse (coronal) sections made at 1.0- to 1.5-cm intervals
(depending on the size of the brain) from the anterior lobe through the occipital lobe (eFigure 1A-46). If possible,
these sections (often only five or six in infants but as many as 12 to 15 in older children) should include ones
through the stalk of the pituitary, the mammillary bodies, the apex of the interpeduncular fossa, and the top of the
cerebral peduncles. This allows close examination of the numerous nuclei of the deep gray matter.
CNS Microscopic sections. Routine sections of the central nervous system include, but are not limited to, the
following (Figure 1A-27):
1. Brainstem--pons
2. Cerebellum including dentate nucleus
3. Frontal (or occipital) cortex and white matter
4. Hippocampus
5. Internal capsule/posterior limb/thalamus
6. Cervical, thoracic, and lumbar spinal cord
7. Additional sections of specific lesions—for example, tumor, necrosis, hemorrhage
Examination of the inside of the cranium
Removal of the pituitary. The pituitary can easily be removed from the hypophyseal fossa of the sella turcica
after the brain has been removed. The gland is usually quite soft and delicate, and the best approach is made by
using a pair of small curved scissors to dissect around and beneath the gland.
Opening of middle ear. The middle ear can be visualized by removing the petrous portion of the temporal bone
with a pair of heavy (bone) scissors or with saw cuts on either side of the petrous protrusion (Figure 1A-28). With
removal of the bone, the middle ear can be examined for infection (pus or cloudy fluid) and a culture performed if
indicated. The bones of the middle ear (maleus, incus, and stapes) can also be seen.
FIGURE 1A-27 ▪ Standard histological sections from the brain include portions of the cerebrum, hippocampus,
and basal nuclei (arrows) along with sections of cerebellum and brain stem.
FIGURE 1A-28 ▪ The middle ear is exposed by removing the petrous portion of the temporal bone.
Removal of eye/s can be performed from beneath the eyelids by cutting around the orbital septum and palpebral
ligaments holding the eyeball to the bones of the orbit, then dissecting posteriorly to separate the ocular muscles
and transect the optic nerve. Care must be taken to avoid damage to the eyelid and skin of the face.
A less potentially damaging approach is through the opened skull following removal of the brain. Access to the
eye is made by cutting an opening in the superior surface of the orbital plate of the frontal bone (Figure 1A-29).
In a newborn, this can often be done with a scalpel and a pair of scissors but may require a saw in older patients.
When the opening is large enough to accommodate the size of the eyeball, the optic nerve and orbital muscles
can be dissected and visualized, then transected. As the eye is moved posteriorly, the ligaments holding the eye
to the orbit can be cut across
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(with special care taken to avoid cutting the eyelid) and the eye pulled through the opening in the orbital plate.
FIGURE 1A-29 ▪ The opened calvarium provides access to the eyes through the roof of the orbits (indicated
with squares).
Preparing the Remains for Disposition

Following the completion of the gross autopsy examination, all tissues NOT taken for microscopic examination,
longterm storage, or teaching purposes (in accordance with the autopsy permit) should be returned to the body
in a plastic bag of appropriate size. This includes the chest plate and vertebral column. The body and scalp over
the calvarium can be sewn closed, as might be the custom in your area. Consulting with morticians as to their
preference is often helpful. The outside of the body should be appropriately tagged for identification, washed,
dried, and wrapped in appropriate material for transfer to the funeral home.
REFERENCES
1. College of American Pathologists Pamphlet. Autopsy: aiding the living by understanding death. Northfield,
IL.
2. Marshall WA, Tanner JM. Variations in pattern of pubertal changes in girls. Arch Dis Child 1969;44:291-
303.
Chapter 1B
Fine-Needle Aspiration
John J. Buchino
Robert F. Debski
Fine-needle aspiration (FNA) was first reported in the early 1930s by Martin and Ellis (10) and Stewart (12), but
the procedure did not gain widespread acceptance until after Zajicek published his monograph in 1974 (15).
Although several studies and monographs have established the usefulness of FNA in pediatrics, many pediatric
centers have been slow to adopt this technique. However, those that have adopted this technique have found it
to be a relatively easy, low-cost diagnostic procedure that can provide a great deal of information (9). Several
important advantages of FNA are listed in Table 1B-1. It is important that clinicians recognize that FNA is most
applicable in mass lesions, generally not in diffuse processes such as a pulmonary infiltrate. However, Their et
al. have advocated the use of FNA for the evaluation of rejection in children after liver transplantation (13).
Indications for the use of FNA in children are summarized in Table 1B-2. For several reasons, we strongly
believe that a pathologist should perform FNA of all palpable lesions, and that a pathologist should be present
when a radiologist performs image-guided FNA of deep-seated lesions. The pathologist is able to obtain an
accurate history and observe the exact size and location of the lesion. The person performing the aspiration is
best able to evaluate whether the lesion has been penetrated. As one gains experience, the texture of the lesion
and consistency of the aspirated material help in the formulation of a differential diagnosis. The pathologist is
also best able to prepare the smears and triage the aspirated material for other studies.
The equipment required for FNA is listed in Table 1B-3. If need be, it can easily be carried in a phlebotomy tray
to the patient's bedside or, preferably, to a treatment room. For outpatient FNA, we recommend a setting with
adequate room for the patient, parents, and assistants as well as the pathologist. An adjacent area in which rapid
staining and microscopic evaluation can be performed is highly desirable. Although untoward complications are
extremely rare when a superficial lesion is aspirated, the area in which the procedure is performed should be
equipped to handle emergencies, just like any other area in which clinical procedures are carried out.
Relatively few complications are associated with FNA. The most common is bruising or swelling at the site.
Inadvertent puncture of a vessel may result in a small hematoma. However, a history of a possible bleeding
diathesis should always be obtained. A vasovagal response or a light-headedness may occur in a small
percentage of patients. (We have also experienced parents feeling faint when observing the procedure.) A
pneumothorax is possible when a chest wall lesion or a lesion in the supraclavicular space is aspirated. Seeding
of tumor in the needle tract is a markedly rare occurrence (11).
The technique of FNA is outlined in Figure 1B-1. This is essentially the same as the technique used for adults. It
should be noted that use of an aspiration gun and the specific type of gun are optional. In children, the standard
size of the needle for superficial FNA is 1 inch, 23 gauge. A 23-gauge needle recovers adequate material for
diagnosis in more than 90% of cases and is unlikely to cause any significant organ or vessel trauma. A 22-gauge
needle may facilitate the drainage of purulent material but should not be used in regions where a major vessel in
an infant might be sheared (e.g., near the carotid artery). One must also be mindful of spatial differences, such
as decreased chest wall thickness in infants and small children.
Table 1B-1 ▪ ADVANTAGES OF FNA

Can be performed on outpatient basis
Low cost
Rapid diagnosis
No general anesthesia necessary
Minimal trauma with little morbidity
Can be used in conjunction with other diagnostic modalities
(e.g., immunocytochemistry, electron microscopy, microbiologic culture, flow cytometry)
Allows pathologist to have direct patient interaction
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Table 1B-2 ▪ INDICATIONS FOR FNA
Indication Example
Mass lesion of unknown cause Any tumor >0.5 cm
Alternative to surgery High-risk surgical candidate

Child in respiratory distress secondary to an anterior
mediastinal mass
Documentation of a nonresectable Large neuroblastoma-tissue diagnosis necessary before

tumor chemotherapy
Confirmation of a metastasis Pulmonary lesion in a child with previous Wilms tumor
Support of a clinical diagnosis Persistent lymphadenopathy
Preoperative planning Salivary gland lesion
When FNA is performed in children, adequate control of the patient must be maintained so that the need to
attempt to aspirate a moving target does not arise. A skilled assistant is invaluable in this situation. The assistant
is usually able to hold infants less than 1 year of age in the desired position. Children older than 6 years can
generally cooperate well when talked through the procedure. However, children between 12 months and 6 years
can be difficult because of their lack of comprehension of what is happening and their strength. We advocate the
use of sedation whenever possible. Most tertiary-care pediatric services now have sedation teams that are adept
at sedating children for procedures. The choice of sedatives may vary and is somewhat dependent on the
personal preference of the anesthesiologist and/or the pathologist. When sedation is used, the child must be
monitored in the appropriate fashion. If sedation is not available, a papoose wrap may be employed to immobilize
the child. We also use a local anesthetic whenever possible. The only exceptions to the use of a local anesthetic
are a known allergy or a lesion so small that the injected anesthetic will make it difficult to palpate the lesion.
FIGURE 1B-1 ▪ Aspiration technique. A: Insert needle attached to syringe/gun into mass while stabilizing mass
with the other hand. B: Create negative pressure while moving needle back and forth until aspirate is present on
the needle hub. C: Release negative pressure. D: Remove needle from mass. E: Detach needle from syringe
and fill syringe with air. F: Attach needle to syringe and express aspirate onto slide or into medium.
Table 1B-3 ▪ EQUIPMENT NEEDED FOR FNA
Needles (22- to 25-gauge)
Local anesthetic
Syringe
Betadine, alcohol
Aspiration gun (optional)
Gauze
Glass slides
Paper clips
Fixatives
Adhesive bandage
Gloves
Nonbacteriostatic normal saline solution
Flow cytometry medium with heparin
Prior to actually performing the aspiration, we recommend a “time out.” During this time out, the pathologist and
assistant should verify patient identification, the site to be
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aspirated, and that a consent form for the procedure has been signed. The site to be aspirated should have
been marked in the presence of the patient's parents/guardians.
Typically, we perform three separate aspirations to obtain adequate material for cytologic evaluation. The
number of aspirates may vary somewhat depending on the amount and type of material obtained. One of the
significant advantages of FNA is that it can be used to obtain material for studies in addition to cytomorphology.
However, each ancillary study usually requires an additional aspiration, which may be difficult in a child.
The most common condition for which children are referred for FNA is persistent lymphadenopathy. In contrast to
the adult population, malignancy is present in only a very small percentage of children with enlarged lymph
nodes (7). Because infections are the most common cause of enlarged lymph nodes in children, microbiologic
culture, including culture for acid-fast bacilli, of the aspirate can have a significant positive yield (3). Other
studies, such as flow cytometry for the immunophenotyping of lymphoid populations or the determination of the
ploidy of tumors, immunocytochemistry, cytogenetics, electron microscopy, and polymerase chain reaction, may
be performed if warranted by the clinical situation (1, 4).
Several articles and monographs have described various lesions encountered in the pediatric population and
diagnosed by FNA (2, 6, 14). Although it is helpful to be familiar with these, the algorithmic approach offered by
Howell et al. (8) serves as an excellent starting point in the evaluation of FNA smears. For those pathologists
considering initiating an FNA service, practicing on fresh surgical specimens can be helpful to gain experience
with little risk.
One should be mindful of common pitfalls in the diagnosis of lesions by FNA in children. These include (a)
obtaining inadequate material because of inadequate patient control; (b) attempting to aspirate an ill-defined
swelling rather than a discrete, palpable mass; and (c) lacking familiarity with the differential diagnosis of lesions
in children.
The accuracy of an FNA diagnosis in pediatrics varies depending on the type of lesion aspirated and the
experience of the pathologist obtaining and interpreting the specimen. In several published series of pediatric
FNA, the sensitivity has been greater than 90% and the specificity greater than 95% in distinguishing benign
from malignant lesions (5). The percentage of samples inadequate for diagnosis typically ranges from 5% to
10%.
Finally, essential to achieving a sound diagnosis by FNA is clear communication between the clinician and the
pathologist, both before and after the procedure. This was best stated by Dr. Fred Stewart in 1933: “Diagnosis
by aspiration is as reliable as the combined intelligence of the clinician and the pathologist make it” (12).
REFERENCES
1. Barroca H, Carvalho J, Gil da Costa M, et al. Detection of N-myc amplification in neuroblastomas using
Southern blotting on fine needle aspirates. Acta Cytol 2001;45:169-172.
2. Buchino JJ. Cytopathology in pediatrics. In: Wied GL, ed. Monographs in clinical cytology, Vol. 13. Basel,
Switzerland: Karger, 1991:1-7.
3. Buchino JJ, Jones VF. Fine-needle aspiration in the evaluation of children with lymphadenopathy. Arch
Pediatr Adolesc Med 1994;48: 1327-1330.
4. Buchino JJ, Lee HK. Specimen collection and preparation in fineneedle aspirations in children. Am J Clin
Pathol 1998;109:54-58.
5. Drut R, Drut R, Pollono D, et al. Fine-needle aspiration biopsy in pediatric oncology patients. J Pediatr
Hematol Oncol 2005;27:370-376.
6. Geisinger KR, Silverman JF, Wakely PE. Pediatric cytopathology. Chicago, IL: ASCP Press, 1994:4-5.
7. Handa U, Mohan H, Bal A. Role of fine needle aspiration cytology in evaluation of paediatric
lymphadenopathy. Cytopathology 2003;14:66-69.
8. Howell L, Russell LA, Howard PH, et al. The cytology of pediatric masses: a differential diagnostic
approach. Diagn Cytopathol 1992;8: 107-115.
9. Howell L. Changing role of fine-needle aspiration in the evaluation of pediatric masses. Diagn Cytopathol
2001;24(1):65-70.
10. Martin HE, Ellis EB. Biopsy by needle puncture and aspiration. Am Surg 1930;92:169-181.
11. Postovsky S, Elhasid R, Weyl Ben Arush M, et al. Local dissemination of hepatocellular carcinoma in a
child after fine-needle aspiration (Letter to the Editor). Med Pediatr Oncol 2001;36:667-668.
12. Stewart FW. The diagnosis of tumors by aspiration biopsy. Am J Pathol 1933;9:801-812.
13. Their M, Lautenschlager I, Willenbrand E, et al. The use of fine-needle aspiration biopsy in detection of
acute rejection in children in after liver transplantation. Transpl Int 2002;15:240-247.
14. Vielh P, Howell LP. Techniques. In: Kline TS, ed. Guides to clinical aspiration biopsy. Pediatrics. New
York, NY: Igaku-Shoin; 1994:5-8.
15. Zajicek J. Aspiration biopsy cytology. Part I: cytology of supradiaphragmatic organs. New York, NY:
Karger, 1974.
Chapter 1C
Molecular Techniques in Pediatric Pathology
Jason A. Jarzembowski
D. Ashley Hill
INTRODUCTION
Rapid advances in the understanding of the molecular and genetic basis of disease have led to an increasingly information-rich and complex
working environment for the pediatric pathologist. In addition to providing insight into the pathology and biology of disease, these advances have
led to improvements and refinements in diagnosis, risk stratification, prediction of outcome, determination of eligibility for new targeted therapies,
and gene-based screening for disease risk. Molecular techniques have become the standard of care in the pathologic evaluation of hematopoietic
diseases, pediatric tumors, infectious diseases, immunodeficiencies, metabolic diseases, and chromosomal/genetic disorders. Pediatricians and
surgeons, armed with the latest literature on the gene expression profile of a given set of tumors or a newly described mutation associated with a
congenital defect, are anxious to apply these new discoveries to their patients’ specimens. With a solid understanding of disease morphology and
pathogenesis coupled with access to advanced technology and tissue resources, pediatric pathologists are in an advantageous position to utilize
this wealth of information in a manner that is clinically important to today’s patients. Here, we discuss several molecular techniques focusing on
relevance to the standard practice of a pediatric pathologist. We include a broad overview of the technical aspects of each methodology with the
utility of each method illustrated by applications to specific pediatric diseases. Because detailed descriptions of all techniques and all relevant
diseases are beyond the scope of this chapter, we refer the reader to key references and helpful websites for a more in-depth discussion.
Tissue Handling
The appropriate management of complicated pediatric specimens submitted to the pathology laboratory begins well before the slides cross the
microscope stage. Even before the child is in the operative suite, it is fundamental that pathologists participate in the preoperative treatment
planning. Establishing an open line of communication with the referring physicians and surgeons will ensure that the pathology team is well
prepared for special handling requirements. This is a good opportunity to consider the differential diagnosis and plan ahead for appropriate
specimen transport; intraoperative assessment of tissue adequacy, preliminary diagnosis, and margin evaluations when necessary; and tissue
requirements for potential ancillary testing and clinical trial enrollment (Table 1C-1). Debski and colleagues have written an excellent review on the
approach to handling pediatric tumors that applies to other specimen types as well (Figure 1C-1) (9).
Specific Molecular Techniques

Most pathology laboratories today have a sizeable arsenal of molecular tools that can be deployed to assist in diagnosis, predict treatment
efficacy, or provide other pertinent clinicopathologic information. The first group of assays described—flow cytometry, immunohistochemistry (IHC),
and immunofluorescence—are all protein based and predicated on the specific recognition of antigen by antibody. Those in the second group are
all nucleic acid dependent and include traditional cytogenetics, in situ hybridization, polymerase chain reaction (PCR), and the recently developed
and continually evolving worlds of microarray technology and next-generation sequencing (NGS).
FLOW CYTOMETRY
Background
Since its inception in the 1970s, flow cytometry has gained widespread acceptance and is today considered an essential component of the
diagnostic workup of hematopoietic neoplasms and immunodeficiency disorders. This methodology allows for rapid identification of cell surface
molecules and
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their coexpression patterns, thus separating subpopulations of cells such as monocytes from lymphocytes, B- from T-lymphocytes, or CD4+ from
CD8+ T-lymphocytes. Aberrant marker profiles or absolute cell counts can also be easily determined. The diagnosis and classification of leukemias
and lymphomas are thoroughly discussed elsewhere in this book. Herein we describe the general theory and method with special attention to
specimen processing and practical applications.
Table 1C-1 ▪ TYPICALTISSUE REQUIREMENTS FOR COMMONLY USED MOLECULAR DIAGNOSTICTECHNIQUES
Methodology Tissue Format Tissue Amount
Fresh Frozen FFPE
Flow cytometry <1 cm3 ✓
FISH/ISH 4-10-μm sections ✓

Immunofluores-cence 4-10-μm sections ✓
Immunohis-tochemistry 4-10-μm sections Some ✓
Cytogenetics <1 cm3 ✓
PCR Varies ✓ ✓ ✓
Microarray <1 cm3 ✓ ✓
FFPE, formalin-fixed paraffin-embedded; PCR, polymerase chain reaction
Method
The most common specimens submitted for flow cytometry are peripheral blood, bone marrow aspirates, cerebrospinal fluid, and lymph nodes. For
peripheral blood (except for paroxysmal nocturnal hematuria [PNH] studies) and bone marrow specimens, erythrocytes are removed by lysis or
differential centrifugation. A portion of the sample is spun onto a slide for assessment of cell viability and possible contaminating debris. From this,
total cell counts can be estimated, which determines how many analysis tubes can be run; approximately 106 cells are needed for optimal results
from a typical reaction tube. When testing will be applied to solid samples, the tissue should be immediately transported on saline-moistened
gauze or in fresh RPMI medium to the laboratory. Touch preparations for cytological evaluation are useful in guiding the triage of the sample for
light microscopy, flow cytometry, cytogenetics, and storage in a -80°C freezer for subsequent studies. Flow cytometry requires a small (3 to 5 mm3)
piece of viable tissue placed in fresh RPMI or similar medium. If subsequent processing will be delayed for more than an hour or so, the tissue
should be finely diced to maximize exposure to the nutritive medium and prolong viability (10). Once at the flow cytometry laboratory, the tissue is
carefully teased apart and separated into a single-cell suspension (23, 24, 57).
FIGURE 1C-1 ▪ Recommended triage and sampling protocol for pediatric specimens.
For the next step, cell aliquots are mixed with surface antigen-specific antibodies that have been conjugated to fluorescent dyes, such as
phycoerythrin (PE), fluorescein isothiocyanate (FITC), and phthalocyanines (PC5, PC7). After a short incubation to allow the conjugated
antibodies to bind their target surface antigens on the cells in the aliquot, the sample is loaded into the flow cytometer (Figure 1C-2). The cell
suspension flows through capillary tubing, eventually streaming single file through the detection chamber. Here,
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one or more lasers “interrogate” each cell, determining the forward scatter (roughly correlating with size), the side scatter (roughly correlating with
cytoplasmic complexity or granularity), and a measurement of fluorescent dyes reflecting expression of a particular protein by the cell. Multiple
antibodies can be combined in each tube to the extent that they each have a distinct fluorescent tag. For example, a single tube for profiling
lymphocytes might contain CD3-PE, CD19-PC5, and kappa-FITC; the expression profile of these four surface markers can be quantitated
separately because each has a different associated dye. Detection of each fluorescent signal requires a separate channel on the device, so that a
four-channel flow cytometer can analyze four markers per tube, and a five-channel machine can observe five molecules in concert. In addition to
demonstrating the coexpression patterns of these markers on distinct cellular populations, multichannel technology also minimizes the necessary
sample size and shortens analysis time by reducing the number of tubes needing to be run. Nonetheless, modern cytometers can analyze cells at
flow rates exceeding 1,000 cells/second.
FIGURE 1C-2 ▪ Overview of a typical four-channel flow cytometry setup. The antibody-bound cell suspension flows through the cytometer with
cells passing singly through the laser beam(s). Forward scatter (to the right) is determined by the intensity of the light passing directly through the
cell, whereas side scatter (to the bottom) depends on the intensity of reflected light. A series of filtering mirrors (left) selectively reroute light of
the desired wavelength toward the detectors and allow the remainder of the beam to pass. Each antibody under investigation is conjugated to a
tag that fluoresces at a different wavelength. Thus, each detector effectively measures a specific antibody and, together, four antibodies, forward
scatter, and side scatter are measured simultaneously for each cell. Data are compiled by a computer processor and presented as interactive
scattergrams.
The data obtained from each cell are recorded as an event, theoretically allowing the user to look at the individual profile of each cell in the
specimen. By selecting populations of cells with a certain range of expression for a particular marker (“gating”), the relative frequency and
coexpressed markers can be visualized (see Figure 1C-3, panel D). For example, one might initially gate on CD45+ cells (leukocytes only), and
then observe the CD3+ and CD20+ cell populations to assess the relative B- and T-cell numbers. T-cells might be gated into CD4+ and CD8+
groups. If the T-cells coexpressed both markers, one might suspect an immature T-cell neoplasm such as pre-T-ALL, and would then investigate
other markers such as CD5, CD10, and TdT. In such a stepwise fashion, each cell subpopulation can be examined for abnormalities.
The interpretation of flow cytometric data is a dynamic process (19). Tabular reports that list the markers analyzed and the percentage of cells
positive are unable to capture the complexity of such data. It is good clinical practice to review the expression patterns of the cells of interest,
seeing where they lie on each plot and correlating the flow cytometric patterns with the microscopic appearance and results of other ancillary
studies.
Applications
Flow cytometric analysis is invaluable, not only in the diagnostic workup of leukemias and lymphomas, but in a myriad of other applications, as
well. For example, in lieu of the traditional Kleihauer-Betke test, the degree of fetomaternal hemorrhage can be accurately determined using flow
cytometry with antibodies directed against fetal hemoglobin (7, 12). The diagnosis of PNH can be made by demonstrating an absence of GPI-
linked proteins such as CD55 and CD59 (25, 39). For some diseases, it is important to enumerate classes of lymphocytes such as monitoring
CD4+ cell counts in HIV-infected patients, or other specific subtypes that may be lacking (such as in various immunodeficiencies) or present in
excessive numbers (such as CD3+, CD4-, CD8-, “double negative” T-cells in autoimmune lymphoproliferative disorder) (47, 51).
IMMUNOHISTOCHEMISTRY
Background
Over the course of a single decade, IHC has rapidly gained acceptance and became standard of care in most anatomic pathology laboratories.
IHC boasts high sensitivity, specificity, and resiliency. Unlike immunofluorescence or many molecular techniques, IHC can be performed on
formalin-fixed, paraffin-embedded tissue (FFPE). This greatly enhances its utility, especially on cases with limited material and in retrospective
studies. Finally, automated stainers can easily perform IHC with minimal human hands-on time. Such machines have reduced the relative cost of
IHC in many laboratories.
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FIGURE 1C-3 ▪ Burkitt lymphoma. Although just a small round blue cell tumor at first glance, the H&E-stained section (A) shows uniform cells with
scant cytoplasm and a high mitotic rate along with interspersed tingible body macrophages. The lesional cells are positive for CD19 (B), identifying
them as B-lymphocytes with a near-100% proliferation rate (C, MIB-1 IHC). Flow cytometry (D) shows lymphoblasts that are negative for CD34 and
CD117 (blastic and myeloblastic markers, respectively), but positive for the B-cell markers CD 10 (moderate), CD 19, and CD20 (bright) and
kappa-restricted (suggesting monoclonality). Conventional cytogenetic analysis (E) revealed an abnormal karyotype with a t(8; 14) characteristic of
Burkitt lymphoma, as well as an extra copy of 1q attached to the short arm of 21. FISH was performed on both metaphase (F) and interphase
nuclei (G) using MYCC (chr 8, red) and IGHprobes (chr 14, green) and demonstrates several fusion genes (in yellow). Extra green signals
indicate occasional gain of chromosome 14. (Flow cytometry plots kindly provided by Dennis W. Schauer, Clinical Immunodiagnostic and
Research Laboratory, Department of Pediatrics, Medical College of Wisconsin. Karyotype and FISH analysis courtesy of Dr. Peter vanTuinen,
Dynacare Clinical Cytogenetics Laboratory, Medical College of Wisconsin.)
Method
The principle of IHC is simple enough and involves a primary antibody specific for the antigen of interest, a secondary antibody that not only binds
the first antibody but is also conjugated to an enzyme, and a colorimetric indicator such as a dye that is formed or changes color via the action of
the aforementioned enzyme. Thus, a molecular linkage is formed that localizes a readily discernible color (typically, brown or red) to the vicinity of
the antigen of interest (Figure 1C-4) (31).
For most diagnostic and research applications of IHC, FFPE tissue is used (3, 14). The application of microwave or steam heat to the tissue prior
to the staining process (“antigen retrieval” or “unmasking”) can improve sensitivity of the technique by reducing or reversing formalin-induced
crosslinking of proteins (which modifies or blocks some epitopes required for antibody recognition). A few special antibodies have been optimized
for use with frozen tissue sections, and these require the forethought to reserve a piece of the specimen for this purpose.
The primary antibody is the primary determinant of IHC reactivity. The choice of antibody is guided not only by the target antigen, but also by the
desired sensitivity/specificity, the reaction conditions, and the cost and reliability of product from a given manufacturer. Different antibody clones
react with different portions of proteins and may yield strikingly different IHC results. Monoclonal antibodies (where all antibody molecules
recognize a single epitope) are usually more specific than polyclonal sera (where the antibody molecules recognize a variety of antigenic epitopes
on a single protein); however, the former are therefore more vulnerable
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to false-negative results when the epitope is obscured (by protein interactions or misfolding) or absent (via mutations). Perhaps the most
pronounced difference between monoclonal and polyclonal antibodies is seen with carcinoembryonic antigen (CEA); fewer than 5% of all
hepatocellular carcinomas stain with monoclonal CEA antibodies, whereas roughly 70% stain with polyclonal CEA antibodies in a cytoplasmic or
canalicular pattern (34).
FIGURE 1C-4 ▪ Overview of immunohistochemical staining. Tissue slides (A) are incubated with a primary antibody specific for the antigen of
interest (B), to which a secondary antibody conjugated to a marker is then bound (C). Detection is then achieved by a colorimetric or light-
producing assay (D).
The optimal reaction conditions, such as antibody concentration and incubation time, must be determined for each new antibody and existing
protocols should be tested with each new lot of an established antibody. Excessive concentrations of antibody or prolonged incubation times may
allow nonspecific binding, whereas insufficient antibody or time can yield false-negative results (21).
The choice of secondary antibody is in large part dictated by the primary antibody; the two must originate from different species so that the
secondary antibody recognizes the constant portions of the primary antibody. Some manufacturers offer a secondary antibody that reacts against
primary antibodies from multiple species. The variety of signaling methods and permutations thereof is too numerous to describe here. Suffice it to
say, the secondary antibody is conjugated to a signal molecule—a dye, an enzyme, or a fluorescent marker—such that the location of the antibody
“sandwich” can in some way be visualized.
One of the most common detection reactions involves horseradish peroxidase (HRP), which can convert a chromogen (such as TMB or DAB) into
a brown-colored product. This method is susceptible to high background signal created by endogenous peroxidases found especially in
erythrocytes and granulocytes; these cytoplasmic enzymes can react with the dye precursors to produce a signal indistinguishable from the
intended one, except for its localization. In order to quench these enzymes and lower the background noise, pretreatment with methanol and dilute
hydrogen peroxide (or other related methods) is used to denature these culprits without significantly inhibiting the subsequently used HRP.
However, endogenous peroxidase activity may still hinder interpretation in enzyme-rich tissues such as spleen and bone marrow. Regardless of
the specific methods employed, the end result is a color change localized to the antigenic sites of interest. A light counterstain, such as
hematoxylin alone, hematoxylin and eosin, methyl green, or periodic acid Schiff, is often employed to allow background architecture and unstained
cells to be discerned.
The final step is careful interpretation. The positive control should show strong, specific staining on a section of tissue known to contain the
antigen of interest. Ideally, this positive control tissue should be present on the same slide as the case tissue section. Likewise, the negative
control, usually performed on an additional slide of the actual case material, omits the primary antibody and should demonstrate the lack of
nonspecific binding of secondary antibody. Each case tissue section has internal controls built in, as well, in the form of adjacent normal
constituents—blood vessels, connective tissue, or epithelium. The staining patterns of these should be confirmed before interpreting the staining
of the areas of interest. Interpretation should include consideration of the quality, quantity, and patterns of staining. Proteins can be nuclear,
cytoplasmic, and/or cytoplasmic membranous. Knowledge of the expected pattern of antibody staining in particular tissues is important for quality
control.
As an ancillary diagnostic method, IHC results are usually reported as part of a more comprehensive report. Within the “Microscopic Description”
or “Comment” section, the performed IHC stains should be described (in tabular or textual format) including the name of the stain, the results with
lesional cells, and verification of controls. For example, “Properly controlled immunohistochemical stains demonstrate that the lesional cells are
positive for a, b, and c, but negative for x, y, and z.” Depending on personal and institutional preference, an explanation of these results and how
they support the diagnosis may then be appended. Most laboratories automatically add a note detailing whether these stains are FDA approved
for clinical use or are investigational only, which may affect the interpretation of, or payment for, these services.
Applications
The most common use for IHC is as an ancillary method in the diagnosis and detection of tumors and identification of tissue. Sometimes, its
purpose is to detect or highlight a single population of cells—for example, ganglion cells (for the ret protein, in biopsies of suspected Hirschsprung
disease) or endodermal sinus tumor components (by a-fetoprotein staining) of mixed germ cell tumors or teratomas (44, 48, 50). Some IHC stains
lend insight into the origin and portend outcome of tumors, such as EGFR and p53 expression patterns, which distinguish de novo pediatric and
adult glioblastomas, and differ between de novo and progressive glioblastoma in adults (35, 56). The results of other IHC tests can direct and
optimize treatment by confirming the presence of target molecules, for example, estrogen and progesterone receptor status for antihormonal
therapy in adult breast carcinoma, and CD20 surface expression for rituximab in leukemia/lymphoma and autoimmune disease
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(52, 58). As with any ancillary technique, the results of IHC alone should not determine the diagnosis or treatment but, rather, should be
interpreted in the context of the morphologic appearance and clinical history.
ANTIGENS
Cytoskeleton
Three main groups constitute the cytoskeleton of human cells: thin, intermediate, and thick filaments. Thin filaments (5 to 6 nm) are composed of
α-, β-, and γ-actins; the former are exclusively found in muscle cells and can be distinguished by antibodies such as muscle-specific actin (MSA;
HHF35), smooth muscle actin (SMA), and smooth muscle myosin heavy chain (SMMS-1). For example, the IHC staining pattern differentiates
between nonmuscle cells and tumors (MSA- SMA- SMMS-1-), skeletal muscle myocytes and rhabdomyosarcomas (MSA+ SMA- SMMS-1 —), and
smooth muscle myocytes and leiomyosarcomas (MSA+ SMA+ SMMS-1+). Myoepithelial cells and myofibroblasts also stain positively for all three
markers, although to varying degrees.
Diagnostically speaking, the most useful cytoskeletal proteins are the intermediate filaments (10 nm). The relative composition of intermediate
filaments varies by cell type and allows distinction by IHC. The major intermediate filaments include vimentin, desmin, glial fibrillary acidic protein
(GFAP), and cytokeratins.
Vimentin can be found in all mesenchyme-derived cells— fibroblasts, myocytes, osteocytes, chondrocytes, Schwann cells, endothelial cells, and
hematopoietic elements—often leading to its dismissal as “nonspecific.” Nonetheless, a vimentin stain serves well to distinguish sarcomas and
lymphomas from carcinomas. Even with the most poorly differentiated neoplasms, this distinction can usually be made. Vimentin IHC is also of
great utility in confirming that tissue antigenicity has been preserved; most sections have at least focal areas of vimentin-positive cells. Necrotic
tissue can be surprisingly informative, as it often maintains some degree of reactivity, often in the original pattern of distribution. In these cases,
careful comparison with control tissue and nontumoral tissue in the section is necessary to ensure accurate interpretation.
Desmin shares sequence homology with vimentin and is likewise restricted to mesenchymal cells. However, unlike vimentin, desmin is only
expressed at significant levels in smooth, skeletal, and cardiac myocytes. Thus, in a sense, desmin is a marker of myogenic differentiation;
although the aforementioned cells contain desmin, primitive mesenchymal cells and neoplasms do not. Desmin-positive tumors include
leiomyomas, leiomyosarcomas, and rhabdomyosarcomas. Of special note, desmin expression in most cardiac myocytes is limited to the
intercalated discs, whereas the Purkinje fibers show diffuse cytoplasmic staining.
Glial fibrillary acidic protein (GFAP) is relatively specific for astrocytes and their corresponding neoplasms— astrocytomas, glioblastomas, and
other gliomas (5). Reactive astrocytes are markedly positive and care must be taken to ensure that such a population of cells is not mistaken for
the actual neoplasm (5, 11, 60). Ependymal cells and their derivative neoplasms show variable reactivity for GFAP. Neurofilament is actually a set
of three related proteins that form fibers within the cell bodies and processes of neurons; the main diagnostic utility of a neurofilament IHC stain is
to highlight neurons within tissue or tumor.
Epithelial cells are easily distinguishable by the presence of distinct cytokeratin profiles. Carcinomas are positive when using broad-spectrum
cytokeratin antibody “cocktails” such as AE1/AE3 or CK7/CK20, which can rule out most lymphomas and sarcomas. Important exceptions include
the glandular component of biphasic synovial sarcoma (Figure 1C-5, panel C) and the characteristic cytoplasmic inclusions of malignant rhabdoid
tumors; the latter stain for cytokeratin, not muscle markers, belying their epithelial origin. More specific antibodies can help highlight organ-specific
epithelium, for example, CK19 in breast or biliary tract.
Thick filaments (20 to 25 nm) are composed of β-tubulin and are ubiquitous to all cell types. Thus, their diagnostic utility is limited.
Cell Surface Markers

Cell surface antigens have proven utility not only in IHC, but also in flow cytometry and cell sorting. However, while flow cytometry requires fresh
tissue or cell-rich fluid, the same markers can be evaluated on FFPE tissue by IHC. These antigens are indispensable in the diagnosis of
hematopoietic neoplasms, and such use is detailed elsewhere in this book (see Chapter 22).
Many of these cell surface molecules are numerically designated as a “cluster of differentiation,” or “CD.” For example, the normal constituent cells
of the immune system can be roughly grouped by their expression of these proteins: B-lymphocytes (positive for CD19, CD20 [L26], CD79a;
Figure 1C-3), T-lymphocytes (positive for CD3, CD4, CD8), and natural killer cells (positive for CD56). Myeloblasts stain for CD34 and CD117 (c-
kit), two markers also associated with other neoplasms; a CD34 stain is positive in synovial sarcoma and some vascular tumors, and CD117
positivity is an important finding in gastrointestinal stromal tumors (26).
Macrophages and histiocytes exhibit granular staining in their cytoplasm for CD68, a component of lysosomal membranes (more accurately, a
lysosomal surface marker); CD 1a is specific for Langerhans cells and T-lymphoblasts (6, 22, 29, 46). Mast cells have membrane positivity for CD
138, as well as cytoplasmic positivity for a characteristic enzyme, tryptase (30). This enzyme can also be detected by histochemical methods that
require frozen tissue.
Endothelial cells, both nascent and tumoral, can be marked with CD31 and CD34, although these antibodies mark some other cells, as well.
GLUT-1 is a relatively specific marker for the endothelial cells of infantile hemangiomas (45).
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Pathogens
A wide variety of viruses can be detected by antibodies specific for well-conserved antigens, including adenovirus, cytomegalovirus (CMV), herpes
simplex virus (HSV) types I and II, parvovirus B19, human herpesvirus 8 (HHV8), human papilloma virus, BK virus (via large T antigen), hepatitis B
(via surface antigen), and Epstein-Barr virus (EBV, via the latent membrane protein [LMP]). Typically, clinical suspicion, positive serologic testing,
or viral cytopathic change seen on routine H&E-stained slides serves as a trigger for further workup by IHC. Companion in situ hybridization tests
are available for both HPV and EBV (Figure 1C-6, panel D); these tests can identify the former as high- or low-risk subtypes. Whenever possible,
viral IHC should be performed in tandem with culture and serologic testing (64).
IHC is less commonly used to identify bacteria, both because of these organisms’ patchy and often sparse distribution in tissue and the greater
detection sensitivity of microbiologic culture methods (2, 64). However, antibodies against Helicobacter pylori have supplanted more traditional
Steiner, Giemsa, or Alcian Yellow stains at some institutions (16, 33). Also, Pneumocystis jiroveci, once thought to be protozoal but now formally
classified as a fungus, can be easily highlighted by appropriate antibodies in IHC (38, 62).
FIGURE 1C-5 ▪ Biphasic synovial sarcoma. The H&E-stained sections (A,B) demonstrate a spindle cell sarcoma with areas of glandular
differentiation; the latter are immunohistochemically positive for mixed cytokeratins (C) and the majority of the tumor cells are positive for bcl-2 (D).
Conventional cytogenetic analysis (E) demonstrated t(X;18) pathognomonic for the SYT-SSX fusion gene of synovial sarcoma. Breakapart FISH
probes (one red, one green from opposite ends [5’ and 3’, respectively] of the SYT gene) are seen separately instead of together as a single intact
yellow signal (as seen in the surrounding normal cells) (F). (Karyotype and FISH analysis courtesy of Dr. Peter vanTuinen, Dynacare Clinical
Cytogenetics Laboratory, Medical College of Wisconsin.)
Hormones
IHC can assist in confirming the diagnosis and hormone secretion profile of many endocrine tumors. For example, pancreatic endocrine neoplasms
(islet cell tumors) can be categorized as derived from alpha, beta, delta, or G cells based on immunohistochemically verifiable expression of
glucagon, insulin, somatostatin, or gastrin, respectively. Likewise, VIP-producing tumors and serotonin-secreting carcinoids can be demonstrated
by IHC.
In conjunction with clinical presentation, pituitary adenomas can be easily classified by IHC profiling for prolactin, adrenocorticotrophin hormone
(ACTH), thyroidstimulating hormone, growth hormone, follicle-stimulating hormone, and luteinizing hormone (1, 32). This approach is especially
useful with silent adenomas, which have
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detectable hormone(s) in the tumor cells’ cytoplasm, but not in the patient’s serum.
FIGURE 1C-6 ▪ Classic Hodgkin lymphoma.The H&E-stained section (A) reveals scattered large cells with atypical, convoluted nuclei in a
mixed inflammatory background. At lower power, fibrous bands were seen entrapping nodules of tumor. By IHC, the Hodgkin cells are positive for
CD15 (B) and CD30 (C). In situ hybridization for EBV encoded RNA (EBER) is positive in many of these cells (D, red staining).
Medullary thyroid carcinoma stains positively for calcitonin, as do normal C-cells and the hyperplastic foci of multiple endocrine neoplasia
syndrome. More generally, thyroid epithelial cells can be highlighted by antibodies to thyroglobulin or thyroid transcription factor-1 (TTF-1).
Parathyroid hormone stains are useful in identifying parathyroid tissue, although normal, hyperplastic, and neoplastic tissues react identically.
Although less often useful in the pediatric realm, IHC for estrogen and progesterone receptors has become standard of care in the evaluation of
breast cancer, serving to guide the choice of chemotherapy. Germ cell and sex cord tumors can express a-inhibin and β-human chorionic
gonadotrophin (β-hCG), and the serum levels of these markers are sometimes used to monitor patients for recurrence.
Embryonal and Cancer Markers

Fetal tissues and neoplasms share expression of primitive traits, including a subset of proteins normally restricted to developmental periods. For
example, a-fetoprotein can be found in fetal liver as well as hepatoblastomas, hepatomas, and endodermal sinus tumors. Placental alkaline
phosphatase (PLAP) stains germ cell tumors and some carcinomas. The stem cell marker OCT4 is now replacing PLAP as a more sensitive and
specific marker of germ cell neoplasms, specifically seminoma/germinoma, embryonal carcinoma, and intratubular germ cell neoplasia. Among the
so called cancer markers, CA-125 is more specific for genitourinary neoplasms, whereas CA19-9 is preferentially expressed in gastrointestinal
cancers. Both these markers are also detectable in patient serum.
Protooncogenes
Anaplastic lymphoma kinase-1 (ALK-1) is the fusion product of a characteristic t(2;5) translocation found in most anaplastic large cell lymphomas
and inflammatory myofibroblastic tumors; its expression in the former is thought to portend favorable prognosis. Tyrosine kinases, such as c-kit
(CD117), can help diagnose tumors such as gastrointestinal stromal tumors, as well as predict which lesions might respond to monoclonal
antibody therapy (in this case, imatinib).
Cell Cycle and Apoptotic Markers

The most commonly used antibody in this category is MIB-1 (Ki67), a proliferation marker frequently used to assess the
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proliferative activity of lesions. Although a high MIB-1 index does not define something as neoplastic, it can be used as a corroborating piece of
evidence in making such a decision, or in determining the histologic grade of a tumor (Figure 1C-3, panel C). Some apoptotic markers, such as
Bcl-2 and Bcl-6, have utility in identifying the lineage of hematopoietic neoplasms and other tumors (Figure 1C-5, panel D). β-Catenin, a molecule
involved in the Wnt signaling pathway, is highly expressed in desmoid tumors, as well as colorectal lesions with aberrations of the APC pathway.
Other
Alpha-1-antitrypsin (A1 AT) is expressed in normal and neoplastic liver tissue, as well as in yolk sac tumors. Its primary diagnostic utility, however,
is in identifying A1 AT deficiency manifest as strong cytoplasmic positivity in the setting of hepatic or pulmonary disease; recall that the disorder
affects A1AT export, not production, so the mutated protein accumulates intracellularly. Alpha-1-antichymotrypsin is also a serine protease inhibitor
(serpin), and is found in histiocytes and pancreatic and salivary duct epithelium.
Limitations
As mentioned previously, immunohistochemical stains are only useful when done properly and in a well-controlled fashion. Evaluation of
appropriate positive and negative controls, as well as internal controls, is required every time a stain is run in order to ensure validity. Minor
changes in reaction conditions or the antibody supplier can lead to major changes in results.
IHC staining patterns can only be interpreted in the context of the H&E morphology, the clinical scenario, and the results of other ancillary tests.
Basing a diagnosis on a single immunostain can be risky. Performing a panel of five stains that are 80% specific is bound to result in at least one
stain with spurious results.
IMMUNOFLUORESCENCE
Background
Direct immunofluorescence (DIF) is a molecular technique that provides ancillary information in the diagnosis of dermatologic, renal, and transplant
organ disease. DIF relies on the same antibody-antigen recognition as flow cytometry and IHC. The most common uses in pathology include
detection of immunoglobulins, complement proteins, and fibrinogen in patient tissue sections or infectious organisms in other samples
(Pneumocystis spp.) (40).
Method
DIF requires fresh or snap frozen tissue as formalin and other aldehyde-derived fixatives alter the antigenicity of immunoglobulins, complement
proteins, and other molecules of interest (27). Ammonium sulfate-based buffers that inhibit tissue proteases can be used to transport specimens
from the procedure areas to the testing laboratory (13, 20, 41, 43, 54, 59). Tissue is then snap frozen in OCT, sectioned in a cryostat, air dried,
rehydrated, and incubated with the appropriate antibody solution. If DIF is not needed immediately, it is helpful to know that tissue antigens are
stable in OCT for up to 4 months at either -20°C or -70°C (40).
FIGURE 1C-7▪Overview of immunofluorescent staining. For DIF (A), slides bear tissue with native antibody of interest already bound (1). A
secondary antibody conjugated to a fluorescent tag is then bound to the original antibodies (2), and then detected by laser-induced fluorescence
(3). DIF (B) is nearly identical to IHC (see Figure 1C-4), except detection requires laser excitation.
For most renal and skin biopsies, a typical immunofluorescence panel will include antibodies directed against IgG, IgA, IgM, C1, C3, fibrinogen,
C4d (in renal transplant), properdin, and albumin—the latter as a positive control. Antibody binding and staining is similar to that for IHC but uses a
fluorochrome rather than a dye for detection (Figure 1C-7). Stained slides are stored in a dark refrigerator until reading, which should occur as
promptly as possible (within 24 hours) as fluorescence intensity diminishes with time.
Applications
The most common specimens that routinely involve immunofluorescence staining are for medical kidney disease and autoimmune skin disorders
(Figure 1C-8). In addition to the particular stains that are positive (e.g., IgG versus IgM), the pattern (linear versus granular) and the location
(basement membrane versus dermal-epidermal junction) of staining are also important in distinguishing between specific entities. (See Chapters
17 and 25 for detailed information about DIF in kidney and skin lesions.) Ultimately, the simultaneous consideration of all available information—
clinical presentation, laboratory studies, H&E-stained sections, and DIF—is required to maximize diagnostic sensitivity and accuracy.
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FIGURE 1C-8▪DIF for IgA in a renal core biopsy from an 11-year-old girl with gross hematuria and nephrotic-range proteinuria. Glomeruli showed
diffuse IgA deposits in the mesangium and granular deposition in the capillary loops which, in conjunction with the light and electron microscopic
findings, was consistent with IgA nephropathy.
FLUORESCENCE IN SITU HYBRIDIZATION (FISH/ISH)

Background
Increasing numbers of tumors and congenital disorders are being identified as monogenic in nature, and their underlying genetic alterations are
being discovered. In turn, this has yielded a large number of genetic targets that can be used in the diagnosis, prognosis, and therapeutic
selection of these diseases. As such, a variety of molecularly based genetic assays have been developed to investigate and analyze these targets.
Instead of relying on the specificity of an antibodyantigen reaction, as in IHC and IF, FISH and ISH instead utilize the base complementarity
between a labeled oligonucleotide probe and a DNA or RNA sequence of interest.
One of the main advantages of FISH/ISH is its in situ nature—target sequences of interest are precisely localized within specific cells, the
nuclear/cytoplasmic distribution can be compared, and the relative chromosomal location of two genes can be identified. This is in contrast to
techniques such as PCR, microarrays, or cytogenetics, which by necessity destroy the architecture to get at the DNA, RNA, or chromosomes,
respectively. Another benefit of FISH/ISH is the high specificity of nucleic acid strand interactions, which can easily distinguish between closely
related gene products. Alternatively, probes can be designed against conserved sequences to detect multiple isoforms or products, for example,
multiple strains of a single virus or common population variants of a polymorphous locus. Linking the probes to a fluorescent tag (in FISH) amplifies
the signal and increases the sensitivity of the assay without affecting the specificity. Finally, these methods are usually suitable for use with
formalin-fixed, paraffin-embedded tissue, allowing their application in cases with limited material as well as in retrospective research studies.
Methods
The optimal tissue source for FISH/ISH assays depends on the target molecule; frozen tissue is preferable for RNA studies, whereas formalin-
fixed, paraffin-embedded tissue is suitable for DNA work as well as for some small, stable RNAs. Standard 4- to 10-μm sections are cut on a
cryostat or microtome and, for FFPE, deparaffinized and rehydrated. Other potential specimen sources include touch preparations and nuclear
spreads from conventional cytogenetics (Figures 1C-3, 1C-5, and 1C-9); in both instances nuclei are fixed by alcohol- or aldehyde-based methods.
Slides are overlaid with a small amount of buffer containing the desired probe, and incubated from several hours to overnight. Probes can be RNA
or DNA, and can be synthesized in-house from templates or purchased from a variety of commercial vendors; some of the latter sell kits with
multiple probes and controls appropriately packaged together for a particular disease entity or differential diagnostic workup.
Sections are then washed to remove unbound and nonspecifically bound probe. Then, for ISH, the detection reaction is run to create a color
change at the site of binding, akin to that described for IHC (Figure 1C-6, panel D). For FISH, sections are examined under a fluorescent
microscope to detect the labeled probe (Figure 1C-3, panels F and G; Figure 1C-5, panel F; Figure 1C-9, panel F; and Figure 1C-10, panels B and
C). Appropriate controls include probes against “housekeeping” genes, or the centromeres of uninvolved chromosomes, as well as evaluation of
the analytic probes in “normal” or uninvolved cells.
Applications
FISH and ISH have wide utility in a broad range of applications. The most common may be in the detection of chromosomal translocations in solid
and hematopoietic neoplasms. This analysis can be approached in two ways: looking for the fusion product, or looking for the destruction of the
original gene (“breakapart”). For example, in some cases of Burkitt lymphoma (Figure 1C-3, panels F and G), the MYCC and IGH genes are
involved in a t(8; 14) translocation. In the assay pictured, the MYCC probe is conjugated to a red tag and the IGH probe to a green tag. In normal
cells, two separate red signals and two separate green signals should be present. However, in Burkitt cells harboring the translocation, one set of
red and green signals overlaps producing a yellow fusion signal. For the breakapart strategy, the principle is reversed: normal cells should have
two intact signals, but translocation will destroy one of those signals and create two new signals. For example, in synovial sarcoma (Figure 1C-5,
panel F), the central tumor cell has two separate SYT signals (one red and one green, from opposite
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ends of the gene) instead of a single intact yellow signal as seen in the surrounding normal cells.
FIGURE 1C-9 ▪ Neuroblastoma. On an H&E-stained section (A), small round blue cells with occasional early gangliocytoid differentiation, copious
neuropil, and a low mitotic-karyorrhectic index are seen. The tumor cells are immunohistochemically positive for PGP9.5 (B), NB84 (C), and
synaptophysin (D), confirming the diagnosis of neuroblastoma. A metaphase chromosomal spread (E) shows numerous double minutes in the
background, which are FISH-positive for MYCN (F), indicating amplification. (Chromosomal and FISH analyses courtesy of Dr. Peter vanTuinen,
FISH can also be used to detect copy number changes of a gene or locus, such as MYCN amplification in neuroblastoma (Figure 1C-9, panel F)
or loss of one copy of the hSNF5/INI1 locus in malignant rhabdoid tumor (4). In the case of MYCN amplification, this method is able to detect
amplification whether via intrachromosomal sequence duplication or extrachromosomal double minutes (as shown).
FISH is invaluable in the examination of stillbirth and intrauterine death, especially in cases of delayed delivery where tissue quality is
compromised. In the example case of a fetal demise with dysmorphic features (Figure 1C-10), the bottom two panels (B and C) show a standard
FISH workup for common cytogenetic abnormalities in this setting— analysis of chromosomes 13, 18, 21 (most commonly implicated in stillborn
trisomies), and X and Y. As shown, the fetus is triploid with three clear signals for chromosomes 13,
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18, 21, and X. Not only is this study diagnostic, but essential because cells did not grow for conventional cytogenetics and karyotyping (see
Chapter 3).
FIGURE 1C-10 ▪ Fetaltriploidy. A 29-year-old G1P0 female underwent medical pregnancy termination at 20 weeks’ gestation following ultrasound
diagnosis of intrauterine growth retardation, Dandy-Walker malformation, and ventricular septal heart defect (A) (Courtesy of Pat Rogers,
Children’s Hospital of Wisconsin, Audio Visual Services). Amniocentesis and FISH analysis had been performed, demonstrating triploidy (69, XXX).
FISH probes in (B) include chromosomes 13 (red) and 21 (green) and, in (C), chromosomes 18 (aqua) and X (green); three copies of each
chromosome are present, and no copies of the Y chromosome [redprobe in (C)] are identified. (FISH analysis courtesy of Dr. Peter vanTuinen,
Nonfluorescent (colorimetric) ISH is often used as a nucleic acid version of IHC with otherwise similar techniques and applications. Most
commonly, ISH is used for detecting nucleic acids of infectious agents, such as HPV, HSV, and EBV (Figure 1C-6, panel D). ISH tends to have
higher sensitivity and specificity than IHC for the companion viral proteins (18).
CYTOGENETICS
Background
Conventional cytogenetic analysis, or karyotyping, is a wellestablished technique that gives a broad genetic overview at a chromosomal level. It
can identify constitutional disorders and demonstrate abnormalities that aid in diagnosis with or without providing insight into prognosis and
therapeutic effectiveness. Despite the development of increasingly sophisticated and sensitive molecular assays, cytogenetics maintains a crucial
diagnostic role because of its capability to detect a wide range of abnormalities at once using a simple and cost-effective procedure.
Method
Typical specimens submitted for cytogenetic analysis include tumor or lymph node tissue, skin biopsy as a source of fibroblasts, whole blood or
peripheral blood mononuclear cells, and prenatal samples from chorionic villus sampling or amniocentesis. For conventional cell culture and
karyotyping, fresh viable tissue is required. For tumors and lymph nodes, a small (1 cm3) piece of grossly viable lesional tissue usually suffices.
For fetuses or neonates, a placental biopsy, taken superficially from the cleansed fetal surface, is usually an acceptable surrogate; however, care
must be taken to prevent contamination of the sample with maternal cells (decidua or blood).
Samples should be immediately placed in standard tissue culture medium (such as RPMI) and kept at room temperature until transport to the
cytogenetics laboratory. Often, testing can be delayed until initial workup of the case is completed—for example, with most tumors we routinely
save a tissue sample in medium until the H&E-stained slides can be reviewed, and the necessity of cytogenetic testing can be
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evaluated. Most tissue is stable and viable for several days in culture medium at 4°C, although the risk of bacterial or fungal contamination
increases over time unless antibiotics are included in the formulation of the medium. Another alternative is to submit the tissue to the cytogenetics
lab for tissue culture and to make the decision about proceeding with karyotyping at a later date.
Upon receipt in the laboratory, specimens are disaggregated and the cells are allowed to grow in culture for several days until they reach a
sufficient number of actively dividing cells. At this point, a mitotic inhibitor is added that arrests the cells in metaphase with chromosomes neatly
condensed and separated. Cells are cultured long enough for as many cells as possible to reach the stage of mitotic arrest, without reducing
viability. The cells are then chemically treated to preserve the chromosomal integrity, fix the nuclei, and remove the cell membrane and cytoplasm.
The nuclear preparation is then placed onto slides for staining and analysis.
Several different stains and procedures can be employed, but the most commonly used method is Giemsa staining (G-banding; see Figure 1C-3,
panel E, and Figure 1C-5, panel E), which utilizes a limited trypsin digestion before staining with the same DNA-binding dye used elsewhere in
histology, thereby producing light-and-dark bands across each chromosome. Other protocols utilize other chemical treatments and other dyes to
specifically stain telomeres (T-banding), heterochromatin (C-banding), or AT-rich sequences (Q-banding). Each method produces a characteristic
banding pattern that can be compared to known reference standards. G-banding typically yields roughly 400 bands across the genome for
analysis, but higher resolution banding can discriminate smaller regions but requires preparation of less condensed chromosomes, such as those
in prometaphase instead of metaphase, and involves a more lengthy analysis. Standard G-banding has a detection limit of about four megabases;
deletions or additions of smaller amounts of DNA may not be identified by this method.
Regarding terminology, the pattern produced by a particular method is compared to a standard reference (the Paris nomenclature) where bands
are numbered according to their location on the chromosome — arm, region, band, sub-band, sub-sub-band, etc. The short arm is dubbed p (petit)
and the long arm is q (queue). Bands (p11, p12, p13,…), sub-bands (p12 divided into p12.1, p12.2, p12.3,…), and sub-sub-bands (p12.2 divided
into p12.21, p12.22, p12.23,…) are numbered from the centromere outward (the centromere can be considered to be both p10 and q10); p21.23
would be more telomeric than p21.22. Note that this terminology places band p3.25 between p3.1 and p3.3, as a sub-sub-band belonging to sub-
band p3. A band’s location is preceded by its host chromosome, such as 5q23.1, which is an area located roughly halfway out on the long arm of
chromosome 5, and is properly described as “five q two three point one.” Karyotypes are denoted in writing as the total diploid number of
chromosomes, followed by the identities of the sex chromosomes, and then details regarding abnormalities. For example, boys and girls would
usually have constitutional karyotypes of 46,XY, and 46,XX, respectively, while a female patient with Cri du chat syndrome might instead have a
46,XX,del(5p) karyotype.
Chromosomes are examined under a microscope and their banding patterns are compared within each chromosomal pair (22 autosomal pairs and
2 sex chromosomes in each examined mitotic spread) and to the reference standards looking for aberrations in number, size, and/or composition.
Standard karyotyping can detect numerical changes in chromosomes (e.g., monosomy or trisomy), duplication or loss of chromosomal material,
translocations, and other disorders. Typically, the chromosomes from 20 different nuclei are examined, assuring a representative sampling in order
to exclude mosaicism or a small percentage of abnormal cells, such as occasional tumor cells within a preponderance of normal cells.
Chromosomal abnormalities detectable by conventional cytogenetics are numerical or structural in nature. The former includes common
constitutional disorders such as Down syndrome (trisomy 21), Edwards syndrome (trisomy 18), and Patau syndrome (trisomy 13), in all of which
patients have a third, additional copy of an autosome (e.g., a girl with Down syndrome would have a 47,XX,+21 karyotype). Constitutional triploidy,
three copies of all chromosomes (e.g., 69,XXX) is much less common than isolated trisomies, and is almost always embryonic lethal (Figure 1C-
10). Monosomies, such as Turner syndrome (45, XO), are readily detected. Again, standard analysis includes 20 cells because sporadic loss of a
chromosome (or other aberration) can occur during sample preparation; an abnormality should be consistently seen in multiple cells before it is
considered real. Tumors often show aneuploidy with varying numbers of each chromosome, in addition to structural aberrations.
Structural chromosomal problems occur in many different forms and appear to be the result of DNA damage and/or faulty repair. Some are
balanced, in which no net material is lost, but sequences are simply rearranged; this includes translocations and inversions. Others are
unbalanced, with net loss (deletions) or gain (duplications) of genetic material. These include inversions, deletions, additions, ring chromosomes,
translocations with loss of one derivative chromosome and combinations thereof.
Translocations involve swapping of material between two or more chromosomes, usually without net loss (reciprocal and balanced). However, the
rearranged genes can be separated from their regulatory sequences and be aberrantly expressed, or can instead be combined to produce a novel
fusion protein. The former is exemplified by common translocations involving the MYC proto-oncogene in Burkitt lymphoma, such as t(8;14)
(q24;q32), which places MYC under constitutive expression of the immunoglobulin heavy chain promoter, instead of its usual tightly controlled
regulation (Figure 1C-3). A classic example of the latter mechanism is the Philadelphia chromosome, t(9;22)(q34;q11), seen in a subset of adult
and pediatric leukemias, which juxtaposes
P.34
the BCR and ABL genes to create a novel BCR-ABL fusion protein with dysregulated tyrosine kinase activity that drives oncogenesis. Note the
terminology used for such events: “t” for translocation, followed by the involved chromosomes (9 and 22), and the regions or bands involved (q34
from chromosome 9 and q11 from chromosome 11); thus, the karyotype of a Philadelphia chromosome-positive pediatric ALL might be
46,XY,t(9;22)(q34;q11). Some balanced translocations are constitutional, but because the overall genetic content of the cells is unchanged, there
is no problem detected in the carrier of a particular translocation. The problem occurs when offspring inherit only one of the abnormal parental
chromosomes incurring an unbalanced genotype and, therefore, disease. A good example of this is a subset of Robertsonian translocations
implicated in some cases of Down syndrome.
Applications
Probably the most common application of conventional cytogenetics is analyzing constitutional karyotype. This usually occurs prenatally, using
fetal cells obtained by chorionic villus sampling or amniocentesis, or in the neonatal period using a blood sample. Such information may guide
prenatal care, anticipate difficulties in the neonatal period, portend outcomes, or guide future family planning. In instances of fetal loss or stillbirth,
samples of skin or placenta should be sent for cytogenetics as part of the standard workup, especially if dysmorphic features are noted. In all
cases with abnormal genetic results, and in many cases with normal karyotypes, parental referral to a genetic counselor is helpful.
Cytogenetic analysis can also provide important information in the evaluation of many neoplasms. A particular translocation may be identified that
is pathognomonic for a given tumor (Figures 1C-3 and 1C-5), while other genetic aberrations may provide information on prognosis or therapeutic
efficacy. For example, 95% of cases of acute promyelocytic leukemia bear a t(15;17)(q22;q12) abnormality that, besides being a diagnostic finding,
can be used in molecular tests to monitor recurrence and also offers a therapeutic target—the fusion protein that results from this translocation,
PML-RARa, appears to convey sensitivity to all-trans retinoic acid. (37) Other prognostic genetic markers include 1p/19q loss in
oligodendrogliomas, 1p/16q loss in Wilms’ tumor, 6q/17q loss in medulloblastoma, and 1p loss and the previously mentioned MYCN amplification
in neuroblastoma.
Limitations
The major limitations of conventional cytogenetic analysis are threefold: the requirement for fresh, viable tissue with cells that can grow in a culture
environment, the variable length of time for cells to grow and be analyzed, and the relatively low resolution of detection (four megabases). Assays
such as FISH and PCR can circumvent the need for growing cells, can be done in 1 day, and provide higher resolution than cytogenetics. On the
other hand, they are considerably more expensive and are designed for targeting precise molecular abnormalities. In many cases, the methods
may be more complementary than competing. Despite continued methodological advances in molecular pathology (see below), karyotyping still
has a major role as a simple, cost-effective method of examining the entire genome at low-resolution for numerical or structural abnormalities.
POLYMERASE CHAIN REACTION

Background
The purpose of PCR is to create millions of copies of a specific segment of DNA so that it can be analyzed by its sequence, size, and
complementarity to other sequences. It is used regularly in molecular diagnostics, forensic science, and research laboratories. Variations of the
basic principles of PCR have led to numerous advancements in our ability to quickly and cost-effectively detect DNA sequence variants associated
with specific diseases.
Methods
PCR sensitivity is best on fresh, snap frozen samples but the technique can also be applied to formalin-fixed paraffin embedded tissue. First, DNA
is extracted from the sample of interest. If starting with RNA, total RNA is extracted and then converted into cDNA (complementary DNA) by an
enzyme called reverse transcriptase (RT), the so-called RT-PCR. This DNA or cDNA is then mixed with free nucleotides (dNTPs), buffer,
thermostable DNA polymerase, and two short, sequence-specific oligonucleotides called primers (Figure 1C-11). Primers are approximately 20
base pairs long and are designed so that one primer is complementary to the top strand of DNA at one end of the target segment and a second
primer is complementary to the bottom strand of DNA at the other end of the target segment. Within this mixture, the target DNA or cDNA is then
amplified through cycles of denaturation (at high temperatures such as 95°C), annealing (at lower temperatures defined by primer-template
nucleotide sequence, 55°C to 65°C), and elongation (70°C). The denaturation stage separates the DNA into single strands, to which the primers
can then bind during the annealing phase. During elongation, the polymerase uses the target DNA strand as a template to lengthen the primers,
creating complementary double-stranded molecules; these products then serve as additional targets in the next round, allowing exponential
amplification.
The amplified product is then subjected to gel electrophoresis and staining (with ethidium bromide or fluorescent analogues) where the relative
size of the DNA can be determined by comparison to known standards. Confirmation that this product is the sequence of interest can be done by
transferring the DNA from the gel to a nylon or nitrocellulose membrane, applying a radioactively or fluorescently labeled probe
P.35
P.36
(similar to a primer, a probe is a short sequence of DNA designed to match a sequence internal to that of the primer pairs), and then placing the
membrane on x-ray film. If the probe matches the sequence on the membrane, the label will expose the x-ray film at the location of the band. This
process is known as Southern blotting. Some laboratories choose to clone and sequence PCR products for confirmation rather than blotting.
Other laboratories do not perform either of these confirmatory steps; these laboratories may be at risk for reporting false positives.
FIGURE 1C-11 ▪ Schematic of PCR methodology. A comparison of conventional PCR (left column) and real-time PCR (right column).
Conventional PCR involves denaturing of double-stranded DNA (or cDNA), annealing of primers, and elongation steps (A,B). The detection of
amplified product typically involves agarose gel electrophoresis (C). D, E: Real-time PCR uses the same features of denaturation, annealing of
primers, and elongation but adds a probe complementary to sequence in between the two end primers. This probe is labeled with both fluorescent
reporter and quencher dyes that when in close proximity do not emit a signal. As the strand elongates from the 5’ primer, the probe is disrupted
and cleaved, releasing the reporter dye into the solution. Once the reporter is no longer in proximity to the quencher dye, its fluorescent signal is
detectable. Additional reporter dye will be released with each cycle and is proportional to the accumulated amount of amplified product. A detector
measures fluorescence in real time and the results are viewed in graphical form with quantity of signal on the y-axis and number of PCR cycles on
the x-axis. The horizontal line indicates a threshold level beyond which there is exponential accumulation of signal, confirming that the specific
DNA product is obtained. (D and E adapted from Applied Biosystems’ TaqMan literature.)
Real-Time Polymerase Chain Reaction

Real-time or quantitative PCR is a variation on standard PCR that was first described by Holland et al. (28, 55). It follows the same basic principles
of standard RT-PCR, but utilizes the 5’ exonuclease activity of the Thermus aquaticus (Taq) DNA polymerase coupled with fluorescence energy
transfer (Figure 1C-6). First DNA is extracted from the sample of interest. If starting with RNA, the RNA is extracted and reverse transcribed into
cDNA. The target cDNA or DNA is amplified in a mix containing not only a set of forward and reverse oligonucleotide primers designed to amplify
sequences specific to the gene of interest but also a probe designed to match a sequence internal to that of the primer pairs. The probe is labeled
with a reporter fluorescent dye at the 5′ end and a quencher fluorescent dye at the 3′ end. The quencher dye acts to decrease emission of the
reporter dye as long as they are in close proximity to each other (on the ends of the same molecule). During the elongation phase of PCR, both
primers and probe anneal to the target sequence if present. As the 5′ primer is extended, the 5′ exonuclease activity of the Taq DNA polymerase
releases the fluorescent reporter dye once it reaches the 5′ end of the annealed probe; now separated from the 3′ quencher, fluorescence from the
5′ dye increases. As the specific product accumulates, additional probes anneal and then release more fluorescent signal with each PCR cycle.
Rather than visualizing the PCR product after agarose gel electrophoresis and staining, the product is visualized in real time on a computer that
plots the intensity of the reporter’s fluorescent signal. Signal intensity is directly proportional to the amount of specific amplicon produced with each
cycle of amplification. This method is capable of providing highly sensitive detection of target DNA and rapid results. Because of the elimination of
postprocessing steps such as nested PCR or Southern blot confirmation, the risk of cross contamination in association with carryover PCR
product is reduced and there is no need for radioactive materials.
Applications
PCR has numerous applications for pediatric pathologists primarily in tumor pathology, microbiologic speciation of organisms, genetic testing for
mutations and forensic identification.
Detection of Fusion Gene Transcripts

One of the first applications developed for pathology was detection of fusion gene transcripts resulting from translocations in hematopoietic and
solid tumors (Table 1C-2). Although many of these translocations are also detectable by cytogenetics, the latter technique requires fresh tissue
and growing cells. FISH is another method for detecting gene fusions/translocations and may perform superiorly to RT-PCR when only FFPE is
available. The benefit of RT-PCR over both those techniques is that it preserves the ability to obtain sequence information from the fusion gene
product. A few studies have suggested that in some tumors, the fusion type may have prognostic importance, thus designing the test to distinguish
between variants may have some additional clinical utility (36, 55). Also, RT-PCR can detect fusion genes in the setting of complex translocations
involving small amounts of DNA (<400 kb) below the resolution of conventional chromosomal banding and karyotyping.
Molecular Microbiology
The use of PCR technology has transformed the clinical microbiology laboratory. For many microbial infections, PCR techniques have replaced
standard culture or immunoassay identification (17). Real-time PCR is particularly appealing for use in microbiology for its speed over current
methods (results in hours rather than days), efficacy (the ability of an organism to grow in culture is not an issue with PCR), and accuracy for
speciation. Further, because real-time PCR is performed in a closed system, meaning that there is no open handling of amplified DNA products,
there is a greatly decreased risk of crosscontamination. PCR-based sequencing can add additional utility to the microbiology laboratory. The
sequence of 16S ribosomal RNA appears to be unique among microbial species and can be used to accurately identify organisms such as
mycobacteria as well as to identify new pathogens (53). Further, PCR-based assessment of antimicrobial resistance genes may become more
commonplace as information from microbial research laboratories makes its way to clinical application (63).
Genetic Testing for Mutations

In the last decade, hundreds of diseases have been attributed to abnormalities in the DNA sequence code. Base substitutions and insertions and
deletions of coding sequences alter the production of or change the nature of proteins in a manner that is associated with particular diseases.
Duchenne muscular dystrophy, cystic fibrosis, and neurofibromatosis type 1 are common examples of monogenic diseases that are amenable to
PCR-based genetic testing, but there are many more in the categories of metabolic diseases, neurologic and muscular diseases, and cancer
predisposition syndromes. Sequencing for commonly mutated exons (socalled mutational hot spots) in affected children or offspring of affected or
carrier parents provides important information
P.37
P.38
P.39
P.40
P.41
P.42
P.43
P.44
to the treating physician and parents, but full sequencing of large genes can be complex and arduous with routine PCR technology.
Table 1C-2 ▪ MOLECULAR TESTING IN PEDIATRIC DISEASES
Immunohistochemistry
System Tumor Genetic Pertinent Positives Pertinent Notes

Aberration Negatives
Head and Neck Congenital epulis — — S100 S100(−)

unlike other
granular cell
tumors
Sinonasal papillomas — HPV, in a subset |β- — —

Nasopharyngeal Some associated catenin — —
angiofibroma with APC mutations
and/or FAP
Osteomas May be associated — — —

with Gardner
syndrome
Teratoma — Varies by component AFP to rule out —
yolk sac tumor
component
Nasopharyngeal carcinoma HLA associations EBV/EBER; mixed Cytokeratins 7 —

Adenoid cystic carcinoma cytokeratins and 20
Adenoid cystic carcinoma Some have t(6;9) Ductal cells: — IHC helps
(q21-24;p13-23), cytokeratin, EMA, identify
others have LOH at and CEA. different
6q. Myoepithelial cells: components.
cytokeratin, p63,
S100.
Pleomorphic adenoma FLAG 1 t(3:8) Ductal cells: — IHC helps

(p21;q21) cytokeratin, EMA, identify
and CEA. different
Myoepithelial cells: components.
cytokeratin, p63,
S100.
Salivary gland anlage tumor — Epithelial cells: — —

cytokeratin; stromal
cells: vimentin, actin,
and cytokeratin
Mucoepidermoid carcinoma MECT1/MAML2 Cytokeratin, — —

Sialoblastoma translocations including CK7 — —
— Ductal cells:
cytokeratin; basaloid
cells: S100 and actin
Cardiovascular fibroma May be associated — — —

with Gorlin
syndrome
Rhabdomyoma May be associated Myoglobin, actin, S100 —

with Gorlin desmin
syndrome
Myxoma May be associated with Vimentin — —

Carney syndrome
Teratoma — Varies by component AFP to rule out —

yolk sac tumor
component
Juvenile hemangioma — GLUT1,LeY — —
Kaposiform — CD31, CD34 GLUT1, LeY —

hemangioendothelioma
Epithelioid — CD31, vWF; EMA —

hemangioendothelioma substantial fraction
show cytokeratin
staining
Lymphangioma — D240 — —
Respiratory Juvenile papillomatosis — HPV 6 and 11, — —

cytokeratins
Pleuropulmonary blastoma Germline loss-of- Primitive stromal Loss of Dicerl —
function DICER1 component similar to staining in
mutations in familial embryonal RMS: epithelium
cases desmin, myogenin,
MyoD1, myoglobin
Pulmonary blastoma — cytokeratin, EMA; — —

morule positive for
CGA
Midline poorly differentiated NUT translocation — — —

carcinoma t(15;19)(q14;p13.1)
Inflammatory myofibro-blastic ALKrearrangements ALK; — —

tumor in some myofibrobleastic
markers
Gastrointestinal Granular cell tumor — S100 c-kit (CD117), — —

Gastrointestinal stromal tumor Mutations in KIT or vimentin, bcl-2, — KIT staining
PDGFRA CD34 may identify
tumors
suitable for
monoclonal
antibody
therapy
aclenocarcinoma — Cytokeratins, CEA, — —

cA19.9
Adenocarcinoma — Cytokeratins, CEA, — —

Inflammatory myofibroblastic ALK CA19.9 ALK;
tumor rearrangements in myofibroblastic
some markers
schwannoma — S100, GFAP KIT desmin, —

smooth muscle
actin
Leiomyoma — Desmin, smooth S100, KIT —

muscle actin
Fundic gland gastric polyps Some associated — — —

with FAP (APC
mutations)
Juvenile polyps Cronkhite-Canada — — —

syndrome; some
with SMAD4/DPC4
mutations
Carcinoid tumors — Chromogranin, NSE, — —

PGP 9.5, specific
polypeptide
hourmones
hepatobiliary Adenoma None Known Hep Par 1, CAM5.2, AFP IHC does not
polyclonal CEA; help dis-
CD34 in endothelial tinguish
lining adenoma
from
carinoma
Focal nodular hyperplasia None Known Hep Par 1, CAM5.2, — —
polyclonal CEA;
CD34 in endothelial
lining
Hepatocellular carcinoma Gains of 1q, 7q, 8q; Hep Par 1, CAM5.2, — —

losses of 16q polyclonal CEA;
CD34 in endothelial
lining
Hepatoblastoma Variable; some AFP, hCG, Hep Par — —

associated with 1, polyclonal CEA
BWS
Infantile hemangioen- — CD31, CD34, factor — —

dothelioma VIII
Mesenchymal hamartoma Translocations Cytokeratins in — —

involving 11, 17 and ductal component;
19 t(11;19) smooth muscle actin
(q11;q13.4) in stromal cells
Desmoplastic small round cell t(11;22) Cytokeratin, EMA, — —

tumor (p13;q12)creating WT1, CD99, NSE,
EWS-WT1 fusion PLAP
gene
Undifferentiated embryonal May have same Not much: — —

sarcoma translocation as sometimes vimentin
mesenchymal and bcl-2
hamartoma
Pancreatic Acinarr cell carcinoma — Enzymes: lipase, Chromogranin, —

trypsin, chymotrypsin synaptophysin
Solid pseudopapillary tumor Mutations in β- Vimentin, NSE, Cytokeratins —

catenin CD10, CD56, α1- usually
antitrypsin; nuclear negative
β-catenin expression
Pancreatoblastoma Mutations in β- Enzymes: lipase, — —

catenin/APC trypsin,
pathways chymotrypsin; also
CEA and CA19.9
Genitourinary Nephroblastome (Wilms Deletions or WT1 — —

tumor) mutations in WT1,
WT2, WT3, or WTX
Cellular mesoblastic t(12;15)(p13;q25) — — Same genetic

nephroma creating ETV6- aberra-tion
NTRK3 fusion as congenital
infantile
fibrosarcoma
Ewing sarcoma/PNET Translocations of CD99 — —

EWS on 22q11;
partners vary
Clear cell sarcoma t(10;17) and del — — —

14q have been
described
Malignant rhabdoid tumor hSNF5 Cytokeratin Loss of nuclear —
mutations/deletions INI1 staning,
(22q11) BAF47
“Translocation” carcinomas t(x;1)(p11.2;q25) Cytokeratins; — —

and APSL-TFE overexpression of
fusion or t(6;11) TFE
(p21;q12) involving
TFEB
Renal medullary carcinoma Sickle cell carriers Cytokeratin Loss of nuclear —

(11p 15.5 mutation INI staining
=HbS); possible
22q11 involvement
Angiomyolipoma TSC1 and TSC2 HMB45, smooth — —

genes—9q34 or muscle actin, Melan-
16p13.3 mutations A
Inflammatory myofibroblastic ALK ALK,vimentin,smooth — —

tumor rearrangements muscle actin,
such as t(2;5) desmin; variable for
cytokeratin
Embryonal rhabdomyo- LOH at 11p15 Muscle markers: Rule out other Myogenin
sarcoma desmin, myogenin, SR-BCTs: usually <50%
MyoD1, myoglobin PGP9.5, WT1, (as opposeed
CD99, CD45 to alveolar
RMS)
Female condyloma accuminata — HPV 6,11 — —

Reproductive
System
Clear cell adenocarcinoma — Cytokeratin — —
Embryonal rhabdomyo- LOH at 11p15 Muscle markers: Rule out other Myogernin
sarcoma desmin, myogenin, SR-BCTs: usually <50%
MyoDe, myoglobin PGP9.5, WT1, (as opposed
RMS)
dysgerminoma I(12p) PLAP, hCG — —
Embryonal carcinoma — CD30, cytokeratin; — —

less often PLAP and
AFP
Endodermal sinus tumor (yolk I(12p)± AFP — —

sac tumor)
Mature teratoma <95% karyotypically Component-specific Rule out youlk —

normal sac tumor
component:
AFP
Immature teratoma 60% with Component-specific Rule out yolk —

nonrecurrent sac tumor
cytogenic component:
abnormalities AFP
Struma ovarii — Thyroglobulin, TTF1 — —
Granulosa cell tumor — Inhilbin, vimentin, — —

Cd99, cytokeratin
Sertoli-Leydig cell tumor — CD99, WT1, inhibin, — —

calretinin,
cytokeratin
Gonadoblastoma Phenotypic females — — —

with 46 XY
karyotype
Small cell carcinoma — Cytokeratin, NSE, — —

chromogranin
Complete hydatidiform mole 46,XX or 46,XY by — Negative for Cytogenetics

dispermy p57/KIP2 in the and p57
cytotrophoblast staining are
far superior
to histology
for
distinguishing
partial versus
complete
moles
Choriocarcinoma — hCG Only weak hPL —
Male Condyloma accuminata — HPV 6,11 — —

Reproductive
System
Intratubular germ cell I(12p) PLAP, OCT4, NSE, — —

neoplasia p53, ferritin, CD117,
D2-40
Embryonal LOH at 11p15 Muscle markers: Rule out other Myogeninn

rhabdomyosarcoma desmin, myogenin, SR-BCTs: usually <50%
MyoD1, myoglobin PGP9.5, WT1, (at opposed
RMS)
Seminoma I(12p) PLAP, focal — —

cytokeratin, vimentin,
CD30, CD44
Embryonal carcinoma — CD30, cytokeratin, — —

OCT4, less often
PLAP and AFP
Endcdermal sinus tumor (volk — AFP, SALL4 PLAP± —

sac tumor)
Mature teratoma <95% karyotypically Component-specific Rule out yolk —

normal sac tumor
component:
AFP
— Granulosa cell Inhibin, vimentin, — —

tumor CD99, cytokeratin
Sertoli tumot — Inhibin, vimentin, PLAP, CEA —
cytokeratin, variably
for S100
Leydig cell tumor — Inhibin, vimentin, PLAP, CEA —

cytokeratin, variably
for S100 Inhibin,
melan A, vimentin,
S100, chromogranin,
synapto-physin;
variably for
cytokeratin, EMA,
desmin
Endocrine Pituitary adenomas — Mono/oligoclonal Decreased —

hormone production type IV
collagen matrix
Papillary thyroid carcinoma RETgene Cytokeratin, TTF1, — —

mutations; also thyroglobulin
BRAF, APC, RAS,
TRK
Medullary thyroid carcinoma RET mutations production — —

Cytokeratin, TTF-1,
thyroglobulin
Calcitonin,
cytokeratin,
chromogranin, TTF-
1, synaptophysin,
CEA
— Spindle epithelial Cytokeratin — —

tumor with thymus-
like differentiation
(SETTLE)
Parathyroid MEN1(11q13), Cytokeratin, PTH — —

adenomas/hyperplasia RET(10q11), or
HRPT2(1q25)
Pheochromoytoma RETin MEN2- NSE, chromogranin, Cytokeratin, —

related cases; 1p synpato-physin; EMA
losses S100 in su
stentacular cells
Neuroblastoma MYCN PGP9.5, NSE, CD99, CD45 —

amplification, ALK synpatophysin,
amplification or NB84
mutation
Pancreatic endocrine tumors Variable Chromogranin, — —

synaptophysin, NSE,
specific hormones
Skin Verruca vulgaris — HPV — —
Tricholemmoma Multiple in Cowden CD34 in lesional — —

syndrome cells
Epidermoid cyst Multiple in Gardner — — —

syndrome
Sebaceous adenoma Muirlorre syndrome — — —
Melanocytic bnevi — S100, Melan A, — —

HMB4
Cellular blue bnevus Camey complex HMB45 S100 —
Dermatofibroma — Factor XIIIa CD34 —
Dermatofibrosarcoma Translocation CD34, p53 Factor XIIIa Same

protuberans COLIA1 — PDFGB translocation
t(17;22) (q22;q13) as giant cell
fibroblastoma
— Juvenile CD68, vimentin, CD1a —

xanthogranuloma S100 (variable)
Langerhans cell histiocytosis S100, CD1a — — —
Neurothekeoma — S100, vimentin; CD68 —

cellular variant
positive for NKI/C3
and negative for
S100
Soft tissue Lipoblastoma PLAG1 — — —

rearrangements
Liposarcoma FUS-CHOPius\on — — —
gene from t(12;16)
or variant
translocation
Gardner fibroma APCmutations β-catenin — —
Desmoid fibromatosis APCmutations β-catenin — —
Inflammatory myofibro-blastic ALKrearrangements ALK; myofibroblastic — —

tumor in some markers
Infantile fibrosarcoma t(12;15)(p13;q25) — — Same

creatinETV6- translocation
NTRK3 fusion as cellular
mesoblastic
nephroma
Low-grade fibromyxoid Some with t(7;16) — — —

sarcoma (q34;p11)
translocation and
FUS-CREB3L2
fusion
Embryonal LOH at 11p15 Muscle markers: Rule out other Myogenin

rhabdomyosarcoma desmin, myogenin, SR-BCTs: usually <50%
MyoD1, myoglobin PGP9.5, WT1, (as opposed
Cd99, CD45 to alveolar
RMS)
Alveolar rrhabdomyosarcoma t(2;13) ort(1;13) Muscle markers: Rule out other Myogenin
translocations desmin, myogenin, SRBCTs: usually <50%
fusing PAX3 or MyoD1, myoglobin PGP9.5, (as opposed
PAX7, respectively, WT1,CD99, to alveolar
with FOXO1 CD45 RMS)
Ossifying fibromyxoid tumor — S100; rarely, — —

desmin, GFAP, and
cytokeratins
Soft tissue myoepithelioma — Cytokeratin, S100, — —

calponin
Synovial sarcoma t(X;18)(p11.2;q11.2) Cytokeratin and — —

fusing SVTand EMA in the epithelial
SSX1 or SSX2 phase
Epithelioid sarcoma — Vimentin, keratin, Loss of nuclear —

and EMA INI expression
Alveolar soft part sarcoma TFE3-ASPL fusion — — —

gene
Clear cell sarcoma t(12;22)(q13;q13) S100, HMB45 — —

(melanoma of soft parts) with EWS-ATF1
Ewing sarcoma/PNET Translocations of CD99 — —

EWS on 22q11;
partners vary
Osteochondroma EXT1 and EXT2 — — —

gene mutations in
multiple hereditary
exostoses
Chondromyxoid fibroma 6q13 — — —

rearrangements
Osteomas May be associated — — —

with Gardner
syndrome
Chordoma — S100 and epithelial — —

markers
fibrous dysplasia Seen in McCune- — — —

Albright syndrome
and with GNAS
mutations
Central Medulloblastoma i(17q) most — — —

Nervous common, other
System various ones. Some
cases associated
with Gorlin
syndrome and
PTCH gene.
Retinoblastoma Rb loss-of-function — — —
Atypical teratoid/rhabdoid hSNF5 gene—22q1 — Loss of nuclear —

tumors mutations/deletions INI1 staining,
baf47
Hemangioblastoma von Hippel-Lindau Vimentin, NSE, Epithelial —
syndrome GFAP, inhibin A markers
dysplastic gangliocytomas Cowden syndrome — — —
Peripheral Schwannoma Monosomy 22 or S100 — —

Nervous 22q loss (NF2
System gene)
Neurofibroma NF1 S100 — —
MPNST Associated with Only a minority are — —

NF1 S100-positive
Lymph nodes Hodgkin lymphoma — CD15, CD30, EBER ALK —

ISH
Anaplastic large cell — ALK, CD30 — —

lymphoma
Burkitt lymphoma MYCtranslocations CD10, CD19, near- — —

100% MIB-1
positivity
Precursor B-or T-cell lym- — TdT, lineage specific — —

phoblasticlymphoma/leukemia markers (may be
mixed)
Forensic Identification
PCR is commonly used to amplify specific segments of DNA for forensic analysis such as for identification of victims of natural disasters, victims of
crimes, and also identification of perpetrators leaving DNA evidence at a crime scene. PCR-based identification methods include analysis of
sequence and length polymorphisms and mitochondrial DNA sequences. The most common method used to identify individuals is examination of
13 different loci that show variability among humans to create a “DNA fingerprint.” (49).
Limitations
While PCR has become one of the most commonly used tools in molecular medicine, it is important to note its limitations. The assay is extremely
sensitive and care must be taken to avoid contamination from nucleic acids in the environment, particularly in the microbiology laboratory.
Laboratory technicians performing PCR testing should have adequate training and experience and understand the importance of good technique.
PCR detection of sequence variants in mutation analysis is limited to base substitutions and small insertion/deletions. Detection of larger intragenic
deletions currently requires other supporting methodology (8, 15). PCR applications in detecting and identifying new organisms can lead to
dilemmas about whether or not a newly sequenced isolate is clinically relevant. Finally, PCR testing is not “agnostic.” It is not a screening test for
unknown abnormalities; rather, it is applied in a target-specific manner.
ARRAY TECHNOLOGY
DNA microarrays are used as a tool to evaluate and quantify sequence information for tens of thousands to a million sequences in a single
experiment. The development of microarrays required the advances of miniaturization and computer technology coupled with the knowledge of
DNA sequence among multiple species. The basic methodology involves thousands/millions of small chemically generated oligonucleotide
sequences representing portions of the genomic DNA sequence that are fixed to a platform such as a glass slide or silicon wafer. These
sequences are “arrayed” in a manner such that the location and sequence of each probe is known and millions of probes can fit in a small area.
The patient DNA (or cDNA reverse transcribed from RNA) is then labeled with a fluorescent dye and hybridized to the array. Patient DNA that has
sequence identity to a probe on the array binds there and can be detected by a fluorescence detector.
There are currently three main applications of microarray technology: (a) comparative genomic hybridization (CGH), which compares the amount
of patient DNA at a given locus to a reference standard, (b) Single nucleotide polymorphism (SNP) detection, which assays the genotype of an
individual at hundreds of thousands of sites known to be polymorphic among individuals, and (c) gene expression, which is a reflection of which
genes are actively transcribed in a given sample in a relatively quantifiable manner. CGH arrays can provide information on genomic gains and
losses just as in standard karyotyping albeit at a much higher resolution and without the requirement for growing cells. Karyotyping has one big
advantage over CGH in that karyotyping can detect balanced translocations whereas CGH cannot. SNP arrays can detect SNPs or mutations that
can link someone to a specific disease state, determine suitability to targeted therapy, or determine individual variations in drug metabolism. SNP
arrays can also measure copy number changes including uniparental disomy. It is clear that microarray technology has transformed molecular
biology and genetic research and for the same reasons it is valuable in research, there is no shortage of potential applications in clinical molecular
diagnostics.
NEXT-GENERATION SEQUENCING
For the last three decades, the Sanger method of sequencing has been the favored method of reading the base code sequence of DNA. This
method using capillary sequencer machines has high fidelity and is still considered the gold standard. It was used to sequence the first human
genome in a 13-year effort ending in 2003. Since the completion of that first human genome sequence, new powerful technology has emerged.
These so-called next-generation sequencers can perform massively parallel DNA sequencing of clonally amplified or single DNA molecules. This
technology has made sequencing entire genomes possible in a matter of days to weeks rather than years and has also substantially brought down
the price of sequencing large areas of DNA.
NGS may soon replace standard PCR-based methods for mutation detection and screening. Advances in the preparation and enrichment of
specific regions of DNA for subsequent sequencing will facilitate the use of this technology in disease-specific manner both for diagnosis and
management. Sequencing large numbers of genes for clinical conditions such as hypertrophic cardiomyopathy (42), and neuromuscular diseases
becomes possible with this new technology. Testing genomes of viral populations in sera for therapeutic sensitivity or resistance can guide choice
of antiretrovirals in HIV infection (61). One can envision routine testing of tumor cells for genes predicting responses or lack thereof to common
chemotherapeutic agents as well as targeted therapies.
More advances are needed in streamlining and automating the technical procedures and data analysis steps before a complete transition of NGS
from research to clinical laboratories is possible. In addition, at the present in 2010, its cost
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remains prohibitive for routine clinical use. But, considering how powerful this technology is and how rapidly it has evolved, it is likely only a matter
of time before these technical issues are addressed and these platforms become more affordable and ready for use in molecular diagnostics.
FUTURE DIRECTIONS FOR MOLECULAR METHODS IN PEDIATRIC PATHOLOGY

Technology and its applications to medical sciences will inevitably continue to advance at an extremely rapid pace. The era of personalized
medicine is coming, but that does not mean that the hematoxylin and eosin stain is no longer sufficient and cost effective for diagnosing the vast
majority of diseases. Pathologists have an important role ensuring that new methodologies are subjected to validation, quality control, and quality
assurance measures just as one would naturally expect from any clinical chemistry test or immuno-histochemical stain. And, pathologists and
laboratory managers are especially qualified to determine the cost-benefit ratio of introducing new tests. These are very important responsibilities.
Far from being at risk of replacement by technological machinery, as pathologists we are uniquely positioned to determine which new technologies
will be beneficial to the patient in terms of improving accuracy or timeliness of diagnosis, reducing costs, improving quality, or providing added
benefit over currently used diagnostic methods.
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FURTHER READING
Arch Pathol Lab Med 2008; 132(3). A special issue devoted to immunohistochemistry, with individual articles devoted to various organ
systems.
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633.
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cytogenetics laboratory manual, 3rd ed. Philadelphia, PA: Lippincott-Raven, 1997.
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Springer, 2010.
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Voelkerding KV, Dames SA, Durtschi JD. Next generation sequencing: from basic research to diagnostics. Clin Chem 2009;55;4:1-18.
Chapter 1D
Electron Microscopy
Gary W. Mierau
Electron microscopy remains an essential tool for today's pediatric pathologist. The technique continues to
provide for a significant number of childhood diseases the best, and sometimes the only, means of establishing a
definitive diagnosis. Offering a direct morphologic approach, it is arguably the most powerful and least
treacherous of the many ancillary diagnostic techniques currently available. This having been said, it must also
be stressed that each special technique has its relative strengths and weaknesses in particular situations. These
should not be regarded as competitive techniques but rather as complementary tools, which are best employed
using a highly selective but fully integrated approach.
We have found ancillary electron microscopic studies to be warranted in approximately 5% of the surgical
specimens submitted for histologic examination. In contrast to our experience with the adult population, where
renal specimens predominate, a very broad mix of specimens is received from pediatric patients. Presented in
Figure 1D-1 are workload distribution statistics derived from an analysis of 1,000 consecutive diagnostic studies
performed on patients from our institution. Tumors comprise the largest proportion of cases, followed closely by
muscle and cilia, not too distantly by liver and skin, and then in gradually diminishing numbers by a wide variety
of other tissue types.
Increasing recognition of the fact that immunohistochemical studies, even when properly performed and
interpreted, will sometimes produce misleading information (5, 9, 12, 13, 14 and 15, 18, 21, 24, 31) has led to a
resurgence in the popularity of electron microscopy for tumor diagnosis. Ultrastructural studies of muscle biopsy
specimens are of particular utility in diagnosing mitochondriopathies, storage diseases, and causes of infantile
hypotonia. With respiratory tract specimens, electron microscopy offers the only readily available means of
demonstrating defects in ciliary structure, and is also useful in the diagnosis of surfactant deficiency states,
pulmonary interstitial glycogenosis, and some infectious diseases. Liver specimens are examined, among other
things, to look for early evidence of metabolic disease. Electron microscopy often offers the fastest, cheapest,
easiest, and sometimes the only means of screening for metabolic storage diseases. Skin biopsies are utilized
for diagnosis of the inherited epidermolyses. Bowel biopsies are examined to diagnose microvillous inclusion
disease and to detect furtive organisms such as microsporidia. A substantial number of renal diseases (e.g.,
minimal change lesion, thin basement membrane nephropathies, dense deposit disease) can only be diagnosed
with confidence using this technology. Making up the remainder of the workload is a smattering of almost every
type of specimen imaginable. Specific examples demonstrating some of the many applications of electron
microscopy will be found in the chapters that follow.
It is not just in surgical pathology, however, that electron microscopy has a role to play. Collaborative endeavors
involving the clinical pathology services account for a substantial portion of our total workload. During that same
period when the previously alluded to 1,000 surgical pathology specimens were examined, 3,206 stool
specimens were received for viral diagnosis. As a component of our departmental quality assurance program, we
also at that time performed 266 ultrastructural studies on a random selection of the tumor specimens for which
special studies had not been considered necessary for diagnosis, with the resulting information being used to
help establish appropriate levels of test utilization and to stimulate a review of the diagnosis in cases with
discordant findings. An additional 37 cases were studied to support the autopsy service, where the technique
can be useful with questions arising after the option to perform alternative procedures (e.g., virus culture) has
already been lost. Research activity was at an ebb during this interval, with only 21 such specimens being
examined, but it is worth emphasizing that opportunities do abound for the use of electron microscopy in this
setting.
THE ELECTRON MICROSCOPY LABORATORY

The central element in any electron microscopy laboratory is, of course, the electron microscope itself. A basic
transmission electron microscope is all that is needed for diagnostic applications. Ease of operation, reliability,
and
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optimal performance at low magnifications are of far greater importance in this setting than are the advanced
features offered by the more expensive and more demanding “analytical” microscopes. Electron microscopes
have achieved a relatively mature state of technological development, and several manufacturers produce
excellent (100 to 120 kV) instruments suitable for this application. In contrast to the situation with respect to
electron microscopes, the technology associated with integrated digital imaging systems presently remains in a
state of rapid evolution. Digital cameras offer many advantages over film cameras. They are, however,
comparatively expensive and tend to perform least well at the lower magnifications generally employed for
diagnostic studies. Alternatively, most of the benefits afforded by this new technology can also be attained by the
means of scanning negatives produced by a film camera. Other than the electron microscope, the only
specialized equipment needed is an ultramicrotome. Because ultimate success with this technique depends so
heavily upon the capacity to obtain quality ultrathin sections in a reliable and rapid manner, it is more important to
possess a modern ultramicrotome than a modern electron microscope.
FIGURE 1D-1 ▪ Distribution of pediatric surgical pathology workload by specimen type.
The space requirements for an electron microscopy facility are quite modest. Following the changeover from
photographic to electronic image processing, which eliminated the need for a darkroom, just 185 square feet of
floor space in our current facility remained dedicated to this enterprise. Some activities, such as specimen
processing, can be integrated into the workflow of the routine histology laboratory. This sharing of equipment,
space, and personnel serves not only to reduce costs but also to enhance overall productivity. If occasional
assistance can be provided by other team members, one dedicated technologist for every 250 specimens
examined annually would seem a reasonable guide for staffing the EM laboratory. Generating results within a
clinically relevant time frame is crucial to a successful operation. It is a reasonable expectation to have an
interpretive report, complete with illustrations, available within two working days of specimen receipt.
In providing the interpretive component of the ultrastructural studies, a number of organizational models have
been shown effective. Which is best in a given situation will depend upon the particular circumstances and
personnel available. In some institutions, each pathologist while on service assumes full responsibility for the
submission, examination, and interpretation of their cases. In others, a designated pathologist carries the
responsibility for the examination and interpretation of all cases. In the majority of laboratories, however,
technical personnel (with appropriate training and guidance) do much of the examination and may even assist
with the interpretation of results.
It is often assumed that the electron microscopy laboratory will be a financial liability for its parent institution. This
need not be true. With some attentiveness to basic business practices, an electron microscopy laboratory can be
a profitable enterprise. Electron microscopy is sometimes still thought of as being a very expensive and
extremely slow technique, but its modern-day cost and speed is actually quite comparable to that of most other
ancillary diagnostic techniques. The cost savings to be derived from using the most powerful techniques
available to obtain a fast and accurate diagnosis, necessary for minimizing the length of a hospital stay, should
be obvious. Certainly for health care facilities already maintaining an electron microscopy laboratory, there is no
economic reason not to use the technique to its fullest advantage. The major expenses associated with this
endeavor are fixed rather than incremental, so its actual cost to the institution will remain virtually the same
whether it is used a little or used a lot.
THE ELECTRON MICROSCOPY TECHNIQUE

While most other ancillary techniques (e.g., IHC, FISH, PCR) are restricted in application to hypothesis testing,
electron microscopy can provide the right answer even when the wrong question, or no specific question, is
being asked. Preserving the option to perform electron microscopy is therefore an important habit to develop.
Placing a bit of tissue into an appropriate fixative as a matter of routine takes little extra time and costs next to
nothing—but provides excellent insurance should a diagnostic issue arise later.
Electron microscopy is not so much a different technology as a modification, and extension, of a most familiar
one. Specimens for electron microscopy are handled in almost the same way as for light microscopy. Here the
standard fixative, instead of formaldehyde, is glutaraldehyde (to better preserve proteins) followed by osmium
tetroxide (to better preserve lipids). Ordinary formalin preparations, if they are properly buffered (pH 7.2 to 7.4
range) and adjusted to moderate hypertonicity (400 to 500 milliosmols), actually serve quite well as a primary
fixative for electron microscopy and can be substituted when necessary. The tissues are dehydrated in a similar
fashion but then are embedded in an epoxy resin to enable the cutting of thinner sections
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than would be possible with the softer paraffin wax used for routine histology. To cut the necessary (∽80-nm
thick) ultrathin sections requires a similar but more refined “ultra” microtome and the use of a diamond blade. An
electron beam cannot penetrate glass, so the sections are mounted on a finemeshed screen (referred to as a
grid) rather than on a glass slide. The sections are not stained in the true sense of the word (as color reactions
cannot be detected with an electron microscope) but are incubated in similar fashion in heavy metal solutions
(usually of uranium and lead) to selectively add contrast to various substructural components. Methodological
details for all these procedures can be found in many standard texts (3, 17). Except in its use of a beam of
electrons, rather than a beam of light, the electron microscope is not very different in design or operation from
that of an ordinary light microscope. It is the shorter wavelength of electrons that enables the superior point-to-
point resolution and, thus, higher working magnifications offered by this instrumentation. The two techniques
form a strong partnership, with the light microscope being best suited for the study of collections of cells and the
electron microscope being best suited for the study of individual cells.
Under ideal conditions, specimens submitted for electron microscopy will consist of a representative sampling of
appropriately sized (∽1 mm3) tissue cubes placed, upon removal from the patient, into the most suitable fixative
immediately. Real-life conditions will not always be ideal but all is not necessarily lost if they are not. Just as an
experienced automobile mechanic can still usually identify the make and model of a car after it has been involved
in an accident, so can an experienced electron microscopist still usually identify the cell type and disease
process involved in a partially wrecked tissue specimen. One has to be more cautious when dealing with
suboptimal specimens, however, as there is a strong inverse relationship between the quality of specimen
preservation and the probability of making a significant interpretive error. The safest strategy when dealing with
suboptimally preserved specimens is to restrict electron microscopy to the search for some particular feature(s)
predetermined to be of diagnostic relevance.
A demonstration of the deleterious effects associated with suboptimal specimen processing is presented in
Figure 1D-2, which shows subsamples from the same case of Langerhans cell histiocytosis after being subjected
to progressively harsher treatments. Here it can be seen that, though the diagnosis can still be made, the degree
of difficulty increases (and the degree of confidence decreases) as the quality of cellular preservation is
diminished. With optimal processing (Figure 1D-2A), the richness of cytoplasmic detail almost obscures the
diagnostic Birbeck granules. Substitution of formaldehyde for glutaraldehyde as the primary fixative (Figure 1D-
2B) results in a significant loss of cytoplasmic detail but, at least in this instance, this does not interfere with
identification of the critical feature. Often, it is the case that in order to perform additional specialized procedures,
such as the immunohistochemical reaction for S100 protein shown here, it becomes necessary to trade-off some
degree of cellular preservation to maintain an adequate degree of tissue reactivity. Such applications, however,
will fall mainly within the domain of research. For general purposes, when using formalin fixed tissues for electron
microscopy, it is best to refix the specimen in glutaraldehyde before proceeding with the tissue processing. As a
last resort, one can retrieve and reprocess for electron microscopy tissue that has already been embedded in
paraffin. The technique is simple (20). Following removal of a carefully selected appropriately sized tissue
sample from the paraffin block using a sharply pointed scalpel blade, the specimen is dewaxed overnight in
xylene. Best results are obtained if, following rehydration in graded alcohols, the tissue is refixed both with
glutaraldehyde and with osmium tetroxide prior to further processing. Because much of the lipid will have been
extracted during the earlier processing events, one can expect poor preservation of membranes and other
structures of high lipid content. Nevertheless, as shown in Figure 1D-2C, the features of key interest may still
remain clearly identifiable. Structures composed largely of proteins (e.g., filaments, granules, intercellular
junctions, immune deposits) are most likely to remain identifiable but sometimes membranous structures also are
preserved. We were, for example, in a correlative study able to demonstrate Birbeck granules in deparaffinized
material from 11 of 14 cases in which they were known to exist (25). In some situations, for example in attempting
to identify a focally distributed virus, this approach may actually prove more efficacious than would an unfocused
search through optimally preserved tissue. Utilization of deparaffinized tissue preserved with a nonaldehyde type
fixative (e.g., alcohol, B5, Bouin's) will generally be unrewarding for, without crosslinking of proteins, nearly all
substructural features are lost during processing (Figure 1D-2D).
It is often assumed that autopsy specimens will not be suitable for electron microscopic study. This is not always
the case. While some autolytic degradation is inevitable, its severity and speed of occurrence is not entirely
predictable. Sometimes, as illustrated in Figure 1D-3, cellular preservation remains surprisingly good even after
an extended postmortem interval. The appearance of certain organelles, such as mitochondria, is very quickly
altered by anoxic conditions. One would not, therefore, want to attempt assessment of a mitochondrial disorder
using autopsy material. On the other hand, many structures are quite durable and can be confidently identified
even in a specimen that is very degenerate. Here again, the denser the structure, and the greater its protein
content, the more likely it is to remain recognizable.
Frozen tissue as well can be used for electron microscopy. Best results are obtained when the frozen tissue is
placed directly into cold glutaraldehyde and allowed to fix as it thaws. The quality of cellular preservation,
particularly with respect to ice crystal damage, varies regionally within a specimen. Examination of multiple sites
may therefore prove beneficial. This recovery technique is especially useful in renal pathology, in circumstances
where the initial specimen
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submitted for electron microscopy happens not to contain any glomeruli. The technique may serve other
purposes as well, as demonstrated in Figure 1D-4.
FIGURE 1D-2 ▪ Subsamples from the same case of Langerhans cell histiocytosis subjected to variations in
processing technique. A: Routine processing, utilizing glutaraldehyde fixation, results in optimal preservation of
Birbeck granules (arrow) and other subcellular components. B: Substitution of formaldehyde for glutaraldehyde
results in swelling of the mitochondria and some loss of cytoplasmic detail but has little effect on the Birbeck
granules (arrow). The less intense fixation enables immunogold labeling of S100 protein (arrowheads). C:
Birbeck granules (arrow) display an altered appearance but remain clearly identifiable in formalin-fixed tissue
retrieved from a paraffin block. The mitochondria are reduced to smudges and most other cytoplasmic detail is
lost. D: In B5-fixed tissue retrieved from paraffin, the Birbeck granules (arrow) are hardly recognizable and nearly
all other components have been lost.
FIGURE 1D-3 ▪ Autopsy specimen of brain showing perivascular cell with large cytoplasmic inclusion (asterisk).
The mitochondria (arrows) exhibit degenerative changes but the material within the inclusions remains well
enough preserved to enable a confident diagnosis of Krabbe disease.
Electron microscopy is very well suited for the examination of fine-needle aspirate specimens (30, 35). The
aspiration biopsy technique is not as frequently employed in pediatric medicine but has been shown useful in this
setting as well (4, 32, 33 and 34). We, along with others (1), have found an electron microscopic approach to the
examination of fine-needle aspirates to be especially useful in the diagnosis of childhood round-cell tumors.
Since with electron microscopy it is normally the situation that relatively small numbers of cells are examined
individually for identifying characteristics, the technique is not much compromised by the small disrupted samples
produced by the aspiration procedure. Figure 1D-5A shows how a confident diagnosis can be established even
with just a few neoplastic cells being present. Demonstrated in Figure 1D-5B is an ultrastructural “special stain”
for glycogen that can be of particular usefulness in circumstances like these (7), as it can be applied directly to
an existing ultrathin section and does not require the processing of any additional material.
With lower risk of loss during the embedding process, and within approximately the same time frame, tiny
specimens of other sorts (for instance, from an endomyocardial biopsy procedure) can be embedded in epoxy
resin instead of paraffin wax. More specimen detail than usual will be observed by light microscopy because of
the enhanced resolution offered by the 1-μm-thick resin-embedded sections. The array of special stains utilizable
with these sections is somewhat limited, but this strategy does preserve the option to use electron microscopy,
which might be considered the most powerful “special stain” of all.
FIGURE 1D-4 ▪ Snap frozen specimen of liver thawed in chilled glutaraldehyde shows excellent preservation of
mitochondria (arrows) and other cellular structures, and allows demonstration of the “granular” bile (asterisk)
characteristic of Byler disease.
The introduction of flow cytometric, immunocytochemical, and molecular diagnostic techniques has greatly
diminished the role for electron microscopy in the diagnosis of hematological disorders. Nevertheless, it remains
for the diagnosis of certain conditions (e.g., platelet storage pool disorders) an indispensable tool. Ultrastructural
studies continue furthermore to be helpful in the diagnosis of leukemias, especially when routine cytochemical
and/or flow cytometric studies produce conflicting, confusing, or equivocal results; insufficient or aberrant
leukemic cell differentiation causes diagnostic uncertainty; or where an unusual or uncommon diagnosis is under
consideration (11, 29). To meet the challenges presented by the minuscule specimens obtainable from pediatric
patients, we have developed the following procedure (22). Specimens for electron microscopy are procured in
two or three heparinized glass microhematocrit tubes. Immediately upon transport to the laboratory (or the next
morning, in
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the case of late arriving specimens), the hematocrit tubes are centrifuged and then scored and broken just below
the buffy coat layer (Figure 1D-6A). The buffy coat samples are then gently expelled into a vial of glutaraldehyde.
As the droplet settles through the fixative, it consolidates into a single small firm pellet, which can subsequently
then be processed with the ease of a solid tissue specimen. After about an hour, the fixative is replaced with a
buffer “holding” solution and, at this point, further processing can be suspended. We normally proceed next to
performing the Graham/Karnovsky reaction (16) for myeloperoxidase (MPO) and then embedding the specimen,
holding in reserve whenever possible a bit of the fixed tissue for additional or repeat studies. The technical
performance of the MPO reaction can be satisfactorily evaluated simply by examining the accompanying
granulocytes that will almost invariably be present within the specimen. For our basic morphologic studies, we
simply perform the customary uranyl acetate/lead citrate stain over the peroxidase stain. This produces no
interpretive difficulty (Figure 1D-6B). Neither does subjecting the tissue to the peroxidase reaction interfere with
a subsequent tannic acid stain that might be employed for the demonstration of glycogen. Performing the MPO
reaction (Figure 1D-6C) enables us at least to determine whether it is a case of lymphogenous or
nonlymphogenous leukemia that we are dealing with. The majority of cases can be confidently identified at this
point but, occasionally, we do find it necessary to proceed with some additional techniques (11). The tannic acid
procedure for demonstration of glycogen mentioned previously (Figure 1D-5B), which is more sensitive than the
light microscopic PAS stain, can be useful in a number of situations and is of particular value in establishing a
diagnosis of erythroleukemia. The acid phosphatase stain, which can be performed on the remaining
unprocessed fixed tissue, is sometimes helpful in identifying immature or aberrant granules (2). The fixed tissue
being held in reserve can also be used for the NTA (nonspecific esterase) reaction, which is a bit capricious but
sometimes useful in the identification of monocytic precursors (Figure 1D-6D) (26). The routine MPO technique
will sometimes enable the identification of platelet peroxidases (PPO) but, generally, when a diagnosis of
megakaryoblastic leukemia is being considered another specimen must be procured to perform the more
sensitive PPO procedure (Figure 1D-6E) (19). Cryopreserved cells can be used for this or any other purpose
requiring special fixation or handling, such as the immunogold/MPO procedures that are helpful in dealing with
“mixed” or “hybrid” cell leukemias (Figure 1D-6F) (17).
FIGURE 1D-5 ▪ Fine-needle aspirate specimen displaying focal deposits of cytoplasmic glycogen (asterisk)
characteristic of Ewing sarcoma. That the “moth-eaten” areas represent glycogen deposits (A) is easily
confirmed by incubating the sections in a weak tannic acid solution prior to staining with uranyl acetate and lead
citrate (B).
In the diagnosis of peroxisomal disorders, ultrastructural studies are often needed to determine whether these
organelles are normal, abnormal, reduced in number, or absent (6). Usually, this can be accomplished without
the application of any special techniques. Occasionally, however, we have found it necessary to employ the
alkaline-diaminobenzidine reaction for catalase activity (10) to verify the identity of morphologically abnormal
peroxisomes.
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FIGURE 1D-6 ▪ Special procedures for the diagnosis of acute leukemias. A: The capillary tube technique
(described in text) provides an easy and utilitarian means of specimen collection that yields consistent
highquality results. B: Performing the MPO reaction prior to routine staining with uranyl acetate and lead citrate,
as illustrated with this case of acute myeloid leukemia, does not affect the morphology. This example
demonstrates also that, provided the capillary tubes are not first subjected to centrifugation, these specimens
suffer little from an overnight delay in fixation. C: Detection of MPO (arrows), as shown in this case of acute
“undifferentiated” leukemia, can be achieved by electron microscopy when not possible by light microscopy.
Note that accompanying normal platelet (arrowhead) is not stained. D: Ultrastructural demonstration of
nonspecific esterase activity (arrows) can be useful in the identification of early monocytic precursors. E:
Identification of megakaryoblasts is enabled by the demonstration of PPO (arrow) within the endoplasmic
reticulum. Note that the accompanying abnormal platelet (arrowhead) shows similar staining. F: Cryopreserved
cells offer a convenient source of material for procedures with special processing requirements. Shown is a
leukemic cell that, after retrieval, was subjected both to immunogold labeling with MY7 cell surface marker
(arrowheads) and the MPO reaction (arrows).
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FIGURE 1D-7 ▪ Brush biopsy specimen of nasal respiratory epithelium showing numerous favorably oriented cilia
(asterisks).
Another easily performed ultrastructural special stain that has upon occasion proved very helpful is the uranaffin
reaction. It can be used, among other things, to establish the identity of neuroendocrine granules in tumors (28)
and serotonin granules in platelets (27).
Evaluation of cilia morphology requires electron microscopy. Nasal brush or curette specimens are
recommended for this purpose because they generally produce a better yield of favorably oriented cilia than do
traditional biopsy methods (Figure 1D-7). These techniques also offer the advantages of being less expensive to
perform and easier for the patient to endure. We have found little need here for the use of any special fixatives or
techniques (23).
Electron microscopy, while losing popularity as a means for detecting viruses in solid tissue specimens, has
been gaining in utilization for detection of viruses in body fluids and fecal specimens. The techniques used for
this purpose are fast and easy to perform, and need not be very elaborate. We routinely use a Beckman Airfuge
ultracentrifuge to help concentrate the virus onto the grid surface but have found the agar diffusion method,
which requires no special equipment, to work almost as well (8). The more cumbersome immunological
techniques have not performed as well for us in this regard but have occasionally proved useful in confirming the
identity of a detected virus. The negative staining technique can be used with a variety of specimens (e.g., urine,
blood, vesicle fluid, cerebrospinal fluid, amniotic fluid, respiratory tract secretions), but its major application in
pediatrics lies in the diagnosis of acute viral gastroenteritis. This very practical and cost-effective approach is
being employed as the primary method for detection of stool viruses in a growing number of institutions. Not only
does it provide the most reliable means for detecting rotavirus but concurrently enables detection of all the other
viral pathogens, which together account for nearly as many cases of pediatric gastroenteritis as does rotavirus.
Multiple agent infections are readily detected using this methodology (Figure 1D-8), which can be important
when isolation procedures to stem a nosocomial outbreak are being implemented. The technique can be
performed in just a matter of minutes, which may be of importance when initiation of therapy awaits
establishment of a firm diagnosis or, in the event of a bioterrorism attack, an infectious organism requires quick
identification.
FIGURE 1D-8 ▪ Negative stained stool specimen from a patient infected simultaneously with rotavirus (open
arrow), coronavirus (curved arrow), and a small round virus (solid arrow).
It is emphasized in concluding that electron microscopy remains today an extremely powerful, highly versatile,
absolutely indispensable diagnostic technique for the practice of pediatric pathology. Ideally, every pathologist
would have an electron microscope located just down the hall. Fortunately, when such is not the case, modern-
day transportation and communication systems allow consultative arrangements to be developed, almost
anywhere in the world, that are virtually as fast, convenient, and effectual. Barriers to its utilization do not exist.
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(peroxisomes). J Cell Biol 1969;43:275-288.
11. Favara BE, Mierau GW, McCarthy RC, et al. The leukemias of childhood. In: Rosenberg HS, Berstein J,
Newton WA Jr, eds. Neoplasia in infancy and childhood, Vol. 9. Basel, Switzerland: Karger, 1987:75-132.
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13. Friedman HD, Tatum AH. HMB-45-positive malignant lymphoma. A case report with literature review of
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1994;7:317-321.
15. Gown AM, Boyd HC, Chang Y, et al. Smooth muscle cells can express cytokeratins of “simple”
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16. Graham RC, Karnovsky MJ. The early stages of absorption of injected horseradish peroxidase in the
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17. Hayat MA. Principles and techniques of electron microscopy. Biological applications, 3rd ed. Boca
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18. Heyderman E, Warren PJ, Haines AMR. Immunohistochemistry today—problems and practice
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19. Heynen MJ, Tricot G, Verwilghen RL. A reliable method with good cell preservation for the demonstration
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871.
23. Mierau GW, Agostini R, Beals TF, et al. The role of electron microscopy in evaluating ciliary dysfunction:
Report of a workshop. Ultrastruct Pathol 1992;16:245-254.
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electron microscopy in the diagnosis of childhood round cell tumors. Ultrastruct Pathol 1996;20: 507-517.
25. Mierau GW, Favara BE, Brenman JM. Electron microscopy in histiocytosis X. Ultrastruct Pathol
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electron microscopy laboratory. Arch Pathol Lab Med 1989;113:1354-1356.
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soft tissue. A reappraisal of cytokeratin as a reliable marker in diagnostic immunohistochemistry. Appl
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to clinical aspiration biopsy. New York, NY: Igaku-Shoin, 1992:1.
Chapter 2
First and Second Trimester Pregnancy Loss
Deborah E. McFadden
Pathologic examination of the products of embryos and fetuses, both from spontaneous abortions (SAs) and
terminations of pregnancy, has become increasingly important over the past few decades. While such
examination was once performed primarily for the purpose of furthering scientific understanding of prenatal
human development, the practical medical applications of this knowledge have become clear and now form an
integral part of the medical assessment and management of fertility issues (30, 52, 65, 68). As an understanding
of the factors involved in successful pregnancies has developed and as patient demand for information has
increased, the role of pathologic examination has grown. Increased use of assisted fertilization techniques has
heightened the interest of physicians and patients alike in understanding why pregnancies fail. This chapter will
address the examination of disorders encountered in those pregnancies that end spontaneously in the first and
second trimesters of gestation; the pathology of fetuses delivered after pregnancy termination after prenatal
ultrasound diagnosis is beyond the scope of this chapter.
It is recognized that many conceptions do not end in livebirths but, rather, that there is a high rate of loss,
especially early in gestation. It is estimated that 10% to 20% of recognized pregnancies end as SAs, with most
losses occurring in the first trimester or first 12 to 14 weeks of gestation. With the demonstration of fetal cardiac
activity, the miscarriage rate drops somewhat to approximately 3% to 12% (38). In a study of women who had
had a normal prenatal visit at 6 to 11 weeks of gestational age (GA), the risk of subsequent SA was 1.6% or less,
considerably lower than for pregnancies overall (69). After the first trimester, approximately 1% to 2% of
pregnancies are spontaneously aborted (56). The incidence of stillbirth at term gestation is in the order of 0.1 %
to 0.5%. This high loss rate, together with changing or changed societal approaches and expectations of
pregnancy such as delaying childbearing until later in a woman's reproductive life and increased access to
assisted reproduction methods, has led to an intense interest in understanding the cause of pregnancy loss and
the implications for future reproductive success.
GA refers to the number of weeks since the last menstrual period (equivalent to menstrual dates), while
developmental age (DA) refers to the age as determined from the time of fertilization, generally considered to be
approximately 2 weeks after the last menstrual period. Embryos are assessed by developmental features that
correlate with age, usually given as DA. Thus, in a normal gestation, GA is DA plus 2 weeks.
The first trimester of pregnancy is the period of implantation and embryogenesis, with the completion of
embryogenesis by 8 weeks of DA (10 weeks of gestational age). Upon completion of embryogenesis with
development of all organ systems, the conceptus is referred to as a fetus. Definitions of fetus and infant vary with
locale; in Canada, a fetus is considered an infant once it has reached the GA of 20 weeks or is liveborn at any
GA. Stillbirth is defined as delivery of a deceased infant at or after 20 weeks of GA.
CAUSES OF EARLY SPONTANEOUS ABORTION

It is well recognized that the major cause of early SA is chromosome abnormality, usually aneuploidy. The use of
techniques such as comparative genomic hybridization (CGH) and quantitative fluorescence-polymerase chain
reaction (QF-PCR) to supplement conventional cytogenetic analysis has increased the detection of chromosome
abnormalities because these techniques do not require cell culture and can address the issue of maternal cell
contamination.
Numerous studies have shown that at least half of early SAs are chromosomally abnormal; in our laboratory,
70% of cases are chromosomally abnormal, somewhat higher than reported rates. Some of this may be
attributable to our routine use of cytogenetic analysis supplemented by CGH with flow cytometry in cases in
which tissue culture for cytogenetic analysis has failed or in which maternal cell contamination is suspected (36)
and some is attributable to the fact that the average maternal age in our population is higher than in other
published reports.
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The distribution of chromosome abnormalities is consistent between various studies, with trisomy accounting for
nearly half of all chromosomally abnormal SAs, triploidy accounting for 6% to 8%, and monosomy X accounting
for another 8% to 10%. Autosomal monosomy (primarily monosomy 21) and structural rearrangements account
for an additional 5% of abnormal cases. The structural abnormalities are an important subgroup because of the
possibility that they have arisen from a parent who carries a rearrangement predisposing to an unbalanced
karyotype in offspring. Studies of couples who have had recurrent miscarriages show that 5% have balanced
rearrangements such as reciprocal translocations (67). These individuals have an increased risk of having
chromosomally abnormal pregnancies, with the actual risk depending on the type of rearrangement and the
chromosomes involved (66).
Thus, the vast majority of early pregnancy loss is due to trisomy. Studies of parental origin of trisomies have
demonstrated that the largest proportion of trisomy is maternal in origin. There is a strong association with
maternal age, with the births of trisomic infants rising with increasing maternal age. Trisomy affects 3% of
pregnancies in 25-yearold women but affects 35% of pregnancies in women aged 42 years (58). Most trisomies
are the result of errors in meiosis I (26), although this varies for individual chromosomes. For example, trisomy 18
is more typically the result of errors in the second meiotic division, while trisomy 21 is predominantly the result of
errors in the first meiotic division.
With the predominance of chromosome abnormality in SA, it is clear that in order to ensure clinical relevance of
examination of SAs, the examination must include determination of the karyotype. In the event that the
examination proves normal, with normal karyotype and normal villus histology, management of persons having
SAs, especially recurrent SAs, shifts and other etiologies for pregnancy loss must be considered.
The investigation of those who have had chromosomally normal miscarriages with no other pathology is
dependent upon the proposed nonchromosome mechanisms for fetal loss. These proposed associations include
exogenous environmental exposures (77), skewed X-inactivation (5, 28), disorders of endocrine function, immune
disorders including conditions with autoantibodies, and thrombophilic conditions (56). The roles of these factors
in miscarriage remain under investigation, and the significance of each has not been established with certainty.
Antiphospholipid antibodies are found more in women who have recurrent miscarriages (RSA) than in other
women. The mechanism by which an antiphospholipid antibody causes pregnancy loss is not known. There are
no specific pathologic features identified in the first trimester SA from women who are positive for this antibody
(61, 63, 73). There is some evidence to suggest that those with recurrent miscarriages are more likely to have
thrombophilia mutations such as antiphospholipid antibody, factor V Leiden deficiency, prothrombin gene
mutations, or methylene tetrahydrofolate reductase (MTHFR) gene, although the studies of small series of
affected individuals have shown conflicting results (15, 21, 33, 56, 59). Some studies have demonstrated that the
incidence of factor V Leiden, MTHFR, and prothrombin gene mutations is no different in a population with
recurrent miscarriages than in parous controls (15, 31), and one review points out that while there is an
association between thrombophilia and pregnancy loss, a causal relationship has not been established (59).
Others suggest that there are insufficient data to recommend that all women who have recurrent pregnancy loss
should be screened for a broad range of inherited thrombophilias in the absence of other clinical features (1).
There is possibly less controversy about the role of antiphospholipid antibody in recurrent pregnancy loss, and
some advocate screening for this antibody (only) in those with recurrent miscarriages (9). The management of
those diagnosed with one of the thrombophilic disorders remains controversial. This area of investigation in
recurrent pregnancy loss is confounded by the fact that many reported series and treatment trials are
compromised by methodological problems (13).
With changing reproductive patterns such as women starting their families later in life and having fewer children,
there is a desire to diagnose and manage causes of miscarriage. Given that cytogenetic abnormalities account
for the majority of first trimester abortions, it has been suggested that the evaluation of first trimester SA with
cytogenetic analysis may prove more cost effective than a standard battery of tests such as thyroid function
tests, endometrial biopsy, or thrombophilia testing (35). Such examination also serves to identify those conditions
not associated with abnormal karyotype that may require additional investigation or treatment to increase the
chance of successful pregnancy and to diagnose conditions in which there is a risk of neoplasia, as with
complete hydatidiform mole (CHM) and its attendant risk of gestational trophoblastic neoplasia (GTN) (30).
Examination of fetal losses in the second trimester is similar to the investigation of intrauterine death in later
gestation and requires a complete autopsy examination. This examination is performed in the same way as
autopsies in older fetuses and infants, with the intent of identifying a cause of intrauterine death, making a
diagnosis, and assessing risks of recurrence. The rate of chromosome abnormality in the second trimester is
approximately 5% to 10%, less than observed in first trimester SAs, and an indication that other processes play a
more significant role in the second trimester intrauterine death or S A. The identification of these other causes of
pregnancy loss can not only provide some understanding and comfort to the affected individual but also be
important in assessing risks of adverse outcomes in future pregnancies and in determining management options.
FIRST TRIMESTER SPONTANEOUS ABORTION

Indication for Cytogenetic Analysis
Given that chromosome abnormality accounts for the majority of first trimester SAs, an argument can be made
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for performing cytogenetic analysis in all cases. The proportion of cases in which morphological abnormalities
that account for the SA are identified is small. To assist in the reproductive counseling regarding cause of the SA
and risks for recurrence, karyotype is a vital piece of information. Where embryopathology examination was once
performed only in cases of recurrent SAs, changes in reproductive patterns and practices have altered, resulting
in a broader range of cases referred for embryopathologic examination. Those who treat women who have had
difficulty conceiving or who have had previous miscarriage(s) are anxious to know whether the pregnancy failure
was the result of chromosome abnormality or if there is perhaps another etiology necessitating further
investigation. Increasingly, assisted reproductive technologies (ARTs) such as in vitro fertilization (IVF) or
intracytoplasmic sperm injection (ICSI) are utilized. There are concerns that ARTs are associated with increased
incidence of chromosome abnormalities at prenatal diagnosis and at birth, specifically for sex chromosome
abnormalities in pregnancies that are the result of ICSI (3, 22). In a comparison of 133 cases of SAs occurring
after ARTs with 144 cases of SAs in naturally conceived pregnancies, there was no significant difference in the
rate of chromosome abnormality between the two groups, with 63.2% of the ART group abnormal as compared
to 71.5% of the naturally conceived group (8). This suggests that the increased rate of chromosome abnormality
in the ICSI population does not translate to an increased rate of miscarriage of chromosomally abnormal
pregnancies.
Until the natural history of ART pregnancies is delineated, the use of these technologies should be considered
as an indication for cytogenetic analysis in cases of SA. In some laboratories, the fact that the majority of first
trimester SAs are the result of chromosome abnormality is sufficient indication to perform cytogenetic analysis of
all cases examined morphologically. In other laboratories, constraints imposed by funding structures may impose
the necessity of specific clinical indication before cytogenetic studies will be funded. These other indications
include a history of recurrent miscarriages (variably defined as two or more losses or three or more losses),
abnormal villus morphology, abnormal or normal embryo, parental chromosome rearrangement, and maternal
age 35 years or greater.
Examination
Examination of the early pregnancy loss or embryo specimen is quite different from that of a fetal specimen as
the latter represents an autopsy examination of a fetus and its placenta. Examination of the products of an early
pregnancy loss (spontaneous or missed abortion) is performed to identify pregnancy-related tissues (embryo
and/or placental tissue) to confirm intrauterine pregnancy and to assess their morphology. This examination
includes sampling of tissues for additional studies, including for cytogenetic analysis or other means of
determining the chromosome complement of the conceptus. Thus, it is imperative that all specimens for
embryopathology examination are submitted in the fresh state, not in fixative.
An assessment of the products of conception is best accomplished by examining the specimen under a
dissecting microscope equipped with a camera. The presence of any placental or embryonic tissue allows
confirmation of intrauterine pregnancy. In the absence of pregnancy-related tissues, intrauterine pregnancy
cannot be confirmed, and the report must reflect that. Decidualized endometrium may be seen in estrogen effect,
including with ectopic pregnancy, and is therefore insufficient for confirmation of intrauterine pregnancy.
The morphology of the chorionic villi is characterized— their individual morphology and their distribution over the
chorionic sac. Attention to whether the villi appear overly abundant and/or cystic is important because of
concerns for CHM or partial hydatidiform mole (PHM). Other features of embryonic development, such as
presence of amnion, yolk sac, and umbilical cord, are assessed.
Tissues are sampled for cytogenetic analysis and to be retained frozen for additional studies as required. Our
practice is to submit amnion and chorion for cytogenetic analysis as the amnion is thought to be most reflective
of the embryo itself. Cytogenetic cultures from chorion are more likely to be complicated by maternal cell
contamination, necessitating further examination by other means, such as CGH, to confirm the karyotype of the
conceptus, but amnion is not present in all cases and chorion must be sampled. Chorion and amnion seem to
grow in culture more readily than do chorionic villi and are preferred. In all cases, chorionic villi are frozen and
are available in the event that the tissue submitted for cytogenetic analysis fails to grow in culture and additional
testing such as CGH is required, for assessment in cases where maternal cell contamination is of concern, or for
additional genetic studies as indicated. In the case of pronounced maceration, the decision is made to proceed
directly to CGH, rather than attempting tissue culture for cytogenetic analysis. With the introduction of array CGH
techniques, subtle abnormalities that cannot be detected by cytogenetic analysis will be diagnosed; an argument
can be made to utilize array CGH in all cases of SAs to eliminate the labor and risk of culture failure associated
with conventional cytogenetic analysis (7, 36, 62).
The presence of embryonic tissue confirms intrauterine pregnancy and is a feature in favor of a diagnosis other
than CHM, a frequent concern as edematous villi are often identi-fied on ultrasound or at gross examination. In
determining the developmental stage and thus age of the embryo, standard developmental criteria are used (24,
48). Embryos may be normally developed (Figure 2-1) according to established criteria, but this does not exclude
chromosome abnormality. Most developmental tables were established without karyotype determination. Some
cases do not show regularly
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developed embryos but rather embryos or embryonic tissues in which normal developmental features are not
present, a state referred to as growth disorganization. In our experience, when embryos are identified, slightly
less than half of them show features of growth disorganization. Growth disorganization has been divided into four
categories: a type I growth-disorganized embryo (GDI) refers to an intact empty sac (Figure 2-2), type II refers to
a nodular embryo in which cranial and caudal ends cannot be distinguished (Figure 2-3), type III refers to a
cylindrical embryo in which there is some cranial-caudal differentiation with retinal pigment (Figure 2-4), and type
IV refers to an embryo in which there is more recognizable embryonic development but delayed growth of limbs
and other developmental features (Figure 2-5). While growth disorganization is readily identified and classified,
the findings are nonspecific—in all types of growth-disorganized embryos, the incidence of chromosome
abnormality is similar to that encountered in SAs in general, with the same types of chromosome abnormalities
identified. Ultrasound detection of an embryo does not strictly correlate with the morphological detection of
embryonic tissue, but it has been demonstrated that the rates of abnormal karyotypes are not
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significantly different between SAs in which an embryonic pole is identified on ultrasound examination and those
that appear anembryonic (35). This corresponds to our experience: 63% of anembryonic specimens are
chromosomally abnormal and 71% of embryonic specimens are chromosomally abnormal. Of embryonic
specimens, 58% of growth-disorganized embryos are abnormal and 79% of regularly developed embryos are
abnormal.
FIGURE 2-1 ▪ Normally developed human embryo, stage 14 of development.

FIGURE 2-2 ▪ Growth-disorganized embryo, GDI—intact empty amniotic sac (AS). Opened chorionic sac (arrow).
FIGURE 2-3 ▪ Growth-disorganized embryo, GD2—1 mm nodular embryo in opened amniotic and chorionic sac.
FIGURE 2-4▪Growth-disorganized embryo, GD3—cylindrical embryo with retinal pigment (arrow).
FIGURE 2-5 ▪ Growth-disorganized embryo, GD4—delayed development of head, trunk, and limbs relative to
crown-rump length.
FIGURE 2-6 ▪ Stage 20 embryo with parietal and occipital encephaloceles.
FIGURE 2-7 ▪ Monosomy X embryo with parietal encephalocele.
Embryos may show isolated or focal abnormalities such as neural tube defects, facial clefts, or limb anomalies
(Figures 2-6,2-7 and 2-8). Many of these abnormalities, such as neural tube defects, occur in the setting of
chromosome
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abnormality (41). In the setting of a normal karyotype, the focal defects likely have the same significance as in
later gestation, and genetic counseling to discuss the findings and possible recurrence risks is indicated.
Chromosomally abnormal embryos may show a number of nonspecific abnormalities, such as delay of normal
limb development, abnormal tan deposits, and various types of growth disorganizations. Embryos with the
trisomies more commonly encountered in later gestation and livebirths, such as trisomy 13, 18, and 21, may
show some features in common with the phenotypes observed in the fetal period. Most often, however,
embryonic phenotypic manifestations of these trisomies are nonspecific (Figure 2-9).
FIGURE 2-8 ▪ Stage 18 embryo with cleft lip, absent digit in the right hand, and coloboma.
Triploidy is encountered in approximately 6% of early SAs, and embryos are often identified. A phenotype
thought to be characteristic of triploidy has been described by Harris et al. (25) (Figure 2-10). With the
identification of imprinting effect in fetal triploid phenotypes, the possibility of imprinting effect in the triploid
embryo population has been assessed and there has been no correlation with embryo phenotype and parental
origin of the triploidy. In triploid embryos, a variety of appearances are encountered, ranging from growth-
disorganized to apparently normal embryos. These apparently normal embryos are most often at approximately
stage 16 of development (37 to 42 days), equivalent to approximately 7 to 8 weeks of GA (Figure 2-11). The
normal phenotype and the growth-disorganized phenotypes were seen in triploids of both maternal origin and
paternal origin. In this series of triploids with embryonic tissue present, digynic triploidy accounted for 67% of
cases. Of the nine cases of diandric origin, eight showed features of PHM (43).
Histological examination of placental tissues and decidua is routinely performed in all cases. Microscopic
examination of the chorionic villi allows detection of infection, including viral infections such as cytomegalovirus
(CMV) and bacterial infections such as listeriosis. In addition, disorders of uncertain etiology such as
intervillositis or conditions with increased intervillus fibrin are occasionally detected. Villus infarction is distinctly
unusual and should raise concerns of maternal vascular/thrombophilic disease. Routine histological examination
of decidua allows for assessment of decidual (maternal) vasculature. In a review of the histopathology of SAs
with known karyotype, 19% of SAs with a normal karyotype showed evidence of chronic inflammation or
perivillus fibrin deposition in contrast to 8% of those with an abnormal
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karyotype. The findings were even more frequent (31%) in the subset of SAs that were chromosomally normal
and occurred in a population with recurrent SAs (55).
FIGURE 2-9 ▪ Embryo with trisomy 13—postaxial Polydactyly of feet.

FIGURE 2-10 ▪ Triploid embryo showing dysplastic face, delayed limb development, and defect in lumbosacral
region (arrow).
FIGURE 2-11 ▪ Triploid embryo showing normal stage 18 phenotype, approximately 41 days DA.
FIGURE 2-12 ▪ Chronic (mononuclear) intervillositis in SA at 10 weeks' GA.
Intervillositis is a disorder of unknown etiology in which there is either focal or diffuse increase in mononuclear
cells within the intervillus (maternal) space (Figure 2-12). The lesion is thought to be possibly an immune
disorder and may recur in subsequent pregnancies (11, 16). With focal intervillositis, it may be difficult to
distinguish between focal intervillositis and an infectious process characterized by intervillus inflammation and
villus abscess; special stains for organisms should be performed to exclude Listeria and syphilis.
Increased perivillus fibrin is another disorder of unknown etiology in which it has been suggested that immune
disorder may play a role. Perivillus fibrin may be increased as a degenerative change in response to intrauterine
death of the embryo; distinguishing between degenerative changes and subtle increases in perivillus fibrin is
difficult. When there is obvious increase in perivillus fibrin, the lesion may be considered to account for the loss;
some suggest that an arbitrary threshold of 50% villus involvement be used to make this diagnosis (75). Although
probably etiologically heterogeneous, this entity may also recur and has been associated with recurrent SAs.
Infection is a clear cause of pregnancy loss, with viruses, spirochetes, and bacteria all playing significant roles.
Syphilis has increased in frequency over the past few years, and it has been encountered with increasing
frequency in pediatric pathology, including in the pregnancy loss specimens. Although first trimester loss may
occur with syphilis, it is seen more often in losses occurring in later gestation.
Listeriosis, by contrast, causes pregnancy loss throughout gestation. Listeriosis may occur as outbreaks in a
community related to improper food handling or may occur as sporadic events related to ingestion of foods
known to be at higher risk of containing Listeria, such as soft or unpasteurized cheeses (10). Listeriosis in the
first trimester SA is characterized, histologically, by acute villus abscesses, with abundant neutrophils in the
intervillus spaces (Figure 2-13). There is usually also an acute chorioamnionitis. Gram-positive bacilli may be
demonstrated on Gram stain; the histology is usually sufficiently characteristic to allow diagnosis.
FIGURE 2-13 ▪ Villus abscesses of listeriosis. SA at 14 weeks' GA.
Excluding the small number of cases in which infectious, immune, or vascular causes of first trimester SA are
identified, the majority of SAs are shown to be chromosomally abnormal. Although there are histological features
that have been suggested as being more commonly observed in aneuploid pregnancies, such as irregular villus
outlines, trophoblast inclusions, or invaginations, in general, the predictive value of these findings is low (20, 47,
57, 74). Our experience, similar to that of others (54), is that some trisomies, such as trisomy 22, are more likely
to show these features (Figure 2-14).
The most commonly encountered chromosome abnormalities are trisomy, and there are reports for trisomies of
all chromosomes encountered in SAs. Trisomy 16 is the single most commonly encountered trisomy. Trisomy for
two chromosomes (double trisomy) is seen in 3% of the chromosomally abnormal SAs.
Concern for GTN is heightened in the SA population as CHM may present as spontaneous or missed abortion. It
has been shown that fewer than 44% of CHMs or PHMs are detected at routine first trimester ultrasound (18),
providing
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an indication for the necessity of histological examination of S As, even when a gestation is apparently normal at
ultrasound or at the time of evacuation. The risk of GTN requiring chemotherapy is 15% to 28% after diagnosis
of CHM (78), making the diagnosis imperative. The risk of GTN after triploid PHM is less well-defined; there are
case reports of choriocarcinoma occurring after triploid PHM (12, 39, 45, 64), but others have shown that the risk
of persistent GTN is rare, occurring in fewer than 5% of cases (22). Given the risks, some recommend that these
cases be managed as would women who have had a CHM (76).
FIGURE 2-14 ▪ Irregular (“busy”) appearing trophoblastic epithelium in trisomy 22.
The diagnosis of early CHM and PHM can be difficult in specimens from early SAs, perhaps more so than in the
past when these pregnancies presented later in gestation. Gross examination of early hydatidiform moles may
show cystic change of chorionic villi—grossly this may be impossible to differentiate from the cystic change in
partial moles and the hydropic degeneration occurring in nonmolar SAs. Histological diagnosis is readily
achieved by pathologists experienced with this type of pathology, but there have been studies that demonstrate
considerable interobserver and intraobserver variability in the diagnosis of both CHM and PHM (19), and
concerns about the ability to consistently diagnose these entities have been raised by those practicing in a less
specialized environment (53). With recognition that the features in early CHM may be subtle and with the
availability of karyotype determination, ploidy determination, and immunohistochemical staining for p57kip2,
diagnostic accuracy is increased (20). A major problem that occurs in routine practice is to distinguish between
hydropic abortion (degenerative change) and molar gestation.
CHMs are diploid, with both haploid complements being paternal in origin. Thus, CHMs are androgenetic, with
no maternal contribution present. The abnormal development in this situation is considered to be a reflection of
abnormal imprinting (see Chapter 3), since both maternal and paternal genetic contributions are required for
normal embryo and placenta development.
The histopathological features of CHM are diffuse villus edema (hydropic change), cistern formation, and
circumferential trophoblastic hyperplasia. Rudimentary fetal vessels may be identified, but ordinarily fetal blood
cells are not seen within such vessels. Stromal karyorrhexis is a feature of early CHM, thought to be related to
increased stromal proliferation and apoptosis (Figure 2-15) (76). Immunohistochemical staining for p57kip2 is
useful in the assessment of possible molar gestations because of its expression from the maternal allele only.
Thus, in a CHM by definition androgenetic, the normal p57kip2 staining of cytotrophoblast and villus stroma is
absent (46). p57kip2 staining of triploid PHM is normal because of the maternal haploid contribution.
Triploidy may be either paternal (diandric) or maternal (digynic) in origin. Older studies demonstrated that
diandry was the predominant origin of triploidy, while more recent studies have shown that the distribution of
diandric triploidy and digynic triploidy is somewhat more complex than that. In very early gestation, digyny is at
least as common as diandry, while in cases presenting as later missed abortion with grossly cystic villi, diandry is
more common; in the fetal and infant population, digyny is clearly predominant. In early pregnancy, the incidence
of diandric triploidy is in the range of 50% to 65% (40, 42, 79). Of the two origins, it is diandric triploidy that
presents as PHM.
FIGURE 2-15 ▪ Early CHM with stromal karyorrhexis.
PHM is characterized, classically, by two populations of villi, some with hydropic change and cistern formation
and others that are small and not hydropic. The trophoblastic profile is irregular and has been described as fjord-
like. Invaginations or inclusions of trophoblast are common. There may be a lacey appearance to the
syncytiotrophoblast and the trophoblast hyperplasia is focal. Unlike CHM, there may be extensive fetal
vasculature with fetal blood cells present.
In our practice, any case in which cystic villi are identified grossly is submitted for histological examination, flow
cytometry, and cytogenetic analysis. Tissue is retained frozen at -70°C in the event that additional studies are
required. The slides are examined, and if a diagnosis of CHM is made on morphological grounds, a p57 stain is
ordered to support that diagnosis. The case is reported on histopathological grounds, and the results of
additional studies are reported as they become available. Similarly, if the histological diagnosis is of PHM, the
diagnosis is issued and flow cytometry and/or cytogenetic results are added later. If the case appears to be a
hydropic abortus, with no evidence of trophoblastic hyperplasia, p57 is ordered and the results of flow cytometry
and cytogenetic analysis are awaited.
Second Trimester Pregnancy Loss

With completion of the embryonic phase of life, all organ systems are developed. The forces that lead to loss of
pregnancy during fetal life are somewhat different than during embryonic life—while chromosome abnormality
remains a significant factor, it is considerably less so than in first trimester losses, and the types of chromosome
abnormalities more closely resemble those seen in third trimester losses and neonates. Nonchromosomal factors
such as twinning and placental pathology assume a larger role, and thus the causes of second trimester loss are
more heterogeneous with a broader range of implications for counseling and management of future pregnancy.
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As many of the cases examined in the fetal pathology service come after diagnosis of intrauterine fetal death,
maceration of tissues is a problem that must be addressed. The characteristic features of various disorders are
present but may be altered or obscured by the effects of maceration. Maceration is not a diagnosis and should
not be considered a limitation to the examination of affected fetuses. The extent of examination varies between
institutions, with external examination of a formalin-fixed fetus sufficing in some laboratories, while others provide
complete autopsy examination. The latter is the only way of adequately assessing such specimens, and
examination other than complete autopsy with examination of the placenta should be considered incomplete.
The autopsy of fetal specimens is conducted exactly as in all other perinatal cases: a complete external
examination is performed, skeletal survey is done as indicated, and internal examination with dissection of all
organ systems, including central nervous system (CNS), is performed. The external examination includes an
assessment of all growth parameters and comparison to established normal values for the determination of DA
and detection of intrauterine growth restriction (IUGR). Sections from all organ systems are submitted for
histological examination. Cytogenetic studies are initiated in all cases of intrauterine death, abnormal maternal
serum screening, hydrops fetalis, fetal anomaly, cystic placental abnormality, and maternal age 35 years or
more. Fetal and placental tissues are frozen in all cases in the event that the tissue is required for CGH or other
genetic studies. When indicated, tissues are submitted for molecular genetic analysis for disorders such as
hemoglobinopathies and for viral cultures. In cases of suspected skeletal dysplasia, skeletal survey is performed,
fibroblast cultures are initiated, and the resultant cell line is frozen and retained.
It is not possible to outline all of the disorders encountered in the pathology of second trimester losses. Suffice it
to say that with the possible exception of a greater proportion of cases showing chromosome abnormality in the
second trimester, one has to be prepared to see all of the pathology encountered in later gestation intrauterine
deaths and pregnancy losses (see Chapter 4).
In general, the losses occurring in the second trimester can be considered as fetal deaths (missed abortions) or
SAs. In the latter setting, the fetuses are well preserved and the findings raise concerns for uterine anomalies,
cervical incompetence, and/or ascending infection.
Uterine anomalies are present in approximately 15% of women who have recurrent miscarriages (14). Pathologic
examination of the aborted fetus and placenta cannot make this diagnosis, but the finding of a nonmacerated,
anatomically and chromosomally normal fetus with no evidence of ascending infection may lead to clinical
consideration of anatomic or mechanical uterine factors.
Ascending infection is a common cause of second trimester pregnancy loss (27, 65). There may be no
antecedent history. Examination of the fetus shows a well-preserved, anatomically normal fetus. Histological
examination of fetal organs may show neutrophils within the gastrointestinal tract and lungs, with pulmonary
neutrophils seen more often in later gestation. Gross examination of the placenta may show opaqueness of the
fetal membranes, and histological examination shows neutrophils in the chorion and amnion. Fetal response may
be present in the form of neutrophils in the fetal surface vessels and in the vessels of the umbilical cord.
Ascending infection has been associated with intrauterine death though this presentation is less common before
20 weeks of GA. It is not clear what the mechanism of intrauterine death is in these cases; death has been
reported to be more likely when there is fetal response to intrauterine infection (34, 60).
Although a broad range of organisms is responsible for chorioamnionitis, routine culturing of the products of SA
is not performed. When there is clinical concern for specific notifiable diseases, such as listeriosis or syphilis,
confirmatory cultures are indicated. Listeriosis may occur as outbreaks, and thus knowledge of its role in
infection of pregnancy is important from an epidemiological perspective as well as from a clinical one.
Listeriosis is caused by Listeria monocytogenes. Infection may be caused by exposure to foods such as
unpasteurized or soft cheeses as well as processed meats. Outbreaks may occur and have been related to
contaminated processes such as cheese making or machines used in processing meats. Listeriosis may be
subclinical or may be associated with gastrointestinal diseases such as vomiting and diarrhea or generalized
malaise that includes fever and myalgias. Infection in pregnant women is associated with an increased risk of
intrauterine death and SA, including both first and second trimester SAs.
Pathologically, listeriosis may be suspected when external examination of a fetus shows small white lesions on
the skin, which are confirmed to be small abscesses in which organisms abound. Similar abscesses may be
identified in fetal organs. Gross examination of the placenta may be normal or show characteristic gross features
of ascending infection. Histological examination usually shows chorioamnionitis that may be severe. The
characteristic lesion of listeriosis is acute villus abscess. Gram-positive bacilli are abundant on tissue Gram stain
(Figures 2-16 [skin] and 2-17 [villus abscess]).
FIGURE 2-16 ▪ Listeriosis. A: Gross examination shows small white nodules/plaques on the skin of second
trimester fetus (arrows). B: Histological examination shows necrosis with abundant bacteria, shown to be
Grampositive bacilli, culture positive for Listeria monocytogenes.
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FIGURE 2-17 ▪ Villus abscesses of listeriosis associated with severe, acute chorioamnionitis.
Other infections, including viral infections, can account for intrauterine death in the second trimester, with CMV
being the viral infection most commonly encountered in fetal death (2). Fetuses affected by CMV may be grossly
morphologically normal aside from the effects of retention after fetal death but may also show hepatic
calcification and CNS abnormalities. Histological examination of fetal organs may show mononuclear
inflammatory infiltrates, ranging in severity, and CMV inclusions may be identified. Destructive lesions may be
seen in affected organs. The placenta will show lymphoplasmacytic villitis, and CMV inclusions are often readily
identifiable on routine H&E stains (Figure 2-18). In situ hybridization with appropriate probes can be used for
confirmation, as necessary. There may be a discrepancy between the severity of placental manifestations and
those of the fetal organs—it often appears that the fetal organs are more likely to show inclusions and
inflammation when the placental inflammation is milder.
FIGURE 2-18 ▪ CMV in macerated second trimester fetus. A: Villitis with viral inclusions. B: Viral inclusions in
kidney. C: In situ hybridization for CMV highlights inclusions.
Syphilis is encountered with increasing frequency in the obstetric population, including in fetal deaths.
Spirochetes may be readily identified in fetal organs, and the placenta shows the features described elsewhere
(see Chapter 9), including villitis, villus edema, and vascular changes.
Chromosome abnormality is encountered less often in second trimester losses than in those occurring in the first
trimester, and the type of aneuploidy encountered is less varied, bearing a closer resemblance to the range
observed nearer term (see Chapter 3). The trisomies encountered during life, trisomy 21, 13, and 18, as well as
monosomy X and triploidy are the most commonly identified abnormalities. These abnormalities are expected to
be encountered in second trimester miscarriages because intrauterine survival is profoundly affected, with only a
minority of chromosomally abnormal cases surviving to term; it is estimated that only 20% of trisomy 21
conceptions, 5% of trisomy 18 conceptions, and 1% of monosomy X conceptions survive to be liveborn. The
mechanism allowing some of the chromosome abnormalities, such as trisomy 13 and 18, to survive into later
gestation has been suggested to be the presence of a normal cell line in trophoblast. Placental mosaicism has
not been shown to account for the survival of trisomy 21 concepti.
Trisomy 21
Trisomy 21 syndrome in the fetus shows the same range of developmental anomalies observed in liveborns and
may be associated with abnormalities of maternal serum markers (Table 3-5). In addition, it is common for
trisomy 21 (+21) to present as hydrops fetalis, with generalized subcutaneous edema and nuchal hygroma
(Figure 2-19). The only feature observed more commonly in those +21 cases presenting as intrauterine death as
opposed to those terminated after prenatal cytogenetic diagnosis is hydrops fetalis; the other anomalies do not
appear to be different between the two
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groups and thus do not provide an explanation to account for the survival of only some +21 conceptions until
later gestation. The presence of features such as atrioventricular cardiac defect suggests trisomy 21 syndrome,
but cytogenetic analysis is required for confirmation. Occasionally, myeloproliferative syndrome with hepatic
fibrosis is identified in a hydropic +21 fetus, but this does not account for all hydrops fetalis observed in trisomy
21. Identification of the specific chromosome abnormality is necessary as diagnosis will affect management of
subsequent pregnancies. There is an empiric risk of recurrence of trisomy on the order of 1% after a pregnancy
(second trimester or later) is affected by trisomy, whereas other chromosome abnormalities, such as monosomy
X, are not associated with the increased risk of recurrence. In addition, if the trisomy 21 is the result of a
robertsonian translocation carried in balanced form by one parent, the risk for recurrent trisomy 21 is even
higher.
FIGURE 2-19 ▪ Trisomy 21 syndrome. Hydropic fetus confirmed by cytogenetic analysis to have trisomy 21. No
other internal anomalies.
Trisomy 18
Fetuses with trisomy 18 syndrome may present as intrauterine death with no external anomalies and may have
been associated with abnormal maternal serum screening, including very low estriol levels. The assessment of
IUGR can be difficult in a case where there is maceration, as retention after fetal death may account for some of
the discrepancy in fetal growth parameters. Trisomy 18 syndrome fetuses often show a somewhat rounded
appearance to the head with a small face (Figure 2-20). The hands show flexion of the fingers, with the second
and fifth fingers clasped over the third and fourth, respectively. Feet may show prominent heels and rocker
bottom feet, though these features are subjective and often overstated. Internal examination may be normal or
may show the internal abnormalities described in liveborns, with renal anomalies such as horseshoe kidney
being one of the most commonly observed (See Chapter 3, Table 3-6). Dysplasia of cardiac valves is
encountered in most cases and has been referred to as “diaphanous dysplasia.” Although it has been suggested
that there are more female than male fetuses with trisomy 18, a review of our data of trisomy 18 fetuses, either
spontaneously or therapeutically aborted, showed no variation from the expected sex chromosome ratio and no
difference between those miscarried and those therapeutically aborted.
FIGURE 2-20▪Trisomy 18 syndrome. Fetus showing rounded head and rather small face with bilateral cleft lip
and palate. Hands show characteristic clenched appearance. Internal examination showed horseshoe kidney,
single umbilical artery, ventricular septal defect (VSD), and dysplasia of the cardiac valves.
Trisomy 13
Trisomy 13 may also present as otherwise unanticipated fetal death, with only 5% of all trisomy 13 conceptions
surviving to be liveborn. There is often cleft lip and palate, and the facial abnormalities may include those
reflective of the characteristic brain anomaly, holoprosencephaly, and include proboscis and synophthalmia
(Figure 2-21). There is often postaxial polydactyly. There may be an omphalocele and internal anomalies
affecting a variety of systems including the kidneys, which may be enlarged and show cystic change, and the
heart, which characteristically shows a tetralogy of Fallot or truncus arteriosus (See Chapter 3, Table 3-7). At
gross examination, the differential diagnosis includes Meckel-Gruber and pseudo-trisomy 13 syndromes.
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FIGURE 2-21 ▪ Trisomy 13 syndrome. Macerated fetus showing synophthalmia with proboscis. Bilateral postaxial
polydactyly of feet. Internal examination showed VSD.
Monosomy X
Monosomy X is also known as Turner syndrome. In the fetal period, this most commonly presents as hydrops
fetalis, often with a very large cystic hygroma. Accentuation of the subcutaneous edema on the dorsal aspects of
the hands and feet is characteristic but nonspecific. These fetuses are female and show normal female genitalia,
internally and externally. Characteristic anomalies include left-sided cardiac anomalies such as hypoplasia of the
aortic arch and/or left ventricle. Renal anomalies include horseshoe kidney (Figure 2-22).
Triploidy
Triploidy is the presence of an entire extra haploid set of chromosomes, which may be of maternal (digynic) or
paternal (diandric) origin. In the fetal period, digynic triploidy predominates, accounting for the majority of cases
(42). Although the chromosome abnormality is numerically the same, an epigenetic phenomenon known as
imprinting causes the fetal and placental phenotypes to vary quite significantly from each other.
In general, triploidy is characterized by anomalies that affect almost every organ system and can be present in
both digynic triploidy and diandric triploidy. Complete syndactyly of the third and fourth fingers is a characteristic
feature of triploidy, independent of parental origin. Thus far, only adrenal hypoplasia has been shown to be
dependent on parental origin, being found in digynic triploids. The parental origin effects appear to be limited to
growth patterns in both the fetus and the placenta. Digynic triploidy is characterized by marked asymmetric
IUGR, with the head size being relatively well preserved compared to the trunk and extremities, which are very
thin (Figure 2-23). There is marked adrenal hypoplasia as observed in other cases of
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severe IUGR, consistent with the role of placental function in intrauterine adrenal growth and development. Other
anomalies are varied and affect all organ systems. The placenta is abnormally small and shows no villus edema
or trophoblastic hyperplasia. In diandric triploidy, growth is better preserved, but there may be symmetric IUGR
(Figure 2-24). The placenta shows changes of PHM with villus edema and cistern formation, with focal
trophoblastic hyperplasia involving the syncytiotrophoblast, which can have a lacey appearance with
invaginations into the villus stromal core. The growth and placental differences are reflected in the abnormalities
observed in maternal serum screening with digynic triploids showing markedly decreased estriol and human
chorionic gonadotropin (hCG), while the diandric triploids can show markedly increased levels of alpha-
fetoprotein (AFP) and hCG.
FIGURE 2-22▪Monosomy X syndrome. Macerated female fetus showing hydrops fetalis with large cystic nuchal
hygroma. Internal examination showed hypoplasia of the aorta.
FIGURE 2-23▪Digynic triploid phenotype—the phenotype most often encountered in triploid fetuses. Asymmetric
IUGR, with relative sparing of the head and thin extremities. No molar change is seen in the placenta.
FIGURE 2-24▪Diandric triploid phenotype—the phenotype seen only rarely in triploid fetuses. Growth parameters
better preserved. Large placenta shows changes of PHM.
Hydrops Fetalis
Hydrops fetalis is a common presentation in second trimester fetal deaths and warrants complete evaluation for
diagnosis, as in those cases diagnosed as stillbirths (Figure 2-25). The differential diagnosis is extensive and
includes chromosome abnormality, infection such as CMV and parvovirus B19, hemoglobinopathies such as
thalassemia, antibodies such as Rh isoimmunization, fetal arrhythmias, congenital pulmonary airway
malformations of the lung, tumors, and metabolic disorders (see Chapter 4) (37, 44). Accordingly, the approach
to hydrops fetalis includes complete autopsy examination with cytogenetic analysis, viral cultures, PCR for
parvovirus, initiation of fibroblast cultures, and retention of a variety of tissues for freezing at -70°C in the event
that additional studies such as alpha-thalassemia gene studies are required. With the exclusion of these entities,
one is left with a diagnosis of hydrops fetalis, etiology undetected. Because of the possibility of an undetected
metabolic condition leading to the hydrops, genetic counseling considers the risk of an undiagnosed autosomal
recessive condition; thus, the risk of recurrence may be as high as 25% for each subsequent pregnancy.
FIGURE 2-25▪Hydrops fetalis, cause not determined, after extensive investigation. Genetic counseling should
include possibility of undiagnosed genetic conditions, with recurrence risks as high as 25%.
Twinning
Monozygous twinning is associated with an increased risk of intrauterine fetal death and may occur on the basis
of vascular anastomoses, leading to twin-twin transfusion syndrome (Figure 2-26) or twin reversed arterial
perfusion (TRAP)
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sequence (23, 70, 71 and 72). The former cannot be diagnosed conclusively but can be suggested if there are
growth and/or perfusion discrepancies between the two fetuses. TRAP is a condition in which the umbilical cords
of monochorionic monoamniotic twins are implanted very close to one another, establishing large vascular
anastomoses. It is hypothesized that some event leads to an imbalance in the shunting of blood, resulting in
reversed perfusion such that one twin receives deoxygenated blood from the other via retrograde flow through its
umbilical artery. This results in hypoxia in the recipient twin with resultant tissue necrosis, most severe in the
cranial aspect. Thus, the tissues of the perfused twin regress, leading to an acardiac, acephalic twin (Figure 2-
27). This perfusion abnormality may result in the death of both twins, or the acardiac twin may be delivered at
term with the coexisting twin. Monoamniotic twins are also more likely to have cord entanglement that can lead to
compromise of umbilical cord blood flow, resulting in the death of both twins.
FIGURE 2-26▪Twin-twin transfusion syndrome in intrauterine death. Monochorionic twin fetuses show size
difference as well as differences in the degree of congestion, consistent with circulatory imbalance.
FIGURE 2-27▪TRAP sequence, with normal pump twin and acardiac recipient twin.
Umbilical Cord Compromise

Umbilical cord compromise can occur in a variety of settings and results in the death of the fetus (4, 6, 29, 32,
51). In some cases, there may be no gross features to suggest the cause of death, whereas other cases show
features, such as entanglement that could not have occurred postmortem, that suggest the diagnosis (Figure 2-
28). Some have identified histological features that support a diagnosis of cord blood flow restriction (50); some
of the features are difficult to assess in the very macerated fetus. There has been considerable controversy as to
whether the twist at the junction of the umbilical cord with the abdominal wall often observed in macerated
fetuses is a cause of cord compromise or a postmortem artifact; we have observed this finding in cases of
pregnancy termination in which death has been caused by potassium chloride injection prior to delivery, and thus
we consider it usually to be a postmortem artifact.
FIGURE 2-28▪Probable cord entanglement in intrauterine fetal death.
Limb-Body Wall Complex

Limb-body wall complex (LBWC) is a disorder within the spectrum of short cord or placental adhesion sequence,
is characterized usually by limb anomalies, often absence of an entire limb, associated with a large body wall
defect and an abnormally short umbilical cord, and is usually chromosomally normal (Figure 2-29) (70, 71). With
the routine use of detailed ultrasound examination in pregnancy, these cases are now more often encountered
as products of pregnancy termination but may present as early intrauterine death, presumably on the basis of
compromise of umbilical cord blood flow.
Postprocedure Pregnancy Loss

Loss of pregnancy can occur after invasive prenatal procedures such as chorionic villus sampling and
amniocentesis. The rate varies with institution, usually being on the order of 0.5%, although some studies show
no significant increase in fetal loss in those who had amniocentesis than in those who did not (17, 49). The loss
can take the form of SA or fetal death. Ascending infection is one cause of SA, while placental circulatory
abnormalities have been hypothesized to account for fetal death. For quality assurance purposes, losses
occurring within a month of an invasive procedure are considered postprocedure losses.
FIGURE 2-29▪LBWC in intrauterine death. Large body wall defect, absence of limb, and abnormally short
umbilical cord.
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Chapter 3
Chromosomal Abnormalities
Raj P. Kapur
Joseph R. Siebert
Chromosomal abnormalities are defined as alterations that can be resolved by microscopic examination of banded chromosome
preparations. The primary means to recognize chromosomal disorders is a karyotype—organization of all the individual
chromosomes from largest to smallest with the shorter chromosomal arms oriented upward. Karyotype analysis has led to the
recognition of a wide range of chromosomal abnormalities and corresponding clinical-pathological features. The growth of this
discipline is apparent from the rich lexicon that is used to characterize specific types of chromosomal disorders and their
consequences. Some of the terms commonly encountered in the practice of pediatric pathology are defined in Table 3-1.
A traditional definition distinguishes chromosomal abnormalities from more subtle genetic alterations (e.g., single base pair
changes, microdeletions, epigenetic modifications), despite the fact that chromosomes are the common substrate for all these
events. The use of fluorescent molecular probes to interrogate specific genetic sequences blurs this distinction because methods
such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY) are
being used to identify and/or clarify chromosomal rearrangements that frequently encompass more than an individual gene but
cannot be resolved in a routine karyotype. At present, it seems reasonable to subclassify genetic defects into microscopically
visible chromosomal abnormalities and “submicroscopic” alterations. In addition to changes that affect the nucleotide sequence,
submicroscopic alterations also include epigenetic DNA modifications (e.g., methylation, histone acetylation), which influence gene
expression without changing the primary sequence. These covalent modifications are involved intimately in parental imprinting, X-
chromosome inactivation, and the physiological silencing/activation of genes. Defects in epigenetic regulation are associated with
developmental disorders, neoplastic transformation, and other disease states.
The focus of this chapter is traditional karyotypic and selected submicroscopic disorders, which are particularly relevant to the
practice of pediatric and surgical pathology. Emphasis is placed on conditions that arise during gametogenesis or prenatally, most
of which have developmental consequences. Related topics that are not covered in this chapter include chromosomal
rearrangements associated with pediatric neoplasms and mutations that affect mitochondrial DNA. Cytogenetic changes
characteristic of childhood tumors are introduced as part of the discussion of specific neoplasms in other chapters and have been
the subject of several excellent reviews (42, 67, 100, 105). For information about the mitochondrial genome and related diseases,
the reader is referred to the review by Schapira (108).
TRADITIONAL CYTOGENETIC ANALYSIS (THE KARYOTYPE)

Routine cytogenetic studies require mitotically active cells. The goal is to visualize chromosomes in their most elongate forms
(prophase or early metaphase), identify each one, and assess its integrity. Distinctive features for each chromosome include size,
centromere position, and banding pattern, which refers to characteristic zones of GCor AT-rich sequences that are resolved by
specific stains (Table 3-2) (89). Giemsa (G)-banding is the routine procedure employed by most laboratories. Recognition of subtle
structural defects is limited ultimately by the number of chromosomal bands that can be unambiguously identified, which varies with
cell type, staining procedures, culture conditions, and laboratory technique (Table 3-3) (64). The conventional range of reportable
karyotypes extends from 300-450 (low resolution) to 650-850 (high resolution) bands. Explicit statement of the band resolution is an
integral part of a cytogenetics report.
An international standardized nomenclature exists for cytogenetic results (111). The format and abbreviations are designed to
encompass the broad range of observable chromosomal aberrations and the methods by which they are resolved. Some of the
more frequently used abbreviations are summarized in Table 3-4 with some examples of their use.
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Table 3-1 ▪ GLOSSARY OF TERMS USED FREQUENTLY IN CYTOGENETICS
Aneuploidy—numerical deviations of just one or a few chromosomes. Usually includes trisomies and monosomies, but
not polyploidy. Does not encompass other types of genetic imbalance (e.g., partial deletions or duplications of a specific
chromosome).
Chimerism—coexistence, within one conceptus, of more than one cell lineage due to the union of two originally separate
embryos.
Chromosomal mosaicism—coexistence, within one conceptus, of two or more chromosomally distinct cell lines that
derived from a single zygote.
Deletion—loss of a portion of a chromosome.
Duplication—gain of a portion of a chromosome.
Insertion—intercalation of a portion of one chromosome into a second chromosome (interchromosomal) or into a new
location on the original chromosome (intrachromosomal).
Inversion—180-degree rotation of an intrachromosomal segment.
Pericentric—the inverted segment encompasses the centromere.
Paracentric—the centromere is excluded from the inverted segment.
Nondisjunction—failure of homologous chromosomes or sister chromatids to segregate properly during cell division.
Parental imprinting—differential expression of alleles acquired from each parent.
Polyploidy—one or more complete extra set of chromosomes (triploidy, tetraploidy, etc.).
Ring chromosome—circular chromosome, which is formed from a chromosome by end-to-end fusion of either the
telomeres or subtelomeric sites which are exposed by chromosomal breaks in the long and short arms.
Translocation—recombination of nonhomologous parts of two chromosomes.
Balanced—reciprocal translocation with no net gain or loss of the diploid chromosomal content.
Unbalanced—net gain and loss of translocated portions of specific chromosomes due to segregation of a balanced
translocation during meiosis.
Robertsonian—centric or pericentric translocation of acrocentric chromosomes (e.g., t[13q14q]).
Uniparental disomy—both chromosomes of a homologous pair are derived from the same parent.
Heteroisodisomy—UPD in which the two homologues differ.
Isodisomy—UPD in which the homologues are identical.
Common tissue sources for routine cytogenetic study include blood (phytohemagglutinin-stimulated T lymphocytes), amniotic fluid
(amniotic epithelial cells), chorionic villous biopsy (trophoblast and/or fibroblasts), and skin (fibroblasts). For most samples, days to
weeks of tissue culture is required to produce a cohort of cells that can be pharmacologically arrested in metaphase, harvested,
stained, and analyzed. These cell culture preparations differ from direct preps, which can be obtained within 48 hours from tissue
samples with high basal rates of proliferation (e.g., leukemic blasts, chorionic trophoblast). It is important to realize that tissue
culture may select for subsets of mitotically active cells in the original sample and/or cytogenetic changes that arise in vitro. Biased
selection in cell culture may yield a karyotype that does not represent particular cells of interest. In this respect, direct preps are
more reliable.
Table 3-2 ▪ CYTOGENETIC BANDING TECHNIQUES
Principle
Technique Reagent Target Properties
Q (Quinacrine)-banding Quinacrine fluorophore AT-rich areas Fades with time
C (Constitutive Acid/alkali-pretreatment prior to Stains

heterochromatin)-banding Giemsa stain heterochromatin Permanent
Proteolytic pretreatment prior to

G (Giemsa)-banding Giemsa stain AT-rich areas Permanent
Permanent (inverse of Q- or
R (Reverse)-banding Hot alkali prior to Giemsa GC-rich areas G-banding)
Sampling also affects interpretation of cytogenetic results. Some tissue sources contain a mixture of cell types with different
chromosomal compositions. Depending on the clinical situation, it may be critical to obtain karyotypic information from one or more
of the sampled populations. For example, some individuals are mosaics, whose tissues contain chromosomally different lineages. In
the case of a diploid:aneuploid mosaicism, sampling and successful culture of both cell populations are essential to establish the
diagnosis cytogenetically. Placental samples can be particularly confusing in this regard. The placenta contains a mixture of cell
types that are closely (chorionic stroma, amniocytes), remotely (trophoblast), or not (decidualized endometrium) related to cell
lineages of the fetus. Decidualized endometrium is most concentrated at the maternal surface of the placenta. Stromal cells, not
trophoblast, are propagated selectively in cultures of chorionic villus samples. Appropriate tissue sampling (fetal versus maternal
surface) and culture methods (direct versus prolonged growth in vitro) can bias cytogenetic studies toward desired cell types.
Because routine cytogenetic analysis requires successful cell culture, autolysis or contamination by microorganisms may also
compromise results. Rapid handling and sterile technique minimize these risks, but for postmortem specimens, particularly stillborn
fetuses, significant autolysis may be unavoidable. Several studies have shown that the likelihood of successful culture is inversely
related to postmortem interval, particularly the period of time a dead fetus is retained in the uterus (65, 74). It is unlikely that a
karyotype can be obtained from fetal samples acquired more than 3 days after intrauterine demise (116). Refrigeration slows
autolysis considerably, and Macpherson et al. reported
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successful cultures from the tissues of a refrigerated neonate 144 hours after demise (74). In general, fibroblasts seem to be the
heartiest cells in most organs. Therefore, fibroblastrich tissues (e.g., dermis, fascia) are favored sites. Chondrocytes also are
reported to fare well despite generalized fetal autolysis (34). Organs such as lung and gastrointestinal tract are less reliable since
they may be colonized by microorganisms. If subcutaneous tissue is sampled, it is generally best to procure a sample immediately
after the skin has been incised to avoid contamination during subsequent dissection. The skin does not need to be sterilized, but
the sample should be taken with a sterile blade, deep to the epidermis and away from the initial incision to exclude surface
microbes. If chemicals are used to sterilize the skin, care should be taken not to contaminate the transport medium with toxic agents
that may prevent cell culture. For very autolyzed stillborn fetuses, placenta may be the tissue of choice, because it is kept viable by
the maternal circulation after the fetus dies (3, 27).
Table 3-3 ▪ TISSUE SOURCES, CELL TYPES, APPROXIMATE INCUBATION TIMES, AND BAND RESOLUTION
FOR TRADITIONAL CYTOGENETIC ANALYSES
Tissue Source Cell Type Approximate Incubation Time (64) Typical Band Resolution
Amniotic fluid Amniocytes 5-21 days 450-500
Chorionic villi
Direct preparation Trophoblast 0-2 days 400-450
Culture 5-21 days
Peripheral blood Lymphoblasts 2-4 days 500-650a
Skin or soft tissue Fibroblasts 1-6 weeks 450-500
aHigher resolution (>650 bands) is possible with prometaphase preparations.
Table 3-4 ▪ CYTOGENETIC NOMENCLATURE
Common abbreviations:
del, deletion; inv, inversion; dup, duplication;

mar, marker chromosome; t, translocation; rob,
Robertsonian translocation; pter, terminus of
short arm; qter, terminus of long arm; der,
derivative (of abnormal recombination)
Normal karyotype
Autosomes are specified only when an abnormality is present:
Symbols and band numbers are used to denote complex rearrangements between chromosomes:
KARYOTYPIC DISORDERS
Constitutive karyotypic disorders are extremely common during all stages of development. The results of several studies suggest
that 5% to 25% of conceptions are aneuploid, of which 99% are spontaneously aborted (46). Rates and types of chromosomal
abnormalities detected in spontaneous abortions differ through gestation: 78% at 2 weeks post conception, 35% to 62% between
the first missed menses and 20 weeks, and 4% to 6% for stillborn infants (Table 3-5). The pathology of early embryonic loss and its
poor correlation with cytogenetic findings are discussed in Chapter 2. The rate of aneuploidy among all liveborn infants is
approximately 0.5%, although the rate in malformed infants is significantly higher (4, 73).
The most common karyotypic anomalies are forms of chromosomal aneuploidy (Table 3-6) in which one or a few complete
chromosomes are lost or gained during cell division. The underlying basis for aneuploidy is nondisjunction, failure of paired
chromatids or homologous chromosomes to segregate appropriately during mitosis or meiosis (17). Premature separation of a
chromatid pair during the first meiotic division is another theoretical mechanism to produce an aneuploid gamete, but
nondisjunction has received more experimental support (83). Meiotic nondisjunction produces an aneuploid gamete and zygote,
which is the basis for most nonmosaic aneuploid conceptuses (33). Autosomal nondisjunction is far more common during
oogenesis than spermatogenesis and typically occurs during the first meiotic division (when bivalent chromosomes segregate). By
contrast, sex chromosome nondisjunction occurs more commonly during male gametogenesis in the second meiotic division
(separation of chromatids). Postzygotic chromosomal nondisjunction during mitosis is the basis for diploid:aneuploid mosaicism,
either because an aneuploid clone arises from a diploid zygote or vice versa. Examination of human embryos conceived by in vitro
fertilization suggests that rates of spontaneous postzygotic nondisjunction are very high (20% to 50% of preimplantation embryos)
(8, 46).
Chromosomal segregation and cytokinesis are synchronized by meiotic and mitotic checkpoints. The molecules that influence
these cellular events have been partially characterized (17). Not surprisingly, genetic or pharmacologic alterations
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that disrupt these proteins predispose to nondisjunction and aneuploidy (33). However, most instances of human meiotic
nondisjunction are sporadic; in one population-based study, aneuploid spontaneous abortion was not associated with an increased
risk of aneuploidy in a subsequent spontaneous abortion (102).
Table 3-5 ▪ INCIDENCE OF ANEUPLOIDY DURING DEVELOPMENT
Gestation
0 6-8 20 40
(weeks):
Preimplantation Preclinical Spontaneous

Sperm Oocytes Embryos Abortions Abortions Stillbirths Livebirths
Incidence of 1%-2% ∽20% ∽ 20% ? 35%-54% 4%-6% 0.3%-0.6%

aneuploidy
Most Various Various Various ? 45,X; +16, 45,X, +13, 45X, +13,
common +21, +22, +18, +21, +18, +21
aneuploidies polyploidy polyploidy XXX, XXY,
XYY
From reference (49), modified to include data from references (12, 62, 92).
Karyotypic disorders other than aneuploidy involve structural rearrangements (deletions, duplications, and translocations) that alter
the normal banding pattern, but not necessarily the total number, of chromosomes. Most of these structural changes arise from
recombination between or within individual chromosomes, as discussed later in this chapter (33).
Mosaicism
In the context of human chromosomal disorders, mosaicism refers to the presence of more than one karyotypically distinct cell
lineage, derived from a single zygote. Usually, two cell populations are present, one with a normal, diploid chromosome content
and another aneuploid cohort. In cytogenetic reports, mosaicism is denoted by a slash that separates the karyotype of each cell
population and brackets that indicate the number of cells observed with each karyotype (e.g., 45,X[15]/46XY[5]). Occasionally, both
populations are aneuploid. The relative abundance of the two cell populations is highly variable, between individuals and between
organs. Interorgan differences may reflect origin of a cytogenetic abnormality in a cell lineage with restricted embryological fates
and/or selective pressures that favor a cytogenetically distinct cell population. Because of interorgan differences and the possibility
that cytoge-netically distinct cell populations may have very different growth properties in vitro, routine cytogenetic studies cannot
completely exclude low level mosaicism. Evaluation of multiple tissues (skin, blood, and amniocytes) or application of sensitive
techniques (e.g., FISH) increases the likelihood of detecting mosaicism. Because aneuploid populations can arise during tissue
culture, accepted standards for the diagnosis of mosaicism require 3/100 monosomic cells and 2/100 trisomic cells (118).
Table 3-6 ▪ CHROMOSOMAL ANOMALIES IN SPONTANEOUS ABORTIONS
Abnormality Reported Rate of Occurrence (%)a
Autosomal trisomy 50-60
Polyploidy 20-25
Monosomy X 10-20
Translocations 2-5
aPooled results from references (7, 12, 57, 62, 73).
Chromosomal mosaicism is a consequence of postzygotic mitotic errors, which can occur at any stage of prenatal development.
Empirical data suggest that such errors occur frequently in mitotically active cell populations (e.g., hematopoietic precursors), but
the majority of aneuploid cells are eliminated by unknown mechanisms (17). Many neoplasms are complex mosaics, often
comprised of several cytogenetically distinct cell populations.
Conceptuses with aneuploid:diploid mosaicism can arise from either a diploid or aneuploid zygote. In a diploid embryo,
nondisjunction of a single chromatid pair during mitosis will give rise to daughter cells that are trisomic and monosomic.
Aneuploid:diploid mosaicism results from survival and clonal expansion of either the trisomic or monosomic lineages. Excluding
monosomy X, trisomy:diploid mosaicism is far more common than monosomy:diploid mosaicism, probably because autosomal
monosomies are not compatible with cell lineage survival. The other origin for aneuploid:diploid mosaics is a nondisjunction event
during cell division in an aneuploid embryo. In the case of a trisomic or monosomic embryo, nondisjunction may restore a diploid
chromosome content (trisomic rescue) to one of the daughter cells and its descendants. The most frequent mosaics are
trisomy:diploid and 45X:diploid combinations. However, monosomy:diploid, polyploid (one or more complete extra set of
chromosomes):diploid mosaics and mosaicism involving structurally abnormal chromosomes also occur.
The phenotype of mosaic individuals is largely influenced by the relative number of aneuploid and diploid cells in each tissue
lineage. Large numbers of aneuploid cells are more likely to alter development. For monosomy X or trisomies associated with
syndromes, longer survival or mild clinical features are often an indication of underlying mosaicism. If
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mosaicism arises in the cleavage-stage embryo, both aneuploid and diploid cell populations are likely to contribute to the
embryonic (embryo, amnion) and extraembryonic lineages. Mitotic errors occurring later in development produce more restricted
mosaicism that may be confined to either the fetus or placenta.
Confined Placental Mosaicism

Confined placental mosaicism (CPM) appears to be a relatively common phenomenon. Discrepant fetal and placental karyotypes
have been observed in 0.5% to 2% of chorionic villus samples, with a placental aneuploid population in 80% to 90% (118). CPM
can be subclassified into three types depending on whether the aneuploid population is confined to the trophoblast (type I),
chorionic stroma (type II), or both cell lineages (type III) (59). Specific trisomies are more commonly associated with particular types
of CPM (70). It is important to understand that chorionic stromal cells, but not trophoblast, replicate efficiently in cell culture.
Therefore, type I CPM will not be detected by routine karyotype of indirect preparations. Type I CPM can be ascertained by direct
analysis of cytotrophoblast cells or by in situ methods like FISH that target specific chromosomes.
Prospective studies indicate that the vast majority of placentas with confined aneuploid mosaicism detected by chorionic villus
sampling early in gestation have no clinical significance (118). However, the remaining cases represent important causes of
miscarriage, intrauterine growth restriction (IUGR), intrauterine demise, and postnatal morbidity. Risks for each of these outcomes
differ depending on the type of CPM (Figure 3-1) (70).
Lestou and Kalousek recommend testing the placenta for CPM in cases of idiopathic IUGR (birth weight < 5th percentile) without
obvious maternal, fetal, or placental causes (70). The contemporary approach of most perinatal services is to perform traditional
indirect cytogenetic analysis of one or more biopsies of the placenta, an approach that will only detect CPM types II or III. CGH or
array-based CGH is less expensive and can detect all three forms of CPM, but is not widely available. Although CGH will not detect
low-level mosaicism, only high levels of mosaicism (CGH-detectable) are thought to be clinically significant. For this reason, a
single placental biopsy may be adequate to exclude “clinically relevant” mosaicism by traditional cytogenetics or CGH. The practice
of screening all placentas from newborns with idiopathic growth restriction has yet to be adopted as the standard of care in the
United States or many other countries.
A strong reason to test for CPM in the placentas of liveborn growth-restricted infants is to identify uniparental disomy (UPD) (128).
Some infants with constitutive UPD appear to arise from a trisomic zygote when an early progenitor of the embryonic lineage
undergoes a nondisjunction event that restores a diploid chromosome content. Descendants of the diploid lineage populate the
entire embryo, but the placenta retains some of the original aneuploid cell lineage. In this scenario, the aneuploid zygote contains
two chromosomal homologues that were derived from one parent and a third homologue derived from the other parent. In theory, a
onein-three chance exists that postzygotic nondisjunction will eliminate the latter chromosome, creating a diploid daughter cell with
a chromosome pair from a single parent—UPD. If embryonic tissues descend from the diploid cell lineage, the fetus/infant is at risk
for syndromes or other complications of UPD for the chromosome in question (70). In some instances, the clinical features may be
nonspecific and/or not apparent until childhood, at which point screening for UPD is impractical using contemporary methods.
Therefore, testing for placental CPM in infants at risk (growth restricted) may be worthwhile, even though the majority of studies will
exclude CPM and only a small fraction of those with CPM will have UPD (128).
FIGURE 3-1 ▪ Three types of CPM in which the fetus is diploid are distinguished based on whether aneuploid cells are either
restricted to the trophoblast (type I), chorionic stroma (type II), or both trophoblast and chorionic stroma (type III). Trisomies
commonly or rarely associated with each type and their clinical correlates are indicated in the table. (Modified from Tyson RW,
Kalousek DK. Chromosomal abnormalities in stillbirth and neonatal death. In: Dimmick JE, Kalousek DK, eds. Pathology of the
embryo and fetus, JB Lippincott, Philadelphia, PA, 1992, with permission.)
Trisomy 16 may be the most common and best studied aneuploidy associated with CPM, long-term fetal survival, and UPD.
Langlois and colleagues provided follow-up data for 36 cases of trisomy 16 mosaicism, 19 diagnosed by chorionic villus sampling,
and 17 by amniocentesis (66). UPD was only observed in a subset (10/18 tested) of cases diagnosed by chorionic villus sampling,
six of which had major
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anomalies. None of the ten chromosome 16 UPD patients in this series exhibited developmental delay.
No specific gross or microscopic placental findings exist for CPM, although very few case series include these data. Although
Lestou and Kalousek do not recommend testing for CPM if maternal or placental disorder can explain fetal growth restriction,
maternal hypertension sometimes complicates CPM pregnancies and corresponding placental pathology can be present (57, 70,
128, 139).
Mosaic Variegated Aneuploidy

As described previously, mitotic nondisjunction can lead to somatic aneuploidy. Premature chromatid separation with mosaic
variegated aneuploidy (PCS-MVA) is a fascinating rare disorder that highlights this mechanism (15). Individuals with PCS-MVA
have several different aneuploid cell populations in their tissues. Their clinical findings include IUGR, neurological deficits, ocular
malformations, and high rates of neoplasia. Cytogenetic studies demonstrate premature separation of chromatids in ≥50% of
stimulated lymphocytes from these patients due to mutations in the BUB1B gene, which encodes a mitotic checkpoint protein (45,
79).
Autosomal Trisomies
Most nonmosaic autosomal trisomies lead to early embryonic demise. Only trisomies 21, 13, and 18 appear to be compatible with
survival to term, though each has a high rate of embryo and fetal wastage (Table 3-5). Although trisomies for each of the nonsex
chromosomes have been identified in spontaneous abortuses, the pattern is not random. Trisomies 16, 21, and 22 are particularly
common. Most trisomic abortuses manifest as highly disorganized embryos. The reader should consult Chapter 2 for a discussion
of the anatomic pathology of early abortuses.
The vast majority of nonmosaic autosomal trisomies arise from errors during the first meiotic division in the maternal germline (83).
In human female fetuses, oogenesis arrests in late prophase of the first meiotic division and oocytes remain in a “dormant” state
from the second trimester until one to five decades after birth. During this period of meiotic arrest, recombination between
homologous chromosomes occurs (46). The physical sites of recombination, termed chiasmata, stabilize the chromosomal pairs
through metaphase (103). It is believed that the prolonged first meiotic division increases the risk of nondisjunction, possibly due to
age-related loss of chiasmata.
The three autosomal trisomies compatible with postnatal survival (trisomies 21, 18, and 13) are associated with clinically defined
syndromes that include malformations of multiple organ systems. Tables 3-7, 3-8, and 3-9 list many of the phenotypic features of
these “surviving” trisomies, some of which are illustrated in Figures 3-2,3-3 and 3-4. Some of the findings (e.g., appendiceal
diverticula in trisomy 13) are fairly specific, though relatively insensitive markers of a particular trisomy (31). Mental retardation is
common to all three.
Two theories have been developed to explain the syndromes associated with specific trisomies (98). The “critical region”
hypothesis asserts that a subset of syndromic anomalies are due to a 1.5-fold increase in the dose of a small set of genes on the
trisomic chromosome. The resemblance of individuals with partial trisomies (due to duplication or translocation of portions of a
chromosome) as well as transgenic mouse models, support this model. Based on such data, a 1.6 to 2.5 megabase critical region
in chromosome 21 has been postulated to contain all of the genes necessary to produce the Down syndrome (122). Critical regions
have been proposed for trisomies 18 and 13 as well (10, 49, 130). However, the critical region theory for Down syndrome remains
controversial and is not consistent with data from other sources (94). An alternative hypothesis, termed “amplified developmental
instability,” contends that developmental defects are the effect of triplicated genes in general, related more to the number of
triplicated genes than their specific functions (98).
A significant fraction of nonmosaic trisomy 21 and possibly all nonmosaic trisomy 13 or 18 conceptions die spontaneously in utero
(Table 3-10). A retrospective examination of fetuses with trisomy 21 or trisomy 18 established a relatively constant rate of
spontaneous demise in each group after diagnostic amniocentesis (134). In total, 10% of trisomy 21 and 32% of trisomy 18 died in
utero, although higher rates of demise were observed in a different series of fetuses with trisomy 21 (50). In an independent study,
analysis of placentas from fetuses and infants with apparent nonmosaic trisomy 13 or 18 suggests that those surviving to term are
placental mosaics (56). It is possible that fetal demise is a consequence of placental trisomy 13 or 18 and that formation of diploid
placental cells, presumably by postzygotic rescue in the cytotrophoblast lineage, permits survival.
Prenatal diagnosis and elective termination of pregnancy also impact the rate of liveborn trisomies. Definitive prenatal diagnosis
requires amniocentesis, chorionic villus sampling, or another invasive procedure to obtain tissue for cytogenetic studies (karyotype,
FISH, or another approach). To reduce unnecessary risks and cost associated with prenatal diagnosis, screening methods have
been developed, which identify pregnancies at greatest risk (Table 3-10). Contemporary studies suggest that an “integrated test”
affords an 80% to 85% detection rate of trisomy 21, with a false positive rate of less than 1% to 5% (13, 129). The integrated test
includes quantitative measurement of pregnancy-associated plasma protein A, alpha-fetoprotein, unconjugated estriol, free β-
human chorionic gonadotropin, and/or inhibin A in maternal serum, early second trimester ultrasound evaluation of nuchal
translucency, and maternal age. Similar sensitivity and specificity have been reported for trisomy 18 based on a two-stage
screening approach using maternal serum markers.
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Table 3-7 ▪ MALFORMATIONS AND POSTNATAL DISORDERS ASSOCIATED WITH TRISOMY 21/DOWN
SYNDROME (DS)
% %
of of
Malformation DSa Postnatal Disorder DSa
Central nervous
Craniofacialb system
Mental
Upslanted palpebral fissures >50 retardation 100
Early-onset
Alzheimer
Ear anomalies >50 dementia >50c
Epicanthal folds >50 Seizures
Cancer (relative
Flat midface >50 risk)
Acute
25- lymphoblastic
Hypertelorism 50 leukemia (22)
Acute myeloid
leukemia (17)
Other: brachycephaly, midline parietal hair whorl, mild microcephaly,
choanal stenosis, cleft palate without cleft lip Lymphoma (3)
Cardiovascular >50 Colon (3)
10-
Atrioventricular canal 25 Testicular (12)
10- Transient
Patent ductus arteriosus 25 myeloproliferative
10-
Tricuspid valve defects 25 disease 5-10
5- Autoimmune
Ventricular septal defect 10 (relative risk):
5- Crohn disease
Atrial septal defect 10 (3) 1-5
Ulcerative colitis
Tetralogy of Fallot 1-5 (3)
Other: coronary valve defects, hypoplastic right heart, hypoplastic left heart, Celiac disease
anomalies of coronary circulation, coarctation of the aorta, other aortic (5)
anomalies, pulmonary artery stenosis, anomalies of great veins, single
umbilical artery Early-onset
diabetes mellitus
(3)
Thyroiditis (44)
Autoimmune
hepatitis (47)
10-
Digestive tract 25 Psoriasis (4)
5-
Duodenal stenosis 10 Musculoskeletal
Hirschsprung disease 1-5 Hypotonia >50
Joint
Anal atresia/stenosis 1-5 hyperextensibility >50
Other: tracheoesophageal fistula, esophageal atresia/stenosis, Other

nonduodenal intestinal atresia/stenosis, intestinal malrotation, ectopic anus,
annular pancreas Testicular 25-
microlithiasis 50
Enlarged thymic
Hassall 10-
corpuscles 25
Abnormal
lymphocyte 10-
Respiratory subsets 25
10-
Anomalies of larynx, trachea, or bronchi Ocular: 25
Pulmonary anomalies Glaucoma 5-10
25-
Genitourinary Strabismus 50
Obstructive defects of renal pelvis, ureter, bladder neck, or urethra 1-5 Nystagmus
5-
Cryptorchidism 10 Scoliosis
Hypospadias/epispadias 1-5 Hearing lossd
Central nervous system
Hypoplastic superior temporal gyrus

Flat frontal poles, retarded myelination
Hydrocephalus
Limb
25-
Clinodactyly (fifth finger) 50
25-
Single transverse palmar crease 50
Syndactyly 1-5
Other: clubfoot, polydactyly, limb reduction defects, rhizomelic shortening
Ocular
Brushfield spots 1-5
Cataract 1-5
5-
Keratoconus 10
aData pooled from multiple references (1, 26, 36, 38, 44, 47, 55, 60, 76, 77, 88, 96, 109, 114, 121, 125, 126, 138,
140).
bMany of the craniofacial features are less distinct in fetuses than in infants or children.
cOnsetof Alzheimer dementia is age dependent. Hundred percent have pathological changes by age 40 years
and >50% have clinical findings by age 50 years.
d The incidence of hearing loss varies with age and aggressive treatment of middle ear infections.
Approximately 50% of infants born with either trisomy 13 or trisomy 18 die within the 1st year and less than 5% survive to 10 years
(6). Most deaths are secondary to cardiac malformations, and these infants have significant neurocognitive deficits and multiple
other medical complications. By contrast, the life expectancy of infants with trisomy 21 is much better. In developed countries, more
than 90% of Down syndrome children born after 1990 live beyond 10 years and the average lifespan for present-day populations of
Down patients is approximately 60 years (9). In addition
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to mental retardation and malformations, postnatal health issues often associated with trisomy 21 include acute leukemia, early-
onset Alzheimer disease, hearing loss, and other conditions (Table 3-7).
Table 3-8 ▪ MALFORMATIONS ASSOCIATED WITH TRISOMY 18/EDWARDS SYNDROME (ES)
%
of
Malformation ESa
General
25-
50b
Intrauterine growth restriction 5-
Fetal hydrops 10
Craniofacialb
Microcephaly 25-
Choroid plexus cyst 50b
Other: triangular facies, abnormal calvarial shape (“strawberry” skull), hydrocephalus, micrognathia,
hypotelorism, cleft lip/palate, small ears, wide fontanels, narrow nasal bridge, microstomia, short sternum
Cardiovascular
Ventricular septal defect 25-

Atrioventricular communis 50
Other: ectopia cordis (pentalogy of Cantrell), hypoplastic left or right heart, overriding aorta, single umbilical
artery, patent ductus arteriosus, tetralogy of Fallot, double-outlet right ventricle, transposition of the great
arteries, mitral valvular disease
Digestive tract
Omphalocele 10-
Meckel diverticulum 25
Other: anorectal atresia, esophageal atresia, pyloric stenosis, ectopic pancreas, abnormal liver lobation >50
Respiratory
Abnormal lung lobation, tracheal stenosis, tracheoesophageal fistula
Genitourinary
Abnormal genitalia, cloacal exstrophy, obstructive uropathy, horseshoe kidney, renal a/hypoplasia,
renal/ureteral duplication, cryptorchidism, bifid uterus
Cerebellar and pontine hypoplasia >50

Meningomyelocele ± Chiari malformation 10-
Other: anencephaly, craniorrhachischisis, hippocampal dysplasia, agenesis of the corpus callosum, neural 25
migration defects
Limb
Clenched hand with overlapping digits >50
Radial ray defects 5-
Rocker-bottom feet 10
Other: arthrogryposes, polydactyly, phocomelia, syndactyly, hypoplastic nails, ectrodactyly 25-
50
Ocular
Coloboma, cataract, cloudy cornea, retinal hypopigmentation, microphthalmia, iridial hypoplasia
Musculoskeletal
Other: diaphragmatic defect, absent 12th ribs, malformed occipital bones
Other viscera
Hypoplasia of adrenals, thymus, thyroid, and/or gallbladder, accessory spleen
Placenta/Cord
Umbilical cord cysts

Small placenta
aData pooled from multiple references (12, 14, 24, 37, 61, 75, 92, 93, 113, 115, 131, 137).
bFrequency as a second trimester ultrasound finding.
Autosomal Monosomies
In theory, meiotic nondisjunction events that lead to trisomic embryos should produce an equal number of monosomic embryos.
This is not the case because monosomic embryos, with the exception of monosomy 21 (1/1,000 karyotyped abortions) or
monosomy X, die prior to implantation (91). Empiric data to support this concept come from studies of embryos conceived in vitro
(91, 106). Mosaic autosomal monosomy:diploidy is compatible with long-term survival, and phenotype/genotype correlations have
been established for some autosomes (81, 97).
Sex Chromosome Aneuploidies

At least one X chromosome is required for survival of the preimplantation embryo. Therefore, monosomy Y conceptuses are not
observed. However, other forms of sex chromosome aneuploidy, including monosomy X or extra copies of either the X or Y
chromosomes, are compatible with long-term
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survival and account for the majority of liveborn aneuploid infants.
Table 3-9 ▪ MALFORMATIONS ASSOCIATED WITH TRISOMY 13/PATAU SYNDROME (PS)
%
of
Malformation PSa
General
10-
Intrauterine growth restriction 50b
Fetal hydrops 5-10
Craniofacial
Microcephaly 10-
Holoprosencephalic facies (cyclopia, ethmocephaly, cebocephaly, premaxillary agenesis/dysgenesis) 50
Cleft lip/palate (midline/bilateral > unilateral) >50
Ocular hypotelorism
Other: malformed ears, absent ear canal, aplasia cutis of scalp, choanal stenosis or atresia; hemangiomas,
receding forehead, sparse curled eyelashes, natal teeth, micrognathia
Cardiovascular >50
25-
Ventricular septal defect 50
Patent ductus arteriosus 25-
Echogenic intracardiac foci (myocardial calcifications) 50
Other: dextrocardia, tetralogy of Fallot, atrial septal defect, truncus arteriosus, aortic coarctation, pulmonary 10-
atresia/stenosis, bicuspid aortic valve, single umbilical artery 25
Digestive tract
Pancreatic-splenic fusion
Appendiceal diverticulum
Other: omphalocele, abnormal liver lobation, intestinal atresia, Meckel diverticulum
Respiratory
Abnormal lung lobation
Genitourinary
Obstructive dysplasia 25-

Renal/ureteral duplications 50
Other: cryptorchidism, double vagina, bicornuate uterus, abnormal Fallopian tubes, small penis, abnormal 25-
scrotum 50
Holoprosencephaly 25-
Arrhinencephaly 50
Cerebellar malformations >50
Other: anencephaly, meningomyelocele, agenesis of the corpus callosum, hydrocephaly, hippocampal 25-
dysplasia, neural migratory defects, choroid plexus cyst 50
Limb
Postaxial polydactyly ∽50

Other: syndactyly, rocker-bottom feet, hypoplastic nails, clubbed feet, hypoplastic nails, single transverse
palmar crease, radial aplasia
Ocular
25-
Microphthalmia 50
Coloboma of iris or retina 25-
Other: retinal dysplasia, aniridia, anophthalmia, cataract, premature vitreous body, hypoplasia of optic nerve 50
Musculoskeletal
Dysplastic/fused lumbosacral ± thoracic vertebra, absent 12th ribs, hypoplastic sphenoid bone, diaphragmatic
defect >50
Hematologic
Irregular neutrophil nuclei

Increased fetal and Gower-2 hemoglobin
aData pooled from multiple references (32, 37, 62, 69, 115, 131).
bReported rates of IUGR appear to be higher in populations that were studied later in gestation.
Monosomy X (Turner Syndrome)

A 45,X karyotype is one of the most frequently encountered forms of aneuploidy in spontaneously aborted embryos. In contrast with
autosomies, for which maternal meiotic nondisjunction events predominate, loss of either the maternal or paternal sex chromosome
appears to occur more often during postzygotic mitosis. Hence, mosaic monosomy X is very frequent, and occult mosaicism has
been speculated to exist in all “pure” 45,X patients (124). Pure 45,X or high 45,X mosaicism produces a fairly distinct syndrome
characterized by female genitalia, short stature, and a high prevalence of specific anomalies (Table 3-11). Intelligence is usually in
the normal range.
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FIGURE 3-2 ▪ Trisomy 21: A: 35-week fetus with typical late gestation facies (epicanthal folds, broad nose, bulging tongue). B:
Similar facial changes are apparent in 2-month-old infant (note increased ‘Mongoloid’ slant of palpebral fissures). C: Single palmar
crease. D: Lateral view of brain showing small superior temporal gyrus and enlarged middle temporal gyrus. E: Duodenal atresia.
F: Atrioventricular canal, with large primum atrial septal defect, large ventricular septal defect in position of AV canal, and cleft
septal leaflet of the tricuspid valve.
FIGURE 3-3 ▪ Trisomy 18. A: Late gestational fetus with omphalocele and rocker bottom feet. B: Young infant with widely
separated eyes and mild trigonocephaly. C: Infant shown in (B), with dysplastic ear and micrognathia. D: Infant with triangular
facies, ocular hypertelorism, and bilateral cleft lip. E: Overlapping digits in pattern common to trisomy 18. F: Horseshoe kidney
(with ureters and urinary bladder).
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FIGURE 3-4 ▪ Trisomy 13. A: Infant with cebocephaly (ocular hypotelorism and single nostril nose), one of the facial changes
associated with holoprosencephaly. B: Aplasia cutis of the scalp. C: Postaxial polydactyly. D: Alobar holoprosencephaly. E:
Appendiceal diverticula (“dinosaur tail”) are pathognomonic for trisomy 13, although not present in every case. F: Fusion of spleen
(left) and tail of pancreas (note tiny splenic islands within the pancreas).
Only 1% of 45,X embryos survive to term. Many are lost in the first trimester, but late fetal loss is also common. Severe hydrops
with massive nuchal edema is common in utero (Figure 3-5) and usually portends a poor outcome. Extravascular fluid in the neck
collects as a multiloculated cystic hygroma, a lymphatic malformation characterized by thin membranous septa and inconspicuous
endothelial linings (16). Gross and microscopic studies of cystic hygroma in Turner syndrome suggest hypoplasia/agenesis of
lymphatic vessels and failure of the lymphatics to connect to the venous system (127). Resolution of transient nuchal edema is
proposed as the basis for the webbed neck commonly observed in Turner syndrome. Other common malformations (Table 3-11)
include aortic coarctation, hypoplastic left heart, “horseshoe” kidney, and streak ovaries (Figure 3-5).
Table 3-10 ▪ PRENATAL SCREENING MARKERS FOR COMMON TRISOMIES
Rate of Percentage of
IUFD or Liveborns Surviving
Prenatal Screening Markersa Stillbirthb,c tob
Maternal
serum 1 1 10
Trisomy analytesa Ultrasoundd month year year
21 ↓ AFP 1st trimester: nuchal translucency, nasal bone 10%-30% >95 95 >90
↓ PAPP-A, hypoplasia
↑ fβ-HCG 2nd trimester: echogenic intracardiac foci,
↑ inhibin echogenic bowel, rhizomelic limb shortening; mild
pyelectasis
18 ↓ AFP 1st trimester: nuchal translucency 45%-70% 50% 5%- 1%

↓↓ PAPP- 2nd trimester: choroid plexus cyst, clenched hands, 30%
A echogenic bowel, IUGR, mild pyelectasis, mild
↓↓ fβ-HCG ventriculomegaly
↓ uE3
13 ↓ AFP 2nd trimester: mild pyelectasis, echogenic 20%-40% 50% 15%- 1%

↓↓ PAPP- intracardiac foci, IUGR, mild ventriculomegaly 30%
A
↓↓ fβ-HCG
aReferences (100, 109, 123).
bReferences (6, 9, 29, 51, 58, 73, 90, 95, 104, 117, 134, 135 and 136).
cAfter
diagnosis by amniocentesis or ultrasound; does not include first trimester losses or elective terminations of
pregnancy.
dNonspecific findings that significantly increase the risk for trisomy.

AFP, α-fetoprotein; PAPP-A, pregnancy-associated plasma protein A, increased inhibin A; fb-HCG, free β-human chorionic
gonadotropin; uE3, unconjugated estriol; IUGR, intrauterine growth restriction; VSD, ventricular septal defect; IUFD,
intrauterine fetal demise.
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Table 3-11 ▪ LFORMATIONS AND POSTNATAL ABNORMALITIES ASSOCIATED WITH MONOSOMY

X/TURNER SYNDROME (TS)
% %
of of
Malformation TSa Postnatal Abnormality TS
General External
Intrauterine growth restriction >80 Short stature >90

Fetal hydrops (may be transient) >80 Webbed neck >80
Broad chest Short neck/low hairline >70
Widely spaced nipples Cubitus valgus
Inverted and/or hypoplastic nipples Infantile external genitalia
Scant pubic/axillary hair
Failure to develop secondary sex
characteristics
Craniofacial Endocrine
Nuchal cystic hygroma Low estrogen and progesterone 15-

Triangular face Elevated follicle stimulating hormone (FSH) 30
Down-slanted palpebrae Autoimmune thyroiditis
Other: epicanthus, ptosis, high-arched narrow Diabetes mellitus
palate, micrognathia, hypertelorism, low-set ears,
dysmorphic ears
Cardiovascular 17- Other

45
Coarctation of the aorta Aortic dissection, myopia. deafness,

Hypoplastic left heart hypertension, Crohn disease, cardiac
Bicuspid aortic valve conduction defects, chondrodysplasia of the
Cystic medial necrosis of aorta distal radius (Madelung deformity)
Other: mitral valvular dysplasia, ventricular septal
defect, anomalous pulmonary venous return
Genitourinary Increased risk for neoplasiab >50
Gonadal dysgenesis (streak gonads) >90 Melanocytic nevi

Horseshoe kidney or other renal malformations 40 Gonadoblastoma (if portion of Y
Hypoplastic uterus chromosome is present)
Clitoral hypertrophy
Mild cortical dysplasia

Neuroglial heterotopia
Hydrocephalus
Limb
Short 4th and 5th metacarpals >50

Other: narrow hyperconvex nails
Ocular
Cataracts
Musculoskeletal
Raised semilunar carpal bones >60

Inferior displacement of inner tibial growth
Other: bone dysplasia with coarse trabeculae,
scoliosis, spina bifida, vertebral fusion, cervical
rib, abnormal sella turcica, dislocated hip
aData pooled from multiple references (37, 54, 80, 119, 120, 133).
bAlthough a study suggested that Turner syndrome patients may

be at risk for a wide variety of nongonadal neoplasms (143), this
has not been supported by recent large reviews (126).
One half of all individuals with Turner syndrome have a 45,X karyotype; various forms of 45,X mosaicism account for most of the
rest (120). Those with 45,X/46,XY mosaicism or retained portions of a Y chromosome exhibit a range of phenotypic features from
normal male to Turner syndrome. Within this continuum, genitalia and gonads may show ambiguous differentiation. Ovotestes or
other forms of gonadal dysgenesis are common and 7% to 30% of patients develop gonadoblastoma (43).
Sex Chromosome Polysomy
In general, all the genes of only one X chromosome remain active beyond the blastocyst stage (48). After this stage, most genes of
all but one X chromosome in each cell are inactivated (silenced) by epigenetic modifications. The process of X-inactivation appears
random in each cell of the inner cell mass. Therefore, each cell in a 46,XX embryo has an equal chance of inactivating the paternal
or maternal X chromosome.
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In embryos that carry three or more X chromosomes, genes on all but one are silenced, except for the subset of X-linked genes that
normally escape X inactivation. The latter seem to have little impact on the development of females with a 47,XXX karyotype.
However, dose-related effects are observed in males and females with tetrasomy or pentasomy X, who exhibit mental deficiency
and mild dysmorphic features (Table 3-12). A 47,XXY karyotype causes Klinefelter syndrome (mild neurocognitive deficits,
behavioral problems, hypogonadism, and hypogenitalism) (132).
FIGURE 3-5 ▪ 45X. A: Fetus with massive cystic hygroma and hydrops. B: Left anterior oblique view of heart in situ; arrow
indicates region of narrowing (coarctation) of the preductal aorta (Asc Ao, ascending aorta; MPA, main pulmonary artery; Desc Ao,
descending aorta)—17 weeks. C: In situ view of low-set horseshoe kidneys and small but histologically normal ovaries—18 weeks.
D: Streak ovaries in specimen from newborn; prominent cervix is normal for age. E: Small horseshoe kidney—17 weeks.
One of every thousand liveborn males has an extra Y chromosome (47,XYY). Associated features are highly variable, but
aggressive behavior, mild cognitive defects, and minor dysmorphic features have been reported (54).
Polyploidy
Polyploidy refers to complete extra haploid sets of chromosomes, as with triploidy (69 chromosomes) or tetraploidy (96
chromosomes). Triploidy is common (1% of human embryos), and usually leads to spontaneous abortion between 7 and 17 weeks
(23). The extra chromosomal set is more often of maternal (digyny) than paternal (diandry) origin (6). However, diandry
predominates in triploid early spontaneous abortions. Most, if not all, diandric triploid conceptions result from dispermic fertilization
of a single oocyte (84, 123). Maternal origin of the extra chromosomes in triploid conceptuses (digyny) is caused by errors in the
first, or less often the second, meiotic division.
Most triploid embryos are miscarried (141). Liveborn triploid infants are rare and generally die within a few hours (25). Diandric and
digynic triploid conceptuses exhibit distinct phenotypes, which are referred to as type I and type II
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triploidy, respectively (85). Diandric fetuses that survive into the second trimester typically show normal fetal growth to moderate
symmetrical growth restriction and partial molar transformation of their placentas, with large cystic villi and trophoblast hyperplasia
(18). Survival of digynic embryos into the second and third trimesters is associated with severe asymmetrical growth restriction and
small placentas that do not exhibit molar change. These phenotypic differences have been attributed to parental imprinting of
genes that influence placental and fetal growth (21).
Table 3-12 ▪ SEX CHROMOSOME POLYSOMIES
Polysomy Clinical/Pathological Featuresa
47,XXX Normal
47,XYY Accelerated growth, prominent glabella, “dull” mentality, behavioral problems, severe acne
47,XXY Klinefelter syndrome: hypogonadism, hypogenitalism, infertility (testicular fibrosis), long limbs,
gynecomastia, mental retardation (15%-20%), neoplasia (1%-2%) including breast cancer,
leukemia/lymphoma, testicular tumors, and extragonadal germ cell tumors
48,XXXX Mental retardation, mild facial anomalies, 5th finger clinodactyly
49,XXXXX Penta X syndrome: microcephaly, mental retardation, small hands with 5th finger clinodactyly, abnormal
facies, growth deficiency, patent ductus arteriosus
48,XXXY Klinefelter syndrome with mental retardation, growth deficiency, radioulnar synostosis
48,XXYY Klinefelter-like syndrome with higher incidence of mental retardation and behavioral abnormalities
aReferences (2, 37, 54)
Common malformations observed in triploid fetuses include adrenal hypoplasia, syndactyly (particularly digits 3 and 4),
hydrocephalus, and other defects (Table 3-13). Apart from partial mole formation, no specific malformations have been found to
distinguish diandric versus digynic triploidy (18, 86). If triploidy is suspected, the diagnosis can be confirmed less expensively and
faster by flow cytometry than traditional cytogenetics.
Partial Chromosomal Aneuploidies

A variety of structural chromosomal anomalies result in partial duplication (trisomy) or deletion (monosomy) of portions of
chromosomes (Figure 3-6). The initiating event for most of these aberrations is abnormal recombination between homologous and
heterologous chromosomes during gametogenesis. As a consequence, portions of chromosomes may be translocated, duplicated,
or lost. Reciprocal translocations between heterologous chromosomes are often “balanced,” with two abnormal chromosomes but
no net gain or loss of genetic material. Individuals who constitutively harbor balanced translocations are likely to be normal, unless
a translocation breakpoint results in a microdeletion/duplication or disrupts coding or regulatory elements of a particular gene.
However, carriers of balanced translocations are at risk for transmitting an unbalanced translocation (one of their abnormal
derivative chromosomes) to their offspring, who will be partially monosomic and partially trisomic for portions of the two
chromosomes involved in the translocation.
Table 3-13 ▪ DIGYNIC VERSUS DIANDRIC TRIPLOIDYa
Digynic Diandric
Fetus
Growth Asymmetric IUGR Usually normal or symmetric IUGR
Craniofacial Macrocephaly, hypertelorism, ventriculomegaly, Usually normal or microcephaly, ventriculomegaly

low-set ears, microphthalmia, coloboma
Cardiac Normal, ventricular septal defect Normal, ventricular septal defect
Extremities Syndactyly between 3rd and 4th digits Syndactyly between 3rd and 4th digits
Other Adrenal hypoplasia, micropenis, renal Adrenal hypoplasia, ambiguous genitalia

malformations, pulmonary hypoplasia, Leydig
cell hyperplasia
Placenta Hypoplastic with no features of partial mole Partial mole edematous with cisternae in terminal
villi trophoblast hyperplasia scalloped villus
contours
Common Error in meiosis II Fertilization of normal oocyte by two spermatozoa

mechanism
Usual outcome Spontaneous abortion or stillbirth Spontaneous abortion or stillbirth
aReferences (18, 21, 25, 46, 53, 123, 141).
Schinzel has cataloged numerous translocations, duplications, and deletions in an effort to identify phenotypes associated with the
gain or loss of specific chromosomal segments (110). As expected, a myriad of unbalanced chromosomal rearrangements have
been reported, many of which are unique with regard to the specific DNA sequences that are lost or gained. However, partial
chromosomal aneuploidies that involve overlapping portions of the genome have been correlated with recognizable syndromes,
including those listed in Table 3-14.
Robertsonian translocations are those that involve the diminutive short (p) arms of acrocentric chromosomes (33). The acrocentric
chromosomes in humans are numbers 13, 14, 15, 21, and 22. The product of a Robertsonian translocation is a composite
chromosome containing two closely spaced centromeres and q arms of both “donor” chromosomes, but lacking portions of the
donor p arms. Robertsonian translocations can be homologous (e.g., 13q13q) or heterologous (e.g., 13q14q). Loss of DNA from
the miniscule short arms of these chromosomes is not clinically significant, so carriers of “balanced” Robertsonian translocations
are generally normal. Diploid cells of heterologous Robertsonian translocation carriers also contain one normal copy of each
chromosome involved in the translocation. If one of these normal chromosomes segregates with the Robertsonian translocation
product during meiosis, the resulting zygote will be trisomic. Therefore, carriers of Robertsonian translocations are at significant
risk for producing a conceptus with trisomy of either chromosome involved in the translocation, as well as recurrent trisomy.
Sites of the interchromosomal and intrachromosomal recombination events that occur during gametogenesis and
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underlie many constitutional translocations are not randomly distributed (28). Instead, “hot spots” appear to exist that are prone to
recombination. In some instances, these produce submicroscopic duplications or deletions that are below the resolution of standard
cytogenetic analysis. Application of array-based CGH and other methods to screen for these rearrangements is likely to uncover
hitherto unrecognized disorders that have their basis in structural chromosomal anomalies (112).
FIGURE 3-6 ▪ Examples of chromosomal rearrangements.
Table 3-14 ▪ SELECTED SYNDROMES ASSOCIATED WITH PARTIAL ANEUPLOIDY
Partial
Aneuploidy Clinical and Pathological Featuresa,b
del 4p16-ter Wolf-Hirschhorn syndrome: IUGR, microcephaly, hypotonia, seizures, “Greek warrior helmet” facies
(prominent glabella, ocular hypertelorism, high-arched eyebrows, broad nasal bridge), cleft lip ± palate
(47%), strabismus, epicanthal folds, micrognathia, “fish” mouth, short upper lip and philtrum, preauricular
skin tag or pit, talipes equinovarus, single transverse palmar crease, hypospadias (50% of males), renal
hypoplasia, malformed toes, cryptorchidism, cardiac malformation (33%), mental retardation
del 5p15.2- Cri du chat syndrome: IUGR (72%), catlike cry (100%), mental retardation (100%), hypotonia (78%),
ter microcephaly (100%), abnormal facies, cardiac malformation (30%), single transverse palmar crease,
CNS malformations (arachnoid cyst, hydrocephalus, cerebellar hypoplasia), renal malformations
del 9p21-ter Mental retardation, trigonocephaly, abnormal facies (upslanting palpebrae, midfacial hypoplasia,
anteverted nares, depressed nasal bridge, long philtrum), hypoplastic ear lobes, long middle and short
distal phalanges, cardiac defects (33%-50%), scoliosis, abnormal external genitalia
dup 10q24- Mental retardation, IUGR, microcephaly, abnormal facies (flat with high forehead, high-arched eyebrows,
ter ptosis, microphthalmia, broad nasal bridge, bow-shaped mouth, cleft palate, posteriorly rotated ears),
camptodactyly, proximally placed thumbs, two to three pedal syndactyly, cardiac (50%) and renal (50%)
malformations, absent 12th ribs, kyphoscoliosis
tetrasomy Cat eye syndrome: iris coloboma, mild mental retardation, ocular hypertelorism, preauricular skin tags or
22q11-pter pits, micrognathia, downslanting palpebral fissures, cardiac malformations (>33%), anal atresia, renal
agenesis
aNumbers in parentheses are frequencies of selected findings
bReferences (15, 22, 54, 82).
UGR, intrauterine growth restriction; CNS, central nervous system
Chromosomal Instability Disorders

Several human syndromes and diseases are due in part to chromosomal instability that can be resolved by karyotype analysis
using normal or specialized culture and/or staining techniques (Table 3-15). Most chromosomal instability disorders are genetic
conditions that predispose to congenital malformations and/or cancer because of an underlying
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defect in molecular systems that mediate DNA replication or repair. Medical professionals who deal with these conditions need to
be aware that cytogenetic evaluation of specimens for chromosomal instability is somewhat specialized and not available in every
laboratory. Many of the assays require quantitative measurements with appropriate controls. For many of the conditions listed in
Table 3-15, mutational analysis of specific genes has replaced cytogenetic studies.
Table 3-15 ▪ CYTOGENETICS AND MOLECULAR GENETICS OF SELECTED CHROMOSOMAL INSTABILITY

SYNDROMES
Cytogenetic Genetic
Syndrome Clinical Features Finding Defect(s) Comment
Fanconi (FA) Pancytopenia, Increased FANCA,FANCB, Mutations in at least 12 different genes

IUGR, abnormal chromosomal FANCC, (complementation groups) can cause
skin pigmentation, breakage after FANCD1/BRCA2, Fanconi anemia. Molecular genetic
radial ray defects, exposure to a FANCD2, testing for every complementation
VACTERL- like clastogenic FANCE, FANCF, group is not available. Diagnosis and
malformations, agent FANCG, determination of complementation
increased cancer FANCJ/BRIP1, group by cytogenetic analysis of
risk (AR) FANCL, somatic cell hybrids facilitate targeted
FANCM/Hef DNA testing.
Robert-SC (R- Symmetric limb Repulsion of ESCO2

SC) reduction defects, heterochromatic
IUGR, cleft regions near
lip/palate, other centromeres
craniofacial (“puffing”) and
malformations, delayed
mental retardation progression
(AR) through
metaphase
(“anaphase
lag”)
Nijmegen Characteristic Increased NBS1 Nijmegen breakage syndrome is

breakage (NB) facies, chromosomal observed primarily in patients of Slavic
microcephaly, breakage with descent due to the founder effect of a
immunodeficiency, normal culture single mutation. NBS1, FANCD2, and
growth or after ATM may interact with one another.
retardation, exposure to a
radiosensitivity, clastogenic
increased cancer agent
risk (AR)
Fragile X Macrocephaly, “Fractured” FRM1 Clinical disease is associated with

(FRAX) large testes, appearance of expansion of a trinucleotide repeat
mental retardation the X (>200 repeats) in the 5′-untranslated
(X-linked) chromosome region of exon 1. Molecular genetic
induced by one testing has replaced cytogenetic
of several testing.
culture methods
Ataxia- Ataxia, Radiation- ATM AT-like disorders have been described

Telangiectasia telangiectasia, induced due to mutations in other genes (e.g.,
(AT) dysarthria, nonrandom MRE11).
abnormal ocular rearrangements
movements, of
recurrent chromosomes
infections, 7, 14, and X ±
increased cancer nonspecific
risk (AR) chromosome
breakage in
fibroblasts
AR, autosomal recessive; IUGR, intrauterine growth restriction; VACTERL,

vertebral-anorectal-cardiac-tracheo-esophageal-renal-l imb malformation
association.
References: FA (83), R-SC (116, 135), NB (138), FRAX (26, 128), AT (36,
127).
For Fanconi anemia, however, chromosomal breakage studies remain diagnostically useful. Fanconi anemia, a multigene disorder,
is manifest by pancytopenia, IUGR, and high incidence of congenital malformations (Figure 3-7) that overlap with the anomalies of
the VACTERL ( vertebral- anorectal- cardiac tracheoesophageal- renal- l imb) association (35). Because of this phenotypic
overlap, some authors have recommended testing for Fanconi anemia of any individual with VACTERL association including a
radial ray defect (30). At least 12 different “complementation groups” for Fanconi anemia are recognized and the specific genes
responsible for many of these groups have been identified (Table 3-15) (78). To determine a
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patient's complementation group, his/her cells are fused with cells of established complementation groups and then chromosomal
breakage rates are examined in the somatic cell hybrids (Figure 3-8). The patient is assigned to the complementation group that
fails to rescue the breakage phenotype in these somatic cell hybrid assays. Approximately 66% of cases fall into complementation
group A (FANCA) (78).
FIGURE 3-7 ▪ Fanconi syndrome. A: Fetus with bilateral radial aplasia. B: Close view of right arm and hand, showing marked
deviation of wrist, secondary to radial aplasia, and tiny remnant of thumb. C: Posterior view of viscera, showing proximal atresia of
esophagus (*) with distal tracheoesophageal fistula (arrow). Thoracic aorta is reflected to left.
Most of the genes that are disrupted in patients with Fanconi syndrome encode proteins that mediate DNA repair. Mutational
analysis of one or more of these genes is a potential alternative to complementation studies, but is less efficient and will miss cases
of Fanconi anemia due to complementation groups for which mutational analysis is not yet possible. Novel techniques have been
introduced to replace somatic cell hybridization for complementation group determination, but at present these are only available for
select complementation groups.
FIGURE 3-8 ▪ Identification of Fanconi complementation group by somatic cell hybridization. Cells from the patient with Fanconi
anemia are fused with cells from established complementation groups to create somatic cell hybrids. The hybrids are exposed to
clastogenic agents in vitro and chromosomal breakage rates are observed. Only hybrids of the same complementation group retain
the high rate of chromosome breakage that characterizes Fanconi anemia.
SUBMICROSCOPIC DISORDERS
Development of sensitive methods to resolve small chromosomal aberrations has expanded the scope of cytogenetic disorders to
include microdeletions, microduplications, and other structural changes that cannot be elucidated by traditional karyotype analysis.
This is a rapidly evolving field in which targeted FISH probes and targeted or global arraybased CGH are important techniques.
Some of the more common applications in fetal and pediatric medicine are listed in Table 3-16. As examples of this class of
disorders, the 22q11 microdeletions associated with velocardiofacial (VCF)/DiGeorge syndrome and subtelomeric deletions are
discussed in more detail.
Velocardiofacial/DiGeorge Syndrome
VCF/DiGeorge syndrome (also referred to as the 22q11 deletion syndrome) is a convenient designation for two clinical entities that
share pathogenic and phenotypic features including craniofacial and cardiovascular anomalies (39, 63). In addition to these
common elements, DiGeorge patients classically exhibit agenesis or hypoplasia of the thymus and parathyroid glands.
VCF/DiGeorge syndrome is an autosomal dominant disorder with variable penetrance and expressivity. Palatal insufficiency or
clefts are common. Cardiac malformations usually involve the conotruncal region (truncus arteriosus, tetralogy of Fallot, interrupted
aortic arch, ventricular septal defect, or others). In addition, the 22q11 deletion is frequently detected in patients with the same
spectrum of isolated heart anomalies (40).
The VCF/DiGeorge syndrome critical region (DGCR), a 3-MB segment on chromosome 22q11, is deleted in 90% of patients with
DiGeorge syndrome. The overwhelming majority of these deletions are not detected by routine karyotype, but are readily identified
by FISH. The DGCR contains more than 20 genes. Of these, TBX1 is afforded considerable
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attention because disruption of the murine homologue is associated with conotruncal defects, and some humans with
VCF/DiGeorge syndrome and no 22q11 deletion have point mutations in TBX1 (41). TBX1 encodes a transcription factor that is
expressed in embryonic pharyngeal arch mesoderm and the cardiac outflow tracts. The current standard of care is to perform FISH
testing for the DGCR in any patient with VCF/DiGeorge syndrome or an isolated conotruncal cardiac malformation (63).
Table 3-16 ▪ SELECTED SUBMICROSCOPIC CHROMOSOMAL ANOMALIES
Genetic Locus [affected

Syndrome or Disorder gene(s)] Alteration
Velocardiofacial syndrome (VCFS)/DiGeorge 22q11.2 (TBX1 and contiguous Microdeletion

genes)
X-linked congenital adrenal hypoplasia/Glycerol kinase Xp21 (DAX1, GK, DMD) Microdeletion
deficiency/Duchenne myopathy
Tuberous sclerosis/Autosomal dominant polycystic kidney 16p13 (TSC2, PKD1) Microdeletion

disease
Maturity-onset diabetes of the young type 5 17q12 (TCF2) Microdeletion
WAGR 11p13.3 (PAX6, D11S2163, PER, Microdeletion

WT-1)
Smith-Magenis 17p11.2 (RAI1) Microdeletiona
Rubinstein-Taybi 16p13.3 (CBP) Microdeletiona
Alagille 20p12 (JAG1) Microdeletiona
Williams 7q11.23 (ELN + >20 contiguous Microdeletion

genes)
Prader-Willi/Angelman 15q11-13 (SNURF-SNRPN and Microdeletion (70%

contiguous genes) of cases)
Miller-Dieker 17p13.3 (LIS1 and contiguous Microdeletion
genes)
Monosomy 1p36 1p36 (KIAA1273 and contiguous Microdeletion

genes)
Microduplication 22q11.2 22q11.2 (same as Microduplication

VCFS/DiGeorge)
Hereditary neuropathy with liability to pressure palsies 17p11.2 (PMP22; same as Microdeletion
(HNPP) CMT1A)
Charcot-Marie-Tooth 1A (CMT1A) 17p11.2 (PMP22; same as HNPP) Microduplication
aPoint mutations have also been observed in a small subset of patients, but FISH is often used as an initial diagnostic test.
Subtelomeric Deletions
Telomeres are short repetitive sequences found at the very ends of chromosomes. The adjacent subtelomeric regions are gene-
rich areas that are difficult to evaluate by traditional cytogenetic methods because subtelomeres contain indistinct G-bands that
look similar from one chromosome to the next. Therefore, small subtelomeric deletions or terminal translocations are often
undetectable in routine or high-resolution karyotypes. In many instances, subtelomeric rearrangements are heritable and a subset
appears as benign variants (99). Therefore, the genetic implications are quite variable.
FISH with subtelomere-specific probes and array-based CGH permit screening for deletions or translocations involving
subtelomeres (52). These methods have shown that deletions are relatively common, particularly among individuals with
unexplained mental retardation and/or malformations. For some chromosomal arms, subtelomeric deletions result in well-known
syndromes that are associated with larger deletions (e.g., Wolf-Hirschhorn (4p-) syndrome; Table 3-14); thereby narrowing the
critical regions responsible for these phenotypes. In other instances, new syndromes are being defined based on large series of
patients with the same subtelomeric deletions and similar phenotypic features. An outstanding review of specific subtelomeric
deletions and their associated findings was written by de Vries et al. (19).
The “del 1p36 syndrome” is a prime example of how FISHbased diagnosis of subtelomeric deletions has helped define a clinical-
pathological syndrome and shown it to be more common than believed (5). The current literature suggests that 1p36.3 deletions
account for up to 0.5% to 0.7% of idiopathic mental retardation. The syndrome also includes high rates of structural heart defects
(>50%), dysmorphic facies, hypothyroidism (15% to 20%), deafness, and other neurological anomalies. Brains of these patients
show a variety of nonspecific findings including cortical dysplasia, hydrocephalus, and leukodystrophy. This syndrome poses a
difficult and more frequently encountered problem for perinatal pathologists because only nonspecific findings (e.g., isolated heart
defect) may be evident in a fetus. The yield for 1p36 deletion studies is likely to be very low in this scenario, and FISH analysis is
not the standard of care at present. However, the situation is likely to change as array-based CGH or other techniques for targeted
or global microdeletion analysis become less expensive and more readily available.
EPIGENETIC CHROMOSOMAL MODIFICATIONS AND ASSOCIATED DISORDERS

Each human chromosome is a linear sequence of nucleotides surrounded by proteins and RNA. Nucleotide sequence is crucial to
genetic integrity, and gain, loss, and alteration of nucleotides are the primary bases for many pediatric disorders. However, a large
and growing number of disease states result from epigenetic chromosomal modifications that influence gene expression without
mutations of genes themselves (101). Pediatric disorders associated with abnormal parental imprinting are listed in Table 3-17.
Recent case reports
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suggest that disorders of imprinting may be more common among individuals conceived in vitro by assisted reproductive
technologies (72). However, more data is needed to substantiate this hypothesis.
TABLE 3-17 ▪ PEDIATRIC IMPRINTING DISORDERS

Chromosomal
Locus
[Affected
Disorder or Syndrome Gene(s)] Phenotypic Features
Beckwith-Wiedemann 11.15.5 (IGF2, Macrosomia, large tongue, hemihypertrophy, omphalocele, ear

H19, KCNQ1, pits, adrenocytomegaly, placental mesenchymal dysplasia,
and others) neoplasia (Wilms tumor, hepatoblastoma)
Pseudohypoparathyroidism/Albright 20q13 (GNAS) Developmental delay, mental retardation, obesity, short

hereditary osteodystrophy stature, ± hypocalcemia
Prader-Willi 15q11.2 Mental retardation, obesity, short stature, behavioral problems

(MKRN3,
MAGEL2, NDN,
and others)
Angelman 15q12 (UBE3A, Mental retardation, dysmorphic facies, behavioral and speech
ATPC10C) problems
Transient neonatal diabetes 6q24 (PLAG1) Growth retardation, low fetal/infantile insulin levels
mellitus
Russell-Silver syndrome 7p11.2 Short stature, asymmetric skull, triangular facies, incurved 5th
fingers
Epigenetic chromosomal modifications affect the secondary and/or tertiary structure of chromosomes, but do not alter the
chromosomal bands that are visualized by traditional cytogenetic methods. For example, chromosomal DNA is intimately wound
around histone proteins, which are particularly dense in transcriptionally silent portions of the genome. Histones are subject to
phosphorylation, methylation, and acetylation, which modify their interaction with the DNA and other proteins to facilitate or reduce
transcriptional activity (87). The nucleotides of chromosomes are also subject to covalent modifications that correlate with local
gene activity, including methylation of cytosine bases. Epigenetic modifications differ between individuals, cell types, and
maternally versus paternally derived chromosomes (11). Gene silencing, such as X-chromosome inactivation, is often correlated
with high levels of cytosine methylation. The molecular bases for X-chromosome inactivation are partially worked out, but the
mechanisms that regulate epigenetic modification of many autosomal genes remain poorly understood (71).
Parental imprinting, a type of epigenetic regulation already referred to in this chapter, refers to differential “marking” of genes in the
maternal and paternal germlines, which influences gene expression in tissues of resulting offspring. Imprinting generally involves
clusters of closely spaced genes, which are regulated coordinately by discrete DNA elements termed imprinting centers (71). The
IGF2/H19 imprinting cluster, one of the best studied, is located on chromosome 11p15, where improper imprinting has been
associated with Beckwith-Wiedemann syndrome (20). Paternal imprinting is associated with methylation of the IGF2/H19 imprinting
center, expression of IGF2, and silencing of H19, whereas the reverse methylation and expression patterns occur for the maternal
allele. In a subset of patients with Beckwith-Wiedemann syndrome, IGF2 is overexpressed due to either paternal uniparental
isodisomy of 11p15 (replacement of the maternal locus by a paternal locus) or loss of imprinting (activation of the normally silent
maternal locus).
Unfortunately, neither UPD nor other alterations of imprinting are detected by traditional cytogenetic methods. Instead,
sophisticated molecular genetic studies are required.
Future Directions
For several decades, karyotype analysis has been the basis for defining chromosomal disorders. However, the advent of molecular
diagnostic approaches is revolutionizing this field to encompass smaller and more subtle chromosomal alterations, a small subset
of which are discussed above. In the future, approaches such as array-based CGH are likely to replace the karyotype as a screen
for cytogenetic defects (7). Pilot studies suggest that CGH is faster, as sensitive to most and more sensitive to some unbalanced
chromosomal alterations, and may be more economical than traditional cytogenetic methods (68, 107). As these approaches are
applied, recognition of submicroscopic abnormalities will expand the range of chromosomal disorders and probably define
genotype-phenotype correlations. As in the past, accurate and comprehensive anatomic pathology, including pathology of the
embryo and fetus, will be vital to the evolution of this field.
ACKNOWLEDGMENTS
The authors thank Dr. Kent Opheim for his thoughtful comments.
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Chapter 4
Congenital Anomalies and Malformation Syndromes
Joseph R. Siebert
The study of congenital anomalies continues to be hampered by misunderstandings at a number of levels. In

many circles, for example, the statement that “the baby was born with a genetic deformity” is often heard. In fact,
this is often not the case, for many congenital anomalies are not genetic in origin nor do they constitute a
physical deformation per se. Another common, but erroneous, opinion is that hundreds or even thousands of
substances in the environment cause birth defects. In fact, only 30 to 40 exogenous substances (i.e., teratogens)
have been proven to have this potential. But if these issues continue to hamper our dealings professionally, they
also tug at the souls of grieving parents who ask “What caused my baby's problem?” “How did this happen?” or
“Will it happen again?” These questions take on added complexity when multiple anomalies are encountered in a
single patient. It can fall to the pathologist, as well as clinical specialists, to help explain these findings. The
purpose of this chapter, then, is to provide a broad context for understanding the basic patterns of anomalies. As
such, descriptions will emphasize gross features over microscopic appearances or discussions of intricate
pathologic processes. For help with these latter matters, the reader is referred to chapters that cover specific
organ systems.
ETIOLOGY AND PATHOGENESIS

The question of causation—etiology—is not at all simple, for etiology may be heterogeneous, that is, multiple
factors may bring about a given defect. Holoprosencephaly is a powerful example, for it arises sporadically or is
associated with several gene mutations, aneuploidies (i.e., trisomy 13), and teratogens (e.g., ethyl alcohol).
Robin sequence (micrognathia, cleft palate, glossoptosis) is another example, in which causes may be
chromosomal, teratogenic, monogenic, disruptive (i.e., amniotic bands), or unknown (45).
The mechanism—or pathogenesis—responsible for a defect may be varied as well. In Robin sequence, for
example, deformations may occur secondary to intrauterine constraint produced by oligohydramnios. However,
reduced amniotic fluid volume may occur from premature rupture of membranes (particularly chronic leakage),
renal anomalies, placental, or maternal factors.
CONCEPTS AND TERMS OF MORPHOGENESIS

In 1982, a set of standardized terms for describing human developmental abnormalities was established (235).
The definitions are essential for pediatric pathologists, pediatricians, medical geneticists, and others dealing with
congenital anomalies. Several discussions of the terminologic, historic, diagnostic, nosologic, and morphologic
aspects of congenital anomalies in humans are available (45, 180).
Hypoplasia refers to underdevelopment and hyperplasia to overdevelopment of an organism, organ, or tissue
and result from a change in cell number. Hypotrophy and hypertrophy refer to a decrease and increase,
respectively, in the size of an organ, tissue, or cells. Agenesis is the absence of a part of the body caused by a
presumed absence of the anlage, or primordium. Aplasia refers to absence of a rudimentary structure caused by
failure of the anlage to develop completely. Aplasia can be regarded as an extreme degree of hypoplasia.
Atrophy describes the shrinkage of a previously normally developed tissue mass or organ because of a decrease
in cell size or cell number.
A developmental field is the portion of the embryo that reacts as a coordinated unit to inductive effects with
differentiation and growth (178). Developmental fields represent, then, the major branches on the morphogenetic
tree. It has been suggested that the embryo itself constitutes the primary developmental field (177) and that
other more constricted ones are operational at later stages of development. A monotopic field defect represents
a defect in organogenesis and includes contiguous anomalies (e.g., cyclopia and holoprosencephaly; cleft lip
and cleft palate). Such alterations are more likely to arise late in gestation and produce more
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confined defects (151). By contrast, a polytopic field defect is thought to result from an earlier defect during
blastogenesis—the first 4 weeks of development—and occurs if abnormal inductive processes produce more
distantly located and diverse defects (151, 152).
The midline also acts as a developmental field (179). It represents the normal plane of cleavage in monozygotic
twinning and the plane around which symmetry of visceral position is determined. It is an especially vulnerable
site in terms of developmental anomalies. Morphogenetic events involving the midline include fusions,
segmentation, programmed cell death with morphogenetic “necroses” or resorptions, rotations, and other
developmental movements. In some anomalies involving the midline, the incidence of monozygotic twinning may
be increased (e.g., sirenomelia, cloacal anomalies). Other examples of midline anomalies include the
holoprosencephaly complex, agenesis of the corpus callosum, cleft lip, cleft palate, midface cleft complex, spina
bifida, omphalocele, congenital heart defects, hypospadias, and imperforate anus.
A malformation is “a morphologic defect of an organ, part of an organ, or larger region of the body resulting from
an intrinsically abnormal developmental process” (235).
A disruption, or secondary malformation, is “a morphologic defect of an organ, part of an organ, or a larger
region of the body, resulting from the extrinsic breakdown of, or interference with, an originally normal
developmental process” (235). Disruptions are causally heterogeneous and may bear close resemblance to
malformations anatomically. In a given case, the distinction between a disruption and a malformation may be
made on the basis of the associated malformations or the history of gestational exposure to a teratogenic agent
or event. The general prevalence of birth defects is given as 3% to 5%.
A deformation is “an abnormal form, shape, or position of a part of the body caused by mechanical forces.” It
may be extrinsic, due to intrauterine constraint (e.g., lack of amniotic fluid), or intrinsic, due to a defect of the
nervous system that causes hypomobility (235). Examples of deformities are talipes equinovarus and
arthrogryposis. About 1% to 2% of newborn infants have deformations of some sort.
Dysplasia represents “an abnormal organization of cells into tissue(s) and its morphologic result(s)” (235).
Dysplasia is therefore a process and the consequence of dyshistogenesis, an abnormal differentiation of tissue
structure. This is in contrast to a malformation, which is a defect in morphogenesis of the organ structure.
Dysplasias may or may not be metabolically induced, may involve one or several germ layers, and may be
generalized or localized; they often demonstrate a sporadic pattern of occurrence (235).
Mild dysplasias, common in the normal population, include freckling, capillary hemangioma over the glabella and
metopic suture area of the forehead, café au lait spots, moles, and nevi. If they are Mendelian traits, they usually
represent autosomal dominant mutations. Dysplasias are components of every aneuploidy syndrome and
probably are one reason for the increased incidence of associated cancers. Dysplasias can be induced
environmentally by radiation, viruses, and carcinogens.
Anomalies sometimes occur as groups of defects, which require additional classification. The terms described
below help in categorizing anomalies, but are only aids. Placing a name on a cluster of anomalies helps in
organizing thoughts about a given condition, but does not identify cause or mechanism or suggest recurrence
risk. In general, “the classification and terminology of infants with birth defects remain confusing. Terms such as
syndromes, associations, phenotypes, patterns, fields, and spectra often appear to be used as convenient labels
and do not help clarify the underlying cause or pathogenesis” (125).
That being said, a syndrome is “a pattern of multiple anomalies thought to be pathogenetically related and not
known to represent a single sequence or a polytopic field defect” (235). No structural component anomaly of any
malformation syndrome is obligatory, and no one component is pathognomonic of any syndrome.
A sequence is a “pattern of multiple anomalies derived from a single known or presumed prior anomaly or
mechanical factor” (235). In the Potter sequence, the pathogenetic event is oligohydramnios arising from a
genetic or nongenetic cause; the causal event represents a malformation (e.g., renal agenesis or dysplasia, as in
polycystic kidney) or a mechanical factor (e.g., amniotic fluid leakage). Lack of amniotic fluid restricts fetal
movement and causes fetal compression, producing the typical changes of Potter sequence (Figure 4-1).
A malformation complex consists of “those groups of heterogeneous disorders with overlapping characteristics
that are difficult to separate into specific conditions,” for example, facio-auriculo-vertebral spectrum and
hypoglossiahypodactylia.
An association consists of “a nonrandom occurrence in two or more individuals of multiple anomalies not known
to be a polytopic field defect, sequence, or syndrome” (235). Associations have also been defined as the results
of “disruptive events acting on developmental fields” (144). However, the diversity of findings in such
associations as CHARGE highlight how much remains unknown about possible developmental fields. In a sense,
the term “association” is a temporary category that should change as conditions become better understood.
Jones has offered a valuable policy for naming patterns of malformations (118):
1. When the etiology is known and easily remembered, the appropriate term should be used to designate the
disorder.
2. Time-honored designations should be continued unless there is good reason to change.
3. In the absence of a reasonably descriptive designation, eponyms, some of them multiple, may be used until
the basic defect for the disorder is recognized. However, use of an eponym should thereafter be limited to one
proper name.
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4. The use of the possessive form of an eponym should be discontinued, because the author neither had nor
owned the disorder.
5. Designation of a disorder by one or more of its manifestations does not necessarily imply that they are either
specific or consistent components of that disorder.
6. Names that may have an unpleasant connotation for the family or affected individual should be avoided.
7. The syndrome should not be designated by the initials of the originally described patients.
8. Names that are too general for a specific syndrome should be avoided.
9. Unless acronyms are extremely pertinent or appropriate, they should be avoided.
FIGURE 4-1▪Potter sequence. A: 22-week fetus with history of severe oligohydramnios (renal system normal; no
history of premature rupture of membranes). Note the blunt nose, small mandible, and flattened ear. B: Marked
skin webbing (pterygium) of right elbow developed secondary to prolonged immobilization of joint. C: Medial
rotation of foot at ankle joint (talipes equinovarus) resulted from intrauterine constraint. D: Fetal surface of
placenta shows amnion nodosum, a finding common in cases of oligohydramnios.
DEFORMATIONS
Amniotic Fluid Volume
Oligohydramnios, or anyhdramnios, effectively reduces the space available to the fetus and is associated with a
wide variety of fetal deformations involving the limbs and the craniofacial complex. With reduced inhalation of
fluid comes pulmonary hypoplasia, which is lethal when severe. Reduced fluid volume comes about primarily
from leakage (i.e., premature rupture of membranes) or renal anomalies with reduced production of fetal urine.
Uterine and Placental Implantation Abnormalities

A bicornate uterus may cause fetal compression and constraint, resulting in a deformed fetus. Uterine
malformations may also predispose the fetus to malformations arising from abnormalities in implantation,
placentation, body stalk formation, and late fetal cord compression or torsion. With these occurrences comes an
increased risk of stillbirth (see Chapter 18).
Neurogenic, Skeletal, and Other Causes of Deformations

Central nervous system (CNS) and skeletal muscle defects (e.g., amyoplasia) may result in deformations. The
most common congenital limb deformities are tibial bowing, mild metatarsus varus, talipes equinovalgus and
varus, and the flexural contractures of arthrogryposis (Figure 4-2). Skeletal dysplasias may be associated with
deformities of prenatal or postnatal onset. Twins and multiple fetuses may effectively interfere with each other's
physical development and manifest deformities.
DISRUPTIONS
Ionizing Radiation
Studies of radiation exposure to pregnant women during medical treatments or warfare have provided valuable
information regarding radiation-induced fetal anomalies.
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It is commonly held that pregnant women should avoid all unnecessary radiation exposure. However, data
regarding exact doses of radiation are often unavailable, and so fears and actions based upon those fears (i.e.,
elective abortion after an exposure or suspected exposure) are often unwarranted. Counselors must use extreme
caution when dealing with questions regarding radiation exposure.
FIGURE 4-2 ▪ Severe arthrogryposis in 23-week fetus. Extraordinary flexion and contracture deformities and
marked flattening of the face are apparent. Autopsy revealed no other fetal anomalies (karyotype 46,XX).
Etiology is heterogeneous in this condition.
Guidelines are widely available for this purpose (9, 13, 32, 255). During pregnancy, the acceptable cumulative
dose of ionizing radiation during pregnancy is 5 rads. With few exceptions, diagnostic studies produce dosages
less than this level. A two-view radiograph of mother's chest, for example, exposes the fetus to just 0.00007 rads.
Therefore, a mother would need the equivalent of 500 chest examinations before the fetus would be exposed to
a harmful level of radiation. Because 8 to 25 weeks, and especially 10 to 17 weeks, of gestation is a highly
sensitive period for CNS teratogenesis, unnecessary exposures directly to the fetus should be avoided during
this time. Prenatal radiation exposure may produce a slight increase in the risk of childhood leukemia or small
change in the frequency of gene mutations, but these are quite rare and not an indication for pregnancy
termination.
Fetal irradiation is associated with generalized growth retardation, microcephaly, skull defects, spina bifida,
microphthalmia, cleft palate, micromelia, clubfoot, and other anomalies following maternal exposure to high-dose
radiation (83). Altered mental status, ranging from reduced intelligence quotient (IQ) to frank mental retardation
and seizures, are recognized; MRI examinations are suggestive of neuronal migration defects (184, 185, 198).
Teratogenic Disruptions
A list of teratogenic agents in humans is shown in Table 4-1 and the specific time of action in embryonic
development is shown in Table 4-2. Excellent resources on this topic are available (73, 225).
Thalidomide Embryopathy
Thalidomide was first recognized as a teratogen by Lenz and McBride in separate reports in 1961. Maternal
administration of thalidomide during the critical period (day 23 to 28 of gestation) results in a number of defects,
the most notable of which are limb defects ranging from triphalangeal thumb to tetra-amelia or phocomelia of the
upper and lower limbs, at times with preaxial Polydactyly of six or seven toes per foot. Congenital heart defects,
urinary tract anomalies, genital defects, gastrointestinal anomalies, eye defects, ear malformations, and dental
anomalies have been observed. The mechanism of action continues to be studied. Some have suggested that
defective angiogenesis in developing limb buds may be operational (238). This hypothesis may also have
application to the sensitivity of certain neoplasias to thalidomide.
Table 4-1 ▪ TERATOGENIC AGENTS IN HUMANS
Radiation
Atomic weapons
Radioiodine
Therapeutic
Infections
Cytomegalovirus
Herpes simplex virus 1 and 2

Lymphocytic choriomeningitis virus (LCMV)
Parvovirus B-19 (erythema infectiosum)
Rubella virus
Syphilis
Toxoplasmosis
Varicella virus
Venezuelan equine encephalitis virus
Maternal and Metabolic Imbalance
Alcoholism
Amniocentesis, early
Chorionic villus sampling (before day 60)a
Cretinism, endemic
Diabetes mellitus
Folic acid deficiency
Hyperthermia
Myasthenia gravis
Phenylketonuria
Rheumatic disease and congenital heart block
Sjogren syndrome
Virilizing tumors
Drugs and Environmental Chemicals
Aminopterin and methylaminopterin

Androgenic hormones
Captopril (renal failure)
Carbamazepinea
Chlorobiphenyls
Cigarette smoke (nicotine)
Cocaine
Corticosteroidsa
Coumarin anticoagulants
Cyclophosphamide
Diethylstilbestrol
Diphenylhydantoin
Enalapril (renal failure)
Etretinate
Fluconazole, high dose
Iodides and goiter
Lithiuma
Mercury, organic
Methimazole (scalp defects and choanal atresia)a
Methylene blue via intra-amniotic injection
Misoprostola
Penicillamine
Phenobarbitola
1,3-c/s-Retinoic acid (Isotretinoin and Accutane)
Sartans
Tetracyclines
Thalidomide
Toluene abuse
Trimethadione
Valproic acid
aAgents produce less than 10 defects per 1,000 exposures From Shepard and Lemire (225), with
permission.
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Table 4-2 ▪ TIME OF ACTION OF HUMAN TERATOGENS
Teratogen Gestational Age Malformation

(Days)
Rubella virus 0-60 Cataract or heart defect more likely
0-129+ Deafness
Thalidomide 21-40 Reduction defects of extremities
Hyperthermia 18-30 Anencephaly
Male hormones (androgens, Before 90 Clitoral hypertrophy and labial fusion

tumors) only
After 90 Clitoral hypertrophy
Coumadin anticoagulants Before 100 Hypoplasia of nose and stippling of

epiphyses
After 100 Possible mental retardation
Diethylstilbestrol After 14 Vaginal adenosis (50%)

After 98 Vaginal adenosis (30%)
After 126 Vaginal adenosis (10%)
Radioiodine therapy After 65-70 Fetal “thyroidectomy”
Goitrogens, iodides After 180 Fetal goiter
Tetracycline After 120 Dental enamel staining of primary teeth
After 250 Staining of crowns of permanent teeth
From Shepard (224), with permission
Folic Acid Deficiency

Deficiency of folic acid results in up to 70% of neural tube deficits (NTDs), particularly anencephaly.
Preconceptional intake of 0.4-mg folic acid daily reduces the incidence of NTDs by approximately 60% (140,
271). The fortification of wheat flour with folic acid in the United States has resulted in a decrease in the
incidence of NTDs; a similar success might also be expected with similar fortification and surveillance worldwide
(14, 25). In addition to aiding in the prevention of NTDs, prenatal supplementation with folic acid prevents
pregnancy-induced megaloblastic anemia (245).
Folic Acid Antagonists and Derivatives

Aminopterin and methotrexate, its methyl derivative, are folic acid antagonists that may cause a variety of
anomalies. Because of the use of methotrexate to end an unwanted or ectopic pregnancy, exposure during early
pregnancy is possible (3). Craniofacial anomalies include severe hypoplasia of frontal, parietal, temporal, or
occipital bones; wide fontanelles; upsweep of frontal scalp hair; broad nasal bridge; shallow supraorbital ridges;
prominent eyes; cleft palate; apparently low-set ears; micrognathia; maxillary hypoplasia; and epicanthal folds.
The limbs are relatively short, and dislocation of hips, short thumbs, partial syndactyly of third and fourth fingers,
dextroposition of the heart, and hypotonia may occur (163, 223, 252).
Fetal Iodine Deficiency

The pregnant woman and her developing fetus both have an increased need for iodine. The woman deficient in
dietary intake of iodine therefore puts both herself and her fetus at risk (81). The use of iodized salt has done
much to reduce the risk of associated mental retardation worldwide but is of little help to women who do not have
access to this food or those who must reduce their salt intake during pregnancy.
Fetal iodine deficiency results in mental retardation, spastic diplegia, deafness, and strabismus (48, 106). It
develops from severe maternal iodine deficiency (<20 mg/day) during the first half of gestation, which occurs
primarily in a number of European countries and in some mountainous areas, such as New Guinea, the
Himalayas, and the Andes (169). The World Health Organization recommends a daily iodine intake of between
150 and 300 mg (12).
Trimethadione Syndrome
In one report of 53 cases, 87% were associated with pregnancy loss or abnormalities in offspring, most
commonly delayed growth and mental development, skeletal, cardiac, or urogenital anomalies, malformed ears,
and cleft palate (71). Infants with the trimethadione or paramethadione syndrome may also have changes that
include unusual eyebrows, higharched palate, and irregular teeth (76, 124, 279).
Valproic Acid Embryopathy

The teratogenicity of valproic acid has been well documented (43, 51, 172, 206). Although valuable as an
antiepileptic drug, valproic acid administration during pregnancy is associated with a host of anomalies, including
microcephaly, porencephaly, spina bifida, and other CNS defects, facial anomalies, cardiac defects, limb
reduction anomalies, and hypospadias (17). Dosages associated with malformations have generally been 750 to
1,000 mg per day and exposures verified during the first trimester.
Warfarin Embryopathy
Although the contraindications of Warfarin during pregnancy are well recognized, women may take the drug
during the first trimester, before pregnancy is recognized. Such exposure produces an embryopathy
characterized by reduced growth, hypoplastic nose, limb defects (shortening, brachydactyly,
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nail hypoplasia), gastroschisis, cardiac defects, and stippled epiphyses or chondrodysplasia punctata (21, 36,
190, 218, 222). If the drug is administered late in pregnancy, brain damage with mental retardation may be
caused by CNS bleeding in the fetus (268). Death may occur from respiratory failure.
Synthetic Progestin Embryopathy

Exposure to synthetic progestins (e.g., 17-α-ethinyl-19-nortestosterone) early in gestation can induce
enlargement of the clitoris or labioscrotal fusion in female fetuses and hypospadias in boys (2, 273). The
incidence of ectopic pregnancy is increased in women who experience contraception failure from either oral
progestins or implants (74). Diethylstilbestrol may cause vaginal adenosis in prenatally exposed girls and
reproductive anomalies in similarly exposed boys (98, 272). The use of a variety of exogenous sex hormones is
not associated with increased risk of major malformations, with the exception of esophageal atresia, which
carries a risk ratio of 2.87, which translates to approximately 6 per 10,000 live births (134).
Mercury Embryopathy
Exposure of the developing human to mercury compounds has serious effects, most notably an increased
incidence of growth retardation, microcephaly, and CNS damage, with consequent deficits that include
blindness, hypotonia or spasticity, deafness, dysarthria, chorea, athetosis, and strabismus. Both maternal
ingestion and occupational exposure are recognized routes of exposure. The classic condition is Minamata
disease, an epidemic that affected women living on the island of Minamata, Japan, who ingested shellfish
contaminated with methyl mercury (153, 225). Women continue to be exposed by this route, especially those
living in areas of heavy industrial pollution, where contamination of soil and water occurs (154), or those
ingesting contaminated marine food in the Arctic (92). In one study of maternal exposure to inorganic mercury,
significant increases were noted in structural anomalies of the CNS, but not miscarriage or stillbirth (68).
Isotretinoin Embryopathy
Isotretinoin (of which the drug Accutane is a prime example) is a synthetic vitamin A analog, 13-cis-retinoic acid;
because it inhibits sebaceous gland function, the drug is valuable in the treatment of cystic acne (133, 192).
Administration to pregnant women is associated with a variety of serious anomalies. Miscarriage, perinatal
mortality, and premature birth are reported, and survivors may have a variety of malformations or decreased
mental status. Ear anomalies are common, including dysplastic, hypoplastic, or absent ears; agenesis of the
external ear canal is variable. CNS abnormalities (microcephaly, hydrocephalus, porencephaly, Dandy-Walker
malformation, neuronal migration defects) and conotruncal congenital heart defects have been reported (211).
The association of isotretinoin administration with adverse psychiatric effects has been described, but remains
controversial (240).
Alcohol Embryopathy
Alcohol is a common and important teratogen in humans, but its influence was not fully appreciated until 1968
(138). In 1973, Jones and Smith named the condition “fetal alcohol syndrome” (FAS) (120). Effects are broad,
including structural, behavioral, and neurocognitive deficits, and so a number of other designations have been
used, including the earlier “fetal alcohol effect” and current “fetal alcohol spectrum disorders” (34, 111). In a
sense, the term “fetal alcohol syndrome” is unfortunate, for, although popular, it implies that alcohol exerts its
primary influence on the fetus; in fact, teratogenic damage to the embryo is far more significant, hence the term
“alcohol embryopathy.”
A maternal history of alcohol consumption is often difficult to ascertain, but nevertheless, clinical criteria for
making the diagnosis are available (111). Major characteristics of affected infants and children include distinctive
facies (epicanthal folds, short palpebral fissures, midface hypoplasia, thin vermilion border of the upper lip,
absent to indistinct philtrum, and short, upturned nose), growth retardation, malformations, and psychomotor
abnormalities (42, 120). Patients generally present with prenatal and postnatal growth retardation and CNS
dysfunction, including mental retardation, hyperactivity, sleep disorders, spastic tetraplegia, seizures, and
behavioral difficulties (Table 4-3). Joint, limb, and conotruncal cardiac anomalies are often present; limb defects
include shortness of the metatarsals and metacarpals or severe ectrodactyly (101). The unusual hirsutism that is
present at birth may disappear with age. Structural brain malformations, chiefly hypoplasia or agenesis of the
corpus callosum, lissencephaly, and holoprosencephaly, as well as ocular abnormalities, have been described
(41). Cystic hygromas are found in patients with FAS, but also with a number of other conditions (Table 4-4).
FAS has been reported in both monozygotic and dizygotic twins; the higher incidence in the former has
suggested a genetic influence (40, 242). Despite small head circumference and initially slow psychomotor
maturation, some infants with FAS may progress and develop intelligence within the normal range. Endocrine
investigations usually show normal or near-normal levels of growth hormone, Cortisol, and gonadotropins (see
Chapter 21) (99, 258). FAS is also a carcinogenic syndrome and is associated with tumors virtually identical to
those seen in the fetal diphenyl-hydantoin (Dilantin) syndrome.
Diphenylhydantoin Embryopathy
Diphenylhydantoin (Dilantin) is associated with a syndrome of microcephaly and mental retardation, cleft palate,
congenital heart defect, and a characteristic facial appearance (93). Human exposure during the 5th to 6th week
results in cleft lip and maxillary hypoplasia (270). Changes produced experimentally are due to embryonic
bradycardia or other arrhythmia and resulting hypoxia, stemming from phenytoininduced blockage of potassium
ion channels and delayed cardiac repolarization (19, 52).
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Table 4-3 ▪ CHARACTERISTICS OF FAS
Somatic and Cutaneous Findings
Prenatal and postnatal growth retardation, with diminished adipose
Hirsutism
Cutaneous hemangiomas
Micrencephaly
Neuronal migration defects (heterotopia)
Absent or hypoplastic corpus callosum
Ventriculmegaly
Holoprosencephaly
Hypoplastic cerebellum
Dysplastic brainstem
Lissencephaly
Craniofacial
Microcephaly
Ocular hypertelorism
Short palpebral fissures, sometimes downslanting or with epicanthal folds
Microphthalmia, other eye anomalies
Posteriorly rotated ears, with hypoplastic concha
Low nasal bridge
Hypoplastic midface, with hypoplastic maxillae
Retro- or micrognathia
Cleft lip and/or palate
Smooth vermillion border
Long, indistinct philtrum
Small teeth
Cardiovascular
Congenital heart disease, often conotruncal (e.g., tetralogy of Fallot)
Atrial and/or ventricular septal defects
Gastrointestinal tract
Esophageal, duodenal, or anal atresia
Tracheoesophageal fistula
Pyloric stenosis
Urogenital System
Hypospadias
Hypoplastic labia
Small rotated kidneys
Hydronephrosis
Musculoskeletal Systems
Abnormal palmar creases
Hypoplastic nails
Reduction defects of limbs and digits
Pectus excavatum or carinatum
Scoliosis
Klippel-Feil anomaly
Diaphragmatic hernia
Umbilical hernia
Behavioral
Developmental delay, mental retardation
Irritability (in infancy)
Hyperactivity (in childhood)
Hypotonia, reduced coordination
From Clarren etal. (41, 42); Potter and Hetzel (198)
Fetal exposure to diphenylhydantoin is also known to be carcinogenic. Neuroblastoma, ganglioneuroblastoma,

and malignant mesenchymoma have been observed in individuals exposed to diphenylhydantoin in utero (77). A
newborn infant has been described with fetal hydantoin syndrome and extrarenal Wilms tumor (248).
Table 4-4 ▪ CONDITIONS ASSOCIATED WITH CYSTIC NUCHAL HYGROMA
Single Gene Disorders
Familial neck webbing (autosomal dominant)
Lymphedema distichiasis syndrome (autosomal dominant)
Roberts syndrome (autosomal recessive)
Bieber syndrome (autosomal recessive?)
Chromosome Disorders
45X (Ullrich-Turner syndrome or monosomy X)
X-chromosome polysomy
13q-
18p-
Trisomy 18
Trisomy 21
Trisomy 22 mosaicism
Teratogenic Disorders
Alcohol embryopathy
Fetal amethopterin syndrome
Fetal trimethadione syndrome
Disorders of Unknown Cause
Noonan syndrome (autosomal dominant?)
Adapted from Gilbert-Barness and Opitz (78)
Metabolic Disruptions
Phenylketonuria
Maternal phenylketonuria (PKU) leads to intrauterine and postnatal growth retardation, microcephaly and mental
retardation, cardiovascular defects, dislocated hips, and other anomalies. The incidence of fetal defects is
greatly decreased in mothers whose PKU is well controlled during pregnancy. It has been suggested that
impaired accretion of two fatty acids, arachidonic and docosahexaenoic acids (structural components of the
CNS), contributes to the small head, reduced vision, and mental retardation (114, 115). Infants of
phenylketonuric mothers are heterozygous, and because phenylketonuric heterozygotes are generally normal,
the defect in the fetus must be attributed to the maternal metabolic disturbance.
Diabetes Mellitus
A large number of complications are recognized in pregnant women suffering from diabetes mellitus. Stillbirth and
perinatal mortality in insulin-dependent women occur at five times the background rate; neonatal mortality is
increased 15 times and infant mortality three times over the general population (230). Macrosomia complicates
vaginal delivery. Type I maternal diabetes is also associated with an increased incidence of preeclampsia and
pregnancy-induced hypertension (135). The effects of gestational diabetes remain under scrutiny.
Maternal diabetes mellitus is associated with a number of fetal anomalies, with an incidence variably estimated at
two to eleven times that of the normal population (64, 84, 276).
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In diabetic embryopathy, defects include those of the CNS (anencephaly, holoprosencephaly, arhinencephaly,
and myelomeningocele), congenital heart defect, caudal regression anomaly, sirenomelia, imperforate anus,
radial aplasia, and renal abnormalities, including renal agenesis and dysplasia (Figure 4-3). Malformations
(Table 4-5) are the most important cause of mortality in infants of diabetic mothers (127).
The exact role of glucose metabolism in diabetic embryopathy is unclear, and workers continue to discuss the
possible effects—and interrelationships—of both hyperglycemia and hypoglycemia (251). Hyperglycemia is
associated with a number of metabolic derangements, including myo-inositol and arachidonic acid deficiency and
altered prostaglandin metabolism, which in turn influences the formation and function of cell membranes (276).
The dramatic influx of glucose through faulty membranes induces the generation of free oxygen radicals, altered
mitochondrial function, and increased peroxidation of lipids, all of which can cause malformations in the
developing embryo.
The correlation between hemoglobin A1C (HbA1C), maternal microvascular disease, and the incidence of major
congenital anomalies in infants of diabetic mothers is high (147). HbA1C is a normal, minor hemoglobin, whose
glycosylation depends upon glucose concentration. Measurement of HbA1C thus provides an index of glucose,
and therefore,
of diabetes control; a higher incidence of major anomalies has been observed in the offspring of women with
elevated HbA1C (see also Chapter 27).
FIGURE 4-3 ▪ Infant of diabetic mother. Pelvic girdle is reduced noticeably in this 31-week-old male with absent
lumbosacral spine and malformed pelvis (caudal regression syndrome).
Table 4-5 ▪ FETAL ANOMALIES ASSOCIATED WITH MATERNAL DIABETES
Anencephaly
Holoprosencephaly
Arhinencephaly
Occipital encephalocele
Cardiovascular System
Atrial, ventricular septal defect
Transposition of the great vessels
Tetralogy of Fallot
Single ventricle, hypoplastic left heart
Ebstein anomaly of tricuspid valve
Pulmonic stenosis, mitral atresia
Other Abnormalities
Bilateral auricular atresia
Cleft lip
Omphalocele
Unilateral renal agenesis
Hypoplastic lungs
Caudal regression
Amelia of upper limbs
Adapted from Gilbert-Barness and Opitz (78).
Infectious Disruptions
Infections, particularly toxoplasmosis, rubella, cytomegalovirus (CMV), herpes simplex, varicella, syphilis, and
others (TORCHS) may cause fetal disruptions (see Chapter 6). The earlier in pregnancy the infection occurs, the
greater is the likelihood of embryonic death or fetal anomalies. The most frequent fetal abnormalities are
intrauterine growth retardation, microcephaly and mental retardation, deafness, cataracts, retinopathy,
microphthalmia, glaucoma, myopia, and congenital heart defects.
Periventricular calcifications and chorioretinitis are frequent in toxoplasmosis. Other organisms that may be
implicated in human congenital anomalies are herpes hominis type 2, which is associated with a severe
congenital brain defect, varicella (119), Venezuelan equine encephalitis, coxsackie virus, and syphilis. Acquired
immune deficiency syndrome (AIDS) is transmitted transplacentally or during labor, delivery, or breast feeding
and constitutes an enormous problem worldwide (160). In 2005 in the United States, 92% of cases of children
with AIDS were attributed to maternal transmission of the human immunodeficiency virus (HIV) (1). The incidence
of neonatal HIV infection has fallen substantially in the United States with the implementation of prenatal testing,
antiretroviral therapy, C-section, and avoidance of breast feeding (1).
Amnion Rupture Disruption Sequence

Early amnion rupture (or ADAM complex) may result in severe defects of the fetus, including asymmetric clefts,
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body wall defects, with extrusion of viscera, and highly variable amputations (Figure 4-4). When amnion adheres
to the head, marked distortions of craniofacial structures are found, with widely separated eyes, displacement of
the nose onto the forehead, and exencephaloceles; swallowing of amniotic bands may produce bizarre orofacial
clefts. Marked deformations, growth deficiency, and a short umbilical cord are also observed in this condition
(161). The fetus may also be adherent to the placenta, making diagnosis straightforward. However, when
strands of amnion are not identified, diagnosis is hampered, although the pattern of defects may still imply this
mode of pathogenesis. In the macerated fetus, strands of tissue resembling sloughed epidermis may be identified
as amnion by microscopy. Amnion may also be absent from the fetal surface of the placenta, or free membranes.
The least severe form of amniotic band disruption is a constriction groove (Streeter band) on a limb. The
temporal relationship of abnormalities in early amnion rupture sequence is shown in Table 4-6.
The phenomenon of amnion rupture is thought to be rather common, affecting perhaps 1 of every 1,200 liveborn
and stillborn fetuses. If this is the case, many cases apparently have few or no sequelae. Rare families with
amniotic bands in relatives have been reported, but the recurrence risk appears to be negligible (145). Causes
for premature rupture are not understood. The forces of uterine contraction have been implicated, but recent
studies have suggested that a process of programmed weakening of membranes may operate prior to delivery
(165). This observation could help explain familial recurrences (see Chapter 27 for additional details).
FIGURE 4-4 ▪ Amnion rupture sequence. A: Close-up view of fetal surface of placenta shows tiny remnant of
amnion. B: 22-week male fetus with multiple amputation defects. C: Face with unilateral cleft lip. D: Right foot
with syndactyly and multiple amputations of the digits. E: Exposed radius and ulna and necrosis of hand reflect
the evolution of a band-induced amputation. F: Radiograph corresponding to E. G: Right hand with multiple
amputation defects. H: Radiograph corresponding to G.
Chorion and Yolk Sac Rupture Sequence

While rupture of the amnion is well recognized, others have hypothesized that similar defects might arise from
rupture of the chorion or yolk sac. Rupture during the 3rd week of gestation and the subsequent mechanical
compression of the fetus could interfere with normal cardiac descent, resulting in cleft sternum, ectopia cordis,
and thoracic and pulmonary hypoplasia (122). Such cases reflect the complex nature of embryogenesis in the
region. Another published example involved an infant with rudimentary occipital meningocele and transverse
defects of the hands, who, by microscopy, had intestinal mucosa adherent to the scalp (250). Possible
explanations included a genetic defect similar to disorganization in the rodent; homeotic transformation; abnormal
juxtaposition of epidermis and yolk sac remnant (or omphaloenteric duct); or adhesion of endoderm and
ectoderm to the embryo.
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Table 4-6 ▪ TEMPORAL RELATIONSHIP OF ABNORMALITIES IN EARLY AMNION RUPTURE
Fetal Age at Occurrence Craniofacial Defects Limb Defects Other Abnormalities

3 weeks Anencephaly Eye defects Placenta adherent to
Encephalocele head or abdomen
Meningocele Short umbilical cord
Facial distortion
Clefting
Proboscis
5 weeks Cleft lip

Choanal atresia Limb deficiency Abdominal wall defect
7 or more weeks Cleft palate Polydactyly Thoracic wall defect

Micrognathia Syndactyly Scoliosis
Ear deformities Amniotic bands Short umbilical cord
Craniostenosis Amputation Omphalocele
Hypoplasia
Pseudosyndactyly
Distal lymphedema
Foot deformities
Dislocation of hip
Third trimester Oligohydramnios, with

associated deformations
Ischemic and Vascular Disruptions

Interference with blood supply may result in ischemic disruptions. Cutis marmorata telangiectatica congenita is a
vascular disruption characterized by atypical capillaries, venules, and veins in different cutaneous layers.
Clinically, the lesions manifest as telangiectasia, capillary hemangiomata, cutis marmorata, venous
hemangiomata, and varicose veins, depending on the type of vessels involved and the layer of skin affected.
Secondary thrombosis with subsequent localized atrophy and ulceration may occur. Cutis marmorata
telangiectatica congenita occurs sporadically, with female preponderance and occasional minor manifestations in
close relatives.
In the Klippel-Trenaunay-Weber syndrome (see below), which usually occurs sporadically, dysplasia and
capillary or cavernous hemangiomatosis and phlebectasia and varicosities with oligodactyly, syndactyly, and
gigantism of digits have been observed. Congenital or postnatal hypertrophy of one or more limbs is frequent.
Visceral hemangiomata may occur.
In addition to the well-recognized difficulties that arise in singletons, twins or other multiple gestations are
especially at risk. Cord entanglement occurs in twins and may disrupt blood flow. Because of the variability in
distance between cord insertion sites, more complications are observed in monochorionic monoamniotic than in
monochorionic diamniotic placentas (20). Two additional examples of vascular disruption involve monochorionic
twinning, namely twin-twin transfusion syndrome and twin reversed arterial perfusion (TRAP).
Twin-Twin Transfusion Syndrome

Twinning within the context of a shared placental disc is complicated by the presence of intraplacental vascular
anastomoses. These may be small and mild or quite large, allowing significant sharing of blood between fetuses.
Problems arise when blood flow is unbalanced and unidirectional, creating “pump” and “recipient” twins (Figure
4-5A). In such circumstances, the pump twin is pale and anemic, while the recipient or perfused twin is
congested, possibly hydropic, and polycythemic. Differences in amniotic fluid volume can create the “stuck twin”
phenomenon, with oligohydramnios in one amniotic sac (with consequent fetal deformation) and polyhydramnios
in the other. The death of one twin quickly affects the wellbeing of the other, resulting in death or embolization of
decay products, resulting in disseminated intravascular coagulation or visceral infarcts (155).
Twin Reversed Arterial Perfusion

Artery-to-artery anastomoses have a particularly striking effect when flow in one umbilical artery and aorta is
reversed, a phenomenon that can be diagnosed in utero by Doppler flow studies. In such a circumstance, the
lower body is perfused, but upper regions are not (Figure 4-5B). The heart may fail to develop (acardia);
absence of the head (acardia-acephalus), upper limbs, or other viscera lungs often occur.
Dysplastic Disruptions
Dysplastic disruptions include the presacral teratoma that may be associated with anencephaly, spina bifida,
meningocele, or imperforate anus; duplication of the lower intestinal tract, uterus, vagina, and ureter/renal pelvis;
patent urachus; cleft palate; and esophageal and duodenal atresia. Imperforate anus and sacral defects may be
inherited on an autosomal dominant basis.
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FIGURE 4-5 ▪ Complications of monochorionic twinning. A: Pale, donor twin (left) and congested, recipient co-
twin (right) in twin-twin transfusion syndrome. B: Acardiac cotwin in TRAP. Note the absence or malformation of
structures of the upper body, omphalocele, and more normal lower extremities (but with anomalies of numerous
digits).
Hyperthermia as a Disruption
Smith and colleagues were the first to make a systematic study of the effects of hyperthermia caused by
infections or sauna bathing during pregnancy (232). Hyperthermia is an antimitotic teratogen that interferes
mostly with CNS development, producing neural tube defects (NTDs), microcephaly, micrencephaly,
microphthalmia, and neurogenic contractures (66). Other anomalies associated with hyperthermia include
neuronal heterotopia, polymicrogyria, small midface, micrognathia, cleft lip and palate, ear defects, and limb
defects (e.g., arthrogryposis and syndactyly). Severe mental deficiency and seizures in infancy have also been
described (232).
The presence and severity of anomalies depend upon the duration of hyperthermic episode, maximum
temperature reached, and stage of development (66). Both mild temperature elevation during the preimplantation
period and more significant elevations during embryonic and fetal development may manifest as anomalies (67).
At weeks 7 to 16 of gestation, hyperthermia may be associated with hypotonia, neurogenic arthrogryposis, or
CNS dysgenesis. In one study, some 18% of women who delivered anencephalic embryos had experienced
hyperthermia at a critical embryonic stage (226). Most mothers experienced febrile illnesses with temperatures of
38.9°C or higher, commonly 40°C or above. Embryonic studies in a number of animal species have highlighted
the sensitivity of brain development to elevated temperatures and identified NTDs, microphthalmia, cataract,
craniofacial clefts, and defects of the body wall, skeleton, heart, and teeth (67).
NONMETABOLIC DYSPLASIA SYNDROMES

The most common dysplasia syndromes are the autosomal dominant conditions: neurofibromatosis 1, von
Hippel-Lindau disease, Marfan syndrome, and the osteochondrodysplasias, most of the lethal forms of which are
autosomal recessive traits. These conditions are discussed in various chapters including Chapters 12, 24 and
27.
Beckwith-Wiedemann Syndrome
In the early 1960s, Beckwith and Wiedemann reported a syndrome of exomphalos (i.e., omphalocele),
macroglossia, and gigantism. In one review, Beckwith-Wiedemann syndrome (BWS) accounted for nearly 12% of
all cases of omphalocele. Craniofacial abnormalities (Figure 4-6) include microcephaly, macroglossia (which may
interfere with respiration or swallowing), prominent eyes with relative infraorbital hypoplasia, capillary nevus
flammeus of the central forehead and eyelids, metopic ridge in the central forehead, large fontanelles, prominent
occiput, and malocclusion, with a tendency toward mandibular prognathism. A marker for the
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syndrome is the unusual linear fissures or pits in the lobule of the external ear and semilunar indentations of the
posterior rim of the helix. Hemihypertrophy, clitoromegaly, large ovaries, hyperplastic uterus and bladder,
bicornuate uterus, hypospadias, and immunodeficiency are recognized. Interstitial cell hyperplasia of the testis,
pituitary hyperplasia, neonatal polycythemia, diastasis recti, posterior diaphragmatic eventration, and
cryptorchidism may also occur. BWS also includes neonatal hypoglycemia, organomegaly, and cytomegaly of the
adrenal cortex and islet cells of the pancreas. The placenta in BWS may exhibit mesenchymal dysplasia, a rare
change that may be mistaken for partial hydatidiform mole. In one study, over 20% of placentas with this change
were from patients with BWS (195).
FIGURE 4-6 ▪ Beckwith-Wiedemann syndrome. A,B: Ear pits were identified in this 9-month-old infant, who had
a large omphalocele excised shortly after birth (46,XY, no deletion recognized). C: Note the distorted architecture
in this dysplastic kidney. D: Microscopic view of adrenal gland, showing marked cytomegaly
The predisposition to the development of malignant tumors such as Wilms tumor, adrenocortical carcinoma,
hepatoblastoma, gonadoblastoma, and brain stem glioma is widely recognized. Wilms tumor may be bilateral
when it is associated with this syndrome (see Chapter 17). Even when free of tumor, the kidneys may be
strikingly enlarged, and their surfaces traversed by numerous, irregularly disposed, shallow fissures that
markedly increase the number of lobulations. The parenchyma is disorganized; minute lobulations crowd one
another, each with a distinctly demarcated cortex and medulla. Other renal changes include persistent
glomerulogenesis, medullary dysplasia, diffuse bilateral nephroblastomatosis, metanephric hamartomas,
hydronephrosis and hydroureters, and duplications.
The incidence of polyhydramnios and prematurity is relatively high in BWS. Most cases are sporadic, but familial
and dominantly inherited cases have been reported. BWS is caused by disruption of the cycle of genomic
imprinting (i.e., germline erasure and establishment, somatic maintenance) within the 1 1p15 region (16). This
mechanism has been exhibited in dramatic fashion by the increased incidence of BWS in families utilizing
assisted reproductive technologies, namely in vitro fertilization and intracytoplasmic sperm injection (8).
Perlman Syndrome
Perlman syndrome is an autosomal recessive disorder comprising macrosomia, nephromegaly with renal
dysplasia (persistent fetal lobation, nephrogenic rests, immature glomeruli, sclerotic glomeruli, primitive tubular
structures, and medullary hamartomatous dysplasia), Wilms tumor, hyperplasia of the endocrine pancreas with
resultant hypoglycemia, cryptorchidism, multiple congenital anomalies (mostly infrequent and nonspecific ones,
such as facial dysmorphia, cleft lip, and cardiac anomalies), and mental retardation. The frequent occurrence of
Wilms tumor has led to the speculation that persistent foci of renal dysplasia, blastema, or nephroblastomatosis
constitute predisposing lesions (100). The condition resembles BWS, but is distinguished on the basis of
inheritance (e.g., BWS is autosomal dominant), differences in specific anomalies or appearance, and different
natural histories and associated malformations. Death by 1 year of age is common.
METABOLIC DYSPLASIA SYNDROMES

Williams Syndrome
Williams syndrome is an autosomal dominant disorder manifest by characteristic facial features, supravalvular
and aortic stenosis, infantile hypercalcemia, and behavioral and neurological abnormalities. Specific
characteristics include growth and mental retardation, microcephaly, congenital hypotonia, and elfin face with
short palpebral fissures, depressed nasal bridge, epicanthal folds, and anteverted nares. Many neonates have a
symptom complex of irritable failure to thrive with spitting up; in more severely affected infants, manifestations of
hypercalcemia may be life threatening or lethal. Cardiovascular defects include supravalvular aortic stenosis,
peripheral pulmonary artery stenosis, pulmonary valvular stenosis, and ventricular and atrial septal defect. Renal
artery stenosis with hypertension, hypoplasia of the aorta, and other arterial anomalies have been reported.
Culler and colleagues studied the hormonal control of calcium metabolism in patients with Williams syndrome,
noting delayed calcium clearance following intravenous loading. No abnormalities of vitamin D metabolism were
found either before or after parathyroid hormone stimulation. Immunoradioactive studies suggested that patients
with Williams syndrome may have a defect in the synthesis or release of calcitonin. A heterozygous deletion of a
region on chromosome 7q1 1.23, the Williams syndrome critical region, encompasses genes which encode for
proteins that regulate the cellular cytoskeleton; defects in the cytoskeleton are thought to relate to the
neurological symptoms of the syndrome (110).
Zellweger Syndrome
Zellweger syndrome (cerebrohepatorenal syndrome) belongs to a group of some 17 inherited peroxisomal
disorders (266). Genetic diseases involving peroxisomes (single-membranebound organelles involved in multiple
metabolic processes) include those in which only a single peroxisomal function is impaired—acatalasemia, X-
linked adrenoleukodystrophy, and the adult form of Refsum disease—and those with impaired peroxisome
biogenesis—the so-called Zellweger spectrum (consisting of Zellweger syndrome, infantile Refsum disease, and
neonatal adrenoleukodystrophy) and rhizomelic chondrodysplasia punctata (267).
An autosomal recessive trait, Zellweger syndrome results from mutations in at least 12 PEX genes encoding for
peroxins (33). The syndrome is lethal in infancy and dominated clinically by severe CNS dysfunction (234).
Affected infants are usually born at term and do not manifest intrauterine growth retardation. The clinical
manifestations are listed in Table 4-7 and include a pear-shaped or light bulb-shaped head, large fontanelles, flat
occiput, high forehead with shallow supraorbital ridges, a flat face, minor ear anomalies, inner epicanthal folds,
Brushfield spots, mild micrognathia, and redundant neck skin.
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Table 4-7 ▪ CLINICAL FINDINGS IN ZELLWEGER SYNDROME
Craniofacial Anomalies
Macrocephaly; high forehead; dolichocephaly
Large anterior fontanel; open metopic suture
Mongoloid slant of palpebral fissures; hypertelorism; shallow supraorbital ridges; epicanthal folds
High-arched palate; posterior cleft of palate
Minor anomalies of ears
Limbs
Talipes equinovarus
Camptodactyly
Contractures
Hypotonia, rarely hypertonia
Severe mental retardation
Seizures
Nystagmus; oculogyric fits
Absent neonatal reflexes
Eyes
Cataract
Glaucoma
Corneal clouding
Brushfield spots
Pigmentary retinopathy
Optic nerve “dysplasia” or hypoplasia
Skeletal Anomalies
Chondrodysplasia calcificans (especially of the patellae)
Delayed skeletal maturation
Bell-shaped thorax
Large fontanels
Other Abnormalities
Cardiac defect
Jaundice with hepatomegaly
Cryptorchidism; clitoromegaly
Single palmar crease
DiGeorge anomaly
The infant with Zellweger syndrome is severely hypotonic, with an inability to suck, reduced deep tendon
reflexes, and total lack of psychomotor development (31, 247). Because of the hypotonia and physical
appearance, infants are sometimes thought to have Down syndrome. Other manifestations include congenital
heart defects (e.g., anomalies of aortic arch, patent ductus arteriosus, ventricular septal defect), stippled
calcification of the epiphyses, and hepatomegaly with signs of hepatic dysfunction and occasional jaundice.
Increased serum iron and tissue siderosis aid diagnosis, but do not appear to be related to disease progression
(264). Death before 1 year of age usually occurs from respiratory complications.
Autopsy findings of patients with Zellweger syndrome are listed in Table 4-8. Brain abnormalities include focal
lissencephaly and other cerebral gyral abnormalities, heterotopic cerebral cortex, olivary nuclear dysplasia,
defects of the corpus callosum, numerous lipid-laden macrophages and histiocytes in cortical and periventricular
areas, and dysmyelination (265). The liver is characterized by hepatic lobular disarray, or micronodular cirrhosis,
biliary dysgenesis, and siderosis. The kidneys show persistent fetal lobulations with cortical cysts.
Albuminuria and aminoaciduria may be observed. Other abnormalities include hypoglycemia, elevated serum
iron, siderosis, hyperpipecolic acidemia, hepatic and cerebral glycogen storage, elevated very long chain fatty
acids, abnormal bile acids, dicarboxylic aciduria, and hypocarnitinemia. Renal cysts have been a consistent
finding and may be a pathologic marker for this condition. They are often macroscopic and both glomerular and
tubular by microscopy. Occasionally, cysts appear to connect directly to terminal ends of collecting tubules
without an intervening tubular segment, suggesting focally deficient metanephric differentiation. More classic
cystic dysplastic changes may also be observed (26), and horseshoe kidneys and ureteral duplication have been
noted (79). Immunodeficiency may develop, and some patients have been diagnosed mistakenly with DiGeorge
syndrome (109). Atypical cases of Zellweger syndrome (Versmold variant) have hypertonia and may live longer
(262). (See Chapter 5.)
Table 4-8 ▪PATHOLOGIC FINDINGS IN ZELLWEGER SYNDROME
Brain
Cerebellar, olivary hypoplasia
Abnormal cerebral convolutions (microgyria, pachygyria)
Partial lissencephaly
Agenesis or hypoplasia of the corpus callosum
Cerebral or cerebellar heterotopias
Enlarged lateral ventricles
Sudanophilic leukoencephalomyelopathy
Gliosis
Heart
Ventricular septal defect
Patent ductus arteriosus
Patent foramen ovale
Liver
Biliary dysgenesis
Cirrhosis
Siderosis
Absent peroxisomes
Abnormal mitochondria
Diminished smooth endoplasmic reticulum
Kidney
Multiple cortical microcysts; glomerular and tubular cystic dysplasia
Hydronephrosis
Horseshoe kidney
Pancreas
Islet cell hyperplasia
Thymus
Thymic hypoplasia
Adapted from Gilbert-Barness and Opitz (78)
SEQUENCES
Robin Sequence
The defects in Robin sequence include micrognathia, glossoptosis, and cleft soft palate. Hypoplasia of the
mandibular area before week 9 of gestation causes the tongue
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to be posteriorly located, presumably preventing closure of the posterior palatal shelves. It may also be a result
of early mechanical constraint in utero, limiting growth before palatine closure. The Robin sequence should alert
the clinician to the possible presence of the Stickler syndrome (see below) and the possibility of blindness due to
high myopia.
Prune Belly Sequence

Prune belly sequence occurs as a triad of absent or hypoplastic abdominal muscles, urinary tract defects, and
cryptorchidism (65). The umbilicus may be displaced cephalad, with flaring of rib margins, Harrison groove, and
pectus deformities, all apparently secondary to the muscle defect. Nearly three quarters of patients have
additional defects of the cardiac, pulmonary, gastrointestinal, or musculoskeletal systems. Most cases are
sporadic, but some familial cases have been autosomal recessive.
Renal anomalies are a critical part of the spectrum. With urethral or bladder neck obstruction, more proximal
segments of the urinary tract become dilated, resulting in megalourethra, megacystis, hydroureter, and
hydronephrosis, the latter with consequent renal hypoplasia. Urinary ascites may occur from overdistention and
prenatal rupture of the bladder. Thus, the abdomen tends to be tense and glassy in the fetus, and, after collapse
of the bladder and/or absorption of intraabdominal fluid, lax and wrinkled in the newborn (Figure 4-7). Neonatal
death occurs in 20% of infants, usually from pulmonary hypoplasia, a sequel of oligohydramnios or abdominal
pressure on the diaphragm. However, long-term survival is possible, especially in patients with mild or no
changes of the abdominal musculature or urinary tract. Complications in survivors include decreased
spermatogenesis/absence of spermatogonia and salt-wasting nephritis (49).
Pathogenesis continues to receive attention. One hypothesis is that an early insult to developing mesenchyme is
responsible for the condition (94). A different view is that massive distention of the urinary bladder causes
stretching and thinning of abdominal skeletal muscles (186). Some have attributed a major role to hypoplasia of
the prostate gland (107).
FIGURE 4-7▪ Prune belly sequence. A: Anterior view of 32-week male fetus with marked distention of the
abdomen secondary to megacystis and bladder outlet obstruction from posterior urethral valves. Used with
permission (231). Note flattened face, a result of intrauterine constraint. B: Posterior view of fetus,
transilluminated to demonstrate fluid-filled abdomen. Abdominal skin takes on a very wrinkled appearance
when/if fluid is resorbed.
ASSOCIATIONS
Because of shared molecular determinants, spatial contiguity, and close timing of morphogenetic events during
histogenesis, it is thought that most malformations arising during this period are polytopic, that is, involving two or
more developmental fields. Some have suggested, therefore, that associations (e.g., VATER schisis association)
be designated polytopic field defects (152). Regardless of nomenclature, it is evident that certain malformation
complexes, of which VATER and schisis feature prominently, develop from a widespread insult or insults during
early development.
VATER Association
In 1973, Quan and Smith coined the acronym VATER to represent the association of vertebral defects, anal
atresia, tracheoesophageal fistula, esophageal atresia, and radial and renal abnormalities (Figure 4-8).
Genitourinary defects
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include renal dysplasia or agenesis, renal ectopia, persistent urachus, hypospadias, and caudally displaced,
hypoplastic penis. Prenatal growth deficiency, ear anomalies, large fontanels, cleft palate, cloacal exstrophy, and
rib anomalies are also recognized. This pattern of malformations occurs sporadically.
FIGURE 4-8 ▪ VATER/VACTERL association. A: Marked deviation of wrist and hand; thumb and radius are
absent. B: Cystic renal dysplasia. C: Esophageal atresia (without tracheoesophageal fistula) from infant with
VACTERL association. D: Radiograph of excised vertebral column with hemivertebrae. Latter image used with
permission (231).
The phenotypic variability of VATER association complicates both diagnosis and classification. VACTERL is an
expansion of VATER that includes cardiac and limb defects. The overlap with Müllerian duct, renal, and
cervicothoracic somite malformations (MURCS) with tracheal agenesis, hemifacial microsomia, and other facial
asymmetry syndromes has been recognized (63). The VATER phenotype also overlaps with Fanconi syndrome
and to a lesser degree with sirenomelia. Some have recommended performing chromosomal breakage studies
on patients with features of both VATER association and Fanconi syndrome (70).
The etiology and pathogenesis of VATER association remain unknown. It has been hypothesized that anomalies
derive from a common pathogenetic mechanism, namely a defect of blastogenesis prior to day 35 of gestation.
Evidence comes from the fact that several critical tissues develop before 35 days, including the septa that divide
rectum/anus and trachea/esophagus, radial limb bud, and mesoderm that form the vertebral bodies (77). The
adriamycin animal model may contribute to future understanding of these issues (167) (see Chapters 12, 17 and
27 for additional details).
MURCS Association
MURCS is an acronym for Müllerian duct aplasia, renal aplasia, and cervicothoracic somite malformations, which
cause cervicothoracic vertebral defects, especially from C5 to T1 (63). This condition is sporadic and not
associated with abnormal karyotype. Absence of the vagina, absence or hypoplasia of the uterus, and renal
abnormalities (in up to 40% of patients), also occur (23). A variety of additional anomalies, including those
involving the skeletal, cardiac, and renal systems, complicate diagnosis (196). A male form of MURCS has been
postulated; findings are azoospermia, renal anomalies, and cervicothoracic spinal abnormalities (156).
Schisis Association and Variants

Midline defects such as NTDs (i.e., anencephaly, encephalocele, meningomyelocele), oral clefts, omphalocele,
and diaphragmatic hernia occur more frequently than expected (50). This so-called schisis association is
frequently a lethal abnormality. It occurs more often in girls, in twins (4.6%), and in breech presentations (13.7%),
and it is associated with lower mean birth weight and a shorter gestational period. Congenital cardiac defects,
limb deficiencies, and defects of the urinary tract, mainly renal agenesis, are defects that have a high association
(50). Schisis-type abnormalities appear to occur in a nonrandom fashion and have been postulated to arise
during blastogenesis (105).
AUTOSOMAL DOMINANT CONDITIONS

Nail-Patella Syndrome
In nail-patella syndrome, or hereditary onycho-osteodysplasia, fingers and toes, especially thumbs and great
toes, show onychodysplasia, hypoplasia, longitudinal ridging, and hemiatrophy. The patellae are small or absent
and the elbows are dysplastic; small osseous spurs or horns on the iliac bones are pathognomonic for the
condition. Glaucoma may develop or progress after birth, and a peculiar heterochromia may be seen in the iris.
Subtle impairment of hearing and peripheral neurological symptoms such as sensory dysfunction are also
recognized. A nephropathy is more frequent in females and varies from proteinuria, which may be transient and
asymptomatic, to renal failure (29). Thickening of the glomerular basement membrane that contains focal
collections of collagen fibers and mesangial thickening is present. Immunofluorescence shows a nonspecific
focal distribution of IgM or complement (additional details are found in Chapters 17 and 27).
One gene identified to date, LMX1B, on chromosome 9q34.1, is a transcription factor important to limb patterning
and morphogenesis of the glomerular basement membrane (28). However, the interfamilial and intrafamilial
variability in phenotypes is highly suggestive of additional genetic involvement (29, 30, 149).
Orofaciodigital Syndrome Type I (0FD1)

Orofaciodigital syndrome is an extremely variable constellation of congenital disorders, and as such has
engendered both attention and debate. Major changes in the group of conditions include hypertrophic frenula,
lingual hamartomas, cleft lip or palate, ocular hypertelorism, brachydactyly, Polydactyly, and syndactyly. Recent
investigations have emphasized the OFD1 protein, a core component of the centrosome and thus influential to
development in numerous ways. OFD1 is an X-linked dominant trait, lethal in hemizygous males prenatally and
characterized by webbing between the buccal mucous membrane and alveolar ridge, partial clefts in the mid-
upper lip, hypoplasia of nasal cartilages, absent lateral incisors, asymmetric shortening of the digits with
clinodactyly, bifid hallux with or without syndactyly, and variable mental deficiency (175).
Branchiootorenal Syndrome
The branchiootorenal syndrome is an autosomal dominant disorder characterized by branchial arch anomalies
(i.e., preauricular pits, branchial fistulas, anomalies of the external ear), hearing loss, and renal hypoplasia and
dysplasia. A preauricular pit at birth is a marker for this syndrome and suggests 1 chance in 200 of severe
hearing loss. The renal anomalies range from minor defects to marked hypoplasia with renal failure. Mutations in
the EYA1 gene have been identified in families with the complete syndrome, but not those lacking branchial
fistulae, suggesting genetic heterogeneity (208, 209).
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Townes-Brocks Syndrome
Townes-Brocks syndrome is an autosomal dominant disorder with variable expressivity (199). Major changes
include thumb anomalies (i.e., triphalangeal thumb), preaxial Polydactyly, auricular anomalies, imperforate anus,
cardiac defects, and anomalies of other internal organs, including renal hypoplasia and cysts. Mental retardation
has been reported in a minority of patients (199). Anomalies overlap with the VATER association and hemifacial
microsomia. It may be particularly difficult, but also important, to distinguish Townes-Brocks syndrome from the
latter condition, which occurs sporadically from mutations in SALL1 (123).
Holt-Oram Syndrome
This syndrome is characterized by certain skeletal and cardiovascular abnormalities (108) and appears as an
autosomal dominant trait with variable expressivity. Skeletal abnormalities in the upper limbs range from thumb
hypoplasia to phocomelia and have a preponderance of left-sided involvement. Hypoplasia or absence of the
first metacarpal and radius, and defects of the ulna, humerus, clavicle, scapula, and sternum may be present.
The most frequently described cardiac anomaly is a secundum-type atrial septal defect. However, a variety of
other cardiac defects and anomalies of the coronary arteries have been recognized. To date, some 37 mutations
of the gene responsible for HoltOram syndrome, TBX5, have been identified (112); missense mutations are
associated with distinct phenotypes. Variability within affected families suggests that the genetic background,
environmental or stochastic modifiers, or modifier genes may be important (112).
Mandibulofacial Dysostosis
Mandibulofacial dysostosis, also known as Treacher Collins or Franceschetti-Klein-Zwahlen syndrome, is also
viewed as a nonspecific developmental field defect that is inherited as an autosomal dominant condition. The
main characteristics of this disorder are malar hypoplasia with downslanting palpebral fissures, defects of the
lower lid, mandibular hypoplasia, and malformations of the external ear (228). Other abnormalities include partial
to total absence of the lower eyelashes, external ear canal defects, conductive deafness, cleft palate,
incompetent soft palate, and a projection of scalp hair onto the lateral cheek. Pharyngeal hypoplasia,
microphthalmia, macrostomia or microstomia, choanal atresia, blind fistulas and skin tags between the auricle
and the angle of the mouth, absence of the parotid gland, congenital heart defects, and cryptorchidism are
occasionally reported. Because the majority (over 60%) of cases arise de novo and expression is highly variable,
diagnosis and counseling can be challenging (60). Some 51 mutations in the TCOF1 gene, which encodes the
protein “treacle,” have been identified, and the Treacher Collins locus mapped to chromosome 5q31.3-32 (150).
Opitz-Frias Syndrome
A heterogenous condition, Opitz-Frias syndrome, also known as hypertelorism-hypospadias or GBBB syndrome,
was described and named using the initials of the surnames of the three families (183). Affected males usually
have ocular hypertelorism and hypospadias, but affected females have only hypertelorism. Cardiac anomalies,
cleft lip or palate, cranial asymmetry, strabismus, and downslanting palpebral fissures may be present. Because
of the overlap between G and BBB syndrome manifestations, some investigators have suggested that they are
the same entity and should be called Opitz or Opitz-Frias syndrome. Neonatally, infants can be recognized by
their hypertelorism, hypospadias, and other anomalies, such as cleft lip or palate and congenital heart defects.
The syndrome is genetically heterogeneous, with both X-linked and autosomal dominant forms recognized. The
former maps to Xp22 and is designated type I; the latter maps to 22ql 1 and is designated type II. Mutations in the
MID1 gene have been demonstrated in X-linked cases. Patients with the two forms are not easily differentiated
by phenotypic means.
ACROCEPHALOSYNDACTYLY SYNDROMES
Acrocephalosyndactyly syndromes are caused by autosomal dominant mutations. The numeric designations of
these entities derive from earlier classifications and are more commonly known by their proper names. The
abnormalities that occur in these syndromes are listed in Table 4-9.
Apert Syndrome
Apert syndrome, or acrocephalosyndactyly type I (27), was formerly called acrocephalosyndactyly type II, Vogt
cephalodactyly, or Apert-Crouzon disease. The disorder is characterized by irregular craniosynostosis
(especially of coronal sutures), midface hypoplasia, syndactyly, and a broad distal phalanx of the thumb and
hallux. Patients may have mental retardation. Craniofacial anomalies include short anteroposterior skull diameter
with a high, full forehead and flat occiput, flat face, supraorbital horizontal groove, shallow orbits, ocular
hypertelorism, downslanting palpebral fissures, small nose, and maxillary hypoplasia. Cutaneous syndactyly of
all toes occurs with or without osseous syndactyly. Synostosis of the radius and humerus, pyloric stenosis,
ectopic anus, pulmonary aplasia, anomalous tracheal cartilages, pulmonary stenosis, cardiac malformations,
cystic kidneys, hydronephrosis, and bicornuate uterus may occur.
The condition is easily diagnosed at birth, although the possibility has been raised that infants with Apert
syndrome with Polydactyly, especially of the toes, represent a nosologically different entity. Two point mutations
in the fibroblast growth factor receptor 2 gene (FGFR2) are the cause of most cases, even though they differ
phenotypically (113). It has been suggested that changes in the composition of extracellular matrix could be
responsible for such variability (35). (See Chapter 27 for additional details.)
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Table 4-9 ▪ MAJOR FEATURES OFTHE ACROCEPHALOSYNDACTYLY SYNDROMES
Apert Syndrome (Type I) Saethre-Chotzen Pfeiffer Syndrome (Type V)

(Type III)
Irregular thumb and toe Brachycephaly, high Craniosynostosis of coronal and

Mental retardation or normal forehead sagittal sutures
intelligence Synostosis of coronal Cloverleaf skulla
Short anteroposterior skull sutures
diameter, with Maxillary hypoplasia Ocular hypertelorism
high forehead and flat occiput
Supraorbital horizontal groove Facial asymmetry Antimongoloid palpebral fissures

Flat face Shallow orbits Small nose
Shallow orbits Ocular hypertelorism Radiohumeral synostosisa
Ocular hypertelorism Small ears Broad distal phalanges of thumb and
Downslanted palpebral fissures Large fontanels great toe
Small nose Ptosis of eyelids Partial syndactyly of fingers and toes
Maxillary hypoplasia Cutaneous syndactyly
Cutaneous syndactyly of all toes, Single upper palmar
with or crease
without syndactyly
Synostosis of the radius and Broad thumbs, great

humerus toes
Pyloric stenosis Mental deficiencya
Ectopic anus
Small staturea
Pulmonary aplasia
Anomalous tracheal cartilages Deafnessa
Pulmonic stenosis, other cardiac Vertebral anomalya
malformations Cryptorchidisma
Cystic kidneys Renal anomaliesa
Hydronephrosis
Bicornuate uterus
aOccasional abnormality. Table adapted from Reference (77).
Pfeiffer Syndrome
Pfeiffer syndrome, also known as acrocephalosyndactyly type V, arises on an autosomal dominant basis, with
most cases representing new mutations. Mutations in FGFR1 have been identified (168), as well as FGFR2
(213). Craniosynostosis of coronal or sagittal sutures, ocular hypertelorism, downward slant of palpebral
fissures, small nose, broad distal phalanges of thumbs and big toes, partial syndactyly of fingers and toes, and
sometimes radiohumeral synostosis and cloverleaf skull (Kleeblattschadel) characterize this syndrome.
Craniofacial abnormalities tend to improve with age. Intelligence is usually normal, although severe secondary
brain defects occur with the Kleeblattschädel anomaly (see Chapter 27 for additional details).
Other Related Conditions

Crouzon Craniofacial Dysostosis
Inherited as an autosomal dominant trait, Crouzon craniofacial dysostosis occurs from a FGFR2 mutation (204). It
is a relatively common disorder that includes craniofacial anomalies with shallow orbits and ocular proptosis,
hypertelorism, frontal bossing, and maxillary hypoplasia with a curved parrotlike nose (61). Craniosynostosis may
involve coronal, lambdoid, and sagittal sutures. The teeth are peg-shaped and widely spaced, and associated
with a large tongue, deviated nasal septum, atretic auditory meatus, and deafness. Facial operations may be
required to correct extreme midface hypoplasia and proptosis.
Robinow Syndrome
Robinow syndrome, or “fetal face” syndrome, is heterogeneous, with both autosomal dominant and autosomal
recessive forms recognized. The gene for the recessive form is ROR2 and located on chromosome 9q22; the
relationship of this gene to the dominant form is unclear (189). Abnormalities include macrocephaly, large
anterior fontanelle, frontal bossing, hypertelorism, small upturned nose, small mouth, and micrognathia. The
latter findings resemble a fetal face (207). The forearms are short with brachydactyly; pectus excavatum, rib
anomalies, hemivertebrae, inguinal hernia, and cardiac anomalies may occur, along with a small penis and
cryptorchidism in boys and small clitoris and labia amajora in girls.
Stickler Syndrome
Stickler syndrome, or hereditary arthroophthalmopathy, is characterized by depressed nasal bridge, epicanthal
folds, midface hypoplasia, cleft of hard palate, micrognathia, deafness, and myopia complicated by frequent
retinal detachment or cataracts (239). Hypotonia, marfanoid habitus, prominence of large joints, and
spondyloepiphyseal dysplasia are also present in Stickler syndrome. Fifty percent of girls and 40% of boys have
mitral valve prolapse. The syndrome should be considered in every newborn infant with the Pierre Robin
sequence—in one study, one-third of patients with Robin sequence were diagnosed subsequently with Sticker
syndrome (261). Stickler syndrome is heterogenous and autosomal dominant with highly variable expression
(139).
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Two genes have been mapped, and two COL2A1 mutations identified. Only mutations in the COL2A1 lead to the
full syndrome with recognizable features.
Noonan Syndrome
Edema of the dorsum of the hands and feet in the newborn may simulate that seen in the infant with Ullrich-
Turner (45X) syndrome. Webbing of the neck, pectus excavatum, cryptorchidism, and pulmonic stenosis
characterize this syndrome (173). Short stature, epicanthal folds, ptosis of eyelids, ocular hypertelorism, myopia,
low-set or abnormal ears, anomalous vertebrae, and mental retardation are common. The condition is genetically
heterogeneous, with mutations in the gene PTPN11 identified in approximately 40% of patients (176). Somatic
PTPN11 mutations are also found in several childhood malignancies, including juvenile myelomonocytic, acute
myeloid, and acute lymphoblastic leukemias (246).
Brachmann-de Lange Syndrome

Cornelia de Lange, or Brachmann-de Lange syndrome (BDLS), has as major manifestations growth deficiency,
profound mental retardation, synophrys, hirsutism, and thin, downturned vermilion borders (Figure 4-9). Other
common anomalies are microcephaly, micrognathia, limb anomalies, dental abnormalities, such as late eruption
of widely spaced teeth, and male genital abnormalities, such as cryptorchidism and hypospadias (97). Less
common anomalies involve the eye (myopia, microcornea, astigmatism, optic atrophy, coloboma of the optic
nerve, strabismus, and proptosis), choanal atresia, low-set ears, cleft palate, congenital heart defects (most
commonly a ventricular septal defect), hiatus hernia, gastrointestinal anomalies (e.g., duplication of the gut,
malrotation of the colon, short esophagus, and pyloric stenosis), inguinal hernia, small labia majora, and absent
second to third interdigital triradius. BDLS, dup(3q), and FASs show some phenotypic overlap, but are
distinguishable (24, 275).
FIGURE 4-9▪ Brachmann-de Lange syndrome. This 30-week fetus was born spontaneously and lived several
hours, dying of respiratory failure secondary to congenital diaphragmatic hernia. A: Characteristic facies, with
hirsutism, synophrys, ocular hypertelorism, and elongated philtrum. B: Lateral view showing elongated
eyelashes, blunt nose, and small mandible. C: Severe reduction anomaly of right arm (absent ulna, third, fourth,
and fifth fingers), with pterygium at elbow. D: Syndactyly of second and third toes.
Both dominant and X-linked forms are recognized. Dominant cases are associated with NIPBL (130). To date,
about 50% of patients with BDLS or BDLS-like phenotypes have had heterozygous mutations in the NIPBL gene
(219); about one-half of cases of X-linked BDLS are estimated to occur from SMC1L1 mutations (170).
AUTOSOMAL RECESSIVE CONDITIONS

Meckel Syndrome
Meckel first described this syndrome in 1822, and in 1934, Gruber coined the term dysencephalia
splanchnocystica (181). Meckel, or Meckel-Gruber, syndrome is recessively inherited and generally leads to
death in the perinatal period or early infancy from respiratory or renal failure; prolonged survival to 28 months
has been reported (143). The sex ratio is equal, and incidence estimated at 1 in 13,250 to 1 in 140,000 live births
worldwide; regional incidences can be considerably higher, for example, 1 in 3,000 in Belgium and 1 in 9,000 in
Finland (216).
The classic diagnostic triad is occipital encephalocele, cystic kidneys, and Polydactyly (Figure 4-10). Cranial
rachischisis, Chiari malformation, hydrocephalus, polymicrogyria, ocular anomalies, cleft palate, congenital heart
defects, hypoplasia of the adrenal glands, pseudohermaphroditism in males, and other malformations may be
present. Excessively large, cystic, dysplastic kidneys cause marked abdominal distension. The cysts are
spherical, glomeruli are absent, and interstitial fibrosis is prominent. The cysts display an orderly, progressive
increment in size from capsule to calyx (10). Other genitourinary anomalies include agenesis, atresia, hypoplasia,
and duplication of ureters and absence or hypoplasia of the urinary bladder (see Chapter 17). Cysts of the liver
and pancreas are encountered; hepatic fibrosis and proliferation of bile ducts (i.e., ductal plate malformation) are
seen in portal tracts and the pancreas may exhibit fibrosis as well. Severe hypoplasia of male genitalia with
cryptorchidism, epididymal cysts, and ductal dilatation are common (202). Bilateral multicystic kidneys, fibrotic
changes in the liver, and occipital encephalocele or other CNS malformation have been offered as minimum
diagnostic criteria (7, 214).
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FIGURE 4-10▪ Meckel-Gruber syndrome. A: Occipital defect marks location of encephalocele, absent at autopsy
(secondary to autolysis) but identified by prenatal ultrasound. B: Microscopic view of cystic renal dysplasia,
associated with atretic ureter; large cysts are mostly collecting tubules; other changes include tubular loss and
peritubular and medullary fibrosis.
Maternal serum alpha-fetoprotein levels may be elevated due to the encephalocele. Prenatal diagnosis may also
be made by ultrasonography, often before the 11th to 12th week (7, 217). Clinical and genetic heterogeneity are
recognized, and three loci, MKS (or MKS1), MKS2, and MKS3, have been localized to 17q, 11q, and 8q
respectively. Mutations in a gene designated MKS1 have been identified at 17q (132) (see Chapter 10 for
details).
Smith-Lemli-Opitz Syndrome
This autosomal recessive disorder was the first true malformation complex to be associated with a metabolic
derangement and the first associated with abnormal synthesis of cholesterol. Deficient cholesterol levels result
from reduced activity of the final enzyme in the synthetic pathway, 7-dehydrocholesterol reductase (DHCR7). As
a result, plasma concentrations of intermediate products are elevated (e.g., 7-dehydrocholesterol).
A distinctive craniofacial appearance with microcephaly, anteverted nostrils, ptosis of eyelids, inner epicanthal
folds, strabismus, micrognathia, syndactyly of second and third toes, hypospadias, cryptorchidism, growth
retardation, and mental deficiency are the main characteristics of Smith-Lemli-Opitz syndrome (75, 233). Defects
in brain morphogenesis include micrencephaly, holoprosencephaly, hypoplasia of the frontal lobes, hypoplasia of
cerebellum and brain stem, dilated ventricles, and irregular gyral patterns and neuronal organization.
Less frequent anomalies are rudimentary postaxial hexadactyly, congenital heart defect, and defects of renal and
spinal cord development. Cystic renal disease, hypoplasia, hydronephrosis, and abnormalities of the ureters are
frequent. Rarely, severe perineoscrotal hypospadias may be seen. The reported higher frequency of boys
affected than girls may be related to a bias in ascertaining the genital anomaly. A number of mutations have been
identified in the delta-7-dehydrocholesterol reductase gene (DHCR 7), which is localized to 11q12-q13 (117,
278).
Leprechaunism
Individuals with leprechaunism, also known as Donohue or Donohue-Uchida syndrome, have a strikingly
characteristic (“elflike”) facial appearance with prominent ears, hirsutism, excessive skin folding with decreased
subcutaneous adipose, acanthosis nigricans, skeletal involvement (large hands and feet), enlarged genitalia,
andhyperinsulinemia (69). Intrauterine growth restriction, failure to thrive, and postnatal mental retardation are
recognized, and marked hyperplasia of pancreatic islet cells is apparent by microscopy. Leprechaunism is an
autosomal recessive congenital disorder of extreme insulin resistance. Some patients have had a limited
response to growth hormone administration, suggesting that other defects account for growth failure. Mutations
in the insulin receptor gene (INSK), located at 19p, are the cause of this condition. Prenatal diagnosis is thus
possible. Another condition with phenotypic similarity, termed leprechaunoid syndrome or pseudoleprochaunism,
is poorly understood (56).
Cockayne and Related Syndromes

Cockayne syndrome (CS) is an autosomal recessive disorder characterized by retarded growth and
development, short stature, premature aging, neurological impairment (e.g., ataxia, spasticity, dementia), hearing
loss, chorioretinitis, dental abnormalities, and photosensitivity (15, 44). It generally becomes manifest in early
infancy and leads to death from intercurrent infections or development of hypertension and atherosclerosis
before adulthood. CNS abnormalities include microcephaly, hydrocephalus, patchy irregular loss of myelin,
axons in a tigroid pattern, focal calcification (especially in basal ganglia), cerebellar atrophy, peripheral
neuropathy, bizarre astrocytosis, and oligodendroglial dysplasia (166).
Two forms of the disorder are recognized. CSA is the classic form described above; CSB is a more severe, early-
onset form that progresses rapidly, leading to death at 6 or 7 years. Pathogenesis is understood incompletely.
The growth of patient fibroblasts is decreased markedly following ultraviolet (UV) irradiation. However,
subsequent DNA synthesis is normal, demonstrating that the defects are not due to abnormal DNA excision
repair (as is the case in xeroderma pigmentosum). Cells do fail to recover RNA synthesis post irradiation (11).
Prenatal diagnosis can be made on the basis of sensitivity of amniocytes to UV light (136). Mutations in two
genes, the CSA gene (excision-repair cross-complementing gene, ERCC8, type A or I, located on 5q) and the
CSB gene (ERCC6, type B or II, on 10q) are most common, but others in the xeroderma pigmentosum genes
XPB, XPD, stadXPG are recognized (15, 137).
Other conditions bear some resemblance to CS and may in fact constitute a Cockayne spectrum. CAMFAK
syndrome (congenital cataracts, microcephaly, failure to thrive,
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kyphoscoliosis) is an autosomal recessive disease with central and peripheral demyelination that is similar to that
seen in CS (244). A less severe variety, without failure to thrive, is termed CAMAK (57). MICRO syndrome, also
autosomal recessive, is characterized by microcephaly, cataracts, and microcornea, should be distinguished from
Cockayne and CAMFAK syndromes, but also cerebro-oculo-facial-skeletal (COFS) syndrome (87). Cultured cells
from patients with COFS and Cockayne syndromes manifest hypersensitivity to UV light (in contrast to those with
CAMFAK, CAMAK, or MICRO syndromes), and mutations in CSB and XPD have been reported in patients
diagnosed with COFS (86).
Seckel Syndrome
Seckel syndrome, or “bird-headed” dwarfism, is inherited as an autosomal recessive trait. It is associated with
severe prenatal growth and mental deficiency with microcephaly and premature synostosis, hypoplasia of maxilla
with prominent nose, malformed ears, sparse hair, clinodactyly of fifth finger, hypoplasia of proximal radius,
dislocation of hip and hypoplasia of proximal fibula, 11 pairs of ribs, and cryptorchidism in boys (95, 221).
Malignant hypertension has been reported to cause rupture of a cerebral aneurysm in one patient (59). Loci for
three forms of the syndrome are recognized: SCKL1 (caused by mutations in the gene ATR, which maps to
3q22-24), SCKL2 at18p11-q11, and SCKL3 at 14q (126).
Dubowitz Syndrome
Dubowitz syndrome is an autosomal recessive, but possibly heterogeneous, disorder characterized by an
unusual facial appearance, infantile eczema, small stature, and mild microcephaly (62). Infants with this
syndrome are usually small for their gestational age and demonstrate retarded osseous maturation. The clinical
manifestations include mild mental deficiency, mild microcephaly, small face, shallow supraorbital ridges, ocular
hypertelorism, and micrognathia. In this regard facial characteristics may resemble those of FAS. Other
abnormalities include submucous cleft palate, pes planus, metatarsus adductus, hypospadias, cryptorchidism,
clinodactyly of the fifth finger, and pilonidal dimple (274). Multiple chromosome breakage and malignancy are
complications (5). A number of behavioral changes have been described including hyperactivity and shyness;
some patients like music, rhythm, and the vibrations produced by music; others dislike crowds (257). No gene
has been identified.
Orofaciodigital syndrome Type II (Mohr Syndrome)

The orofaciodigital syndrome type II is characterized by shortness of stature, conductive deafness, midline partial
cleft lip, midline cleft of the tongue, hypoplasia of the maxilla and mandible, relatively short hands, partial
duplication
of the hallux and first metatarsal, cuneiform and cuboid bones, and normal intelligence (256). The condition is
autosomal recessive.
Pena-Shokeir Phenotype
Pena-Shokeir Type I Sequence (Fetal Akinesia Deformation Sequence)
In 1974, Pena and Shokeir first described early lethal neurogenic arthrogryposis and pulmonary hypoplasia
(PenaShokeir I syndrome or fetal akinesia deformation). Facial abnormalities include prominent eyes,
hypertelorism, telecanthus, epicanthal folds, malformed ears, depressed tip of the nose, small mouth, high
arched palate, and micrognathia (194). Polyhydramnios, small placenta, and relatively short umbilical cord are
frequent findings. Infants are small for their gestational age; approximately 30% are stillborn. Most die from the
complications of pulmonary hypoplasia within the first few weeks.
The sequence has an estimated frequency of 1 in 12,000 births, with a heterozygote frequency of 1 in 55. The
phenotypic malformations appear to be nonspecific and caused by decreased or absent in utero movements,
resulting in the fetal akinesia deformation sequence. Genetic heterogeneity is recognized. One-half of the cases
are sporadic, and onehalf are familial and autosomal recessive or X-linked (193). Hall proposed the term Pena-
Shokeir “phenotype,” because the condition is not a specific syndrome but rather a physical change produced by
lack of movement in utero (89).
Polyhydramnios occurs due to failure of normal deglutition. Neuromuscular deficiency in the function of the
diaphragm and intercostal muscles causes pulmonary hypoplasia. Multiple ankyloses at elbows, knees, hips, and
ankles, rocker-bottom feet, talipes equinovarus, and camptodactyly are present. Absence of the flexion creases
on the fingers and palms, and sparse dermatoglyphic ridges are frequent. The phenotype may resemble that of
trisomy 18, from which it should be distinguished.
Neuropathologic findings include thin cerebral and cerebellar cortices, polymicrogyria, and multiple foci of
encephalomalacia, with loss of neurons and gliosis. The spinal cord is usually involved, with reduction in anterior
motor horn cells. Skeletal muscles show diffuse and group atrophy consistent with neurogenic atrophy.
Prenatal diagnosis may be possible with prior occurrence and a high index of suspicion. Pterygium formation is
one of the manifestations of the Pena-Shokeir phenotype. The lethal form of recessive multiple pterygium
syndrome may represent a severe form of the Pena-Shokeir phenotype (38). (see chapter 27 for additional
discussion).
Pena-Shokeir Type II Sequence (Cerebro-Oculo-Facio-Skeletal Syndrome)

COFS syndrome is recognized as an autosomal recessive disorder with degenerative brain and spinal cord
defects that are
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usually manifest at birth. Reduced white matter of the brain with mottling of the gray matter associated with
generalized hypotonia and hyporeflexia or areflexia are characteristic. The usual postnatal course is
characterized by progressive psychomotor deterioration and death before 5 years of age (200).
Robert Syndrome
Robert syndrome has been described under the names pseudothalidomide or SC syndrome, SC-phocomelia
syndrome, total phocomelia, hypomelia-hypotrichosis-facial hemangioma syndrome, and others (104). This
malformation syndrome includes as the most prominent characteristics nearly symmetric phocomelia-like limb
deficiency, often with radial defects, prenatal and postnatal growth retardation, microbrachycephaly, eye
abnormalities (i.e., shallow orbits, prominent globes, cloudy cornea), cleft lip with or without cleft palate, and
prominent premaxilla (Figure 4-11). The upper limbs may be affected more severely than the lower ones, the
latter sometimes being altered by absent or hypoplastic fibulae (249). Minor craniofacial abnormalities include
sparse, silver-blond hair, extensive hemangiomas, micrognathia, hypoplastic nasal cartilages, and malformed
ears with hypoplastic lobules (102). Nuchal cystic hygromas have been described (85). Autopsy studies have
shown cystic dysplastic kidneys, horseshoe kidney, and ureterostenosis with hydronephrosis. The condition is
inherited as an autosomal recessive trait. Infants are stillborn or die in early infancy. Premature centromere
separation with puffing and splitting and heterochromatin repulsion are diagnostic markers for this syndrome
(249).
FIGURE 4-11 ▪ Robert (pseudothalidomide) syndrome. A: Fetus with multiple limb malformations. B: Lateral
view. C: Agnathia and severely hypoplastic ear. D: Phocomelia and syndactyly of upper limb. E: Radiograph of
foot. F: Metaphase spread, showing prominent centromeres.
Familial Agnathia: Holoprosencephaly

Agnathia may occur alone or in association with other anomalies. The association with holoprosencephaly has
been described in siblings, suggesting autosomal recessive inheritance; other cases are due to unbalanced
translocations (131, 197) or appear to occur sporadically (191, 197). Associations of agnathia with situs inversus,
renal agenesis, ectopia cordis, rib, and vertebral anomalies have been reported (191), as have occurrences with
tetramelia (4) and anal atresia/situs inversus (158). Some of these associations raise fundamental questions
regarding embryogenetic control of the midline (18).
Thrombocytopenia and Absent Radius Syndrome

The thrombocytopenia absent radius (TAR) syndrome is inherited as an autosomal recessive trait; almost half of
patients die during early infancy. Limb defects include absence or hypoplasia of the radius, despite the presence
of thumbs. These defects are usually bilateral and occur with associated ulnar hypoplasia and defects of the
hands, legs, and feet. Other abnormalities include congenital heart
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defect, spina bifida, brachycephaly, strabismus, micrognathia, syndactyly, short humerus, and dislocation of the
hips. In one review of 34 patients, a host of additional anomalies were recognized: lower limb, renal, and cardiac
anomalies, capillary hemangiomas of the face, intracranial vascular malformations, sensorineural hearing loss,
and scoliosis (88). Mental retardation occurs in a small percentage (i.e., 7%) of patients. Prenatal diagnosis can
be suggested by ultrasonography when defects of the upper limbs are recognized. Thrombocytopenia with
absence or hypoplasia of megakaryocytes, leukemoid granulocytosis, eosinophilia, and anemia comprise the
major hematologic abnormalities. A pronounced intolerance to cow's milk is probably related to disturbances in
eosinophils. Leukemoid granulocytosis is present in over half of the patients, particularly during bleeding
episodes. The development of acute myeloid leukemia has been described in an adult patient (82). (see Chapter
27 for details).
Hydrolethalus Syndrome
Hydrolethalus syndrome is characterized by hydrocephalus, midline defects of the brain (e.g., absent or
hypoplastic corpus callosum, absent pituitary gland), micrognathia, limb anomalies including Polydactyly,
abnormal lobation of the lungs, microphthalmia, cleft lip or palate, small or absent tongue, wide or bifid nose, and
low-set, malformed ears. The occipital bone may be altered by a keyhole-shaped defect at the posterior margin
of the foramen magnum. Bilateral pulmonary agenesis and renal anomalies including unilateral agenesis and
hypoplasia or tubular cysts are associated manifestations (215). The syndrome is autosomal recessive and
tends to be lethal in the fetal or newborn period (227). It occurs with increased frequency in Finland, where the
gene has been mapped to 11q23.25 in a number of affected families (263).
HETEROGENOUS AUTOSOMAL DOMINANT AND RECESSIVE DYSPLASIAS

Osteochondrodysplasias and Other Skeletal Dysplasias
Spranger et al. (237) have identified three basic constitutional errors of bone development: dysostoses
(“malformations of single bones, alone or in combination”), disruptions (“secondary malformations of bones”),
and skeletal dysplasias (“developmental disorders of chondro-osseous tissue”). Pathologic diagnosis of these
conditions requires both radiographic and histologic techniques (277). The latter should involve samples of
affected bone and cartilage, and generally includes rib (to include costochondral junction), vertebral body, and
proximal and distal ends of major long bones (again to include osteochondral junctions).
Dysostoses may arise from defects in signaling factors, expressed only temporarily during development.
Examples include those described elsewhere in this chapter, that is, Holt-Oram and Smith-Lemli-Opitz
syndromes, as well as the brachydactylies, Greig polysyndactyly, and Pallister-Hall syndrome. Lesions may be
asymmetric, and in general do not lead to dwarfism unless the axial bones are involved.
Disruptions may arise from the action of teratogens or infectious agents. Thalidomide and warfarin
embryopathies are examples presented in this chapter.
Dysplasias comprise a larger group of disorders, and may be subcategorized as “primary dysplasias,” which
result from mutated genes that are expressed in cartilage or bone, or “secondary dysplasias,” abnormalities
arising from hormonal disease (e.g., hypothyroidism) or metabolic errors (e.g., hypophosphatasia). In these
conditions, effects are widespread and sufficiently severe to cause dwarfism.
The osteochondrodysplasias, in order of increasing lethality, include osteogenesis imperfecta, thanatophoric
dysplasia, achondrogenesis, and the short rib Polydactyly syndromes (Figure 4-12). Even combined, these
disorders are encountered infrequently, on the order of 16 per 100,000 births (237). However, because of the
often striking changes in bones, the conditions are rather easily recognized by prenatal ultrasound, undergo
therapeutic termination of pregnancy, and are seen with some frequency by fetal pathologists.
Chondrodysplasias
These are defects of collagen synthesis, principally type 2 collagen. The classification of chondrodysplasias has
been approached in somewhat different manners by various authors: early clinical manifestations (lethal versus
nonlethal), gross phenotype (short trunk versus normally proportioned trunk with platyspondyly versus short rib
Polydactyly), and predominant site of bone involvement (epiphysis, metaphysis, or spine) (237, 269). The
chondrodysplasias are associated with short stature; the major cause of death among lethal forms is pulmonary
hypoplasia, resulting from rib anomalies and reduced intrathoracic volume.
Those chondrodysplasias with predominant metaphyseal involvement of tubular bones and, in some cases, the
spine, also include many of the same disorders that cause death in utero or shortly after birth (236). The physis,
which is composed of resting and proliferating cartilage, enlarged chondrocytes, and calcified regions within the
zone of enchondral ossification, is the site of the major histologic abnormalities. Deficiency of chondroid matrix,
disorganization of chondrocytes, deviations in individual chondrocytic cytology, absence of proliferating
chondrocytes, degeneration of matrix, and absence or alteration of chondrocytic columnation are some of the
specific microscopic features that, in different combinations, represent the principal histopathologic findings
among the various types of short trunk and non-short trunk chondrodysplasias. Nodules of immature
mesenchymal tissue are interposed at the disorganized and attenuated physeal growth zone in thanatophoric
dysplasia. (see Chapters 12 and 27 for additional discussion).
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FIGURE 4-12 ▪ Skeletal dysplasia. A: Full-term infant with thanatophoric dwarfism type I. B: Radiograph of 24-
week male fetus with the same condition. Note short limbs, flat vertebral bodies, short ribs, and curved long
bones, especially humeri and femora. C: Excised “telephone-receiver” femur is characteristic of thanatophoric
dwarfism type I. D: 22-week male fetus with osteogenesis imperfecta, type 2. E: Radiograph of same fetus. Note
multiple telescoping fractures of long bones, multiple rib fractures, and poorly mineralized calvaria. F:
Microscopic section of femur, showing multiple compression fractures.
Other Osteochondrodysplasias
Except for some very general phenotypic similarities, the nonchondrodysplasias constitute a heterogeneous
group of conditions due to defects in collagen. Osteogenesis imperfecta represents a group of inherited
connective tissue disorders associated with fragile bones and a number of other nonosseous abnormalities of
connective tissues (148). Its prevalence is approximately 1 case per 100,000 births. The most severe form of
osteogenesis imperfecta is type II, which is typically lethal in the perinatal period.
Osteopetrosis is heterogenous and either an autosomal recessive or dominant condition that is characterized by
a generalized increase in bone density, especially affecting the pelvis and skull. A defect in osteoclast function
has been demonstrated, particularly in the “malignant” or autosomal recessive form, with death occurring in the
first decade of life. The histologic findings are diagnostic in most cases.
X-LINKED MUTATIONS
Lowe Syndrome
Hypotonia, congenital cataract, renal tubular dysfunction, and mental retardation manifest as Lowe syndrome or
oculocerebrorenal syndrome of Lowe (141). The disorder may represent an inborn error of inositol phosphate
metabolism, for such metabolism is abnormal in cultured cells (142). The renal tubular defect causes limited
ammonium production, hyperchloremic acidosis, phosphaturia, hypophosphatemia, generalized aminoaciduria,
albuminuria, osteoporosis, sometimes rickets, and organic aciduria (205). Protein trafficking between endosomes
and the trans-Golgi network is disrupted and may be responsible for some of the phenotypic changes (39).
Death is usually due to renal failure. Mutations in the Lowe syndrome gene OCRL1 (mapped to Xq24-26) are
recognized (142).
Menkes Syndrome
Menkes, or Menkes kinky hair, syndrome is distinguished by progressive cerebral deterioration with seizures,
twisted and fractured hair (pili torti), and systemic copper deficiency (53, 159). Affected infants have pudgy
cheeks and sparse, coarse, and lightly pigmented hair that, when magnified, shows pronounced twisting and
breakage. Hair changes are thought to be due to defective disulfide bonds in keratin (which are copper
dependent). Nervous system findings include reduced numbers of noradrenergic fibers in the forebrain and
peculiar torpedo-like swellings of catecholaminecontaining axons in peripheral nerve tracts, which may relate to
vascular disturbances, deterioration of the viscera, and eventual death (260). Skeletal changes in Menkes
syndrome include wormian bones, metaphyseal widening, particularly of ribs and femora, and lateral spurs.
Arteriograms show widespread arterial elongation and tortuosity due to reduced copper-dependent cross-linking
in the internal elastic membrane of vessel walls. The condition is X-linked recessive. The gene for Menkes
disease (MNK) codes for
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a copper-transporting ATPase that controls copper homeostasis in virtually every tissue except the liver (96).
Copper transporters are impaired, limiting copper uptake, primarily in the small intestine. First-trimester prenatal
diagnosis is possible with a DNA probe.
Lesch-Nyhan Syndrome
Lesch-Nyhan syndrome is caused by an X-linked recessive trait that produces a deficiency of the enzyme
involved in purine synthesis—hypoxanthine guanine phosphoribosyl transferase. Patients produce excessive
amounts of uric acid and suffer from profound mental retardation, characteristic self-mutilation, and motor
disability, the latter primarily a severe action dystonia overlying a baseline hypotonia (116). The gene has been
cloned and mapped to the long arm of the X chromosome at Xq26; a large number of mutations are recognized
(174).
Opitz-Kaveggia (FG) Syndrome

This syndrome is X-linked and associated with mental retardation, hypotonia, and anal malformation, sometimes
with constipation (58, 182). Other prominent findings are megalencephaly, midline fusion of the mammillary
bodies, heterotopia of cranial nerve nuclei, and pachygyria or other dysgenesis of the cerebral cortex (182).
Affected patients have lived up to 18 years of age (182). Five loci (FGS1-5) have been mapped to the ×
chromosome. × inactivation has been reported but not correlated with specific loci (203).
Pallister (W) Syndrome
Pallister and colleagues described two brothers with mental retardation and unusual appearances including
frontal prominence, anterior cowlick, hypertelorism, antimongoloid orbital slant, broad, flat nasal bridge, midline
notch of the upper lip, submucosal cleft of the hard palate, absent upper central incisors, elbow subluxation,
camptodactyly, and pes cavus (187). These children had grand mal seizures. The mother and a sister were
mildly afflicted, consistent with heterozygous manifestations of an X-linked trait. The sister is known to have had
an affected boy.
SPORADIC ABNORMALITIES
Klippel-Trenaunay-Weber Vascular Malformation
Features of this malformation complex include limb hypertrophy; hemangiomata that may be capillary; cavernous
phlebectasias; and varicosities (128, 188). The legs, buttocks, abdomen, and lower trunk are the usual sites of
vascular lesions. Less common abnormalities include arteriovenous fistulas, lymphangiomas, macrodactyly,
syndactyly, Polydactyly, hyperpigmented nevi, and telangiectasia. Craniofacial abnormalities include asymmetric
facial hypertrophy, hemangiomata, intracranial calcifications, and eye abnormalities. Visceromegaly and
hemangiomata of the intestinal tract, urinary system, and mesentery may be present. Mental deficiency and
seizures may occur with facial hemangiomatosis. A susceptibility gene, VG5Q (formerly AGGF1), encodes for an
angiogenic factor, that, when mutated, enhances angiogenic activity (253, 254).
Sturge-Weber Dysplasia
The association of hemangiomata in the facial skin, eyes, and meninges in this condition may be related to an
early defect in vascular morphogenesis. Aberrant vascular innervation and expression of vasoactive and
extracellular matrix molecules may play important roles in pathogenesis (46). Cutaneous hemangiomata may
occur in the trigeminal distribution, but this is not obligatory; meningeal hemangiomata may present in occipital
and temporal areas (6, 37). Progressive neurological deficits are complicated by seizures and may develop from
impaired cerebral perfusion (47). Pathologic findings include cerebral cortical atrophy, sclerosis, and
calcification.
Hallermann-Streiff Syndrome
Oculomandibulodyscephaly with hypotrichosis was first reported by Audry in 1893; Hallermann and Streiff
independently described three cases later (91, 241). The syndrome is rare, with only 150 cases reported, and
characterized by microphthalmia, a small, pinched, birdlike nose, and hypotrichosis (55, 72, 121, 164). Infants
with this syndrome have proportionately small stature, brachycephaly with frontal and parietal bossing, thin
calvaria, malar hypoplasia, micrognathia, and anterior displacement of the temporomandibular joint. Other facial
anomalies are microstomia and high, narrow, arched palate. Hair is sparse, and skin is thin and atrophic, most
prominently over the nose and suture areas of the scalp. Additional ocular manifestations include spherophakia,
blue sclerae, nystagmus, strabismus, colobomata, glaucoma, and various chorioretinal pigment alterations;
cataracts may resorb spontaneously. Nasal and mandibular anomalies may compromise respiration and feeding,
requiring rhinoplasty, facial augmentation, or mandibular surgery (55). Reported karyotypes have been normal,
and possible inheritance patterns remain unknown.
Hypomelanosis of Ito
Hypomelanosis of Ito (systematized achromic nevus or incontinentia pigmenti achromians) appears to be a
manifestation of mosaicism rather than a distinct entity, most likely involving a number of chromosomes, that
disrupts pigmentary genes (243). Thus, the condition has been termed “pigmentary mosaicism” and consists of a
triad of scattered, streaked, whorled, or mottled areas of cutaneous hypopigmentation that fluoresce, mental
deficiency, and severe intractable seizures present from birth (201, 220). Skin manifestations bear a
resemblance to those of incontinentia pigmenti and the ash
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leaf macule of tuberous sclerosis. Pathologic changes in the brain are variable and include cortical dysplasias,
heterotopias, and hamartomas (201, 220). Recognition of the cutaneous changes may alert clinicians to defects
in other organ systems (220).
Rubinstein-Taybi Syndrome
The Rubinstein-Taybi mental retardation syndrome (RTS) is a rare condition characterized by mental retardation
and a number of physical anomalies. RTS is characterized by broad thumbs and toes, bulbous fingers, slanted
palpebral fissures, and hypoplastic maxilla (212). Other abnormalities include short stature and small cranium,
mental retardation, beaked nose with nasal septum extending below nasal alae, epicanthal folds, strabismus,
low-set or malformed auricles, excess dermal ridge patterning in the thenar and first interdigital areas of the palm,
cryptorchidism, and cardiac defects (particularly ventricular septal defect and patent ductus arteriosus). Cataract,
colobomata, ptosis of eyelids, long eyelashes and hypertrichosis, polydactyly, simian crease, and renal
anomalies have been described (103). A large number of mutations in CBP, the gene encoding the cyclic AMP
response element binding protein (CREB), a coactivator important to gene transcription and cognitive functioning
(90). A second gene, EP300, has also been identified in patients (210).
ABNORMALITES OF UNKNOWN ORIGIN

Short-Cord Syndrome
The length of the normal umbilical cord varies, but averages about 60 cm (±13 cm) at term (171). Cord length is
static during the third trimester, presumably because the fetus is constrained during this period. This observation
supports the notion that cord length is determined in large part by fetal activity and the tension placed on the
cord during growth (162). Both short and long cords are associated with an increased risk of complications
during labor and delivery. The former may complicate delivery (129, 229), while the latter is more easily
obstructed. Short umbilical cords are associated with a variety of severe fetal anomalies. Factors that retard fetal
movement (e.g., intrauterine compression, uterine anomaly, amniotic bands, CNS or musculoskeletal anomaly,
other fetal malformation) are associated with a short umbilical cord. While some cases appear to be due to
reduced fetal motion, others are thought to develop as part of an early defect involving the body stalk or the cord
itself (22, 54, 259).
Nonimmune Hydrops Fetalis

Fetal hydrops is a generalized increase in fluid accumulation in serous cavities, causing edema of the soft
tissues in a fetus (Figure 4-13). At birth, the affected infant shows gross edema and may be difficult to
resuscitate because of pleural effusions, ascites, and associated lung hypoplasia. Fetal hydrops is divided into
immune and nonimmune types. The most common cause of immune hydrops was once Rh isoimmunization.
Since the advent of anti-D globulin (i.e., RhoGAM), most cases of fetal hydrops are nonimmune in nature (Table
4-10). The incidence of nonimmune hydrops is thought to be between 1 in 2,500 and 1 in 3,500 newborns. The
pathogenetic mechanisms leading to fetal hydrops are increased intracapillary hydrostatic pressure, decreased
intracapillary osmotic pressure, and damage to the peripheral capillary or vascular integrity. Machin has
extensively reviewed the differential diagnosis and pathogenesis of hydrops fetalis (146).
Chronic and severe anemia leading to hydrops may be caused by a variety of conditions, most commonly
homozygous alpha-thalassemia, twin-to-twin transfusion, chronic fetomaternal transfusion, or infection by
parvovirus B19. Severe, progressive anemia leads to congestive heart failure. Fetomaternal transfusion is
thought to occur in at least half of all pregnancies, but the quantity of transfused blood is usually small. A
Kleihauer-Betke acid elution test of the mother's blood is used to demonstrate the presence of fetal erythrocytes.
The presence of fetal cells indicates bleeding from the fetal circulation into maternal circulation; by knowing the
percentage of fetal cells in the maternal circulation, one can estimate the amount of blood lost by the fetus. If this
quantity reaches significant proportions, it can be a cause of
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fetal death. In twin-to-twin transfusion, nonimmune hydrops may be seen in the donor twin secondary to anemia
(where it causes congestive heart failure), but more often the condition occurs in the recipient twin, secondary to
volume overload.
FIGURE 4-13 ▪ Nonimmune hydrops fetalis. This 25-week female fetus suffered intrauterine fetal demise. The
heart was enlarged and showed biventricular endocardial fibroelastosis. The cause of fetal hydrops was not
ascertained but may have been related to maternal antiphospholipid antibody syndrome.
Table 4-10 ▪ CONDITIONS ASSOCIATED WITH NONIMMUNE HYDROPS FETALIS

CAUSAL CONDITIONS
Fetal conditions
Severe chronic anemia in utero
Fetomaternal transfusion
Twin-to-twin transfusion
Homozygous thalassemia
Acardius
Atrioventricular shunts
Hemorrhage or thrombosis
Maternal drugs (e.g., chloramphenicol)
Placental conditions
Chorionic vein thrombosis
Umbilical vein thrombosis
Angiomyxoma of umbilical cord
Aneurysm of umbilical cord
Chorioangioma of placenta
Maternal conditions
Maternal diabetes mellitus
Maternal nephritis
EFFECTS ON BODY SYSTEMS
Cardiovascular system
Severe congenital heart disease
Large arteriovenous malformation

Premature closure of foramen ovale
Hypoplastic left heart
Hypoplastic right heart
Cardiopulmonary hypoplasia with bilateral hydrothorax
Premature closure of ductus arteriosus, with pulmonary hypoplasia
Fetal arrhythmias
Myocarditis
Cardiac tumors (rhabdomyomas)
Pulmonary system
Congenital cystic adenomatoid malformation of the lung
Pulmonary hypoplasia
Pulmonary lymphangiectasia
Intrathoracic mass
Gastrointestinal system
Bowel atresia
Duplications of the gut
Peritonitis
Liver
Congenital hepatitis
Kidney
Congenital nephrosis
Renal vein thrombosis
ASSOCIATED CONDITIONS
Developmental or genetic disorders
Ullrich-Turner syndrome
Trisomy 18
Meckel syndrome
Pena-Shokeir syndrome
Noonan syndrome
Neu-Laxova syndrome
Multiple pterygium syndrome
Skeletal dysplasias (lethal congenital short limb dysplasias)
Multiple congenital abnormalities
Intrauterine infections
Syphilis
Toxoplasmosis
Cytomegalovirus
Coxsackie B virus pancarditis
Chagas disease
Leptospirosis
Lysosomal storage diseases
Mucopolysaccharidosis
Gaucher disease
Gangliosidosis
Sialidosis
Other conditions
Fetal neuroblastoma
Hemangioendothelioma
Tuberous sclerosis
Dysmaturity
Amniotic band syndrome
Fetal tumors (teratoma, neuroblastoma, angiomas)
From McGillivray and Hall (157), with permission
Cardiovascular causes of fetal hydrops include transient arrhythmias during pregnancy. When arrhythmias
persist, they lead to fluid accumulation and congestive failure in the fetus. Congenital heart block with
bradycardia should suggest a possible diagnosis of autoimmune disease in the mother (especially systemic
lupus erythematosus). A number of cardiovascular abnormalities can lead to intrauterine congestive heart failure,
including septal defects, premature closure of the foramen ovale, premature closure of the ductus arteriosus,
agenesis of the ductus venosus, and hypoplastic left ventricle.
Respiratory causes of fetal hydrops include diaphragmatic hernia, pulmonary lymphangiectasis, and congenital
pulmonary airway malformation of the fetal lung. Within the context of mediastinal shift, obstruction of the
lymphatic duct and major blood vessels occurs, producing excess accumulation of fluid. Gastrointestinal atresia,
midgut volvulus, and duplication are thought to cause hydrops because of decreased intravascular colloid
osmotic pressure. Obstruction of the fetal urinary tract at the ureteropelvic junction or by posterior urethral valves
or renal abnormalities causing nephrosis may also be associated with fetal hydrops. In this latter case,
hypoalbuminemia develops, with subsequent cardiac failure and fluid accumulation.
Chromosomal defects may be associated with cystic hygroma and often with generalized hydrops fetalis. This is
commonly seen with Ullrich-Turner syndrome. Bieber syndrome manifests as a familial cystic hygroma simulating
an encephalocele. Incomplete formation of the lymphatic system delays drainage into the thoracic duct. The
cause of nuchal cysts and hydrops is not clear, but the condition is relatively common and its associated
conditions have been reported.
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Placental abnormalities, including chorioangioma, torsion of the cord, or umbilical vein thrombosis, may also be
the primary cause of fetal hydrops. Intrauterine infection, particularly the toxoplasmosis, rubella, CMV, and
herpes simplex (TORCH) syndrome, may also have associated hydrops. The likely mechanism for ascites and
more extreme fluid collections is usually the severe hepatic injury caused by the infection and consequent
hypoalbuminemia and portal hypertension.
The lethal chondrodysplasias may be associated with hydrops. Syndromes involving absent or abnormal fetal
movement, including syndromes exhibiting the Pena-Shokeir phenotype, may exhibit fetal hydrops. The
mechanism leading to fluid collection is unknown. Transient in utero hydrops early in the second trimester is
characteristic of the Noonan syndrome. Lysosomal storage diseases, including Gaucher disease, GM1
gangliosidosis, mucopolysaccharidoses, disorders of sialic acid, and others, have been described with fetal
hydrops (80). Hypoproteinemia and sinusoidal obstruction of the liver by Kupffer cells swollen with storage
material have been suggested as causes for the fluid accumulation.
ACKNOWLEDGMENT
The contributions of Drs. Enid Gilbert-Barness and John Opitz to the previous edition are gratefully
acknowledged.
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284.
Chapter 5
Inborn Errors of Metabolism
Carole A. Vogler
David S. Brink
Dorothy K. Grange
In the early 1900s, Garrod defined a group of four inherited disorders, each characterized by blocked metabolic pathways, as
inborn errors of metabolism (IEM) (98). Since that time, knowledge of the human genome and understanding of IEM have
increased dramatically. This, along with improved technology, has resulted in refinement in diagnosis and classification of IEM
(285, 302). Early diagnosis is increasingly important as treatments, such as dietary management and enzyme replacement,
become a reality for these disorders (303). Although individually rare, the collective incidence of IEM is approximately 1/1,500
persons (304).
Online genetic/metabolic disease databases such as OMIM (Online Mendelian Inheritance in Man http://www.ncbi.nlm.
nih.gov/entrez/query.fcgi?db=OMIM), MetaGene (http://www.metagene.de/index.html), Human Genetic Disease Data base
(http://life2.tau.ac.il/GeneDis/), Japan Metabolic Disease Database (http://www.jmdbase.jp/JmdBaseExt/Top. aspx), Genetests
(http://www.genetests.org/), Gene Reviews (www.genere-views.org) and NORD (http://www.rare diseases.org/) are ideal sources
for current information on IEM.
CLINICAL PRESENTATION OF INBORN ERRORS OF METABOLISM

Most metabolic diseases are autosomal recessive disorders, some are X-linked, and a few are inherited as dominant traits (102).
Heterozygotes are usually asymptomatic, while homozygous (autosomal) or hemizygous (X-linked) patients are symptomatic. IEM
may present at any age and in a variety of ways (Table 5-1). Symptoms may begin before birth (for example with hydrops fetalis or
fetal ascites), at birth, with sudden death in infancy, or with deterioration after a symptom-free interval (80, 255). Symptoms may
suggest sepsis and infection can lead to decompensation in IEM (80).
IEM can cause dysmorphism. Peroxisomal biogenesis disorders such as Zellweger syndrome, disorders of lipid metabolism such
as Smith-Lemli-Opitz and Conradi-Hunermann syndromes, desmosterolosis, mevalonic aciduria, and glutaric acidemia type II are
examples of metabolic dysplasias. Lysosomal storage diseases (LSD) can cause abnormal facies and skeletal abnormalities that
are present at birth (80, 259).
DIAGNOSIS OF INBORN ERRORS OF METABOLISM

Biochemical studies may allow definitive diagnosis: Evaluations of blood glucose, lactate, ammonia, and ketones
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may provide important information (80). Enzymes and metabolites including organic, fatty, and amino acids can be evaluated.
Molecular analysis is important for disorders in which a specific mutation is common or has been identified in a family.
Table 5-1 ▪ CLINICAL SYMPTOMS IN PATIENTS WITH INBORN ERRORS OF METABOLISM
Nonimmune hydrops fetalis, fetal ascites
Sudden unexpected death in infancy
Episodic illness
Failure to thrive
Loss of cognitive milestones or motor skills
Lethargy
Vomiting
Respiratory distress and tachypnea
Shock
Coma
Seizures
Macrocephaly or microcephaly
Hepatomegaly
Splenomegaly
Cardiomyopathy and arrhythmias
Acute parenchymal liver disease
Chronic liver disease/cirrhosis
Hypotonia or hypertonia
Exercise intolerance, cramps, fatigue, and rhabdomyolysis
Corneal clouding
Macular and retinal changes
Deafness
Skeletal abnormalities
Dysostosis multiplex
Coarse facial features
Macroglossia
Unusual odor
Burnt sugar—maple syrup urine disease
Mousy—phenylketonuria
Cabbage, fishy—tyrosinemia type 1
Sweaty feet—isovaleric acidemia and glutaric acidemia type II
Cat urine—multiple carboxylase deficiency

Morphology of tissues, including liver, muscle, skin, conjunctiva, or placenta, may show characteristic findings by light microscopy
(LM). Brain, lymph nodes, spleen, kidney, and heart may also show findings in IEM, but biopsies of these sites are not evaluated
as commonly. Transmission electron microscopy (EM) of skin, conjunctiva, rectal mucosa, liver, peripheral nerve, muscle, bone
marrow, or peripheral blood leukocytes is important in evaluation of some IEM. Particularly for LSD, ultrastructural study of these
sites is a sensitive screen that can provide valuable information about stored material (see LSD below) (10, 44, 83, 144, 248, 272,
301, 340).
Many IEM affect the liver; in some cases (cystinosis, metachromatic leukodystrophy, Fabry disease), morphologic alterations have
no apparent clinical consequence. In other disorders—such as tyrosinemia, Wilson disease, and GSD IV—progressive liver
disease is common (70, 94). A variety of histologic alterations—including hepatitis, steatosis, cirrhosis, cholestasis, ductopenia,
ductular proliferation, neoplasia, and storage—can be seen in IEM, and many IEM cause similar morphological alterations (169,
300).
We receive liver biopsies at the bedside on a Telfa-coated pad, and divide and fix the tissue for LM and EM. Placing the liver
biopsy on gauze or handling it with forceps with teeth causes artifacts. For LM, at least a 1-cm core of liver is fixed in 10% buffered
formalin (170, 300). There are many special stains useful for evaluating liver biopsies (254) (Table 5-2). Fixation in 95% alcohol
allows preservation of cystine crystals and glycogen. If indicated, tissue for biochemical or molecular analysis should be snap
frozen in liquid nitrogen and stored at -70°C in a metal-free container. With a 16-gauge needle, a 2-cm core yields approximately
45 mg of tissue, and in general, 20 mg of tissue is adequate for biochemical diagnosis of most IEM (171). Exceptions include
investigation of GSDs of unknown type or nonketotic hyperglycinemia, either of which may require 100 mg of tissue and an open
biopsy for diagnosis (172). For EM, several 1-mm3 samples of tissue are fixed at the bedside in 2.5% glutaraldehyde.
Transmission EM of liver and other tissues, such as skin or conjunctiva, may be useful in the evaluation of patients with a variety
of disorders, particularly the LSD (Tables 5-3 and 5-4) (300). Placenta alterations may suggest an LSD (Table 5-5) (37, 46, 107,
250, 253, 306, 327, 337).
Table 5-2 ▪ LIVER BIOPSY SPECIAL STAINS
Stain Material Highlighted
Periodic acid-Schiff (PAS) Glycoproteins, glycolipids, amylopectin, glycogen
PAS after diastase digestion Glycoproteins, glycolipids, amylopectin
Prussian blue (Perls') Hemosiderin
Fontana Lipomelanin, lipofuscin
Acid fast Lipofuscin
Colloidal iron Glycosaminoglycans
Reticulin Reticulin fibers
Masson trichrome Collagen and bile ducts
Hall Bilirubin
Oil Red O and Sudan black Neutral lipids, triglyceride, cholesterol phospholipid (frozen tissue)
Schultz modification Cholesterol of Lieberman-Burchard stain
Orcein Copper-associated protein

Victoria blue Copper-associated protein
Aldehyde fuchsin Copper-associated protein
Rubeanic acid Copper
Rhodanine Copper
Muscle biopsy is useful for evaluation of mitochondrial myopathies and LSD. Increased mitochondria, with “raggedred fibers,”
subsarcolemmal mitochondria collections, and structurally abnormal mitochondria, occur in mitochondrial myopathies. Increased
muscle fiber glycogen, sometimes in vacuoles, may be present in the glycogen storage diseases. Increased lipid occurs with
abnormal fatty acid metabolism and with mitochondrial dysfunction (85).
Newborn screening began in the 1960s with testing for phenylketonuria, and additional tests have been added since then.
Disorders evaluated in newborn screening have generally been those that have a treatment available and for which early detection
and therapy can prevent morbidity and mortality (Table 5-6). Congenital hypothyroidism, galactosemia, phenylketonuria, and
hemoglobinopathies are now screened for in all states in the United States, and congenital adrenal hyperplasia (21-hydroxylase
deficiency form) has been added in 46 states thus far. With the development of tandem mass spectrometry (MS/MS) for newborn
screening, it has become possible to detect many more metabolic disorders via a rapid-throughput methodology. As of April, 2007,
47 states in the United States have added expanded newborn screening using MS/MS. Information on each state's IEM screening
program is available through Genes-R-Us, http://genes-r-us.uthscsa.edu. The March of Dimes and the American College of
Medical Genetics have recommended that all newborns should be screened for 29 core conditions, with detection of an additional
22 secondary target conditions.
Table 5-3 ▪ TRANSMISSION EM IN DIAGNOSIS OF INBORN ERRORS OF METABOLISM
Lysosomal storage diseases (LSD)
Glycogen storage diseases (GSD)
Hereditary fructose intolerance
Peroxisomal disorders
Hyperammonemia/Urea cycle disorders
Mitochondriopathies
Wilson disease
Alpha-1-antitrypsin deficiency
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Table 5-4 ▪ ULTRASTRUCTURAL APPEARANCE OF LYSOSOMAL STORAGE IN SKIN AND CONJUNCTIVAL

BIOPSIES
Ultrastructural
Appearance
of
Predominant
Disorder Storage Stored Material Cells Affected
Disorders with ultrastructurally characteristic storage
Pompe disease Electron-dense Glycogen Lymphocytes,

glycogen endothelium, fibroblasts,
granules epithelium, nerves
Cholesterol ester storage disease, Cholesterol, Cholesterol ester Fibroblasts

Wolman disease lipids
Infantile ceroid lipofuscinosis Granular Saposin A, D Perithelial and endothelial

osmiophilic cells, smooth muscle,
sweat gland epithelial
cells, lymphocytes
Late infantile ceroid lipofuscinosis Curvilinear Mitochondrial subunit c of Perithelial and endothelial
ATPase synthase cells, smooth muscle,
sweat gland epithelial
cells, lymphocytes
Juvenile ceroid lipofuscinosis Curvilinear and Mitochondrial subunit c of Perithelial and endothelial
fingerprint ATPase synthase cells, smooth muscle,
profiles sweat gland epithelial
cells, lymphocytes
Cystinosis Rectangular, Cystine Fibroblasts

rhomboid,
polymorphic
crystals
Metachromatic leukodystrophy Tuffstone or Sulfatide, cholesterol, Peripheral nerve Schwann

herringbone phosphatides cells, histiocytes
Krabbe disease (globoid Crystals with Galactosylceramide Peripheral nerve Schwann

leukodystrophy) sharp corners cells
Fabry disease Pleomorphic Globotriaosylceramidecontaining Epithelium, fibroblasts,

leaflets, substrates endothelium, lymphocytes
lamellae,
tubular
structures
Disorders with ultrastructurally nonspecific storage
Mucopolysaccharidoses (MPS), Lucent or fine Oligosaccharides, Eccrine gland epithelium,

Mucolipidoses, fibrillogranular glycosaminoglycans endothelial cells (may be
Aspartylglycosaminuria, Alpha- spared in MPS),
mannosidosis, Beta- fibroblasts, lymphocytes,
mannosidosis, GM1 macrophages, pericytes,
Gangliosidosis, I-cell disease Schwann cells, smooth
muscle cells. Mucolipidosis
III affects primarily
fibroblasts with normal
lymphocytes.
Fucosidosis, GM1 and GM2 Fine lamellated Gangliosides and glycolipids Peripheral nerve Schwann
gangliosidosis, galactosialidosis, membranous cells, endothelial cells,
MPS, Mucolipidosis IV and cytoplasmic pericytes, smooth muscle
sialidosis bodies, zebra cells in Fabry disease. In
bodies Niemann Pick and ML IV
lymphocytes have storage
Table 5-5 ▪ PLACENTAL LYSOSOMAL STORAGE
Lysosomal Storage Disease Affected Cells Hydrops Fetalis
Sialidosis (Mucolipidosis type I) Syncytiotrophoblasts, Hofbauer and stromal cells yes
Mucolipidosis II (I-Cell Disease) Syncytiotrophoblasts, Hofbauer and stromal, X-cells yes
Mucolipidosis IV Hofbauer and stromal cells yes
Sialic acid storage disease Syncytiotrophoblasts, Hofbauer cells, endothelium, amniocytes yes
Galactosialidosis Syncytiotrophoblasts yes
MPS I (Hurler) Villous stromal cells yes
MPS III (Sanfilippo) Syncytiotrophoblasts, Hofbauer and stromal cells
MPS IVA (Morquio A) Villous stromal cells yes
MPS VII (Sly) Villous stromal cells yes
GM1 gangliosidosis Syncytiotrophoblasts, Hofbauer cells, yes
GM2 gangliosidosis (Tay Sachs) Syncytiotrophoblasts
Cholesterol ester storage disease Syncytiotrophoblasts
Wolman disease Syncytiotrophoblasts yes
GSD II Amniocytes, endothelial cells, stromal cells
GSD IV Amniocytes
Niemann-Pick A Syncytiotrophoblasts, Hofbauer cells, fibrocytes in cord yes
Gaucher disease type 2 Vessels yes
Fabry disease Endothelial cells, perithelial cells, vascular smooth muscle cells yes
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Table 5-6 ▪ DISORDERS COMMONLY SCREENED FOR IN NEWBORNS

Cystic fibrosis (immunoreactive trypsinogen (IRT), CFTR DNA analysis)
Hemoglobinopathies, including sickle cell disease, sickle/beta-thalassemia disease and sickle-C disease (hemoglobin
electrophoresis)
Congenital adrenal hyperplasia (17-OH-progesterone for 21-hydroxylase deficiency)
Congenital hypothyroidism (T4, TSH)
Galactosemia (total galactose level and/or galactose-1-phosphate, galactose-1-phosphate uridyl transferase activity)
Biotinase deficiency (biotinidase activity)
Amino acid disorders (MS/MS technology used)
Phenylketonuria
Maple syrup urine disease
Tyrosinemia I and II
Homocystinuria
Urea cycle defects (MS/MS technology used)
Citrullinemia (argininosuccinate synthase deficiency)
Argininosuccinic aciduria (argininosuccinate lyase deficiency)
Argininemia (arginase deficiency)
Organic acid disorders (MS/MS technology used)
Methylmalonic acidemia (mutase deficiency)
Methylmalonic acidemia due to cobalamin A or cobalamin
B defect
Propionic acidemia
Isovaleric acidemia
Glutaric acidemia type I
3-Methylcrotonyl CoA carboxylase deficiency (3-MCC)
HMG CoA lyase deficiency
Beta-ketothiolase deficiency
Multiple carboxylase deficiency
FAO defects (MS/MS technology used)
MCAD
SCAD
LCHAD/trifunctional protein deficiency
VLCAD
Glutaric acidemia type II (MADD)
CPT I deficiency
CPT II deficiency
Carnitine transporter deficiency
Carnitine acylcarnitine translocase deficiency
HMG = hydroxy-methyl-glutaryl; MCAD = medium chain acylcoenzyme deficiency; SCAD = short chain acyl-CoA
dehydrogenase deficiency; LCHAD = long chain 3-hydroxyacyl-CoA dehydrogenase deficiency; VLCAD = very long chain
acyl-CoA dehydrogenase deficiency; MADD = multiple acyl-CoA dehydrogenase deficiency; CPT = carnitine
palmitoyltransferase.
LYSOSOMAL STORAGE DISEASES

The LSD are a group of some 50 genetic disorders with a combined incidence of approximately 1/5,000 to 8,000 live births (375).
In the United States, 500 to 800 patients with LSD are born each year (354). As a group, LSD are among the most commonly
diagnosed metabolic disorders. Most LSD result from a mutation in a gene that encodes a single hydrolyzing lysosomal enzyme
(Table 5-7). A few are caused by mutations in genes coding for an activator or transport protein or for an enzyme required for
posttranslational processing of lysosomal enzymes (11, 357).
In LSD, the enzyme's substrates progressively accumulate in lysosomes in many tissues, resulting in progressive cellular and
organ dysfunction by virtue of disruption of cytoplasm, metabolic imbalance, or metabolite toxicity. Perturbation in complex cell
signaling mechanisms with secondary structural and biochemical changes may be central to the pathogenesis of LSD (339).
Phenotypic variability is common in LSD, and clinical features depend on what cell or organ is dependent on the deficient enzyme
for normal function. For most LSD patients, symptoms begin in the first months of life and are progressive. Symptoms can include
nonimmune hydrops fetalis, dysmorphic facies, dysostosis multiplex, organomegaly, psychomotor delay, and progressive loss of
developmental milestones (16, 41, 46, 252, 374).
Six to fifteen percent of cases of nonimmune hydrops fetalis are due to LSD (37, 227, 250, 253). The pathogenesis of hydrops
fetalis in LSD is debated. Storage in liver, spleen, and marrow may cause decreased hematopoiesis and/or hypoproteinemia.
Visceromegaly and myocardial damage by storage may cause decreased venous drainage and ascites. In hydrops due to LSD, the
placenta may be pale and bulky (327).
Classification of LSD is based on the character of the stored material. Individual disorders are defined by the deficient enzyme.
Most LSD are autosomal recessive traits. The exceptions—Hunter (MPS II), Fabry, and Danon diseases— are inherited as X-
linked traits.
Diagnosis of Lysosomal Storage Diseases

Diagnosis is established by a biochemical assay for the deficient enzyme. Leukocytes, fibroblasts, or amniocytes can be assessed
for enzyme levels, and the degree of enzyme deficiency impacts prognosis. These assays are generally based on catabolism of
simple water soluble substrates that incorporate colored or fluorescent groups (373).
Ultrastructural evaluation of tissue for morphological evidence of stored material may be used as a screening tool or if an unusual
or as yet unrecognized enzyme deficiency is suspected. Skin, conjunctiva, peripheral blood lymphocytes, liver, marrow, muscle,
rectal biopsy (including neurons), or placenta can provide confirmation of LSD. The morphological character of the stored material
and its distribution may suggest a specific diagnosis or a group of LSD. Ultrastructurally characteristic storage occurs in some of
the LSD, while others show less specific storage, with diagnosis resting on biochemical analysis (100) (Table 5-4). The presence
of storage may allow for rapid identification of LSD and may help to direct the diagnostic evaluation.
Methods for newborn screening for LSD are being developed. As therapeutic options for these disorders become a reality, early
diagnosis becomes more critical to ensure that treatment is instituted as soon as possible after birth. Enzyme replacement is an
established therapy for Type I Gaucher
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disease, Fabry disease, MPS I, II, VI, and Pompe disease. Stem cell transplantation and substrate inhibition have utility in some
clinical settings.
Table 5-7 ▪ LYSOSOMAL STORAGE DISEASES
Disease Deficient Protein
Gaucher disease Beta-glucocerebrosidase
Fabry disease (angiokeratoma corporis diffusum universale) Alpha-galactosidase A (ceramide trihexosidase)
Mucopolysaccharidosis
MPS I Hurler Alpha-L-iduronidase
MPS II Hunter Iduronate sulfatase
MPS III Sanfilippo A Heparan N-sulfatase (Sulfamidase)
MPS III Sanfilippo B N-acetyl-alpha-D-glucosaminidase
MPS III Sanfilippo C Acetyl-CoA: alpha-glucosaminidase N-acetyl

transferase
MPS III Sanfilippo D N-acetylglucosamine-6-sulfatase
MPS IV Morquio A Galactosamine-6-sulfatase
MPS IV Morquio B Beta-galactosidase
MPS VI Morateau Lamy N-acetylgalactosamine 4-sulfatase (arylsulfatase B)
MPS VII Sly Beta-glucuronidase
MPS IX Hyaluronidase
Neuronal Ceroid Lipofuscinoses (NCL)
Infantile (INCL, CLN1) Palmitoyl-protein thioesterase 1 (PPT 1), Cathepsin D

Late infantile (LICL, LINCL, CLN2) Tripeptidyl peptidase 1 (TPP1)
Juvenile (JNCL, CLN3) Battenin (a lysosomal transmembrane protein)
Adult (ANCL, CLN4) Unknown
Pompe disease (glycogen storage disease type II, GSD-II) Alpha-glucosidase (acid maltase)
Danon disease, X-linked vacuolar myopathy Lysosome-associated membrane protein-2 (LAMP-2)
Disorders of lysosomal enzyme phosphorylation and

localization
Mucolipidoses II (ML II, I-cell disease) N-acetylglucosamine 1-phosphotransferase

(phosphotransferase)
Mucolipidoses III (ML III, pseudo-Hurler polydystrophy) N-acetylglucosamine 1-phosphotransferase

(phosphotransferase)
Mucolipidosis type IV Mucolipidin 1
Disorders of Glycoprotein Degradation (Oligosaccharidoses/
Glycoproteinoses)
Alpha-Mannosidosis Alpha-mannosidase
Beta-Mannosidosis Beta-mannosidase
Fucosidosis Alpha-L-fucosidase
Sialidosis (formally ML I) Neuraminidase
Aspartylglycosaminuria Aspartylglucosaminidase
Gangliosidoses
GM1 gangliosidosis Beta galactosidase
GM2 gangliosidosis
GM2 Type 1, Tay-Sachs disease, B variant Hexosaminidase A
GM2 Type II, Sandhoff disease, O variant Hexosaminidase A and B
GM2 activator deficiency, AB variant GM2 activator protein
Niemann-Pick disease (Sphingolipidoses, sphingomyelin Sphingomyelinase
lipidosis, sphingomyelin-cholesterol lipidosis, NPD) type A,

B
Niemann-Pick disease type C, D NPC-1 or NPC-2 mutation, protein not identified
Metachromatic leukodystrophy (Sulfatide Lipidosis, MLD) Arylsulfatase A or Saposin
Wolman disease and cholesterol ester storage disease Acid lipase

(CESD)
Farber disease (disseminated lipogranulomatosis) Acid ceramidase
Krabbe disease (galactosylceramide lipidosis, globoid cell Galactocerebroside beta-galactosidase

leukodystrophy)
Cystsinosis Cystinosin
Gaucher Disease
Gaucher disease is the most common LSD and is classified as a sphingolipidosis. The three clinical types (Table 5-8) are allelic
disorders due to autosomal recessive mutations in the beta-glucocerebrosidase gene, leading to failure of cleavage of glucose
from ceramide. Glucocerebroside derived from glycolipids in white and red cell membranes accumulates in lysosomes mainly in
reticuloendothelial tissues. There are more than 150 allelic mutations that cause Gaucher disease.
Type 1 Gaucher disease is the most common LSD with an incidence of 1/855 among Ashkenazi Jewish individuals (228, 327).
Patients typically present with painless splenomegaly and pancytopenia (100). Type 2, the infantile, acute neuronopathic type (the
most severe form (228), is panethnic, and patients have virtually no detectable enzyme activity.
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Type 3, the juvenile (Norrbottnian) form, is clinically intermediate between types 1 and 2 (80, 311, 324, 327).
Table 5-8 ▪ CLINICAL FORMS OF GAUCHER DISEASE
Type Onset/Survival Hepatosplenomegaly CNS Storage/Pathology

Disease
1. Chronic Childhood to + to +++ None Limited to phagocytes,

nonneuronopathic adulthood hepatosplenomegaly, pancytopenia
(due to marrow storage,
hypersplenism), destructive
osteoporotic bone disease
2. Infantile, acute Infancy to <2 + +++ Neuronal loss, gliosis, perivascular

neuronopathic years cell storage, hydrops fetalis rarely
3. Juvenile, chronic Childhood to + to +++ + to ++ 3a: primarily neurologic disease 3b:

neuronopathic second to fourth marked visceral disease
(Norrbottnian) decade
Glucocerebrosides accumulate in phagocytic cells in the three types. Characteristic Gaucher cells are large, 20-to 100-mm
eosinophilic phagocytes with wrinkled or striated cytoplasm (Figure 5-1A to C) and are present in liver, marrow, spleen, nodes,
tonsils, thymus, Peyer patches, alveolar septa and airspaces, and Virchow-Robin space. Gaucher cells are capable of
erythrophagocytosis, are acid phosphatase positive, and label with antibody to CD68. The striations can be highlighted with
Masson trichrome, aldehyde fuscin, and PAS after diastase (228). By EM, lysosomal rod-shaped or tubular lipid bilayer stacks with
a diameter of up to 4 mm distend cytoplasm (Figure 5-1D,E).
The liver is enlarged in all three types, and storage accumulates in Kupffer cells, most prominent in zone 3, but hepatocytes are
not affected (69, 220). Fibrosis may progress to cirrhosis. The spleen is enlarged, weighing as much as 10 kg, and may be
uniformly pale or mottled due to storage accumulation. In marrow, infiltrating Gaucher cells lead to osteopenia, sclerosis, necrosis,
and pathologic fractures (80). Erlenmeyer flask deformity of the distal femur is considered diagnostic of Gaucher disease (100).
The brain has storage in cells in Virchow-Robin space but no neuronal storage, although neurons are progressively lost. It is
suspected that lipids that accumulate in brain phagocytes are toxic to neurons in patients with type 2 and 3 Gaucher disease. The
placenta may be involved with Gaucher cells in villous vessels (80).
Diagnosis is based on quantitating beta-glucocerebrosidase activity in leukocytes or fibroblasts or by DNA analysis. Enzyme
replacement therapy effectively treats the pancytopenia and hepatosplenomegaly in Type 1 patients, but the bone disease
responds slowly, if at all (39).
Fabry Disease (Angiokeratoma Corporis Diffusum Universale)

Fabry disease is due to alpha-galactosidase A (ceramide trihexosidase) deficiency leading to disordered glycosphingolipid
metabolism with accumulation of globotriaosylceramide (ceramide trihexoside, ceramide digalactoside, blood group B glycolipid)
containing substrates (56, 91). Fabry disease is X-linked, and 1:40,000 to 1:60,000 males are affected. Over 150 mutations have
been identified (56, 92). Clinical manifestations include extremity pain and paresthesias, skin and mucous membrane
angiokeratomas, and corneal opacities; renal impairment leads to end-stage renal disease by 20 to 40 years of age. Death is due
to renal failure, cardiac disease, or cerebrovascular disease. Female heterozygotes have an intermediate level of alpha-
galactosidase A; they may be asymptomatic or have corneal opacity, though rarely they are as severely affected as hemizygous
males (56, 80). Hemizygotes and heterozygotes with B or AB blood type are more severely affected due to the additional
accumulation of B-specific glycolipid (100).
Renal, cardiac, and cerebral dysfunction relates to endothelial storage. PAS-positive glycolipid and cholesterol accumulate in
lysosomes in endothelial cells, reticuloendothelial cells, and macrophages, and, by EM, osmiophilic lamellated leaflets and tubules
are seen in endothelial, perithelial, and smooth muscle cells (7, 56, 87, 263). Glomerular podocytes, endothelial, mesangial,
interstitial, and tubular epithelial cells all can contain storage. Podocyte storage causes cellular injury, followed by glomerular
capillary wall thickening, progressive mesangial matrix expansion, glomerulosclerosis, and, eventually, end-stage renal disease
(Figure 5-2A to C) (93). Some patients with Fabry disease develop hypertrophic obstructive cardiomyopathy. Liver lysosomal
storage is of little clinical significance. Kupffer cells have a tan appearance in H & E sections, and storage is birefringent and
crystalline in frozen sections stained with the Schultz method (219).
Diagnosis is based on identifying decreased alpha-galactosidase A in leukocytes or fibroblasts or by DNA analysis for the
mutation. Enzyme replacement therapy may have benefit in Fabry patients.
Neuronal Ceroid Lipofuscinoses (NCL, Batten Disease)

These progressive encephalopathies affect patients at any age causing seizures, blindness, psychomotor deterioration, and
premature death. The incidence is 1/10,000, and carrier frequency is approximately 1%. Collectively, the NCL are the most
common inherited progressive encephalopathies of childhood. The NCL have been divided into nine forms based on age of onset,
character of storage, and deficient enzyme.
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FIGURE 5-1 ▪ Gaucher disease. A: The liver of a patient with Gaucher disease has prominent Kupffer cells due to pale
eosinophilic expansion of the cytoplasm by lysosomal glucocerebroside storage material (H&E). B: Enlarged phagocytes in the
spleen have a “wrinkled tissue paper” appearance of their cytoplasm because of the glucocerebroside storage (H&E). C: Wright
stained bone marrow aspirate from a patient with Gaucher disease showing “Gaucher cells” with a “wrinkled tissue paper”
appearance of cytoplasm due to glucocerebroside storage (Wright). D: Ultrastructural appearance of Gaucher cell from the spleen,
obtained at autopsy, showing cytoplasmic storage in upper middle and lower middle of the image, to the left of the nucleus (Uranyl
acetate, lead citrate). E: Ultrastructurally, glucocerebroside storage material in Gaucher disease comprises rod-shaped or tubular
lipid bilayer stacks with diameter of up to 4 mm (Uranyl acetate, lead citrate).
FIGURE 5-2 ▪ Fabry disease. A: By LM, glomeruli in Fabry disease show mesangial expansion with prominence of palestaining
visceral epithelial cells (podocytes) (H&E). B: At higher magnification, podocyte cytoplasm is markedly expanded by PAS-positive
storage material (PAS). C: Ultrastructurally, visceral epithelial cell cytoplasm is expanded by osmiophilic, lamellated leaflets and
tubules, representing glycolipid and cholesterol storage (Uranyl acetate, lead citrate). (Images' courtesy of Helen Liapis, M.D.,
Washington University Department of Pathology & Immunology, St. Louis, Missouri.)
Table 5-9 ▪ THE MORE COMMON NEURONAL CEROID LIPOFUSCINOSES
Type Eponym Age at Predominant Storagea Protein Defect

Presentation
Infantile (INCL, Santavuori- 6-12 months Granular osmiophilic (GROD), saposin Palmitoyl-protein
CLN1) Haltia A, D thioesterase 1 (PPT 1),
Cathepsin D
Late infantile Jansky- 2-3 years Curvilinear, (in some cases also Tripeptidyl peptidase 1
(LICL, LINCL, Bielschowsky fingerprint), mitochondrial subunit c of (TPP1)
CLN2) ATP synthase
Juvenile (JNCL, Batten- 4-9 years Fingerprint, mitochondrial subunit c of Battenin (a lysosomal
CLN3) Speilmeyer- ATP synthase transmembrane
Vogt protein)
Adult (ANCL, Kufs, Parry 30 years Granular osmiophilic, finger print Unknown
CLN4) bodies,bmitochondrial subunit c of
ATP synthase
aThere is overlap in character of storage among
these disorders.
bStorage may be sparse outside the CNS.
The most common and best characterized are infantile, late infantile, juvenile, and adult NCL (Table 5-9). The infantile and the late
infantile forms have deficient lysosomal enzyme activity; other types have abnormal lysosomal membrane proteins (6, 108, 116,
322).
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Autofluorescent PAS-positive glycolipid accumulates in lysosomes in lymphocytes, cells in skin (particularly pericytes, endothelial,
smooth muscle, and sweat gland epithelial cells), conjunctiva, skeletal muscle, and rectal mucosal neurons (322). As many as 10%
to 20% of lymphocytes may have storage in late infantile NCL, but these cells are generally normal in adult NCL (Figure 5-3A)
(322). Although the stored lipopigment is ultrastructurally different in each NCL type, there is morphological overlap (Figure 5-3B to
H).
FIGURE 5-3 ▪ Neuronal ceroid lipofuscinosis. A: Enlarged cells in a lymph node of a patient with neuronal ceroid lipofuscinosis
have cytoplasmic glycolipid storage within a background of normal-appearing lymphocytes. A cluster of cells with prominent
eosinophilic cytoplasm is easily identified in the center of the field (left image). Epifluorescence of the same microscopic field
(right image) highlights the glycolipid storage, with a central cluster of storage cells as well as additional, scattered storage cells
(H&E, autofluorescence). B,C: In infantile neuronal ceroid lipofuscinosis, lysosomal storage is typified by osmiophilic granular
bodies. D,E: In late infantile neuronal ceroid lipofuscinosis, lysosomal storage is typified by osmiophilic curvilinear material.
FIGURE 5-3 ▪ continued) F,G: In juvenile neuronal ceroid lipofuscinosis, storage material is typified by osmiophilic “fingerprint
bodies.” H: Despite the association of granular bodies with infantile neuronal ceroid lipofuscinosis, curvilinear bodies with late
infantile neuronal ceroid lipofuscinosis, and fingerprint bodies with juvenile neuronal ceroid lipofuscinosis, there is overlap in the
morphologic appearance of storage material. In this image, the bulk of the storage material has the curvilinear appearance typical
of late infantile neuronal ceroid lipofuscinosis; however, some of the darker material approaches the morphology of fingerprint
bodies typical of juvenile neuronal ceroid lipofuscinosis (B-H Uranyl acetate, lead citrate).
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Cerebral and cerebellar atrophy with neuronal loss, apoptosis, and gliosis occurs in the central nervous system with neural and
extraneural lysosomal storage (116, 274). Neuronal storage and loss is severe in the CA2 sector of the hippocampus (109). The
heart has myocardial, valvular, and conduction system storage (116). Diagnosis can be made using DNA analysis.
Pompe Disease (Glycogen Storage Disease Type II, GSD-II)

A range of phenotypes occurs in GSD II patients, reflecting the variety of mutations in the alpha-glucosidase (acid maltase) gene,
residual enzyme activity, and tissue-specific isoenzymes (80) (Table 5-10). All are autosomal recessive. Patients have hypotonia
and cardiomegaly but differ in age of onset, extent of organ involvement, and rate of progression (84, 115, 222, 300). Infants with
classical Pompe disease die of cardiac or respiratory failure in the first several years of life. The “muscular variants” are generally
less severe (80).
Table 5-10 ▪ CLINICAL FORMS OF GLYCOGEN STORAGE DISEASE II
Type Symptoms
Infantile Hypotonia, cardiomegaly, macroglossia; Hepatomegaly is mild or absent; hypoglycemia and acidosis are
(Pompe uncommon; death from respiratory or cardiac failure in 1st years of life
disease)
Childhood, Onset after early infancy, predominant skeletal muscle involvement, usually without heart involvement,
juvenile, slowly progressive course, exercise intolerance, myalgia, weakness (in some cases rhabdomyolysis),
or impaired respiratory function; death usually from respiratory failure
muscular
Adult Slowly progressive proximal myopathy, respiratory insufficiency
PAS-positive, diastase-digestible glycogen lysosomal storage is generalized but most severe in the skeletal muscle, heart, liver,
and brain. Increased acid phosphatase activity indicates lysosomal distention and secondary elevation of other lysosomal enzymes
(Figure 5-4A). A vacuolar myopathy with disruption of cytoplasmic structure affects skeletal,
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cardiac, and smooth muscle (Figure 5-4B,C). Cardiac hypertrophy (Figure 5-4D) and endocardial fibroelastosis occur, and the
conduction system may be involved (40).
Glycogen is increased in Schwann cells, anterior horn cells, brain stem motor nuclei and spinal ganglia, myenteric plexus,
astrocytes, oligodendroglia, endothelial cells, and pericytes with relative sparing of cortical neurons (115). Hepatocytes are only
slightly enlarged with delicate glycogen-containing vacuoles. Liver lacks the mosaic pattern and nuclear glycogenation seen in
other GSD (Figure 5-4E) (80). In kidney, glycogen accumulates in epithelium of loops of Henle and collecting tubules (221, 300),
and the adrenal zona fasciculata has prominent storage (80).
FIGURE 5-4 ▪ Pompe disease (type II glycogen storage disease). A: Glycogen storage in Pompe disease is lysosomal (in
contrast to glycogen storage in the other types of glycogen storage disease). The distended lysosomes also contain abundant acid
phosphatase activity, which can be demonstrated histochemically, here in skeletal muscle by acid phosphatase staining (acid
phosphatase stain). B: Vacuolar myopathy, though not specific for Pompe disease, is nonetheless characteristic and often striking
in this LSD. The pale vacuoles in skeletal muscle fibers (representing glycogen storage) seen with H&E stain can be highlighted
by PAS stain (not shown) (H&E). C: Histologically, cardiac myocytes are enlarged due to sarcoplasmic expansion by pale, often
vacuolar material (H&E). D: Cardiac myocyte enlargement can lead to a hypertrophic gross appearance of myocardium, as seen in
this image of the left ventricle from an infant who died with Pompe disease.
FIGURE 5-4 ▪ (Continued) E: The histologic appearance of hepatocytes in Pompe disease is usually less striking than that of
skeletal muscle. Hepatocytes are slightly enlarged with somewhat rarefied, vacuolar cytoplasm. Note the absence of glycogenated
nuclei, which are typically not seen in the liver in Pompe disease but are observed in several other types of glycogen storage
disease (H&E). F,G: Though glycogen storage in Pompe disease (and morphologically indistinguishable Danon disease) is
lysosomal (in contrast to other glycogen storage diseases), ultrastructural analysis of skeletal muscle often shows both widespread
extra-lysosomal and lysosomal glycogen accumulation. H: Endomysial capillaries in skeletal muscle biopsy material as well as in
other tissues, such as skin and conjunctiva, typically reveal membranebound glycogen. I: Most cells in conjunctival biopsy show
lysosomal storage. In the left image, an axon contains a distended lysosome filled with glycogen granules. The right image shows
prominent membrane-bound glycogen storage within a myelinated axon (F-I Uranyl acetate, lead citrate). (Image I: Used from,
American Journal of Medical Genetics, with permission.)
Skin, conjunctiva, liver, muscle, lymphocytes, and placenta can show diagnostic lysosomal glycogen accumulation by EM (Figure
5-4F to I). Glycogen in muscle is both lysosomal and cytoplasmic. Diagnosis is confirmed by demonstrating absent enzyme in dried
blood spots, leukocytes, muscle, liver, or fibroblasts.
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Danon Disease, X-Linked Vacuolar Cardiomyopathy and Myopathy
Originally described as lysosomal glycogen storage disease with normal acid maltase (90), this disorder is characterized by
mental retardation, hypertrophic cardiomyopathy, skeletal myopathy, and death due to heart failure in the third decade (89, 275).
X-linked dominant mutation in the LAMP-2 gene encoding lysosome-associated membrane protein-2 leads to failure of fusion of
endosome and lysosome (13). Some children with hypertrophic cardiomyopathy (especially if skeletal myopathy is also present)
have been found to have LAMP-2 deficiency (377).
Muscle fibers have degeneration, size variation, and PASpositive vacuoles that contain glycogen and autophagic material; the
amount of vacuolization correlates with clinical disease. LAMP-2 is not identifiable immunohistochemically in leukocytes, skeletal
muscle, and myocardium in affected patients (84, 88, 376), and definitive diagnosis is based on DNA testing for the mutation (8).
FIGURE 5-5 ▪ Mucopolysaccharidosis. A,B: MPS patients have a characteristic facial appearance with coarse facial
appearance, thick doughy skin, coarse hair, flattened midface, wide nasal bridge, and macroglossia, here seen in two children who
died with MPS. C: The hands in MPS patients have joint stiffness and are held in a flexed position, a function of periarticular
altered connective tissue and altered bone formation.
FIGURE 5-5 ▪ continued) D: In mucopolysaccharidosis, stored GAGs (previously called mucopolysaccharides) have the
ultrastructural appearance of fine fibrillogranular material and clear membrane-bound vacuoles. Distinguishing different types of
mucopolysaccharidosis based on ultrastructural morphologic characteristics is not possible. E,F: The heart in patients with MPS
typically has thickened sclerotic valves, due to GAG storage in heart valve stromal cells and altered extracellular connective tissue
in the valve. Endocardial thickening is also frequent. G,H: The femoral head of a patient from MPS shows articular synechiae and
thick, poorly pliable periarticular connective tissue. These joint changes cause marked joint stiffness and make normal movement
impossible. The vertebral column from an MPS patient shows characteristic anterior inferior beaking of the lower thoracic and
upper lumbar areas caused by hypoplasia of the anterior superior aspect. This change results in the dorsal kyphosis or gibbus
deformation often seen in MPS patients and it is part of the widespread dysostosis multiplex.
FIGURE 5-5 ▪ (continued) I: Though storage material in neurons can resemble that seen in other organs, it can also take the form
of “zebra bodies,” as shown in this case of Hurler syndrome (D,I: Uranyl acetate, lead citrate). J: Note the presence of cortical
atrophy, loss of white matter and hydrocephalus.
Mucopolysaccharidoses
The mucopolysaccharidoses (MPSs) are systemic diseases due to deficiency of an enzyme needed for catabolism of
glycosaminoglycans (GAG) including dermatan, heparan, chondroitin, and keratan sulfate, with resultant storage of undegraded
GAG in lysosomes in a variety of cell types (279). The clinical course is variable; MPS patients may have progressive psychomotor
delay, coarse facial features, short stature, and bone and joint abnormalities (dysostosis multiplex), hepatosplenomegaly, corneal
clouding, macroglossia, and airway narrowing (Figure 5-5A to C) (Table 5-11). All MPS are autosomal recessive traits except X-
linked Hunter syndrome; Hurler (Type I) and Hunter (Type II) syndromes are the most common types. Scheie and Hurler-Scheie
syndromes are subtypes of MPS I with a milder disease. Some infants, particularly with MPS VII, present with hydrops (Table 5-5).
In MPS, many organs have lysosomal storage. Grossly, the liver is enlarged and firm. Vacuolization is more prominent in Kupffer
cells than hepatocytes. Fibrosis of Disse's
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space occurs late in disease; rarely, more severe fibrosis can develop in older patients (73, 188, 300). The stored GAG can be
highlighted with colloidal iron and Alcian blue stains and are digested by hyaluronidase. Adding 10% acetyltrimethylammonium
bromide to formalin may help preserve tissue GAG (300). By EM, visceral lysosomal storage is fine fibrillogranular material (Figure
5-5D). Vessel walls and heart valves are often affected with storage with resultant sclerosis (Figure 5-5E,F) and endocardial
fibroelastosis may occur (80). In bone, in most patients, storage in osteocytes and chondrocytes alters bone growth (Figure 5-
5G,H). Neurons store both GAG and gangliosides, with membranous cytoplasmic bodies, zebra bodies, and fibrillogranular storage
(Figure 5-5I). Neuronal loss and gliosis are seen in some patients, and meningeal storage may contribute to hydrocephalus (Figure
5-5J).
Table 5-11 ▪ MUCOPOLYSACCHARIDOSIS
MPS Eponym Enzyme Deficient Clinical and Pathology Findings
I Hurler, Alpha-L-iduronidase Corneal clouding, dysostosis multiplex, hepatosplenomegaly, cardiac

Scheie, valve sclerosis, mental retardation, premature death (Scheie has milder
Hurler- phenotype without mental retardation), rarely hydrops fetalis
Scheie
II Hunter Iduronate sulfatase Dysostosis multiplex, hepatosplenomegaly, cardiac valve sclerosis,

mental retardation, X-linked
IIIA Sanfilippo Heparan N- Mental retardation, mild somatic disease

A sulfatase
(sulfamidase)
IIIB Sanfilippo N-acetyl-alpha-D- Similar to IIIA

B glucosaminidase
IIIC Sanfilippo Acetyl-CoA:alpha- Similar to IIIA

C glucosaminidase N-
acetyl transferase
IIID Sanfilippo N- Similar to IIIA

D acetylglucosamine-
6-sulfatase
IVA Morquio A Galactosamine-6- Skeletal abnormalities, corneal clouding, odontoid hypoplasia, hydrops
sulfatase fetalis
IVB Morquio B Beta-galactosidase Similar to IVA
VI Maroteaux- N- Dysostosis multiplex, corneal clouding, normal intelligence

Lamy acetylgalactosamine
4-sulphatase
(arylsulfatase B)
VII Sly Beta-glucuronidase Dysostosis multiplex, hepatosplenomegaly, mental retardation, hydrops

fetalis
Diagnosis is suggested by increased urine GAG and the presence of vacuoles and metachromatic Adler-Reilly granules in
peripheral blood leukocytes. EM of skin, conjunctiva, buffy coat, or liver can show characteristic fibrillogranular lysosomal storage.
LM of thick sections of tissue prepared for EM are useful for identifying the multiple clear cytoplasmic vacuoles indicative of
lysosomal storage. Enzyme assay of serum, leukocytes, or fibroblast culture provides definitive diagnosis, and carrier testing using
DNA analysis is practical (80, 100).
Mucolipidoses
I-cell disease (ML II) and pseudo-Hurler polydystrophy (ML III) are autosomal recessive traits due to altered lysosomal
enzyme phosphorylation and localization (252).
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Both have disordered lysosomal enzyme targeting to lysosomes due to defective N-acetylglucosamine 1-phosphotransferase
activity in Golgi. In normal cells, lysosomal targeting of enzymes is mediated by receptors that bind mannose 6-phosphate
recognition markers on the enzymes. The recognition marker is synthesized in the Golgi, catalyzed by the phosphotransferase
(252). Phosphotransferase deficiency results in abnormal lysosomal enzyme transport with synthesized enzyme being secreted out
of the cell instead of being targeted to lysosomes. Elevated plasma lysosomal enzymes result.
Affected patients have features of both MPS and sphingolipidoses, hence the designation mucolipidoses. Clinical and radiologic
findings (coarse facial features, psychomotor retardation, failure to thrive, hepatomegaly, dysostosis multiplex) are similar to those
seen in MPS I, but earlier onset, a more rapid course, marked gingival hyperplasia, and absence of mucopolysacchariduria help
distinguish ML II and III clinically from MPS I (252).
The term I-cell disease was coined because cultured fibroblasts from affected patients contain dense inclusions (252, 300). PAS-
positive and Hale's-colloidal-iron-positive vacuoles are prominent in endothelial cells and fibroblasts and occur in lymphocytes,
Kupffer cells, glomerular visceral epithelial cells, satellite cells in muscle, myocardium, and pancreatic acinar cells (37, 300, 305).
Storage in stromal fibroblasts in heart is associated with valve thickening. Granulomas with finely vacuolated histocytes may occur
in lung and portal areas as well as portal tract fibroblasts. Hepatocytes are normal or only mildly altered and contain triglyceride
droplets (300).
The CNS may be normal morphologically, except for lamellar bodies in spinal ganglia neurons and anterior horn cells, or may have
cerebral cortical atrophy with neuronal loss (100, 252). Storage may be apparent in affected fetuses and their placentas (252). I-
cell disease can present as nonimmune hydrops (Table 5-5) (327). By EM, storage is electron lucent or fibrillogranular and
includes oligosaccharides, mucopolysaccharides, and lipids (252); EM of skin or conjunctiva can be used for diagnostic evaluation
(Figure 5-6). Increased serum lysosomal enzymes and decreased N-acetylglucosamine 1-phosphotransferase provide biochemical
confirmation (100).
ML III (pseudo-Hurler polydystrophy) symptoms are similar to ML II but milder with growth retardation, coarse facial features,
cardiac valve disease, dysostosis multiplex with stiff joints, and corneal clouding (80). ML II and III are distinguished on clinical
findings and progression of disease (252). The pathology of ML III is not as well documented as that of ML II patients (80, 100,
252). Storage is identified in skin fibroblasts, but lymphocytes are normal (82).
Mucolipidosis Type IV
Mucolipidosis type IV (ML IV, sialolipidosis, gangliosidesialidase deficiency) results from mutations in the gene MCOLN1, which
codes for the TRP family ion membrane channel, mucolipidin 1, a transient receptor potential protein important in endocytosis
(323). As a result, there is abnormal intracellular membrane trafficking (12, 104). This disorder is classified as a mucolipidosis
because of the storage of both lipids and mucopolysaccharides (36). Although panethnic, ML IV is more common among Ashkenazi
Jewish individuals (104, 111). Patients have severe psychomotor retardation, ophthalmologic abnormalities with corneal clouding,
retinal degeneration, and optic nerve atrophy, but they do not have dysostosis multiplex (80).
FIGURE 5-6 ▪ I-cell disease. In I-cell disease, fibroblast cytoplasm is expanded by numerous membrane-bound vacuoles
containing electronlucent to fibrillogranular material (Uranyl acetate, lead citrate).
Widespread storage affects brain and viscera including liver, pancreas, kidney, marrow, conjunctiva, cornea, skin, muscle,
peripheral nerve, rectum, and placenta (300). In neurons and glia, ganglioside, phospholipid, and GAG accumulation is variably
PAS-positive and Sudanophilic and is associated with neuronal loss and astrocytosis (100). By EM, lysosomes contain
heterogeneous material with fibrillogranular and concentric membranous bodies.
Hypergastrinemia and achlorhydria are described (264). Chronic atrophic gastritis and enterochromaffin-like cell hyperplasia are
seen along with cytoplasmic vacuolization of parietal cells due to lysosomal storage (104, 265). Confirmatory diagnosis of ML IV
should include screening for mutations in MCOLN1 (104).
Disorders of Glycoprotein Degradation (Oligosaccharidoses/Glycoproteinoses)

These autosomal recessive disorders are due to deficiency of a lysosomal enzyme that degrades glycoprotein oligosaccharide
side chains of glycoproteins (173, 300, 334). The phenotype resembles that of MPS, and tissues accumulate glycoproteins and
oligosaccharides (Table 5-12).
Alpha-mannosidosis. Affected patients have deficient alpha-mannosidase and increased plasma levels and excretion of small
mannose-rich oligosaccharides (334). Phenotype is variable (Table 5-12). Frequent infections may relate to a defect in leukocyte
chemotaxis (334), decreased serum IgG, or an impaired leukocyte membrane recognition
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process that results from defective catabolism of substrates with alpha-D-mannose residues. Hepatocytes have granular or foamy
cytoplasm, and Kupffer cells and hepatocytes contain reticulogranular, amorphous, or membranous storage by EM (300). In brain,
neurons have marked and widespread ballooning with membrane-bound vacuoles containing reticulogranular material (334).
Diagnosis can be based on ultrastructural morphology of skin, conjunctiva, or peripheral blood lymphocytes; demonstration of
oligosacchariuria; and measurement of tissue alpha-mannosidase activity (57, 334).
Table 5-12 ▪ DISORDERS OF GLYCOPROTEIN DEGRADATION

(OLIGOSACCHARIDOSES/GLYCOPROTEINOSES)
Disorder Enzyme Deficiency Clinical Findings
Alpha-Mannosidosis Alpha-mannosidase Mental retardation, coarse facies, dysostosis multiplex,

hepatosplenomegaly frequent infections
Beta-Mannosidosis Beta-mannosidase Angiokeratomas, mental retardation, hearing loss, respiratory

infections, seizures, quadriplegia, death in early childhood
Fucosidosis Alpha-L-fucosidases Mental retardation, hepatosplenomegaly, angiokeratomas, thick

skin, cardiomegaly, respiratory infections, dysostosis multiplex,
mental retardation, increased sweat sodium chloride
Sialidosis Neuraminidase Cherry-red spot-myoclonus syndrome, decreased visual acuity,

(Mucolipidosis I) corneal clouding, seizures, hyper-reflexia, ataxia, dysostosis
multiplex, hepatosplenomegaly, nephrotic syndrome, infantile form
may have hydrops fetalis
Aspartylglycosaminuria Aspartylglucosaminidase Onset by 1 year, hypotonia, coarse facies, thick calvarium,

osteoporosis, seizures, abnormal gray and white matter
differentiation, delayed myelination.
Beta-mannosidosis. This LSD, due to deficiency of betamannosidase, has a variable phenotype (Table 5-12). Cytoplasmic
vacuoles are described in skin and bone marrow in isolated patients, and the diagnosis rests on measurement of beta-
mannosidase in leukocytes or fibroblasts (334).
Fucosidosis. This LSD, due to deficient alpha-L-fucosidase, causes accumulation of fucoside moiety-containing glycolipids,
glycoproteins, and oligosaccharides (Table 5-12). Most patients are of Italian or Spanish descent or from the southwestern United
States (100, 334). Some patients have a rapidly progressive course with death in the first decade, while others have a milder
course with survival into the teen years and beyond. Angiokeratoma corporis diffusum occurs with fucosidosis and is essentially
identical in appearance and distribution to that seen in Fabry disease (334). Hepatocytes, Kupffer cells, and bile duct epithelial
cells are vacuolated. The CNS also may have lysosomal storage (334). Conjunctiva, muscle, skin sweat gland epithelium, and
peripheral blood lymphocytes all show granular lysosomal storage by EM (Figure 5-7). Diagnosis is based on demonstrating
deficient alpha-L-fucosidase in leukocytes and fibroblasts. Some clinically normal individuals have low alpha-L-fucosidase levels in
plasma (334).
Sialidosis. This LSD, previously called Mucolipidosis I, results from a recessively inherited deficiency of neuraminidase, an
enzyme that cleaves terminal sialic acid residues from oligosaccharides and glycoproteins. The deficiency results in lysosomal
accumulation of sialylated glycoproteins and oligosaccharides (Table 5-12) (80, 327). Patients may present with hydrops, facial
dysmorphism, psychomotor retardation, dysostosis multiplex, hepatosplenomegaly, and cardiomegaly (100). Hepatomegaly and
portal fibrosis may be present. Kupffer cells, endothelial and stellate cells, lymphocytes, glomerular visceral epithelial cells,
neurons in myenteric plexus and brain, fixed tissue macrophages in marrow and lung, biliary epithelium, chondrocytes, and
placental stromal and trophoblast cells have cytoplasmic vacuolization (Figure 5-8A,B) (80, 229). By EM, lysosomes contain
osmiophilic droplets and lamellar and fibrillogranular material (Figure 5-8C) (300, 334). Vacuolated lymphocytes and
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increased urine sialyloligosaccharides can suggest the need for enzymatic evaluation (80). Definitive diagnosis is based on
measurement of neuraminidase activity in cultivated fibroblasts or leukocytes. If tissue is used for analysis, it cannot have been
frozen or exposed to prolonged sonication (334).
FIGURE 5-7 ▪ Fucosidosis. Granular storage material distends the cytoplasm of this endomysial endothelial cell from a muscle
biopsy of a 2-yearold girl with fucosidosis (Uranyl acetate, lead citrate).
FIGURE 5-8 ▪ Sialidosis (mucolipidosis I). A: Foamy macrophages in lung tissue are present in this case of sialidosis (H&E). B:
Cells of the reticuloendothelial system in sialidosis are vacuolated, as seen in chorionic villi (left and middle images) and a
peripheral blood monocyte (right image) (H&E and Wright's). C: Ultrastructurally, storage material in sialidosis can manifest as
lamellar inclusions (left image) or fibrillogranular material (right image) (Uranyl acetate, lead citrate).
Aspartylglycosaminuria. This autosomal recessive glycoprotein degradation defect occurs predominantly in Finland (100) and is
due to lack of aspartylglucosaminidase, an enzyme important in liver and brain (Table 5-12). Enlarged lysosomes contain
aspartylglucosamine that appears as fibrillogranular storage in skin, conjunctiva, rectal mucosa, peripheral blood lymphocytes, and
viscera including liver (34, 118). Despite normal liver function, hepatocytes and Kupffer cells have abundant storage (34, 117, 297,
300). In the CNS, delayed myelination, white matter gliosis, and gray matter atrophy are seen; storage affects cortical and deep
gray matter neurons and is variably lucent, dense granular, or lipofuscin (35). Fetuses can have storage in liver, kidney, skin, and
placenta as early as 20 weeks' gestation (34). Diagnosis is based on enzyme assay or DNA molecular analysis (34).
Gangliosidoses
These autosomal recessive disorders all have lysosomal accumulation of glycosphingolipids (gangliosides).
GM1 gangliosidosis. Beta-galactosidase is deficient with accumulation of gangliosides in CNS, and galactosyl oligosaccharides
and keratan sulfate in viscera (5). Beta-galactosidase is also deficient in MPS IVB but presumably with some residual enzyme
activity allowing sparing of the CNS (80, 327, 328). Patients resemble those with MPS with coarse facies, dysostosis multiplex,
hepatosplenomegaly, rapid neurological deterioration, and seizures; infants may have hydrops fetalis, and the disease is fatal
generally by 2 years of age. A late infantile/juvenile GM1 gangliosidosis presents at 1 year of age, is clinically similar to the early-
onset form but with milder dysostosis, and leads to death by 5 years of age.
Sudanophilic gangliosides accumulate in CNS neurons with ballooning, neuronal loss, gliosis, and atrophy; by EM the storage
includes membranous cytoplasmic bodies. Peripheral nerve is also affected (80). PAS-positive GAG accumulation causes
vacuolization of Kupffer cells, hepatocytes, glomerular visceral epithelial cells and endothelial cells, placental syncytiotrophoblasts,
marrow histiocytes, lymphocytes and cells in spleen, nodes, thymus, lung, intestine, pancreas, pituitary, thyroid, salivary gland,
skin (including sweat glands), and conjunctiva (119, 180, 328). By EM, visceral storage is fibrillogranular. Definitive diagnosis rests
on demonstrating beta-galactosidase deficiency in leukocytes, fibroblasts, or amniocytes or on DNA analysis (80, 328).
GM2 gangliosidosis. These gangliosidoses are due to autosomal recessive defects in lysosomal hexosaminidase with resultant
accumulation of GM2 gangliosides mainly in neurons.
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GM2 Type 1, Tay-Sachs disease, B variant. This form of GM2 gangliosidosis is due to hexosaminidase A deficiency;
GM2containing gangliosides accumulate particularly in the CNS. The incidence is increased in Jewish populations (105, 179).
Psychomotor deterioration, seizures, blindness, and death by 3 to 5 years of age characterize most patients, although milder
juvenile and adult forms are recognized. The liver appears normal by LM, but, by EM, there is granular and zebra body storage
(178, 300). The brain is atrophic with neuronal loss and secondary gliosis; cholesterol, phospholipid, and GM2 ganglioside
accumulate as sudanophilic storage in essentially all neurons (80, 105). The stored material is PAS-positive in frozen but not in
paraffin sections (105). By EM, stored material in the CNS is concentrically lamellated, membranous, and granular (80). More
pleomorphic inclusions are present in glia. Diagnosis is based on hexosaminidase A (decreased) and B (normal) assay in
leukocytes or fibroblasts (4).
GM2 Type II, Sandhoff disease, O variant patients have no hexosaminidase A or B activity (hence “O” variant) and are clinically
indistinguishable from Tay-Sachs disease patients. The cerebral cortex is atrophic and yellowed by accumulated
asialoganglioside. PAS-positive sphingolipids and glycoprotein accumulate in liver, both in hepatocytes and Kupffer cells; in
histiocytes of the spleen, lymph nodes, and bone marrow; and in lymphocytes and pancreatic acinar cells (80, 105, 300). By EM,
storage is similar to that of Tay Sachs disease with prominent membranous cytoplasmic bodies in brain and heterogeneous
material in viscera (80, 105). Diagnosis can be determined by enzyme assay or DNA analysis (3, 105).
FIGURE 5-9 ▪ Gangliosidosis. A,B: Membranous cytoplasmic bodies in GM2 (AB variant) gangliosidosis are heterogeneous and
can show concentric or parallel structure, here in peripheral nerve axons. Though the morphology of the stored gangliosides is
often not helpful in distinguishing the gangliosidoses, location of the storage material can be helpful (Uranyl acetate, lead citrate.
(Image B used from, American Journal of Medical Genetics, with permission.)
GM2 activator protein deficiency, AB variant (indicating normal hexosaminidase A and B activity) is due to a mutation in the
GM2A gene. Patients cannot form a functional ganglioside GM2/GM2 activator complex to interact with hexosaminidase A and GM2
ganglioside to facilitate the hydrolysis of GM2 ganglioside (105, 353). Clinically, they resemble infantile Tay-Sachs and Sandhoff
diseases but with normal total hexosaminidase A and B levels. By LM, neuropathologic findings are identical to those of other GM2
gangliosidosis. Visceral organs are not involved. Zebra and membranous cytoplasmic bodies accumulate (Figure 5-9A,B), and
heterogeneous storage affects glial cells (105). Diagnosis is based on increased GM2 ganglioside in cerebrospinal fluid and
reduced activator protein level in fibroblasts (356).
Niemann-Pick Disease (sphingolipidoses, sphingomyelin lipidosis, sphingomyelin-cholesterol lipidosis, NPD). There are at least
6 types (A to F) of NPD; all are autosomal recessive. NPD A and B, both due to sphingomyelinase deficiency, have lysosomal
sphingomyelin, cholesterol, glycolipid, and acylglyceropyrophosphate. Residual sphingomyelinase level is less and the phenotype
more severe in Type A than Type B patients. Type A is the most common (85% of cases) and most severe, infantile,
neuronopathic form of NPD. Hydrops fetalis, failure to thrive, hepatosplenomegaly, hypotonia, and progressive neurological
deterioration end with death by 3 to 4 years of age (312). Ashkenazi Jewish populations have a higher incidence: 1:80 in this
population are carriers. Heterozygote detection, unreliable by enzyme assay, requires molecular studies (312). Type B is
phenotypically variable, more chronic, and nonneuronopathic; the disease presents
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in older infants with hepatosplenomegaly, and progressive pulmonary disease may become a major complication. This form does
not show an increased prevalence in Jewish patients (312).
The pathologic hallmark of NPD is the Niemann Pick (NP) cell (Figure 5-10A), though NP cells may be infrequent in the very young
child (133, 181). These 25 to 75 mm in diameter foamy lipid-laden histocytes have pale yellow or tan cytoplasmic pigmentation with
H&E stain, the result of lipofuscin, sphingomyelin, ganglioside, and cholesterol storage. The vacuoles are birefringent with
polarized light and stain with Sudan black B, oil red O (ORO), and Schultz reaction (312) but stain poorly with PAS and for acid
phosphatase (312). The blue green cytoplasm of histiocytes with storage stained with Wright-Giemsa stain led to the term sea-
blue histiocytes.
FIGURE 5-10 ▪ Niemann-Pick disease. A: Several vacuolated “Niemann-Pick” cells, the pathologic hallmark of types A and B
Niemann-Pick disease, are present and show “sea blue” coloration by Wright-Giemsa stain in this smear of a bone marrow
aspirate. Niemann-Pick cells are capable of erythrophagocytosis and emperipolesis. B: Enlarged, foamy Kupffer cells in Niemann-
Pick disease, as shown here, may be absent in very young children but become more prominent with time (H&E). C-F:
Ultrastructurally, storage material in Niemann-Pick disease is a heterogeneous mix of membranous lamellar material, concentrically
lamellated myelin-like material, and lipofuscin (C-F Uranyl acetate, lead citrate).
FIGURE 5-10 ▪ (continued).
Kupffer cells (Figure 5-10B) (and, in some cases, hepatocytes) have progressive increase in foamy cytoplasm, and portal fibrosis
and cholestasis are observed, but cirrhosis is rare (177). Infants with NPD A may have cholestasis, bile duct paucity,
pseudoglandular formation, and giant cell transformation with a neonatal hepatitis pattern (100, 300). The spleen may be as much
as ten times normal size with extensive infiltrate and replacement of red pulp by NP cells, some of which show
erythrophagocytosis. By EM, liver, spleen, lung, marrow, kidney, and lymph node storage is lipid with membranous lamellar or
concentrically laminated myelinlike and lipofuscin storage (Figure 5-10C to F). Brain is atrophic with neuron loss, gliosis, and
demyelination. Vacuolated neurons have sudanophilic, ORO-positive, and Luxol-fastblue-positive storage (80), and foam cells and
lipid-laden glia are in brain parenchyma and Virchow-Robin space.
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Diagnosis rests with identifying sphingomyelinase deficiency in leukocytes or fibroblasts. In families with a known molecular lesion,
heterozygote status can be determined by DNA analysis (312).
NPD C, a cholesterol esterification and intracellular trafficking defect, leads to lysosomal accumulation of sphingomyelin and
unesterified cholesterol and secondary reduction in sphingomyelinase activity (298, 312). NPD C and D are allelic and are due to
mutations in the NPC-1 and NPC-2 genes; NPD D is thus a variant phenotype of NPD C rather than a separate entity (298, 312).
NPD C is most commonly caused by mutations in the NPC-I gene. The protein product of NPC-1 is thought to facilitate the egress
of cholesterol and other lipids from the late endosomes and lysosomes to other cellular compartments. Protean manifestations can
begin any time from intrauterine life to adulthood. Patients may present with fetal ascites or with transient neonatal jaundice and
hepatitis. Hepatosplenomegaly may occur in some patients but usually regresses over time, and in general, is less severe than that
seen with NPD A or B. Neurologic disease is progressive with spasticity and seizures (80). NPD D occurs in Nova Scotian
Acadians with neurological disease beginning in childhood, generally later than in NPD C (80, 298, 312, 327).
Neurovisceral storage is prominent with vacuolated cells in viscera and storage in neurons and glia (298). Vacuolated cells stain
with Luxol fast blue, PAS, and Sudan black B and are positive for cholesterol with the Schultz reaction and for acid phosphatase.
EM identifies membrane-bound whorled and dense osmiophilic lysosomal storage in skin and conjunctival cells, endothelial and
perithelial cells, keratinocytes, retinal ganglion cells, retinal pigment epithelium, Schwann cells, smooth muscle cells, and
fibroblasts (298).
NPD C and D may cause a neonatal hepatitis-like histology with giant cell transformation, fibrosis, or cirrhosis (80, 176). The
pathogenesis of this injury is unknown (175). Storage in liver is inconspicuous and easily overlooked, particularly in the setting of
hepatitis. With time, whorled and irregular lamellar inclusions, clefts, and lipid storage accumulate in macrophages and Kupffer
cells and to a lesser extent in hepatocytes (174). Neuronal storage occurs throughout the nervous system with neurofibrillary
tangles, meganeurites, and axonal spheroids (298). Cerebral atrophy is generally severe, and neuronal loss may occur by
apoptosis (298). A screening test involves staining cultivated cells with filipin to detect free cholesterol (359). Diagnosis is based
on measurement of cholesterol esterification in fibroblasts during LDL uptake (15) and molecular analysis of the NPC-1 or NPC-2
genes.
Metachromatic Leukodystrophy (Sulfatide Lipidosis, MLD)

Autosomal recessive deficiency of arylsulfatatse A, which hydrolyzes galactocerebroside sulfate to galactocerebroside, leads to
accumulation of sulfated glycolipids primarily in the CNS but also in extraneural sites. There are several clinical forms with infantile,
juvenile, and adult types recognized; multiple mutations have been described, and patients have a variable course with
progressive neurological disease. The central and peripheral nervous system have demyelination, and the cerebellum is atrophic
with Purkinje and granule cell loss. Accumulation of 15 to 20 mm in diameter spherical masses of metachromatic material occurs in
oligodendrocytes and macrophages in Virchow-Robin space and Schwann cells. This material comprises sulfatide, cholesterol,
and phosphatides, and in frozen sections it stains positive with PAS, Alcian blue, and colloidal iron, is brown metachromatic (with
1% cresyl violet at low pH), and stains purple with toluidine blue (341). By EM, storage in oligodendrocytes, astrocytes, Schwann
cells, and endoneurial cells in peripheral nerve is closely packed, lamellar, amorphous, or prismatic material with alternately
leaflets and tubules giving it a “herringbone” or “tuffstone” pattern (Figure 5-11A,B) (80).
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FIGURE 5-11 ▪ Metachromatic leukodystrophy. A: This unmyelinated nerve from a conjunctival biopsy contains an inclusion of
variable electron density. In some foci (arrow), closely approximated osmiophilic lamellae contribute to a subtle herringbone
pattern. B: Myelinated nerve with pleomorphic lysosomes of variable density, “tuffstone” inclusions from a sural nerve of a patient
with metachromatic leukodystrophy. (A, B: Uranyl acetate, lead citrate, A: Used from American Journal of Medical Genetics, with
permission.)
The gallbladder may be small and fibrotic with multiple mucosal papillomas and radiolucent choleliths; lamina propria
macrophages, gall bladder epithelial cells, and intrahepatic bile ducts have storage. However, patients only rarely present with
cholecystitis or pancreatitis (341). Liver macrophages, Kupffer cells, hepatocytes, and renal tubular epithelial cells also contain
metachromatic storage (58, 182).
Diagnosis is based on measuring arylsulfatase A activity. However, a low level does not prove MLD nor does a normal level
exclude the diagnosis (341, 355). A deficiency of the sphingolipid activator protein saposin B (80) can result in a normal or
heterozygous range arylsulfatase A level in an affected patient. Pseudo-arylsulfatase A deficiency occurs when an abnormal allele
that encodes only 5% to 15% of residual activity leads to low arylsulfatase A activity in a person who does not have MLD (341).
Excessive urine sulfatides can confirm the diagnosis of MLD (358); a sulfatideloading test allows distinction between patients
homozygous for the pseudodeficiency allele and MLD patients (341).
Wolman Disease and Cholesterol Ester Storage Disease (CESD)

These autosomal recessive phenotypic variants, due to absence or reduction in acid lipase, have accumulation of cholesterol
esters and triglyceride. Wolman disease patients have complete deficiency of acid lipase. Death often occurs in infancy, preceded
by hydrops, steatorrhea, hepatosplenomegaly, jaundice, abnormal neurological development, and failure to thrive. The enlarged
liver is a distinctive bright orange-yellow with a greasy consistency. Bile duct proliferation and cholestasis are described, and
periportal fibrosis with portal bridging may progress to cirrhosis (183). In viscera— including liver, spleen, adrenal, lymph nodes,
lymphocytes, marrow, and intestine—cholesterol esters and triglycerides accumulate as cholesterol crystals in foamy histiocytes
(Figure 5-12A to C) (184). This storage can be identified by viewing sections of unfixed frozen tissue with polarized light (Figure 5-
12D). Cholesterol and triglycerides in these cells can also be highlighted histochemically with the Schultz modification of the
Lieberman-Burchard reaction (59, 300). EM shows lipid droplets and membrane-bound angular cholesterol clefts in hepatocytes,
Kupffer cells, fibroblasts, and macrophages (Figure 5-12E) (14, 185). The mucosa of the small intestine, particularly duodenum
and ileum, is velvety yellow (300) due to lamina propria storage. Adrenal glands are large, hard, and bright yellow, with dystrophic
calcification and necrosis of the inner fasciculata and residual fetal cortex (80, 300). Oligodendroglia, ganglia neurons of the CNS,
and Schwann cells of the peripheral nervous system contain lipid. Placental syncytiotrophoblasts may be affected. Demonstration
of acid lipase deficiency in tissue, cultivated fibroblasts, or leukocytes confirms the diagnosis (33).
Since CESD patients have 3% to 8% residual acid lipase activity, their phenotype is similar but more benign than that seen in
Wolman disease, and diagnosis may not be made until childhood or early adulthood (33). Hyperbetalipoproteinemia and premature
atherosclerosis may complicate CESD (60, 80). The liver morphology is indistinguishable from that of Wolman disease.
Hepatomegaly may be the sole clinical feature; cirrhosis is unusual, although periportal fibrosis may be present (186, 300). Storage
affects hepatocytes, bile duct epithelium, and endothelium (61). Unlike in Wolman disease, adrenals may not be calcified, but
lymphocytes and histiocytes in intestinal lamina propria and marrow contain storage (38, 80).
Farber Disease (Disseminated Lipogranulomatosis)

A rare autosomal recessive deficiency of acid ceramidase leads to accumulation of ceramide, which is formed from turnover
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of sphingolipids in lymph nodes, liver, kidney, and lung. Mucopolysaccharides and gangliosides also accumulate (80, 276).
Symptoms begin in infancy and include failure to thrive; vomiting; painful, progressively deformed joints; subcutaneous nodules,
particularly near joints; and laryngeal involvement with hoarseness and respiratory insufficiency (276). Clinically, histocytosis is
often in the differential diagnosis (80). Farber disease may present in utero with hydrops fetalis (187, 327).
FIGURE 5-12 ▪ Cholesterol ester storage disease. A: In cholesterol ester storage disease, there is widespread vacuolization of
hepatocytes (H&E). B,C: Widespread cytoplasmic lipid can be demonstrated in frozen section analysis of liver tissue (B) and the
lamina propria of gut (C) in cholesterol storage disease, here stained with oil red O. D: Hepatocellular cholesterol ester crystals
are birefringent in frozen sections when viewed with polarized light. E: This conjunctival macrophage does not show needle-
shaped clefts but does show many sharply demarcated electron-lucent vacuoles, some of which have peripheral osmiophilia,
characteristic of lipid following fixation. (E: Uranyl acetate, lead citrate, Used from American Journal of Medical Genetics, with
permission)
Lymph node, lung, larynx, spleen, liver, heart, subcutaneous, and periarticular nodular lipogranulomas contain PASpositive storage
in foam cells and multinucleated giant cells. Storage is also present in endothelial cells, pericytes, Schwann cells, hepatocytes,
renal tubular epithelium, and glomerular visceral epithelial cells. Brain and spinal cord neurons are distended with PAS-positive
ceramides and gangliosides (276). By EM, storage is membrane-bound, comma-shaped
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curvilinear tubular profiles, termed banana-bodies or Farber bodies, along with concentric lamellar, zebra-body, and fibrillogranular
material (62). Diagnosis is confirmed by demonstration of decreased acid ceramidase activity in leukocytes, fibroblasts, or
amniocytes (276).
Krabbe Disease (Galactosylceramide Lipidosis, Globoid Cell Leukodystrophy)

Autosomal recessive deficiency of galactocerebroside betagalactosidase activity results in rapidly progressive neurological
deterioration in affected infants (360). The pathology is limited largely to the nervous system (360): The brain has atrophy, myelin
loss, neuronal degeneration, and gliosis. Distinctive “globoid cells” derived from monocyte-macrophage marrow stem cells are
distended by PAS-positive and acid phosphatase-positive undigested psychosine (galactosylsphingosine), and galactosylceramide
and accumulate in white matter and perivascular spaces; gray matter is generally less affected (360). Psychosine accumulation
causes oligodendroglia destruction (80). By EM, storage comprises electron-dense, straight or curved, hollow tubular profiles in
longitudinal section with crystalloid profiles in cross section (Figure 5-13A,B) (360). Peripheral nerves have endoneural fibrosis,
demyelination, and infiltration of PAS-positive macrophages, similar to CNS globoid cells (360). Storage also occurs in sweat gland
epithelium (360). Diagnosis is based on identifying galactocerebroside beta-galactosidase deficiency in leukocytes, fibroblasts,
amniotic, or chorionic villous cells.
Cystinosis
In cystinosis, cystine accumulates because of defective transport of cystine out of lysosomes into the cytoplasm. This transport
defect is due to an autosomal recessively inherited deficiency of cystinosin, a lysosomal membrane protein (97, 228). Of the
several forms of cystinosis, the most severe, nephropathic cystinosis, presents in the 1st year of life with Fanconi syndrome,
rickets, photophobia, and short stature and can result in renal failure if untreated (80).
FIGURE 5-13 ▪ Krabbe disease. A,B: Electron-lucent, angulated, and needle-shaped inclusions in conjunctival myelinated nerve
Schwann cells, characteristic of Krabbe disease. (A,B: Uranyl acetate, lead citrate; B: Used from American Journal of Medical
Genetics, with permission.)
Rectangular, rhomboid, or polymorphic cystine crystals accumulate in lysosomes in most tissues, particularly in the fixed tissue
macrophage system in liver, marrow, kidney, liver, lung, pancreas, intestine, appendix, spleen, conjunctiva, cornea, retina, lymph
nodes, thyroid, thymus, muscle, brain, gingiva, and placenta (Figure 5-14) (9, 80, 97). The crystals are apparent in unfixed frozen
or alcohol-fixed tissue examined with polarized light (80), which gives them a brilliant silvery birefringence (300). Kidney is the most
severely affected organ, and cystine crystals may be present in interstitial, glomerular, and tubular cells (80). A “swan neck”
deformity with atrophy of proximal tubule segments adjacent to cystine-containing interstitial cells is seen early in the disease.
Progressive interstitial fibrosis and inflammation with tubular atrophy is associated with end-stage renal failure. Other organs also
are affected, particularly after renal transplantation. Hepatomegaly is not associated with significant liver dysfunction. Perivenular
Kupffer cells accumulate refractile crystals in clusters, and spaces left by crystals can be seen by EM. Pancreatic endocrine and
exocrine insufficiency is due to long-standing cystine accumulation. Skeletal muscle fiber atrophy, ring fibers, and cystine crystals
in endomysial cells lead to a myopathy (97). CNS involvement may cause nonobstructive hydrocephalus, demyelination, and cystic
necrosis with calcification and spongy change (97). Diagnosis is based on the presence of ophthalmologic demonstration of
cystine crystals; identification of cystine crystals in bone marrow, cornea, fixed tissue macrophages, or amniocytes; and
measurement of leukocyte or fibroblast cystine content (97).
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FIGURE 5-14 ▪ Cystinosis. Electron-lucent, pleomorphic, polygonal, and rectilinear cystine crystals (C) in dermal macrophage
from a 22-year-old with cystinosis. (Uranyl acetate, lead citrate; Usedfrom American Journal of Medical Genetics, with
permission.)
AMINOACIDOPATHIES
In these disorders, amino acid catabolism is blocked because of an enzyme deficiency with resultant accumulation of a specific
amino acid (80).
Phenylketonuria (PKU, hyperphenylalaninemia) is usually due to a mutation in the gene encoding for hepatic phenylalanine
hydroxylase (PAH), which converts phenylalanine to tyrosine. Both deficient PAH and exposure to dietary phenylalanine are
necessary for expression of the phenotype (313). The biochemical consequence is accumulation of phenylalanine and its
metabolites and a relative deficiency of tyrosine, which becomes an essential amino acid in PKU patients (112, 231). Clinical
features are the result of tyrosine deficiency and elevated phenylalanine (313). The main clinical effect is in the brain with
microcephaly, severe mental retardation, seizures, and progressive motor dysfunction. Affected patients have a mousy odor,
eczema, and light skin and hair due to deficiency of tyrosine, a precursor of melanin. A strictly reduced phenylalanine diet begun in
infancy can prevent severe neurological damage, although treated patients may have a lower IQ, neuropsychological or
neurological abnormalities, and abnormal cerebral white matter; adults who relax their diet may have motor or cognitive decline
(112). Some patients respond to treatment with BH4, the cofactor for PAH, with reduction of phenylalanine levels, allowing a less
restricted diet.
Pregnant women with PKU must keep phenylalanine concentrations low to prevent toxic embryopathy/fetopathy. Microcephaly,
callosal hypoplasia, mental retardation, growth restriction, and heart malformations (aortic coarctation with hypoplastic left heart
syndrome, tetralogy of Fallot, patent ductus arteriosus) are seen in heterozygous infants of PKU mothers with
hyperphenylalaninemia during pregnancy (313).
Some patients with hyperphenylalaninemia have a milder form of PKU with residual PAH activity; they may not require dietary
therapy or may only need general protein restriction. However, even women with mild PKU need to keep phenylalanine levels in a
safe range for the fetus during pregnancy.
The brain injury in untreated PKU patients is secondary to phenylalanine accumulation in blood (which increases brain
phenylalanine), combined with deficiency of other large neutral amino acids (especially tyrosine and methionine). This results in
abnormal brain protein synthesis, myelin turnover, and biogenic amine neurotransmission (112). Untreated patients have variable
white matter alterations with spongiosis, delayed myelination or demyelination, focal myelin pallor, or breakdown with deposition of
neutral fat, gliosis, and neuronal loss. Diagnosis is based on blood phenylalanine level. MS/MS has recently become the main
method of screening for PKU (80).
PKU variants are caused by deficiency of the PAH cofactor tetrahydrobiopterin (BH4), due to one of several defects in the
biosynthesis or recycling of BH4. These patients respond to oral BH4 treatment with normalization of serum phenylalanine. BH4 is
also a cofactor for tyrosine and tryptophan hydroxylases and nitric oxide synthase (112), and BH4 deficiency results in
neurotransmitter deficiencies, in addition to hyperphenylalaninemia. These patients also need treatment for their CNS dopamine
and serotonin deficiencies, with L-dopa and 5-hydroxytryptophan, respectively, as well as carbidopa, since BH4 does not
adequately cross the blood-brain barrier. Deficiencies of GTP cyclohydrolase I (GTPCH), 6-pyruvoyltetrahydropterin synthase
(PTPS), dihydropteridine reductase (DHPR), and pterin-4a-carbinolamine dehydratase (PCD) have been described. GTPCH and
PTPS are involved in synthesis of BH4 from GTP, and DHPR and PCD are involved in recycling BH4. Patients with DHPR
deficiency also need folinic acid supplementation. All newborns with hyperphenylalaninemia should be screened for these less
common disorders by testing for abnormal urine pterins and DHPR enzyme activity on a dried blood spot.
Tyrosinemia Type I (Hepatorenal Tyrosinemia, Congenital Tyrosinosis)

This autosomal recessive trait due to deficient or defective fumarylacetoacetate hydrolase (FAH), the last enzyme of tyrosine
degradation, is due to one of a number of mutations
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at 15q23-q25. Tyrosine degradation by FAH normally primarily occurs in hepatocytes and renal tubular epithelium (132, 273).
Tyrosinemia I has an incidence of approximately 1/100,000 with an increased prevalence in French Canadians. Symptoms vary,
but, in general, the earlier the presentation, the worse the prognosis (336). Patients may have failure to thrive; a distinctive boiled
cabbage or fishy odor; hepatomegaly, and acute liver failure, cirrhosis, renal Fanconi syndrome, rickets, proteinuria, and peripheral
neuropathy; hepatocellular carcinoma may develop as early as 15 months of age (145, 273).
Liver, kidney, and peripheral nerve damage result from tyrosine degradation products fumarylacetoacetate and maleylacetoacetate
that may act as alkylating agents, disrupt sulfhydryl metabolism, and inhibit transport function (146, 273). In the chronic form of
tyrosinemia I, symptoms develop later and are less severe but include mental retardation, rickets, and hepatocellular carcinoma.
Liver lesions can begin in utero. The liver is generally enlarged with sharply demarcated regenerative nodules with variegated
colors, ranging from yellow to deep green. Microscopically, zones of hepatocellular collapse, cirrhosis, steatosis, intracanalicular
and ductal cholestasis, cholangiolar proliferation, pseudoacinar transformation, and giant cell change are seen. Alpha fetoprotein is
demonstrable in hepatocytes in cirrhotic areas. Sinusoidal collagen deposition is often present and may be prominent (228).
Dysplasia, adenomas, and hepatocellular carcinoma can be seen (Figure 5-15) (80, 121, 228, 273, 300). Hepatocellular carcinoma
occurs in a third of patients who survive beyond 2 years of age, may occur as early as the 1st year of life, and may be
accompanied by normal or increased alpha fetoprotein level (100, 300). Transplantation by 2 years of age is recommended by
some to prevent carcinoma (147). Iron accumulates in hepatocytes and Kupffer cells and other organs including spleen, pancreas,
thyroid, and peritracheal mucous glands. Kidneys are enlarged, with cortical tubular ectasia, tubular calcification,
glomerulosclerosis, interstitial nephritis, and fibrosis. Half of affected patients have islet hyperplasia, but hypoglycemia is unusual.
Hypertrophic obstructive cardiomyopathy is also described (230). Axonal degeneration with demyelination similar to that seen in
porphyrias occurs, and a third of patients have white matter spongiosis (80, 228, 273, 300, 310).
Diagnosis is based on increased levels of succinylacetone in dried blood samples, plasma, or urine (148, 228, 273). FAH can be
assayed in lymphocytes, fibroblasts, or liver. Demonstration of two mutant alleles known to cause FAH deficiency confirms the
diagnosis.
Treatment includes phenylalanine and tyrosine dietary restriction and liver transplantation. Treatment with the herbicide 2-(2-nitro-
4-trifluoromethylbenzoyl)-1,3 cyclohexanedione (NTBC), which blocks tyrosine's degradative pathway and prevents accumulation
of maleylacetoacetate and fumarylacetoacetate, may improve liver and renal function and reduce mortality from liver failure. Its
impact on the risk of hepatocellular carcinoma is uncertain (80, 120, 149, 273).
FIGURE 5-15 ▪ Tyrosinemia. Hepatocellular carcinoma in liver from a young child with tyrosinemia.
Tyrosinemia Type II (Oculocutaneous Tyrosinemia, Richner-Hanhart Syndrome)

Oculocutaneous tyrosinemia is due to deficient cytoplasmic tyrosine aminotransferase (237, 273). Patients have palmoplantar
keratosis, corneal erosions, photophobia, and variable mental retardation but no liver dysfunction. Skin shows hyperkeratosis,
acanthosis, and parakeratosis (236). Conjunctival and skin biopsies may have large lipid-like inclusion bodies with filaments and
myelin-like figures in epithelium, fibrocytes, and endothelium (273).
Homocystinuria
Classical homocystinuria is due to cystathionine beta-synthetase (CBS) deficiency, inherited as an autosomal recessive disorder.
Affected patients have increased urine and serum homocysteine and methionine (235, 277). This multisystem disorder affects eye,
skeleton, liver, vessels, and CNS. Extra-CNS complications of CBS deficiency are secondary to accumulation of homocysteine
(277). CNS complications may be due to the metabolic defect as well as cerebrovascular disease (112). The risk of venous (and
less likely arterial) thromboembolism increases with age. Thromboemboli can occur in children, particularly with dehydration, and
can be multiple and recurrent (112, 234).
Ischemic lesions due to occlusive thromboemboli in veins, arteries, and the dural sinus can cause multifocal CNS infarction.
Approximately 50% of untreated patients die as young adults, often due to a thromboembolic event (233). Leukoencephalopathy
with focal perivascular demyelination may also occur. The liver shows zone 3 steatosis, mild to moderate periportal fibrosis, and
portal arteriole thickening with intimal hyperplasia. By EM, liver mitochondria are pleomorphic, and there are increased smooth
endoplasmic reticulum and pericanalicular lysosomes (112). Newborn screening by MS/MS for hypermethioninemia is useful in
identifying patients with CBS deficiency (232).
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Nonketotic Hyperglycinemia (NKH)
This is an autosomal recessive error of glycine degradation by an intramitochondrial enzyme complex. Patients have undetectable
or low glycine cleavage system activity (110, 112). Affected children have a broad range of phenotypes. Hypotonia, lethargy,
abnormal eye movements, mental retardation, seizures, and death in the first 6 months of life occur in more severely affected
patients (80, 112, 114). There is no ketosis or organic acid excretion, unlike with the hyperglycinemia that occurs in methylmalonic
aciduria or propionic acidemia (80). Glycine accumulates in all body fluids and all tissues, including brain; it is preferentially
elevated in the cerebrospinal fluid (112).
In the CNS, abnormal myelination, callosal agenesis or thinning, cerebellar hypoplasia, and gyral defects, related to abnormal
neuronal migration, and spongiform myelopathy (particularly of cerebellar white matter, corticospinal and optic tracts) are described
(110). These CNS abnormalities are thought to reflect brain amino acid imbalance (which interferes with myelin synthesis) or
increased spinal fluid glycine (which may impair neuronal function) (113). Liver may be steatotic, and skeletal muscle may have
intranuclear filamentous inclusions and abnormal mitochondria (2).
The diagnosis is suggested by a cerebrospinal fluid/plasma glycine concentration ratio of greater than 0.08 (55). Confirmation is
based on measurement of glycine cleavage system activity in liver (53). The normal hyperglycinuria in newborns makes
measurement of the urine glycine not useful for diagnosis (55).
Maple Syrup Urine Disease (MSUD, Branched-Chain Ketoaciduria)

This autosomal recessive disorder is due to a mutation in a gene encoding any subunit of the a-ketoacid dehydrogenase complex
(69). Branched-chain amino acids (leucine, isoleucine, and valine) accumulate in plasma. MSUD is the most common inborn error
of metabolism among Mennonites; in some communities, MSUD occurs in approximately 1 in 176 newborns. In non-Mennonites,
the incidence is 1 in 185,000 (20, 69). In the classical phenotype, neonates present in the 1st days of life with poor feeding,
alternating hyper-and hypotonia, ketoacidosis, seizures, and sudden unexpected death. Less severe, later-onset forms also occur
(55).
Sotolone imparts a maple syrup, burnt sugar, or curry odor to urine, sweat, and saliva (20, 29, 55). Brain abnormalities include
edema, astrocytosis, and delayed myelination without myelin destruction. Gray matter is unaffected. Loss of cerebellar granule cell
layer has been described (20, 55). The liver contains increased glycogen (43). Evaluation of plasma amino acids with MS/MS,
urine organic acids measurement, and leukocyte or fibroblast branched-chain ketoacid decarboxylase measurement can provide
the diagnosis.
CARBOHYDRATE METABOLISM ABNORMALITIES

Galactosemia
Affected patients have deficiency of one of three enzymes that convert galactose to glucose (99, 130). Deficiency of erythrocyte
galactose-1-phosphate uridyl transferase (GALT), galactokinase (GALK), or uridine diphosphate galactose-4-epimerase (GALE)
can cause galactosemia. Newborn screening is usually aimed at identifying the classical form, due to decreased GALT, and
characterized by toxic accumulation of galactose, galactose-1-phosphate, and galactitol that damage liver, kidneys, and lungs. A
number of mutations have been identified; incidence is 1/35,000; and the disease shows autosomal recessive inheritance (29).
Severe disease clinically mimics hereditary fructose intolerance but follows galactose feeding, usually after milk, and is
characterized by feeding intolerance, failure to thrive, vomiting, diarrhea, lethargy, hypotonia, jaundice, and hypoglycemia (64).
Galactosemia can present with sepsis (often with E. coli ) due to depressed neutrophil function (64, 99).
Cataracts, caused by galactitol accumulation in the lens, are typical at presentation but are mild and may be detected only by slit
lamp examination in the first few weeks of life (29, 69, 121, 130). Hemolysis, coagulopathy, aminoaciduria, proteinuria, and renal
failure occur (43). Extensive liver damage may be prevented or reversed by a galactose-free diet, though CNS complications may
not be avoided by dietary restriction (65, 99). Patients may have serious long-term neurological complications such as tremor and
ataxia, possibly due to endogenous galactose production (130). Hypergonadotrophic hypogonadism and ovarian failure with
amenorrhea and delayed puberty occur in females.
In infants with galactosemia, the liver is enlarged and yellow with panlobular macrovesicular steatosis, followed by periportal
ductular reaction with bile-plugged cholangioles surrounded by acute inflammation. The early liver lesions resemble those of
hereditary fructose intolerance (29). By 1 to 11/2 months of age, pseudoacinar transformation of hepatic plates occurs, and
hepatocytes surround dilated canaliculi that may contain bile. Extramedullary hematopoiesis and iron deposition may be prominent.
Fibrosis, apparent as early as 2 weeks of age, progresses to cirrhosis by 3 to 6 months. In some cases, giant cell transformation
and regenerative or dysplastic nodules occur (63, 99). The most severe hepatic abnormalities occur during episodes of sepsis;
endotoxin may contribute to liver injury (121). Pancreatic islets are hyperplastic, and vacuolization of renal tubular epithelium
(similar to that seen in hereditary fructose intolerance and tyrosinemia) is accompanied by tubular dilatation and necrosis. Ovarian
histology in ovarian failure is variable: oocytes may be absent or reduced in number (42, 130).
Diagnosis may be suspected if non-glucose-reducing substances are present in urine, although this test is neither sensitive nor
specific (69). Diagnosis of classical galactosemia
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is based on red cell GALT assay (65) and can only be done if red cells have not been transfused in the last 3 months. GALK and
GALE deficiencies can be identified by newborn screening if galactose is measured on a blood spot, and specific enzyme assays
then confirm the diagnosis (69).
Hereditary Fructose Intolerance (HFI)
Deficiency of fructose-1-phosphate aldolase (fructoaldolase B), inherited as an autosomal recessive disorder, leads to fructose-1-
phosphate accumulation. This toxic substrate damages liver, kidney, and brain, and inhibits glycogenolysis and gluconeogenesis,
resulting in hypoglycemia, phosphate sequestration, and ATP depletion (65). It has been proposed that acute lesions in Hfiare due
to ATP depletion and osmotic effects of fructose-1-phosphate accumulation (60). Fructose-1,6-diphosphatase deficiency can
cause a similar inhibition of gluconeogenesis with fructose- and fastinginduced hypoglycemia (65).
Symptoms develop when fructose in fruits and some vegetables or sucrose in candy is introduced into the diet, and clinical
improvement occurs if fructose, sorbitol, and sucrose are withdrawn from the diet (65, 99). Older children with HFI may avoid sweet
foods (65, 99). Liver, kidney, and intestine are affected; symptoms vary but include failure to thrive, vomiting, hepatomegaly,
coagulopathy, and renal failure with renal tubular acidosis, aminoaciduria, and proteinuria. Acute liver failure may occur if fructose
or sucrose is ingested in the newborn period.
Liver lesions resemble those of neonatal hepatitis and galactosemia with giant cell transformation, steatosis (Figure 5-16), ductular
proliferation, cholestasis, and portal fibrosis. Acute hepatic necrosis with little inflammation may be seen in the acute phase (63).
Progression to cirrhosis with portal hypertension, ascites, and splenomegaly occurs but is rare (60, 65, 99). Older infants may have
less severe liver damage with variable steatosis and portal fibrosis (63). By EM, characteristic but not pathognomonic changes
include “fructose holes” in hepatocytes: lucent spaces with sparse glycogen and membranous arrays surrounded by a single
membrane. These lesions may result from dilated degranulated rough endoplasmic reticulum and relate to intracellular
accumulation of enzyme substrate or ATP depletion (65). Pancreatic islet hyperplasia is seen, and, in kidney, proximal tubule
epithelium is granular and vacuolated with slight tubule dilatation (116).
FIGURE 5-16 ▪ Hereditary fructose intolerance. In hereditary fructose intolerance, the liver shows microvesicular and
macrovesicular steatosis. The histologic appearance of the liver can also resemble neonatal hepatitis with giant cell
transformation, ductular proliferation, cholestasis, fibrosis, and necrosis (not shown here) (H&E).
Hypophosphatemia, metabolic acidosis, and elevated transaminases are typical but not diagnostic (99). Fructose tolerance test is
not recommended for diagnosis because of potential danger to the patient. Analysis of leukocyte DNA for the aldolase B gene is
generally performed first, and, if DNA is normal, measurement of aldolase B activity in liver or intestinal tissue can be done (99,
116).
Glycogen Storage Diseases (GSD)

Glycogen catabolism is an important energy source. The many forms of GSD are generally associated with glycogen accumulation
and deficiency of an enzyme important in glycogen synthesis or degradation (Table 5-13) (18, 64, 67). Many organs, including
liver, heart and skeletal muscle, kidney, erythrocytes, and intestine, are affected by GSD (67). GSD can cause muscle fatigue,
cramps, progressive weakness, rhabdomyolysis, hypoglycemia, acidosis, failure to thrive, or hepatomegaly. Overall incidence is
approximately 1/20,000 (18). The various GSD have specific treatments, so early identification of an enzyme defect is important
(67, 99).
Subtle differences in liver morphology in GSD have been described (82) but, in general, pathological features are clearly distinctive
in only a few GSD, such as the light microscopic findings in GSD IV and the ultrastructural appearance of hepatocytes in GSD II
and IV (64, 99). In general, hepatocytes are enlarged with clear or vacuolated cytoplasm (67) and resemble plant cells in that cell
membranes appear thick, due to peripheral displacement of organelles by glycogen. Cytoplasm is PAS-positive and diastase
digestible. Some glycogen is lost due to its water solubility with formalin fixation; optimal glycogen preservation can be achieved by
alcohol fixation or by using fresh frozen tissue. Nuclei may be glycogenated, particularly in types I and III; types VI and IX typically
do not have glycogenated nuclei (64). Increased collagen in Disse's space occurs in GSD I, III, IV, VI, and IX. By EM, cytoplasmic
glycogen pools in hepatocytes, and variably sized lipid droplets occur in most GSD but are particularly abundant in GSD I, II, and VI
(60, 99).
GSD 0 is not true a GSD but is an autosomal recessive deficiency of glycogen synthase that results in ketotic hypoglycemia
without hepatomegaly or muscle symptoms (132). Steatosis and a slight decrease in liver glycogen content with normal glycogen
structure are seen (29, 94, 99, 132).
GSD I, von Gierke disease. In classical GSD I, GSD Ia, glucose-6-phosphatase enzyme complex in endoplasmic reticulum is
defective, and diagnosis is based on glycogen
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quantitation and glucose-6-phosphatase analysis. In GSD Ib, glucose-6-phosphatase is normal, but a defect in glucose-
6phosphatase translocase transporter protein results in failure of enzyme transport (29). Patients with GSD Ia present in infancy
with hepatomegaly, recurrent ketotic hypoglycemia with acidosis, hyperuricemia, hyperlipidemia, seizures, liver failure, and failure
to thrive. Truncal obesity, short stature, aminoaciduria, phosphaturia, muscle atrophy, and a bleeding tendency due to
hypoglycemia-induced platelet dysfunction are also described (29, 99). Patients with Type 1b additionally have neutropenia and
impaired neutrophil function, with recurrent bacterial infections and oral and intestinal mucosal ulcerations indistinguishable from
Crohn disease (18, 64, 99, 133).
Table 5-13 ▪ GLYCOGEN STORAGE DISEASES
Type Eponym Clinical Tissues Affected Enzyme Deficient
O Fasting ketotic hypoglycemia, short Liver Glycogen synthase

stature, osteopenia, without
hepatomegaly or weakness
I von Gierke 1a Ia-most severe of GSD, recurrent Liver, kidney, hepatic 1a-glucose-6-
and 1b (non-a) hypoglycemia, hepatomegaly, adenoma, phosphatase 1b-
nephromegaly, proteinuria, muscle hepatocellular glucose-6-phosphatase
atrophy, failure to thrive, xanthomas, carcinoma (1b: translocase
1b also has recurrent bacterial neutropenia,
infections inflammatory bowel
disease)
II Pompe Hypotonia, cardiomyopathy, Muscle, heart, CNS, Alpha-1,4-glucosidase

hepatomegaly lymphocytes, liver, (acid maltase)
kidney, adrenal
III Forbe, Cori, limit Hypotonia, hypoglycemia, ketosis, Muscle, heart, liver, Amylo-1,6-glucosidase,
dextrinosis growth failure, infections, WBC 4-
hepatosplenomegaly, alphaglucanotransferase
cardiomyopathy (debrancher enzyme)
IV Amylopectinosis, Hepatosplenomegaly, cirrhosis, Liver, heart, muscle, Amylo (1, 4 and 5, 6)
Andersen muscle wasting, gastroenteritis, CNS, PNS transglucosidase
osteoporosis, cardiomyopathy, (brancher enzyme)
hydrops
V McArdle Exercise intolerance, cramps, Muscle Muscle
fatigue, myoglobinuria myophosphorylase
VI Hers Growth retardation, hepatomegaly, Liver Hepatic phosphorylase

hypoglycemia
VII Tarui Exercise intolerance, cramps, fatigue Muscle, hemolytic Phosphofructokinase

myoglobinuria anemia
IX Exercise intolerance, stiffness, Liver, heart, blood Phosphorylase kinase

weakness, includes GSD VIII and X cells, muscle complex
XI Fanconi-Bickel Hepatorenal glycogen accumulation, Liver and kidney Glucose transporter 2

(GLUT2)
CNS, central nervous system; PNS, peripheral nervous system; WBC, white blood cell.
In GSD Ia, liver involvement is prominent with uniformly increased hepatocellular glycogen, nuclear glycogenation, and steatosis
with small and medium-sized lipid droplets (Figure 5-17A to D). In the GSD 1b liver, minimal or no nuclear glycogenation is seen,
unlike in GSD Ia (Figure 5-17E,F) (29). Sinusoids are compressed by distended hepatocytes (60, 64). Mallory bodies and zone 3
and periportal fibrosis have been reported (Figure 5-17C) (60). Focal nodular hyperplasia, adenomas (often multiple with atypical
cytologic features including dysplasia and hepatocellular carcinoma), may occur in patients with GSD 1a, particularly with the
G727T mutation (Figure 5-17G,H). Adenomas may arise because of glucagon stimulation and can regress if hypoglycemia is
reduced with diet (29). They are more common in boys than girls and are seen as early as 3 years of age (99). Hepatoblastomas
have also been described in GSD I (29).
Nephromegaly, increased glycogen in tubular epithelium, focal segmental glomerulosclerosis, and interstitial fibrosis occur in GSD
1. Nephrocalcinosis relates to hypercalcuria due to tubular acidosis (29). Xanthomas and chronic pancreatitis may reflect
hyperlipidemia (29).
GSD II is included above in the section on LSD.
GSD III (Cori-Forbe disease, Forbe disease, limit dextrinosis) patients have amylo-1,6-glucosidase,4-
alphaglucanotransferase (debrancher enzyme) deficiency, which can be measured in liver, fibroblasts, skin, or lymphocytes (29,
111). This clinically and genetically heterogeneous disorder has symptoms similar but less severe than those seen in GSD I.
Progressive weakness may be the predominant feature in adults, and cardiomyopathy occurs in some patients (29). Liver failure
can occur, though liver function often improves with age (18, 29). GSD IIIa has liver and muscle involvement; GSD IIIb has liver
involvement only; and GSD IIIc is an isolated muscle disease (111).
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FIGURE 5-17 ▪ Glycogen storage disease, type I. A: The liver in type I glycogen storage disease shows diffuse steatosis with
hepatocyte distension obscuring sinusoids (H&E). B: Despite diffuse hepatocellular involvement, the liver in type I disease shows
little (if any) fibrosis (trichrome). C,D: In type Ia disease, the liver shows steatosis (C) and hepatocellular Mallory's hyaline (D); note
the presence of glycogenated nuclei (C), typical of type Ia disease (H&E).
FIGURE 5-17 ▪ (continued). E,F: This example of type Ib disease shows steatosis and occasional pigment-laden macrophages
(H&E). G,H: Patients with type I GSD are at increased risk for the development of hepatocellular adenoma (with varying degrees
of dysplasia), which can evolve into hepatocellular carcinoma. In (G), the interface between the hepatocellular adenoma (lower
right) and nonneoplastic liver (upper left) is shown; in (H), the cells of the adenoma show dysplastic cytologic features (H&E).
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If restricted to muscle, enzyme assay of muscle is required for diagnosis. Muscle biopsy may show slight fiber size variation with
little vacuolization or increased glycogen. Some patients have vacuolar myopathy with glycogen accumulation at the periphery of
fibers (31). Liver has delicate reticular septal fibrosis or, less commonly, micronodular or mixed cirrhosis, and glycogenated nuclei
can occur (67). Hepatic adenomas occur in up to 25% of GSD III patients, but hepatocellular carcinoma is rare (60, 65, 99).
GSD IV (Andersen disease, branching enzyme deficiency, amylopectinosis). Amylo (1, 4 and 5, 6) transglucosidase
(brancher enzyme) deficiency can be identified in liver, muscle, leukocytes, and fibroblasts (29). Abnormally long, relatively
insoluble amylopectin-like glycogen chains with reduced branch points accumulate in all tissues, particularly liver, skeletal muscle,
and heart (46, 64). GSD IV is autosomal recessive, and there are multiple mutations causing the enzyme deficiency, reflected in
clinical variability (99). Infants may appear normal or have nonimmune hydrops and failure to thrive. In classical GSD IV,
hepatomegaly occurs in the first months of life and progresses to cirrhosis and liver failure without hypoglycemia by 2 to 5 years
(46, 61, 72). A neonatal neuromuscular form has been identified, and some infants present with dilated cardiomyopathy and
arthrogryposis. Later-onset nonprogressive hepatic disease with hypotonia and cardiomyopathy may also occur (110).
The liver is tan with a waxy or a tough leathery consistency and tiny nodules that may aggregate into larger nodules (61). The liver
resembles that seen in Lafora disease but with progression to fibrosis and cirrhosis. Only rarely do hepatic neoplasms occur with
GSD IV (65). Pericellular fibrosis encircles clusters of hepatocytes with round or oval ground-glass intracytoplasmic inclusions
primarily in periportal hepatocytes (Figure 5-18A). These PAS-positive, diastase-resistant inclusions stain green with colloidal iron;
stain either brown, blue, or not at all with Lugol's iodine; are removed by pectinase or alpha-amylase; and have an artifactual
space around them (60, 65, 99). By EM, inclusions are non-membrane-bound with undulating random delicate fibrils up to 5 nm in
diameter surrounded by glycogen rosettes (65). Similar inclusions are seen in heart, skeletal muscle, skin, CNS neurons, and
lymph node macrophages (Figure 5-18B) (29, 46, 61, 65).
GSD V, McArdle disease is due to autosomal recessive inherited myophosphorylase deficiency. Children with GSD V typically
have exercise intolerance, and this disease rarely presents as respiratory failure in infancy (29, 111). Patients have no rise in lactic
acid after ischemic exercise. By LM, muscle may appear normal or may have mild alterations, with occasional degenerating fibers
and subsarcolemmal glycogen-containing vacuoles. EM highlights subsarcolemmal and sarcoplasmic glycogen, and
histochemically demonstrable myophosphorylase activity is absent (31).
GSD VI, Hers disease due to hepatic phosphorylase deficiency is a relatively benign autosomal recessive disorder that causes
hypoglycemia and growth failure. The disease improves with age: hepatomegaly decreases after puberty, and adults are typically
asymptomatic (99). A nonuniform mosaic pattern of distended hepatocytes without nuclear glycogenation is accompanied by mild
fibrosis and steatosis (Figure 5-19). Heart and skeletal muscle are not altered.
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FIGURE 5-18 ▪ Glycogen storage disease, type I V. A: In type IV disease, hepatocytes are enlarged with ground glass
cytoplasmic inclusions (H&E). B: Skeletal muscle involvement in type IV disease, with rarefaction of myofibers due to non-
membrane-bound glycogen accumulation (H&E).
GSD VII, Tarui disease is characterized by absent phosphofructokinase activity, easy fatigability, and exercise intolerance in
children. Severe infantile cases with respiratory failure also occur (18, 31, 49, 111). Patients have hemolytic anemia due to
absence of a muscle isoenzyme in red blood cells, hyperuricemia, and myoglobinuria (111). Muscle has extensive subsarcolemmal
and sarcoplasmic glycogen, and a few fibers contain hyaline, PAS-positive, diastase-resistant inclusions with a filamentous fine
structure resembling amylopectin. Histochemical staining suggests these are an insoluble form of glycogen (1).
GSD VIII and GSD IX have been grouped together (18), both with mutations in genes that encode subunits of phosphorylase
kinase; a defect in one of the four subunits of phosphorylase kinase result in a variable clinical presentation with hepatomegaly
and growth failure (18). GSD IXa is X-linked, and the autosomal recessive forms (IXb, IXc) have more severe liver disease that can
progress to cirrhosis (99).
FIGURE 5-19 ▪ Glycogen storage disease, type VI. Mosaic pattern of nondistended (lower middle) and distended hepatocytes,
characteristic of type VI disease, with mild steatosis and absence of glycogenated nuclei (H&E).
GSD XI, Fanconi-Bickel patients have failure to thrive, rickets, hepatomegaly, nephromegaly, hyperglycemia when not fasting,
glucosuria, and aminoaciduria. Hepatorenal glycogen accumulation is secondary to nonfunctional glucose transport due to a
mutation in the GLUT2 gene.
FATTY ACID OXIDATION DEFECTS

A number of enzymes and transporters are involved in intramitochondrial fatty acid oxidation (FAO), and defects in these pathways
lead to a heterogeneous group of disorders (32). Inherited disorders of fatty acid transport and mitochondrial oxidation can present
with acute liver failure and sudden unexpected death in children (Tables 5-14 and 5-15) (64).
Acyl-CoA Dehydrogenase Deficiencies

Before beta-oxidation of fatty acids occurs, fatty acids must be converted first to their coenzyme A (CoA) thioesters. This
conversion is catalyzed by acyl-CoA dehydrogenases, of which there are at least four types—classified based on chain lengths as
short-, medium-, long-, and very-long-chain synthetases. Any of these four forms of acyl-CoA dehydrogenases can be deficient,
and these deficiencies are the most frequently identified abnormalities of FAO (32). Where known, inheritance is autosomal
recessive.
In general, patients with acyl-CoA dehydrogenase deficiency have hypoketotic hypoglycemia, liver and skeletal muscle
abnormalities, cardiomyopathy, and sudden unexpected death in childhood (63). Patients with short-chain
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defects may have ketotic hypoglycemia. Panlobular microvesicular or macrovesicular steatosis may disappear between crises
when the patient is well (63). Fat accumulation may also be seen in the myocardium and skeletal muscle. EM shows enlarged
mitochondria with increased matrical density, crystals, and widened intercristal space (64). Establishing a diagnosis may allow for
successful treatment, and these disorders are included in newborn screening programs (Table 5-6) (137).
Table 5-14 ▪ FATTY ACID OXIDATION DEFECTS
Acyl-CoA dehydrogenase deficiencies
Medium-chain acyl-CoA dehydrogenase deficiency
(MCADD, MCAD)
Short-chain acyl-CoA dehydrogenase deficiency (SCAD)
Long-chain hydroxylacyl-CoA dehydrogenase deficiency
(LCHAD)/Trifunctional protein deficiency (TFP)
Very-long-chain acyl-CoA dehydrogenase (VLCAD)
Glutaric acidemia type II (Multiple acyl-CoA dehydrogenase
deficiency, MADD)
Substrate Transport Defects
Carnitine palmitoyltransferase (CPT) deficiency
CPT I and CPT II deficiency
Primary systemic carnitine deficiency (carnitine transporter
deficiency)
Carnitine acylcarnitine translocase deficiency
Medium-chain acyl-CoA dehydrogenase deficiency (MCADD, MCAD). Affected patients are often homozygous for the A985G
mutation, and carriers are particularly common in northwest Europe (29). This is one of the most common IEM, with an incidence of
1/5-8,000; it is the most common FAO defect in central Europe (69, 104). Patients present between 3 and 15 months of age with
hypoketotic hypoglycemia after fasting, lethargy, vomiting, and sudden unexpected death (69). Fatal cases may resemble SIDS or
Reye syndrome (69). They can also present in later life with exercise-induced muscle pain, and rhabdomyolysis, and there is
marked clinical variability even in the same family.
Liver may be normal grossly or have minimal steatosis, and the heart may show lipid accumulation (16, 63). Mitochondria may be
enlarged with crystals, increased matrix density, and dilated cristae (63). Encephalopathy and cerebral edema are due to
accumulation of fatty acids in the CNS (69). Octanoylcarnitine is elevated on plasma acylcarnitine analysis, and further
confirmatory testing may include urine acylglycine analysis and molecular testing (69).
Short-chain acyl-CoA dehydrogenase deficiency (SCADD, SCAD). There are two clinical forms: (a) a myopathic form limited
to muscle (with progressive weakness and exercise-induced pain) and (b) a systemic form with neonatal onset of vomiting,
lethargy, acidosis, ketotic hypoglycemia, hepatomegaly, hypotonia, seizures, and microcephaly. However, the vast majority of
patients with SCAD deficiency detected through newborn screening programs do not demonstrate any symptoms. There are two
common mutations in the SCAD gene that are associated with mild disease. Increased lipid in muscle and liver are described (43).
Table 5-15 ▪ METABOLIC CAUSES OF SUDDEN UNEXPECTED DEATH IN INFANCY
Inherited defects of fatty acid oxidation (FAO)
Medium-chain acyl-CoA dehydrogenase deficiency (MCAD)
Very-long-chain acyl-CoA dehydrogenase deficiency (VLCAD)
Long-chain 3-hydroxy acyl-CoA dehydrogenase deficiency (LCHAD)/Trifunctional protein deficiency
Glutaric acidemia type 2 (multiple acyl-CoA dehydrogenase deficiency (MADD)
Carnitine palmitoyltransferase II deficiency (CPT II)
Primary carnitine deficiency (carnitine transporter deficiency)
Carnitine acylcarnitine translocase deficiency (CAT)
Hyperammonemia/urea cycle disorders
Ornithine transcarbamylase (OTC) deficiency
Argininosuccinate synthetase deficiency (citrullinemia)
Argininosuccinate lyase deficiency (argininosuccinic aciduria)
Carbamoylphosphate synthetase deficiency
Lysinuric protein intolerance
Organic acidemias
Methylmalonic acidemia
Propionic acidemia
Isovaleric acidemia
Glutaric acidemia type 1
3-hydroxy-3-methyl-glutaryl-CoA lyase deficiency
3-methylcrotonyl-CoA carboxylase deficiency
Congenital lactic acidosis
Pyruvate dehydrogenase deficiency
Pyruvate carboxylase deficiency
Phosphoenolpyruvate carboxykinase (PEPCK) deficiency

Amino acid disorders
Maple syrup urine disease
Tyrosinemia type 1
Carbohydrate disorders
Galactosemia
Glycogen storage disease type I
Hereditary fructose intolerance
Fructose-1,6-bisphosphate deficiency
Long-chain hydroxylacyl-CoA dehydrogenase deficiency (LCHADD, LCHAD) and trifunctional protein deficiency.
LCHAD is part of the trifunctional protein (TFP) complex, comprising LCHAD, long-chain enoyl-CoA hydratase and long-chain beta-
ketoacyl-CoA thiolase activities. Most patients have isolated LCHAD deficiency, but some are also deficient in the other two
enzymes and have generalized TFP deficiency. Similar clinical and biochemical manifestations occur in affected individuals.
About 75% of LCHAD-deficient patients carry a G-to-C mutation at nucleotide position 1528 (Glu474Gln, E474Q) on both
chromosomes, while up to 25% are compound heterozygotes for E474Q on one allele and a second different LCHAD mutation on
the other allele. LCHAD patients have episodic nonketotic hypoglycemia, cardiomyopathy, and liver
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dysfunction (29, 64). Infants can present with hydrops (72), cardiac involvement, coma associated with fasting, and death; a later-
onset form causes myalgias with myoglobinuria.
The HELLP syndrome (hemolysis, elevated liver enzymes, and low platelet count) with hypertension can occur in mothers of
fetuses with LCHAD and at least one E474Q allele. Why HELLP occurs with this mutation is not known (37). TFP deficiency is also
associated with maternal HELLP syndrome. Acute fatty liver of pregnancy and prolonged hyperemesis can also occur in mothers of
affected fetuses (78). The liver in LCHAD patients has bile duct proliferation, cholestasis, steatosis, fibrosis, and cirrhosis, and fat
accumulates in skeletal and cardiac muscle (29, 65, 104).
Very-long-chain acyl-CoA dehydrogenase (VLCADD, VLCAD). Affected patients may have a lethal childhood form with early-
onset, hypertrophic or dilated cardiomyopathy, and hypoglycemia (32, 16). A milder childhood form with hypoglycemia and
dicarboxylic aciduria is less common, and some patients resemble those with CPT II deficiency with rhabdomyolysis and
myoglobinuria (32). Muscle pathology is generally mild with increased variation in fiber size and a mild increase in muscle fiber lipid
(32). Bile duct proliferation, hepatic fibrosis, and cirrhosis can occur (65).
Glutaric acidemia type II (Multiple acyl-CoA dehydrogenase deficiency, MADD). Glutaric acidemia type II is caused by
deficiency of the electron-accepting protein electron transfer flavoprotein (ETF) or ETF-ubiquinone oxidoreductase (ETF-QO).
Patients may present in early infancy with hypotonia, hepatomegaly, sweaty feet odor, acidosis, and nonketotic hypoglycemia;
death may occur in the first few weeks of life in the most severe form. Congenital anomalies include cerebral cortical dysgenesis
with abnormal neuronal migration, facial dysmorphism, genital defects, and renal cystic dysplasia (Figure 5-20A) as well as
irregular glomerular basement membrane can be seen (43). Later-onset disease is variable. It can present in infants with episodic
vomiting, acidosis, and hypoglycemia or in adults with similar biochemical findings, hepatomegaly, and proximal myopathy. Hepatic
steatosis, intracytoplasmic cholestasis, paucity of intrahepatic bile ducts, mild portal fibrosis, and hepatocellular necrosis are
described (Figure 5-20B), and some patients respond to riboflavin (32, 63).
FIGURE 5-20 ▪ Glutaric acidemia, type II. A: Renal dysplasia in an infant with glutaric acidemia, type II (H&E). B: Hepatic
steatosis in glutaric acidemia, type II (as well as widespread extramedullary hematopoiesis, reflective of the patient's neonatal
state) (H&E).
Substrate Transport Defects

Carnitine palmitoyltransferase (CPT) is an enzyme that catalyzes the reaction of carnitine and long-chain fatty acyl groups for
transport into mitochondria. There are two forms of this enzyme, CPT I and CPT II, associated with the outer and inner
mitochondrial membranes, respectively. Either can be deficient leading to a myopathy. Most patients are males, although the
defective CPT genes are on autosomes (32). Beginning in childhood, patients have recurrent myoglobinuria, often after exercise or
fasting, and recurrent myalgia but no muscle cramps or intolerance to short spells of exercise. Respiratory muscles may be
affected (32). Between attacks, patients are normal without weakness. CPT II deficiency can result in sudden unexpected death
(16).
Muscle biopsy obtained when the patient is without symptoms may be normal or have only mild lipid accumulation. Biopsies
obtained after an episode of myoglobinuria have fiber necrosis. At autopsy, fatty infiltration of myocardium, liver, kidneys, and
muscle has been described (16).
Carnitine deficiency. Carnitine is an essential cofactor for transport of medium- and long-chain fatty acids across the inner
mitochondrial membranes, where they undergo betaoxidation (32). Carnitine is derived either from the diet or via biosynthesis; it is
made in liver and transported to other tissues, including muscle, which has the highest concentration of free carnitine, followed by
liver and heart.
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Decreased concentration of carnitine in skeletal muscle occurs in two biochemically and clinically distinct syndromes (32). Primary
systemic carnitine deficiency (OMIM 212140), or carnitine uptake deficiency, is an autosomal recessive disorder caused by a
defect in a sodium-dependent transporter protein OCTN2, which is encoded by the gene SLC22A5. In systemic carnitine
deficiency, liver, muscle, and serum carnitine concentrations are all reduced. This disorder can present with hydrops fetalis and
has been linked to sudden unexpected infant death (29). Generally, beginning in childhood, patients have muscle weakness,
progressive cardiomyopathy, and recurrent acute hepatic encephalopathy with hypoglycemia, hyperammonemia, nausea, emesis,
confusion, or coma. In some cases, hypoglycemia occurs with metabolic acidosis with increased lactate and ketoacids (32). Death
due to cardiorespiratory failure occurs in many before 20 years of age. The muscle has severe lipid storage and panacinar
microvesicular steatosis in the liver mimicking that seen in Reye syndrome. Fat may not be seen during asymptomatic periods or
may be present in sinusoidal lining cells. Mild periportal fibrosis can occur (60). In some patients, there is cardiomegaly and
endocardial fibroelastosis (16, 32), and myocardium and renal tubular epithelium can contain increased lipid. This disorder
responds very well to treatment with carnitine supplementation.
Myopathic carnitine deficiency (OMIM 212160) is an autosomal recessive disorder, associated with progressive weakness
beginning in childhood. Cardiac involvement can occur (32). Carnitine is decreased in muscle but is normal or only slightly
decreased in serum and liver. Lipid storage myopathy is seen with increased lipid, often adjacent to mitochondria (32).
MITOCHONDRIAL DISORDERS
The mitochondriopathies are a heterogeneous group of neuromuscular and multisystemic disorders due to altered mitochondrial
metabolic function. This altered function is the result of an abnormal enzyme complex involved in energy production; patients have
impaired respiratory chain function or oxidative phosphorylation. Some mitochondriopathies are inherited as autosomal dominant or
recessive traits and others are maternally inherited due to mutations in mitochondrial DNA. The same molecular abnormality can
cause very different clinical presentation, and even in a single family there may be a wide range of phenotypes (Table 5-16) (32).
Some mitochondrial disorders affect only a single organ, while others involve multiple organ systems and have complex clinical
features. Likewise, some individuals will have a distinct cluster of symptoms that fall into a specific clinical syndrome, such as
Kearns-Sayre syndrome, while others will have a variety of problems that are more difficult to categorize. Nuclear gene defects are
more likely to present in infancy or early childhood, while mitochondrial DNA defects often become symptomatic later in childhood
or in adult life, although there are exceptions.
There are over 70 different polypeptides on the inner mitochondrial membrane that form the enzyme complexes I, II, III, IV, and V of
the respiratory chain. Thirteen of these subunits are encoded by the mitochondrial DNA, but all of the rest are encoded by nuclear
genes. In addition, the mitochondrial DNA encodes 22 transfer RNAs (tRNAs) and 2 ribosomal RNAs (rRNAs) that are essential for
normal intramitochondrial protein synthesis. Of the 13 mitochondrial DNA-encoded polypeptides involved in the formation of the
enzyme complexes, 7 are in complex I, 1 is in complex III, 3 are in complex IV, and 2 are in complex V. All of the complex II
polypeptides and the remaining mitochondrial proteins are encoded by nuclear genes.
Patients with mitochondriopathies may present with ptosis, ophthalmoplegia, exercise intolerance, increased lactate level, or
abnormal brain MRI. Onset may be from birth to adulthood and may be rapidly progressive or static; weakness may be generalized
or proximal. There may be associated mitochondrial alterations including abnormal number or altered cristal structure (32).
Secondary multiorgan damage occurs, with steatosis, cardiomyopathy, and, in the CNS, spongiosis, neuronal loss, gliosis, and
demyelinization.
Subacute necrotizing encephalopathy (Leigh syndrome) can occur as a result of multiple different molecular defects (Tables 5-16
and 5-17). Brain lesions in Leigh syndrome include focal, symmetric necrotizing lesions in the brainstem and thalamus, basal
ganglia, cerebellum and spinal cord with associated demyelination, astrocytosis, and vascular proliferation. Hypertrophic
cardiomyopathy with concentric left ventricular hypertrophy is also associated with Leigh syndrome, and abnormal mitochondria
have been described in lymphocytes in children with Leigh syndrome (43, 62).
Ragged-red fibers (Figure 5-21A) may be seen with the Gomori trichrome stain in mitochondriopathies. With H&E, these fibers are
basophilic and granular (Figure 5-21B); they react with NADH and may lack cytochrome oxidase activity (32). The presence of
ragged-red fibers indicates ultrastructural abnormalities in mitochondria or increased number of morphologically normal
mitochondria (32). Ragged-red fibers are more common in patients with mitochondrial gene mutations than nuclear gene
mutations, but the number of these fibers is variable and does not correlate with phenotype (32). In some cases, ragged-red fibers
are not apparent by LM (Figure 5-21C) and EM is required to see mitochondrial alterations: abnormalities in number, size, or
shape; giant or bizarre mitochondria: altered cristae; or crystalline or osmiophilic intramitochondrial inclusions (Figure 5-21D to G).
Such structurally abnormal mitochondria are not specific for a single clinical syndrome or molecular defect (32).
The liver in Complex IV and I deficiencies (the most common generalized enzyme deficiencies) has microvesicular and
macrovesicular steatosis, cholestasis, and giant cell transformation. Portal fibrosis and cirrhosis are common. The number of
hepatocyte mitochondria may be increased, and they may be pleomorphic with abnormal cristae and intramitochondrial inclusions
(63, 65).
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Table 5-16 ▪ MITOCHONDRIAL DEFECTS (MITOCHONDRIOPATHIES)
Inheritance
Primary Mitochondrial DNA Disorders Clinical Features Pattern
Rearrangements Chronic progressive external External ophthalmoplegia Ptosis Sporadic or

(large-scale ophthalmoplegia (CPEO) mitochondrial
partial deletions
and
duplications)
Kearns-Sayre syndrome (KSS) PEO before age 20 years Pigmentary Sporadic or
retinopathy Heart block Cerebellar ataxia mitochondrial
Deafness Myopathy Dysphagia Diabetes
mellitus Hypoparathyroidism Dementia
Pearson marrow-pancreas syndrome Sideroblastic anemia Exocrine pancreatic Sporadic or

dysfunction Neonatal or infantile onset mitochondrial
Survivors may develop KSS symptoms
later in life
Diabetes mellitus and deafness Sporadic
Point mutations Genes encoding structural

proteins
Leber hereditary optic neuropathy Sudden loss of vision, usually bilateral, Mitochondrial
onset 18 to 30 years
LHON (G11778A, T14484C, G3460A)
Males: females 4:1
NARP (T8993G/C)(ATPase6 gene) Neuropathy, late childhood or adult onset Mitochondrial
Ataxia
Retinitis pigmentosa
Leigh syndrome (T8993G/C) Onset of symptoms in 1st year of life Mitochondrial

(ATPase6 gene)
Hypotonia and motor retardation
Seizures
Lactic acidosis
Pigmentary retinopathy
Necrotizing encephalomyopathy
Genes encoding transfer RNAs
MELAS (A3243G, T3271C, A3251G) Seizures and/or dementia Lactic acidosis Mitochondrial
Stroke-like events Ragged red fiber
myopathy
MERRF (A8344G, T8356C) Myoclonic epilepsy Ragged red fiber Mitochondrial

myopathy
CPEO (A3243G, T4274C) Chronic progressive external Mitochondrial

ophthalmoplegia
Myopathy (T14709C, A12320G) Mitochondrial
Hypertrophic cardiomyopathy Mitochondrial

(A3243G, A4269G, A4300G)
Diabetes and deafness (A3243G, Mitochondrial

C12258A)
Encephalomyopathy (G1606A, Mitochondrial

T10010C)
Genes encoding ribosomal RNAs
Nonsyndromic sensorineural deafness Mitochondrial

(A7445G)
Aminoglycoside induced Mitochondrial

nonsyndromic deafness (A1555G)
Nuclear Gene
Disorders
Disorders of Autosomal dominant progressive

mtDNA external ophthalmoplegia
maintenance
Mutations in adenine nucleotide PEO, muscle weakness, bipolar disease AD

translocator (ANT1)
Mutations in DNA polymerase PEO, muscle weakness, psychiatric AD or AR

(POLG1) disease, neuropathy, ataxia
Mutations in Twinkle helicase PEO, myalgia, exercise intolerance, AD

(C10ORF2) peripheral neuropathy, psychiatric
disease
Mitochondrial neurogastrointestinal Onset in infant to adult ages AR

encephalomyopathy (MNGIE)(2° Gastrointestinal dysmotility (pseudo-
multiple mtDNA deletions): Mutations obstruction) Leukoencephalopathy
in thymidine phosphorylase (ECGF1) Neuropathy Ophthalmoplegia Myopathy
Myopathy with mtDNA depletion: AR

Mutations in thymidine kinase (TK2)
Encephalopathy with liver failure and Neonatal or infantile onset Progressive AR

mitochondrial DNA depletion: liver disease
Mutations in deoxyguanosine kinase
(DGUOK), MPV17 or POLG1
Mitochondrial DNA depletion due to Hypotonia Muscle atrophy Hyperkinesia AR

ATPforming beta subunit of the Krebs Severe hearing impairment Postnatal
cycle enzyme succinyl-CoA ligase growth retardation Methylmalonic
(SLUCA2) acidemia
Primary Leigh syndrome (Subacute necrotizing
disorders of the encephalomyopathy)
respiratory chain
Complex I deficiency—mutations in AR
complex I subunits (NDUFS2,4,7, 8
and NDUFV1)
Complex II deficiency—mutations in AR
complex II flavoprotein subunit
(SDHA)
Leukodystrophy and myoclonic AR

epilepsy: Complex I deficiency —
mutations in complex I subunit
(NDUFV1)
Cardioencephalomyopathy: Complex I AR
deficiency—mutations in complex I
subunit (NDUFS2)
Optic atrophy and ataxia: Complex II AD

deficiency — mutations in complex II
flavoprotein subunit (SDHA)
Disorders of Dystonia-deafness: Mutations in XLR

mitochondrial deafnessdystonia protein DDP1
protein import (TIMM8)
Disorders of Leigh syndrome (Subacute necrotizing

assembly of the encephalomyopathy)
respiratory chain
Complex IV deficiency—mutations in Most common cause of Leigh syndrome AR

COX assembly protein (SURFI)
Complex IV deficiency—mutations in AR
COX assembly protein (COX10)
Cardioencephalomyopathy: Complex AR
IV deficiency—mutations in COX
assembly protein (SCO2)
Hepatic failure and encephalopathy:
COX assembly protein (SCO1)
protein affecting COX mRNA stability
(Leucine rich pentatricopeptide repeat
cassette, LRPPRC)
Tubulopathy, encephalopathy, and AR

liver failure: Complex III deficiency—
mutations in complex III assembly
(BSC1L)
Encephalopathy: Complex I deficiency AR

— mutations in the complex I
assembly protein (B17.2L)
Disorders of Leigh syndrome (Subacute necrotizing

RNA metabolism encephalomyopathy)
Complex IV deficiency (LRPPRC) AR
Multiple complex defects with AR

mutations in mitochondrial elongation
factor G1 (EFG1)
Disorders of the Coenzyme Q10 deficiency (COQ2) Ataxia, seizures, encephalomyopathy AR

lipid membrane
Cerebellar atrophy Renal dysfunction

Treatable with coenzyme Q10
Barth syndrome (Tafazzin) Cardiomyopathy XLR
Noncompaction of left ventricle
Neutropenia
3-methylglutaconic aciduria Skeletal

muscle myopathy
PEO, progressive external ophthalmoplegia; AD, autosomal dominant; AR, autosomal recessive; XLR, X-
linked recessive.
Mitochondrial DNA (mtDNA) Abnormalities

In these disorders, heteroplasmy is common, with a mixture of mutant and normal mtDNAs in the same patient, varying from
mitochondrion to mitochondrion, cell to cell, and tissue to tissue, rather than the normal identical mtDNA (homoplasmy) in all cells
and tissues (29). Heteroplasmy impacts clinical symptoms and organ dysfunction. Diagnosis may be made with histochemistry,
EM, respiratory chain enzyme analysis, or mtDNA mutation analysis using muscle tissue. In families with mtDNA mutations, there is
marked phenotype variability (Table 5-16) (29).
Mitochondrial DNA Rearrangements

Chronic progressive external ophthalmoplegia (CPEO) with ragged-red fibers is generally a benign disorder with slowly
progressive ophthalmoplegia, ptosis, and proximal limb weakness beginning in adolescence. Most cases are sporadic and have
deletions or duplications of mtDNA (29). Muscle biopsy shows ragged-red and cytochrome oxidase (COX)-negative fibers.
Kearns-Sayre syndrome (KSS) patients have a severe mitochondriopathy and may have retinitis pigmentosa, progressive
external ophthalmoplegia, and heart block beginning before 20 years of age. Ptosis, cerebellar involvement, ataxia, and
endocrinopathies (diabetes mellitus,
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hypoparathyroidism, growth hormone deficiency, Addison disease, and hypogonadism) also occur. The brain may have basal
ganglia calcification. Muscle biopsy shows numerous or infrequent ragged-red fibers and COX-negative fibers. Structurally
abnormal mitochondria are apparent by EM (32).
Table 5-17 ▪ METABOLIC CAUSES OF LEIGH SYNDROME (SUBACUTE NECROTIZING

ENCEPHALOMYELOPATHY)
Pyruvate dehydrogenase deficiency
Pyruvate carboxylase deficiency
Complex I
Mutations in NDUFS 1,2,3, 4,7,8, NDUFV1, MTND2,3,5,6
Complex II
Mutations in SDHA
Complex III
Mutations in BCS1L
Complex IV
Mutations in SURFI, COX10, COX15, LRPPRC, MTCO3,
SCO2
Complex V
Mutation of MTATP6 (T8993G/C)
Mitochondrial tRNA protein mutations
MTTV, MTTK, MTTW, MTTL1(A3243G)
Coenzyme Q10 deficiency (COQ2)
Mitochondrial elongation factor G1 (EGF1)

FIGURE 5-21 ▪ Mitochondrial disorders. A,B: Many mitochondrial disorders are characterized by peripheral red granularity on
Gomori trichrome stain, frequently described as ragged red fibers (A); by H&E (B) a similar pattern of basophilic granularity is
present in myofibers [Gomori trichrome (A) and H&E (B)]. C: The absence of “ragged red fibers” does not exclude mitochondrial
disease. In this case of Complex 1 deficiency, there is a subtle increase in red granularity of myofibers (i.e., mitochondrial
prominence) but no frank “ragged red fibers” (Gomori trichrome). D-G: Though mitochondria can assume a variety of, often
striking, morphological abnormalities— including crystalline/paracrystalline inclusions (D-G: uncharacterized mitochondrial
disease;
FIGURE 5-21 ▪ (continued) H-J: MELAS) sometimes with a “parking lot appearance (J: MELAS), concentric cristae (K: MELAS),
and other variable abnormalities in size and arrangement of cristae (L-O: Complex 1 Deficiency)—the specific morphologic
features of abnormal mitochondria cannot reliably determine which mitochondrial disease is present (D-O: Uranyl acetate, lead
citrate).
FIGURE 5-21 ▪ (continued).
Pearson marrow-pancreas syndrome is a sporadic disorder that is generally fatal in early childhood due to sepsis with bone
marrow failure with pancytopenia, exocrine pancreatic failure, and refractory sideroblastic anemia (105). This disorder may cause
hydrops fetalis (78), renal tubular disease, diarrhea, and liver failure. Neuromuscular symptoms resembling those seen in KSS may
develop in older children. The marrow shows vacuolization of marrow precursors (29).
Point Mutations in Genes Encoding Mitochondrial Structural Proteins

Leber hereditary optic neuropathy (LHON) patients have painless vision loss as teens or young adults. Only a few have
neurological symptoms or dystonia and lactic acidosis is absent. Mutations in ND4 complex I are associated with this disorder.
Optic nerve pathology with ganglion cell atrophy and loss and retina nerve fiber atrophy may be seen, but ragged-red fibers are
not present (29, 31).
Neuropathy, ataxia, and retinitis pigmentosa (NARP) and Leigh syndrome (subacute necrotizing encephalomyopathy)
are two syndromes that are phenotypic variants due to variation in the ratio of wild type to abnormal mitochondrial
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DNA. They are both associated with T8993G/C mutations in the ATPase6 gene that causes a deficiency in complex V (32). The
Leigh syndrome clinical presentation with this mutation is severe (91). Ragged-red fibers are not typically seen on muscle biopsy
(32).
Point Mutations in Genes Encoding Mitochondrial Transfer RNA (tRNA)

Mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) is one of the most common respiratory
chain disorders. Patients have sudden onset of stroke-like episodes, usually in adulthood, along with myopathy, ataxia,
headaches, deafness, cardiomyopathy, and diabetes mellitus (29). Ragged-red fibers are seen in muscle biopsy, but these fibers
have cytochrome oxidase activity unlike in most other cases of mitochondrial myopathy with ragged-red fibers (32).
Myoclonic epilepsy with ragged-red fibers (MERRF) patients have myoclonic epilepsy, cerebellar ataxia, dementia, myopathy,
deafness, short stature, and increased lactate and pyruvate levels (32). Ragged-red fibers are present in muscle biopsy along with
numerous COX-negative fibers.
Disorders of Mitochondrial DNA Maintenance

These disorders are caused by defects in nuclear-encoded factors required for maintenance of mtDNA stability and replication.
Mutations in these genes result in depletion of mtDNA and/or multiple mtDNA deletions.
Multiple mitochondrial DNA deletions. Mutations in adenine nucleotide translocator-1 (ANT-1) and the mitochondrial helicase
Twinkle (C10ORF2) can lead to autosomal dominant or recessive progressive ophthalmoplegia and in some cases multisystemic
symptoms.
Mitochondrial neurogastrointestinal encephalopathy (MNGIE) disease. This autosomal recessive condition usually presents
before age 20 years and is characterized by severe gastrointestinal dysmotility (pseudo-obstruction), poor weight gain,
gastroesophageal reflux, vomiting, diarrhea, abdominal pain, and abdominal distension. Patients may also have hearing loss,
ptosis, external ophthalmoplegia, sensorimotor neuropathy, and leukoencephalopathy on brain MRI scan. Demyelinating peripheral
neuropathy is associated with paresthesias and distal limb weakness. The gene encoding thymidine phosphorylase, ECGF1, is the
only known gene associated with MNGIE disease. Both mitochondrial DNA depletion and multiple mitochondrial DNA deletions are
found in patients with MNGIE disease.
Muscle biopsy shows ragged-red fibers; eosinophilic cytoplasmic inclusions, representing enlarged mitochondria, occur in rectal
submucosal ganglia and smooth muscle cells (97). The duodenum may demonstrate focal atrophy or absence of the muscularis
propria with increased number of nerves and focal loss of Auerbach's plexus with fibrosis (122).
Diagnosis may be made by demonstrating elevated plasma thymidine and deoxyuridine or by demonstrating less than 10% of the
control mean thymidine phosphorylase activity in buffy coat leukocytes. Respiratory chain enzyme assays on tissues demonstrate
defects in single or multiple complexes. The most common defect is in cytochrome c oxidase (complex IV). Molecular genetic
testing of the ECGF1 gene detects essentially 100% of affected individuals.
Mitochondrial DNA depletion syndrome patients have disease onset in the early months of life with progressive liver failure and
encephalopathy. Three nuclear-encoded mitochondrial genes associated with hepatocerebral syndrome have been identified:
deoxyguanosine kinase (DGUOK), MPV17, and DNA polymerase gamma (POLG1).
Mutations in the POLG1 gene have been associated with autosomal recessive Alpers syndrome characterized by progressive
encephalopathy, ataxia, and liver failure; intractable seizures may be the predominant clinical feature. Liver histopathology in
patients with POLG1 mutations is variable, ranging from steatosis and mild fibrosis to marked fibrosis and cirrhosis, hepatocyte
degeneration, and bile duct proliferation. Muscle biopsy shows COX-deficient fibers and ragged-red fibers (71).
Liver failure is the major presenting symptom in patients with DGUOK or MPV17 mutations, although there is overlap in these
defects. In DGUOK deficiency, progressive liver disease leads to steatosis, siderosis, canalicular and hepatocellular cholestasis,
multinucleated giant cells, cirrhosis, and, in some patients, hepatocellular carcinoma; fatal infantile liver disease may occur with or
without encephalopathy (39). By EM, there is accumulation of mitochondria with reduced cristae. Mutations in MPV17, encoding
an inner mitochondrial membrane protein, have been reported in patients with infantile hepatic mtDNA depletion.
Diagnosis may be made by demonstration of depletion of mitochondrial DNA by real-time PCR analysis on liver or muscle tissue.
Also, DNA analysis of the POLG1, DGUOK, and MPV17 genes is available.
Other mitochondrial DNA depletion disorders include a myopathy associated with thymidine kinase (TK2) deficiency and
encephalomyopathy and anemia associated with mutations in SUCLA2 encoding the beta subunit of the ADPforming succinyl-CoA
synthetase ligase.
Nuclear DNA Mutations That Cause Primary Disorders or Disorders of Assembly of the Respiratory Chain
These disorders are generally inherited as autosomal recessive traits (91).
Complex I deficiency. NADH-coenzyme Q (CoQ, ubiquinone) reductase is the largest complex of the respiratory chain and
includes many polypeptides and several nonprotein components. Patients with abnormalities of complex I can present with an
infantile multisystem disease with lactic acidosis, psychomotor delay, hypotonia, exercise
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intolerance, and weakness. Cardiomyopathy occurs with childhood or adult-onset forms. Others have encephalomyopathy
beginning in childhood or adulthood, accompanied by ophthalmoplegia, seizures, dementia, ataxia, deafness, and sensory
neuropathy.
Complex II deficiency. Patients with succinate CoQ reductase defect have encephalomyopathy with exerciseinduced
myoglobinuria and lack of SDH activity in muscle biopsy. Some patients with complex II defects may present with the more severe
Leigh syndrome phenotype of subacute necrotizing encephalomyopathy.
Complex III deficiency. Defects in succinate cytochrome c reductase and NADH cytochrome c reductase (reduced CoQ-
cytochrome c reductase) present clinically with encephalomyopathy and myopathy or with cardiomyopathy (91). There is also an
autosomal recessive defect in complex III assembly caused by mutations in the BSC1L gene that presents with renal tubulopathy,
encephalopathy, and liver disease.
Complex IV (cytochrome c oxidase, COX) deficiency. Patients may present with myopathy or encephalopathy. The
encephalopathic form of cytochrome oxidase deficiency is the most common biochemical abnormality in Leigh syndrome (32), and
a number of mutations in genes encoding proteins with a role in cytochrome oxidase assembly have been identified (Table 5-16).
Defects of the Inner Mitochondrial Membrane

Coenzyme Q10 (CoQ10, ubiquinone) deficiency. CoQ10 is a lipid-soluble component of essentially all cell membranes and
functions as an electron and proton carrier. CoQ10 deficiency is caused by mutations in the COQ2 gene and is inherited as an
autosomal recessive disorder. CoQ10 deficiency can present in infancy with encephalomyopathy and renal dysfunction; later in life
as a myopathic form with myoglobinuria, exercise intolerance, and weakness; or may be associated with cerebellar atrophy, ataxia,
and seizures. It is treatable with CoQ10 supplementation. Muscle biopsy shows increased lipid and ragged-red fibers (32).
Barth syndrome is due to defects in the TAZ gene that encodes the protein tafazzin, which influences incorporation of cardiolipin,
an essential part of the inner mitochondrial membrane. This X-linked disorder causes mitochondrial myopathy with ragged-red
fibers, dilated cardiomyopathy with abnormal left ventricular compaction, and neutropenia (32).
HYPERAMMONEMIAS/UREA CYCLE DISORDERS

The urea cycle, important in liver and intestine, converts toxic nitrogenous waste into water-soluble urea. Blocks in this pathway
lead to hyperammonemia with neurotoxicity and cerebral edema. Presentation is variable: neonates can have hypotonia, seizures,
respiratory alkalosis, hyperammonemia, coma, and death (65). Cerebral or pulmonary hemorrhage may be the terminal event (78).
Alternatively, children or even adults with urea cycle defects can present when highprotein diet or infection precipitates symptoms.
Liver biopsy may be normal or show mild steatosis, glycogenated nuclei, occasional necrotic hepatocytes, or interface hepatitis,
and mild portal or bridging fibrosis. Mitochondria may be normal or have nonspecific pleomorphism and swollen or shortened
cristae (63, 65). Diagnosis is based on plasma or urine amino acid analysis.
Ornithine Transcarbamylase (OTC) Deficiency

This is the most common urea cycle disorder. It is inherited as an X-linked dominant disease with variably symptomatic
heterozygotes (55). A large number of mutations at Xp21 have been described. In the neonatal form, patients have emesis,
hyperammonemia, progressive encephalopathy, and focal neurological defects. Hemizygous boys typically present in the first days
of life, but heterozygous girls may be intermittently symptomatic. All patients with OTC deficiency patients are vulnerable to
valproate-associated hepatotoxicity (61), and OTC-deficient heterozygote mothers may have postpartum hyperammonemic
encephalopathy (65).
Liver may be normal even in fatal disease. Microscopically, islands of pale, water-clear hepatocytes, with lipid accumulation, are
characteristic but not unique (being seen in a variety of urea cycle defects) (61). Centrilobular microvesicular steatosis,
glycogenated nuclei, mild hepatitis, focal necrosis, cholestasis, mild periportal fibrosis, and, in severe cases, interface hepatitis and
bridging fibrosis have been described (65). Heterozygote girls may have portal fibrosis or a hepatitic pattern with inflammation and
piecemeal necrosis, steatosis, and fibrosis (64). By EM, lipid and pleomorphic, elongated, enlarged, or branching mitochondria
(some with paracrystalline inclusions and peroxisomal swelling and matrix rarefaction) are seen (29, 60, 61).
The brain is edematous with Alzheimer type II astrocytosis. Spongiosis with hypomyelination or cystic destruction of the cerebellar
hemispheres and mineralization of deep gray matter neurons occur in severely affected girls (29, 55). Others have described
ulegyria and atrophy of the cerebellar granular layer (73). Diagnosis is confirmed by mutation analysis, urine amino acid analyses
to identify increased orotic acid, and low leukocyte, fibroblast, or liver OTC.
Carbamoyl Phosphate Synthetase I Deficiency

Carbamoyl phosphate synthase is a mitochondrial enzyme responsible for the first part of urea synthesis (61). Deficiency is
inherited as an autosomal recessive disorder, and there are a lethal neonatal onset form and a delayed onset milder form with
episodic hyperammonemia (61). The liver is normal or has mild steatosis with focal necrosis and slight portal fibrosis (29).
Mitochondria may be normal or dilated
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and pleomorphic with increased matrix density (29, 61). The brain shows Alzheimer type II astrocytosis and spongiosis (29).
Diagnosis is confirmed by enzyme assay on liver tissue (29).
Citrullinemia
This disorder, due to autosomal recessive deficiency of argininosuccinic acid synthetase, leads to steatosis, focal hepatocellular
necrosis, and patchy cholestasis. By EM, numerous small mitochondria with increased matrix density and paracrystalline and
electron-dense bodies may be due to increased citrulline. Rough endoplasmic reticulum has a concentric profile and peroxisomes
may be increased (29, 140). Diagnosis is based on enzyme assay of fibroblasts or amniocyte cultures (29).
Argininosuccinic Aciduria
This autosomal recessive defect in argininosuccinate lyase (argininosuccinase) is associated with a nodular liver with severe
septal portal fibrosis or cirrhosis, steatosis, and focal hepatocellular necrosis. Ultrastructural mitochondrial abnormalities are not
seen (29). Older infants may have brittle hair with trichorrhexis nodosa, particularly if protein in the diet is low (29).
Argininemia
This autosomal recessive disease, due to arginase deficiency, presents later than other urea cycle disorders with spastic
paraplegia, dementia, dystonia, ataxia, and mental retardation (55). Hepatomegaly with steatosis and periportal fibrosis but with
normal mitochondria may be seen (43).
Hyperornithinemia, Hyperammonemia, Homocitrullinuria (HHH Disease)

This rare autosomal recessive disorder, due to mutations in the SLC25A15 gene, is characterized by impaired transfer of ornithine
from cytoplasm to mitochondria where it is normally converted to citrulline (29). Affected patients have increased plasma ornithine
concentration, homocitrullinuria, and hyperammonemia (124). Increased mitochondrial number and giant pleomorphic hepatocyte
mitochondria contain crystalline inclusions (29, 124). Abnormal mitochondria may also be found in muscle and leukocytes (43).
Lysinuric Protein Intolerance (LPI)

This disorder, due to a mutation in amino acid transporter gene SLC7A7, is inherited as an autosomal recessive trait (112). The
mutation results in lack of normal absorption and excessive loss of the cationic amino acids lysine, ornithine, and arginine with
resultant low plasma levels of these amino acids and increased urine lysine. Affected patients may have failure to thrive, feeding
difficulty, hepatomegaly, mental retardation, sparse hair, osteoporosis, hypo-tonia, and, in some cases, sudden unexpected death
in infancy (24). Episodic hyperammonemia is due to deficient ornithine.
Pathologic alterations include interstitial pneumonia, alveolar proteinosis, and pulmonary hemorrhage, and immune complex-
mediated IgA mesangial proliferative or membranous glomerulonephritis. In liver, periportal steatosis, fibrosis, and micronodular
cirrhosis, with decreased glycogen and nuclei with PAS-positive inclusions are described (83, 112). These changes may reflect
protein malnutrition (112). Pancreatic atrophy and fibrosis and hemophagocytic lymphohistiocytosis with macrophage activation
also occur (33, 112).
ORGANIC ACIDEMIAS
In these disorders, catabolism of amino acids, carbohydrates, or fatty acids is blocked due to an enzyme or cofactor deficiency with
resultant accumulation of organic acids, which can be identified by MS of urine (29). Clinically, the organic acidemias are
characterized by severe, progressive encephalopathy with coma, seizures, and death (Table 5-18). Liver changes are nonspecific
and include hepatomegaly and mild steatosis (65).
Propionic Acidemia
Actual or functional deficiency of the biotin-dependent enzyme propionyl-CoA carboxylase, inherited as an autosomal recessive
disorder, leads to tissue propionic acid accumulation. Patients can present with ketotic hyperglycinemia, and most have neonatal
progressive encephalopathy with coma, myoclonus, and early death. Later-onset forms have acute encephalopathy, anorexia,
failure to thrive, and developmental delay (55). Neutropenia, thrombocytopenia, acidosis, hyperammonemia, decreased free
carnitine, increased propionyl carnitine, and increased urine excretion of propionyl glycine and methylcitrate characterize this
disorder (55).
Hepatomegaly and steatosis are seen. By EM, liver mitochondria are enlarged with decreased cristae and amorphous matrix
material (43). In early-onset forms, spongy white matter degeneration occurs, particularly in the globus pallidus (55). In later-onset
patients, the basal ganglia have perivascular rarefaction in caudate with bilateral symmetrical encephalomalacia in the lentiform
nucleus, along with neuronal loss (55).
Methylmalonic Acidemia
Accumulation of methylmalonic acid in body fluids and urine is due to actual or functional deficiency of methylmalonylCoA mutase
activity. Clinically, patients are similar to those with propionic acidemia, and the pathology is nonspecific. Diffuse white matter
gliosis with Alzheimer type II astrocytosis has been described (43).
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Table 5-18 ▪ ORGANIC ACIDEMIAS
Common
Clinical Enzyme Substrate Laboratory
Name Presentation Deficiency Accumulation Evaluation Pathology
Propionic Neonate, Actual or Propionic acid (U, Ketoacidosis, Hepatomegaly,

acidemia vomiting, functional biotin- B) Hyperammonemia, steatosis,
respiratory dependent Hypoglycemia or spongy white
distress, propionyl-CoA Hyperglycinemia matter
hypotonia, carboxylase degeneration,
seizures, death particularly of
globus pallidus
Methylcitrate (U)
Tiglylglycine (U)
3- Neutropenia
hydroxypropionate Thrombocytopenia
(U)
Methylmalonic Similar to Actual or Methylmalonic Ketoacidosis, Spongy white

acidemia propionic functional acid (U, B) Hyperammonemia, matter
acidemia methylmalonylCoA Hypoglycemia degeneration
mutase
Methylcitrate (U)
Tiglylglycine (U) Hyperglycinemia
3- Neutropenia
hydroxypropionate Thrombocytopenia
(U)
Isovaleric Similar to Isovaleryl-CoA Isovaleric acid (B) Acidosis, Spongy white

acidemia propionic dehydrogenase matter
acidemia; May degeneration,
have later age steatosis
of onset
Isovalerylglycine Sweaty feet odor
(U) Pancytopenia
Glutaric Developmental Glutaryl-CoA Glutaric acid (U) Acidosis Frontotemporal

acidemia type delay, dehydrogenase atrophy,
1 macrocephaly, striatal
choreoathetosis, degeneration,
dystonia white matter
spongiosis,
subdural
hematoma,
steatosis,
myocardial and
smooth muscle
and renal
tubular
epithelium fat
deposition
3-OH-glutaric acid
(U)
U, urine; B, blood.
Isovaleric Acidemia
Deficiency of isovaleryl-CoA dehydrogenase is inherited as an autosomal recessive disorder and causes patients to be acidotic
and have a sweaty feet odor (43). Patients may have pancytopenia, white matter spongiosis, and hepatic steatosis (43).
Glutaric Acidemia Type I

Glutaryl-CoA dehydrogenase (GCDH) deficiency is inherited as an autosomal recessive trait and results in choreoathetosis and a
severe dystonic-dyskinetic syndrome. Rarely, this organic acidemia can present with subdural hematoma and bilateral retinal
hemorrhage that mimic shaken baby syndrome (55, 93). The CNS is remarkable for frontotemporal atrophy, striatal degeneration,
and white matter spongiosis (55). Steatosis and lipid deposition in myocardium and renal tubular epithelium are seen. Diagnosis is
based on increased glutaric and hydroxyglutaric acid in urine and decreased GCDH in fibroblasts; molecular diagnosis is possible
(43).
HYPERLACTATEMIA, LACTIC ACIDEMIA

The normal lactate:pyruvate ratio in human plasma is 10:1 to 25:1. Increased lactate can be due to nongenetic disease (including
tissue necrosis and sepsis), but there are also primary genetic disorders of lactate metabolism.
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Pyruvate dehydrogenase (PDH) deficiency. The clinical spectrum of PDH deficiency ranges from fatal lactic acidosis in the
newborn to chronic neurologic dysfunction with structural abnormalities of the brain without systemic acidosis. The most common
defect affects the X-linked E1-alpha subunit of the PDH complex. Affected males may have a thin callosum or callosal agenesis,
polymicrogyria, and facial dysmorphism resembling fetal alcohol syndrome. Liver shows diffuse microvesicular steatosis, and the
brain may show Leigh encephalopathy (43). Female heterozygotes may have as severe a presentation as affected males (Figure
5-22A to C) or may be normal. The E1-beta, E2, and E3 subunits of the complex are encoded by autosomal genes, and
deficiencies are inherited as autosomal recessive traits. If low PDH activity is found in leukocytes or fibroblasts, the activities of the
individual PDH components—E1, E2, and E3—can be assayed for further classification, and DNA analysis is available for the E1-
alpha gene.
Pyruvate carboxylase deficiency. This autosomal recessive disorder results in persistent lactic acidosis and hepatomegaly.
There is hepatocyte swelling with prominent nucleoli, steatosis, cholestasis, ductular proliferation, and mild portal fibrosis with
acinar transformation (43, 63). Other clinical features can include mental retardation, developmental delay, hypotonia, and
seizures. Some patients present with cystic lesions of the brain consistent with Leigh syndrome, along with neuronal loss in the
cerebral cortex, poor myelination, astrocytosis, thin callosum, and periventricular leukomalacia (43).
FIGURE 5-22 ▪ Pyruvate dehydrogenase deficiency. A-C: Magnetic resonance imaging of a girl who was heterozygous for
pyruvate dehydrogenase deficiency. Note atrophy, abnormal gyral formation, and absence of the corpus callosum.
PEROXISOMAL DISORDERS
Although peroxisomes were named for their peroxide-based reactions, they have many other important metabolic biosynthetic and
degradative functions (131). Peroxisomes are spherical with a diameter of 0.1 to 1 mm, bound by a single lipid bilayer. They are
larger and more abundant in liver and kidney than in other tissues. In general, peroxisomal disorders can be identified by increased
tissue and body fluid very-long-chain fatty acids (VLCFA), decreased plasmalogen, and increased phytanic acid levels (43). EM
may be useful in defining the number and size of peroxisomes and in identifying trilaminate inclusions thought to be related to
VLCFA deposition (29). Peroxisomal disorders can be divided into two groups (Table 5-19) (131).
Peroxisomal Biogenesis Disorders

In patients with abnormal peroxisomal biogenesis, there is a reduced number of peroxisomes because of defective peroxisomal
assembly, and there is defective function of multiple peroxisomal enzymes (43, 65). The Zellweger
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syndrome spectrum is caused by defects in any of the PEX genes required for normal peroxisome assembly (15, 99, 131) and
includes the first three diseases listed below. These disorders have similar biochemical findings. At least 10 genetic defects may
be responsible for these disorders.
Table 5-19 ▪ PEROXISOMAL DISORDERS
Peroxisomal biogenesis disorders
Zellweger syndrome (cerebrohepatorenal syndrome)
Neonatal adrenoleukodystrophy
Infantile Refsum disease
Rhizomelic chondrodysplasia punctata type 1

Single peroxisomal enzyme deficiencies
X-linked adrenoleukodystrophy
Acyl-CoA oxidase deficiency (pseudo-neonatal adrenoleukodystrophy)
D-bifunctional protein deficiency
2-Methylacyl-CoA racemase deficiency
Sterol carrier protein X deficiency
Refsum disease
Hyperoxaluria type I (oxalosis 1).
Zellweger (cerebrohepatorenal) syndrome is the most severe of the biogenesis disorders. An autosomal recessive disease, it
presents in neonates with metabolic abnormalities; distinctive facial dysmorphology; severe hypotonia; failure to thrive; mental
retardation; seizures; ocular, genital, and cardiovascular malformations; renal glomerular cysts; and calcific stippling of the
patellae.
In the brain, premature arrest of migrating neuroblasts during development results in site-specific cerebral microgyria and
pachygyria with neuronal heterotopia, an abnormal convolution pattern and olivary dysplasia. Liver initially shows a hepatitic
pattern, with hepatocellular unrest, focal necrosis, steatosis, and canalicular and cytoplasmic cholestasis with pseudoacinar and
giant cell transformation. Lymphocytes and macrophages accumulate in sinusoids and portal spaces. Intrahepatic bile ducts may
be normal, decreased, or hyperplastic. With time, the liver becomes firm and fibrotic with micronodular cirrhosis (65). By EM,
peroxisomes are absent or rare in liver and kidney (60, 65). The pathogenesis of the liver injury is unknown but may relate to injury
by abnormal bile acids (65).
Neonatal adrenoleukodystrophy is an autosomal recessive disorder that is less severe than Zellweger syndrome and is
characterized by hypotonia, craniofacial dysmorphism, adrenocortical atrophy, and psychomotor deterioration. The brain shows
progressive dysmyelination/demyelination of cerebral and cerebellar white matter, and polymicrogyria. Hepatic peroxisomes are
reduced in number and size (43, 60). PAS-positive macrophages with angulate lysosomes are present in viscera and brain.
Infantile Refsum disease patients resemble those with Zellweger syndrome with hypotonia, seizures, mental retardation, hearing
loss, and dysmorphic facies. Their course is milder than seen in Zellweger syndrome or neonatal adrenoleukodystrophy (84).
Hepatomegaly with fibrosis and portal-to-portal bridging is seen and, by EM, peroxisomes are deficient or very small (60).
Lysosomal PAS-positive trilaminate inclusions with two dense outer and an inner lucent lamellae and an outer thickness of 6 to 14
mm accumulate first in macrophages and then in hepatocytes and suggest VLCFA storage (63, 84). An elevated phytanic acid,
trihydroxycoprostanoic acid, pipecolic acid, and VLCFA and decreased phytanic acid oxidase are laboratory abnormalities (60).
Rhizomelic chondrodysplasia punctata type 1 is a peroxisomal disorder that is genetically and biochemically distinct from
Zellweger syndrome and is due to a defect in a peroxisomal targeting gene that affects importation of enzymes into peroxisomes
(29). Patients have impaired plasmalogen synthesis and phytanic acid oxidation, severe proximal limb shortening, calcific stippling
in hyaline cartilage, cataracts, renal dysfunction, facial dysmorphism, ichthyosis, and death in childhood. Hepatocytes may have
absent or large irregularly shaped peroxisomes (43). Plasma phytanic acid is increased, and RBC and tissue plasmalogen are
decreased (29) (see Chapter 27).
Single Peroxisomal Enzyme Deficiency

In these disorders, peroxisome structure is intact (43).
X-linked adrenoleukodystrophy. Very-long-chain fatty acyl-CoA synthetase is impaired and plasma and fibroblast VLCFA are
increased due to impaired capacity to degrade these molecules (43). Progressive behavioral, cognitive, and neurologic
deterioration occurs with a 1/21,000 incidence in boys. Adrenal glands are small, and zona fasciculata cells contain cytoplasmic
lipid inclusions with a lamellar pattern (44). Lipid accumulation in CNS causes demyelination with a perivascular lymphocytic
infiltrate that resembles that seen in multiple sclerosis (131). Rarely, sheaves of thin hollow needles in Schwann cells in skin are
seen (30).
Acyl-CoA oxidase deficiency (pseudo-neonatal adrenoleukodystrophy) presents clinically with hypotonia, psychomotor
retardation, sensory deafness, hepatomegaly, retinopathy and with or without mild craniofacial dysmorphism. Atrophy of skeletal
muscle, brain stem ganglia, and cranial nerves along with hepatic fibrosis and large PASpositive, sudanophilic adrenal
macrophages, and brain astrocytes are described in this disorder (131). Hepatic peroxisomes are heterogeneous in size and
increased in number.
Rhizomelic chondrodysplasia punctata type 2, due to dihydroxyacetonephosphate acyltransferase deficiency, presents with
craniofacial abnormalities, hypotonia, cataracts, mental retardation, eczema, cerebral atrophy, and dwarfism with rhizomelic
shortening of the upper arms but no stippled patellar calcifications.
Rhizomelic chondrodysplasia punctata type 3, due to alkyl dihydroxyacetonephosphate synthase deficiency, is clinically
similar to rhizomelic chondrodysplasia punctata type 1.
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D-bifunctional protein deficiency presents with neonatal hypotonia, seizures, failure to thrive, visual abnormalities, psychomotor
delay, and dysmorphism that resembles that seen in Zellweger syndrome (131).
2-Methylacyl-CoA racemase deficiency has a variable clinical course, with adult-onset sensory motor neuropathy and fulminant
liver disease described (131).
Sterol carrier protein X (SCPx) deficiency. Dystonic tremor, hypergonadotropic hypogonadism, and sensory motor neuropathy
are seen in this rare disorder (131).
Refsum disease is due to autosomal recessively inherited phytanoyl-CoA hydroxylase deficiency. Patients have deterioration of
night vision, retinitis pigmentosa, anosmia, deafness, ataxia, and arrhythmias.
Hyperoxaluria type I (oxalosis 1). Hepatic peroxisomal alanine:glyoxylate aminotransferase is deficient (61). High urine oxalate,
progressive oxalate nephrocalcinosis, and oxalate urolithiasis are seen, and calcium oxalates deposit in many sites including bone,
soft tissue, eye, and cardiac conduction system (61). The patients may have renal failure, recurrent fractures, and cardiac
arrhythmias, and die before 20 years of age (43). The liver is grossly normal, but oxalate crystals may be present in the hepatic
arterioles and gallbladder (60, 61). The crystals have a rosette pattern with radial striations and a star-burst appearance seen with
polarized light (43).
METAL METABOLISM ABNORMALITIES

Neonatal Hemochromatosis (Neonatal Iron Storage Disease)
This is one of the most commonly recognized causes of liver failure in the neonate. The disease is inherited as an autosomal
recessive or codominant trait. Liver injury begins in utero and can result in stillbirth, hydrops, or severe neonatal liver disease, and
extrahepatic siderosis. Infants can have intrauterine growth restriction, polyhydramnios, or oligohydramnios and prognosis is poor
(43, 61, 63). The underlying metabolic defect is unknown, but some cases may be an alloimmune gestational disease (136).
Alternatively, iron may be a marker for massive intrauterine liver destruction and iron redistribution (61).
FIGURE 5-23 ▪ Neonatal hemochromatosis. A,B: Widespread hemosiderin accumulation in hepatocytes as shown by H&E (A)
and Prussian blue (B) stains in neonatal hemochromatosis. (Images' courtesy of Elizabeth Brunt, M.D., Washington University,
Department of Pathology and Immunology, St. Louis, Missouri).
The liver morphology is that of extensive hepatocellular loss. Cirrhosis occurs in a small bile-stained liver (136). Central veins may
have obliterative fibrosis extending into sinusoids (61, 136). Regenerative nodules may be present and, in other cases, almost no
hepatocytes remain. Residual hepatocytes show giant cell or pseudoacinar transformation with canalicular bile plugs and acute
and chronic inflammation. Hepatocytes may have coarsely granular siderosis (Figure 5-23A,B), but Kupffer cells do not accumulate
iron (136). By EM, hemosiderin is present in hepatocyte lysosomes.
Extrahepatic iron storage is not associated with organ dysfunction, and the fixed tissue macrophage system is spared (61, 136).
Hypertrophy and hyperplasia of the islets of Langerhans accompanies pancreatic acinar and islet cell iron accumulation. Iron also
accumulates in myocardium, oropharyngeal and respiratory submucosal glands, renal tubule epithelium, adrenal cortex, thyroid
follicular epithelium, and other sites (136). Biopsy of oral submucosal glands can be used to demonstrate siderosis.
Wilson Disease (Hepatolenticular Degeneration, ATP7B Disease)

This autosomal recessive inherited mutations of the ATP7B gene result in copper retention in liver and other organs. Incidence is
approximately 1:30,000, and 1:90 are heterozygous carriers. Over 200 mutations in the ATP7B gene have been identified. Most
Wilson disease patients are compound heterozygotes (5). Copper homeostasis is controlled by the gene product ATP7B-ATPase,
expressed in the canalicular membrane and involved in incorporation of copper into apoceruloplasm to form ceruloplasmin with
subsequent biliary excretion (65). Absent or reduced ATP7B-ATPase function leads to hepatic copper retention by interfering with
biliary copper excretion and incorporation into ceruloplasmin (5).
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Progressive copper ion accumulation is hepatotoxic and also injures kidneys, brain, and cornea. The unbound copper ion may act
as a free radical, damaging cell and organelle membranes, causing oxidative injury to membranes, depletion of mitochondrial
glutathione, and DNA-copper, and DNAprotein complexes that may result in DNA mutations (65).
Patients present in the 1st or 2nd decade with hepatomegaly, acute or chronic hepatitis, cirrhosis, or acute fulminant liver failure
often accompanied by hemolytic anemia; hepatic symptoms are more common in children (5). Copper measurement confirms the
diagnosis — the copper content is greater than 250 μ/g dry weight (normal <50 μg/g). It is important to select for analysis tissue not
affected by fibroconnective tissue scar (65). Tissue copper quantitation can be done on 1 to 2 mg of fresh or frozen tissue (about
0.5 cm of a liver biopsy obtained with a 14-gauge needle or 1 cm of a biopsy obtained with an 18-gauge needle) or on tissue
excised from the paraffin block (5, 81). Copper levels may be normal in advanced Wilson disease due to variability in copper
distribution in fibrotic liver or due to release of copper from necrotic hepatocytes in fulminant hepatic failure (5, 61). Serum
ceruloplasmin levels should be measured if Wilson disease is suspected. The level is less than 20 mg/dL in most affected patients.
However, ceruloplasmin is an acute phase reactant, and an elevated or normal level does not rule out Wilson disease (5).
The liver initially shows glycogenated nuclei (in periportal hepatocytes) and steatosis, followed by periportal mononuclear
inflammation with focal hepatocyte necrosis, fibrosis, hepatocyte swelling, and cholestasis. Chronic active hepatitis with marked
portal inflammation and interface hepatitis may occur with progression to cirrhosis with cholestasis and small neocholangiole
proliferation around the nodules (Figure 5-24A). Mallory bodies may be present in periportal hepatocytes (5, 61, 65). In some
children, fulminant acute hepatitis with submassive or massive hepatocellular necrosis can occur (61). Kupffer cells may contain
hemosiderin, particularly if there is a hemolytic crisis.
FIGURE 5-24 ▪ Wilson disease. A: Micronodular cirrhosis in late-stage Wilson disease: regenerative hepatocellular nodules (red)
contrast with diffuse bridging fibrosis (blue) (trichrome). B: In late-stage Wilson disease, copper accumulation is apparent as
cytoplasmic pigment within periportal hepatocytes (H&E).
Cytoplasmic copper is soluble and does not stain with copper and copper-associated protein stains (Table 5-2). Thus, special
stains may be negative for copper in the early precirrhotic stage of disease, becoming positive when lysosomal copper
accumulates (5, 65). When identified by stains, copper is generally in periportal hepatocytes in children and in panlobular
hepatocytes in patients with more advanced disease (Figure 5-24B,C) (5, 65). Copper accumulates in Kupffer cells as it is
released from hepatocytes and nodules may have a variable amount of copper (64).
By EM, mitochondria are enlarged, pleomorphic, with increased matrix density, separation of inner and outer membranes, and
widened intracristal spaces with dilatation and microcyst formations at the tips of cristae (Figure 5-24D). Electron-dense granular
copper accumulates in mitochondria and lysosomes (65). Nonimmune hemolytic anemia is associated with macrophage
erythrophagocytosis and fulminant liver failure (5). Copper deposition in the brain leads to neuropsychiatric symptoms, and corneal
Kayser-Fleischer rings are characteristic. Neurologic symptoms are more common in adults and reflect basal ganglia cavitary
degeneration, gliosis, neuronal loss, and copper accumulation. The mechanisms causing basal ganglia damage with relative
sparing of the cortex in Wilson disease, despite the diffuse increase in copper throughout the CNS, are unknown (22).
Menkes Disease (Menkes Kinky Hair Syndrome)

This X-linked recessive disorder of copper transport is caused by mutations in the ATP7A gene at Xq13.3. It is rare, with an
estimated incidence of 1:250,000, and is characterized by profound neurodevelopmental deterioration, seizures, poor growth,
hypothermia, brittle and kinky hair, hypopigmentation, and connective tissue abnormalities. Most affected boys die within the first 3
years of life with
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progressive neurological deterioration. Carrier females may have abnormal hair. ATP7A encodes an intracellular
coppertransporting ATPase, and patients have defective intestinal copper absorption, leading to copper deficiency.
FIGURE 5-24 ▪ (continued) C: Copper stain showing abundant hepatocellular copper accumulation in late-stage Wilson disease
(Rhodanine). D: Ultrastructurally, Wilson disease shows mitochondrial abnormalities including widening of space between cristae
and dilatation with microcystic expansion of the tips of cristae (D: Uranyl acetate, lead citrate).
Multiple copper-dependent enzymes become secondarily deficient, resulting in the multiple phenotypic features. One of the
deficient copper-dependent enzymes, lysyl oxidase, is involved in collagen and elastin crosslinking; the multiple connective tissue
defects in Menkes disease presumably are due to deficiency of lysyl oxidase and include loose and redundant skin,
hyperextensible joints, vessel (including intracranial vessel) tortuosity and ectasia, emphysema, hypoplasia of arteries (including
aorta and pulmonary arteries), bladder and bowel diverticula, gastric polyps, and bone fragility (with recurrent fractures and
osteopenia) (22). The skeletal changes have been confused with those seen in child abuse.
Hair is normal at birth but is replaced by about 6 weeks of age with sparse, hypopigmented, brittle, twisted hair. Microscopic hair
examination shows pili torti. Hypopigmentation is related to deficiency of tyrosinase, with decreased melanin production. Deficiency
of the copper-dependent enzymes cytochrome c oxidase, dopamine β-hydroxylase, and peptidyl a-monoxygenase is thought to be
responsible for the neurodevelopmental problems in Menkes disease.
The cerebral cortex and the cerebellum have abnormal myelination, progressive gliosis, and atrophy. Blood vessels have
disruption of elastic lamina. Brain at autopsy may show subdural hematomas and diffuse atrophy, focal gray matter degeneration,
and cerebellar neuronal loss. The Purkinje cells show abnormal dendrite arborization and axonal swelling.
Both serum copper and ceruloplasmin levels are low in Menkes disease. Deficiency of dopamine-b-hydroxylase leads to increased
plasma dihydroxyphenylalanine (DOPA), dihydroxyphenylacetic acid (DOPAC), and dopamine, and to reduced norepinephrine and
dihydroxyphenylglycol (DGPG). Since copper and ceruloplasmin are relatively low in normal newborns and young infants,
measurement of these plasma catecholamines can provide an earlier definitive diagnosis of Menkes disease.
Treatment with subcutaneous copper histidine injections and other forms of copper replacement have been attempted with limited
success. Serum copper and ceruloplasmin levels are corrected, but unless started very early in life, copper supplementation has
not been able to prevent progressive neurological complications.
DISORDERS OF THE ENDOPLASMIC RETICULUM

These disorders include alpha-1-antitypsin deficiency, congenital disorders of glycosylation, and GSD I (29).
Alpha-1-antitypsin (A1AT) Deficiency

A1AT, synthesized primarily by hepatocytes, is an acute phase reactant glycoprotein that accounts for most alpha1-globulins in
serum. Its principal function is inhibition of proteases in lung. In A1AT-deficient patients, proteolytic enzymes released during
inflammation are not inhibited normally, leading to lung damage (67, 117). A1AT deficiency is the most common pediatric genetic
liver disease with an incidence of 1/600 to 1/2,000 (5, 67, 99). More than 100 alleles of the A1AT gene have been identified, each
inherited as an autosomal codominant (5, 99).
Serum A1AT level should be determined in children with neonatal hepatitis or undefined liver disease. Levels in A1AT
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deficient children are usually 10% to 40% of normal (65). Diagnosis and genotype are confirmed by protease i nhibitor (Pi)
phenotyping. The abnormal proteins are designated alphabetically, based on isoelectric focusing electrophoresis relative to the
normal protein, PiM. The two most common deficiency variants are the S and Z alleles (5). PiZZ genotype patients have liver and
lung disease with serum A1AT levels less than 20% of normal. The Z allele is present in 1% to 2% of Caucasians of northern
European ancestry and is virtually absent in African-Americans and Asians (99).
FIGURE 5-25 ▪ Alpha-1-antitrypsin deficiency. A: PAS-positive, diastase-resistant globules of alpha-1-antitrypsin expand the
cytoplasm of periportal hepatocytes in a patient with alpha-1-antitrypsin deficiency (PASdiastase). B: Immunoperoxidase staining
for alpha-1-antitrypsin highlights enzyme accumulation (red-brown) in periportal hepatocytes in a patient with alpha-1-antitrypsin
deficiency (alpha-1-antitrypsin immunoperoxidase).
In patients with ZZ phenotype, a point mutation in the SERPINA 1 gene leads to aggregation and polymerization of the abnormal
protein (5). The misfolded protein cannot be secreted normally, and A1AT accumulates in the endoplasmic reticulum, leading to
serum A1AT deficiency with resultant lung damage. Liver accumulation and delayed degradation of misfolded A1AT leads to
hepatocyte injury, and, in some patients, hepatocellular carcinoma; the mechanism of hepatotoxicity is unknown (65). The lack of
antiprotease function of bile in A1AT deficiency may make patients prone to epithelial injury during ascending infection (29).
Several patterns of liver injury are seen in A1AT-deficient infants (5). Only 10% to 20% of PiZZ infants develop cholestatic liver
disease, and the prognosis is variable (61, 121). Intense lymphocytic portal and lobular inflammation with bile plugs, acinar
formation, and mild giant cell transformation resembles neonatal hepatitis. Giant cell transformation is generally not as prominent in
A1AT deficiency as in viralrelated neonatal hepatitis or biliary atresia (67). Extensive bile duct proliferation, bile plugs, and varying
fibrosis may suggest biliary atresia. A less common ductopenic pattern has paucity of bile ducts (5). With progression, inflammation
may resolve, periportal steatosis is common, and cirrhosis develops with large hyperplastic regenerative nodules (61).
Numerous eosinophilic, 1 to 40 mm PAS-positive, diastase-resistant round hyaline-like globules with peripheral clearing in
periportal hepatocytes, hepatocytes adjacent to fibrous septa, and bile duct epithelium are seen, generally after 3 to 4 months of
age, although they can be identified immunohistochemically earlier (but hepatocytes in unaffected infants may also stain similarly)
(Figure 5-25A,B) (5, 65, 67). These globules are abnormal A1AT retained in rough endoplasmic reticulum. In young infants, diffuse
staining without distinct globules occurs in periportal hepatocytes (67). The globules can also occur in heterozygotes (e.g., PiMZ)
with no liver disease and are not specific for the diagnosis of A1AT deficiency (5, 67). By EM, electron-dense finely granular
material distends endoplasmic reticulum even in infants.
Several forms of glomerulonephritis occur in patients with A1AT deficiency-associated liver disease. Immunoglobulin, complement,
and A1AT accumulate in a subendothelial location in glomeruli, and IgA deposition is also seen (23, 99, 120).
Replacement therapy with purified human A1AT is helpful for progressive lung disease but is ineffective in treatment of liver
disease (which is due to accumulation of A1AT rather than its deficiency). Liver transplantation cures the liver disease and corrects
the deficiency (5).
Congenital Disorders of Glycosylation (CDG) (Formerly Known as Carbohydrate-Deficient Glycoprotein

Syndromes)
Congenital disorders of glycosylation (CDG) are a heterogeneous group of multisystem disorders caused by abnormal
glycosylation of N-linked oligosaccharides. The wide range of phenotypes associated with CDGs has resulted in underrecognition
of these disorders. All known CDGs are inherited as autosomal recessive traits and are due to enzymatic defects. Because of the
abnormal assembly or processing of carbohydrate moieties of glycoconjugates, there is abnormal glycosylation of glycoproteins
(25, 29, 38). Diagnosis is usually made by analyzing the glycosylation status of transferrin, most commonly by isoelectric focusing,
chromatography, or capillary electrophoresis. Newer methodology using mass spectrometry is likely to be more informative.
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Subsequent confirmatory testing by enzyme assay, DNA analysis, glycan analysis on fibroblasts, or complementation assays is
required if the screening test is abnormal. At least 25 subtypes due to deficiencies in enzymes that function in the endoplasmic
reticulum and Golgi have been identified (Table 5-20) (25, 55). The CDGs are classified into two major groups, CDG-I and CDG-II.
CDG-I patients have a defect in assembly of N-glycan moieties in the cytosol and endoplasmic reticulum. CDG-Ia is the most
common CDG and is caused by deficient phosphomannomutase (25, 55). Patients have a variable phenotype with developmental
delay, hypotonia, ataxia, stroke-like episodes, seizures, and facial dysmorphism (Table 5-20). Nonimmune hydrops has also been
described in CDG-Ia (25). In CDG-Ib, the liver is affected; CDG-Ib patients may have cyclic vomiting, hypoglycemia, failure to
thrive, liver fibrosis, and protein-losing enteropathy, without brain involvement (Table 5-20). This form responds to oral mannose
supplement and is the only currently successfully treatable type of CDG.
CDG-II patients have a defect in processing of N-glycan moieties of glycoconjugate in the Golgi (38, 55). Clinical findings are
variable depending upon the type and include dysmorphism, microcephaly, liver dysfunction, hypotonia, severe mental retardation,
seizures, lipodystrophy, hypertrophic cardiomyopathy, peripheral neuropathy, and hydrops fetalis (Table 5-20) (38). CDG-IIb, IIc,
and IIf have normal transferrin glycosylation and thus are more difficult to diagnose.
In CDG patients, there is often severe neonatal onset olivopontocerebellar atrophy with Purkinje and granule cell depletion;
atrophy of cerebellar white matter; loss of pontine nuclei, inferior olives, and middle and inferior cerebellar peduncles with relative
sparing of dentate neurons (55). Outside the CNS, pleural and pericardial effusions, hypertrophic obstructive cardiomyopathy,
cystic dilatation of renal tubules and collecting ducts, steatosis, ductal plate abnormalities, portal fibrosis, cirrhosis, and retinal
dystrophy with pigment epithelium degeneration are seen (21, 55). Many patients have a coagulopathy due to low levels of clotting
factors, although it is usually not clinically significant except during surgery or following trauma.
LIPID METABOLISM DISORDERS

Smith-Lemli-Opitz Syndrome
This autosomal recessive disorder of cholesterol biosynthesis is due to deficiency in 7-dehydrocholesterol reductase. The defect in
cholesterol synthesis leads to abnormal development with a variable phenotype that can include microcephaly, mental retardation,
hypotonia, dysmorphic facies, cleft palate, ambiguous genitalia, and congenital heart disease (44). Plasma cholesterol is reduced;
patients with very low cholesterol levels have a more severe phenotype, and cholesterol deficiency may have a role in
dysmorphogenesis (29).
Morphological findings include reduced myelination of the cerebral hemispheres and cranial and peripheral nerves, absent corpus
callosum, cerebellar hypoplasia, abnormal gyral pattern, and altered neuronal migration. There is pancreatic enlargement and islet
cell nuclear hyperchromasia (44), and liver has nonspecific changes (Figure 5-26A,B) with cholestasis.
Conradi-Hunermann Syndrome (CDPX2)

This X-linked dominant disorder of sterol metabolism is caused by a deficiency of sterol-D8-isomerase that converts 8-
dehydrocholesterol to 7-dehydrocholesterol, one step before the enzymatic step that is abnormal in Smith-LemliOpitz syndrome.
Typical features include chondrodysplasia punctata, bilateral and asymmetrical limb anomalies, joint contractures, ichthyosiform
skin lesions, patchy alopecia, and short stature. The skin lesions are most severe at birth and histologically show hyperkeratosis,
parakeratosis, and marked acanthosis. They improve with age, leaving behind follicular atrophoderma (“orange peel” lesions) and
hypopigmented streaks that follow Blaschko's lines. It was originally thought that this defect would be lethal in males; however,
there have been two affected males reported (one of whom was a somatic mosaic for the genetic defect, and the other of whom
had a very severe phenotype). Plasma sterol analysis shows elevated 8-dehydrocholesterol and 8(9)-cholestenol. Blood
cholesterol levels are typically normal (see Chapter 27).
CHILD Syndrome (Congenital Hemidysplasia, Ichthyosis, and Limb Defects)

This is another rare X-linked dominant disorder of sterol metabolism characterized by unilateral ichthyosiform skin lesions, often
sharply demarcated at the midline of the trunk; limb deficiencies; and punctate calcifications of the epiphyses and other
cartilaginous structures. In addition, ipsilateral visceral anomalies can be present, including brain, renal, lung, and cardiac defects.
While some of the skin lesions follow Blaschko lines, many patients have more extensive patches of abnormal skin. It is presumed
generally lethal males. In most patients, it is caused by a defect in the NSDHL [NAD(P)H steroid dehydrogenase-l ike] gene, which
encodes the 3β-hydroxysteroid dehydrogenase component of the sterol-4-demethylase protein, one step above the defect for
CDPX2 in the cholesterol biosynthesis pathway. A few patients with the CHILD phenotype have been found to have a defect in
sterol-D8-isomerase as in CDPX2.
The skin abnormality in the NSDHL defect is typically a persistent scaly, raised, and sometimes verrucous lesion. Skin biopsy in
the NSDHL defect shows marked ichthyosiform epidermal hyperplasia, inflammation, and sometimes foamy histiocytes within the
dermal papillae (referred to as verruciform xanthoma). The lesions may regress with age. Sterol analysis of plasma, lymphocytes,
and fibroblasts shows abnormally increased levels of 4-methylsterols.
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Table 5-20 ▪ CARBOHYDRATE-DEFICIENT GLYCOPROTEIN DISORDERS
CDG Gene Protein Major Clinical Comments

Subtype Features
CDG-Ia PMM2 Phosphomannomutase II Mental retardation Most common form of CDG

(MR) Incidence may be 1:20,000
Seizures Enzyme and mutation analysis
Hypotonia clinically available
Strabismus No treatment available
Abnormal fat Occasionally normal transferrin
distribution electrophoresis
Inverted nipples
Coagulopathy
Cerebellar
hypoplasia/atrophy
Ataxia
Hydrops
CDG-Ib MPI Phosphomannose isomerase Hepatic fibrosis Treatment with oral mannose
Coagulopathy resolves symptoms
Protein-losing No brain involvement; normal
enteropathy development
Hypoglycemia
Cyclic vomiting
CDG-Ic ALG6 Glucosyltransferase I Dol-p-Glc: MR Second most common form of

Man(9)GlcNAc(2)-PP-dolichyl Seizures CDG
glucosyltransferase Hypotonia
Strabismus
CDG-Id ALG3 Dolichyl-P- Severe MR

Man:Man(5)GlcNAc(2)PP- dolichyl Seizures;
mannosyltransferase hypsarrhythmia
Optic nerve
atrophy
Iris colobomas
CDG-Ie DPM1 Dolichol-phosphate mannose Severe MR

synthetase I Seizures
Hypotonia
Coagulopathy
CDG-If MPDU1 Mannose-P-dolichol utilization defect MR Episodes of hypertonia

1/Lec 35 Pigmentary
retinopathy
Short stature
Ichthyosis
CDG-Ig ALG12 Dolichyl-P- MR

Man:Man(7)GlcNAc(2)PP-dolichyl Hypotonia
mannosyltransferase Microcephaly
Frequent
infections
CDG-Ih ALG8 Glucosyltransferase II Dolichyl-PGlc: Hepatomegaly

Glc(1)Man(9)GlcNAc(2)-P-PDol Protein losing
glucosyltransferase enteropathy
Coagulopathy
Renal failure
CDG-Ii ALG2 Mannosyltransferase II MR

GDPMan:Man(1)GlcNAc(2)-P-P-Dol Severe seizures
mannosyltransferase Coloboma of eye
Coagulopathy
CDG-Ij DPAGT1 UDP-GlcNAc: Dolichol phosphate N- Severe MR

acetylglucosamine-1 phosphate Seizures
transferase Hypotonia
Microcephaly
CDG-Ik ALG1 Mannosyltransferase I GDPMan: Severe MR

GlcNAc(2)-PP-Dol Hypotonia
mannosyltransferase Microcephaly
Coagulopathy
Nephrotic
syndrome
CDG-Il ALG9 Mannosyltransferase Dol-P-Man: Severe
Man(6)- and Man(8)-GlcNAc(2)-PP- microcephaly
Dol mannosyltransferase Seizures
Hypotonia
Hepatomegaly
CDG-IIa MGAT2 GlcNAc transferase II MR Normal cerebellum

Seizures
Dysmorphic facies
CDG-IIb GCS1 Glucosidase I Hepatomegaly; Normal transferrin isoelectric

hepatic fibrosis focusing
Seizures
Hypotonia
Dysmorphic facies
CDG-IIc SLC35C1 GDP-fucose transporter 1 MR Normal transferrin isoelectric

Hypotonia focusing May be treatable with
Microcephaly fucose supplementation
Frequent
infections;
persistently
elevated
peripheral
leukocyte count
CDG-IId B4GALT Beta-1,4-galactosyltransferase 1 Hypotonia due to

1 myopathy
Spontaneous
hemorrhage
Dandy-Walker
malformation with
hydrocephalus
CDG-IIe COG7 Conserved oligomeric Golgi complex Severe seizures Fatal in infancy
subunit 7 Hepatomegaly
Progressive
jaundice
Frequent
infections
Cardiac failure
Dysmorphic facies
CDG-IIf SLC35A1 CMP-sialic acid transporter Thrombocytopenia Normal transferrin isoelectric

Abnormal platelet focusing
glycoproteins No neurological abnormality
Sudden Death in Infants with Inborn Errors of Metabolism

Sudden unexpected death in infancy (SUDI) is defined as sudden unexpected death occurring before the age of 12 months (137).
Many metabolic disorders can cause SUDI or acute metabolic crisis in infants, in some cases without preceding clinical symptoms
(Table 5-15) (93). Key to the clinical history of a metabolic error presenting in the young infant is deterioration in clinical status
after a symptom-free interval of hours to days (29). Findings that may indicate an IEM include family history of a similar sudden
death, particularly in a sibling; dysmorphic features; enlarged liver, spleen, or heart; fatty or pale liver, heart, muscle, or kidney; or
cerebral edema (16).
FIGURE 5-26 ▪ Smith-Lemli-Opitz syndrome. A,B: Hepatocellular disarray in Smith-Lemli-Opitz syndrome (H&E).
FIGURE 5-26 ▪ (continued) C,D: Hepatocytes with multiple cytoplasmic whorled structures, lamellar structures, lipid droplets, and
lipofuscin in Smith-Lemli-Opitz syndrome (C,D: Uranyl acetate, lead citrate).
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Inherited defects of FAO have been shown to cause 4% to 5% of SUDI cases, and these are the most common disorders
presenting as SUDI (93, 137). Of these, medium-chain acylCoA dehydrogenase deficiency (MCAD) is the most common IEM that
causes SUDI. As many as a third of affected infants die during the initial presentation, often without previous clinical evidence of a
FAO defect (137). Hypoglycemia following birth or hypoketotic hypoglycemia suggests the diagnostic possibility of MCAD.
Autopsy of Child with Suspected Inborn Errors of Metabolism

The autopsy may include photography, radiography, histology, histochemistry, ultrastructure, fibroblast culture, biochemical
analysis, and DNA analysis. It is important to do the autopsy as soon as possible after death, preferably within 2 hours.
Photographs and whole-body radiographs will document dysmorphology that can be seen in a number of IEM. Hydrops suggests
another group of disorders (Tables 5-5 and 5-21). There are several suggested protocols for sample collection in cases of
unexpected metabolic death (16, 77, 93).
Biochemical studies will be directed based on the autopsy findings, but it is important to collect tissue and fluids that may be useful
for further evaluation (Table 5-22). Since sepsis and intoxication can mimic IEM, cultures and toxicology may be necessary. Urine
should be collected by catheterization or suprapubic tap, placed in a preservative-free container and frozen at -20°C for amino and
organic acid analysis (37, 93). Blood, serum, and plasma are useful for wholeblood acylcarnitine analysis by MS/MS, which is
compared to postmortem sample reference ranges (93). Spotting a few drops of whole blood obtained by cardiac puncture onto
filter paper (i.e., Guthrie card) is the most efficient collection method and suitable for many analyses (77, 93, 137). If more blood is
available, collect heparinized blood, separate, and store plasma at −20°C (16, 37, 77). Red blood cells can be stored at +4°C (93).
If DNA is to be analyzed, collect whole blood in EDTA and keep at room temperature or +4°C until DNA can be extracted (37).
Genomic DNA (25 to 60 mg) can be obtained from l to 2 mL of blood, and 5 to 10 mg of DNA is sufficient for a single analysis.
CSF can be obtained by cisternal puncture by hyperflexing the neck and inserting a needle between the atlas and the axis
vertebrae. CSF may only provide reliable information if collected before death, but it is useful in certain
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circumstances, especially for mitochondrial respiratory chain diseases and organic acid and acylcarnitine analyses (93). Collect
two 1-mL samples, one in a plain tube and one in fluoride oxalate, and store at -70°C (93). Vitreous humor can be collected into a
fluoride tube and stored at -20°C for glucose and electrolyte analysis.
Table 5-21 ▪ INBORN ERRORS OF METABOLISM THAT CAUSE HYDROPS FETALIS
Lysosomal storage disease
Mucopolysaccharidosis types I, IVA and VII
I-cell disease (mucolipidosis II)
Mucolipidosis IV
GM1 gangliosidosis
Wolman disease
Fabry disease
Farber disease
Gaucher disease type II
Sialidosis
Galactosialidosis
Niemann Pick disease type A, C
Glycogen storage disease type IV
Long-chain hydroxylacyl-CoA dehydrogenase deficiency
Primary carnitine deficiency
Pearson marrow-pancreas syndrome
Congenital disorders of glycosylation (CDG)
Neonatal hemochromatosis
Table 5-22 ▪ SPECIMENS TO BE TAKEN IN AUTOPSY OF AN INFANT WITH POSSIBLE INBORN ERROR IN
METABOLISM
Specimen Store
Urine -20°C
CSF -70°C
Whole blood in EDTA for DNA Room temperature or +4°C
Serum -20°C
Vitreous -20°C
Erythrocytes +4°C
Bile -20°C
Skin for fibroblast culture Room temperature or +4°C
Brain, heart, kidney, liver, skeletal muscle, adrenal -70°C
Brain, heart, kidney, liver, skeletal muscle In 2% glutaraldehyde
Sources: Byard RW. Sudden death in infancy, childhood and adolescence. Cambridge, UK: Cambridge University Press,
2004; Fitzpatrick D. Genetic metabolic disease. In: Keeling JW, ed. Fetal and neonatal pathology. London, UK; New York,
NY: Springer, 2001:153-174.
Bile may be the only analyzable fluid in cases where the interval between death and autopsy is long. In all cases where there is a
possibility of underlying metabolic disease, a sample of bile should be obtained (93). Bile can be collected on filter paper or a
Guthrie card for acylcarnitine testing or collected in a plain tube for storage at -20°C.
Tissues must be taken promptly if accurate results are to be obtained. One cubic centimeter of tissue from brain, kidney, muscle,
liver, and other viscera can be snap frozen in liquid nitrogen, wrapped in foil, and stored at -70°C. Liver and skeletal muscle can be
obtained at the bedside after death (93). Fresh-frozen muscle is the tissue of choice for diagnosis of mitochondrial respiratory
chain disorders. Complexes I, II, III, and IV of the respiratory chain can be measured (93). Some enzymes of intermediary
metabolism are more stable, and tissue analysis may provide essential diagnostic information, even when obtained at the time of a
routine autopsy (93). Both MCAD and LCAD in liver may be stable up to 100 hours after death if the body is refrigerated and for 5
years if tissue is kept at -70°C (16).
Fibroblast culture is essential for evaluation of many IEM, and obtaining skin for fibroblasts should be part of any autopsy on an
infant or child who dies from unknown cause; this may be the only tissue on which a suspected diagnosis can be confirmed (93).
Fibroblasts can be used for studies of DNA, enzymes, and metabolites and for karyotype and can be saved for future studies (29).
Achilles tendon, kidney, pericardium, and fascia may also be used for a source of fibroblasts (29). Skin is not a good choice for cell
culture for a macerated fetus; in that case, placental villi, kidney, lung, or heart could be used for culture. Take two pieces of tissue
from different sites, using sterile technique, place in separate sterile vials containing culture transport medium (Ham's F10, Eagle's
MEM, Dulbecco's medium, or sterile normal saline if the only solution available). Taking samples at the beginning of the autopsy is
recommended because of the lower risk of bacterial contamination (93). Skin fibroblasts remain viable for up to 9 days after death,
but they should be obtained as soon as possible as this increases chance of successful culture (16). Store specimen at 4°C (not
below 0°C) until it can be delivered to the cell culture lab. Cultivated fibroblasts can be cryopreserved for indefinite period for future
studies.
Histologic, histochemical, and ultrastructural findings can be a guide to diagnosis but are unfortunately often nonspecific. Liver and
kidney frozen can be used to look for lipid. Although not specific for and not always present in FAO defects, steatosis can occur in
SUID due to these disorders (137). Increased glycogen suggests altered glycogen metabolism. If membrane bound, it suggests
GSD Type II.
Unless tissue is obtained minutes after death, ultrastructure is seldom well preserved; however, storage may remain identifiable
even in autolyzed tissue (Figure 5-1D,E) (29). EM requires mincing tissues into 1-mm3 pieces and fixation in 2% glutaraldehyde.
LM and histochemistry can be performed on skeletal muscle up to 24 hours after death in children using a 1 to 2 mm in diameter,
1-cm long strip of muscle frozen in mountant in isopentane cooled to -170°C in liquid nitrogen. An infant with unexplained
nonimmune hydrops may show characteristic lysosomal material suggesting a storage disease in viscera, brain, placental villi, or
amnion cells (103).
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Chapter 6
Congenital and Acquired Systemic Infectious Diseases
Haresh Mani
Thomas J. Stocker
As it takes two to make a quarrel, so it takes two to make a disease, the microbe and its host.
—Charles V. Chapin, 1856-1941
Infections are the leading cause of death in the pediatric population (e333). The optimism that accompanied the advent of antibiotics in the mid-20th century
was premature, and even advances such as immunization and improved sanitation have not stopped microbial reemergence time and again. The importance
of infectious diseases is underscored by the ever-increasing antimicrobial resistance and the resurgence of infections such as tuberculosis (TB), malaria,
and syphilis; once thought eradicated from developed countries. Further, international travel has removed boundaries from the spread of infection, as
exemplified by the rapid spread of infections such as severe acute respiratory syndrome (SARS) across countries. There is voluminous literature on this
subject, with numerous heavy tomes devoted to individual infections. In this chapter, we aim to provide an overview of systemic infections that the pediatric
pathologist is likely to encounter. Infections that are predominantly confined to a single organ system, (e.g., poliomyelitis, hepatitis) are not considered in this
chapter, even though they may occasionally cause systemic manifestations. The reader is directed to chapters dealing with specific organ systems for
information on such infections. Further, since it is impossible to comprehensively detail all facets of various systemic infections in a single chapter, we have
generously referenced resources for the reader with specific interests. Detailed information is also available in standard textbooks including Feigin and
Cherry's Textbook of Pediatric Infectious Diseases (56), Connor and Chandler's Pathology of Infectious Diseases (32) and the American Academy of
Pediatrics Red Book (www.aapredbook.com).
THE PATHOGENESIS OF INFECTIOUS DISEASES

An infection is the result of an encounter between an infectious agent and a susceptible host. The microorganism is the sine qua non, but the occurrence of
the disease, its pathophysiology, and its outcome are determined by host and environmental factors. A discussion of microbial virulence factors is beyond the
scope of this chapter; various reviews cover the topic in considerable depth (130, 184) (e37,e107).
HOST FACTORS
Host genetic factors, immune status, age, and geographic location determine exposure to and invasion by microorganisms. The apparent heritability of
infectious disease susceptibility is determined by developmental and maturational changes in host defense, from embryo through adolescence, with resultant
differences in response to infection (e65). The contribution of host genetics to infection susceptibility is complex (179) (e340). Genes responsible for simple
or complex control of susceptibility to infection with different pathogens have been recently identified and characterized. Polymorphisms in genes coding for
proteins that recognize bacterial pathogens [such as toll-like receptor 4, CD14, Fc(gamma) RIIa, and mannose-binding lectin] and the response to bacterial
pathogens [with elaboration of cytokines such as tumor necrosis factor-a, interleukin (IL)-1α, IL-1β, IL-1 receptor agonist, IL-6, IL-10, heat shock proteins,
angiotensin I converting enzyme, plasminogen activator inhibitor-1] can influence response to bacterial stimuli (33).
Immunologic maturity and immunodeficiencies (quantitative and qualitative) also determine susceptibility to invasive microbial infections. Neonates and
infants are at a relative immunologic disadvantage since they have developmentally immature immune systems. The immaturity of the fetal immune system
helps prevent “premature rejection” by the host (the mother). Paradoxically, this potential benefit also increases the risk of infections for the fetus and the
prematurely born neonate. Term newborns have a higher frequency of microbial infections than older children and adults; extremely premature newborns
(<28 weeks of gestation)
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have a five to tenfold higher frequency than even term newborns (e179). Immunological immaturity also obscures clinical symptoms in neonatal sepsis.
Recent advancements in developmental immunology provide a framework for understanding the mechanisms underlying the propensity of infections in the
preterm, near-term, and term newborn (28). The immune environment during early life favors innate over acquired immunity. Innate immunity against
pathogens represents the critical first-line barrier of host defenses, as newborns have a naïve adaptive immune system. However, innate immune
mechanisms are also relatively impaired in neonates as compared to older children and adults, thereby increasing neonatal susceptibility to infections (94)
(e211). Further, the neonate is unable to produce antibody to thymus-independent antigens such as bacterial polysaccharides owing to multiple factors,
although transplacentally acquired maternal antibodies confer some protection for the first few months of life. Neonatal B-cells are of an immature phenotype,
the neonatal spleen has a different cellular composition and neonatal accessory cells (macrophages and dendritic cells) appear to produce lesser amounts
of stimulatory cytokines and an overabundance of inhibitory cytokines (101).
Children with immunodeficiencies (primary or acquired) have an increased risk of infections (Tables 6-1 and 6-2). Impaired splenic function (due to asplenia,
disease, or splenectomy) significantly increases the risk of life-threatening bacterial sepsis, especially with capsulated organisms, necessitating
pneumococcal and meningococcal immunizations. Secondary factors such as comorbidities, medications, and nutritional status also dictate clinical course
(Table 6-3). Organisms causing disease in a setting of immunodeficiency are, for the most part, “opportunists”; they are already on the scene, either as
normal flora of skin, upper respiratory tract, or gastrointestinal tract (GIT), or they are ubiquitous in the environment where they ordinarily do no harm.
Table 6-1 ▪ INFECTIONS AND PRIMARY IMMUNODEFICIENCIES
General associations
Recurrent respiratory and pyogenic infections by extracellular bacteria: Streptococcus pneumoniae, Antibody deficiencies
Haemophilus influenzae, Staphylococcus aureus
Chronic or severe infections with intracellular pathogens: viruses, mycobacteria, Pneumocystis carinii, Deficiencies of T
Toxoplasma gondii , and others lymphocytes
Specific associations
Chronic viral encephalitis X-linked

Echo virus dermatomyositis agammaglobulinemla
Polio vaccine-induced paralysis
Severe parainfluenza infection Severe combined

Severe varicella immunodeficiency
Severe Epstein-Barr virus infection Cartilage-hair hypoplasia
Recurrent meningococcal sepsis X-linked lymphoproliferative
Disseminated gonococcal infection syndrome
Complement deficiencies
Staphylococcal skin infections Neutrophil abnormalities

Mucosal and periodontal infections
Infections with Aspergillus sp., S. aureus, Pseudomonas cepacia, Chromobacterium violaceum Chronic granulomatous
BCGosis and atypical myocobacteria disease
Persistent mucocutaneous candidiasis NF-γ and IL-12 deficiencies
Chronic/recurrent giardiasis Chronic/recurrent giardiasis
IgA deficiency
BCG, bacille Calmette-Guérin; INF, interferon; IL, interleukin; IgA, immunoglobulin A.
ENVIRONMENTAL FACTORS
Over 50 years ago, Haldane proposed that the prevalence of thalassemia in malaria-endemic areas was due to the heterozygotic advantage it conferred
against malaria, despite its otherwise deleterious effects. Table 6-4 outlines examples of the influence of geographic, political, and socioeconomic factors on
infectious diseases that account for a major part of the world's infant morbidity and mortality. Infectious disease risks associated with international travel are
diverse and depend on the destination, planned activities, and baseline medical history. Children have special needs and vulnerabilities that should be
addressed when preparing for travel abroad (109).
On a less global scale, certain local environments must frequently be considered as contributors to disease. Such nosocomial environments as intensive
care units, neonatal nurseries, day care centers, schools, and summer camps play a role either by serving as reservoirs for pathogenic microbes or by
facilitating their spread in a susceptible population. Finally, hospitalized or chronically ill children are exposed additionally to equipment and pharmaceutical
agents that may be the source of iatrogenic infections.
Infections and Teratogenesis

In order to act as a teratogen, an agent must be capable of crossing the placenta at an early stage of embryogenesis or organogenesis and either inhibit cell
growth and differentiation, or produce destructive fetal lesions. The spectrum of resulting defects depends both on the timing and the severity of the insult.
The dysmorphogenetic syndromes produced by fetal infection are remarkable in their clinical and morphologic variability. The first such association was the
recognition of congenital rubella by Sir Norman Gregg in 1941 (e126). Of the large number of agents causing fetal infection, only rubella, cytomegalovirus
(CMV), varicella-zoster virus (VZV), herpes simplex virus (HSV), toxoplasma, and syphilis are firmly established as human teratogens. Early reports of a
dysmorphic syndrome associated with human immunodeficiency virus (HIV) infection, distinguishable from the effects of drug/alcohol abuse or concomitant
opportunistic infection (e156), have not been confirmed (e263). The evidence linking coxsackie viruses and mumps to congenital heart defects and
endocardial fibroelastosis, respectively, is also inconclusive.
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Table 6-2 ▪ CONGENITAL IMMUNODEFICIENCIES WITH AN INCREASED RISK OF SEPSISa
Immunodeficiency Characteristic Susceptibilityb Estimated Sepsis Occurrence, %c
Innate immune defects
Complement deficiency Neisseria 28

Mannose binding lectin Neisseria
NEMO deficiency Klebsiella, S. pneumoniae, mycobacteria 86

IRAK4 deficiency Gram-positive bacteria 75
TLR-4 Gram-negative bacteria
Caspase-12 defect
CGD Salmonella, Burkholderia, Candida 21 (X-linked)
10 (autosomal)
Leukocyte adhesion def ciency Pseudomonas aeruginosa 28

Specific granule deficiency
Severe chronic neutropenia 3d
Type-1 cytokine axis defects Mycobacteria, Salmonella
Adaptive immune defects
SCID 5e
Agammaglobulinemia
Hyper-IgM
Pseudomonas 10f
Pseudomonas, pneumococcus, Escherichia 13
CVID 1g
Transient hypogammaglobulinemia of infancy

IgG subclass deficiency
Ataxia telangiectasia Gram-positive bacteria 5

IPEX Enteric bacteria
Wiskott-Aldrich syndrome 36
aSepsis, caused by bacteremia, fungemia, or viremia.
bCharacteristic infectious susceptibility that can be useful in considering a particular diagnosis.
cOccurrence of sepsis within a particular population.
dBacteremia in additional 15%.
eAs high as 30% in reticular dysgenesis and 16% in Omenn syndrome.
fAs a presenting manifestation before immunoglobulin replacement therapy.
gHigher in the Good syndrome variant (16%).
NEMO, nuclear factor_B; TLR, toll-like receptor; CGD, chronic granulomatous disease; SCID, severe combined immunodeficiency; Ig,
immunoglobulin; CVID, common variable immunodeficiency; IPEX, immunodysregulation, polyendocrinopathy, enteropathy, X-linked.
Source: Orange JS. Congenital immunodeficiencies and sepsis. Pediatr Crit Care Med 2005;6(Suppl.):S99-S107.
Table 6-3 ▪ PRIMARILY NONIMMUNE DISORDERS INFLUENCING INCIDENCE AND SEVERITY OF INFECTION
Site Predominant Organisms Proposed Mechanism References
Metabolic disorders
Diabetes mellitus Skin, GU tract S. aureus, E. coli, yeasts, Impaired phagocytosis, neutrophilic 17
Galactosemia Bacteremia, Zygomycetes chemotaxis, and opsonization 117, 281
Uremia meningitis E. coli , group D streptococci Impaired phagocytosis due to 314
Iron deficiency Pneumonia, Unspecified hypoglycemia 8, 356, 482
Nephrotic syndrome septicemia Unspecified Impaired macrophage function 499
Intravenous lipid Unspecified S. pneumoniae, enteric bacilli Impaired bacterial killing 369, 370,
Peritonitis Malassezia furfur Unknown, protein loss (?) 384
Pulmonary arteritis, Lipophilic organism
fungemia
Circulatory alterations
Congenital/rheumatic Endocarditis, Viridans streptococci, S. Endocardial damage due to jet effects, 231, 308
heart disease pericarditis aureus turbulence 25, 123,
Sickle cell disease Meningitis, systemic S. pneumoniae, Salmonella Ischemia, functional asplenia, defective 211, 346,
Exudative enteropathy osteomyelitis sp. opsonization 357, 492,
Pneumonia, S. pneumoniae, enteric bacilli, Intestinal loss of immunoglobulins and 504
gastroenteritis Giardia lymphocytes 156, 157
Obstructive phenomena
Cystic fibrosis Bronchitis, S. aureus, Pseudomonas Defective ciliary movement, mechanical 272, 298,
Immobile cilia bronchiectasis, H. influenzae, Neisseria, obstruction due to hyperviscosity of 341, 446,
syndromes pneumonia staphylococci, streptococci, mucus 460
GU Otitis, sinusitis, Pseudomonas Defective ciliary motility 376, 461
obstruction/malfunction bronchitis, E. coli, Proteus, Urinary stasis, instrumentation, trauma 93, 256, 266
bronchiectasis Enterobacteria 267, 382,
Pyelonephritis, 427
cystitis
Barrier defects
Eczema, exfoliative Impetigo, sepsis Staphylococci, β-hemolytic Mechanical loss of skin barrier 156, 157
dermatitis Skin, sepsis streptococci Changes in flora, physiochemical 156, 157
Burns Meningitis Pseudomonas, S. aureus, S. properties of the skin 34, 55
Skull fractures Meningitis Epidermidis, fungi, varicella, Direct access to CSF via respiratory 34, 156, 157
Neural tube defects herpes simplex passages and sinuses
S. pneumoniae Direct access to CSF from skin
S. pneumoniae, Gramnegative
enterics, staphylococci
Foreign bodies
Arterial and venous Phlebitis, omphalitis, S. epidermidis, Pseudomonas, Barrier bypass, nidus for infection 51, 277, 300
catheters endocarditis, yeasts Barrier bypass, nidus for infection 336, 415
CSF shunts arteritis, liver S. epidermidis, S. aureus, Nidus for infection 251, 448
Prostheses abscess enteric organisms Aspiration of infected material, 47, 326
Aspiration Meningitis, S. aureus, S. epidermidis bronchial obstruction by foreign bodies,
peritonitis, Anaerobes necrosis of airway epithelium
septicemia,
endocarditis,
phlebitis
Endocarditis
Pneumonia, lung
abscess
Splenectomy Fulminant septicemia S. pneumoniae, Salmonella Defects in opsonization and clearing 146, 436
Malnutrition Pneumonia Measles, herpes simplex, Depression of complement, cell- 215, 253
staphylococci, enteric Gram- mediated immunity, and phagocytosis
negatives, Pneumocystis
CSF, cerebrospinal fluid; GU, genitourinary.

? = Pathogenesis suspected, but largely unknown at present.
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In-utero infections can result in a variety of adverse fetal outcomes. Microorganisms damage fetal cells or tissues (either directly or indirectly by elaborating
toxic substances), interrupt cell division or migration, and/or evoke (or depress) host inflammatory and repair responses. Depending on the organism and the
timing of the insult, intrauterine infection may result in no detectable damage, resorption of the embryo, spontaneous abortion, prematurity, stillbirth,
intrauterine growth restriction, congenital malformation, acute or chronic neonatal infection, or clinically inapparent ongoing or static disease with late
sequelae. Intrauterine rubella infection represents a paradigm of fetal infection because it
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operates by all of these mechanisms to affect virtually all of the possible outcomes. Table 6-5 lists various viral infections associated with adverse fetal
outcomes. (146).
Table 6-4 ▪ INFLUENCE OF ENVIRONMENTAL FACTORS ON INFECTIOUS DISEASES
Example Comment References
Geographic
Climate Falciparum malaria Requires summer average over 21°C 44, 381
Geologic Onchocerciasis Insect vector develops in fast-flowing rivers 381
characteristics Toxoplasmosis Oocysts produced in cats 166
Animal reservoirs Chagas disease Transmitted by triatomid insects 500
Vector availability
Political and economic
Population Tuberculosis Complex relationship, including ease of droplet 270, 278,

density/overcrowding Measles, polio transmission, malnutrition, presence of other chronic 386, 388
Political structure Malaria diseases 163, 207
and stability Tetanus Can be eradicated by effective and sustained public 355
War, refugee status AIDS health and immunization programs 455
Socioeconomic Refugee borne global spread of disease 167, 175,
status Most common cause of neonatal death in countries with 398
Cultural/behavioral lowest per capita income
patterns Sexual and parenteral transmission of disease in
homosexuals and drug addicts
Nosocomial sources of infection
Pediatric ICU S. aureus, E. coli, Klebsiella, Pediatric ICU-acquired infections less common than 24, 37, 389
Neonatal ICU Enterobacter, Serratia adult ICU-acquired 177, 217
Day care Staphylococci, E. coli Susceptibility Increased because of absence of normal 26, 118,
Contaminated Diarrheal illnesses, hepatitis A, flora 483, 502
ventilabory Haemophilus influenzae, upper Close person-to-person contact in a highly susceptible 156, 157
equipment respiratory viruses population with behavior patterns facilitating 363
Contaminated IV Pseudomonas, Serratia transmission 156, 157
fluids E. coli, Erwinia, Pseudomonas Aerosols and nebulizers are reservoirs; cystic fibrosis 139
Contaminated Legionella patients especially affected
water/air supplies Contamination of containers
Reservoirs in drinking water supply, air conditioning
equipment. Rarely seen in normal children
ICU, intensive care unit; IV, intravenous.
Intrapartum or neonatal infections are more limited in scope than those occurring in utero because of the relatively more advanced developmental state of
the infant. Nonetheless, they may produce acute and possibly fatal disease (e.g., neonatal herpes virus infection), persistent infection with ongoing tissue or
organ dysfunction (e.g., postnatal CMV infection), or late complications of the infection and its subsequent repair process (e.g., obstructive hydrocephalus
resulting from neonatal meningitis).
TRANSMISSION
Materno-fetal transmission is specific to the pediatric population and may occur in utero (“vertical transmission”) or during breast feeding. Other routes of
transmission including inhalation, ingestion, and inoculation are similar in adults and children. Routes of fetal and neonatal infection have been thoroughly
reviewed by Blanc (14). His findings are illustrated in Figure 6-1 and summarized in Table 6-6.
VERTICAL TRANSMISSION
Pathways of vertical transmission include in utero (transplacental and ascending), intrapartum (in the birth canal, from maternal genital and GITs), or
immediately postnatally (although, strictly speaking, this is not vertical transmission). Although in-utero infection can occur in any trimester of pregnancy, the
timing of infection significantly affects its clinical course resulting in asymptomatic infection, fetal demise, teratogenicity, prematurity, clinical disease present
at birth, or later presentation. There are two major routes of intrauterine fetal infection. Organisms may ascend from the maternal genital tract through the
cervix to the amniotic sac through either intact or ruptured membranes. This ascending route is the preferred one for HSV, most bacteria, and Candida.
Hematogenous spread of maternal blood-borne organisms across the placenta is the pathway used by most
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viruses and protozoa such as plasmodia and toxoplasma. Intrauterine fetal manipulation, amniocentesis, and chorionic villus sampling represent potential
risks for infection, but this appears to be a very rare event (e145,e219,e223).
Table 6-5 ▪ VIRAL INFECTIONS ASSOCIATED WITH ADVERSE FETAL OUTCOMES
Transmission
Clinical
In During Breast Incidence per Present in AF/Fetus in Consequences at
Virus Utero Delivery Milk 1,000 Live Births Unaffected Cases Birth Postnatal
HSV + +++ ++ 0.04 Yes IUGR, death, Recurrence

multiorgan disease
CMV +++ +++ +++ 5-22 Yes IUGR, CNS Developmental

disease, CID delay, deafness
Adenovirus +++ - ++ Unknown Yes IUGR, fetal hydrops Unknown
AAV + + ++ Unknown Unknown Prematurity —
EV + — +++ Unknown Unknown Myocarditis Neurodevelopmental

delay, dia
HHV6 + + + Unknown Yes Encephalitis Unknown
LCMV + - + Unknown Unknown CNS disease, eye Blindness

disease, death
Parvovirus ++ + + Unknown Unknown Fetal hydrops Anemia
Rubella ++ — + 0.01 Unknown CNS disease, eye Deafness, exanthem

disease
VZV + ++ ++ 0.01 Yes Limb disease, CNS Disseminated VZV

disease
+, rare; ++, frequent; +++, common.

AAV, adenovirus associated virus; A F, amniotic fluid; CID, chronic inflammatory disease; CMV, cytomegalovirus; CNS, central nervous system; E V,
enterovirus; HHV, human herpes virus; HSV, herpes simplex virus; IUGR, intrauterine growth restriction; LCMV, lymphocytic choriomeningitis virus;
VZV, varicella zoster virus.
Modified from Rawlinson WD, et al. Viruses and other infections in stillbirth: what is the evidence and what should we be doing? Pathology
2008;40(2):149-160.
Perinatal infections commonly occur from exposure to blood or body fluids and contact with pathogens from the maternal genitourinary and GITs. The
likelihood that the exposed infant will be infected varies significantly with the specific organism and various host factors (e.g., passively acquired antibody
levels in the infant). Intrapartum transmission is more efficient in a setting of prolonged labor combined with an infected maternal birth canal. Premature
inspiratory movements on the part of the fetus may result in pneumonia occurring soon after birth, with high mortality. Postnatally acquired infections are
transmitted most commonly through contact with caregivers (parents, relatives, visitors, and health care providers), the environment (medical equipment,
other fomites), or breast milk, depending on the organism (97). In most situations of perinatal mother to child transmission, the infant is exposed before the
illness is diagnosed in the mother (e.g., measles, Coxsackievirus infection) and frequently occurs even before the mother becomes ill (e.g., chickenpox,
hepatitis).
FIGURE 6-1 ▪ Routes of fetal infection.
BREAST MILK TRANSMISSION

Human milk protects against specific pathogens as well as separate clinical illnesses (e.g., necrotizing enterocolitis, bacteremia, meningitis, respiratory tract
illness, diarrheal disease, and otitis media). Protective mechanisms of breast milk include improved growth of nonpathogenic flora, decreased colonization
with enteropathogens, enhanced development of the respiratory and intestinal mucosal barriers, providing secretory IgA and functioning immune cells
(neutrophils, macrophages, T and B lymphocytes), preventing gut inflammation and immunomodulation (51, 104) (e123). Although breast milk may be a route
for transmission of infection, organisms that are a concern for transmission through breastfeeding are transmitted more commonly through other
mechanisms. Clinically significant infectious agents that are transmitted through breast milk include HIV-1, human
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T-lymphocytotropic virus-1 (HTLV-1), CMV, measles virus, and streptococcus (104) (e290). Bacterial infections are rarely, if ever, transmitted to infants
through breast milk. However, temporary cessation of breastfeeding has been recommended in certain maternal bacterial infections ( Neisseria
gonorrhoeae, Haemophilus influenzae, Group B streptococci (GBS), and staphylococci; longer period of cessation for others including Borrelia burgdorferi,
Treponema pallidum, and Mycobacterium tuberculosis).
Table 6-6 ▪ PREDOMINANT PATHWAYS OF THE MAJOR FETAL AND NEONATAL INFECTION
Transplacental Ascending Postpartum
Bacteria
Listeriaa Group B streptococci Staphylococcia

Treponema pallidum Enteric bacillb Pseudomonasa
Mycobacterum tuberculosis Hemophilus influenzae
Nongroup B streptococcib
Borrelia Neisseria gonorrhea
Campylobacter fetus (?) Anaerobes
Actinomyces
Fusobacteria
Viruses
Cytomegalovirusc Herpes simplexc Respiratory syncytial virus

Human immunodeficiency virus Coxsackie Ba
Rubella
Mumps
Measles
Variola
Vaccinia
Poliovirus
ECHO
Hepatitis Bc
Western equine encephalitis
Human parvovirus
Varicella zostera
Epstein-Barr virus
Protozoa
Toxoplasma
Plasmodium
Trypanosoma
Babesia
Fungi
Coccidioides Candidac
Aspergillus
Torulopsis
Mycoplasmas Mycoplasma hominis

Ureaplasma urealyticum
aAlso utilize ascending route.
bAlso utilize hematogenous route.
cAlso acquired postnatally.
From a summary of references 46, 130, 167, 184, 196, 306, 345, 400, 414, 437, and 441.
EVALUATION OF SUSPECTED FETAL INFECTION

Given the incredibly wide spectrum of disease produced by fetal infection, the pathologist must be prepared properly to evaluate fetal and neonatal deaths in
order to arrive at a correct diagnosis. Infection should be suspected in newborns exhibiting any or all of the following: intrauterine growth restriction or failure
to thrive, hydrops, jaundice or hepatosplenomegaly, skin rashes (especially vesicular or purpuric), hydrocephalus or microcephaly, and eye lesions such as
microphthalmia, chorioretinitis, and cataract. Wigglesworth has outlined a procedure for autopsy evaluation of such infants using serologic studies, radiology,
careful and complete bacteriologic studies including darkfield examination, viral diagnostic studies including culture, and electron microscopy (e365,e366).
Nucleic acid amplification techniques, many applicable to formalin-fixed tissues, have made possible the identification of infectious agents hitherto difficult or
impossible to detect (62) (e279). It cannot be overemphasized that histopathologic examination, however indispensable, constitutes only one facet of
adequate autopsy examination in suspected prenatal or perinatal infection.
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VIRAL INFECTIONS
Most significant viral infections in neonates or infants occur through transplacental or intrapartum transmission. The risk of transmission is dependant on
whether the maternal infection is primary (e.g., HSV, HIV-1), secondary (reactivation) (e.g., HSV, CMV) or chronic (e.g., hepatitis B, HIV-1, HTLV-1). Fetal
and neonatal viral infections are multifaceted. Many of these agents are teratogens, and most can affect the fetus or infant at any stage of development to
produce fetal death or malformation, acute self-limited infection, ongoing infection, and late sequelae due to destructive, repair, or immune responses. Table
6-7 summarizes the main features of fetal and neonatal viral infections, while Table 6-8 outlines the major pathologic features of commonly seen viral
infections in infants and older children. The number and diversity of the viruses affecting the human host have assumed substantial proportions. Of the more
commonly encountered viral illnesses of children, many involve almost exclusively the nervous system (e.g., poliomyelitis, rabies) and are discussed in
Chapter 10. The respiratory tract pathogens are included in Chapter 12, and the hepatitis viruses in Chapter 15. Of the remainder, relatively few are
encountered with any frequency by the pathologist; many are opportunists in immunocompromised children. In general, these disseminated opportunistic
viral infections resemble their perinatal counterparts. For comprehensive information, the reader is referred to Feigin and Cherry's textbook (56); only
selected infections will be discussed here.
CYTOMEGALOVIRUS
Cytomegalovirus, the largest member of the family Herpesviridae, is encountered in all populations. CMV is ubiquitous and its seroprevalence in adult
populations ranges from 50% to 90%. It is the most common cause of congenital infection in the United States, with frequency ranging from 0.2% to 2.2% of
live-born babies in the United States (e76); 30% to 60% of fetuses of mothers with primary CMV infection during pregnancy are congenitally infected.
However, unlike congenital infections with rubella and toxoplasma, intrauterine transmission of CMV can occur in women who are CMV-seroimmune before
pregnancy, albeit at a much lower frequency. Approximately 1% of all infants excrete CMV in their urine at or within 3 weeks after birth; about 5% of
congenitally infected infants manifest disease at birth and 15% develop late sequelae (137). More children may be affected by congenital CMV than by other,
better known childhood conditions, such as Down syndrome, fetal alcohol syndrome, and spina bifida. CMV is, therefore, one of the most common causes of
birth defects and childhood disability.
Transmission
Infection may be transplacental, perinatal, or postnatal. Early, hematogenous gestational infections are the most devastating. Primary maternal infection is
much more likely to result in fetal infection than is recurrent maternal disease, but rarely congenital CMV infection “repeats” in subsequent pregnancies.
Perinatal infection through body fluid contact at delivery and postnatal infection through breast milk do occur, but are rarely associated with clinical illness in
fullterm infants (160) (e132,212). Transplacental acquisition of maternal antibodies against CMV protects full-term infants of CMV-seropositive mothers.
Rarely, primary CMV infection occurs in the mother around delivery or during lactation, increasing the risk for illness in the infant because of a lack of
available anti-CMV antibodies. Postnatal exposure of susceptible infants (i.e., premature infants, infants of CMVseronegative mothers, and immunodeficient
infants) can lead to severe disease. CMV is also commonly reactivated in a setting of immunodeficiency, either congenital or acquired. Childcare centers are
another significant source of transmission of CMV, propagated by frequent mouthing of hands and toys. Approximately 20% to 40% of toddlers in day care
shed the virus for years. These children function as an important infectious source for other children, parents, and daycare workers (e151). Beyond puberty,
infection is mainly sexually transmitted. Virus is present in urine, oropharyngeal, cervical and vaginal secretions, breast milk, semen, and tears and can be
shed intermittently for years.
Clinical Features
Transplacental transmission can result in congenital infection and neurological sequelae. Perinatal and postnatal transmission does not usually manifest with
clinical disease except in extremely preterm infants (157). Most (˜90%) infants born with congenital CMV infection do not exhibit clinical abnormalities at birth
(so-called asymptomatic congenital CMV infection). Of the 40,000 children born with congenital CMV infection each year, approximately 10% to 15% exhibit
clinical abnormalities (symptomatic congenital infection). Infection involves multiple organ systems, with particular predilection for the reticuloendothelial and
central nervous systems (CNS). The most commonly observed physical signs are petechiae, jaundice, and hepatosplenomegaly (Figure 6-2). Neurologic
abnormalities such as microcephaly and lethargy affect a significant proportion of symptomatic children. Intrauterine growth restriction, chorioretinitis, optic
atrophy, and seizures are other physical signs (157). Postnatal infection in the neonatal period results in an acute sepsis-like picture with apnea,
bradycardia, hepatitis, leucopenia, and prolonged thrombocytopenia. In older children, severe infection occurs in a setting of immunodeficiency. Features of
active disseminated CMV infection include fever, leucopenia, thrombocytopenia, pneumonia, hepatitis, chorioretinitis, adrenalitis, and encephalitis. Infected
infants may have the characteristic “blueberry muffin lesions,” a hemorrhagic purpura with mobile gray-blue skin lesions, which histologically show dermal
extramedullary hematopoiesis. CNS lesions are irreversible and affect prognosis. There is a high incidence of symptomatic liver disease, ranging from mild
cholangitis (with inclusions) to severe cholestatic hepatitis (Figure 6-2). Noncirrhotic portal fibrosis with portal hypertension is a rare but potentially lethal late
sequela (e83,e120). Glomerulonephritis, ascites, and pulmonary hypoplasia are also described (e21,e317). A syndrome of hepatosplenomegaly, respiratory
distress, a peculiar gray pallor, and atypical lymphocytosis occurs in multiple transfused low birth weight infants. Interstitial pneumonitis with inclusions
(Figure 6-2) is the main pathologic feature and is likely responsible for the high (24%) mortality rate in this setting.
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Table 6-7 ▪ VIRAL INFECTION INTHE FETUS AND NEONATE
Intrauterine
Growth Congenital Acute Perinatal
Abortion Stillbirth Restriction Defects Infection Late Effects References
Rubella + + + Cataract, Interstitial Interstitial 182, 183,

retinopathy pneumonitis, pulmonary 321 401,
sensorineural cholestatic hepatitis fibrosis, hepatic 441, 476
deafness, patent with giant cell fibrosis/cirrhosis,
ductus arteriosus, transformation, biliary atresia,
pulmonary anemia, arteriopathy with
stenosis, VSD, thrombocytopenia, infarction,
microcephaly, myocarditis, diabetes mellitus,
mental immunopathy, chronic
retardation osteoporosis, lymphocytic
pancreatitis thyroiditis,
panencephalitis,
autism (?)
Cytomegalovirus ? + + Microcephaly, Necrotizing Deafness, 32, 124,

hydrocephaly, meningoencephalitis neurologic 129, 172
microphthalmia with arterial and deficits, optic
periventricular atrophy,
calcification, noncirrhotic
hepatitis with giant portal
cell transformation, hypertension,
cholangitis, and vascular and
inclusions in lung, periventricular
renal tubules, rare calcifications
pneumonitis and (brain)
interstitial nephritis Hypoganglionosis
of bowel (see
Pediatr Pathol
1984;2:85-102)
Herpes simplex + + + Microcephaly, Hepatoadrenal Psychomotor 36, 218,

hydranencephaly, necrosis, vesicular retardation 390, 391,
microphthalmia skin rash, 437
vesicular/ulcerated
stomatitis,
esophagitis,
necrotizing
pneumonitis,
chorioretinitis
Varicella zoster ? ? + Limb hypoplasia, Typical varicella, Blindness, 4, 447

rudimentary acute disseminated psychomotor
digits, cutaneous varicella with retardation
scars in necrotizing
dermatome cutaneous and
distribution, visceral lesions
chorioretinitis,
microphthalmia
HIV + + + None See Table 6-9A See Table 6-9B

See Table 77
Parvovirus + + - Ocular defects Anemia, hydrops, ? 10,52,164,

hepatic fibrosis, 202, 261,
siderosis 432, 477
Hepatitis B - - - - Acute hepatitis, giant Cirrhosis, carrier 135, 283,

cell transformation, state 420, 435,
chronic active Hepatocellular 443
hepatitis, fulminant carcinoma
hepatitis
Hepatitis A + - + - Rare - 122
Mumps + + - Not proved Perinatal parotitis Endocardial 234

(extremely rare) fibroclastosis (?)
Influenza + + - Unlikely Rare influenza - 200

pneumonia, apnea
Vaccinia + + + - Generalized vaccinia -
Variola + + ? ? Smallpox - 299
Measles + + - ? Measles pneumonia -
Polio + + + - Paralytic polio Paralysis 27, 28
Echo - - - - Meningitis, DIC -
Coxsackie B ? ? - - Disseminated - 258, 438

disease,
meningoencephalitis,
myocarditis
Adenovirus - - - - Pneumonia - 462
RSV - - - - Apnea, bronchiolitis, Asthma (?), 22, 192,

pneumonia COPD 325, 434
DIC, disseminated intravascular coagulation; VSD, ventricular septal defect. +, occurs; -, does not occur.
From references 196, 345, 400, with permission.
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Table 6-8 ▪ COMMON SYSTEMIC VIRAL INFECTIONS IN INFANTS AND CHILDREN
Localized or
Self-Limited Disseminated or Serious
Virus Disease Disease Specific Inclusions Comments References
Adenoviruses Acute respiratory Types 1, 2, 4, 5, 7, 11 1. Large basophilic Easily confused with 269, 402,
illness (Types 1, Hepatitis, massive hepatic indistinctly demarcated disseminated HSV 510
2, 3, 4, 7, 21) necrosis intranuclear inclusion infection
(smudge cells)
Laryngotracheitis, Pneumonia, Hemorrhagic 2. Smaller eosinophilic
pneumonia cystitis, Gastroenteritis, intranuclear with
Meningoencephalitis incomplete halo
Cytomegalovirus Mononucleosis Interstitial pneumonia 1. Large amphophilic or Disease in 196, 239,

Gastroenteritis basophilic nuclear immunocompromised host 320
inclusions with distinct is similar to neonatal
Retinitis, encephalitis, halo pattern
glomerulonephrits 2. Smaller basophilic
PAS positive indistinct
cytoplasmic inclusions
Herpes simplex Localized oral, Hepatitis, hepatoadrenal 1. Type A-eosinophilic Either type I or type II may 151, 228,
skin, or genital necrosis, stomatitis, nuclear inclusions with disseminate: disseminated 263, 312,
vesicular or esophagitis, encephalitis, halo form resembles neonatal 408, 456
ulcerated pneumonia 2. Type B-basophilic or disease
eruption, may be amphophilic nuclear
extensive inclusions filling
nucleus with
peripheral chromatin
rim, often
multinucleate cells
Varicella-Zoster Localized herpes Disseminated zoster Multinuclear or Associated with Reye 158, 334
zoster, acute mononuclear cells with syndrome
varicella, Progressive disseminated nuclear type A
generalized varicella, pneumonia, inclusions,
vesicular eruption meningo encephalitis, indistinguishable from
hepatitis HSV inclusions
Epstein-Barr Mononucleosis Fatal mononucleosis, Noe Implicated in oncogenesis, 77, 205,

virus hepatitis, myocarditis, especially in X-linked 285, 374,
immunodeficiency various lymphoproliferative 422
hematologic phenotypes in syndrome, posttransplant
X-linked lymphoproliferative lymphoproliferative
syndrome syndrome
Rubeola Uncomplicated Progressive measles Cytoplasmic and nuclear Subacute sclerosing 83, 380, 392
primary measles, inclusions in epithelial panencephalitis, late
skin, conjunctiva, and Warthin-Finkeldey
respiratory tract giant cells
Mumps Parotitis Orchitis/oophoritis None Late sequelae include 84

Meningitis pancreatitis deafness, diabetes
Mastitis, nephritis, Arthritis mellitus
Coxsackie Virus Coxsackie Coxsackie viruses B; Noe 154

viruses A; benign, myocarditis
selflimited febrile meningoencephalitis
illness with
respiratory
disease
Echoviruses Mild nonspecific Hepatitis, hepatic necrosis, None 265, 319
febrile illness with meningitis, adrenal and
respiratory renal hemorrhage
disease
Variola Disease declared Cytoplasmic Guamieri 44

(smallpox) eradicated by bodies. Nuclear changes
WHO in 1980 inconsistent
Vaccinia Eczema Disseminated vaccinia Indistinguishable from 44

vaccinatum smallpox
Hantavirus ? Hantavirus pulmonary None Noncarciogenic 32, 234,

syndrome in adolescents pulmonary edema 332, 511
HSV, herpes simplex virus; PAS, periodic acid-Schiff; WHO, World Health Organization.
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FIGURE 6-2 ▪ Congenital CMV infection. A: Body with marked ascites. B: Face with petechiae as well as elsewhere. C: Abdominal cavity at autopsy showing
hepatosplenomegaly. D: Skull x-ray with diffuse calcification secondary to necrosis. (See Malinger G, Lev D, Zahalka N, et al. Am J Neuroradiol 2003;24:28-
32) E: CMV hepatitis with inflammation around a bile duct and within the lobules. F: Lung: CMV immunostain with large nuclear inclusion.
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Pathology
The morphologic hallmark of CMV infection is cytomegaly with extremely large (25 to 40 mm) inclusion-bearing cells with both nuclear and cytoplasmic
inclusions; often the nucleolus is retained within the inclusion, appearing as an “accessory body.” A clear zone around the inclusion with chromatin
margination gives an owl-eye appearance. The inclusion is eosinophilic in some stages of development but the fully developed inclusion is amphophilic to
deeply basophilic. The inclusions are PAS and GMS positive, although immunostains are commonly used to specifically identify the virus. CMV is found in
endothelial cells, epithelial cells (notably the biliary tree, pneumocytes, many exocrine cells, and renal tubular cells), fibroblasts, and histiocytes. Tissue
damage is characterized by patchy and focal necrosis with mononuclear, and occasionally neutrophilic, inflammatory response, with vascular and
parenchymal calcifications, the latter feature is seen especially in the brain. Giant cell transformation of hepatocytes is not a frequent feature. A complete
picture of the morphologic spectrum appears in Becroft's review (10).
Congenital infection causes neurologic and hematologic damage and developmental defects which are evident at birth in 10% of infected babies. During
infancy, sequelae such as sensorineural deafness, psychomotor retardation, and cerebral palsy develop, even in babies who are asymptomatic at birth. As a
result about 20% of all infected neonates suffer sequelae of a congenital CMV infection (e76).
Laboratory Diagnosis
When the large inclusion-bearing cells are present, morphologic diagnosis is straight forward. Sensitivity of histopathologic methods can be improved with
immunocytochemical and molecular virologic techniques. The reference method for diagnosing congenital CMV infection involves isolating the virus in cell
culture from urine collected within 3 weeks of birth. A positive CMV result in urine collected after the third week might well be the consequence of exposure to
infected vaginal secretions at delivery, through breast feeding, or untested transfusions (e76). Detection of CMV in the saliva and urine of infants is
accomplished easily because newborns with congenital CMV shed large amounts of virus into these body fluids. Blood is also a useful specimen to identify
viral DNA in serum or pp65 antigen in peripheral blood leukocytes (e334), but this method has not been evaluated for diagnosing congenital CMV infection.
Tests for viral DNA have proved a valid means of diagnosing congenital CMV infection in neonatal blood dried on paper (DBS). The DBS test is simpler,
faster, and less costly than viral isolation; in addition the samples can be safely stored for long periods, so diagnosis can be made even after several years.
The DBS method is reported to have high sensitivity (71% to 100%) and specificity (99% to 100%) (7).
Laboratory findings in infected children include conjugated hyperbilirubinemia, thrombocytopenia, and elevations of hepatic transaminases in more than half
of the symptomatic newborns, reflecting the involvement of the hepatobiliary and reticuloendothelial systems (157). Prenatal diagnosis of congenital CMV
infection is feasible when maternal CMV infection occurs during pregnancy. Viral culture of amniotic fluid can identify fetal infection but has a high
falsenegative result (e198,e227). Molecular [polymerase chain reaction (PCR)] assays on amniotic fluid may have better sensitivity and specificity. However,
PCR for CMV DNA requires the presence of viremia in the peripheral blood and may not identify every infant with congenital CMV infection (e232).
Prognosis and Outcome

Mortality rate among symptomatic children is now probably less than 5%. Of the symptomatic children who survive infancy, most will suffer mild to severe
psychomotor and perceptual handicaps and approximately half will develop sensorineural hearing loss, mental retardation, and microcephaly (157).
Predictors of adverse neurological outcome include microcephaly, chorioretinitis, presence of other neurologic abnormalities at birth or in early infancy, and
presence of cranial abnormalities on CT scans within the first month of life. Petechiae and intrauterine growth restriction are independently predictive of
hearing loss (157). Although, in general, children asymptomatic at birth have a better longterm prognosis, approximately 10% will develop sensorineural
hearing loss (half of these have bilateral deficit). Other neurological complications can also occur in asymptomatic congenital CMV infection but at a much
lower frequency than in symptomatic infection. The pathogenesis and mechanisms of hearing loss and other neurologic sequelae in children with congenital
CMV infection, especially in those with asymptomatic infection, are not well understood. Furthermore, predictors of adverse outcomes in asymptomatic
children have not been defined. This inability to identify infants at risk for the development of hearing loss and other sequelae necessitates monitoring and
follow-up of all children with congenital CMV infection (154).
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HERPES SIMPLEX VIRUS
First described as hepatoadrenal necrosis by Haas in 1935 (e135), HSV types 1 and 2 cause severe perinatal infections and, less frequently, prenatal and
postnatal infections, with HSV-2 predominating; about 20% are caused by HSV-1 (e309). The risk to the infant is highest in mothers with primary genital
herpes at the time of delivery (e33), but fetal infection may occur in the absence of visible maternal lesions.
Transmission
Most HSV infections in infants are acquired during passage through an infected birth canal. Maternal skin and nipple lesions, as well as paternal lesions
pose a threat to the infant. Intrauterine infection can also occur as a consequence of either primary or recurrent maternal infection, with severe fetal
consequences (e20,e151a,e276,e277). The pathogenesis is not well elucidated, but HSV antigen is demonstrable in endometrium, decidua, and placenta,
suggesting that transplacental passage is possible. Case reports have demonstrated HSV infections in infants related to maternal HSVpositive breast lesions
and inoculation of virus from primary gingivostomatitis in the infant to the mother's breast during breastfeeding (e90,e264,e323).
Clinical Features
Neonatal infections manifest in the first week of life. Although the neonatal form of the disease may be relatively benign, the majority of cases result in death
from disseminated disease with meningoencephalitis (50%), or serious neurologic impairment (30%). Although no specific sign or symptom is diagnostic, the
diagnosis should be strongly considered in the presence of HSV risk factors, atypical sepsis, unexplained acute hepatitis, or focal seizure activity. Neonatal
HSV infection may be either disseminated or relatively localized; in general the younger the patient at presentation, the more disseminated the lesions, and
even infants with encephalitis usually have at least skin and mucous membrane lesions (Figure 6-3A). Conversely, however, at least a third of newborns with
disseminated HSV do not have detectable skin or mucous membrane lesions at the time of presentation (e309). The clinical presentation is variable and
diagnosis may be extremely difficult; seizures, cyanosis, shock, and bleeding diathesis are common manifestations.
Pathology
The pathologic hallmark of disseminated HSV are patchy and focal well-demarcated punctuate areas of yellow-tan to hemorrhagic coagulative necrosis with
little cellular inflammatory reaction at the periphery of irregular zones of necrosis (Figure 6-3B and C). The characteristic inclusions are beautifully illustrated
in Singer's paper (e309) (Figure 6-3D) and are of two types. The early infectious inclusions (Cowdry type B) are variably staining (usually amphophilic,
sometimes basophilic), homogeneous and glassy, occupying the entire nucleus, and pushing the chromatin to the nuclear membrane. The second type
(Cowdry type A) is smaller, deeply eosinophilic, round or polygonal and separated from the nuclear membrane by a clear halo. Multinucleated cells are more
likely to contain Cowdry type B inclusions. Type A inclusions occur later in the infection and reflect excess viral capsid material following extrusion of
encapsidated viral DNA. In 75% to 80% of cases of disseminated HSV, the liver (Figure 6-3B and C) and adrenal glands are involved. Lesions may also be
seen in the lung, brain, spleen, bone marrow, and GIT. Care must be exercised in the evaluation of necrotizing and ulcerated skin or mucous membrane
lesions; HSV inclusions can usually be found at the periphery of such lesions, but secondary bacterial or yeast infection may obscure the underlying viral
lesion.
If vesicular or ulcerated lesions are present, a firm diagnosis is usually possible using smears of vesical fluid or scrapings of the base of the lesion. In
properly stained smears, identification of the characteristic, often multiple inclusions is straightforward; epithelial cells contain one or more large intranuclear
inclusions, described with three Ms as multinucleate, with nuclear molding and chromatin margination (Figure 6-3D). Morphologic distinction from varicella-
zoster inclusions is not possible, but in the usual clinical setting this is not a problem. Both immunohistochemical and molecular biologic techniques are
available and are useful in distinguishing HSV from other viruses (e239).

Neonatal herpes is a potentially devastating illness with 80% mortality without treatment. The mortality rate for disseminated disease remains very high at
over 50%, even with therapy (e160). About 25% of survivors may have neurologic defects and/or blindness. Maternal treatment or prophylactic treatment of
the infant may be reasonable in certain situations to decrease shedding, hasten clinical resolution of the lesions, and protect the infant.
The consequences of neonatal HSV infection can be severe (e175). Disease can be localized to skin, eye, and mouth (SEM disease), involve the CNS or
manifest as disseminated infection involving multiple organs. Most surviving infants in the latter two categories have neurological sequelae. Neonatal herpes
may occur in the absence of skin lesions; if infection is suspected, swabs of the oropharynx, conjunctiva, rectum, skin lesions, mucosal lesions, urine, and
cerebrospinal fluid (CSF) should be promptly submitted for laboratory studies (96).
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FIGURE 6-3 ▪ Herpes simplex infection. A: Herpetic stomatitis. B: Liver (low power) with multifocal areas of coagulative necrosis. C: Liver (high power) with
smooth nuclear inclusions usually at the interface between the necrotic and viable parenchyma. D: Pictorial representation of inclusions—camera lucida
drawings by E. Piotti; each nucleus corresponds to the types of inclusions seen in the first reported case of HSV infection (With permission from Singer DB.
Pathology of neonatal Herpes simplex virus infection. Perspect pediatr pathol 1981;6:243-278.)
HUMAN PARVOVIRUS INFECTION

The B19 parvovirus, although better known as a cause of erythema infectiosum (fifth disease), is a relatively recent entrant to the list of fetal pathogens
(e8,e32). This single stranded DNA virus of the Erythrovirus genus primarily targets erythroid precursors in the bone marrow. Because the erythrocyte P
antigen is the cellular receptor for the virus, individuals lacking this antigen are resistant to infection (e31).
Transmission
Transmission is through contact with respiratory secretions (droplets, saliva) and, less commonly, other body fluids (blood and urine). Seroprevalence data
show peak parvovirus infection occurring in school-age children. Mode of entry into bloodstream and placental invasion is not clearly known.
Clinical Features
Intrauterine infection results in fetal anemia with a pronounced leukoerythroblastic reaction and hepatitis, with excessive iron deposition in the liver.
Parvovirus is a major cause of nonimmune hydrops, possibly accounting for up to 16% of cases of “idiopathic” nonimmune hydrops (54). In Anand's series
(e8), two of six affected pregnancies resulted in fetal hydrops and death; the other four infants were normal. Studies of fetuses and newborns infected with
parvovirus have also described presentations other than fetal hydrops. Ocular lesions include microphthalmia, aphakia, and
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dysplasia of sclera, anterior segment, and retina (e134). In live-born infants, a lethal constellation of anemia, petechial rash, purpuric “blueberry muffin”
appearance and severe liver disease with hepatic fibrosis and siderosis mimics the syndrome of neonatal hemochromatosis (188) (e218,e306,e364). (see
Chapter 15).
Although postnatal infection is frequently asymptomatic, the best known clinical illness is the immune-mediated erythema infectiosum or fifth disease. Fifth
disease is a highly contagious illness with a slapped cheek lacy erythematous exanthem on the face, trunk, and proximal limbs in children; adults manifest
arthralgias and arthritis. Severe disease is seen most often in individuals with hemoglobinopathy, red blood cell abnormalities, and immune deficiency.
Erythroid abnormalities include pure red cell aplasia and aplastic anemia (especially aplastic crisis in a setting of chronic hemolytic anemia). Vasculitis and
hemophagocytic syndrome can also occur.
Pathology
The bone marrow may show erythroid hypoplasia of variable severity. Morphologic abnormalities in red cell precursors include giant pronormoblasts with
vacuoles and multiple nucleoli. Distinctive eosinophilic intranuclear inclusions may be seen in erythroid lineage cells. The inclusions have been shown by
DNA hybridization and electron microscopy (e8,e42,e181) to contain B19 virus, and the virus is readily detected in tissue with immunohistochemical and
PCR techniques (54) (e209). Histologic studies on very young fetuses show ocular malformations and intense inflammatory reactions in all tissues (e348).
The first clue to the infection may be the presence of infected red cells in the fetal capillaries of the placenta (see Chapter 9).
RUBELLA
Rubella is caused by a single stranded RNA virus. Originally described by Gregg as a classic triad of cataracts, deafness, and congenital heart disease
(e126), the “expanded rubella syndrome,” as it is sometimes referred to, is rare in countries where rubella vaccination is the norm. However, the congenital
rubella syndrome (CRS) is unfortunately not just an item of historical interest due to the continued existence of both a nonimmune population of women of
childbearing age and the many survivors of the 1964 to 1965 epidemic. Investigation of these individuals, now adults, has made possible the delineation of
the late effects of congenital infection (156).
Transmission
The rubella virus is capable of infecting the fetus at any time during gestation. The virus reaches the fetus in emboli of necrotic placental tissue and affects
the fetus by at least three mechanisms: (a) inhibition of cell growth, (b) cytolysis, and (c) compromise of blood supply (156). These, in turn, incite necrosis,
inflammation, and scarring in virtually limitless combinations and permutations. Unlike in CMV infection, maternal antibodies to rubella virus protect the fetus
from infection. Postnatal and childhood infections are transmitted through inhalation of droplets of nasopharyngeal secretions.
Clinical Features
The incidence and pattern of fetal disease vary strikingly with gestational age at the time of maternal viremia (e126,342). Congenital heart defects result from
infection in the first trimester of gestation, deafness, and neurologic deficits from infection through the 4th gestational month, and retinopathy through the 5th
gestational month. Infection late in gestation is more likely to produce inflammatory and destructive lesions, without evidence of malformation. The probability
of the fetus suffering significant damage reduces from 80% to 90% in first trimester to negligible beyond 20-week gestation. In either case, the virus is
recoverable for months to years after birth. With the possible exception of microcephaly, most of the CNS abnormalities are the result of meningoencephalitis
and/or necrosis. Necrosis is presumably ischemic and related to the vascular lesions seen in a majority of cases. True developmental malformation is rare
(156). A late-onset chronic progressive panencephalitis is seen in the second decade of life in some survivors of CRS; the neuropathologic changes are
similar to those of subacute sclerosing panencephalitis (SSPE) including meningeal and perivascular infiltrates of lymphocytes and plasma cells with glial
nodules, predominantly in the white matter (e338). Rubella virus has been recovered from these late lesions. Deafness in CRS is related to both CNS
damage resulting in central auditory imperception and also to inflammation and scarring in the cochlea. The disseminated effects of infection may be related
to vascular spread and cytopathic effects on endothelial cells. Interestingly, deafness, cardiovascular and neurological damage, and retinopathy are rare if
infection occurs beyond the second trimester, raising the possibility of a protective role of maternal antibodies in the second trimester. Webster has reviewed
these facets of rubella teratogenesis (192).
Rubella in postnatal life presents as a prodrome followed by a characteristic postauricular lymphadenopathy; a fine maculopapular rash appears 1 to 5 days
later, starting in the face and spreading to limbs and face that lasts for about 3 days. Complications are infrequent and include immune manifestations such
as arthritis, encephalitis, Guillian-Barre syndrome, and thrombocytopenia. Surveillance of postnatally and congenitally acquired infection is an essential
component of CRS prevention since rubella is difficult to diagnose on clinical grounds alone. Laboratory differentiation of rubella from other rash-causing
infections, such as measles, parvovirus B19, human herpes virus 6, enteroviruses in developed countries, and various endemic arboviruses is essential.
Reverse transcriptase PCR and sequencing for diagnosis and molecular epidemiological investigation and detection of rubella-specific IgG and IgM salivary
antibody responses in oral fluid are now available (6).
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Pathology
A wide spectrum of cardiovascular disease is seen in CRS. In addition to the characteristic patent ductus arteriosus, the most common lesions are pulmonary
artery branch stenosis, myocarditis, and systemic arterial hypoplasia and stenosis. Valvular sclerosis has been frequent in some series (156). The arterial
lesion of CRS is distinctive and possibly unique: fibromuscular intimal proliferation, devoid of inflammatory change, leads to patchy and focal vascular
stenosis. The media and adventitia are usually not disrupted, and there is no calcification (except in the brain). Chronic meningeal inflammation, perivascular
lymphocytic infiltrates, gliosis, and mineralization of cerebral arterioles may occur. Bone lesions are transient and consist of focal osteopenia and growth
inhibition. Metaphyseal changes reminiscent of syphilis, in the form of longitudinal radiologic striations are seen in half the patients. Interstitial pneumonitis is
seen in up to 75% of CRS infants and may persist for up to a year after birth. Alterations of the lymphoreticular system are variable; both precocious germinal
centers (from viral antigenic stimulus) and lymphoid depletion are encountered. Histiocytic proliferation and erythrophagocytosis may be seen. Hepatic
changes include cholestatic hepatitis, giant cell transformation, necrosis, extramedullary hematopoiesis, and fibrosis; cirrhosis may ensue. On occasion, bile
duct proliferation mimics extrahepatic biliary atresia; true biliary atresia has been reported anecdotally (e97). Eye changes include cataracts, lens necrosis,
ciliary body inflammation, iridocyclitis, and retinitis. Interstitial nephritis and chronic lymphocytic thyroiditis have also been described. The placenta may show
villitis, villous stromal necrosis, villous stromal sclerosis, and vascular endothelial lesions (92). No specific histopathologic studies are available of postnatal
rubella infection, due to its short and benign course.

Characteristically, complications affect the eyes (cataracts or retinopathy) and hearing (sensorineural deafness). Patients may have multiorgan involvement
including myocarditis, hepatitis, cytopenia, meningoencephalitis, and visceromegaly. Cardiac teratogenic effects include patent ductus arteriosus, pulmonary
artery stenosis, and supra-aortic stenosis. Long-term effects of intrauterine rubella infection have been studied in the original cohort of patients studied by Sir
Norman Gregg and include an increase in the prevalence of diabetes, thyroid disorders, early menopause, and osteoporosis, as also an increased frequency
of HLA haplotypes that are associated with autoimmune disorders (60). Approximately 20% of CRS survivors develop diabetes mellitus, usually in the second
or third decade. The pathogenesis is not understood, but persistent viral infection is implicated; the virus may be recovered from the pancreas and
lymphocytic infiltration of the pancreas has been seen at postmortem. Growth retardation continues postnatally probably because the virus remains active,
as evidenced by its prolonged shedding in nasopharyngeal secretions.
VARICELLA ZOSTER VIRUS

The VZV is a double stranded DNA virus (human herpes virus 3). The existence of a fetal varicella syndrome was suggested in 1974 (e311), although the
first case had been reported years earlier. Several studies have described the defects (e34,e140) associated with fetal varicella infection, and a specific
neonatal syndrome is associated with maternal varicella (but not by maternal zoster) in the first half of pregnancy (e7).
Transmission
Congenital infections are transmitted transplacentally. The risk of embryopathy with maternal infection in the first 20 weeks of gestation is estimated at 0.4%
to 2% (e25,252). Intrauterine insult occurs between 8 and 20 weeks of gestation resulting in a fetal disease with distinctive herpes zosterlike distribution.
Although the virus has not been isolated from affected fetuses or newborns, virus specific IgM has been demonstrated in affected fetus (e68) and VZV DNA
sequences have been recovered from the placenta (e157). Neonatal varicella infection is acquired in utero near term, or postnatally from the mother or other
(household or nursery) contacts. Infants delivered of mothers who were infected more than 5 days before delivery were the best, presumably because there
is time for production and transfer of maternal antibody. Perinatal infection can be severe when the mother presents with the rash of chickenpox from 5 days
before to 2 days after delivery, since the virus is transmitted during maternal viremia and there is not sufficient time for transfer of antibodies to the infant in
this narrow window. Postnatal transmission occurs through respiratory droplets and contact or aerosolization of virus from the skin lesions of either varicella
or zoster. Infection peaks in winter and spring.
Clinical Features
Fetal varicella results in multiple defects of skin, limbs, eyes, and brain (e7,e326), giving an impression of a sudden devastating, but self-limited, herpes
zoster-like illness occurring in utero. The most constant (100% of cases) are cicatricial skin lesions corresponding to the distribution of the affected
dermatome. These are associated frequently with hypoplasia of the underlying bone and soft tissue. Hypoplastic limbs, many seriously deformed by scarring,
are seen in 80% of cases. Calcification of the liver has been reported, suggesting dissemination, and viral-like inclusions have been reported in the lung
(e278). CNS involvement may take the form of necrotizing encephalitis with calcification (e326). Microphthalmia, severe chorioretinitis with scarring and
cataract lead to blindness. Neurologic abnormalities frequently correspond anatomically to the afflicted dermatome including limb paresis, microcephaly,
Horner syndrome, cranial nerve palsies, and cortical atrophy.
Varicella in the newborn may be limited to the skin (Figure 6-4) or disseminate widely; disseminated disease carries a very high mortality rate, largely due to
varicella pneumonia. Older children develop a prodrome for 2 to 3 days
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followed by a transient scarlatiniform rash that may precede or accompany the characteristic varicelliform rash that appears on the trunk and spreads out as
crops of 1 to 4 mm maculopapular lesions that progress to clear fluid-filled vesicles (“dew drop on a rose petal”) and pustules, with accompanying distressing
pruritis. Lesions of various stages are seen in a given patient and the patient remains infectious till all the lesions have crusted. Excoriation may leave
shallow pink depressions that get scarred if complicated by secondary bacterial infection. Vesicles may also develop on mucous membranes leaving multiple
small ulcers. Rarely there may be septic shock, hemolytic-uremic syndrome, necrotizing pneumonia, encephalitis, hepatitis, and/or Reye's syndrome (e111).
FIGURE 6-4 ▪ Varicella zoster virus infection. A: Neonatal varicella with skin lesions. B: Herpes zoster in an older child.
VZV infects sensory nerves and migrates to sensory ganglia during acute infections and remains latent there to later cause herpes zoster (Figure 6-4).
Involvement of nonneuronal satellite cells, which interface with multiple neurons, might allow the virus to involve large geographic areas (e.g., an entire
dermatome) during reactivation (e66). A prodrome of pain, itching, burning, and paresthesia may precede the characteristic zosteriform eruption by 4 to 5
days, as may constitutional symptoms such as headache, fever, and malaise. Lesions may continue to develop within the dermatome over a week and last
for 2 to 3 weeks, but may last longer in debilitated and immunodeficient patients. Ulcers, scaling, hyperpigmentation, and secondary bacterial infection with
resultant scarring may complicate the clinical picture. Herpes zoster is uncommon in childhood.
Pathology
Chicken pox is a clinical diagnosis. However, Tzanck smears of vesicular or pustular fluid (of varicella or zoster) allows rapid identification of infected cells by
demonstrating intranuclear inclusions and giant cells, similar to those seen in HSV infection. Skin biopsies also show features similar to HSV with ballooning
degeneration progressing to acantholytic intraepidermal vesicles; adnexal structures may be involved. Unlike in HSV infection, however, a leukocytoclastic
vasculitis with occasional hemorrhage may be seen in the dermis. Inclusions start as faint basophilic intranuclear bodies with peripheral chromatin
condensation, later becoming eosinophilic with a surrounding halo. An immunostain is available for specific identification. Disseminated VZV may involve a
variety of organs with hemorrhagic necrosis, little or no inflammation and eosinophilic intranuclear inclusions. Pulmonary lesions consist of an interstitial
mononuclear infiltrate with edema, hemorrhage, and hyaline membranes with focal, sharply defined centrilobular areas of necrosis (e111).

Chicken pox is almost always self-limited. Death from chickenpox is distinctly unusual (12 deaths/100,000 cases), except in immunodeficient patients in
whom pneumonia, meningoencephalitis, and hepatitis may develop (141) (e258). Less frequently, other systemic infections may occur, including nephritis,
myocarditis, arthritis, myositis, uveitis, orchitis, and idiopathic thrombocytopenic purpura (177). Reye syndrome has been described following chickenpox that
was treated with salicylate administration, and aspirin is contraindicated in patients who have chickenpox (e324).
HUMAN IMMUNODEFICIENCY VIRUS

The first cases of pediatric AIDS were reported in 1982 to 1983, soon after recognition of this entity in adults (e240,e285,e343). Over 90% of cases of
childhood HIV infection are acquired by vertical transmission. HIV/AIDS in children is similar in many
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respects to adult disease in both the primary viral cytopathic effects of HIV on the lymphoid and nervous systems, and the secondary effects of
immunodeficiency including opportunistic infections and neoplasia. However, beyond the very different mode of transmission, there are important differences
in diagnostic methods, response capability of the developing immune system, etiology of secondary infection, and type and distribution of pathologic lesions.
HIV-2 causes clinical disease similar to HIV-1, but with a significantly slower progression to immune suppression. Ekpini and colleagues documented
infrequent HIV-2 vertical transmission, but no cases of late postnatal seroconversion in a cohort of West African mothers and infants (e93). HIV-2
transmission through breast milk is less common than for HIV-1, but the risk and possible factors contributing to transmission have not been quantified
adequately. Recent volumes have detailed the epidemiology, immunopathogenesis, molecular biology, and clinicopathologic aspects of pediatric AIDS (87,
118, 134). The Center for Disease Control has recently released revised surveillance case definitions for HIV and AIDS (Table 6-9) (161). However, the
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classification system for HIV infection among children aged 18 months to 13 years has not changed and the 1994 guidelines for AIDS-defining conditions
remain valid (Table 6-10) (1).
Table 6-9A ▪ 2008 SURVEILLANCE CASE DEFINITION FOR HIV INFECTION AMONG CHILDREN AGED <18 MONTHSa
Criteria for definitive or presumptive HIV infection
Child born to an HIV-infected mother and laboratory criterion or at least one other criteria met
Laboratory criterion for definitive HIV infection
Positive results on two separate specimens (not including cord blood) using HIV virologic (nonantibody) tests (HIV nucleic acid detection is method
of choice):
Laboratory criterion for presumptive HIV infection
Criterion for definitively HIV infected not met, and
Positive result on one specimen (not including cord blood) using HIV virologic tests AND no subsequent negative results from HIV virologic or
antibody tests
Other criteria (for cases that do not meet above laboratory criteria)
HIV infection diagnosed by a physician or qualified medical-care provider based on the laboratory criteria and documented in a medical record. Oral
reports of prior laboratory test results are not acceptable.
or
When test results regarding HIV infection status are not available, documentation of a condition that meets the criteria in the 1987 pediatric
surveillance case definition for AIDS
Criteria for uninfected with HIV, definitive or presumptive
Child born to an HIV-infected mother is either definitively or presumptively uninfected with HIV if (1) the criteria for definitive or presumptive HIV
infection are not met and (2) at least one of the following laboratory criteria or other criteria are met.
Laboratory criteria for uninfected with HIV, definitive
At least two negative HIV DNA or RNA virologic tests from separate specimens, both of which were obtained at age ≥1 months and one of which
was obtained at age ≥4 months.
or
At least two negative HIV antibody tests from separate specimens obtained at age ≥6 months.
and
No other laboratory or clinical evidence of HIV infectionb
Laboratory criteria for uninfected with HIV, presumptive
Two negative RNA or DNA virologic tests, from separate specimens, both of which were obtained at age ≥2 wk and one of which was obtained at
age ≥4 weeks
or
One negative RNA or a DNA virologic test from a specimen obtained at age ≥8 weeks.
or
One negative HIV antibody test from a specimen obtained at age ≥6 months.
or
One positive HIV virologic test followed by at least two negative tests from separate specimens, one of which is a virologic test from a specimen
obtained at age ≥8 wk or an HIV antibody test from a specimen obtained at age ≥6 months.
and
Other criteria (for cases that do not meet above laboratory criteria)
Determination of uninfected with HIV by a physician or qualified medical-care provider based on the laboratory criteria and who has noted the HIV
diagnostic test results in the medical record. Oral reports of prior laboratory test results are not acceptable.
and
Criteria for indeterminate HIV infection
Child born to an HIV-infected mother if the criteria for infected with HIV and uninfected with HIV are not met.
aThese guidelines are intended for public health surveillance only and are not a guide for clinical diagnosis.
bNo positive results from virologic tests (if

tests were performed) and no AIDS-defining condition for which no other underlying condition indicative of
immunosuppression exists (see Table 6-10).
Modified from Schneider E, Whitmore S, Glynn KM, et al. Centers for Disease Control and Prevention (CDC). Revised surveillance case definitions
for HIV infection among adults, adolescents, and children aged <18 months and for HIV infection and AIDS among children aged 18 months to <13
years—United States, 2008. MMWR Recomm Rep 2008;57(RR-10):1-12.
Table 6-9B ▪ 2008 SURVEILLANCE CASE DEFINITION FOR HIV INFECTION AMONG CHILDREN AGED 18 MONTHS TO <13
YEARSa
Criteria for HIV infection
At least one of laboratory criteria or the other criterion should be met
Laboratory criteria
Positive result from a screening test for HIV antibody (e.g., reactive EIA), confirmed by a positive result from a supplemental test for HIV antibody
(e.g., Western blot or indirect immunofluorescence assay)
or
Positive result or a detectable quantity by a HIV virologic (nonantibody) testsb
Other criterion (for cases that do not meet laboratory criteria)
HIV infection diagnosed by a physician or qualified medical-care provider based on the laboratory criteria and documented in a medical record. Oral
reports of prior laboratory test results are not acceptable.
Criteria for AIDS
Children aged 18 months to <13 years are categorized for surveillance purposes as having AIDS if the criteria for HIV infection are met and at least
one of the AIDS-defining conditions has been documented.
aThese guidelines are intended for public health surveillance only and are not a guide for clinical diagnosis. The 2008 laboratory criteria for
reportable HIV infection among persons aged 18 mo to <13 y exclude confirmation of HIV infection through the diagnosis of AIDS-defining conditions
alone (see Table 6-10). Laboratory-confirmed evidence of HIV infection is now required for all reported cases of HIV infection among children aged
18 mo to <13 y.
bFor HIV screening among children aged 18 mo to <13 y infected through exposure other than perinatal exposure, HIV virologic (nonantibody) tests
should not be used in lieu of approved HIV antibody screening tests. A negative result (i.e., undetectable or nonreactive) by an HIV virologic test
(e.g., viral RNA nucleic acid test) does not rule out the diagnosis of HIV infection.
Transmission
Vertical transmission is the most common mode of acquisition of pediatric HIV. Transmission of virus by breast feeding, sexual abuse, and heterosexual or
homosexual relationships accounts for most of the remaining infection. The risk of transmission through transfusion of blood or blood products has been
almost eliminated. The timing and mechanisms of mother-to-infant virus transmission are imprecisely understood. Vertical transmission can take place
antepartum in utero, intrapartum, or postpartum, through breast feeding. To some extent, these are distinguishable on the basis of culturable virus or HIV
genome in cord and infant blood. The best predictor of transmission risk is maternal viral burden, as measured by maternal plasma HIV-1 RNA level. Levels
under 500 copies per milliliter are associated with minimal risk of perinatal transmission (116). Treatment of HIV-infected mothers with effective antiviral
agents has significantly decreased the rate of vertical transmission (e64). Studies demonstrating lower transmission rates with caesarean section and with
shortened interval between rupture of membranes and delivery indicate that obstetric interventions may also decrease the rate of perinatal infection (102)
(e185,e280).
Breastfeeding by an HIV-1-positive mother increases transmission risk through breast milk by 4% to 22%, in addition to the risk for prenatal and perinatal
transmission (e26,e72,e91). However, the lack of acceptable, feasible, affordable, sustainable, and safe (AFASS) water for breast milk alternatives has
complicated infant feeding practices in less developed nations. Current WHO/UNICEF guidelines recommend exclusive breastfeeding for all infants for at
least the first 6 months (unless AFASS criteria are satisfied) because of reduced infant mortality among exclusively breastfed, HIV-exposed infants (194).
There are many issues related to breast milk HIV-1 transmission including the increased risk for transmission with primary HIV-1 infection in the mother
during lactation, the health of the HIV-1-infected, breastfeeding mother, the presence of the virus and potentially immunologically protective factors in
colostrum and breast milk, factors that contribute to HIV-1 transmission in breast milk, and possible interventions to prevent or limit HIV-1 transmission
through breast milk (149). The avoidance of breastfeeding in maternal HIV-1 infection is an important component of preventing mother-to-child transmission
in the United States and other countries. In resource-poor situations, where the complete avoidance of breast milk can increase morbidity and mortality
because of poor nutrition or other infections, potential interventions can limit HIV-1 mother-to-child transmission, including exclusive breastfeeding, early
weaning, education, and support to decrease the occurrence of mastitis or nipple lesions, antiretroviral therapy for the mother or infant, treating the human
milk to decrease the viral burden (ultraviolet light, freezing, and thawing), and stimulating the infant's immune defenses with active or passive immunization.
Clinical Features
Clinical manifestations include hepatosplenomegaly, lymphadenopathy, failure to thrive, fever of unknown origin (FUO), chronic diarrhea, various infections,
parotitis, chronic otitis media, lymphoid interstitial pneumonitis (LIP), HIV nephropathy, HIV encephalopathy, HIV cardiomyopathy, idiopathic
thrombocytopenia purpura, and lymphoma. Age-specific data suggest that HIV manifestation changes with the child's age, and these may further vary based
on geographic location (103, 166, 172). HIV encephalopathy, HIV cardiomyopathy, idiopathic thrombocytopenia purpura, and lymphoma may occur later than
other manifestations.
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Table 6-10 ▪ AIDS DEFINING ILLNESSES IN THE PEDIATRIC AGE GROUP
Bacterial infections, multiple or recurrenta
Candidiasis of bronchi, trachea, or lungs
Candidiasis of esophagusb
Cervical cancer, invasivec
Coccidioidomycosis, disseminated or extrapulmonary
Cryptococcosis, extrapulmonary
Cryptosporidiosis, chronic intestinal (>1 month's duration)
Cytomegalovirus disease (other than liver, spleen, or nodes), onset at age >1 mo
Cytomegalovirus retinitis (with loss of vision)b
Encephalopathy, HIV related
Herpes simplex: chronic ulcers (>1 month's duration) or bronchitis, pneumonitis, or esophagitis (onset at age >1 mo)
Histoplasmosis, disseminated or extrapulmonary
Isosporiasis, chronic intestinal (>1 month's duration)
Kaposi sarcomab
Lymphoid interstitial pneumonia or pulmonary lymphoid hyperplasia complexa,b
Lymphoma, Burkitt (or equivalent term)
Lymphoma, immunoblastic (or equivalent term)
Lymphoma, primary, of brain
Mycobacterium avium complex or M. kansasii , disseminated or extrapulmonaryb
M. tuberculosis of any site, pulmonary,b,c disseminated,b or extrapulmonaryb
Mycobacterium, other species or unidentified species, disseminatedb or extrapulmonaryb
Pneumocystis jirovecii pneumoniab
Pneumonia, recurrentb,c
Progressive multifocal leukoencephalopathy
Salmonella septicemia, recurrent

Toxoplasmosis of brain, onset at age >1 monthb
Wasting syndrome attributed to HIV
aOnly among children aged <13 years. (CDC. 1994 Revised classification system for human immunodeficiency virus infection in children <13 years
of age. MMWR 1994;43[No. RR-12].)
bCondition that might be diagnosed presumptively.
cOnly among adults and adloescents aged <13 years. (CDC. 1993 Revised classification system for HIV infection and expanded surveillance case
definition for AIDS among adolescents and adults. MMWR 1992;41[No. RR-17].)
Source: Schneider E, Whitmore S, Glynn KM, et al. Centers for Disease Control and Prevention (CDC). Revised surveillance case definitions for
HIV infection among adults, adolescents, and children aged <18 mo and for HIV infection and AIDS among children aged 18 mo to <13 y—United
States, 2008. MMWR Recomm Rep 2008;57(RR-10):1-12.
There is great variation in rapidity of onset, age of onset, and rate of progression in pediatric AIDS. In perinatally infected infants, the onset of symptomatic
disease occurs at 6 to 8 months of age, as compared with a mean of about 18 months in transfusion acquired pediatric AIDS (and years in adults). This
extremely rapid progression undoubtedly reflects early disruption of differentiation in the developing cellular immune system that results from HIV-induced
destruction of CD4 lymphocytes before the establishment of a fully developed immunologic response. There is also marked variation in the rate of
progression of HIV in pediatric patients once they are symptomatic. Some perinatally infected children have onset of disease in the first year of life
characteristically with Pneumocystis jiroveci pneumonia (PCP), HIV encephalopathy, and recurrent severe bacterial infections. Another group is
characterized by onset after the first year and a more indolent and chronic course of mucosal candidiasis, LIP, and cardiovascular disease. The reason(s) for
these differences are, as yet, unclear. Children with AIDS do not show the marked degree of lymphopenia seen in adults but are more likely to have
hyperglobulinemia. Cutaneous anergy is seen in infants. Severe bacterial infections are extremely common in pediatric AIDS, occurring in over 80% of
affected children. Among pediatric opportunistic infections, candidiasis is the most frequent (Figure 6-5), beginning as oral thrush and affecting the entire
GIT; PCP is the most frequent fatal infection in infancy (Figure 6-5). Other common opportunistic pathogens include CMV (Figure 6-5), MAIC, TB,
aspergillosis, cryptococcosis, cryptosporidiosis, histoplasmosis, HSV, adenoviral pneumonia, measles, and RSV depending on the frequency of occurrence
of the organism in a given geographic area and/or population. About 25% of children with AIDS develop a lymphoproliferative syndrome with generalized
lymphadenopathy and splenomegaly.
Children with HIV demonstrate lower motor, cognitive, and adaptive functioning compared to uninfected children. Risk factors that may negatively affect the
development of infected children include neurological abnormalities, progression of the disease, and poor environmental factors (15). Anemia is also a very
common complication of pediatric HIV infection, associated with a poor prognosis. Failure of erythropoiesis may be the most important mechanism for anemia
(18). Survival in children with AIDS is in general shorter than survival in HIV-infected adults.
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FIGURE 6-5 ▪ HIV infection. A,B: Lymphoid interstitial pneumonia (low power and high power) with follicular bronchiolitis and diffuse interstitial
lymphoplasmacytic inflammation. C,D: Pneumocystis jiroveci pneumonia— foamy alveolar material with saucer or cup-shaped organisms that stain heavily
with silver (GMS stain). E: Oral thrush (candidal glossitis). F: Incidental HSV inclusions in thyroid follicular cells.
Pathology
All of the pathologic lesions that occur in adults with HIV infection are seen in children, but there are significant differences in frequency and distribution (87).
Pathologies identified more frequently in children include thymic lesions, pulmonary lymphoid and lymphoproliferative disorders (LPDs), and arteriopathy.
Polyclonal B-cell lymphoproliferative disorders (PBLD) and malignant lymphoma, especially of brain, are the common neoplasms in children; Kaposi sarcoma
is rarely encountered (e63). Table 6-11 outlines the systemic pathology of pediatric HIV infection.
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Table 6-11 ▪ SYSTEMIC PATHOLOGY OF PEDIATRIC HIV INFECTIO
Organ/System References
Placenta
Increased weight 233, 244, 246, 247,

Chorioamnionitis 359, 410
Funisitis/fetal vasculitis
Villitis
Growth and development
Increased fetal wastage, intrauterine demise 143, 197, 275,

Failure to thrive/wasting 377, 423
No increase in malformations
Thymus
Small size, cortical atrophy 18, 180, 235

Lymphoid depletion
Warthin-Finkeldy-type giant cells
Accelerated involution
Fibrosis, plasmacytosis
Calcified, cystic, or small Hassall corpuscles
Spleen 235, 469, 470
Splenomegaly
Immunoblastic proliferation
Lymphoid depletion
Histiocytosis
Hemophagocytosis
Opportunistic infection
“Kaposiform” spindle cell proliferation
Lymph nodes
Follicular hyperplasia, histiocytosis 62, 235, 351,

Plasmacytosis 411, 451
Multinucleate giant cells
Hemophagocytosis
Lymphoid depletion, fibrosis
Lymphoproliferative disorders
“Kaposiform” spindle cell proliferation
Opportunistic infection
Bone marrow
Hypoplasia or hyperplasia 180,451

Myelodysplasia
Plasmacytosis, histiocytosis
Eosinophilia, lymphoid aggregates
Hemosiderosis, fibrosis, granulomas
Serous fat atrophy
Cardiovascular system
Dilated cardiomyopathy 180, 236, 255, 286

Myocarditis, pericarditis
Vasculitis, vascular calcification
Coronary and cerebral aneurysm
Inflammation and fibrosis of conducting system
Lung
Opportunistic infection, especially PCP, CMV, RSV 14, 49, 237,

Lymphoproliferative disorders (PLH, LIP, PBLD) 238, 239,
Malignant lymphoma 463
Giant cell pneumonia
Smooth muscle neoplasms
Gastrointestinal tract
Opportunistic infection, especially 13, 180, 244

Candida, CMV, MAI, Cryptosporidium,
Isospora, Salmonella, Shigella
Lymphoid depletion of MALT
Lymphoproliferative disorders
Neoplasms (lymphoma, smooth muscle tumors, Kaposi sarcoma)
Ulcers of undetermined etiology
Pneumatosis, pseudomembranous enteritis
Liver
Chronic active hepatitis, including HBV and HCV, giant cell hepatitis 233, 244, 246,
Opportunistic infection, especially CMV, adenovirus, MAI 247, 359, 410
Pancreas
Acute and chronic pancreatitis, some associated with pentamidine or dideoxyinosine 65, 245, 248
Opportunistic infection (CMV, MAI, Candida)
Steatonecrosis
Islet hypertrophy and fibrosis
Dilatation of ducts and acini
Nodular lymphoid infiltrates
Kidney
HIV-associated nephropathy: focal and segmental glomerulosclerosis, mesangial hyperplasia, immune complex 68, 107, 180, 235
glomerulonephritis, and minimal change disease
Opportunistic infection (CMV, Candida, MAI)
Nephromegaly
Nephrocalcinosis
Skin
Opportunistic infection (Candida, HSV, VZ, HPV) 180, 235, 282, 458
Molluscum contagiosum
Scabies
Seborrheic dermatitis
Kaposi sarcoma
Nervous system
Cerebral atrophy, multinucleate giant cells, microglial nodules, vascular mineralization 40, 121, 144, 145,
Lymphoma 180, 427
Opportunistic infection (Candida, CMV, MAI, progressive mulfocal leukoencephalopathy)
Nonspecific white matter pallor or gliosis
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Lymphoid Organs
The thymic lesions of childhood HIV infection include precocious or marked involution, marked reduction or absence of Hassall corpuscles, and thymitis
(e165). Thymitis may take the form of follicular, mononuclear, or plasma cell infiltrates. Thymic dysfunction and thymic involution occur during HIV disease
and have been associated with rapid progression in infants infected perinatally with HIV. Perivascular sclerosis is common. Thymic involvement may be due
to direct infection or may represent an autoimmune process. Thymic recovery may be achieved in some patients as a result of potent antiretroviral therapy.
Extensive thymic damage may, however, hamper immune reconstitution, particularly in pediatric patients (198).
Although splenomegaly is commonly seen in HIV-infected children, there is lymphoid depletion, architectural disarray, increased macrophages and functional
hyposplenia. Cytologically, lymphoid organs show many large lymphocytes, immunoblasts, and also giant cells (polykaryocytes). Progression to lymphoma
and Castleman disease may occur in nodal and extranodal sites. Quijano has detailed histopathologic findings in lymph nodes (144).
Lungs
HIV-related pulmonary lymphoid and lymphoproliferative lesions including pulmonary lymphoid hyperplasia (PLH), LIP, and PLBD (86) are more common in
children than in adults. PLH is a peribronchial infiltrate of benign lymphoid follicles, often with germinal centers. LIP is characterized by a significant infiltrate
of lymphocytes, plasmacytoid cells, plasma cells, and the occasional large immunoblastic cell that expand the interstitial septa (Figure 6-5). There is much
overlap between PLH and LIP; they often coexist, hence the designation PLH-LIP complex. These disorders constitute a spectrum of disease related to
Epstein-Barr virus (EBV) infection and may eventuate in PLBD or in malignant lymphoma (e164,e331).

Neurologic manifestations are frequent in children with AIDS (42) (e95); HIV-related encephalopathy is characterized by low IQ, loss of developmental
milestones, microcephaly, progressive weakness, and seizures. Morphologic features include gross brain atrophy, hydrocephalus, diffuse gliosis,
multinucleated giant cells, microglial nodules, basal ganglia mineralization, HIV encephalitis, corticospinal tract degeneration, and siderocalcinosis of blood
vessels. Common lesions not directly related to HIV infection are lymphomas and cerebrovascular accidents. Opportunistic CNS infections are relatively
uncommon, limited predominantly to monilial and cytomegaloviral encephalitides. CNS lymphoma, although less common in children than in adults, is the
most common malignancy in pediatric AIDS and is usually EBV-associated. Myelin abnormalities occur both in the brain and the spinal cord and are
attributed to delayed myelination, myelin injury, and/or Wallerian degeneration. Progressive multifocal leukoencephalopathy (PML) is rare in children. It has
been suggested that most of the CNS effects of HIV infection cannot be attributed to detectable levels of viral antigen, but may be due to circulating
cytokines and other soluble factors. The prevalence of HIV encephalopathy has not decreased despite use of HAART; as patients live longer, the prevalence
of CNS manifestations may actually increase (e118).
Other Viscera
Liver disease manifests as hepatomegaly with altered enzymes, cholestasis, and/or hepatitis. Cholestatic hepatitis may be the first clue to a pediatric HIV
infection. Giant cell transformation of hepatocytes is associated with poor outcomes in these children and is often associated with inflammation and diffuse
fibrosis. Viral hepatitis (HBV, HCV, and EBV-hepatitis) and HAART-induced liver effects may contribute to liver injury (63, 164, 185) (e300). Morphologically,
these may show chronic hepatitis with varying activity and/or cholestatic hepatitis. GI manifestations are similar to that in adults, the pathology including HIV
enteropathy, opportunistic infections, and EBV-associated smooth muscle tumors (185) (e43,e155,e166). Renal lesions include focal segmental
glomerulosclerosis, mesangial hypercellularity, microcystic transformation of renal tubules, immune complex glomerulonephritis, minimal change disease, and
nephromegaly (due to glomerulomegaly, tubular dilatation, and interstitial inflammation) (114, 147) (e5,e268). Secondary changes include drug-related
nephrotoxicity and opportunistic infections. Salivary glands are often affected early giving an appearance of chronic mumps. In HIV-associated arteriopathy,
small and medium-sized vessels in many organs (heart, lung, spleen, kidney, intestine, and brain) show fibrous intimal thickening, fragmentation or loss of
elastica, and calcification. This results in luminal narrowing, aneurysmal dilatation, and distal ischemic lesions (e169). HIV infects the fetus through the
placenta. Although chorioamnionitis, cytotrophoblastic hyperplasia, and other pathology have been identified in placentas of HIV-infected women, no lesion is
specific for HIV infection (37) (e46). HIV antigens have been found in placental Hofbauer cells, trophoblasts, and villous endothelial cells (e16). Infected
placental macrophages may infect fetal circulating cells or fetal endothelial cells.
As in adults, HIV-1 causes the majority of cases of childhood AIDS, but the diagnosis is more difficult in infants. In infants under the age of 18 months, HIV
antibody tests such as the ELISA, western blot, and recently approved rapid tests are not used, since maternal HIV antibodies may persist in the child until 6
to 18 months (e281). In this age group, definitive diagnosis of HIV infection requires two positive viral detection assays on separate specimens, or
documentation of an AIDS-defining illness (148, 161) (e215). For children
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aged 18 months to 13 years, laboratory-confirmed evidence of HIV infection is required in addition to the presence of one or more AIDS-defining conditions,
to meet the surveillance case definition for AIDS (161). The salient differences between the 2007 World Health Organization (WHO) and 2008 CDC revised
surveillance definitions are outlined in Table 6-12 (161).
Table 6-12 ▪ COMPARISON OF WHO AND CDC STAGES OF HIV INFECTIONa
WHO Stageb WHO T-lymphocyte Count and CDC CDC T-lymphocyte Count and Percentage
Percentagec Staged
Stage 1 (HIV infection) CD4+ T-lymphocyte count of Stage 1 CD4+ T-lymphocyte count of ≥500 cells/μL or
≥500 cells/μL (HIV
CD4+ T-lymphocyte percentage of ≥29
infection)
Stage 2 (HIV infection) CD4+ T-lymphocyte count of Stage 2 CD4+ T-lymphocyte count of 200-499 cells/mL or
350-499 cells/μL (HIV
CD4+ T-lymphocyte percentage of 14-28
infection)
Stage 3 (advanced HIV CD4+ T-lymphocyte count of Stage 2 CD4+ T-lymphocyte count of 200-499 cells/μL or
disease [AHD]) 200-349 cells/μL (HIV
CD4+ T-lymphocyte percentage of 14-28
infection)
Stage 4 (acquired CD4+ T-lymphocyte count of <200 cells/mL or Stage 3 CD4+ T-lymphocyte count of <200 cells/mL or
immunodeficiency syndrome (AIDS)
CD4+ T-lymphocyte percentage of <15 CD4+ T-lymphocyte percentage of <14
[AIDS])
aFor reporting purposes only.
bAmong adults and children aged ≥5 years.
cPercentage applicable for stage 4 only.
dAmong adults and adolescents (aged ≥13 years). CDC also includes a fourth stage, stage unknown: laboratory confirmation of HIV infection but no
information on CD4+ T-lymphocyte count or percentage and no information on AIDS-defining conditions.
From Schneider E, Whitmore S, Glynn KM, et al. Centers for Disease Control and Prevention (CDC). Revised surveillance case definitions for HIV
infection among adults, adolescents, and children aged <18 months and for HIV infection and AIDS among children aged 18 months to <13 years—
United States, 2008. MMWR Recomm Rep 2008;57(RR-10):1-12.
MEASLES
The causative agent of measles (rubeola) is a single stranded RNA paramyxovirus virus of the genus morbillivirus. Suboptimum vaccination coverage raises
serious doubts that the goal of elimination by 2010 can be attained (120) (e213,e228). Although global deaths from measles have decreased notably in past
decades, due to both increases in immunization rates and decreases in measles case fatality ratios (CFRs), the values for measles CFR remain imprecise,
resulting in continued uncertainty about the actual toll that measles exacts (195).
Transmission
Measles is highly contagious and is spread by aerosols and droplets of respiratory secretions. The viral receptor is CD46 for viral H and F glycoproteins and
viremia is mediated through infection of lymphoid and endothelial cells. Host innate immune responses are effective in promptly eliminating the virus (70).
Clinical Features
After a 1- to 2-week(s) incubation period, there is a prodrome (of fever, cough, rhinorrhea, and/or conjunctivitis) with the development of Koplik spots
characteristically seen in the oral mucosa. This is followed by an erythematous maculopapular (morbilliform) rash that begins on the face, spreads to the
trunks and limbs (Figure 6-6), and fades about 6 days later in the same order in which it had appeared.
Complications are a result of progressive viral replication, secondary bacterial or viral infections, and/or an abnormal host-immune response. The most
common complications are bacterial pneumonia or otitis media, the former being the most frequent cause of death. Other complications are febrile
convulsions, encephalitis, chronic diarrhea, and liver function abnormalities. Pulmonary complications (secondary pneumonia, giant cell pneumonia, and
atypical measles pneumonia) are the most feared. Prophylactic antibiotics may help prevent respiratory complications (88), although this has been refuted.
While rare, the measles virus can infect the CNS and trigger fatal CNS diseases weeks to years after exposure (200). CNS complications include acute
postinfectious allergic encephalitis, acute progressive measles (inclusion body) encephalitis, pseudotumor cerebri (e328), and SSPE. SSPE has an average
6-year latent period after infection/vaccination and is manifested as progressive mental retardation, motor dysfunction, seizures, coma, and death in 1 to 2
years.
Pathology
The pathology of measles infection is characterized by two types of multinucleated giant cells. Warthin Finkeldey giant cells are seen in lymphoid tissues
throughout the body during the incubation period; while epithelial giant cells occur in the epithelia of all major organs (Figure 6-6). The giant cells may
contain nuclear and/or cytoplasmic inclusions. Interstitial pneumonitis is characteristic, with or without a granulomatous response. Allergic phenomena
(atypical measles
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pneumonia and postinfectious encephalitis) are characterized by vascular injury and necrosis. SSPE may occur as sequelae, following infection of neurons
and glial cells; histopathologically, there is lymphocytic vascular cuffing, gliosis, and demyelination. Both immunohistochemical and in-situ hybridization
techniques are available to demonstrate the virus in tissues. This helps differentiation from respiratory syncitial virus, VZV, and parainfluenza, since all these
agents can cause giant cell pneumonia with a granulomatous response. Although laboratory tests are rarely required to diagnose measles, laboratory
confirmation is an important component of disease surveillance in all settings. The CDC has recently recommended serum-based diagnostics as the “gold
standard” for this purpose, although alternative specimens such as dried blood spots and oral fluid samples are viable alternatives for surveillance (151).
FIGURE 6-6 ▪ Measles. A: Clinical picture of morbilliform rash on the chest. B: Measles pneumonia showing scattered giant cells (low power). C: Warthin-
Finkeldey giant cells in measles pneumonia.
EPSTEIN-BARR VIRUS
The EBV is a gamma-human herpes virus that infects B-lymphocytes and epithelial cells of the pharyngeal mucosa, salivary gland ducts, and uterine cervix.
It has the unique distinction of being the first human tumor virus to be discovered, and has a diverse clinical disease spectrum including infectious
mononucleosis (IM), LPDs, lymphoepitheliomalike (nasopharyngeal) carcinomas, and rare mesenchymal neoplasms. Infection of epithelial cells is lytic
(productive), with resultant full cycle of viral replication and release of infectious virus particles into secretions. On the other hand, infection of B-lymphocytes
is predominantly latent (nonproductive), with the potential for immortalization and activation of infected cells (e293). Only a limited set of genes are expressed
during latent cycle infection [EBV nuclear antigens (EBNAs) and three latent membrane proteins (LMPs)]; these define different latency patterns (30) (e351).
The virus is ubiquitous and is transmitted primarily by saliva, although transmission by blood transfusion and allogeneic bone marrow transplantation is also
documented. Recently, sexual transmission has also been proposed as a route of infection (75) (e371). Very little is known about the risk of congenital EBV
infections, with only one welldocumented case in the literature (e122). Since most adult women have become seropositive during childhood, primary
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EBV infection during pregnancy is rare, whereas reactivation of a latent EBV infection seems to occur more often in seropositive pregnant women as
compared to control subjects (e110). However, only primary infection (and not reactivation) may be harmful to the embryo or fetus. Worldwide, primary
infection occurs within the first few years of life and is usually subclinical; symptomatic IM occurs when infection is delayed to adolescence or beyond.
Infectious Mononucleosis
IM, first detailed by Sprunt and Evans in 1920 (e310), is a self-limiting lymphoproliferative disease with a benign course. The highest rates of IM occurs
between 10 and 19 years of age (6 to 8 cases per 1,000 persons per year) (e115,e138), although mild infections in younger children may often be
undiagnosed. Rates of infection are highest in closeted populations of young adults such as active-duty military personnel and college students (11 to 48
cases per 1,000 persons per year) (19) (e346).
Most clinical symptoms are due to the host's immune response. The incubation period is estimated to be 5 to 7 weeks, followed by a prodrome of 3 to 5 days
(with headache, malaise, and fever) and the characteristic triad of fever, sore throat, and extensive cervical lymphadenopathy/tonsillar enlargement.
Pharyngeal inflammation and transient palatal petechiae are also common. Other frequent clinical manifestations include splenomegaly (identified in all
patients by ultrasonography) and hepatomegaly with transient hepatic dysfunction. EBV infection must be considered in children with FUO (e251). Younger
children may show less typical and less severe clinical disease. The well-known atypical lymphocytes (first described by Downing and McKinley) appear in
circulation from 1 to 4 weeks after disease onset (e82). These atypical cells are mainly activated oligoclonal CD8-positive cytotoxic T-cells, with only a small
proportion representing EBV-infected B-cells; in fact the CD8proliferation may result in a reduction of the CD4/CD8 ratio (e337). The uncomplicated illness
usually lasts for 2 to 4 weeks. Complications of IM involve the hematopoietic system (anemia, thrombocytopenia, neutropenia), heart (pericarditis,
myocarditis), nervous system (meningoencephalitis, cerebellitis, Guillain-Barre syndrome, Bell palsy, transverse myelitis, autoimmune neuropathies), skin
(ampicillin- associated rash, Gianotti-Crosti syndrome), kidneys (nephritis, glomerulopathies), immune system (hypo-, hypergammaglobulinemia, auto-
antibodies), and psychiatric diseases (85) (e293). Although most patients are advised to avoid contact sports to prevent potentitially serious splenic rupture,
this is a rare complication (˜0.1%) (e101). “Virusassociated hemophagocytic syndrome” is an unusual consequence of unknown pathogenesis, characterized
by a benign generalized histiocytic proliferation with marked hemophagocytosis in bone marrow and lymph nodes (e275). Usually, IM is an acute, self-limiting
disease that occurs only once in the host's lifetime. However, some patients suffer from recurrent fever, persistent hepatosplenomegaly, hematological
abnormalities, neuromyasthenia, and the so-called chronic fatigue syndrome (e321). Many of these patients reveal immunological abnormalities such as
deficient natural killer cell activity, and abnormal antibody responses to the different EBV antigens. Prolonged illness after IM may be due to altered immunity
rather than increased viral load (e39).
The differential diagnoses for suspected IM include streptococcal pharyngitis, toxoplasmosis, CMV pharyngitis, acute HIV infection, and other viral
pharyngitis (50). The presence of splenomegaly, posterior cervical adenopathy, axillary adenopathy, and inguinal adenopathy is most useful in considering
the possibility of IM, while the absence of cervical adenopathy and fatigue is most helpful in dismissing the diagnosis. Hoagland's criteria (e142) for the
diagnosis of IM are widely cited: at least 50% lymphocytes and at least 10% atypical lymphocytes in the presence of fever, pharyngitis, and adenopathy, and
confirmed by a positive serologic test. Although specific, these criteria are not highly sensitive; only about one-half of symptomatic patients with a positive
heterophile antibody test meet all the criteria.
Diagnosis rests on viral serology and the detection of the EBV genome, viral antigens, or infectious virus in saliva or lymphoid tissues. The accidental
discovery of elevated heterophile antibody by Paul and Bunnell in 1932 (e253) forms the basis for the heterophile agglutination reaction. Although they are
relatively specific, IgM heterophile antibody tests are somewhat insensitive, particularly in the first weeks of illness. Heterophile antibody tests are less
sensitive in patients younger than 12 years, detecting only 25% to 50% of infections in this group, compared with 71% to 91% in older patients (e195).
Antibodies to viral capsid antigen (i.e., VCA-IgG and VCA-IgM) are produced slightly earlier than the heterophile antibody and are more specific for EBV
infection (e35); in acute infection IgM anti-VCA antibodies are present and anti-EBNA antibodies are absent. The VCAIgG antibody persists past the stage of
acute infection and signals the development of immunity (e9). A past infection is identified by the absence of IgM antibodies and the presence of IgG
antibodies against VCA and EBNA. However, antiEBNA antibodies may not be detected in immunodeficient children. Patients with latent infection have
elevated antibodies against early antigen (EA). Although no evidence-based or consensus guidelines have been proposed to guide the evaluation of patients
with suspected IM, Ebell has proposed an algorithmic approach based on the percentage of atypical lymphocytes and absence of streptococcal pharyngitis
(50). At present, nucleic acid hybridization (by Southern blot, in-situ hybridization or PCR) is the most specific method for the detection of EBV in clinical
material. The laboratory diagnosis of IM has been recently reviewed (69) (e150).
Histologically, enlarged lymph nodes show a predominant paracortical expansion, with atypical cells that are predominantly cytotoxic T-cells (CD8 and TIA-1
positive). EBV infected cells are best identified by in-situ hybridization for EBV-encoded small RNA (EBER) (Figure 6-7). Histologic
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findings of EBV-associated hepatitis include minimal swelling and vacuolization of the hepatocytes, and a peculiar sinusoidal infiltration of T-cells in an
“Indian Bead” pattern, in addition to periportal inflammation. (see Chapter 15).
FIGURE 6-7 ▪ Infectious mononucleosis. AB: Lymph node biopsy shows paracortical expansion and an atypical lymphoid infiltrate with numerous
immunoblasts. C: These activated lymphoid cells are highlighted by CD30 immunostain. D: In-situ hybridization for EBV (EBER probe) shows strong, diffuse
nuclear positivity.
EBV-Associated Neoplasms
Neoplasms associated with latent EBV infection include lymphomas, nasopharyngeal carcinoma, lymphoepithelial carcinomas in various viscera, smooth
muscle tumors, and inflammatory pseudotumor-like follicular dendritic cell tumor (30, 38, 40, 153) (e12,e51). Different latency patterns are associated with
different neoplasms (e.g., type I with Burkitt lymphoma, type II with Hodgkin lymphoma and nasopharyngeal carcinoma, and type III with posttransplant
LPDs), with type III latency expressing more EBV proteins and being more immunogenic and type I being the least immunogenic (30). In lymphomagenesis,
EBV either plays a direct role (such as in posttransplant LPDs and HIV- associated immunoblastic lymphoma occurring in immunodeficient individuals) or as
a cofactor (such as in Burkitt lymphoma and some T/NK-cell malignancies occurring in immunocompetent individuals). EBV-associated T/natural killer (NK)-
cell LPD (EBV-T/NK LPD) of children and young adults is generally referred to with the blanket nosological term of severe chronic active EBV infection
(CAEBV) and overlaps with a unique disease previously described as infantile fulminant EBV-associated T-LPD. This disease is rare, is associated with high
morbidity and mortality, and appears to be more prevalent in East Asian countries. The major signs and symptoms include fever, hepatomegaly,
splenomegaly, liver dysfunction, thrombocytopenia, anemia, lymphadenopathy, hypersensitivity to mosquito bites, skin rash, hydroa vacciniforme, diarrhea,
and uveitis. A classification system for EBV-T/NK LPD of children and young adults has been recently proposed
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based on morphology (polymorphic or monomorphic) and clonality (polyclonal or monoclonal NK or cytotoxic T-cells) (131).
VIRAL HEMORRHAGIC FEVERS

The combination of fever and hemorrhage can be caused by viruses, rickettsiae, bacteria, protozoa, and fungi. However, conventionally, the term
“hemorrhagic fever” refers to fever and hemorrhage caused by viruses transmitted by arthropods (“arboviruses”) and rodents. Viruses implicated in this
syndrome are diverse and include arenaviridae (e.g., lassa virus), bunyaviridae (e.g., Hanta virus, Rift valley fever), flaviviridae (e.g., yellow fever, dengue),
chikungunya, and filoviridae (Ebola, Marburg) (21, 67) (e105,e139,e234,e327). The detailed description of each of these conditions is beyond the scope of
this chapter. Certain general features common to these syndromes will be outlined, using dengue as a prototype (143) (e77,e298). The clinical differential
diagnoses include leptospirosis, rickettsial fevers (e.g., typhus), complicated malaria, and disseminated intravascular coagulopathy (DIC) following severe
sepsis of any etiology. Viral hemorrhagic fevers have become a major concern in the recent past. Agents such as Dengue virus have caused many recent
epidemics; more than 1.2 million cases of dengue fever and dengue hemorrhagic fever (DHF) were reported to the WHO from 56 countries in the 1998
pandemic (106).
Transmission
Many of these arboviral fevers are transmitted to humans by mosquito bites (dengue is transmitted by the female Aedes mosquito) and several ecologic
factors have contributed to a significant increase in the incidence of dengue fever and the emergence of DHF as a major public health problem in America
and Asia. Prenatal or perinatal transmission has been reported in rare instances (e30). There is no evidence for transmission of dengue virus in breast milk,
nor more severe disease in breast-fed infants compared with formulafed infants. There has been no documented person-to-person transmission of dengue
virus without a mosquito vector.
Clinical Features
Dengue viruses cause dengue fever, DHF, and dengue shock syndrome (DSS) in infants less than 1 year of age, but rarely in those younger than 3 months
(e129). The disease spectrum ranges from a mild flu-like illness to life threatening manifestations with severe hypotension (due to vascular dysregulation),
vascular abnormalities (manifested as conjunctival suffusion, flushing, and exanthem), capillary instability (manifested as edema), and hemorrhage (due to a
combination of thrombocytopenia and microvascular damage with DIC). Visceral involvement manifests variably as renal, pulmonary, hepatic, and
neurological dysfunction, and as a result of lymphoid necrosis and depletion. Infection of mononuclear cells leads to cytokine activation and plays a central
role in the pathogenesis of DHF. Antibody-dependent enhancement due to preexisting antidengue IgG against the infecting strain causes more severe
disease.
Infants and young children with dengue usually have only a nonspecific febrile illness, with a rash that is hard to distinguish from other viral illnesses. The
more severe cases usually occur in older children and adults, characterized by a rapidly rising temperature and severe headache, myalgia and arthralgia that
last for 5 to 6 days. Many patients have an initial macular to maculopapular rash that later becomes diffusely erythematous. Minor hemorrhagic
manifestations such as petechiae, epistaxis, and gingival bleeding occur. Although dengue fever may be incapacitating, its prognosis is favorable and most
patients generally recover after 7 to 10 days of illness. DHF, on the other hand, is an acute febrile illness with hemorrhagic manifestations,
thrombocytopenia, and evidence of increased vascular permeability resulting in loss of plasma from the vascular compartment. Hypoproteinemia, an elevated
hematocrit and serous effusion are indicators of plasma leakage, which may progress to circulatory failure, so-called dengue shock syndrome (DSS). The
patient may die within 24 hours, or may recover quickly following appropriate volume replacement and supportive therapy. Neurological manifestations may
occur in the absence of shock (90). Complications such as hepatic dysfunction and fluid overload are more commonly found in infants and the case fatality
rate is also higher in this age group (89).
Pathology
Morphologically there is variably prominent capillary dilatation, endothelial swelling, edema, and/or vasculitis with fibrin thrombi. Target organs are different
in different syndromes; for example, in Hantavirus syndromes the major target organ may be the lung or the kidney with brain, liver, and spleen being
secondary target organs. Each involved organ may show features of severe injury such as diffuse alveolar damage, renal tubular necrosis, medullary
hemorrhage, and features of DIC may be present.
OTHER SYSTEMIC VIRAL INFECTIONS

Disseminated adenoviral disease usually occurs in immunocompromised hosts, posttransplantation or in neonates, and manifests with clinically significant
destructive hepatitis (e349). Rarely, infection may occur in healthy children (e98). The hepatitis is characterized by variable necrosis with intranuclear
amphophilic or basophilic viral inclusions with peri-inclusional clearing or, in late stages, smudge cells. Adenovirus may also cause enterocolitis and
pneumonia.
Echovirus can cause flu-like symptoms in any age group. However, fatal and severe infections are almost
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exclusively reported in neonates and infants. The spectrum of illness includes encephalitis, meningitis, hepatitis, and other unusual manifestations such as
myocarditis, orchitis, postviral fatigue syndrome, and transient erythroblastopenia of childhood (e94).
Infection-associated hemophagocytic syndrome presents as a severe acute systemic illness with prolonged fever, constitutional symptoms,
hepatosplenomegaly, cytopenias, and hepatic dysfunction. Conventionally thought to affect immunocompromised patients, in a relatively large study of 18
pediatric cases, almost 90% of patients were previously healthy; the case fatality rate was 61% with all fatal cases dying within 2 months of disease onset
(23). Formerly thought to be a sequel to a viral infection (especially EBV), it is now known to be also associated with a wide variety of agents including Gram
negative bacilli, mycobacteria, protozoa, and fungi. In the above study, children less than 3 years of age were more vulnerable to neutropenia-associated
bacteremia. The histologic hallmark is widespread proliferation of cytologically benign histiocytes with erythrophagocytosis and lymphophagocytosis.
FIGURE 6-8 ▪ Mumps. A: Bilateral parotitis is seen in this clinical photograph. B: Histological appearance of parotitis is shown with a diffuse lymphocytic
infiltrate. C: Mumps myocarditis is characterized by the presence of a lymphocytic infiltrate and focal myonecrosis. D: Acute epididymoorchitis in mumps is
seen as an active lymphocytic infiltrate in the interstitium.
HTLV infection is endemic in southwest Japan, the Caribbean, South America, and sub-Saharan Africa (66) (e116). Transmission is through sexual contact,
intravenous drug abuse, infected blood and blood products, and breast milk. The frequency of transmission and the contributing factors to sexual and
mother-to-child transmission remain uncertain (e253). HTLV transmission is more frequent in breast-fed than formula-fed infants (e137,e183,e347). Duration
of breastfeeding correlates with transmission rate (e10,e11,e236,e367). Transmission is also associated with higher maternal provirus levels and HTLV-1
antibody titers (76) (e345). The median time of transmission has been estimated at 11 to 12 months of age (e117). Complete avoidance of breastfeeding is
reportedly effective in preventing mother-to-child transmission (e141). HTLV-1 causes adult
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T-cell leukemia/lymphoma (ATLL), a chronic, progressive neuropathy called HTLV-1 associated myelopathy, and tropical spastic paraparesis associated
with various other chronic conditions (uveitis, arthritis, Sjogren syndrome, infective dermatitis, and a persistent lymphadenitis in children). Early life infection
carries the greatest risk for later development of ATLL (e205). In areas of low prevalence, the likelihood of a false positive HTLV-1 test is high; therefore
repeat testing is often indicated. In a pregnant woman, antibody titer testing and proviral load quantification are appropriate to estimate the risk for
transmission to the infant. HTLV- 2 causes at least two forms of chronic ataxia (spastic or tropical) (e199).
FIGURE 6-9 ▪ Coxsackie infection with vesicular oral mucosal lesions.
Mumps has been reported to be resurgent in the United States; over 2,500 cases were reported in 2006 alone (Figure 6-8). The majority of these cases
occurred in college students aged 18 to 25 years, even though most had been vaccinated with two doses of measles, mumps, and rubella-containing
vaccines. Kancherla has reviewed mumps and discussed potential mechanisms for vaccine failure (91).
Coxsackie viruses are implicated in hand, foot and mouth disease (Figure 6-9), myocarditis, and aseptic meningitis.
EMERGING VIRAL INFECTIONS

Every year seems to bring a new “emerging infectious disease” (20, 25, 26, 27, 44, 71, 107) (e58,59e,e106), the H1N1 “swine” influenza being the latest
addition at the time of writing this section (83, 128, 175) (e108,e208,e254,e288). The influenza virus remains an important challenge, given its ability to
mutate at a very high level. Although the common circulating influenza virus strains (H1N1 and H3N2) are not virulent enough to cause mortality, mutated
strains may be lethal, especially in children (95). Apart from these circulating human strains, the avian influenza H5N1, H7, and H9 virus strains have also
been reported to cause human disease. The major threats of emerging infections worldwide are from zoonotic diseases, foodborne diseases, waterborne
diseases, and diseases caused by multiresistant organisms, and challenge clinical acumen, diagnostic armamentaria, and public health systems. Two are
briefly discussed here including SARS and West Nile virus (WNV) infections.
SARS is caused by a coronavirus different from previously studied coronavirus groups (e283). Because the SARS- associated coronavirus was identified
relatively recently, much about it is unknown. The SARS virus is transmitted primarily by respiratory droplets. In the pediatric cases reported in the literature,
children had mild respiratory illness, although the severity of the disease in adolescents seemed more similar to that in adults (13, 80). Infants born to
mothers with confirmed SARS were born prematurely, presumably because of maternal illness. Two of the five infants described developed severe
abdominal disease (coronavirus has been linked to necrotizing enterocolitis), although coronavirus was not identified in any of the infants (169).
WNV infection leads to approximately one case of severe neurologic disease for every 20 cases of nonspecific febrile illness and every 150 to 300 cases of
asymptomatic infection (seroconversion). Clinical illness is rare in infants and children (140). Transmission occurs primarily through the bite of Culex
mosquitoes, but may also occur during pregnancy (82), through organ transplants, following percutaneous exposure in laboratory workers, and possibly
transfusion of blood products. One case of possible WNV transmission through breastfeeding has been reported (e256). However, the absence of illness in
this infant (and most infants/children), the transient nature of maternal viremia with WNV infection, and the rarity of such a transmission event suggest that
there is no reason to avoid breastfeeding or breast milk when a mother is infected with WNV (104).
BACTERIAL INFECTIONS
We will give an overview of neonatal sepsis followed by a discussion of the more common infections caused by individual groups of bacteria.
NEONATAL SEPSIS
Sepsis is a leading cause of death in infants and children, with over 42,000 cases of severe sepsis reported annually in the United States and millions
worldwide. Sepsis is especially devastating in the neonatal population. Neonates significantly differ from adults in multiple respects including their naïve
immune system, pathophysiologic response to sepsis, and response to treatment (108). Half of the children with severe sepsis in the United States are
infants, and half of these are low- or very low-birth-weight babies. Incidence and mortality rates vary by age and the presence of underlying disease, if any
(e360). Attack rates for infants of colonized mothers also vary with the organisms (e40), their serotype (e18), and the presence or absence of maternal
antibody (e17). Sepsis neonatorum denotes fulminant bacterial
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sepsis occurring in the first 30 days of life, characterized clinically by abrupt onset, rapid progression, often without demonstrable anatomic localization, and
very high morbidity and mortality rates even in the face of appropriate antibiotic therapy. The clinical manifestations are protean and include hypothermia,
hyperthermia, respiratory distress, and feeding disturbances. Clinical distinction from noninfectious disease, especially hyaline membrane disease, is
frequently impossible, and thorough microbiologic evaluation is mandatory in all cases of neonatal death. Unfortunately, sepsis is a term that has been, and
continues to be, used very loosely in clinical practice, limiting comparison of studies from around the globe. Although bacteremia, systemic inflammatory
response syndrome (SIRS), and septicemia have been defined for the adult population, these cannot be directly extrapolated to the pediatric population.
Definitions for the pediatric population have only recently been agreed upon in consensus (Table 6-13) (65, 165). SIRS is the body's response to an
infectious or noninfectious insult. The name is only partially accurate since patients who develop SIRS have both an initial proinflammatory state (i.e., initially
hyperimmune) and a later antiinflammatory state (i.e., hypoimmune). The pathophysiology of SIRS is complex and has been recently reviewed (155).
Neonatal sepsis is subclassified based on the timing of infection as early-onset (in the first week of life and especially within the first 24 hours), late-onset (7
to 30 days of age), and very late-onset (beyond 30 days). Early-onset disease is associated with obstetric complications including fever, prolonged labor,
prolonged membrane rupture, and premature delivery. The predominant organisms causing early-onset infections are GBS and enteric bacilli, especially
Escherichia coli . Less common early-onset pathogens include other streptococci, enterococci, Listeria, H. influenzae, Streptococcus pneumoniae,
Chlamydia, and other organisms in the maternal genital flora. However, these organisms can also cause late- or very late-onset bacterial infections.
Antibiotic-resistant strains of Gram negative bacilli and Staphylococci are important nosocomial pathogens in hospital settings. Anaerobic bacteria, Serratia
and N. meningitidis are rare causes of neonatal sepsis. Timely detection and identification of offending organisms are among the most important functions of
the microbiology laboratory. From a diagnostic standpoint, positive blood cultures can establish an infectious etiology for a patient's illness and provide a
microorganism for susceptibility testing and optimization of antimicrobial therapy. Detection of positive blood cultures also has prognostic importance,
providing evidence that the host defenses have failed to contain the infection locally and/or that the physician has failed to remove, drain, or otherwise
eradicate the infection at its primary site. The key principles in obtaining blood cultures include choosing the best available site for culture, paying attention
to aseptic technique, culturing an adequate volume of blood, and obtaining a sufficient number of blood culture sets. Technical variables that can affect
results include culture medium, the ratio of blood to broth, additives to inactivate antimicrobial agents in the blood, and the duration of incubation and testing
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in the laboratory. Special considerations are required for organisms such as mycobacteria, Bartonella, anaerobes, and fungi (e203). Measures of acute
phase proteins, cytokines, cell surface antigens, and bacterial genomes have been used alone or in combination to improve diagnosis of neonatal sepsis,
but are not standardized and many are not available routinely (4, 100). Real-time PCR methods that can simultaneously detect the 25 most important
bacterial and fungal species which cause approximately 90% of all blood stream infections have been proposed for routine assessment of neonatal sepsis
(122).
Table 6-13 ▪ DEFINITIONS OF SYSTEMIC INFLAMMATORY RESPONSE SYNDROME (SIRS), INFECTION, SEPSIS, SEVERE SEPSIS,
AND SEPTIC SHOCK
SIRS
The presence of at least two of the following four criteria, one of which must be abnormal temperature or leukocyte count:
Core temperature of >38.5°C or <36°C.
Tachycardia, defined as a mean heart rate >2 SD above normal for age in the absence of external stimulus, chronic drugs, or painful stimuli; or
otherwise unexplained persistent elevation over a 0.5- to 4-hour time period OR for children <1 year old: bradycardia, defined as a mean heart
rate <10th percentile for age in the absence of external vagal stimulus, b-blocker drugs, or congenital heart disease; or otherwise unexplained
persistent depression over a 0.5-hour time period.
Mean respiratory rate >2 SD above normal for age or mechanical ventilation for an acute process not related to underlying neuromuscular
disease or the receipt of general anesthesia.
Leukocyte count elevated or depressed for age (not secondary to chemotherapy-induced leukopenia) or >10% immature neutrophils.
Infection
A suspected or proven (by positive culture, tissue stain, or polymerase chain reaction test) infection caused by any pathogen OR a clinical syndrome
associated with a high probability of infection. Evidence of infection includes positive findings on clinical exam, imaging, or laboratory tests (e.g.,
white blood cells in a normally sterile body fluid, perforated viscus, chest radiograph consistent with pneumonia, petechial or purpuric rash, or
purpura fulminans).
Sepsis
SIRS in the presence of or as a result of suspected or proven infection.
Severe sepsis
Sepsis plus one of the following: cardiovascular organ dysfunction OR acute respiratory distress syndrome OR two or more other organ
dysfunctions.
Septic shock
Sepsis and cardiovascular organ dysfunction.
From Goldstein B, Giroir B, Randolph A. International Consensus Conference on Pediatric Sepsis. International pediatric sepsis consensus
conference: definitions for sepsis and organ dysfunction in pediatrics. Pediatr Crit Care Med 2005; 6(1):2-8.
Pathologic changes in infants with neonatal sepsis vary little regardless of the agent responsible. Since the organism is commonly acquired from the
mother's genital tract either directly, or through swallowing or aspiration of infected amniotic fluid, the main pathologic finding in early-onset disease is
widespread pneumonia. The lungs are heavy, red, and airless. Histologically there is a widespread, relatively uniformly distributed, intraalveolar
polymorphonuclear exudate. In infants dying within the first few hours of life, there may be little polymorphonuclear infiltrate, with collapse and congestion
predominating. Hyaline membranes may be present, often containing large numbers of bacteria. Interstitial infiltrate may be prominent in GBS sepsis.
Pulmonary hemorrhage is frequently seen. Amniotic squames are present in the alveoli, as they are in virtually all infants dying under 1 month of age.
Systemic lesions are decidedly uncommon; splenitis is seen in 30% of cases, and meningitis is rare.
Late-onset neonatal sepsis, in contrast, has no association with obstetric complications, and the route of acquisition of the organism may be uncertain.
Vertical transmission can be documented in most GBS disease (most commonly subtype III) and in some cases of E. coli infections (e177). Other Gram
negative bacilli and, more recently, nosocomial Acinetobacter infections have also been implicated in late onset neonatal sepsis (e220,e332). Horizontal
transmission from home or nursery contacts is presumed to account for the remainder. The onset is either insidious or fulminant, and mortality is less than
that seen in early-onset disease. Bacteremia results in meningeal seeding in virtually all cases; ventriculitis is the rule and, together with arachnoidal fibrosis,
accounts for the high incidence of hydrocephalus in survivors (e24,e255). The pathologic changes and sequelae are those of neonatal meningitis in general.
Extrameningeal infectious foci were demonstrable in the majority of patients in Berman and Banker's series (e24). Various scoring systems are available to
estimate the severity of illness and organ dysfunction (99).
Sepsis may have distinct characteristics in patients with congenital immunodeficiencies. Congenitally impaired immunity could paradoxically lead to a milder
course of sepsis due to an incomplete inflammatory response, or result in a more severe course, due to a lack of regulatory responses and a higher
pathogen burden. An association is seen between types of immune deficiencies and the class of infecting organisms (Tables 6-1 and 6-2) (132).
STAPHYLOCOCCAL INFECTIONS
Both coagulase-positive and coagulase-negative staphylococci are frequently encountered pathogens in the young. Staphylococcal infection usually occurs
late in the neonatal period; 40% to 90% of infants in the nursery at 5 days of age are colonized with Staphylococcus aureus, skin and nares being the
predominant sites of colonization (e99). The morphologic hallmark of infections by coagulase positive staphylococci is suppurative inflammation with necrosis
(abscess formation) (Figure 6-10), with or without systemic manifestations of inflammation; any organ or organ-system may be involved. Cutaneous and
subcutaneous infections can progress to necrotizing fasciitis, which may become fulminant. Methicillin-resistant S. aureus (MRSA) is now an established
community pathogen with significant morbidity and mortality, and has changed the epidemiology, clinical manifestations, laboratory approach, antibiotic
management, and prevention of staphylococcal infections in children (93). Spread of MRSA has also been documented in the school and daycare settings
(e4). Community acquired MRSA isolates are now also associated with nosocomial infections in neonatal intensive care units (e178). Nursery outbreaks of
S. aureus infections have been traced to postnatal contact with mothers, health care workers, and contaminated, unpasteurized, banked breast milk (e249).
Differentiating between isolates that have the pvl genes and those that are negative for pvl has major therapeutic implications (93).
The most common bloodstream infection encountered in neonatal and pediatric intensive care units is coagulasenegative Staphylococcus (CONS) (186)
(e231,e318,e319). CONS infections are almost always associated with intravenous catheters or invasive procedures. Since the organism is a normal skin
commensal, differentiating infection from colonization and contamination can be difficult. These infections also pose serious concerns because of their high
mortality rates and the frequent presence of the mecA gene, which is associated with b-lactam antibiotic resistance. Colonization rates are as high as 60% to
90% for infants hospitalized at 2 weeks of age.
Staphylococci can also cause diseases due to elaboration of soluble toxins, including food poisoning, the so-called staphylococcal scalded skin syndrome
(SSSS), and toxic shock syndrome (TSS). SSSS (so-called Ritter disease in neonates and staphylococcal toxic epidermal necrolysis in older children) is
caused by an epidermolytic exotoxin produced by phage group II staphylococcus. It is characterized by large intraepidermal bullae that rupture and lead to
exfoliation (Figure 6-10); resultant fluid loss, and/or secondary infection may be fatal. Toxic shock syndrome is rare under age 10, with peak incidence in
teenagers. Predisposing factors include tampon use, surgical procedures, skin infections, and abortions. The toxin (toxic shock syndrome toxin I, or TSST-I)
causes massive intravascular fluid loss leading to edema, diarrhea, and hypotensive shock. Mucous membranes
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are red and edematous; in menstrual cases, erythema, edema, and ulceration involve the cervix, vagina, and perineum. Several autopsy studies reveal little
in the way of specific findings; there is no evidence of bacterial invasion of tissues, and inflammatory reaction is negligible, supporting a toxin- mediated
change. Reported findings include genitourinary tract ulceration, mild lymphoid depletion, a skin lesion remarkably similar to SSSS, and mild and nonspecific
inflammation in kidney, liver, heart, and muscle (e186,e247).
FIGURE 6-10 ▪ Staphylococcal infections. A: Impetigo with bullous features. B: Staphylococcal scalded skin syndrome (toxic epidermal necrolysis) following
MRSA infection. C: Partially “healed” or resolving staphylococcal lung abscess.
STREPTOCOCCAL INFECTIONS
Pathogenic group A streptococci (GAS) are comprised of a number of serotypes based on the M protein, S. pyogenes being the most important. GAS
produce disease by at least three mechanisms: (a) direct tissue invasion of skin and upper airways (impetigo, erysipelas, cellulitis, pharyngitis, tonsillitis,
necrotizing fasciitis, necrotizing pneumonia), (b) toxin elaboration (scarlet fever), and (c) immune-mediated mechanisms (acute glomerulonephritis and
rheumatic fever) (Figure 6-11). The prevalence of invasive GAS disease with resultant bacteremia and/or streptococcal toxic shock syndrome is on the rise
(113) (e80,e224). Nonsuppurative immunologic complications can occur even in the neonate (e222). Maternal carriage is an important factor in neonatal
GAS disease. Early onset disease is associated with concurrent maternal infection and manifests as respiratory distress, pneumonia, and toxic shock-like
syndrome, while late onset disease is associated with soft tissue infections and meningitis.
The nonGAS are mainly encountered in the newborn. S. pneumoniae is a common cause of pneumonia, meningitis,
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and otitis media (77). In asplenic patients, it is the single most common cause of sepsis, accounting for almost 50% of cases. S. pneumoniae has also been
recognized as a cause of invasive soft tissue disease and a toxic shock-like syndrome in previously healthy children (e136). Group B streptococcus (GBS,
e.g., S. agalactiae) is transmitted primarily in utero and during delivery, and is an important cause of early onset neonatal sepsis, meningitis, and pneumonia
(182). Guidelines proposed by the American Academy of Pediatrics Committees on Infectious Diseases and the Fetus and Newborn use several variables to
identify increased risk for GBS infection in the neonate and recommend intrapartum antimicrobial prophylaxis for infants at high risk (152). Other
streptococcal infections, although much less common than GBS, are occasionally encountered; groups D and G have been reported to cause disease similar
to GBS (49, 84, 170, 171). Viridans group streptococci (VGS) are of particular concern in neutropenic children and can result in septic shock with median
mortality of 10% (e301). VGS infection may be accompanied by neurological complications, myocarditis, and acute respiratory distress syndrome (e272). As
is the case with other pathogens, their incidence and severity have increased during the
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past 15 years, and antibiotic resistance is commonplace. S. viridans infection may cause amniotic fluid infection in midgestation with resultant fetal-neonatal
sepsis (e13).
FIGURE 6-11 ▪ Streptococcal infections. A: Streptococcal pharyngitis with erythematous, congested mucosa. B: Congenital streptococcal pneumonia with
diffuse involvement. C: Diffuse alveolar exudates of neutrophils and fibrin in congenital streptococcal pneumonia. D: Scarlet fever with erythematous mucous
membranes and tongue.
ENTEROCOCCI
Enterococci are among the top four causes of nosocomial bacteremia in the United States (17) since they are bacteria that survive for brief periods on hands
and inanimate surfaces. Risk factors for enterococcal bacteremia include prolonged hospital stay, exposure to antibiotics, central venous catheter use, and
necrotizing enterocolitis (e217). They cause urinary tract infections, bloodstream infections (including neonatal sepsis and infections in older children),
catheter-associated bacteremia, endocarditis, intra-abdominal infection, and meningitis. Enterococci are normal inhabitants of the human GIT and infection
may ensue either from the patient's own indigenous flora or dissemination of acquired virulent clones. Virulence is dictated by genes that are clustered on the
genome in distinct regions termed pathogenicity islands (PAIs), transfer and deletion of which are frequent. The management of patients with enterococcal
infections is complex because it requires identifying the susceptibility of the isolate and the site of infection, both of which are key factors in providing optimal
therapy. Glycopeptide-resistant/vancomycinresistant enterococci (VRE) are increasing in prevalence and guidelines have been released for their control
(150).
NEISSERIA INFECTIONS
N. gonorrhoeae can be transmitted in utero, intrapartum, or postpartum. Although gonococcal conjunctivitis is the most frequent clinical manifestation of
neonatal infection (Figure 6-12), septicemia and arthritis can also develop (e6,e154). Gonococcal infections outside of the perinatal period are increasingly
common and are associated with sexual abuse/sexual activity in children and adolescents; their clinical and pathologic features are similar to those in adults.
N. meningitidis is a major cause of childhood morbidity and mortality, causing meningococcal meningitis and meningococcemia. Fulminant meningococcemia
(“purpura fulminans”) is the form most likely to be encountered by the pathologist and is a catastrophic condition with hemorrhagic skin lesions that progress
to gangrene (e56) (Figure 6-13). Lethargy or irritability, petechiae, and purpura are followed by circulatory collapse, shock, and death. The time from first
symptom to death may be only a few hours. The combination of circulatory collapse, purpura, and bilateral adrenal cortical hemorrhage constitutes the
Waterhouse-Friderichsen syndrome. The petechial skin lesions consist of extravasated red cells from small vessels in the absence of vasculitis; fibrin
thrombi may be prominent, and the organism can be identified within endothelial cells or in smears from the lesions. Purpuric lesions show hemorrhagic
infarction of skin and subcutis with vascular thrombi (e133). The pathogenesis of these lesions and the extreme variability of the clinical course are not well
understood. The pathologic picture is reminiscent of generalized Schwartzman reaction and implicates an endotoxin-mediated process. Adrenal hemorrhage
(Figure 6-13B and C), although striking, is unlikely to cause acute adrenal insufficiency and, by itself, does not cause death, given the great reserves of the
adrenal. Acquired deficiencies of proteins C and S are probably more important players in fulminant meningococcemia (e259,e260). The propensity for
severe disease in infants and very young children may be due to the fact that the protein C system is incompletely developed at this age. Early specific
diagnosis is essential for optimal management and is based on CSF microscopy, culture, latex agglutination, and molecular (PCR-based) techniques. Latex
agglutination allows serotyping while molecular methods are rapid and helpful in patients who have already received antibiotics. Traditional clinical
prognostic signs include duration of petechiae, hypotension, presence of meningitis, leucopenia, and lack of elevation of erythrocyte sedimentation rate
(e316). Mortality can be predicted by the pediatric risk of mortality (PRISM) score (e257,370). One must remember that the clinical syndrome of purpura
fulminans may also be caused by infections with other bacteria (e.g., E. coli, S. pneumoniae, P. mirabilis) and viruses (varicella, rubella) (e56).
FIGURE 6-12 ▪ Severe purulent gonococcal conjunctivitis is present in this infant.
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FIGURE 6-13 ▪ Fulminant meningococcemia. A: Numerous petechial and ecchymotic foci with features of purpura fulminans of a consumptive coagulopathy.
B: Bilateral adrenal hemorrhages of the Waterhouse-Friderichsen syndrome. C: The adrenal shows the presence of hemorrhagic necrosis.
ENTEROBACTERIACEAE
Except in the neonate, the enteric bacilli are either GI pathogens (see Chapter 14) or systemic opportunists in the compromised host (e102,e103,e104). As
systemic opportunists they cause pneumonia, septicemia, and localized suppurative reactions depending on the nature of the underlying disease (Figure 6-
14). Although the cellular reaction, when present, is entirely nonspecific, it must be emphasized that in the profoundly leukocytopenic host, the inflammatory
reaction may consist solely of edema, vascular engorgement, hemorrhage, and fibrin deposition without much, if any, cellular
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reaction. Enteric, usually food-borne, infections with verotoxin-producing strains of E. coli (O5l7: H7) are responsible for hemolytic uremic syndrome, which is
discussed in Chapter 17.
FIGURE 6-14 ▪ A: Acute E.coli meningitis with purulent exudate covering convexities. B: Neutrophils filling the subarachoid space.
SALMONELLA INFECTION (TYPHOID)

These Gram negative bacilli can cause localized infection (e.g., gastroenteritis by S. typhimurium) or systemic infection (e.g., typhoid by S. typhi ). Typhoid
and paratyphoid are relatively common infections in developing countries. Transmission is feco-oral and is the result of poor sanitation and fecal
contamination of water and dairy products which is also seen in the United States. In the untreated patient, after an incubation period of 5 to 30 days, a
febrile phase develops with stepwise daily elevations in temperature that may be associated with rose spots on the trunk, bradycardia, and leucopenia.
Fever can be high grade and continuous, lasting up to 3 weeks, followed by a stage of decline and convalescence. In the present antibiotic era, patients may
present with fever, diarrhea, abdominal pain, and hepatosplenomegaly (e204,e221). Specific symptoms and signs may be inapparent in children
(e55,e125,e336). The bacteria gain access to the bowel wall and disseminate via lymphatics to lymph nodes, spleen, and liver. Salmonella proliferate in bile
and amplify by a bacterial enterohepatic circulation. Intestinal typhoid ulcers are a result of necrosis of hyperplastic Peyer patches and are therefore arrayed
in the long axis of the small bowel. Histology reveals numerous macrophages (so-called “typhoid cells” or “Mallory cells”) forming “soft granulomas” with
erythro- and lymphophagocytosis, plasma cells, and activated lymphocytes. Macrophage aggregates (so-called typhoid nodules) may be seen in the spleen,
liver, bone marrow, kidneys, salivary glands, and testes. A fibrinous exudate may be seen on the splenic capsule that later becomes organized to produce
the “sugar-coated” (“zuckergleiss”) spleen. Laboratory diagnosis is by culture (from blood or bone marrow in the first week and stool by the third week) or by
serology (Widal test) (e144) and more specific assays (e54,e161). Complications are numerous and include ileus, acute renal failure, cardiac arrhythmia
(with sudden death), massive intestinal hemorrhage (following Peyer patch necrosis), pancreatic dysfunction, intestinal perforation, peritonitis, Zenker
degeneration of abdominal skeletal muscles, liver necrosis, splenic rupture, and DIC (31) (e28,e73,e168,e174,e243). Atypical manifestations include
pneumonitis, pericarditis, osteomyelitis (especially in sickle cell anemia patients), dactylitis, pericarditis, arthritis, meningitis, cerebellar ataxia, myelonecrosis,
and a generalized hemophagocytic syndrome (78) (e192). A carrier stage may develop and carriers excrete the bacteria in their bile (stool) and urine, posing
a health hazard to society, as exemplified by the infamous Typhoid Mary.
FIGURE 6-15 ▪ Hemophilus influenzae epiglottitis. A: Marked induration and enlargement of the epiglottis are features in this autopsy case. B: Involvement of
the entire epiglottis and larynx by H. influenzae. results in these gross findings.
HEMOPHILUS INFLUENZA
This small Gram negative coccobacillus is a commensal of the upper respiratory tract and a major cause of morbidity and mortality in infants and young
children. Colonization by capsular H. influenza type B is uncommon in a healthy
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individual, but can cause severe disease in patients with respiratory compromise or immunodeficiency. Transmission is through direct contact and by
respiratory droplets; there is no evidence for its transmission through breast milk. In fact, breast milk seems to limit colonization of H. influenzae in the infant's
throat (e146). Most invasive disease outside the neonatal age group results from infection by encapsulated type B organisms. Infections range from mild
(e.g., conjunctivitis and otitis media) to life threatening (epiglottitis, meningitis, pericarditis, pneumonia, septic arthritis, and facial cellulitis) (e71). Until
recently, H. influenzae was the major cause of meningitis in infants accounting for 80% of cases under the age of 2 years and for at least onethird of
bacterial pneumonia in this age group. This has, however, decreased significantly in countries where the use of the H. influenzae conjugate vaccines is the
norm (12). In the upper airway, H. influenzae causes life-threatening acute epiglottitis (Figure 6-15). The larynx and especially the epiglottis are the site of
marked congestion, edema, and leukocytic infiltration, which may completely occlude the small infant airway. The cherry red appearance is helpful in
distinguishing this condition from severe viral laryngotracheitis (croup), which is occasionally severe enough to cause airway obstruction in this age group.
Nontypeable strains commonly cause lower respiratory infections. In children, they are also the most common cause of bacterial conjunctivitis and the
second most common cause (after S. pneumoniae) of otitis media (e121). H. influenzae also causes acute chorioamnionitis. Histopathologic features of H.
influenzae infection are those of any other bacterial infection and are not distinctive.
DIPHTHERIA
Diphtheria is caused by a toxin produced by Corynebacterium diphtheriae carrying a particular lysogenic bacteriophage; all of the gross and microscopic
features of the disease can be produced by purified toxin (e246). The toxin can affect all cells of the body, but is most potent on nerves, kidneys, and heart,
and halts addition of amino acids to elongating polypeptide chains. Humans are the only identified reservoirs and symptom-free carriers. The major sources
of infection are patients in the incubation stage of disease and fomites. The initial reaction appears to be toxin-induced necrosis of upper airway epithelium
with abundant fibrin exudation, leading to the typical fibrinous pseudomembrane overlying mildly inflamed submucosa, accompanied by tremendous edema
of the soft tissues of the neck. Death is related to airway obstruction, to toxin-mediated cardiomyopathy/myocarditis, and to diphtheritic peripheral and cranial
neuropathy (e143).
Despite large scale and, on the whole, successful immunization, rare cases of diphtheria continue to occur in nonimmunized children and in young adults
with nonprotective levels of antitoxin (e171). A massive epidemic in the independent states of the former USSR during the 1990s is a reminder of the
breakdown of public health infrastructure in periods of socioeconomic and political upheaval (e352).
INFECTIONS BY OTHER GRAM-NEGATIVE BACILLI

Pseudomonas aeruginosa is ubiquitous in soil and water, and is seen almost exclusively as an opportunistic pathogen. Infection is frequently associated with
cystic fibrosis. It also causes gangrenous lesions in the skin (pyoderma gangrenosum) and GIT of patients with malignancy. Pseudomonas sepsis is primarily
a nosocomial infection that is seen as a late onset disease in very low birth weight infants and carries a 50% mortality rate (e189). Occasional cases may be
associated with chorioamnionitis (e237). Pseudomonas sepsis is characterized by necrotizing vasculitis (e329), involving both arteries and veins; the vessel
wall is replaced by collections of organisms, often with very little cellular infiltrate, and hemorrhagic infarction of the affected site. Particularly affected are the
skin and mucous membranes (especially of the intestinal tract), manifesting as deep red or violaceous raised plaques, which rapidly undergo necrosis.
Patients may also have a hemorrhagic necrotizing bronchopneumonia (Figure 6-16).
Serratia marcescens is an infrequent pathogen in the newborn and is usually nosocomially acquired through foreign bodies and instrumentation. Serratia
sepsis is late-onset disease with severe meningitis and a high mortality rate (e230).
Citrobacter species (enteric Gram-negative bacilli) are an increasingly recognized cause of neonatal sepsis, meningitis, hemorrhagic encephalitis, and brain
abscess. The majority of cases present in the first 10 days of life (e124). Most cases are apparently nosocomial following surgical manipulation of umbilical
cord stumps (e250), although the existence of early onset sepsis suggests that vertical transmission may also occur (e339).
FIGURE 6-16 ▪ Necrotizing hemorrhagic pneumonia caused by Pseudomonas aeruginosa.
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LISTERIOSIS
Listeria monocytogenes is a small, gram-positive bacillus that acts as a facultative intracellular parasite. The organism is a common intestinal and vaginal
commensal. Human listeriosis presents as one of three clinical forms including febrile gastroenteritis, maternal-fetal/neonatal listeriosis, or bacteremia with or
without CNS involvement. Infections are uncommon with the estimated incidence of listeriosis during pregnancy being 12 cases per 105 (124). Listeriosis
during pregnancy is associated with second-trimester abortion (e184) and infrequently causes premature delivery or stillbirth. Up to two-thirds of surviving
neonates born to infected mothers develop overt neonatal infection due to either transplacental or intrapartum transmission (124). Perinatal infection is an
uncommon cause of severe disease in the neonate.
Neonatal listeriosis is classified as either early (occurring in the first 5 to 7 days following delivery) or late infection (123) (e187). Early-onset infection is due
to types Ia, Ib, and IVb, while type IVb predominates in late-onset infection. Early disease is associated with maternal infection, is often overt at the time of
delivery, and is associated with meconium staining of the amniotic fluid. Clinical presentation resembles neonatal sepsis with pneumonia, bacteremia, and/or
meningitis (124) (e216). In some neonates, the infection manifests as “granulomatosis infantiseptica” with a salmon pink rash (papules surrounded by red
margins) and mucosal nodules; there are widespread microabscesses and granulomata especially in the liver, spleen, and lungs (Figure 6-17). In contrast,
late onset infection usually occurs in full term neonates delivered from uncomplicated pregnancies and presents as meningitis between the second and
eighth week of life. The infection is presumed to be acquired from the maternal vaginal tract, at the time of delivery. Listerial meningoencephalitis cannot be
distinguished from other systemic bacterial or viral infections. In Vawter's series, the organs most frequently involved were the adrenals, liver, GIT, skin, and
tracheobronchial tree (e350). Mortality of untreated early-onset infection is 100%; survivors have CNS sequelae including hydrocephalus and mental
retardation. Factors determining L. monocytogenes virulence have been recently reviewed (48).
FIGURE 6-17 ▪ Listeriosis. A: Lung abscess is seen in this infant with numerous such lesions with minimal inflammation. B: Brown-Hopps stain displays the
rod-shaped bacteria.
Systemic infection is marked by bacterial replication in mononuclear cells in the liver, spleen, and bone marrow (e74,e84,e163). Parasitized monocytes play
an important role in CNS invasion (46). The histopathologic features depend largely on the duration of the disease. Inflammatory foci may consist solely of
necrosis with little cellular reaction (although fibrin thrombi and hemorrhage may be prominent) or miliary abscesses may be seen, particularly in liver and
adrenal glands. Gram, Warthin-Starry, and immunohistochemical stains help identify organisms in tissues (e248). In patients who mount a specific immune
response, the lesions, after several days, assume a granulomatous appearance as mononuclear cells replace polymorphonuclear leukocytes in the
abscesses. Placental infection is common and manifests as intervillous and intravillous microabscesses with necrotizing villitis and chorioamnionitis,
irrespective of route (transplacental or ascending) of infection (e23,e233). A definitive diagnosis of listeriosis is made by culturing L. monocytogenes. Blood
and CSF are the most useful specimens; serologic assays are not useful. (see Chapter 9).
SYPHILIS
Treponemes are microaerophilic spiral gram-negative bacteria that are 6 to 20 mm long and 0.1 to 0.5 mm in diameter. Although this thickness is less than
the resolution limits of conventional light microscopy, the microbe can be visualized by dark-field or phase-contrast microscopy. T. pallidum
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causes multisystem disease in stages similar to Lyme disease. Humans are the sole natural host of syphilis. The WHO estimates that maternal syphilis leads
to 460,000 abortions/stillbirths and 270,000 live-born infants with congenital syphilis yearly (e106). The frequency of congenital syphilis in a specific locale is
determined ultimately by both the prevalence of syphilis among adults and the effectiveness of prenatal screening and treatment programs. HIV-infected
pregnant women are at high risk for having active syphilis. Pediatric syphilis has been the subject of many recent reviews (79, 196).
Transmission
Transmission is through direct sexual contact and contact with open lesions or secretions from the lesions in the skin and mucous membranes. Congenital
syphilis occurs in the fetus through placentitis, while perinatal infection occurs in the neonate through contact with the spirochete during passage through the
birth canal. T. pallidum can cross the placenta and infect the developing fetus throughout pregnancy, from as early as 9 to 10 weeks of gestation. Vertical
transmission during pregnancy occurs more frequently in primary or secondary syphilis than with latent maternal disease. Fetal infection is most efficient
during the early stages of maternal infection when the transmission rate approaches 100%. The risk of transmission diminishes after 4 years of infection,
even when the mother is untreated. Postnatal infection can occur in the infant through contact with open lesions or secretions in the infected mother or
another adult. If syphilitic lesions involve the breast or nipples, then breastfeeding or using expressed breast milk should be avoided until the mother has
completed treatment and the lesions have healed. There is no evidence for transmission of T. pallidum in breast milk without a breast or nipple lesion.
FIGURE 6-18 ▪ Congenital syphilis with its various features. A: Congenital syphilis with hydrops fetalis. B: Congenital syphilis with “barber pole” funisitis.
FIGURE 6-18 ▪ (continued) C: Labial lesions in neonatal syphilis forming condylomata lata. D: Bifid molar as a malformative manifestation in tooth
development. E: Snuffles as a nasal discharge secondary to obstructive nasopharyngitis. The mucopurulent discharge contains viable organisms. F: Mucous
patches representing an ulcerative mucositis.
Clinical Features
Fetal infection can result in spontaneous abortion, stillbirth, early congenital syphilis, and late congenital syphilis (Figure 6-18). Congenital syphilis does not
have a primary stage and may result in perinatal death in more than 40% of affected, untreated pregnancies. Among survivors, manifestations traditionally
have been divided into early and lateonset types with early manifestations appearing in the first 2 years of life. With early-onset disease, manifestations
result from transplacental spirochetemia and are analogous to the secondary stage of acquired syphilis. Late-onset disease is seen in children older than 2
years and is not considered contagious.
Although many features are nonspecific, certain lesions, when present, are pathognomonic, and the disease is recognizable even in severely macerated
stillborns, who represent one-third to one-half of congenital syphilis cases (e167,e242,e372). Infection occurring prior to the fifth month
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of intrauterine life does not cause destructive changes and organogenesis is unaffected, although spirochetes are readily demonstrable in all fetal tissues. In
the macerated second and third trimester stillborn infant, hepatomegaly, and/or hydrops may be the only gross evidence of disease (Figure 6-18A).
Organisms are, however, abundant in all organs, including placenta, even when the internal organs have undergone extensive autolysis. Congenital syphilis
can clinically mimic a number of neonatal conditions including other congenital infections (CMV, HSV, rubella, and toxoplasma), bacterial sepsis and blood
group incompatibility, to name a few. A negative Coombs test in the setting of hydrops is suggestive of congenital syphilis (e36), although parvovirus
infection should also be considered. In early-onset congenital syphilis, most affected infants are asymptomatic at birth and are identified only by routine
prenatal screening. If untreated, symptoms develop within weeks or months. The typical stillborn or highly symptomatic newborn is born prematurely with an
enlarged liver and spleen, skeletal involvement, and often pneumonia and bullous skin lesions. In others, the earliest signs of congenital syphilis may be poor
feeding and snuffles (syphilitic rhinitis; Figure 6-18E). Early manifestations of congenital infection are varied and involve multiple organ systems.
Hepatomegaly is reported in almost 100% of cases, and biochemical evidence of liver dysfunction is usually observed. The most striking lesions affect the
mucocutaneous
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tissues and bones. Mucous patches, rhinitis, and condylomatous lesions are highly characteristic features of mucous membrane involvement in congenital
syphilis (Figure 6-18F). Nasal fluid is highly infectious. Snuffles are followed quickly by a diffuse maculopapular desquamative rash that involves extensive
sloughing of the epithelium, particularly on the palms and soles and about the mouth and anus. In contrast to acquired syphilis, a vesicular rash and bullae
may develop in these areas that may weep and desquamate. These lesions teem with spirochetes and are highly infectious. Bone involvement in the form of
multiple symmetric periostitis and joint osteochondritis occur in 60% to 80% of untreated early congenital cases. Bone involvement can be very painful,
causing the infant to refuse to move the involved extremity (pseudoparalysis of Parrot). Tibial metaphyseal demineralization is seen radiologically
(Wimberger sign). Bone involvement usually resolves spontaneously within 6 months. Neurosyphilis may be present even with normal CSF findings.
Alternatively, it may present as acute meningitis (in the first 6 months) or a chronic meningovascular neurosyphilis (at the end of the first year of life) with
progressive hydrocephalus, cranial nerve palsies, seizures, and neurodevelopmental regression. Cerebral infarction from syphilitic endarteritis may occur in
the second year of life. Anemia, thrombocytopenia, leukopenia, and leukocytosis are common findings. The late manifestations of congenital syphilis are a
consequence of scarring from the early systemic disease and involve teeth (Figure 6-18D), bones, eyes, the eighth cranial nerve, and CNS.
In older children and adolescents, syphilis is sexually transmitted and may be the result of sexual abuse. Manifestations and diagnosis are similar to that in
adults. Neurosyphilis occurs in approximately 30% of patients with secondary syphilis; CSF pleocytosis and proteinosis are typical findings. Neurosyphilis
may be clinically silent or present with meningeal, cranial nerve, or spinal nerve involvement. In addition to mucocutaneous involvement, secondary syphilis
can also manifest with iritis, anterior uveitis, arthritis, and nephrotic syndrome, probably caused by deposition of immune complexes composed of treponemal
antigens, fibronectin, antibodies, and complement. Secondary syphilis lesions resolve without treatment in 1 to 2 months. The infection then enters a latent
period, without overt evidence of disease. The signs of secondary syphilis can recur during the first year (early latency) but not thereafter (late latency).
Relative immunity to reinfection exists during latency, and approximately 60% of untreated patients will not progress from latency to tertiary syphilis, the
remaining progressing after latent periods of 3 to 10 years. This time frame renders acquired tertiary syphilis a very rare occurrence during childhood and
adolescence.
Pathology
In his seminal article Silverstein (e307) suggested that histopathologic changes of syphilis await the development of fetal immune competence. Humoral
immunity is insufficient to control the infection. Cell-mediated immunity is suppressed during the primary and secondary stages of infection. Ultimate
eradication of infection occurs when T-cells infiltrate syphilitic lesions. T. pallidum may escape immune surveillance by antigenic variation. Further, although
a Th1 immune response is elicited in primary syphilis, progression to the secondary stage is accompanied by a shift to a Th2 response, allowing for
incomplete clearance of the pathogen. In pregnancy, intense inflammatory responses and prostaglandins induced by fetal infection may be responsible for
the various manifestations of congenital syphilis (139).
The main pathologic changes are seen in pancreas, liver, GIT, bones and nasal mucosa, and manifest as inflammation, scarring and developmental
retardation (e241). The liver shows diffuse inflammation and fibrosis separating the liver into coarse nodules (hepar lobatum). Osteochondritis and periostitis
of joints, long bones, palate, and nasal cartilage lead to skeletal deformities. Lung involvement leads to pale airless, heavy and fibrotic lungs (pneumonia
alba). The viscera in general appear inappropriately immature for gestational age; there is prominent extramedullary hematopoiesis, persistence of fetal
adrenal cortex, active glomerulogenesis, and persistence of fetal stroma in many organs, notably the pancreas and the pulmonary interstitium. The
inflammatory response is mainly mononuclear and may be difficult to distinguish from extramedullary hematopoiesis. Polymorphonuclear leukocytes occur in
response to tissue necrosis, producing the typical Dubois abscess. Gummata are rare in newborns. Oppenheimer and Dahms provide a complete description
of the pathologic changes of congenital syphilis and describe a pathognomonic triad comprised of interstitial inflammation or fibrosis of the pancreas with
pressure atrophy of the parenchyma, pneumonia alba (sharply defined fibrosing pneumonitis), and thickening of the bowel wall by submucosal infiltrates and
fibrosis (e241). An obliterative endarteritis, consisting of concentric endothelial and fibroblastic proliferative thickening with plasma cell infiltration, should
suggest syphilis. This endarteritis is also found in all stages of acquired syphilis (in the primary chancre, polymorphonuclear leukocytes, and macrophages
often can be seen ingesting treponemes).
Placental examination is mandatory in suspected cases to allow for an early diagnosis. Grossly, the placenta is large and heavy. Syphilitic placentitis is
histologically characterized by histiocytic-predominant villitis, proliferative endovasculitis of the stem villi (perivasculitis with concentric mural vascular
sclerosis) and necrotizing umbilical periphlebitis. The umbilical periphlebitis is pathognomonic and comprises of abscesslike necrotic foci in the Wharton jelly,
with eosinophilic precipitates around the umbilical vein. Other histopathologic findings may include villous dysmaturity, hypercellular villi with variable acute
and chronic inflammation, numerous Hofbauer cells, endarteritis, perivascular fibrosis, numerous nucleated red cells in fetal vasculature, proliferative fetal
vascular changes, chorioamnionitis, necrotizing funisitis, and/or plasma cell deciduitis. Spirochetes may be difficult to demonstrate in the placenta and their
absence does not
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exclude the diagnosis. Spirochetes are easier to demonstrate in the umbilical cord, even if the cord does not show evidence of funisitis (168) (e294). (see
Chapter 9).
Acquired syphilis is almost always by sexual contact. T. pallidum penetrates the skin or mucous membrane at a site of exposure, multiplies locally and
spreads through the perivascular lymphatic system to the systemic circulation, which disseminates infection widely before the primary lesion(s) becomes
evident. During the usual 3-week incubation period (range 10 to 90 days), an intense local inflammatory response develops, consisting of plasma cells,
macrophages, and lymphocytes. This produces the red, indurated, ulcerative, spirochete-filled chancre. The host response likely is initiated by chemotactic
effects of treponemal lipopeptide antigens (e296), but it seems to require proliferation of relatively large numbers of treponemes. Associated cellular
proliferation in regional lymph nodes produces adenopathy. If the immune response is unable to fully eradicate the infection, replication at the site of early
infection leads to dissemination and development of the lesions of secondary syphilis, over the course of 2 to 10 weeks. These lesions occur most commonly
in ectodermal tissues (skin, mucous membranes) and the CNS. The tissue response is similar to that of primary lesions. Condylomata lata (venereal warts)
are characterized by epithelial hyperplasia, hyperkeratosis, and plasma cell infiltrates. Even if untreated, the clinical manifestations of secondary syphilis
resolve, and the disease process enters a period of relative immunologic control; viable organisms remain but in low numbers. Tertiary syphilis can involve
any organ system and typically manifest as gummata, which are focal areas of nonsuppurative inflammatory necrosis surrounded by fibrotic scarring. These
represent a granulomatous hypersensitivity reaction and viable organisms are rare or absent. Tertiary lesions also can take the form of a diffuse, chronic,
noncaseating infiltrate of plasma cells and lymphocytes.
Definitive diagnosis requires demonstration of the spirochete; T. pallidum is a long (15-mm) slender organism that is optimally identified by darkfield
examination, although, if fresh preparations are not available, the Levaditi, Steiner, and Warthin-Starry stains are helpful. An immunohistochemical procedure
has been described, and PCR diagnosis has been useful in selected cases (e235). In the appropriate clinical setting, a diagnosis may also be made if serum
quantitative antibody titer is at least four times greater than the maternal titer. CSF VDRL (Venereal Disease Research Laboratory) test is reactive and/or the
IgM FTA-ABS is positive.

If the newborn survives, progressive inflammation and fibrosis lead to the stigmata of late congenital syphilis: gummatous facial deformities (perforated palate
and saddle nose), skeletal defects [frontal bossing, short maxillae, mandibular protruberance, high palatal arch, scaphoid scapulae, saber shins, Clutton
joints, sternoclavicular thickening (Higoumenakis sign)], dental abnormalities (Hutchinson incisors, mulberry molars), rhagades, meningovasculitis leading to
eighth nerve damage and optic atrophy, interstitial keratitis, and neurosyphilis (e109).
LEPTOSPIROSIS
Leptospirosis is a zoonotic disease caused by a single nontreponemal spirochete species (Leptospira interrogans) with several subgroups. The disease
occurs in epidemic forms in tropical countries of especially South and Southeast Asia, with seasonal trends, and has recently been classified as an emerging
global disease. Although primarily a zoonosis, humans are infected by exposure to water or soil contaminated with animal urine, or by the bite of a rat flea.
Transplacental infection has been documented, as has fetal death due to maternal leptospirosis (e57,e61). Although the majority of leptospiral infections are
either subclinical or result in very mild illness, a proportion of patients develop various complications due to multiorgan system involvement, with CFRs of
over 40% (187). After an incubation period of 2 to 30 days, a flu-like septicemic phase ensues, followed by an immune phase (with involvement of kidneys,
liver, meninges, eyes, skin, pancreas, heart spleen, and lymph nodes). Clinical presentation depends upon the predominant organs involved. Because of its
protean manifestations, leptospirosis it is often misdiagnosed and under-reported. The more severe form (Weil disease) is characterized by jaundice,
coagulopathy, and hematuria (hence the term “icterohemorrhagica”). When fatal, death is usually due to renal failure (22), although pulmonary involvement
has recently emerged as a serious life threat (43). Identification methods include direct (darkfield) microscopy, culture, and the most widely used reference
standard method—the microscopic agglutination test (3). In the first week, blood and CSF cultures are positive, while in the immune phase, leptospires may
be recoverable only from the urine. Pathology reflects organ dysfunction and features of coagulopathy. Mortality is high in fulminant cases. Antibiotic therapy
may cause a Jarisch-Herxheimer type reaction with clinical worsening.
LYME DISEASE
Lyme disease is the most common tickborne infection in both North America and Europe. In the United States, Lyme disease is caused by the spirochete
Borrelia burgdorferi , transmitted by the bite of the deer tick species Ixodes scapularis and I. pacificus.
Transmission
Transplacental spread to the fetus is reported (e344); firsttrimester infection may be followed by premature delivery
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with demonstrable spirochetes in many viscera and severe congenital cardiovascular abnormalities, including hypoplasia of the aorta and endocardial
fibroelastosis (e291,e362). However, whether or not B. burgdorferi directly causes illness in the fetus or congenital abnormalities is debated (e322,e369).
Prenatal transmission is uncommon, even in endemic areas (e305). The case against congenital infection is strong. No inflammation is seen in the placentas
or tissues from children where spirochetes have been identified (e291,e201,e362). Further, longitudinal population studies and serosurveys have not shown
any consistent evidence of adverse fetal effects of Lyme disease during pregnancy (178) (e210,e229,e368), and even when the placenta is infected (e359).
Although B. burgdorferi DNA has been found in breast milk (e292), there is no evidence for transmission of illness through breast milk (167).
Clinical Features
Lyme disease is characterized by multiorgan system involvement (skin, heart, joints, and nervous system) and can occur in three stages (early localized,
early disseminated, and late disease). Following the tick bite, the acute phase is characterized by the erythema migrans rash with or without systemic
manifestations such as fever, headache, photophobia, myalgias, generalized lymphadenopathy and severe fatigue, without localizing signs. Erythema
migrans is a round or oval, expanding erythematous skin lesion that develops at the site of deposition of B. burgdorferi , that typically become apparent
approximately 7 to 14 days after the tick has detached and should be at least 5 cm in largest diameter for a secure diagnosis. The lesion may vary from
erythematous to targetoid to vesicular, and are usually nonscaly and nonpruritic. Secondary skin lesions may arise by hematogenous dissemination from the
site of primary infection. A tick bite hypersensitivity reaction is favored over erythema migrans if the erythematous skin lesion appears while an Ixodes tick is
still attached to the skin, develops within 48 hours of detachment, is urticarial, is less than 5 cm and reduces in size over the 24 to 48 hours following its
appearance. In addition, early Lyme disease manifestations include neurologic [triad of cranial neuropathy, especially Bell's palsy (e299), meningoradiculitis
and encephalitis (e244), carditis (heart block or myopericarditis), and Borrelial lymphocytoma]. Late manifestations include recurrent large joint (typically
knee) arthritis (e313), late neurologic Lyme disease (encephalopathy, encephalomyelitis, and peripheral neuropathy), and acrodermatitis chronica
atrophicans (197). Acrodermatitis chronica atrophicans starts as a doughy bluish-red swelling on the extensor surfaces of the hands and feet and resolves
over months to years with atrophy (“cigarette paper skin”). Most clinical features are immune mediated and are due to elaboration of various cytokines.
There is no well-accepted definition of post-Lyme disease syndrome. Erythema migrans is the only manifestation of Lyme disease in the United States that is
sufficiently distinctive to allow clinical diagnosis in the absence of laboratory confirmation; nearly 90% of children with Lyme disease have erythema migrans
(167). In a community-based prospective study of 201 children with Lyme disease, the average age was 7 years and the initial manifestation was single
erythema migrans (66%), multiple erythema migrans (23%), arthritis (7%), facial palsy (3%), aseptic meningitis (1%), and carditis (0.5%) (167).
Pathology
Histopathologically there is a perivascular and interstitial infiltration of lymphocytes, plasma cells, and histiocytes in involved organs. Borrelia may be
demonstrated in the acute lesions of erythema migrans in hemorrhagic foci. Inflammatory changes are minimal to absent in neonates and, for that reason,
spirochetes may be more numerous (e200). Placental changes range from none to a chorionic villitis with histiocytes, plasma cells, and increased Hofbauer
cells, with intervillous and intravillous spirochetes. Borrelial lymphocytoma is a rare cutaneous manifestation of Lyme disease, which presents as a solitary
bluish-red swelling with a diameter of up to a few centimeters, most commonly on the ear lobe in children and the breast, on or near the nipple, in adults. It
may occasionally be the only sign of Lyme disease and may persist for months. As the name suggests, it is comprised of a dense lymphoid infiltrate in the
cutis and subcutis with or without germinal centers and may suggest the diagnosis of a lymphoma to the unaware; the infiltrate, however, is polyclonal.
Lesions of acrodermatitis chronica atrophicans show a pronounced lymphoplasmacellular infiltration of the skin and sometimes also of the subcutis, with or
without atrophy (121).
Diagnosis is based on serology, and diagnostic testing performed in laboratories with excellent quality-control procedures is required for confirmation of
extracutaneous Lyme disease (197). First-tier testing is most often performed using a polyvalent ELISA. If the first-tier assay result is positive or equivocal,
then the same serum specimen is retested by separate IgM and IgG immunoblots. For patients with symptoms in excess of 4 weeks, reactivity must be
present on the IgG immunoblot, to be considered seropositive. In interpreting the results of serologic tests, it is also important to remember that the
background rates of seropositivity in areas with high endemicity may exceed 4%. False positive results on serology may be due to cross-reaction with other
spirochetes, viruses and autoimmune diseases (e119). Although useful for documentation of B. burgdorferi infection in research studies, amplification of B.
burgdorferi DNA by PCR or culture of specimens of skin or blood for Borrelia species is not recommended for diagnosis of erythema migrans in routine
clinical care (197).
Prognosis and Outcomes

Long-term prognosis is excellent following treatment, irrespective of whether the children present with erythema
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migrans alone, early disseminated disease, or late Lyme disease (e3,e286,e325). Recurrence of arthritis may occur among patients with HLA-DR2, DR3, or
DR4 phenotypes (e325). More recently, children with Lyme neuroborreliosis have been reported to have persistent facial nerve palsy (173). It has been
suggested that therapy of pregnant women with syphilis and lyme borreliosis should follow the same strategy, since both diseases have similar etiologic,
clinical, and epidemiologic characteristics (74).
“Piggyback” Infections Associated with Lyme Disease

The tick I. scapularis that transmits B. burgdorferi may also be infected with and transmit Anaplasma phagocytophilum (previously referred to as Ehrlichia
phagocytophila) and/or Babesia microti , the primary cause of babesiosis. Thus, a bite from an I. scapularis tick may lead to the development of Lyme
disease, human granulocytic anaplasmosis (HGA, formerly known as human granulocytic ehrlichiosis), or babesiosis as a single infection or, less frequently,
as a coinfection (197). Coinfection should be considered in patients who present with more-severe initial symptoms than are commonly observed with Lyme
disease alone, especially in those who have high-grade fever for over 48 hours despite receiving antibiotic therapy appropriate for Lyme disease,
unexplained leukopenia, thrombocytopenia, or anemia. Coinfection may also be indicated by persistence of systemic viremic symptoms even after resolution
of the erythema migrans skin lesion (197). B. microti is an erythrocyte parasitic infection, endemic to coastal New England, which causes a usually mild
hemolytic disease with fever; transplacental babesiosis has been implicated in one perinatal case (e96). On peripheral smears, they may be mistaken for
malarial parasites. HGA is discussed under “Ehrlichiosis.”
Tick-borne lymphadenopathy has been recently described as a new childhood infectious disease. It is probably caused by Rickettsia conorii and R. slovaca,
transmitted by the tick Dermacentor marginatus (142). The tick bite is usually on the scalp and a necrotic eschar surrounded by a perilesional erythematous
halo is reported at the site of bite; there is painful regional lymphadenopathy, as the name suggests.
OTHER SPIROCHETAL INFECTIONS

Nonvenereal treponematoses (and their causative agents) include yaws (T. pertenue), pinta (T. carateum), and bejel (T. endemicum) (55, 136). They are
restricted to the tropics and subtropics, affect children and young adults, and are transmitted by direct personal contact, fomites (bejel), or arthropod vectors
(yaws and pinta). Spirochetes multiply at site of primary infection and disseminate to regional lymph nodes, the adenopathy-mimicking syphilis. Although
bejel and yaws have a systemic spirochetemia with tertiary lesions (in bones and joints), unlike in syphilis, transplacental infection, aortic, and neurologic
involvement do not usually occur. Yaws affects the skin as fissured, verrucous, or oozing lesions; bejel causes mucocutaneous lesions of the mouth and
nasopharynx; and pinta occurs as serpiginous plaques on the foot, hand, or arm.
Abramowsky et al. have described a novel nontreponemal spirochetosis eliciting a pronounced lymphoplasmacytic response in fetal intestine, lung, and
meninges in secondtrimester fetuses that is associated with chorioamnionitis and villitis. The organism is morphologically distinct from that causing syphilis,
leptospirosis, and borreliosis (e2).
CLOSTRIDIAL INFECTIONS
Clostridia are ubiquitous, gram-positive, anaerobic, sporeforming organisms present in the environment, soil, and the GITs of humans, animals, and insects.
Most clostridial syndromes are caused by toxins elaborated by the bacteria and include botulism, tetanus, myonecrosis (gas gangrene), and
pseudomembranous colitis. Clostridial toxins are strongly antigenic and can be neutralized by antisera, a fact that is often used in therapy.
Botulism, an acute neuromuscular paralysis, is an acute systemic toxemia and not strictly an infection (59). It is caused by absorption of preformed
botulinum toxin produced by Clostridium botulinum, usually from the GIT, although the toxin may rarely also be absorbed from infected wounds. The most
common sources of food-borne botulism are home-canned fruits, vegetables, fish, honey, corn syrup, and the skin of fresh fruits such as grapes. It presents
as a descending paralysis (cranial nerves, limbs, and trunk) about 12 to 36 hours after ingestion of toxin. Infant botulism presents with constipation, difficulty
in feeding, weak cry, hypotonia, and drooling, progressing to cranial neuropathy and ventilatory failure. There are no specific morphologic findings. An
association with sudden infant death syndrome has been postulated, but the data are inconclusive. Botulism most frequently occurs between 6 weeks and 6
months of age, with the youngest reported patient being 6 days of age (e15). Breast milk may protect against botulism by causing more acid stools and
increasing the presence of Bifidobacterium species, thereby limiting the intestinal presence of C. botulinum or its spores (e14).
Tetanus, caused by the toxin tetanospasmin produced by C. tetani , is a major cause of neonatal infant mortality in many developing countries (e320).
Neonatal tetanus follows contamination of the umbilical stump due to poor hygiene and certain traditional practices, especially when mothers are not
adequately immunized. Tetanus neonatorum presents as generalized weakness and failure to nurse, progressing to muscular rigidity, spasms, and death (in
over 90% of affected infants). There are no characteristic morphologic features. Older nonimmunized children can develop tetanus following trauma; often
the colonized wound may be trivial.
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Gas gangrene or clostridial myonecrosis caused by clostridial exotoxins (especially lecithinase) follows penetrating or crush injuries contaminated with soil
or feces. Infection may also be nontraumatic in patients with reduced resistance to infections, following an insult to the intestines permitting transmucosal
migration of intestinal clostridia into the blood. The myonecrosis is characterized by severe tissue damage associated with gas and fluid-filled bullae in
necrotic skeletal muscle and surrounding soft tissues. Inflammation in the gangrenous muscle is scant to absent. C. perfringens causes about 80% of these
infections followed by C. septicum and other clostridia (e315). Infection progresses very rapidly in the absence of prompt diagnosis and treatment; higher
mortality rates are seen with C. septicum infections. Neutrophil dysfunction, bowel ischemia, and trauma predispose to C. septicum infection in children
(174).
Pseudomembranous colitis (see Chapter 14) is a toxinmediated condition produced by overgrowth of C. difficile in the large intestine; it may occur in the
newborn (e308).
ZOONOSES
Space does not permit discussion of a large group of bacterial diseases that exist largely in domestic and wild animals; they are acquired only secondarily,
and rarely, by humans. Examples include brucellosis, anthrax, tularemia, and plague. Although the clinical and epidemiologic aspects of these conditions
may be distinctive in the pediatric age group, their pathologic manifestations are not different from those in adults. Recent reviews are comprehensive (24,
119, 129, 176) (e38,e245,e287,e330). Anthrax, plague, and tularemia are potential agents of bioterrorism and are discussed in a later section. Brucellosis is
briefly outlined below.
Brucellosis
Humans are accidental hosts to brucellosis and acquire the disease by direct contact with infected animals (cattle, pigs, or sheep) or by ingesting
contaminated milk/milk products. The bacteria gain entry through skin abrasions, GIT, respiratory tract, or conjunctiva and then localize to the
reticuloendothelial system (lymph node, spleen, liver, and bone marrow). After an incubation period of 3 to 4 weeks the patient has nonspecific symptoms
including fever, chills, profuse sweats, body aches, mental inattention, and depression. Pathologic changes in involved organs include nonspecific
inflammation, lymphoid hyperplasia, and (nonnecrotizing or necrotizing) granulomas. The coccobacillary organisms are rarely, if ever, demonstrable and
culture isolation is also difficult. A presumptive diagnosis is made by demonstrating rising antibody titers in the serum. Complications include sacroiliitis,
osteomyelitis (especially vertebral), neurobrucellosis (meningitis, encephalitis, radiculopathy, myelitis, and peripheral neuropathy), infective endocarditis, and
mycotic aneurysms. (61) (e206, 287).
TUBERCULOSIS
Mycobacterial disease in children is encountered by the pathologist in three clinical forms: TB, pulmonary; disseminated M. avium infection in pediatric HIV
infection; and lymphadenopathy caused by infection with atypical mycobacteria (usually M. fortuitum, M. scrofulaceum, or M. avium-intracellulare).
An estimated one-third of the world's population (2 billion people) is infected with the tubercle bacilli and the WHO estimates that more than 8 million new
cases of TB occur each year, with 3 million persons dying from the disease. Childhood TB, defined as TB in patients less than 15 years of age, accounts for
2% to 40% of all cases (111) (e81). The current WHO practice of reporting only acid-fast bacillus (AFB) smear-positive cases would certainly underestimate
global incidence and prevalence, since 95% of infected children may be AFB smear-negative (126). Difficulties in diagnosis stem from the low yield of
mycobacteriologic cultures and the subsequent reliance on clinical case definitions (57). The epidemiology of pediatric TB continues to be shaped by risk
factors such as age, race, immigration, poverty, overcrowding, and HIV/AIDS. The pathogenesis of disease differs from that in adults, because primary
disease and its complications are more common in children, leading to differences in clinical and radiographic manifestations in pediatric TB. In some
regions, TB accounts for 10% to 15% of all pediatric deaths (e225,226).
Transmission
Pediatric TB occurs in congenital and postnatal acquired forms; congenital TB is rare. Infection of the fetus may be transplacental (50% of cases) or by
aspiration or ingestion of infected amniotic fluid (in maternal tuberculous endometritis or villitis). Transplacental infection leads to primary complex formation
in the liver or lungs, whereas the latter leads to primary disease in the lungs or GIT (e41). Radiologic (CT scan) findings and the time course of the
development of lesions may distinguish the two modes of transmission (e48). Perinatal TB is acquired from postnatal transmission from the mother, adult
caregiver, health care worker, or other infectious source (e.g., M. bovis in cow's milk).
Acquired TB is transmitted by inhalation of infective airborne mucous droplets that are generated by an infected individual or produced by therapeutic
manipulation (aerosol treatments, sputum induction, and through manipulation of lesions). The bacteria may also gain access by ingestion, or through the
skin, mucous membranes, and conjunctiva. The risk of acquiring disease is greatest shortly after initial infection develops and is associated inversely with
age, from birth to 8 years of age. For unknown reasons, a second peak in the risk of developing disease occurs during late adolescence and early adult life
(111). Pediatric patients with TB are usually not infectious; they lack cavities with
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a large number of bacilli, and the relatively weak cough of young children is not conducive to the airborne transmission of organisms.
Clinical Features
Congenital TB presents with nonspecific symptoms during the second or third week of life (poor feeding, poor weight gain, cough, lethargy, irritability, fever)
and may mimic other congenital infections such as syphilis, CMV, or neonatal sepsis. To make a diagnosis of congenital TB, the infant should have proven
TB lesions, exclusion of postnatal transmission by thorough contact investigation of close contacts including health care workers and at least one of the
following: papular or petechial lesions in the first week of life, documentation of TB infection of the placenta or the maternal genital tract, or a primary
complex in the liver (caseating hepatic granulomas) (e41). Hepatosplenomegaly, respiratory distress, fever, lymphadenopathy, and abdominal distention are
the most common signs and symptoms (2) (e41). Most infants have abnormal chest radiographs, usually showing a miliary pattern, hilar and mediastinal
lymphadenopathy, or parenchymal infiltrates and, less commonly, multiple rim-enhancing pulmonary nodules with central hypodense areas (133) (e41). Fetal
involvement is much less common than placental TB. The primary focus may be either in liver or in lung, depending on the route of access, and widespread
miliary disease ensues. Perinatal TB presents at a later time than congenital TB; however, clinical manifestations may be similar to those of congenital TB
(e289).
Clinical features of acquired TB depend on the evolution of disease. In contrast to adults and older adolescents, the clinical manifestations of TB in children
are usually related to primary TB. Inhaled bacilli induce a localized pneumonia at a terminal airway (the Ghon focus), which, with resultant local tuberculous
lymphangitis and hilar adenopathy forms the primary complex. An occult lymphohematogenous spread may disseminate bacilli to a variety of target organs,
where the bacilli may survive for decades (e358). Most children do not develop further disease but instead develop “latent tubercular infection” (LTBI) with a
positive tuberculin skin test result and no clinical or radiographic evidence of TB. Others (especially younger children) develop progressive primary TB,
where the primary focus generally continues to grow even after the development of cellular immunity and may caseate centrally, liquefy, and empty into the
bronchi resulting in further spread (Figure 6-19) (e193). Pleural involvement may result from direct spread of caseous material from a subpleural
parenchymal or lymph node focus, or from hematogenous spread, and may present as pleural effusions or tuberculous empyema. Pleural TB is uncommon
in children younger than 6 years of age and rare before 2 years of age. Mycobacteria disseminated by the bloodstream can cause extrapulmonary disease,
including cervical lymphadenopathy (scrofula) and meningitis. Less common forms of extrathoracic disease are osteoarticular, abdominal, GI, genitourinary,
and/or cutaneous disease. Extrapulmonary TB must be considered when evaluating children with a history of persistent fevers. Meningitis develops when
caseating lesions in the cerebral cortex invade the meninges and disseminate into the subarachnoid space (e274). Children less than 2 years of age are
more likely to experience a rapid progression of meningitis to hydrocephalus, seizures, and cerebral edema, whereas older children have a basal meningitis
that slowly progresses over weeks (41) (e355). Tuberculomas, a less frequent manifestation of CNS disease, form when caseous foci within the brain
enlarge and become encapsulated. Miliary TB occurs when large numbers of bacilli disseminate through the bloodstream and cause simultaneous disease in
two or more organs, typically with millet-sized lesions (Figure 6-19). Miliary disease frequently has an insidious presentation with fever, lymphadenopathy,
and hepatosplenomegaly developing before radiographic abnormalities. As many as 50% of children with military TB have a negative tuberculin skin test at
presentation (e194). Extrapulmonary TB occurs in 9% to 23% of pediatric cases (110, 127). Marais' review of information available from the
prechemotherapy era provides a rich understanding of the natural evolution of childhood TB (112).
Infected children have a comparatively higher risk of progression to active disease than adults: 43% of infants, 24% of 1- to 5-year olds, and 15% of 11- to
15-year olds develop disease if not treated for latent TB (e312). In immune-competent children, the risk of developing TB and the clinical presentation are
highly age-dependent, with younger children being at greatest risk of developing severe manifestations. After reaching the age of 10 years, children are
much more likely to manifest adult-type disease that is primarily pulmonary in focus. Factors that increase the risk of progression from infection to disease
include immunosuppressive therapy, HIV coinfection, malnutrition, medical conditions (e.g., renal and liver failure, diabetes mellitus, or cancer), and
intercurrent viral infections such as measles (e312). Children have a relative deficiency of macrophage and dendritic cell function, and, in contrast to adults,
tend to develop Th2-type T-cell responses to mycobacterial infection characterized by lack of CD8-positive cell response and interleukin (IL)-4 and IL-5
production by CD4-positive cells (105). Although BCG vaccination may not prevent infection (e214), it reduces the hematogenous complications of primary
infection (183) and is reportedly efficacious in preventing tuberculous meningitis (e27, e335).
Pathology
The histopathologic features of TB are similar in children and adults. The classic morphologic feature of TB is the caseating granuloma (Figure 6-19).
Immunocompromised children may have lesions that teem with acid-fast bacilli in macrophages and extracellularly, without granuloma formation.
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FIGURE 6-19 ▪ Tuberculosis. A: Miliary pulmonary disease. B: Fibrocaseous cavitary lesion of secondary/progressive tuberculosis. C: Caseating
granulomas in the lung (low power). D: Tuberculous granuloma, lung with central necrosis and Langhans type giant cells. E: Spleen with military
tuberculosis.
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Diagnostic challenges arise because children have less specific signs and symptoms of disease, have fewer positive mycobacterial cultures and, once
infected, are at increased risk for progression to disseminated disease (57). Traditional techniques like microscopy (acid-fast stains, auramine-rhodamine
fluorescence), and culture techniques (solid, liquid, radiometric, and nonradiometric systems) still remain the mainstay of diagnosis. Molecular amplification
systems (PCR, NASBA, TMA, and LCR) can identify M. tuberculosis as well as nontuberculous mycobacteria and can also identify rifampin (rpoB
gene)/isoniazide (katG gene) resistance. Although molecular assays have high sensitivity and specificity in smear positive sputum, they have variable
sensitivity for sputum negative and extrapulmonary specimens (e361). Unfortunately, congenital and perinatal TB often eludes diagnosis until autopsy.
RICKETTSIAL INFECTIONS
Rickettsiae are arthropod-borne intracellular bacteria (small coccobacillary forms), which cause spotted fevers, typhus, and scrub typhus. A related
organism, Coxiella burnetii , causes Q fever. The clinical and pathologic spectrum of rickettsial disease is discussed thoroughly by Walker et al. (190). The
epidemiologic features of the diseases are listed in Table 6-14. Rickettsial diseases are all characterized by fever, headache, and (except for Q fever) rash.
The pathologic substrate is inflammation of small blood vessels. Rocky Mountain spotted fever (RMSF) is endemic in the southeastern and south-central
United States and in coastal New England, although it is reported from every state. Rickettsiae enter the blood during a tick bite and penetrate blood
vessels. They multiply in endothelial cells and vascular smooth muscle, resulting in a systemic vasculitis, which is the basis for the rash (Figure 6-20),
interstitial pneumonia, myocarditis, hepatic portal triaditis, meningoencephalomyelitis, and interstitial nephritis. Leakage of fluid from the injured vessels leads
to edema and hypovolemia; vascular necrosis and inflammation can initiate consumption coagulopathy (e158,e356). The vasculitis of RMSF involves
capillaries, venules, and arterioles; the cellular inflammatory reaction comprised of mainly macrophages and lymphocytes, with few polymorphonuclear
leukocytes. Eccentric microthrombosis and microinfarction frequently result. Rickettsial organisms are visible, albeit very small (<2 mm), and may be
demonstrated with difficulty using Giemsa or immunostains (e88,e357). Diagnosis, however, is usually accomplished serologically by the Weil-Felix test or by
specific complement fixation. PCR-based methods are also available (e297,e341).
Table 6-14 ▪ RICKETTSIAL DISEASES
Disease Agent Transmission Geographic Distribution
Spotted fever group

RMSF R. rickettsii Tick bite Western hemisphere
Rickettsial pox R. akari Tick bite USA, Russia, Korea
Boutonneuse fever R. conorii Tick bite Mediterranean, Africa, India
Tick typhus Several Tick bite Asia, Australia, central Europe
Typhus group
Epidemic typhus R. prowazekii Louse feces Worldwide
Brill-Zinsser disease R. prowazekii Recrudescent form of epidemic typhus Worldwide
Murine typhus R. mooseri (R. typin) Rat flea feces Worldwide
Scrub typhus R. tsutsugamushi Mite bite Japan, Southeast Asia, Pacific
Q fever Caxiella burnetii Aerosol Worldwide
Ehrlichiosis
Sennetsu fever E. sennetsu Tick bite Japan, Malaysia
Monocytic ehrlichiosis E. chaffeensis Tick bite United States, Portugal, Mali
Granulocytic ehrlichiosis Unnamed species Tick bite United States: Midwest, northeast
RMSF, Rocky Mountain spotted fever.
Summarized and modified from references 134, 141, 178, 484, and 486.
Ehrlichiosis
Ehrlichiae are small pleomorphic coccobacilli in the family Anaplasmataceae; they are tick-borne obligate intracellular parasites and are currently grouped
with the rickettsiae. Human ehrlichiosis is a reportable disease in the United States and its incidence is on the rise. Ehrlichiae infect phagocytes
(macrophages/monocytes and neutrophils). The three genera and the related infections include human monocytic ehrlichiosis (HME), caused by E.
chaffeensis; HGA, caused by A. phagocytophilum; and human ewingii ehrlichiosis, caused by E. ewingii (47) (e159). Patients present with fever and
myalgias, with or without rash and other systemic manifestations, and have leucopenia, thrombocytopenia, and raised transaminases (e114). Complications
are uncommon and include meningitis, pneumonitis, renal failure, and septic shock. Diagnosis is established by blood smear examination for intracytoplasmic
morulae or PCR in the first week
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of infection, with serology being more sensitive beyond 2 weeks (47). Cultures are available in specialized centers. A high index of suspicion is required for
diagnosis, especially in tick-endemic regions. The bone marrow is usually panhypercellular and shows increased histiocytes with granulomas, ring
granulomas, erythrophagocytosis, plasmacytosis, and lymphoid aggregates (e89). The liver shows sinusoidal and portal lymphohistiocytic infiltrates and
hepatocyte necrosis, and the spleen shows focal necrosis on a background of mild histiocytosis (e87). Interstitial pneumonitis and pulmonary hemorrhage
have also been reported (e159). Other organs may show nonspecific perivascular lymphohistiocytic infiltrates. Immunohistochemical stains are available for
diagnosis. Unfortunately, most of the literature pertains to adult infections and the true burden of pediatric infections and the natural course in children is
unclear (162).
FIGURE 6-20 ▪ Rocky mountain spotted fever—lesions on the legs.
HGA, as the name suggests, is a rickettsial infection of neutrophils (e86). Clinical manifestations are nonspecific and may include fever, chills, headache,
and myalgias. The incubation period is 5 to 21 days. In most cases, HGA is a mild, self-limited illness, even without antibiotic therapy. However, serious
manifestations of infection, including a fatal outcome, have been reported in immunocompromised patients. Chronic infection due to A. phagocytophilum has
not been described in humans. Laboratory features may include leukopenia, lymphopenia, thrombocytopenia, and mild elevation of liver enzyme levels. HGA
can be detected in blood samples by smear examination, PCR, or culture using HL60 cells. Identification of the characteristic intragranulocytic inclusions on
blood smear is the most rapid diagnostic method, but such inclusions are often scant in number or sometimes absent; in addition, overlying platelets or other
types of inclusions unrelated to HGA can be misinterpreted by inexperienced observers. The most sensitive diagnostic method is paired (acute-phase and
convalescent-phase) serologic testing using an indirect fluorescent antibody assay (acute-phase testing alone is not sufficiently sensitive). Serologic testing
is often the only way to diagnose a patient who has already begun to receive antibiotic treatment. HGA is rare in children, but perinatal transmission from
mother to child has been reported and is suspected to be transplacental (e147).
MYCOPLASMA INFECTIONS
Mycoplasma and ureaplasma are the smallest free living microorganisms, lacking cell wall peptidoglycans. Mycoplasma hominus and Ureaplasma
urealyticum are frequent inhabitants of the maternal genital tract. They are associated with placental and perinatal pathology including chorioamnionitis (73)
(e302, e314), funisitis (52), diffuse decidual leukocytoclastic necrosis (64), fetal vasculitis (34), fetal demise (e314), prematurity (64), premature rupture of
membranes (e182), chronic lung disease of the newborn (81), and cerebral white matter echolucency (34). However, because both these organisms may be
recovered from perfectly normal infants, a causal relationship to disease may be difficult to establish and requires vigorous exclusion of other pathogens.
Histopathology of infected tissues varies from no pathologic changes to necrosis with or without an associated inflammatory reaction.
In older children, while pneumonia may be the most severe type of M. pneumoniae infection, the most typical syndrome is tracheobronchitis, accompanied
by a variety of upper respiratory tract manifestations (53). The pneumonia is insidious in onset and chest radiographs show bronchopneumonia (often
involving a single lower lobe), plate-like atelectasis, nodular infiltration, and hilar adenopathy (e60). As many as 25% of persons infected with M.
pneumoniae may experience extrapulmonary complications at variable time periods after onset of, or even in the absence of, respiratory illness (158, 180,
189). Extrapulmonary pathology may be due to actual infection of other organs and/or host immune response to infection, and include neurologic
(meningoencephalitis, encephalomyelitis, aseptic meningitis, cerebellar ataxia, isolated abducens palsy, ocular myasthenia, SIADH, transverse myelopathy,
and Guillain-Barre syndrome) (199), dermatologic (maculopapular eruptions, erythema nodosum, erythema multiforme, and Stevens-Johnson syndrome),
musculoskeletal (myalgias, arthritis, and rhabdomyolysis), GI (diarrhea, pancreatitis, cholestatic hepatitis), hematologic (hemolysis, DIC, thrombocytopenia,
thrombocytosis) (135), cardiovascular (vasculitis, pericarditis, and myocarditis),
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renal (glomerulonephritis, renal failure, interstitial nephritis, and IgA nephropathy) (158), and lower genital tract (39) manifestations. Histopathological
findings include bronchial epithelial ulceration with peribronchial and interstitial inflammation (e282) (Figure 6-21). Bronchiolitis obliterans (e190), type II
pneumocyte hyperplasia, diffuse alveolar damage, lung abscess, and fibrinous pleuritis have also been reported, as have long-term sequelae including
pleural scarring, bronchiectasis, and pulmonary fibrosis (e45,e75,e172,e266,e303,e354). Chen et al. have reported active lymphocytic myocarditis in M.
pneumoniae infection (e49). The infection is routinely diagnosed by serological methods, although PCR and culture-based techniques are also available.
Serology is more likely to be positive in children with pneumonia rather than in upper respiratory tract infection or asthma (135).
FIGURE 6-21 ▪ Mycoplasma pneumonia. A: Inflamed bronchiole with epithelial metaplasia hyperplasia with surface necrosis. B: Partial occlusion of
bronchiole.
CHLAMYDIAL INFECTIONS
Chlamydiae are obligate intracellular pathogens; Chlamydia trachomatis and C. pneumoniae are important human pathogens, while C. psittaci is an
important cause of zoonosis. Chlamydial infections in children have been comprehensively reviewed (36, 72) (e131). C. psittaci and C. trachomatis, of which
there are many subtypes, cause several distinct conditions in children (Table 6-15).
C. pneumoniae infects children of all ages. It is a common human respiratory pathogen with asymptomatic nasopharyngeal carriage occurring in up to 5% of
the population. The nasopharynx is probably the most frequent site of perinatally-acquired chlamydial infection, with approximately 70% of infected infants
having positive cultures at that site. Most infections are asymptomatic and may persist for over 2 years. The clinical presentation ranges from mild atypical
pneumonia (similar to that seen with Mycoplasma) to severe disease. Their role in upper respiratory, sinus and middle ear infections is unclear. Infants with
chlamydial pneumonia will usually be symptomatic before the eighth week of life with the insidious development of nasal obstruction and/or discharge,
tachypnea, and a repetitive staccato cough. In very young infants, infection may be more severe and be associated with apnea. Possible laboratory findings
include a distinctive peripheral eosinophilia, mild arterial hypoxemia, and elevated serum immunoglobulins. Untreated disease can linger or recur. Pulmonary
disease takes the form of an interstitial pneumonitis with rare instances of necrotizing bronchiolitis and consolidation (e127). Because the pneumonia is
rarely fatal, pathologic descriptions are few, and no characteristic features have been described. Definitive diagnosis is by cultures in cell lines, but is labor-
intensive and needs special media for collection and transportation. Although serology is commonly used for diagnosis, infection may occur without
seroconversion (45).
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Table 6-15 ▪ CHLAMYDIAL DISEASE IN CHILDHOOD
Organism Disease Pathologic Features Transmission
C. psiltaci Ornilhosis (Psittacosis) Interstitial lobular or lobar pneumonia Aerosol from infected birds
C.
trachomatis
Type A, Trachoma Chronic progressive conjunctivitis with scarring Contact

B, C leading to blindness.
Cytoplasmic inclusions in early stage.
Type Inclusion conjunctivitis Acute follicular conjunctivitis with cytoplasmic Contact

D-M inclusions
Nongonoccal urethritis, proctitis, Nonspecific, but prominent plasma cells and Veneral; rare in prepubertal children and
salpingitis, cervicitis lymphoid nodules suggest sexual abuse
Neonatal pneumonia and Interstitial pneumonitis with rare inclusions Transit through birth canal
conjunctivitis
Type L1, Lymphogranuloma venereum Cutaneous ulcer, granulomatous lymphadenitis Venereal

L2, L3 with stellate abscesses
Modified and summarized from references 44, 189, 194.
C. trachomatis infection is arguably the most prevalent sexually transmitted infection in the United States, with prevalence rates exceeding 10% among
sexually active adolescents (e53). Infection tends to be asymptomatic and of long duration. The rectum and vagina may also be infected at birth; however the
presence of organisms in these sites in older children raises the possibility of sexual abuse (e130, e153). If a pregnant woman has active infection, the infant
may acquire the infection during vaginal delivery, developing either inclusion conjunctivitis (e196) or pneumonitis (e19); the CDC recommends routine
screening of all pregnant women during their first prenatal visit and again during the third trimester if they are at high risk (25 years of age or other risk
factors such as new or multiple sexual partners) (e269). The evidence linking C. trachomatis to premature delivery and fetal loss is inconclusive. Further,
transmission of the organism to other infants in nurseries or intensive care units has not been reported and there is no evidence to suggest that infants with
chlamydial infections should be isolated.
Up to 50% of infants exposed to chlamydiae during vaginal delivery develop conjunctivitis (72). The incubation period for chlamydial conjunctivitis is 5 to 14
days after delivery or earlier if membranes have ruptured prematurely. The severity is variable, ranging from mild injection to purulent discharge with
pseudomembrane formation. Clinical differentiation from gonococcal ophthalmia may be difficult. Inclusion conjunctivitis is characterized by clearly defined
cytoplasmic microcolonies or inclusions in conjunctival epithelial cells. These contain large amounts of glycogen and are readily demonstrated with iodine or
PAS stains. Although the conjunctivitis mostly resolves spontaneously during the first few months even in untreated patients, occasional infants maintain
persistent inflammation with the formation of a micropannus (neovascularization of the cornea) and scarring typical of trachoma. Approximately 70% of
infants who have perinatal chlamydial infection develop asymptomatic nasopharyngeal infection and about 30% of these develop pneumonia (72), usually
presenting between 4 and 12 weeks of age with cough and tachypnea, but no fever. Radiographs do not show any consolidation; laboratory tests reveal
eosinophilia and elevated immunoglobulin levels. Infected adolescents may develop urethritis, epididymitis, bartholinitis, endometritis, subclinical salpingitis,
and perihepatitis (Fitz-Hugh-Curtis syndrome), as in adults.
Except for inclusion body conjunctivitis, early trachoma, and rare cases of neonatal pneumonia with inclusions, the clinical and pathologic features of
chlamydial infections are not specific. Although cell culture techniques are the gold standard for laboratory diagnosis, enzyme immunoassays, direct
fluorescent antibody assays, nucleic acid amplification tests, and microimmunofluorescence serology are more commonly used. However, nonculture
techniques may yield false positive results (72).
ACTINOMYCOTIC INFECTIONS
Actinomycosis is a chronic suppurative inflammatory process caused by an anaerobic gram-positive bacterium, Actinomyces israelii , usually acting in
concert with other bacteria. The organism is a part of the normal flora of the mouth. Actinomycosis is rare in children in the absence of underlying risk factors
(8) (e112). The cervicofacial form of the disease is more likely to be encountered than abdominal and thoracopulmonary disease, and may be seen following
trauma, surgery, or even tooth extraction (especially in a setting of caries). Diagnosis requires a high degree of suspicion (e112). In all locations, the lesion is
an indolent, burrowing suppurative process with large aggregates of organisms forming “sulfur granules” with distinctive peripheral clubbing. The organism is
rather pleomorphic, and diagnosis should ideally be confirmed by culture. Nocardiosis is caused by branching gram-positive coccobacilli; two species,
Nocardia asteroides and N. braziliensis, cause most human diseases. Both are obligate aerobes and are weakly acid fast, the latter helping differentiate
these from Actinomyces species. They are soil inhabitants and act, for the most part, as opportunistic pathogens. Primary infection is pulmonary (e188) and
may take
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many forms including abscesses (often multiple and coalescent), pneumonia, or coin lesions. Extrapulmonary spread occurs most often to brain and kidney.
In any location, the histologic hallmark is liquefaction necrosis and suppuration. The organisms, although small, are readily identifiable on Gram, acid fast or
silver methenamine stains. Nocardiosis is also unusual in children, in the absence of immunosuppression.
FUNGAL INFECTIONS
In-utero fungal infections are decidedly rare. Only Candidiasis occurs with any frequency, the first case having been described in 1958 (e22). Candida
glabrata (previously called Torulopsis glabrata) and Aspergillus have been shown to reach the fetus by the ascending transcervical route, while
Coccidioides spreads by a hematogenous route (14).
Candidiasis ascends from the maternal genital tract and causes chorioamnionitis, from where it can gain access to the fetal skin, upper respiratory and
intestinal tracts. Thus, it causes a generalized cutaneous rash, aspiration pneumonia (Figure 6-22), and intestinal mucositis (35) (e92). Affected infants are
frequently growth restricted, and many are stillborn or abortuses. In recent years, candidiasis has been recognized as a complication of the intensive care of
low birth weight infants; risk factors include parenteral nutrition, central arterial or venous catheters, and a history of broadspectrum antibiotic therapy. These
infants are older than the true congenital cases (e100,e173,e284) and tend to have more visceral dissemination with renal involvement, carditis,
endophthalmitis, arthritis, osteomyelitis, and meningitis. Large aggregates of pseudohyphae may form endocardial vegetations and urinary tract fungus balls
(e70). The lesions bear a striking resemblance grossly to whitish-yellow miliary abscesses of listeriosis and may even be confused with HSV on occasion.
The cellular reaction is suppurative and budding yeast and pseudohyphae are easily demonstrated in the lesions with routine periodic acid-Schiff (PAS),
methenamine silver, or even gram stains. C. glabrata is an occasional cause of neonatal sepsis in premature infants (e271). Candida infections in later
childhood range from relatively mundane cutaneous and mucous membrane infections (diaper rash, thrush, glossitis) to fatal septicemic illness with
widespread miliary abscesses (191). Chronic mucocutaneous candidiasis is a cellular immunodeficiency with defective T-cell response to Candida antigens
(e52). In the immune-deficient or multiple antibiotic-treated patients, the organism enters through intestinal or respiratory tract mucosa, producing local
ulceration and necrosis and hematogenous spread to any organ. The usual suppurative reaction may be modified in the leukopenic host. The usual tissue
form of the organism is a small unencapsulated budding yeast; occasionally blastospores with elongated germ tubes are seen. Confusion with Aspergillus
arises when serially budding organisms are attached end-to-end to form pseudohyphae. A slight “pinching in” of Candida at the site of attachment is a
helpful diagnostic feature. C. glabrata, does not form pseudohyphae. Further confusion with Aspergillus arises when masses of pseudohyphae cause
vascular occlusion, thrombosis, and infarction.
FIGURE 6-22 ▪ A: Candidal pleuritis. B: Candidal pneumonia with yeast and pseudohyphal phase organisms. (PAS stain).
Other fungi are occasionally encountered in the neonatal age group, chiefly as complications of intensive measures in seriously compromised infants.
Malassezia furfur colonization and sepsis in neonates is related to indwelling Broviac catheters and long-term parenteral alimentation using lipid emulsion
(e262). The organism is a lipophilic yeast that localizes in pulmonary vessels, which are the site of lipid deposits associated with parenteral lipid
administration, and causes a pulmonary arteritis (e270). The organism is a tiny (2 to 4-mm) budding yeast with a distinctive “heel and sole” outline seen well
on silver stains. Rare GI zygomycetes infection mimics necrotizing enterocolitis clinically; there is diffuse invasion of bowel wall, with necrosis and fungal
invasion of vessels (e273).
Fungal diseases of older infants and children fall into two categories: the endemic mycoses, characterized by sharply defined geographic boundaries and
occurrence in normal (nonimmunosuppressed) hosts, and the opportunistic mycoses, which are ubiquitous in the environment but do not ordinarily cause
disease in healthy persons (191).
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FIGURE 6-23 ▪ A: Histoplasma nodule, lung. B: Organisms highlighted by a GMS stain.
In North America, the endemic mycoses include blastomycosis, endemic in the eastern states, histoplasmosis in the Mississippi and Ohio River valleys, and
coccidioidomycosis in the southwest. They have several features in common:
1. The organisms are dimorphic fungi; with rare exception, the yeast form is the one seen in tissue.
2. All three are primarily pulmonary diseases with clinical and morphologic features similar to TB.
3. Disseminated disease is the exception and tends to occur in very young children or in immunocompromised patients.
4. All evoke a host response which is primarily granulomatous, but mixed granulomatous and suppurative reactions are seen.
5. All are likely to be misdiagnosed, especially when encountered outside of their usual locale.
Blastomycosis in children ranges from asymptomatic to disseminated forms; most symptomatic disease consists of pneumonic infiltration or consolidation
with or without cavitation (163) (e50,e261). Microscopically, there is a mixed granulomatous and suppurative reaction. Blastomyces dermatitidis is a large
thick-walled yeast easily visible in sections or smears. A characteristic flat-based bud is helpful in distinguishing this organism from other yeasts. The
organism is a rare cause of osteomyelitis (e44,e128). Delayed diagnosis may result in dissemination even in immunocompetent hosts (e67).
Histoplasmosis is asymptomatic in the majority of infected children. Acute pulmonary or disseminated disease may be seen in young infants or
immunocompromised children (16) (e148,e238,e363). Dissemination occurs in pediatric HIV infections, as does cryptococcosis (e85). In the lung,
histoplasmosis provokes a caseating, granulomatous reaction indistinguishable from TB; organisms may be very few (Figure 6-23). In the disseminated form
of histoplasmosis, the organisms are found within macrophages in virtually any site but particularly in the reticuloendothelial system. Sclerosing mediastinitis
is a rare form of thoracic disease. In endemic areas, histoplasmosis is a common cause of hepatic granulomas (e62). Histoplasma capsulatum is a tiny
budding yeast best demonstrated with silver stains. An immunoperoxidase method has been described (e176), and PCR techniques are useful in archival
tissues (e62).
Coccidioides immitis is a soil inhabitant that causes selflimited, often asymptomatic pulmonary disease in well children. Dissemination is unusual and seems
to occur in the very young (e170,e197,e202). The pulmonary disease has many clinical and morphologic similarities to histoplasmosis, but C. immitis exists
in tissues as large double-walled spherules (sporangia), 20 to 100 m in diameter, containing myriads of tiny endospores that are released by rupture of the
spherule (Figure 6-24).
The main opportunists are four: two yeasts (Candida and Cryptococcus) and two mycelial forms (Aspergillus and zygomycetes). All have, in common, an
ability to cause invasive and life-threatening infections in patients who are immunosuppressed or whose normal flora is altered by antibiotic therapy.
Cryptococcus neoformans infection is rarely encountered in immunocompetent children (e304); meningitis, pneumonia, cutaneous lesions (e267), and
disseminated disease have been reported, usually in the compromised host. Two forms of inflammatory reaction are recognized: granulomatous inflammation
and a gelatinous mass composed of large numbers of organisms almost devoid of cellular reaction. The organism is a multiple-budding yeast with a thick
mucoid capsule that in most histologic preparations appears as a clear space. It may be visualized with mucicarmine stain and sometimes shows radial
striations.
Aspergillosis is caused by several species, of which Aspergillus fumigatus is the most common. Four types of disease occur in children: hypersensitivity
pneumonitis, saprophytic colonization of preexisting pulmonary cavities, invasive
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pulmonary aspergillosis, and disseminated aspergillosis (191). Invasive pulmonary and disseminated aspergillosis occurs almost exclusively in
immunocompromised children. Aspergillus is easily identified in tissues as dichotomously branching septate hyphae uniformly 7 to 8 m in diameter. Radial or
sunburst arrangement and a wavelike configuration are characteristic. The inflammatory reaction is suppurative and necrotizing. Aspergillus shares with
zygomycetes (and to a lesser extent, Candida) a propensity for vascular invasion leading to infarction. Aspergillus is present in the sputum in a minority of
patients, and the diagnosis is frequently established by lung biopsy; it is important that the surgeon understand that areas of infarction and necrosis distal to
a fungal thrombus may not contain demonstrable fungi and that care should be used in selecting areas for biopsy.
FIGURE 6-24 ▪ Coccidioidomycosis. A: Cross section of lung with disseminated disease. B: Focal necrosis in the lung with the thick capsules of the
sporangia staining red. C: Endospores in large sporangium. D: Spleen in a case of disseminated infection.
Zygomycosis (or mucormycosis) is caused by several fungi (Rhizopus species, Mucor species, and Absidia species.) All are indistinguishable from each
other in tissues (e207). These organisms are opportunists and are rarely, if ever, seen in normal children. Rhinocerebral and endobronchial zygormycosis
occurs almost invariably in diabetic children (e162). Pulmonary GI and disseminated disease is seen in children with malignancy (e180). The typical pattern
of tissue involvement includes granulomatous or suppurative inflammation, vascular invasion and thrombosis, and infarction. The fungi are coenocytic
hyphae of rather variable
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diameter (5 to 20 mm) that branch at right angles. Folds and wrinkles may mimic septation.
Although histopathology is a major diagnostic tool in diagnosing fungal infections, diagnostic errors may result from morphologic mimics, use of inappropriate
terminology, and incomplete knowledge in mycology (159). Template diagnosis formats have been suggested to minimize errors; species identification
requires microbiology cultures.
PARASITIC DISEASES
The common protozoal and helminthic diseases are listed in Tables 6-16 and 6-17. Although parasitic infections have become more frequent even in the
developed world, space does not permit a detailed review of each entity. Many of these organisms cause disease more or less confined to one organ
system, and are discussed in the appropriate sections. Valuable sources for further details include Pathology of Infectious Diseases by Connor et al. (32),
Atlas of Human Parasitology by Ash and Orihel (5), and Feigen and Cherry's encyclopedic Textbook of Pediatric Infectious Diseases (56). Protozoal
infections of the fetus and neonate will be briefly discussed below.
Table 6-16 ▪ PROTOZOAL INFECTIONS
Category Organism Disease Transmission References
Intestinal protozoa
Amebae Entamoeba Amebiasis liver abscess Fecal-oral 54

histolytica
Flagellates Giardia lamblia Giardiasis Fecal-oral 474, 505
Ciliates Balantidium coli Ciliary dysentery Fecal-oral
Coccidia Cryptosporidium Gastroenteritis Meat, fecal-oral 5, 502
Extra-intestinal protozoa
Amebae Naegleria fowleri Meningoencephalitis Warm water
Acanthamoeba Meningoencephalitis Warm water

culbertsoni
Flagellates Trichomonas Genitourinary infections Venereal 104

vaginalis
Coccidia Toxoplasma gondii Mononucleosis like syndrome, Infected meat oocysts in soil, 166, 238,
lymphadenitis, disseminated sand, cat litter 239
Sporozoa Pneumocystis Pneumonia, rarely disseminated Droplet 215, 238,

carinii 239
Blood borne protozoa
Sporozoa Plasmodium vivax Malaria Arthropod born (mosquito) 381
P. ovale Malaria Mosquito
P. malariae Malaria Mosquito
P. falciparum Malaria Mosquito
Babesia microti Babesiosis Tick bite
Flagellates Leishmania tropica Cutaneous leshmaniasis Arthropod borne (sandflies) 110, 204,
303
L. mexicana Cutaneous leshmaniasis Sandflies
L. braziliensis Sandflies
Mucotaneous
leishmaniasis
L. donovani Visceral leishmaniasis (Kala-Azar) Sandflies
Trypanosoma cruzi Chagas disease Tristomid (reduvid) bugs
T. gambiense African sleeping sickness Tsetse flies 360
T. rhodesiense African sleeping sickness Tsetse flies
From references 44, 155, 297, and 320, in addition to those listed, with permission.
TOXOPLASMOSIS
Toxoplasma gondii is a protozoan of the family Coccidia. The organism is a parasite of worldwide distribution; its definitive host is the cat. Clinical,
epidemiologic, and pathologic features are the subject of recent reviews (9, 117) (e79). Congenital toxoplasmosis, with very rare exceptions, occurs only with
primary maternal infection; the exact route of transmission to the fetus is unknown, but clearly the placenta is involved. The risk to the fetus varies
significantly with gestational age, increasing from 25% in the first trimester to 65% in the third for untreated maternal illness. Prenatal diagnosis is now
feasible and maternal therapy during gestation appears to lessen the ill effects on the fetus (125) (e69,78,113). Among congenitally-infected infants, there is
a wide spectrum of severity; most are asymptomatic, but 10% to 12% have severe morbidity and a few infants die. The characteristic clinical picture consists
of fever, hydrocephalus or microcephaly, hepatosplenomegaly, jaundice, chorioretinitis, seizures, cerebral calcifications, and CSF pleocytosis. Newborns
that die with toxoplasmosis generally have disseminated disease, even when clinical signs are confined to brain and eyes. Organisms may be identified in
brain, eye, middle ear, blood vessels, heart, testes, adrenal,
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lung, kidney, muscle, and occasionally, liver, spleen, and lymph nodes (Figure 6-25). The organisms appear in tissues both encysted and free. The cysts are
round or oval bodies 10 to 30 m in diameter with a thick wall that is certainly visible on hematoxylin and eosin sections but better demonstrated with PAS,
silver, or immunoperoxidase stains. Within the cyst are densely packed tiny trophozoites, 2 mm in diameter. The intact cysts are either intracellular or
extracellular and rarely excite an inflammatory response. They rupture to liberate free trophozoites into the tissues. The host reaction is quite heterogenous
and depends somewhat on the stage of disease (e79). Acute lesions are characterized by suppurative reaction, some eosinophilic infiltrate, and liquefactive
necrosis, which may be extensive and confluent, particularly in the brain. Dystrophic calcification is most conspicuous in the brain. Trophozoites are
numerous in these lesions. At a slightly later stage, the suppurative lesion gives way to a granulomatous one without significant necrosis; the cyst form is
more frequently encountered in and around granulomatous lesions. Healing stages are highly variable in morphology with granulation tissue, gliosis, and
fibrosis. Encysted organisms may persist in many tissues.
Table 6-17 ▪ HELMINTHIC DISEASES
Category Organisms Disease Transmission References
Nematodes
Intestinal Ascaris lumbricoides Ascariasis Egg ingestion 114, 296,

Enterobius vermicularis Pinworm Egg ingestion 488
79
Ancylostoma duodenale Old world hookworm Skin penetration
Necator americanus New world hookworm Skin penetration 302

Strongyloides stercoralis Intestinal and disseminated Skin penetration 375, 421
Trichuris trichiura strongyloidiasis Egg ingestion 296
Whip worm
Tissue Tricbineila spiratis Trichinosis Ingestion of larvae 179,226

Toxicara canis, T. cati Visceral larva migrans Egg ingestion 111
Ancylostoma brasiliense Cutaneous larva migrans Skin penetration
A. caninum
Wuchereria Lymphangitis, elephantiasis Mosquitos

bancrofti Lymphangitis, elephantiasis Mosquitos
Brugia malazi Subcutaneous nodules, Chrysops flies
Loa loa conjunctivitis
Onchocerca volvulus conjunctivitis Black flies 96, 173

Dirofilaria immitis River blindness, subcutaneous Mosquitos 70
Dirofilaria sp. nodules, dermatitis Mosquitos 70
Pulmonary disease (coin lesions)
Subcutaneous nodules, ocular
lesions
Cestodes
Intestinal Diphyllobothrium latum Fish tapeworms Ingestion of raw fish

Hymenolepsis nana Dwarf tapeworms Fecal-oral egg ingestion
Taenia solium Pork tapeworm Ingestion of rare pork
Taenia saginatum Beef tapeworm Ingestion of rare beef
Larval Taenia solium Cysticercosis Fecal-oral 58, 323

Echinococcus Hydatid disease Egg ingestion 72
Diphyllobothrium sp. Sparganosis Copepod ingestion
Trematodes
Intestinal Fasciolopsis buski Giant intestinal fluke Larva ingestion (aquatic plants)
Liver and lung Opisthorchis sinensis Oriental liver fluke Ingestion of raw or undercooked 373
Fasciola hepatica Sheep liver fluke fish
Larva ingestion (aquatic plants)
Paragonimus westermani Lung fluke Larva ingestion (undercooked 63, 213

crabs and crayfish)
Blood Schistosoma mansoni Schistosomiasis (intestinal) Skin penetration 318

S. haematobium Schistosomiasis (urinary tract) Skin penetration 442
S. japonicum Schistosomiasis (intestinal) Skin penetration 493
Modified and summarized from 44, 155, 297, and 320.
OTHER PROTOZOAL INFECTIONS

In endemic areas, malaria and trypanosomiasis constitute a threat to the fetus during episodes of maternal parasitemia. Congenital malaria, however,
appears to be relatively rare (e265). Both Plasmodium vivax and P. falciparum are encountered. The infants develop fever, irritability, jaundice, and
hepatosplenomegaly; the disease is usually not
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suspected, and the diagnosis is almost invariably made by peripheral blood smear. Most reported fetal deaths due to malaria are associated with maternal
falciparum malaria. It is not clear whether maternal anemia or fetal infection plays the most important role in fetal demise, but the parasites and malarial
pigment may be identified in affected fetuses.
FIGURE 6-25 ▪ Toxoplasmosis. A: Brain with microglial nodule and encysted organisms. B: Toxoplasmosis involving the liver with a granuloma-like lesion. C:
Myocarditis with cyst forms within the muscle cells. D: Organisms highlighted by GMS stain.
In Central and South America and Mexico, congenital Chagas disease results from transplacental spread of Trypanosoma cruzi to the fetus (11) (e29). In
endemic areas, T. cruzi is a major cause of abortion and prematurity. Approximately 1% to 10% of pregnancies in women with chronic T. cruzi infection result
in infants born with congenital infection (181). Most infected newborns are asymptomatic or have nonspecific findings such as low birth weight, prematurity,
or low Apgar scores. Other signs include hepatosplenomegaly, anemia, and thrombocytopenia. When the disease follows an acute form in the neonate,
organisms are found in many organs. The acute lesions are mixed granulomatous and suppurative, and T. cruzi is found within histiocytes in the leishmanial
form. Meningoencephalitis, myocarditis, and respiratory distress have a high association with mortality (181).
FIGURE 6-26 ▪ E. histolytica—organisms showing erythrophagocytosis.
Entamoeba histolytica is an important cause of morbidity in the developing countries, and causes amoebic colitis, hepatitis, liver abscess (Figure 6-26), and
related complications. Leishmania species cause cutaneous (Figure 6-27) or systemic disease, with significant morbidity.
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FIGURE 6-27 ▪ Cutaneous leishmaniasis. A: Intracellular organisms (within histiocytes) (H&E). B: Brown-Hopps stain highlights organisms with their nucleus
and kinetoplast.
SYSTEMIC INFECTIOUS AGENTS WITH BIOTERRORISM POTENTIAL

Fears of bioterrorism have exaggerated the importance of various infectious agents to the point of being considered in the routine differential diagnosis of
many human illnesses. These agents have been categorized based, among other things, on their ease of dissemination or transmission, high mortality,
degree of social disruption, and need for special preparation (58). Although any or all of the highest risk biological agents (including inhalation anthrax,
pneumonic plague, smallpox, tularemia, botulism, and viral hemorrhagic fevers) can be seen in the pediatric patient, several agents might closely resemble
some of the more common childhood illnesses (Table 6-18) (138, 177). Selected infections are briefly outlined hereunder.
Table 6-18 ▪ DIFFERENTIAL DIAGNOSIS OF INFECTIONS BY AGENTS WITH BIOTERRORISM POTENTIAL
Agent Differential Diagnosis
Smallpox: variola major Chickenpox-herpes zoster

Herpes
Measles
Mumps
Vaccinia
Epidermolysis bullosa
Impetigo from Staphylococcus aureus, coagulase positive, group 2
Anthrax: Bacillus anthracis Inhalation type: respiratory syncytial virus-“flu/cold”

Cutaneous type: insect bites, cat scratch disease
Gastrointestinal type: rotavirus, Norwalk virus, etc.
Plague: Yersinia pestis Cat-scratch disease: Bartonella henselae

Insect bites
Necrotizing fasciitis: peripheral infarction
Toxic shock syndrome
Stevens-Johnson syndrome
Tularemia: Francisella tularensis “Flu cold”

Hemophilis influenza
Primary pulmonary hemosiderosis
Inhalation toxin with adult respiratory distress syndrome
Botulism: Clostridium botulinum Viral gastroenteritis

Inflammatory bowel disease
Autoimmune disorder: dysarthria, generalized weakness
Drug reaction
Polio: paralysis
Myesthenia gravis
Source: Stocker JT. Clinical and pathologic differential diagnosis of selected potential bioterrorism agents of interest to pediatric health care
providers. Clin Lab Med 2006;26(2):329-344.
Smallpox is a highly contagious infection, caused by the variola virus, which is a strict human pathogen with no carrier state. Although smallpox was the first
human epidemic disease to be eradicated, its high infectivity, ease of person-to-person
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transmission, high mortality, and lack of specific chemotherapeutic agents makes the virus an important biological weapon, especially since the majority of
the world population would be susceptible to infection. Following aerial transmission, the virus spreads and multiplies in the reticuloendothelial system during
the incubation period. A second phase of viremia ensues, associated with prodromal nonspecific symptoms, followed by the characteristic skin lesions. The
differential diagnosis of smallpox includes infection by VZV, HSV, measles, vaccinia, impetigo from S. aureus, and epidermolysis bullosa (177). Unlike in
chicken pox, however, fever precedes the rash by 2 to 3 days, the palms and soles are commonly involved and the vesicles are all in the same stage.
Histologically, in smallpox, the papillary dermis shows signs of inflammation and capillary endothelial swelling, followed by reticulating degeneration of the
overlying epidermis with the presence of basophilic inclusions (Guarneri bodies). Lysis of the infected cells leads to vesiculation; after 4 to 7 days, the clear
vesicles become filled with neutrophils. The pustular fluid is usually bacteriologically sterile, unless secondarily infected. The umbilication characteristic of
the mature smallpox vesicle is due to persistent septa and fixed dermal adnexa. Once the host immune response controls the infection, the vesicular fluid is
resorbed and a scab is formed 10 to 15 days after the appearance of the rash. The scabs fall off by 3 weeks once the basal epithelium is replaced, leaving
scars proportional to the depth of dermal involvement. Mucosal lesions are similar, except that they are covered by slough. The road to smallpox eradication,
the weapon potential of the variola virus, and possible remedies has been reviewed by Raghunath (145). The Advisory Committee on Immunization Practices
recommends not vaccinating pregnant or breastfeeding women or children less than 18 years old in preevent smallpox vaccination programs (193)
Secondary contact vaccinia from smallpox vaccine is rare (Figure 6-28), estimated to occur at a rate of 5 to 7 cases per 100,000 vaccines (e295).
FIGURE 6-28 ▪ Vaccinia. A: Well developed lesions on the hand. B: Disseminated early lesions.
Anthrax, caused by Bacillus anthracis, is a worldwide zoonotic disease. Transmission in humans occurs through contact with animals or animal products
(e.g., wool) and from person to person by way of cutaneous lesions. Anthrax occurs in three forms: cutaneous, GI, and inhalational. The cutaneous form
accounts for nearly 95% of cases in children and adults. Pulmonary and GI infections may be complicated by sepsis and meningitis. In the initial stages, the
cutaneous form may be mistaken for insect bites or cat scratch disease, the GI type for viral gastroenteritis, and the pulmonary type for respiratory syncytial
viral or similar infections, leading to delay in instituting specific therapy (177). The hallmark lesions are edema and hemorrhage, including hemorrhagic
thoracic lymphadenitis, hemorrhagic mediastinitis with radiographic mediastinal space expansion, meningeal hemorrhage/edema (so-called cardinal's cap),
GI submucosa (in over 90% cases) hemorrhagic mesenteric lymphadenitis (in 20% cases) (68, 115) (e1). The causative Gram positive bacilli may be
identified in smear-preparations of lymph node, spleen, or blood. After presumed exposure, antimicrobial prophylaxis is recommended for up to 60 days.
Adverse effects of prolonged antimicrobial use may cause added pathology, although little information is available on effects of such prolonged use.
PLAGUE
Plague is a bacterial zoonosis caused by Yersinia pestis, acquired through infected flea bites, and manifests as bubonic, septicemic, or pneumonic forms
(98). The bubonic form is the most common and presents with one or more enlarged, tender, regional lymph nodes, so-called bubo, as a result of migration
of bacteria from the bite site to the regional lymph nodes. A local skin lesion (papule, vesicle, ulcer, or eschar) may be seen at the bite site. There is marked
neutrophilia (40,000 to 100,000 cells/mL); blood cultures are often positive (50%) and the organism may be identified readily in aspirates of the buboes.
Morphologically, lymph nodes show congestion and edema progressing to hemorrhage and necrosis that spreads outside the nodes. The bacteria resist
phagocytosis to cause
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lymph node necrosis, and may be seen as extracellular aggregates within necrotic foci. Ulceration and cutaneous fistulae may occur. The bacteria may be
recognized in sections and smears by their bipolar “safety pin” morphology or by the identification of monoclonal antibodies to the F1 antigen of Y. pestis
(e47). Destruction of the lymph nodes is followed by bacteremia, septicemia, and endotoxemia. Pneumonic plague, as the name suggests, have dyspnea,
chest pain, and a cough with hemoptysis, while patients with septicemic plague often have prominent GI symptoms and abdominal pain. Gangrene of the
fingers, toes, or the tip of the nose caused by small vessel thrombosis has led to the disease being referred to as the “black death.” All three forms may have
systemic manifestations of gram-negative sepsis. Septicemic and pneumonic plague progress rapidly and are usually fatal without prompt treatment; bubonic
plague has a mortality rate of 50% to 60% (e149). Plague has historically been used as a biological warfare weapon, dating back to at least the early 14th
century, when the Tartar army hurled its plague-infected corpses over the walls of the city during the siege of Caffa. In fact, the United States and the former
Soviet Union were both involved in developing aerosolized Y. pestis before the 1972 convention on prohibition of biologic and toxin weapons (e152).
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Chapter 7
Pediatric Forensic Pathology
Tracey S. Corey
Kim A. Collins
Jurisdictions for forensic deaths vary between states, and often between counties within each state. In the
medical examiner systems of some states, pathologists investigate deaths. In others, the investigator of deaths is
the coroner, an elected lay official who often has no medical background. Some states have dual systems.
Regardless of the particular system, modern death investigation involves forensic science, which is the
application of physical sciences to legal matters. The numerous facets of forensic science include trace
evidence, ballistics, forensic anthropology, forensic odontology, DNA analysis and serology, toxicology and drug
identification, and forensic pathology. Depending on the case, different aspects of forensic science are
employed. Forensic pathology is the study and investigation of bodily disease, injury, and death. The majority of
cases referred to a forensic pathologist are postmortems. In these cases, the cause and manner of death are the
usual focus. The cause of death is the disease or injury that initiates the sequence of events resulting in death.
The manner of death refers to the circumstances under which the disease or injury occurred. Death can be
categorized into five manners: natural, homicidal, suicidal, accidental, and undetermined. Natural deaths are
solely the result of disease. Accidental deaths result from an unforeseen event or action with no harm intended.
Homicides are deaths in which one person takes the life of another with an intended action, even though the
intended result may not be death. Suicide is the taking of one's own life through a deliberate, self-inflicted action.
A death is classified as undetermined when the evidence is insufficient for a manner to be assigned. Pediatric
deaths can fall into any of the five categories. Usually, pediatric deaths are natural, especially during the first
year of life, but a significant percentage is due to accidents or, unfortunately, homicides (63). Accidental deaths
are more prevalent once children reach the toddler stage, and accidents continue to be the leading manner of
death in persons up to the age of 18 (18). Causes of accidental death include asphyxia, as in drowning and
choking, motor vehicle crashes, and recreational drug toxicity. At around the age of fourteen, we see the
percentage of natural deaths decreases with accident, homicide, and suicide as the
common manner of death in descending order (18, 63). Many do not perceive that suicide occurs in this young
age group. However, pediatric suicide rates have been increasing during the past two decades (185). Violent
deaths (accident, homicide, and suicide) are challenging, and their classification requires expertise in the area of
forensic pathology.
The investigation of the death scene is an important component of forensic pathology. Usually, such an
investigation is conducted with law enforcement officials and, when applicable, the coroner or medical examiner.
The death scene investigation provides an opportunity for the pathologist to view the incident site undisturbed,
examine the body in its terminal state, and note its position and postmortem changes. This is also the preferred
time to obtain an accurate history and interview family members or caretakers. At the death scene, the
surroundings can be assessed [e.g., cleanliness, food, appearance of other family members, presence of
animals or infestation, toys, furniture and surfaces (important in cases of falls), sleep location of the child and
other family members, ambient temperature, water supply (in cases of scalds), dangerous objects,
chemicals/drugs/medications]. Photographs should be taken of the immediate surroundings and of the body in its
terminal location. Any items at the scene constituting potential evidence should be procured. Many times, the
body is not at the scene because of prior transportation to a hospital. The scene has been disturbed and
possibly altered, but valuable information may still be obtained by investigation. If present, the body is
photographed and examined. The body position and postmortem changes (described later), any blood or froth,
evidence of the body having been moved, clothing and bedding, nearby objects, and any medical intervention by
emergency medical teams should be documented. Trace evidence, such as blood spots, hair, fibers, particulate
matter, and semen, may be on the body and should be procured before the body is transported for autopsy. All
information gained from the scene investigation will be correlated with the autopsy findings to assign the cause
and manner of death accurately.
The primary investigative tool of the forensic pathologist is the autopsy. The best forensic autopsy is a complete
autopsy,
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The pediatric forensic autopsy may involve procedures that are not performed by most hospital-based
pathologists. One must remember that the focus and purpose of such an autopsy
which includes examination of the brain. The external assessment begins with photographs and measurement of
the growth indices. Special studies such as radiology and ultraviolet photography may be utilized. Postmortem
changes ardocumented and interpreted at this time. These include rigor mortis, livor mortis, algor mortis, and
changes of decomposition. Rigor mortis is the stiffening of the muscles after death secondary to the crosslinking
of actin and myosin to form actomyosin as the ATP levels fall. The process begins soon after death (˜2 hours);
however, most studies of rigor and postmortem intervals have been performed on adults. Livor mortis is the
pooling of blood with gravity when the circulation ceases. The lividity pattern depends on the position of the body
after death. In adults, lividity appears within half an hour after death and becomes “fixed” after approximately 12
hours. Before this time, if the body is moved, the livor pattern can change as the blood redistributes according to
gravity and points of pressure. Algor mortis is the cooling of the body. Determining the postmortem interval by
means of the postmortem temperature is inaccurate. The rate of body cooling is affected by numerous variables,
both between individuals and within the environment. Decomposition is a combination of autolysis and
putrefaction—autolysis from internal cell breakdown and putrefaction from the action of bacteria and fungi. The
rate and appearance of decomposition vary between environments. In utero within the amniotic sac, aseptic
autolysis, or maceration, can occur (274). In cases of maceration, the fetus exhibits erythematous skin, sloughing
epidermis, and overriding skull bones as the brain becomes liquefied. Fetal putrefaction may also be seen if the
amniotic fluid or fetus is no longer sterile (274). Another postmortem change is adipocere, the formation of a
waxy substance of fatty acids derived from the hydrolysis and hydrogenation of body fat. This process is largely
attributed to Clostridium perfringens and most often occurs when a body is immersed. Other postmortem
changes may include insect activity, marine activity, and animal activity (anthropophagy). Postmortem changes,
external and internal gross findings, and the results of histopathology, laboratory, and ancillary studies are
integrated into the final forensic autopsy report (48, 59, 62). The forensic autopsy provides answers to questions
concerning the cause and manner of death, and it also provides an opportunity to determine the time of death
and the body position, gather evidence, procure specimens for toxicology/chemistry/DNA analysis/metabolic
testing, and correlate findings with the history (48, 59, 171, 200). The pediatric autopsy, discussed below, is a
specialized form of the forensic autopsy that is modified for each individual case.
THE PEDIATRIC FORENSIC AUTOPSY: HOW IT DIFFERS FROM THE

HOSPITAL AUTOPSY
The pediatric forensic autopsy may involve procedures that are not performed by most hospital-based
pathologists. One must remember that the focus and purpose of such an autopsy are very different from those of
an autopsy performed after an attended, in-house, natural death. The forensic autopsy is conducted in an
attempt to answer legal as well as medical questions. The main goals of the pediatric forensic autopsy are to
establish the cause and manner of the death and, in certain instances, the identity of the child. Other goals are to
document and interpret traumatic injuries, procure specimens for ancillary studies, and collect trace evidence
(48, 59, 62, 171).
Documentation and Collection of Trace Evidence and Clothing

Unlike the standard in-house autopsy, the forensic autopsy should include a detailed description of the clothing
and personal effects of the decedent. A description of the clothing and its disposition is included in the autopsy
protocol. Any external trace evidence, such as fibers and hairs adherent to the body, should be collected before
the body is transferred to the autopsy table. It is recommended that a sample of scalp hair be removed and
retained because in fatal cases of abuse with head trauma, an impact site containing scalp hair may be
subsequently identified at the scene of injury. A record of chain of custody of any evidence must be maintained,
and persons receiving evidence from the pathologist should sign a receipt for such evidence.
One must keep in mind that trace evidence may take many forms and can even include the presence of maggots.
Good estimates of the time of death of a decomposed body may be obtained from the forensic entomologist who
studies the insects that normally feed on human remains. From the succession of insects (maggots and beetles)
infesting a body, the forensic entomologist can estimate the amount of time that a body has been infested under
specific conditions. The estimate is based on the type and stage of maturity of the insects present. An accurate
estimate requires the proper collection and preservation of evidence. Maggot infestation of a diaper and perineal
region has been successfully used to estimate the time of abandonment of a living child (117).
Documentation of External Evidence of Injury

The forensic autopsy must include a detailed narrative account of any injuries present on the external body
surface. Cutaneous manifestations of abuse may be the most striking visual evidence presented. If documented
properly, such evidence may be used in a court of law and presented to a jury. If improperly documented, the
evidence may be lost forever or considered “too inflammatory” for jury viewing. Therefore, a clear understanding
of the proper documentation of cutaneous evidence of physical abuse is a prerequisite for any medical personnel
who investigate death and injury in children.
Injuries in children that are suggestive or diagnostic of physical abuse should be documented in multiple forms—
narrative, photographic, and diagrammatic. When injuries are described in written form, precise terminology
should be employed. Terms that are used colloquially in an emergency care setting should be avoided.
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FIGURE 7-1▪Abrasion. A blunt force injury on the anterolateral leg. In this example, the direction of force can be
determined from observation of the intact, rolled skin edge anteriorly. The force progressed posteriorly to
anteriorly.
Examples of blunt force injury include abrasion, contusion, and laceration. An abrasion (Figure 7-1) is the blunt
removal of the upper layers of skin. Simply put, it is a scrape. The direction of force of an abrasion may
sometimes be determined by observing a “rolled edge” of intact but displaced epidermis at the far end of the
abrasion. A contusion, or bruise, is bleeding beneath intact skin at the site of a blunt impact. This differs from an
ecchymosis, in which blood dissects through tissue planes to a site distant from the origin of the bleeding. An
ecchymosis commonly occurs in the periorbital area in association with a basilar skull fracture (Figure 7-2). A
laceration (Figure 7-3) is a specific term used to denote a tissue defect created by blunt force. A laceration can
be differentiated from a sharp force injury by the presence of abraded wound margins or tissue bridging within
the wound bed.
FIGURE 7-2▪Periorbital ecchymosis secondary to a fracture of the orbital roof caused by a gunshot wound to the
eyebrow region.
FIGURE 7-3▪Laceration, a blunt force injury.
Special mention should be made of the documentation of bite marks. The surface of a bite mark should be
swabbed immediately with a sterile cotton applicator moistened with sterile water or saline solution to collect any
saliva that may be on the skin surface. A control swab from another area of the body should be prepared at the
same time. Bite marks generally appear as pattern contusions, often with multifocal overlying superficial
abrasions (Figure 7-4). Some bite marks are of sufficient detail that, with proper documentation and subsequent
examination and dental impressions of a suspect, a forensic odontologist may be able to identify a perpetrator or
eliminate a suspect. These injuries must be carefully documented. If possible, the forensic odontologist should
be called to the autopsy suite to view the injury firsthand and document it. If the forensic odontologist cannot
attend the autopsy, the prosecting pathologist should discuss proper and preferred documentation of the injury
with the forensic odontologist. The bite mark should be photographed at 90 degrees, with a linear scale included
in the picture.
FIGURE 7-4▪Human bite mark on the buttock of a toddler.
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FIGURE 7-5▪Incision of the wrist, a sharp force injury that is longer than it is deep.
A sharp force injury is created by a cutting instrument, such as a knife, scissors, or a piece of glass. The two
main types of sharp force injury are the incision, a sharp force injury that is longer than it is deep, and the stab
wound, which is deeper than it is long (Figures 7-5 and 7-6). In general, sharp force injuries have “clean” wound
edges, without abrasions. On inspection, the wound bed displays a uniform, sharp demarcation of injured tissue
on one side or the other, without the bridges of tissue of varying strength that are present in the wound depths of
a laceration. It is important to adhere to a strict and precise use of terminology so that any reader at any time can
recognize immediately the forces that created various described injuries. Such “universality” of terms facilitates
meaningful discussions.
The injury location should be described in relation to a stable anatomic landmark. Examples of stable anatomic
landmarks on the head include the external auditory canal, bridge of the nose, and occipital protuberance.
Examples of stable anatomic landmarks on other areas of the body include the sternal notch, midline of the body,
and heel. The general region of the body should also be noted (e.g., “the left frontal hair-bearing scalp”). When
injuries over the extremities are documented, the body surfaces should be described with the body in the
standard anatomic position. Each injury or injury cluster should be measured, and the number, shape, and color
should be noted. General, nonspecific statements (e.g., “there are bruises on the face”) are unacceptable and
should be avoided.
If possible, the injuries should be documented photographically. Some type of linear scale and case identifier
should be included in the photographs. An identifier may consist of a case number or initials with a date; use of a
full name is discouraged. If available, a color standard may be useful when an attempt is made to delineate
contusion colors at a later date. Areas notable for an absence of injury (e.g., the atraumatic shins of a
preschooler) should also be photographically documented.
FIGURE 7-6▪Stab wound of the chest, a sharp force injury that is deeper than it is long. The contusion adjacent
to the stab wound is consistent with blunt force injury created by the knife handle.
In living children, because variations in color and pattern may be observed as injuries heal, the use of sequential
photography over several days should be considered. This may allow the emergence of faint or subtle patterns
to be identified. Sequential documentation also allows the examiner to observe variations in healing patterns.
When injuries are photographed, attempts should be made to remove or cover extraneous and distracting
objects or body parts (e.g., intravenous tubing, genitalia) from the photographic field. Usually, simple draping of
the surrounding areas with surgical towels or sheets will suffice (Figure 7-7). A ring flash is useful in the
documentation of small areas of injury and provides uniform lighting.
A collection of standard diagrams of the total body and specific anatomic regions should be kept on hand
(Appendices 7 to 10). Quick sketching and notes on these diagrams provide a handy reference when a case is
reviewed. If photographic documentation is not available or fails, these diagrams will be the only visual
documentation of injury. Such diagrams may be useful in an attempt to explain the overall distribution of injury.
FIGURE 7-7▪Multiple blunt impact sites of the scalp. The surrounding body parts are draped with surgical towels.
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Autopsy Techniques and Procedures
The forensic autopsy begins with a thorough inspection of the external body surface. The body is examined from
head to toe three separate times. First, traumatic injuries are described and documented from head to foot. It is
helpful to describe injuries in separate paragraphs based on the anatomic regions of the body (e.g., injuries of
the head listed first in one paragraph, then injuries of the anterior torso in a separate paragraph); such
organization facilitates quick review and understanding at a later date. It must be remembered that all body
surfaces must be viewed, including the intraoral mucosa, axillae, genitalia, posterior aspect of the body, and
anus (Figure 7-8). Next, all evidence of medical treatment is documented. Lastly, a general external description is
recorded.
In a forensic autopsy, it is often necessary to perform dissections and incisions other than the standard “Y
incision” and scalp reflection. The soft tissues are reflected on the dorsal surface of the body, and incisions are
made along the long axes of the extremities. Otherwise, the extent of soft tissue trauma in areas like the buttocks
may not be visible, particularly in children with dark pigmentation (Figure 7-9). Such a dissection does not
interfere with the undertaker's preparations; the incisions are on the posterior aspect of the body and can be
closed at the completion of the autopsy. The dissection may be continued as a posterior neck dissection. The
methodology of such a procedure has been detailed in the forensic literature (3). A posterior neck dissection may
elucidate otherwise occult trauma in victims of abuse, especially those with inflicted head trauma (33, 50).
In suspected abuse cases, a multiple-film “skeletal survey” before the postmortem dissection is strongly
recommended. A standard, single-film “baby-gram” is insufficient and will not elucidate injuries common in cases
of abuse, such as metaphyseal fractures (129, 186, 187, 200). In fact, a skeletal survey should be conducted on
all suspected victims of abuse under the age of 2 years. In older children, “spot films” dictated by history, signs,
or symptoms may be sufficient. If feasible, postmortem neuroradiologic imaging with computed tomography or
magnetic resonance imaging may also be useful (133, 143). During the autopsy, the fractures in question may be
excised for further radiologic and histologic examination (168, 328).
FIGURE 7-8▪Laceration of the intraoral mucosa and frenulum. The external surface of the philtrum region
displayed no evidence of injury.
FIGURE 7-9▪Additional incisions along the dorsal aspect of the body of a child abuse homicide victim are
evidence of blunt trauma to the buttocks, greater on the left than on the right.
The age of contusions is best estimated by histologic examination rather than by gross color determination (177,
266, 281, 318). Therefore, when possible, cutaneous contusions should be sampled for microscopic
examination. Obviously, this is not a feasible procedure for facial contusions. Even with histologic sampling, the
dating of contusions remains an estimation (see “Cutaneous Evidence of Physical Abuse”).
Diffuse, severe hemorrhages of the retina and along the optic nerve sheaths may be found in victims of inflicted
head injury (25, 89, 115, 127, 176, 194, 226). For this reason, the globes of these victims are removed. The
procedure, which is relatively simple, requires freeing of the extraocular muscles from an anterior approach,
followed by globe removal from a superior approach. It is facilitated by removal of a window of bone from the
orbital plate of the basilar skull. If the prosector is not familiar with this procedure and the subsequent fixation and
sectioning of the globes, consultation with an ophthalmologic pathologist is strongly recommended. In cases of a
postinjury survival interval, examination by a pediatric ophthalmologist is helpful. The pediatric ophthalmologist
may be able to document retinal findings photographically.
When the hydration status of the child is in question, vitreous fluid may be aspirated for chemical analysis (59,
62, 171). Although the various electrolytes in the blood undergo rapid changes in the early postmortem interval, it
has been shown that the vitreous compartment is relatively isolated and thus more stable. The chemistry values
of the
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postmortem vitreous fluid mirror those of the antemortem blood in the early postmortem interval (55, 78, 130,
171). With time, the vitreous potassium level rises in linear fashion and so is used by some forensic pathologists
to estimate the postmortem interval. However, this linear increase is affected by factors such as ambient
temperature and antemortem potassium concentration. The relative stability of sodium, urea nitrogen, and
creatinine in the early postmortem interval allows the postmortem identification of antemortem pathologic
processes such as dehydration, hypernatremia, and hyponatremia in low-salt syndrome (55, 171). Vitreous fluid
may be aspirated from the globe with a 20-gauge needle and a 10-mL syringe. The needle is inserted into the
globe from the lateral aspect at an angle of approximately 45 degrees. When the needle is inserted into the
center of the globe, the needle tip is visible through the pupil. Care should be taken to prevent the needle from
being inserted too far and coming into contact with the retina. The fluid should be gently aspirated and placed
into a small sterile red-top vacutainer. The aspirated fluid is clear and colorless. It may be stored in the
refrigerator until it is transported to the chemistry laboratory. Because the fluid may be relatively viscous, it is
helpful to centrifuge the fluid and use the supernatant for testing purposes (55).
In forensic pathology, we are often looking for subtle signs of injury that may have little clinical but considerable
forensic significance. Some of these injury patterns may not be easily visualized when the body is first received.
After the autopsy, intravascular blood has drained away from most cutaneous surfaces, so that faint injuries and
contusions are more easily visualized. Furthermore, as the surface of an abrasion dries, it becomes more
apparent (281). Therefore, in certain cases, it may be helpful to retain the body overnight and reinspect it the
next morning (160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,
179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200,
201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222,
223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244,
245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266,
267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288,
289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310,
311, 312, 313, 314, 315, 316, 317 and 318).
SUDDEN INFANT DEATH SYNDROME

Sudden infant death syndrome (SIDS), a diagnosis of exclusion, is the sudden and unexpected death of a child
under the age of 1 year that remains unexplained after a complete autopsy that includes toxicology studies,
scene investigation, and review of the medical records. Although the exact cause of SIDS is unknown, the
manner of death is presumed to be natural. SIDS accounts for 1 to 6 deaths per 1,000 live births per year, so
that it is the leading cause of postneonatal mortality in the United States (38, 61, 62, 223, 316). Many risk factors
have been reported for SIDS. These include male sex, black race, premature birth or low birth weight, and age
between 2 and 4 months with approximately 80% less than 6 months (61, 153, 223, 316). Most deaths occur
during the winter months and during night sleep (138). The victims often have young parents of low
socioeconomic status (138, 180, 315). Many mothers have used drugs, including tobacco, during pregnancy
(128, 142). Smoking is also associated with low birth weight, so that it compounds the risk for SIDS (170).
Smoking in the household after birth results in passive inhalation by the infant and is also considered a risk
factor (170, 216). Research has shown that many of the victims were not breast fed (103, 180). This finding has
led to the hypothesis of a protective IgA factor in breast milk that bottle-fed infants lack. Overheating and
overwrapping have also been well-documented frequent findings in these deaths and correlate with the
increased occurrence during winter months (15, 101, 215, 223, 228, 286, 303). It is further proposed that
overheating is more likely when a child sleeps in the prone position. Because the face is the main route for heat
loss, thermoregulatory control is likely compromised in the prone position (228, 326). With regard to risk factors,
the greatest focus has been on sleep position and environment of the infant (140, 215, 223, 246, 295, 326).
Epidemiologic studies have shown that infants sleeping on their abdomen (prone) are at a greatly increased risk
for SIDS (91, 140, 215, 223, 246, 295, 316, 326). When back or side sleep was promoted overseas, the
incidence dropped by 50% to 70% (91, 314, 316). In 1992, the American Academy of Pediatrics recommended
that infants sleep on their back or side, and in 1994 it supported the “back to sleep” campaign. This was and
remains a successful effort to reduce the number of deaths. In reviews of the side-sleeping position, it has been
reported that as prone-sleeping death rates have declined following reduction campaigns, side sleeping has
become viewed by some as a risk factor and is not recommended (61, 140). Along with the sleep position, the
sleep environment, in particular fellow sleepers and abundant bedding, is a risk factor that is described below
along with the scene investigation.
To classify a death as SIDS, a scene investigation must be conducted (292). In particular, this is an opportunity
to examine the child's living and sleeping environment. The child's sleep position, location, bedding, room
temperature, fellow sleepers (bedsharing), and clothing are documented at this time. The bedding should be well
described because abundant, fluffy bedding and pillows are often implicated in SIDS (153, 246). Water beds and
adult beds are also a risk for infants (60, 61, 246). Broken cribs, areas of potential wedging, and nearby plastic
should be sought (60). The temperature of the room should be noted, in addition to smoking in the household.
The caretaker should be interviewed at this time to ascertain any recent illness and feeding and to record sleep
patterns, time last seen alive, position the infant was put down to sleep, and time found dead. The infant's
medical records should be reviewed.
The autopsy of a presumptive SIDS victim is essentially negative. Grossly, froth may be seen in the mouth and
nares and is occasionally tinged with blood. The lungs may be congested and edematous. The notable finding in
SIDS is the presence of intrathoracic petechiae (21). These are located on the serosal surfaces of the thymus,
lungs, and heart. Such petechiae are nonspecific and can be seen in other conditions, including asphyxia,
overlaying, and wedging (21, 60, 73). However, the quantity has been noted to be greater in SIDS
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than in cases of overlying, wedging, smothering, or other forms of asphyxia (23, 73). Furthermore, resuscitation
does not affect the number of petechiae (21). It has been theorized that intrathoracic petechiae are caused by
changes in intrathoracic pressure resulting from forceful respiratory efforts against a mechanically occluded
airway (73). Although many support an asphyxial mechanism for SIDS, most researchers do not believe that
SIDS involves a forceful effort to breathe. Instead, hypoxia in and of itself has been regarded as the major
systemic influence predisposing to the formation of petechiae in SIDS (21, 61, 215, 326).
Microscopically, the intrathoracic serosal petechiae can be confirmed. One can see pulmonary edema with some
extravasated red blood cells in the alveolar spaces, correlating with the froth seen grossly. Occasionally, chronic
inflammatory cells are seen around the airways (bronchiolitis), but not to a sufficient extent to cause death.
Hepatic steatosis, a nonspecific finding, has also been noted.
Toxicology must be performed in all cases (48, 59, 62). Vitreous electrolytes should be analyzed to rule out
disturbances such as dehydration. The vitreous hypoxanthine level has been found to be elevated in cases of
SIDS, but no specific causal or diagnostic conclusions can be drawn. Metabolic testing should also be
performed, although not in the formal definition of SIDS. Likewise, microbiologic studies are advised; some
researchers believe the investigation is incomplete without such studies (35).
Numerous theories have been proposed regarding the etiology of SIDS. Asphyxia resulting from partial or
complete airway obstruction is supported by the studies of sleep position (prone) and sleep environment and by
the physical findings of intrathoracic petechiae (153). The associated theory of rebreathing as the face is down
toward the mattress or pressed into the bedding is plausible (140, 153, 326). Supporting this theory is the fact
that the neck muscles of infants are weak, especially in the prone position, so that they are unable to turn and
raise their head in response to hypoxia. Passive smoke inhalation reduces oxygen in the microenvironment and
has been associated with SIDS (128, 138, 170, 216, 265). Apnea and apparent life-threatening events (ALTEs)
have been proposed to result in SIDS. ALTEs, referred to as “near-miss” SIDS, occur when an infant has
episodes of irregular breathing and is aroused. However, only a small number of these infants eventually die of
SIDS, and the majority of SIDS victims never actually experienced a known ALTE (181). Some have proposed an
anatomic variation in the airway of SIDS victims that results in obstruction, but findings have not been consistent
(203). Cardiovascular theories have been proposed, including arrhythmias and a prolonged QT interval (267,
324). However, the studies of prolonged QT interval have yielded conflicting results (125, 309).
Gastroesophageal reflux in infants has received attention (11, 296). Stimulation of the esophageal and laryngeal
receptors in cases of reflux results in apnea, bradycardia, and presumably death (11). However, many SIDS
victims did not have reflux, and many children with reflux survive infancy. Gliosis of the brain stem, in particular
the medulla tegmentum, has been documented in some SIDS victims (294). Gliosis, a reaction to previous
necrosis, is presumed to inhibit vital centers. The immature brain control of cardiorespiratory function is also
under study (66, 181). This particularly correlates with the large number of SIDS victims born prematurely.
Nutritional factors that appear to be associated with SIDS include deficiencies of trace metals (e.g., magnesium,
selenium) and vitamins (e.g., C, D, E, biotin, and thiamine) (206, 323). Infectious agents such as respiratory
syncytial virus, cytomegalovirus, and toxin-producing bacteria have also been investigated without consistent
findings (102, 111, 239). Others believe that SIDS is multifactorial, occurring in an infant at a critical period of
development who is exogenously stressed. Thus, the exact cause of SIDS remains unknown, but through current
research, we have been able to identify risk factors that may be “stressors” to the vulnerable infant.
The diagnosis of SIDS is one of exclusion; however, two other entities can present as SIDS and have similar
autopsy findings: overlying and smothering (60). Intrathoracic petechiae are present in all three entities, although
they are more numerous in SIDS (38). Overlying is an accidental cause of death in which mechanical asphyxia
occurs when a larger person sleeps on top of an infant. At autopsy, one may see pressure marks from the
bedding or the other person's clothing, but otherwise the autopsy findings are negative. The diagnosis of SIDS
should not be designated in a situation that could be overlying. These cases are better designated as
undetermined (i.e., SIDS versus overlying) or Sudden Unexplained Death in Infancy (SUDI). Smothering is a
homicidal death in which the airway is intentionally occluded. If a child has teeth or is of an age to struggle, one
may see lip abrasions, intraoral lesions, or a torn frenulum. Recently, Oehmichen et al. (235) reported the finding
of skin petechiae in victims of smothering, and Meadow (209, 210) reported the presence of respiratory tract
bleeding in cases of smothering. Hemosiderin-laden macrophages in the alveolar spaces have been studied as a
possible indicator of previous hemorrhage secondary to unnatural, inflicted asphyxia (19, 22, 41, 173, 174, 264,
290). Otherwise, the autopsy findings of a smothering victim are negative, and a case of smothering can present
as SIDS.
Multiple SIDS deaths in a single family rarely occur. SIDS does not appear to have a genetic etiology, but certain
situations, such as child abuse, inherited diseases, and unsafe sleeping environments, “run in families” (61).
When a second child dies of SIDS, investigators are often suspicious of infanticide. Some researchers believe
that the occurrence of three SIDS deaths in one family is so unlikely that the manner of death is considered
homicide until proven otherwise (81).
Sudden infant death syndrome is a diagnosis of exclusion with an unknown cause. It is the number one cause of
death in children of this age group. The findings are not specific, so that a thorough autopsy and investigation
must be conducted.
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MUNCHAUSEN SYNDROME BY PROXY
The term Munchausen syndrome by proxy (MSBP) was coined in 1977 by Meadow to describe illnesses in
children produced by their caregivers (207). The syndrome was named as an extension of the disorder known as
Munchausen syndrome, in which a patient “creates” an illness to obtain the attention afforded persons playing a
“sick role.” MSBP was defined as a cluster of four elements:
1. 1. A child's illness is simulated (faked) or produced by a parent or someone who is in loco parentis.
2. 2. The child is presented for medical assessment and care, usually persistently, and medical procedures are
often performed.
3. 3. The perpetrator denies knowledge of the cause of the child's illness.
4. 4. Acute symptoms and signs of the illness abate when the child is separated from the perpetrator (260).
It must be stressed that the above definition excludes physical abuse only, sexual abuse only, and nonorganic
failure to thrive only. In this particular disorder, the perpetrator (most often the mother) creates or feigns illness in
the child to gain attention from the medical community. The methods by which disorders are created in the
victims are often elaborate and almost beyond belief. In the series of 117 cases, common presentations included
bleeding, seizures, central nervous system depression, apnea, diarrhea, vomiting, fever, and rash (260).
Methods of production of various illnesses include forced oral ingestion of drugs or other substances (including
salt), intentional manual suffocation, and intentional injection of nonprescribed substances and bacteria (255).
More recently, because of widespread inappropriate application of the term, Meadow (208) has suggested
further specifications for its use. These include the following actions by and characteristics of the perpetrator:
1. 1. A person intentionally produces or feigns physical or psychological signs or symptoms in someone under
his or her care.
2. 2. The motivation for the perpetrator's behavior is to assume the sick role by proxy.
3. 3. External incentives for the behavior (such as economic gain) are absent.
4. 4. The behavior is not better accounted for by another mental disorder.
Meadow stresses that the key discriminator in the above criteria is the second one—”in relation to the children,
the mother would be harming the child (making the child ill) in order herself to assume the sick role and all its
benefits” (208). It should be stressed that this disorder is not merely a “game” or an act of histrionics on the part
of the mother. It constitutes true physical abuse and may be fatal if not detected by the medical community.
Indeed, Rosenberg's series displayed a mortality rate of 9% (260). All the children who died were under the age
of 3 years; the most common symptoms in these children were apnea and decreased levels of consciousness
(260).
In recent years, covert video surveillance in the rooms of suspected victims of MSBP has proved useful in
detecting and documenting this form of abuse. In such a procedure, the patient is admitted to a hospital room
equipped with a hidden video monitor. Close by is an observation area where designated persons (law
enforcement officers, hospital personnel) monitor the parental activities in the child's room. It is important that
observation be continuous in these cases, so that intervention occurs in a timely fashion if the child is abused or
assaulted. In a series published by Southall et al. in 1997, the use of covert video monitoring led to the
identification and documentation of abuse in 33 of 39 suspected cases (278). Although vocal critics of such
surveillance have emerged, it is certain that many of the cases presented in the article would not have been
confirmed without such evidence, and the children would have remained “in harm's way” with the abusive
caregiver.
When a case of possible MSBP is evaluated, all records should be completely and thoroughly reviewed. It is
important to check multiple sources, including health insurance companies, to make sure that all medical
evaluations have been discovered. It is helpful to construct a time line, as these are usually complicated,
protracted cases. Such a time line is helpful in “keeping the facts straight” and is useful in explaining the
condition and history to law enforcement officers, attorneys, and other lay persons. Information gleaned from
extensive review of the often voluminous medical records should include documentation of admissions,
outpatient and emergency department visits, calls to the physician, consultations, invasive procedures, and
prescribed medications. An issue that should be considered during a review of medical records is the number of
times visits were initiated by the caregiver, as opposed to the number of visits representing physician-ordered
rechecks and specialty consultations. When a suspected victim of MSBP presents to the emergency department,
blood and urine should be obtained for toxicologic analysis because multiple cases of forced ingestion of
medication have been documented in MSBP (97, 106, 260). When a diagnosis of MSBP is considered, one
should always keep in mind that the most common reason why a parent persistently seeks medical attention for a
child is genuine illness of the child.
NEONATICIDE
Before the investigation of neonaticide is discussed, it is helpful to define the term. Neonatal may be defined as
“newborn; relating to the period immediately succeeding birth and continuing through the first 28 days of life”
(288). Based on this definition, neonaticide could be defined as the killing of any baby in the first 28 days of life.
In reality, the term is usually reserved for homicides committed shortly after birth; it is this type of case that will be
discussed here.
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Around the country, state laws vary regarding the circumstances and time at which a fetus becomes a “person”
in the context of criminal homicide statutes. In many states, several facts must be proven before such a death
may be considered a homicide. First, the decedent must be shown to have been “viable”—that is, to have
reached a gestational age at which independent, extrauterine survival is possible. This gestational age is
generally legally considered to be around 24 to 28 weeks, but it varies from state to state. Second, it must be
determined that the fetus was born alive and sustained an existence separate from the mother. Thus, in general,
an intrauterine or intrapartum death of a baby or fetus arising as the result of a criminal act would not be
considered a homicide. Further, the delivery and the subsequent demise of a previable fetus arising as a
consequence of a criminal act also would not be considered a homicide in most courts of law.
When a possible neonaticide is investigated, the autopsy should not be conducted as a “black box” exercise
(217). A complete postmortem investigation includes at least the three following components:
1. 1. Examination of the scene of death and/or body discovery

2. 2. Review of the case history
3. 3. A complete autopsy
The precise incidence of neonaticide cannot be determined because some concealed babies are found decades
after death, and undoubtedly some are never found. However, it appears that neonaticide is more prevalent in
certain areas of the world. The reasons for this are probably multiple and include social customs, economic
factors, and the availability of elective abortions. From the published literature, it appears that Japan has a
higher incidence of neonaticide than the United States; neonaticide accounted for 10% to 24% of medicolegal
autopsies performed in 1 series (272), and 12 cases of 3 or more consecutive neonaticides were described in
another series (105).
Investigation of the Scene

Provided one keeps in mind that it is possible to suffocate an infant intentionally and leave no signs of trauma,
the scene investigation may provide important information for a cause-of-death determination. If the pathologist
does not examine the scene of death or body discovery, then it is important that scene information gathered by
law enforcement officers or lay death investigators be communicated to the prosecting pathologist. Causes of
death that leave little or no evidence on the body of an infant include the common methods of neonaticide—
suffocation, drowning, and exposure (253). Without knowledge of the findings at the scene of death or body
discovery, errors may occur in the determination of the cause and manner of death. If blood stain patterns are
present at the scene, it is wise to have someone experienced in bloodstain pattern analysis examine the scene.
Bloodstain pattern analysis is a scientific area of study based on the physical properties of liquids and the
substances they affect; training seminars are conducted, and a scientific society (the International Association of
Bloodstain Pattern Analysts) has been established. Courts around the country have qualified expert witnesses in
the area of bloodstain pattern analysis based on their education, training, and experience. Among other things,
bloodstain pattern analysis may reveal attempts to clean or conceal the delivery site, or even postpartum
independent movement of the baby.
Case History Review

Historical information concerning the actions and statements of the mother in the months and weeks preceding
the delivery and the actions and statements of the mother following the delivery may provide other “pieces of the
puzzle” in the determination of the cause and manner of death. Ophoven noted “striking similarities in features of
mothers committing infanticide” (238). These may include the following:
Average intelligence
Living at home with parents at the time of delivery
Attempted concealment of pregnancy
Statements on questioning that the child was born dead
Lack of plans for delivery or for care of the infant thereafter
Delivery alone in a high-risk location or circumstance
Concealment of the delivery
Concealment of the infant and placenta
Cases of neonaticide often come to the attention of medicolegal investigators when the mother presents for
medical treatment of birth-related injuries or postpartum bleeding.
Autopsy
The pathologist performing the autopsy on an apparently newborn infant is confronted with three possibilities:
Death in utero
Intrapartum death
Death after delivery
Depending on the time interval between death in utero and delivery, the stillborn infant will show varying degrees
of maceration (274). Maceration is the progressive breakdown of tissues by sterile autolysis. Two of the earliest
macerative changes include red-brown discoloration of the umbilical cord stump and skin slippage (109). The
earliest reliable histologic feature of death in utero is the loss of nuclear basophilia in renal cortical tubular cells
(109). The causes of death in utero are diverse and include maternal diseases, placental disorders, congenital
anomalies, and infection.
Intrapartum death, defined as death occurring during labor and delivery, arises primarily from asphyxia or trauma
(271, 312). The prosecting pathologist must be cautious when attempting to differentiate between postpartum
and intrapartum injuries. The pathologist should attempt to determine the presentation position by physical
evidence. For instance, caput succedaneum identifies the area of the head that presented, whereas a large
fluctuant hematoma over the
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buttock may be seen in breech presentations (Figure 7-10). Other birth injuries include cephalhematoma, forceps
abrasions, and shoulder dystocia.
FIGURE 7-10▪Breech presentation with intrapartum death. Identification of the presentation position. Fluctuant
hematoma of the buttock with associated swelling and congestion of the scrotal sac.
The lungs of an infant succumbing in utero or during delivery generally display primary atelectasis—they are
redpurple, rubbery, and airless. On in situ gross inspection, the lungs do not completely fill the pleural spaces.
Some investigators report that partial pulmonary inflation can occur as a result of attempted resuscitation or
intravaginal breathing. Full-body radiographs taken before the internal examination will document evidence of
aeration within the lungs and gastrointestinal tract. However, it must be remembered that in decomposed bodies,
putrefactive gases may be present (Figure 7-11). The distal edge of the umbilical stump should be examined to
differentiate separation from the placenta by cutting versus tearing. It may be examined microscopically for
histologic evidence of a vital tissue reaction.
After death in utero and intrapartum death have been ruled out, the pathologist is left with but one alternative:
postpartum death. The questions then become: What is the cause of death? What is the manner of death?
Natural causes must be eliminated. The lungs of an infant who has breathed after delivery are well aerated and
light salmon pink in color, and they fill the pleural spaces. To assess aeration of the lungs further, a “flotation”
test, first described in the 1600s, may be performed (238). The lungs, individual lobes, or tissue samples from
each lobe are placed in water or formalin. Simply put, flotation is evidence of aeration once the presence of
putrefactive gases has been ruled out. Evidence of aeration of the gastrointestinal tract should be documented
because, in general, air within the gastrointestinal tract indicates extrauterine swallowing of air and thus
extrauterine existence. Any stomach contents may be retained for possible analysis. The presence of indigestible
vegetable matter representing feces may indicate swallowing of toilet water in cases of toilet water drowning.
FIGURE 7-11▪Putrefactive gases in the soft tissues of an abandoned, decomposed term infant.
If available, the placenta should be thoroughly examined both grossly and microscopically. Examination of the
placenta may shed light on both the cause and the time of death and may confirm or eliminate various factors
contributing to death, such as infection and uterine-placental insufficiency.
No single objective laboratory test allows a diagnosis of neonaticide. Rather, the pathologist must compile and
assess multiple findings before arriving at a conclusion regarding the cause and manner of death. These multiple
findings include, but are not limited to, historical information, evidence from the scene examination, and the
results of a complete autopsy. The pathologist should expect to encounter equivocal cases in which an opinion,
to a reasonable degree of medical certainty, cannot be rendered. Cases may be equivocal for a variety of
reasons, including decomposition of the body, severe natural disease, and indeterminate physical findings.
NEGLECT
Child maltreatment is an intentional act or omission by someone in the role of caretaker that endangers or
impairs a child's physical, mental, or emotional health and development (54, 62, 171). The child from birth to age
18 who suffers maltreatment by parents, guardians, or other caretakers can broadly be defined as a victim of
abuse (299). The four major categories of child maltreatment are physical abuse, sexual abuse, emotional
abuse, and neglect (211). Neglect is the most common form of child maltreatment, three times more common
than physical abuse (54, 62, 93, 104, 171, 211, 242). Neglect accounts for approximately two-thirds of
maltreatment cases (85). Pediatric neglect is defined as the failure of a child's caregiver to provide adequate
safety, food, clothing, shelter, education, protection, medical/dental care, and supervision. Multiple forms of
neglect exist. Physical neglect refers to withholding nutrition, drink, hygiene, clothing, or shelter from a child
(152). Emotional neglect occurs when nurturing or psychological needs are not met or are ignored. A child who is
not immunized, does not attend school regularly, or is allowed to do dangerous things should alert one to a
problem of neglect (242).
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Neglect can be either active or passive. Active neglect involves a deliberate lack of care or the withholding of
necessary components of a child's care. Passive neglect occurs when caretakers inadvertently do not provide for
a child because their focus is elsewhere. The results of neglect range from slight morbidity to death and are the
result of either short- or long-term failure to provide for a child. Lethal neglect usually denotes starvation or
dehydration. Most victims of lethal neglect are under the age of 1 year. Once children are mobile, they are
generally able to obtain drink and food, although the nutritional value is usually suboptimal.
A scene investigation is warranted in all cases of suspected lethal neglect. The caretaker should be interviewed
and interrogated regarding the medical and feeding history. The medical history should include birth and medical
records with chronologic recorded weights and measurements. The feeding history includes schedule and
quantity of feedings. With starvation and dehydration, the history is inconsistent with the physical findings. If
available, any formula given to the child should be procured. The consistency and concentration of the formula
can be compared with the manufacturer's instructions to see if it was diluted and therefore inadequate for proper
nourishment.
At autopsy, full-body radiographs (skeletal survey) should be obtained and interpreted by a pediatric radiologist.
Usually, physical abuse/battering is not present in cases of physical neglect; however, it is not universally
absent. Radiographs will reveal injuries in addition to signs of malnutrition, such as skeletal demineralization and
rachitic changes. Proper external measurements of crown-heel and crown-rump length, head circumference, and
body weight are extremely important, and these must be compared with standard measurements. If a child has
been born prematurely, one can compare its measurements with the expected growth measurements. It is very
useful to examine the aforementioned medical records to observe the chronologic pattern of growth and
development. This can help narrow the time frame of neglect and often aids in ruling out organic disease. The
child should be photographed in color from several views. A color card is useful in highlighting unusual
pigmentation, hypopigmentation, or “blue pallor.” Always back up photography with full-body infant/pediatric
diagrams.
The gross findings at autopsy represent a decreased caloric intake over time and a decrease in total body
adipose tissue, both deep and subcutaneous. The body is underweight for its length. The weight is usually
around or below the fifth or third percentile (depending on the growth chart plotted) (158, 171, 322). The neck is
narrow secondary to the loss of fat, so that the head has a deceptively large appearance. The occiput appears
to protrude because of the decrease in neck adipose tissue and possible atrophy of the neck musculature. The
eyes are sunken within the orbits from a loss of orbital fat and often from associated dehydration. The cheeks
are sunken secondary to loss of the buccal fat pad. The ribs are prominent to the extent that the intercostal
musculature is depressed (concave). The iliac crests are prominent and the abdomen is scaphoid. The skeletal
muscles of the arms and legs are atrophied and the fat decreased, so that the appearance is skeletonized. The
skin about the knees and ankles is wrinkled, and the knees appear “knobby.” Posteriorly, the vertebral spinous
processes are prominent. The scapulae are protuberant because the medial borders are accentuated secondary
to a loss of muscle and adipose tissue. The buttocks are very wrinkled because of the near absence of gluteal
fat. Pressure sores may accompany such a loss of fat over prominent bony planes. The skin is thin and dry and
has a blue pallor. When pinched, the skin remains “tented,” which indicates a loss of turgor resulting from a
decrease of subcutaneous fat and fluid. The hair may be dry, pale, and brittle, with areas of alopecia. The
fontanelles are often depressed as the cerebrospinal fluid pressure drops and the brain shrinks with dehydration
of brain cells (123). Reflection of the scalp demonstrates more clearly the fontanelle depression. Internally, one
sees the decrease in subcutaneous fat and the deeper fat around the gastrointestinal areas (omentum and
mesentery) and kidneys. The serosal surfaces are “sticky.” The organ weights, including those of the lymphoid
organs, are decreased except for the brain, which may be smaller with dehydration, although not substantially.
The organ weights are compared with the expected weights for the body length. The stomach and intestines
have thinned walls, are empty of food material, and are often distended with gas. Any food material present
should be quantified and qualified in regard to location in the tract. Fecoliths may be present secondary to
dehydration. The gallbladder is distended with bile secondary to lack of secretion. Microscopically, the adipose
tissue that remains is atrophied and transformed to brown fat. Brown fat is composed of adipocytes that are
multivacuolated and univacuolated (171). The cytoplasm of the multivacuolated cells appears granular because
of the presence of numerous mitochondria, and the nucleus is centrally located. The brown fat transformation is
a protective mechanism; this type of fat has a higher energy- and heat-producing capacity. Hepatic
microvesicular steatosis may be present, reflecting protein deficiency (62, 171). Thymic involution is common.
Hassall corpuscles undergo degeneration and calcification. The cuff of cortical lymphocytes becomes depleted,
leaving a “starry sky” appearance. Eventually, the gland is replaced by fibroadipose tissue. The adrenal glands
may be atrophic with a thin cortices, lipid depletion, and cortical pseudotubule formation.
To determine a component of dehydration, it is very useful to obtain an electrolyte analysis of the vitreous humor
(57, 59, 171). Dehydration is a loss of fluid from vital tissues, with the potential for circulatory collapse. Infants
are at increased risk for dehydration because their losses are higher (310). Their metabolic rates and surface-to-
volume ratios are higher, and they are more prone to febrile illnesses (310). The three types of dehydration are
isotonic, hypotonic, and hypertonic. Isotonic dehydration is the most common form in children and is usually a
consequence of viral diarrhea (123). It is a loss of water coupled with a proportional loss
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of sodium. Hypotonic dehydration (sodium < 130mmol/L) follows excessive fluid losses through gastrointestinal
tubing and in cystic fibrosis, adrenal insufficiency, and bacillary dysentery. In hypotonic dehydration, the vitreous
levels of sodium and chloride are low, as is the level of potassium, in contrast to the usual postmortem elevation
of potassium (123). Hypertonic dehydration (sodium > 155mmol/L) is seen in salt (sodium) excess, diabetes
mellitus, diabetes insipidus, mental retardation, high environmental temperature, and water deficit/withholding.
This type of dehydration is associated with the highest mortality rate. Dehydration by neglect is usually
hypertonic, with an increase in sodium, potassium (>135mmol/L), and urea nitrogen (>40mmol/L). The exact
numeric levels of the electrolytes vary with the analytic method. In hypernatremic dehydration, the brain cells
become dehydrated, the parenchyma shrinks, and tearing of cerebral vessels with hemorrhage may result (123).
In hypertonic dehydration, the mechanism of death is probably arrhythmia resulting from circulatory collapse and
hyperkalemia or, less commonly, cerebral hemorrhage.
Often, because malnutrition and dehydration depress the immune system, physical neglect is associated with
certain diseases. These include bronchopneumonia, tuberculosis, urinary tract infections, skin infections,
cellulitis, otitis media, meningitis, and intracranial abscesses. The immediate cause of death may be one of the
above, but the underlying cause of death remains physical neglect.
Before a death is classified as having been caused by neglect, one must rule out organic diseases that produce
a wasted appearance (70). Such diseases include partial cleft palate and other oral motor abnormalities,
intestinal malabsorption, cystic fibrosis, protein-losing enteropathies, abetalipoproteinemia, pyloric stenosis,
celiac disease, malignancies, and congenital metabolic disorders (e.g., congenital adrenal hyperplasia and
glycogen storage diseases). In these organic diseases, absorption of the nutrients and calories necessary for
development and the expenditure of energy are inadequate. Other conditions associated with such findings
include congenital heart disease, cerebral palsy, and chromosomal abnormalities. Diseases such as cystic
fibrosis, medium-chain acyl-CoA dehydrogenase deficiency (MCAD), diabetes mellitus, mental
retardation/chromosomal abnormalities, congenital adrenal hyperplasia, and viral gastroenteritis can cause
dehydration (122, 310). Mentally retarded children are at increased risk for dehydration because their intake may
be inadequate as a result of swallowing difficulties associated with neuromuscular incoordination (310). All such
entities must be included in the differential diagnosis for pediatric neglect before such a serious conclusion can
be made.
Other forms of physical neglect besides starvation and dehydration may be seen in forensic pathology.
Hyperthermic and hypothermic deaths in cases of abandonment or exposure are seen in young children unable
to protect themselves from the environment (171). Improper supervision or a lack of supervision combined with a
dangerous environment can result in the death of a child. With the use of recreational drugs in our society,
children are exposed to and may accidentally consume drugs. Another form of neglect is the failure to provide
adequate dental and medical care. Dental caries, periodontal diseases, and other oral conditions, if left
untreated, can lead to pain, infection, and loss of function (240, 261). Infections may lead to meningitis or sepsis,
an inflamed appendix may rupture, or a child may not receive immunizations. Certain cultural and religious
practices that prohibit some types of medical treatment occasionally result in the death of a child. Care must be
taken in the evaluation of such controversial situations. Respect for another person's beliefs must not be allowed
to interfere with the welfare of a child.
Even though neglect is the most common form of child maltreatment, it remains a challenge to investigate and
prove. A careful scene investigation, a review of the medical and feeding histories, a complete autopsy with
radiographic, toxicologic, chemical, and metabolic studies, and a careful elimination of possible organic causes
are all necessary before a death can be classified as resulting from neglect (59).
ACCIDENTAL CAUSES OF DEATH IN CHILDREN

Although this chapter focuses primarily on SIDS and inflicted injuries in children, it is important to remember that
deaths from unintentional injury far outnumber abusive deaths in children. In most abusive deaths, a history of a
minor household accident is often provided as the alleged history of injury. Some types of accidental deaths are
indistinguishable from SIDS by autopsy alone, as are some types of homicide. Asphyxia is one of the more
common causes of death in cases that may be mistaken for SIDS in the absence of a complete investigation.
Thus, these cases require a thorough scene investigation and history review. Finally, it is important to recognize
accidental deaths from a public health standpoint, so that the public may be made aware of such risks and
changes in product design may be undertaken when appropriate. For these reasons, a brief discussion of
accidental deaths in children is warranted. Perhaps the most important fact to remember is that children of
different age groups are vulnerable to different hazards within their environments. Therefore, “childproofing” the
home and discussing safety with parents and other caregivers must be age specific (47, 64).
Infants less than 1 year old represent a unique age group with regard to death from unintentional injury. Unlike
older children, infants are particularly vulnerable to hazards within their sleeping quarters. This is because
infants are unable to extricate themselves from potentially dangerous positions and situations. Indeed, in an 11-
year autopsy series by one of the authors (TSC), asphyxia after placement in unsafe sleeping quarters was the
leading cause of accidental death in infants up to 1 year of age (56, 60). Byard et al. reported similar findings in a
28-year retrospective review of sleeping environment deaths, and they reported wedging/entrapment,
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hanging, nose and mouth occlusion, and external chest compression as common situations conducive to
asphyxia (39). In a follow-up study, Byard stressed the need for scene investigation in cases with relatively
nonspecific autopsy findings (40). Other authors have stressed similar hazardous conditions in the sleeping
quarters of infants (60, 112, 155).
Motor vehicle-related injuries remain the leading cause of death for persons of ages 1 to 24 years in the United
States (18, 95, 126). In a study published in late 1997, it was noted that approximately one-fourth of all collisions
in which children under 15 years of one-fourth age died involved a driver with a blood alcohol concentration
above 0.10%. Furthermore, 60% died while riding with the drunk driver, and only 16% of the children were
restrained. Studies have shown that fatalities among children ages 0 to 4 years have declined with the passage
of child restraint laws (119, 230). Even when caregivers are not driving while drunk and are placing children in
safety restraint seats, the seats themselves are often improperly secured in the vehicle (71). New rules by the
National Highway Traffic Safety Administration became effective November of 2009 that require that all safety
seats be anchored to cars in a uniform way with a single type of anchoring system (250).
Other common causes of accidental death in early childhood include drowning and house fires (150, 171, 218,
219, 234, 243, 327). Contrary to “information” portrayed on television and in the movies, no definitive
postmortem test or finding indicates a drowning death. Drowning is determined by exclusion, based on scene
circumstances and history and the lack of an overt cause of death at autopsy. Drowning is one of the leading
causes of unintentional injury and death in children in the United States; although the circumstances are different
among the various age groups, toddlers and adolescents are at high risk (29). Young infants are at risk for
drowning during bath time if left unattended, either alone or with an older sibling (171, 243). More than 50% of
infant drowning deaths occur in bathtubs. One study found that infants placed in tub rings may be at a greater
risk for drowning because of the caregiver's false sense of security and resultant increased likelihood that an
infant will be left alone. As an infant grows and begins to interact more independently with the environment, new
risks emerge. It is among toddlers that various household hazards such as 5-gallon buckets contribute to
drowning deaths (150, 198). Toddlers are also the age group in which swimming pools become a major
circumstance in drowning deaths. During adolescence, boys are at a higher risk for unintentional drowning than
girls. These drowning deaths often occur in natural bodies of fresh water (29). Thus, as in other categories of
accidental death, prevention measures must be age specific. Caregivers of infants should be reminded of the
dangers of leaving an infant unattended in the tub, “even for a minute.” The dangers associated with tub rings
should also be discussed. For caregivers of toddlers, safeguarding against “attractive nuisances” such as toilets,
buckets, and pools should be emphasized. Adolescents benefit from campaigns alerting the public to common
drowning scenarios. Further public awareness may help to reduce fatalities from these preventable accidents.
Like drowning, accidental strangulation characteristically involves distinct age groups of children. Preschool
children may be strangled when they become entangled in common household items from which they are unable
to extricate themselves. In part because of the actions of the Consumer Product Safety Commission and other
organizations, accidental strangulations in structurally unsafe cribs are becoming less common (98). Some
hazards are rather well-known, such as pacifier strings and cords on Venetian blinds, but others are less well-
known, such as cords to electric devices in a toddler's room (Figure 7-12). Adolescents may succumb to ligature
hanging, either as a suicide or as an accident in association with autoerotic asphyxia (18, 262). Victims of
strangulation generally die of an interruption of blood flow to the brain rather than actual occlusion of the airway.
Significantly less pressure is required to occlude the blood vessels than the airway (79). With the exception of
judicial executions, injury to the cervical spine is very uncommon. Often, the physical findings in deaths from
ligature hanging are few, especially if the victim was extricated from the asphyxiating device within a short period
of time. When physical signs are present, they are generally the consequence of congestion distal to the ligature
site. Such findings may include cephalic congestion, tongue protrusion with discoloration, and petechial
hemorrhage. If the victim has been suspended for a postmortem interval measured in minutes to hours at a
minimum, a ligature furrow may be seen about the neck. This furrow will have a yellow-brown, waxy base and
display a suspension point based on the relative positions of the decedent and the ligature.
House fire deaths remain a significant source of pediatric morbidity and mortality. In fact, in one study of fatal
residential fires, children less than 5 years of age were classified as one of several “high vulnerability” groups
(327). Other highly vulnerable groups included persons aged 64 years
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or older, those with a physical or cognitive disability, and persons impaired by alcohol or other drugs (202). In
most residential fire deaths, the actual cause of death is smoke inhalation. In such cases, the thermal injury to
the body may be described as perimortem. Various fire-related artifacts may be mistaken for trauma by the
uninitiated. Such artifacts include epidural heat hematomas, skin splitting, and heat fractures and disarticulations
(83) (Figures 7-13 and 7-14). In most instances, even when a massive conflagration of the body has occurred,
the various internal organ systems remain well preserved and sufficient blood remains for toxicologic study. Fire
victims may demonstrate carboxyhemoglobin saturations less than the values encountered in pure carbon
monoxide intoxications, but they are still high enough to allow the conclusion to be drawn that death was caused
by smoke inhalation. A carboxyhemoglobin saturation above 10% is highly suggestive of life at the beginning of
the fire. Gross findings in victims of smoke inhalation include black carbonaceous debris adherent to the
respiratory mucosal surfaces and cherry red discoloration of the visceral surfaces, reflecting the
carboxyhemoglobin saturation (Figure 7-15). From various published studies, it appears that prevention efforts
regarding smoke detectors and egress routes should be directed at families of low socioeconomic status with
children less than 5 years of age, as these families represent a high-risk group (202, 234, 327).
FIGURE 7-12▪Accidental ligature asphyxia. A toddler was found hanging by his neck from an electric cord. The
cord connected to a clock radio on the shelf above.
FIGURE 7-13▪Epidural heat hematoma along the inner table of the skull, a fire-related artifact that should not be
confused with antemortem trauma.
FIGURE 7-14▪Fire-related artifacts, including fractures and disarticulations, in a child who died of smoke
inhalation in a house fire.
FIGURE 7-15▪Smoke inhalation. Black carbonaceous material adherent to the laryngeal mucosa.
ASPHYXIA
Asphyxia may lead to death in young children in a variety of circumstances and may represent either an accident
or a homicide. As discussed in the previous section, infants are particularly vulnerable to hazards in their
sleeping quarters that can cause asphyxia (e.g., wedging, which obstructs the nose and mouth or compresses
the chest; strangulation, which occludes the great vessels of the neck) (38, 42, 60, 220, 275). Infants placed on
adult beds may slip between the headboard and the mattress, or between the mattress and the adjacent wall
(Figure 7-16). Infants placed in cribs or bassinets with ill-fitting mattresses may become wedged in a similar
fashion. Infants sleeping together with others may be killed by overlaying (16, 60, 65). Strangulation deaths may
occur when a young child is left unattended in a day cradle, car seat, or swing and becomes entangled in the
safety straps or some other portion of the device (2) (Figure 7-17). Another hazard in sleeping quarters may be
soft bedding and a prone position of the infant, which leads to asphyxia from rebreathing (47, 60, 113, 140, 215,
223, 246, 263, 295, 316, 326). A common feature to all the above causes of death is the fact that they often
leave little, if any, physical evidence of trauma on the body. Strangulation and chest compression may leave
scattered cutaneous petechiae distal to the site of occlusion/compression, but oronasal occlusion by soft
substances and rebreathing may cause death without any evidence of injury. Without proper scene
documentation and gathering of historical information, these cases may be erroneously ascribed to SIDS.
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FIGURE 7-16▪Gingival abrasion in a 1-month-old child who became entrapped between the headboard and
mattress of a standard adult bed.
Even more difficult to detect are cases of homicidal suffocation of an infant or a young child. Even with a careful
scene examination, complete autopsy, and case history review, these cases may be erroneously ascribed to
SIDS. Multiple cases of serial infanticides committed by parents or caregivers over a period of years before
detection are now known (92, 100). Many of these cases were initially erroneously ascribed to SIDS, and they
illustrate the critical importance of strictly adhering to the definition of SIDS and obtaining a complete family
history when investigating an apparent case of SIDS. In the absence of an identifiable metabolic or genetic
defect, some forensic pathologists feel that that a second apparent “SIDS” case within a family should be
classified as “undetermined,” and that a third case should be classified as a homicide. The same thinking is
relevant in alleged cases of “simultaneous SIDS” in twins. Although cases have been reported in the medical
literature, many physicians are skeptical and feel that they represent undetected homicidal or accidental deaths
(17, 276). In the experience of one of the authors (T.S.C.), twins intentionally suffocated on the same night
presented as simultaneous SIDS. One of the babies displayed no evidence of injury whatsoever, and the other
baby displayed small, faint periorbital superficial abrasions about one eye (Figure 7-18). Although the parents
denied use of the apnea monitors present in the home on the night of the infants' demise, the monitors were
collected by the police. Downloading of the monitors provided documentation of the entire double homicide.
When confronted with this evidence, the parents confessed. This case clearly illustrates that it is entirely
possible to suffocate an infant intentionally and leave no evidence of injury whatsoever. Although some feel that
intentional suffocation may account for up to 10% of cases classified as SIDS, most forensic pathologists believe
that homicidal suffocation accounts for only a very small percentage of SIDS cases (80, 94, 233).
FIGURE 7-17▪Strangulation of a 2-monfh-old infant left unattended and unsecured in an infant swing.
FIGURE 7-18▪Homicidal suffocation in a 6-week-old fraternal twin presenting as simultaneous sudden infant
death syndrome. This baby had a small, superficial abrasion on the right upper lid. The other infant displayed no
evidence of trauma.
CHARACTERISTICS OF VICTIMS AND PERPETRATORS OF PHYSICAL ABUSE

Despite the indisputable fact that “violence to children has always existed and is one of the most intractable
aspects of human behavior,” child abuse as a medical diagnosis is a relatively new phenomenon (26). Most of
the data regarding physical abuse have been collected within the last three decades, since the publication of the
landmark articles “Slaughter of the Innocents” in 1961 and “The Battered Child Syndrome” in 1962 (5, 156). First
and foremost, fatal child abuse is an intimate crime—the child is injured by the person or persons to whom
society has entrusted it for care and nurturing. In one study, Starling et al. found that in cases of abusive head
trauma, the perpetrator was most often the biologic father; the mother's boyfriend (unrelated to the child) was the
second most common abuser (287). This same
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prevalence of biologic fathers as perpetrators was found in a study by one of the authors (T.S.C.) of fatally
abused infants, in which the biologic father was identified as the perpetrator in 50% of cases undergoing autopsy
during an 11-year period (65). Other researchers have reported that stepparents are statistically more likely to
abuse children in their care fatally than are biologic or adoptive parents (301). Child abuse crosses all racial and
socioeconomic lines, but common factors in most abusive households include an acceptance of physical
punishment, social isolation, and stress (291). Most victims of fatal abuse are under 2 years of age, and
homicide is the leading cause of traumatic infant death in the United States. Identified risk factors include
childbearing at an early age, second or subsequent infants born to mothers less than 17 years of age, and lack
of prenatal care (241). Common triggering mechanisms include crying, feeding difficulties, and toilet-training
accidents (320). About 50% of fatally abused children display evidence of previous abuse at autopsy; this
evidence may take the form of healing soft tissue injuries or skeletal injuries. In the families of living victims of
maltreatment, maltreatment has been found to recur in more than 50% of families followed for more than 5 years
(72).
Common presenting histories in physical abuse cases involve an account of a minor household fall, such as a
tumble down the stairs or off a bed or couch (161). Other common histories in fatal abuse cases include sudden
respiratory arrest, sudden onset of seizure activity with no previous history of seizure disorder, and simply finding
the baby dead. These last histories are probably partial truths; the caregiver is simply omitting the assaultive act
that precipitated the onset of symptoms described. Inflicted head injury is by far the most common cause of death
in fatal abuse, especially in infants. Abdominal injury is the second most common fatal abusive injury
encountered and is seen more often in toddlers than in infants.
CUTANEOUS EVIDENCE OF PHYSICAL ABUSE

First, it must be understood that a fatally abused child may display no external evidence of injury. The lack of
cutaneous injuries such as abrasions and contusions does not eliminate homicide. Often, children with no
external evidence of injury have massive internal injuries, including fatal head injuries and multiple skeletal
fractures. Conversely, external evidence of contusion or abrasion does not necessarily indicate an abusive
death. Adequate documentation and strict adherence to proper use of terminology in forensic reports is
important.
When the external injuries of a suspected victim of physical abuse are examined, it is important to keep several
points in mind:
Evaluate the distribution of injury.
Evaluate the pattern of injury.
Evaluate the severity of injury.
Compare the injury with the history provided.
FIGURE 7-19▪Toddler “wear-and-tear” injuries over the lower extremities of an active, healthy preschooler.
Evaluating Blunt Trauma

When the distribution of blunt force injuries is evaluated, it is important to recognize common sites of accidental
injuries and the age groups in which these occur. Very young infants are relatively immobile and so do not
commonly have contusions. Unexplained facial contusions in this age group may indicate physical abuse (224).
As babies become more mobile, the likelihood of incurring accidental injury increases. Common sites of
accidental injuries in infants learning to “pull up” and walk include the bony convexities of the anterior head, such
as the forehead and the skin overlying the zygoma. Active, healthy toddlers engaging in play and exploration will
incur a variety of (usually) superficial injuries. These normal toddler “wearand-tear” injuries are distributed in a
characteristic fashion. In toddlers and older children, accidental injuries are commonly encountered over the
elbows, knees, shins, and forearms (308) (Figure 7-19). Isolated injuries in recessed or “protected” areas raise
the index of suspicion of abuse. Accidental injuries may occur in these regions, but usually a correlating history
can be elicited. Examples of recessed or protected areas that may be injured are the philtrum, submental space,
midline of the abdomen, and low back and buttocks (Figures 7-20 and 7-21).
FIGURE 7-20▪Contusions of a recessed area: the submental space in a 17-monfh-old victim of child abuse.
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FIGURE 7-21▪Abdominal contusions in a 5-month-old victim of child abuse.
A pattern injury may be defined as an injury that mirrors at least a portion of the object that caused it, or an injury
that is characteristic of a certain scenario. In cases of physical abuse of a child, most pattern injuries are created
with either the hands of the perpetrator or common household items. The pattern varies depending on the
velocity with which the object strikes the skin. In high-velocity events, such as whippings and slaps, the pattern
often is a linear array of petechiae outlining the dimensions of the object, with a central, unbruised “negative
image” of the object. In such cases, the tissues along the edge have been maximally distorted, with subsequent
rupture of the capillaries in that region. With increasing force, the tissue immediately beneath the impact site is
crushed and also displays bruising. If great forces are applied more slowly, the tissue at the margins of the
impact site may conform and stretch without damage. In this scenario, the force ruptures the vessels directly
impacted and leaves a “positive image” contusion at the site (99). When a strange or an unusual pattern is
encountered, one is encouraged to think about common household items with similar dimensions and shapes. It
is advisable to discuss the pattern and the items that possibly caused it with the investigators, so that the scene
of injury can be examined for such items. A single object may leave many different patterns, depending on which
of its many surfaces impacts the skin. Items often used as “weapons” in physical abuse include belts, electric
cords, coat hangers, and curling irons (Figure 7-22). One may also encounter objects such as brooms, fly
swatters, and kitchen utensils (Figures 7-23 and 7-24). Flexible objects such as belts or cords leave patterns that
vary in length and arc, whereas rigid objects such as broomsticks leave relatively uniform patterns (Figure 7-25).
However, the most common “instrument of injury” remains the human hand, with which the child may be slapped,
punched, pinched, shaken, slammed, or thrown (Figure 7-26).
FIGURE 7-22▪Pattern injuries inflicted with a belt.

FIGURE 7-23▪Multiple diamond-shaped pattern injuries on the leg of a 3-year-old child inflicted by a fly swatter
(see Figure 7-24).
Some patterns are characteristic of a particular method of injury. For example, a “brush-burn abrasion” occurs
when a pedestrian is struck and has tangential contact with
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the pavement (Figure 7-27). A vertical gluteal cleft injury occurs when a child is beaten over the buttocks; the
convex surface flattens, and the regions immediately lateral to the vertical gluteal cleft, which are the interface
between impacted and nonimpacted tissue, are subjected to shearing injury. The resulting pattern consists of
vertically oriented, parallel linear contusions located on either side of the midline (Figure 7-28). A rim of
petechiae may develop along the apex of the ear following direct blunt impact for the same reason. In these two
examples, the pattern is dictated by the shape of the body and the anatomic lines of stress rather than by the
shape of the object (99). When an injury over a joint is examined, it is helpful to move the joint into various
positions. An injury viewed as irregular in the anatomic position may emerge as a pattern injury as the extremity
is flexed or rotated (Figure 7-29).
FIGURE 7-24▪Portion of the fly swatter corresponding to the pattern injuries depicted in Figure 7-23.
FIGURE 7-25▪Pattern injuries inflicted by beating with a broomstick.
When an external examination is conducted, all cutaneous and mucocutaneous surfaces should be inspected.
Specific areas that may be overlooked include the skin surface in and behind the ear (Figure 7-30) and the
axillae, intraoral mucosa, palpebral conjunctivae, buttocks, external genitalia, and anus (Figure 7-31).
FIGURE 7-26▪Open-handed slap mark inflicted by a male adult.

FIGURE 7-27▪Brush-burn abrasion. This injury is commonly seen in pedestrians struck by motor vehicles or
occupants ejected during motor vehicle collisions.
The severity of the injury must be compared with the historical information. Often in cases of abusive injury and
death, a history of a minor household accident, such as a fall from a bed, is given as an explanation (24, 135).
Other common histories include a sudden onset of seizures, choking, or simply discovering the baby dead. A
history that does not agree with the physical findings is a hallmark of child abuse (148). The developmental skills
of the child should be compared with the history to see if the alleged scenario is plausible. Therefore, the
examiner should have at least a rough understanding of the basic developmental milestones, such as rolling
over, crawling, and “cruising” (walking along a piece of furniture while using the hands to maintain balance and
upright position).
FIGURE 7-28▪Vertical gluteal cleft contusions and multiple additional contusions of varying colors and shapes.
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FIGURE 7-29▪AB: Thermal injury over a joint. The injury pattern initially appears irregular, but when the elbow is
flexed, one sees a patterned burn consistent with a curling iron (B). (Courtesy of William Smock, M.D.,
Department of Emergency Medicine, University of Louisville, Kentucky.)
Dating of Contusions
Many texts display charts and illustrations detailing the method of dating contusions by color. However, dating
contusions by color is imprecise. Color may provide a rough estimate of age, but this should not be “set in
stone.” Many factors may affect the color of a contusion on the skin surface. These include the following:
Depth of the contusion within the soft tissue
Location on the body
Amount of bleeding within the tissue
Environmental lighting
Overlying skin color of the patient
Although many texts detail an age range based on color, the descriptions often vary from one text to the next
(318). Studies have shown that contusions do not progress through a predictable color change based on time
(266, 289). In fact, it has been shown that the color of bruises in one person at the same location, with the same
cause, and of the same age may not change color at the same rate. It appears that the most one can say about
the age of a contusion based on color is that a yellow coloration indicates that the bruise is at least 18 hours old
(177). In deceased persons, samples of cutaneous contusions may be excised for microscopic examination,
which allows a more precise estimation of the age of the injury; however, the dating of the injury remains general.
Studies in sheep, calves, and guinea pigs have illustrated that microscopy aids in differentiating acute
contusions from those more than 24 hours old (177, 204, 252). Thornton and Jolly examined 178 experimental
bruises inflicted on sheep and aged from 1 to 72 hours; they found that the model was able to age bruises with
an acceptable degree of accuracy only as 1 to 20 hours old or 24 to 72 hours old (298). In general, perivascular
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polymorphonuclear leukocytes may be visible around 4 hours after injury, with a peripheral infiltration by around
12 hours. Macrophages peak around 16 to 24 hours and may contain hemosiderin by 72 hours. Fibroblasts may
appear at 2 to 4 days (244). Unfortunately, even with the use of microscopy, “pathologic processes seldom
cooperate fully with attempts to date or age them with precise reliability” (136).
FIGURE 7-30▪Inflicted injury behind the ear.

FIGURE 7-31▪Anal lacerations in a victim of fatal child abuse. Extensive associated subcutaneous hemorrhage
was present.
Scald Burns and Contact Burns

Among tap water scald burn victims, 46% are under 5 years of age (8, 69, 84, 214, 236). When examining a
scald burn victim, the pathologist should obtain and document a detailed, specific history of the scalding incident.
As in the examination of contusions, observation, documentation, and correlation of the distribution of the injury
are paramount (8, 69, 84, 214, 236). It is insufficient simply to estimate the total body surface area involved in the
burn.
In incidents allegedly involving scalds from household tap water, scene investigation must be undertaken.
Specifically, the hot water heater should be examined to document the temperature setting. The actual
temperature of the hot water at the tap in question should be measured and recorded over time. Depending on
the alleged history, it may be necessary to measure the tap water temperature with the hot and cold water
running simultaneously, and also the temperature of the standing water in the tub. It may be helpful to utilize an
immersion burn scene investigation worksheet. It is not necessary to purchase expensive medical equipment for
these measurements—a candy thermometer from a retail store will suffice in most instances. When the data are
presented to nonmedical persons, it is recommended that they be expressed in degrees Fahrenheit, as this will
allow a more meaningful interpretation.
In general, accidental scald burns are seen in toddlers— children who are somewhat mobile and thus able to
interact independently with their environment (8, 69, 84, 214, 236, 327). Accidental scalds often involve the
upper extremities and anterior surface of the head, neck, or upper chest. When a scald has been sustained by
pulling a pan of liquid from the stove, the pattern may involve the axilla or submental space in addition to the
face, neck, and upper chest (154). Accidental burns are often asymmetric in distribution and of varying severity
and depth. At times, “flow patterns” may be observed, with the burn lessening in severity as the pattern
progresses inferiorly, or with gravity. Overlying clothing, which holds the hot liquid next to the skin surface, may
alter this pattern (Figure 7-32). In cases of accidental burns, small satellite “splash burns” are often apparent
(Figure 7-33).
FIGURE 7-32▪Scald burns altered by clothing. The patterns of this child's socks and sweat pants are clearly
visible.
FIGURE 7-33▪Satellite splash burns on the medial aspect of the right ankle in an asymmetric scald burn.
In contrast, victims of inflicted burns are generally younger, most being less than 2 years old (8, 69, 84, 236, 258,
327). Inflicted burns are often symmetric and may be characterized by distinct immersion lines without evidence
of splash burns. A glove or stocking distribution is a frequent finding. If a small child has been dipped into hot
liquid, the skin folds of the popliteal fossae and inguinal regions will usually be spared, but the soles of the feet
will not. These areas of sparing occur in regions of skin-to-skin contact, where hot liquid cannot penetrate
(Figure 7-34). The depth of inflicted burns is usually relatively uniform. Occasionally, one may observe a
“doughnut ring” area of sparing over the midportion of the
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buttocks. This is created when that portion of the child's skin surface is in contact with the relatively cooler tub or
basin surface and thus not directly exposed to the hot liquid.
FIGURE 7-34▪Immersion pattern.
The American Academy of Pediatrics recommends a “safe setting” of hot water heaters at 125°F or less. At
125°F, contact with water for 2 minutes is required to produce a fullthickness burn. At 130°F and higher, full-
thickness burns can result with exposure times of 30 seconds or less (74, 225). Of utmost importance in the
investigation of scalds is the correlation of the history with the distribution of the scald burn. Simply put, “Does
the injury pattern fit the history given?”
Contact burns are rarely fatal. Abusive contact burns are usually caused by common household appliances.
Examples include clothing irons, curling irons, hair dryers, and cigarettes (249). Abusive contact burns are often
uniform in depth in all directions. The shape of the burn may delineate the causative object. In accidental contact
burns, the pattern is more irregular and does not mirror the object as faithfully. The burn is uneven, usually more
severe on one side than the other (188). Although rare, abusive microwave burns have been reported (6).
Microwave burns differ from scald or contact burns in that they produce an uneven burn pattern through the
layers of tissue. Tissue with a high water content, such as muscle, heats to a greater degree than tissue with a
relatively low water content, such as subcutaneous fat (255).
HEAD INJURIES
When one attempts to evaluate a head injury in a suspected victim of physical abuse, it may be helpful to refer to
a paradigm such as that described by Hymel et al. (141). In this paradigm, injuries are classified as primary or
secondary, and focal or diffuse. Cranial injuries are divided into three groups—contact injuries, acceleration
injuries, and injuries resulting from hypoxia-ischemia. When a head-injured child is evaluated, the specific cranial
injuries are classified, and then the required causal mechanism for each is defined. Finally, the biomechanical
circumstances required to produce the injury are compared with the history given. Using such a paradigm allows
one to analyze an injury in a systematic, logical, and reproducible way.
Falls
Often, an initial history of a fall is given to account for a young child's head injury. Review of the literature on
witnessed, corroborated falls reveals that children generally tolerate such forces well—better than adults, in fact!
This has been explained by factors unique to children, such as a smaller mass, which reduces the deceleration
force on impact, and a higher proportion of cartilage and subcutaneous fat (307). Several authors have
documented series of children sustaining minor household falls. In 1977, Heifer et al. reviewed a series of 246
children with a history of falling out of bed; 85 of the children were hospitalized at the time of their fall (135). No
child in the study sustained central nervous system damage. The benign nature of falling out of bed was
confirmed by two additional studies of falls in hospitals, one involving 76 children and another involving 207
children falling from beds, cribs, or chairs (196, 231). No serious injuries occurred in either study. Stairway falls
have also been examined and characterized as an initial “moderate impact” fall, followed by a series of minor
impacts. Joffe and Ludwig documented 363 cases of falls down stairs seen in a pediatric emergency department
(145). The majority of the children had only superficial injuries, and no child sustained life-threatening injuries or
required intensive care (145).
Several series of witnessed, corroborated free falls in children have also been published. Barlow and colleagues
examined 61 children during a 10-year period who were admitted to the hospital after falling from a height of one
or more stories (13). Of the children who fell three stories or less, 100% survived. Mortality in those falling from
the fifth and sixth floors was 50%. In one study of 106 witnessed, corroborated free falls in children less than 3
years old, only one death occurred—in a child who fell from 60 ft. The author concluded that falls of less than 10
ft are unlikely to produce serious or life-threatening injury (313). In yet another series, 70 children with a mean
age of 5 years fell from heights ranging from one to 17 stories, and all survived (227). A study of fatal head injury
with a history of a fall revealed only three fatalities from witnessed falls—all from heights greater than 10 ft and
none with evidence of retinal hemorrhage or axonal injury. And yet in this same study, 19 fatalities occurred in
children whose initial history was of a fall of 5 to 6 ft or less; investigation revealed that most of these cases were
actually inflicted trauma with an initially false history (256). A study of 317 children brought to a children's trauma
center with a history of a fall revealed only one death in 117 children falling from 10 to 45 ft, and seven deaths in
children allegedly falling 4 ft or less. In all seven of the fatalities after a short fall, other factors suggested a false
history (51). Compiling the multiple available studies, Chadwick concludes, “Death from a fall is now considered
very unlikely when the fall is less than 20 feet” (52).
Inflicted Head Injuries

Inflicted head injury is the most common cause of traumatic death in infancy (24, 86). Since Caffey first coined
the term “whiplash shaken infant syndrome” in 1974, the medical community has used a variety of terms or
phrases to describe the classic injury pattern. Terms include shaken baby syndrome, shaken/slammed baby
syndrome, shaken impact syndrome, abusive head trauma, inflicted cerebral trauma, and inflicted closed head
injury (43, 49, 86, 144). All these terms describe a constellation of injuries seen with regularity in infants who
have been physically abused; these include diffuse brain injury with altered consciousness, subdural and
subarachnoid hemorrhages over the cerebral convexities, retinal hemorrhages, and scalp contusions. Many
authors also include metaphyseal avulsions in this constellation, as Caffey did (43). It has been debated over the
years whether
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violent shaking alone is sufficient to cause diffuse, severe brain injury, or whether impact must occur (78, 86,
175). Many would now agree that pure shaking injury deaths do occur from time to time. Shaking as a
mechanism of traumatic death has even been documented in an adult (247). However, in a far more common
scenario, evidence of blunt impact to the head is also present (6, 182). Many times, evidence of impact is not
visible externally and is visualized only after reflection of the scalp at autopsy (Figure 7-35). Even at autopsy, the
absence of evidence of cranial blunt trauma does not eliminate the possibility of an impact (283). If the impact
occurs on a soft surface, such as a mattress, then the surface area of the impact could be large enough not to
produce a scalp contusion. The use of terms such as shaken baby syndrome without adding an explanation that
an impact cannot be excluded imparts incorrect information to investigators, who may not consider the possibility
of an impact when examining the scene of injury and interviewing witnesses.
FIGURE 7-35 AB ▪ No external injury could be seen in this 2-month-old victim of fatal abusive head trauma (A).
However, evidence of blunt trauma was identified on reflection of the scalp (B).
Infants and young children have unique characteristics that come into play in central nervous system trauma.
The skull is pliable and unilaminar, with unfused sutures, open fontanelles, and a flat, shallow base. The brain
constitutes a significantly larger percentage of the total body weight in children than in adults (10% to 15% in
children versus 2% to 3% in adults). And this large, heavy head rests on a relatively weak neck. The infant's
brain is less myelinated, has smaller axons, and has a higher water content (49).
Primate studies have shown that rotational acceleration of the head, with the low cervical region as the center of
rotation, causes acute subdural hematomas (110). When the head is subjected to rotational acceleration, diffuse
subdural hemorrhages may be produced over the convexities as a consequence of stretching and tearing of the
bridging veins. These bridging veins travel from the brain surface to the dura. Subdural hemorrhages may be
confined to the parafalcine area or may layer out over the convexities. Rarely are they space-occupying lesions
(Figure 7-36). Rather, subdural hemorrhage is important as a marker of rotational acceleration of the head (141).
Angular or rotational acceleration is poorly tolerated by the central nervous system, and shearing strains cause
primary diffuse brain injury (86, 141, 283). Head acceleration with diffuse brain injury results in widespread brain
dysfunction, which may range from concussion to traumatic coma with or without permanent neurologic sequelae
to sudden death. The unifying feature across the spectrum from concussion to sudden death is the immediate
loss of consciousness (110, 237, 317). In concussion, no pathology is visible; as the severity of the injury
increases, pathologic changes become more apparent. These include subdural and subarachnoid hemorrhage
as markers of the rotational forces that have occurred. Parenchymal pathologic changes may include contusional
tears (slitlike tears at gray-white interfaces) and evidence of diffuse axonal injury, seen as actual axonal
disruption (44) (Figure 7-37). In older children and
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adults, punctate hemorrhages throughout the white matter may accompany axonal disruption.
FIGURE 7-36▪Acute subdural hemorrhages over the cerebral convexities in a victim of fatal abusive head
trauma. These subdural hemorrhages are very thin and are not space-occupying lesions.
FIGURE 7-37▪Schematic representation of abusive head trauma. A: The various structures are defined. B: The
central vein in the sagittal midline. C: Small bridging veins traversing the subdural space. D: Rotation of the
central nervous system in angular acceleration. E: Parafalcine subdural hemorrhage. F: Subdural hemorrhage
over the convexities.
Because of the unique features of the infant brain, it has been difficult to demonstrate actual axonal disruption in
victims of inflicted closed head injury, especially if the infant dies quickly. Even in adults, with good myelination,
larger axons, and a higher fat content, histologic evidence of actual axon disruption in the form of retraction balls
may
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not be apparent without a postinjury survival of at least several hours (4, 50) (Figures 7-38 and 7-39). After
several weeks, light microscopy reveals microglial nodules. Eventually, wallerian degeneration results in a loss of
white matter. The areas most affected include the corpus callosum, fornix, corona radiata, and rostrolateral
quadrants of the brain stem. Beta-amyloid precursor protein (b-APP) has been used to detect diffuse axonal
injury in the early postinjury period. b-APP accumulates in the axon at or near the site of injury. Some
researchers have found b-APP useful to demonstrate diffuse axonal injury in infants with inflicted head injury.
Limitations to this method include the requirement for a postinjury survival of about 2 hours to allow the protein to
accumulate, and for adequate cerebral vascular perfusion during this time (116). Additional limitations for
coroner/medical examiner offices may include the cost and difficulty of routinely performing immunohistochemical
studies.
FIGURE 7-37▪(continued) G: Depiction of the axon system. H: Rotation of the central nervous system with
shearing. I: Resultant diffuse axonal injury. J: Eventual appearance of axon spheroids. (Courtesy of Dan Davis,
M.D., Hennepin County Medical Examiner's Office, Minneapolis, Minnesota.)
“Tin ear syndrome” is a term used to denote a subset of rotational acceleration. It is a triad of unilateral ear
bruising, ipsilateral cerebral edema, and hemorrhagic retinopathy. Each patient in the initial series was a toddler
with thin subdural hemorrhages over the convexities (131).
A history of prehospitalization apnea is common in victims of abusive head trauma and is often the first symptom
reported to emergency services. Apnea arising from angular acceleration may contribute to morbidity and
mortality in these victims through the deleterious effects of ischemia and hypoxia (146). The true morbidity of
abusive head trauma is not known because many children with survivable injuries are misdiagnosed or simply
never present for medical evaluation. Infants with sublethal inflicted head injury may present for medical
evaluation with nonspecific symptoms, including vomiting, fever, irritability, and lethargy. However, these are
common symptoms in a variety of conditions. Because a forthcoming history of trauma is usually absent, the
infant's traumatic injury may be misdiagnosed as a natural disease process, and the misdiagnosis may lead to
further abusive injury and death (144, 279). Some studies have
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shown a “recovery rate” of victims of abusive head trauma of between 20% and 50%, but in one long-term study
of victims of “whiplash shaken infant syndrome,” many infants with what initially appeared to be a “full recovery”
suffered medical, behavioral, and neuropsychological damage 2 to 6 years after the abusive event (27).
FIGURE 7-38▪An axon spheroid (also known as a retraction ball) appearing as an eosinophilic globule.
(Hematoxylin and eosin x400; courtesy of Mitch Morey, M.D., Hennepin County Medical Examiner's Office,
Minneapolis, Minnesota.)
Contact Injuries
Head injuries resulting from direct blunt trauma include skull fractures, epidural hematomas, and, in severe
cases, crush injuries. A focal area of subdural or subarachnoid hemorrhage may be localized under a contact
injury, such as an epidural hematoma. In this case, the focal subdural hemorrhage represents a contact injury. In
contrast, the classic findings in abusive head trauma (parafalcine subdural hemorrhage or thin bilateral subdural
hemorrhages over the convexities) are produced by rotational forces and thus represent diffuse injury. In adults,
epidural hematomas are usually found in association with a fracture of the ipsilateral temporal bone; the epidural
hemorrhage arises from a tear in the middle meningeal artery immediately deep to this bone. In children, because
of the pliant nature of the skull, the temporal bone may not actually be fractured. The bone may be able to bend
in enough to prevent fracture, but the underlying artery may still be lacerated. Epidural hematomas result from
brief, linear contact forces. These injuries can be caused by unintentional falls, especially onto pointed or
protruding surfaces (273). Isolated epidural hemorrhages are not associated with an immediate loss of
consciousness—a finding that makes sense because epidural hemorrhages do not represent primary diffuse
brain injuries. Consciousness may be lost some time after the impact as the epidural hemorrhage becomes a
space-occupying lesion that produces a mass effect.
FIGURE 7-39▪An axon spheroid visualized with a silver stain. (Silver stain, x250; courtesy of Mitch Moray, M.D.,
Hennepin County Medical Examiner's Office, Minneapolis, Minnesota.)
Crushing head injury may be associated with incidents in the home environment, such as driveway runovers and
toppled heavy objects. Such injuries are caused predominantly by static forces rather than the dynamic forces
seen in rotational injuries. In one study of seven cases, including four cases in which the heads of children were
run over by vehicles (usually on concrete or asphalt), only one fatality occurred. All the surviving children made a
good cognitive recovery (88). In contrast, children with inflicted head injuries often have a poor neurologic
outcome (118). Crushing head injuries consist of multiple fractures with deformity of the cranium and cutaneous
pattern injuries that can be correlated with the impacting object (Figure 7-40).
FIGURE 7-40▪Crushing head injury. Pattern injury corresponds to impacting object.
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Retinal Hemorrhage
Retinal hemorrhages have been associated with abusive head trauma since Caffey included them in the
constellation of injuries of the whiplash shaken infant syndrome (43). Although not always present in cases of
abusive head trauma, retinal hemorrhages are disproportionately represented in inflicted head trauma, as
opposed to head trauma caused by other mechanisms of force (50, 88). The mechanism by which retinal
hemorrhages are produced in pediatric inflicted head injury is not fully understood. Proposed mechanisms
include vitreous traction on the retina during angular acceleration and elevated intracranial pressure (36, 114).
Studies indicate that retinal hemorrhages are found in from 50% to 100% of children with abusive head trauma
(24, 36).
Other causes of retinal hemorrhages include sepsis with coagulopathy and vaginal delivery in the newborn.
Retinal hemorrhages have been reported in up to 20% of newborns, especially those with primiparous mothers
or delivered vaginally. Most of the hemorrhages dissipate by the end of the first week of life. Virtually all resolve
without incident by the end of the first month of life (269, 271).
Although retinal hemorrhages may be found in a small percentage of children with accidental injuries, the injuries
have generally been caused by extraordinary force, as in motor vehicle collisions (147). Although many articles
do not describe in detail the pattern of retinal hemorrhages seen in accident victims, it is often different from that
seen in shaken impact syndrome. In cases of accidental trauma, the hemorrhages may be less numerous and
less severe than in abusive head trauma. In shaken impact syndrome, the hemorrhages are often diffuse,
massive, and bilateral. Hemorrhage into the vitreous and traumatic retinoschisis may be seen in inflicted head
trauma (120). Therefore, the often-heard statement that “retinal hemorrhages are nonspecific” is technically
correct but no more meaningful than a statement such as “fractures are nonspecific.” When retinal hemorrhages
are characterized according to type, location, degree, and pattern of associated injuries, the specificity
increases. Cardiopulmonary resuscitation has been offered as an explanation for retinal hemorrhages, but this
hypothesis is not supported in several studies (114, 147).
When the globes of a shaken impact syndrome victim are harvested at autopsy, various changes may be noted.
Grossly, hemorrhage along the optic nerve sheath may be seen. It is relatively standard practice to prepare a
pupillary-optic nerve section for microscopic examination. However, it should be remembered that this represents
only a small surface area of the retina and that the overall pattern of retinal hemorrhage cannot be appreciated in
such a section. Microscopically, hemorrhages frequently are seen in the nerve fiber and ganglion cell layers of
the retina (226, 254). Purtscher retinopathy is another distinct pattern of retinal hemorrhage associated with a
distinct traumatic etiology. Purtscher retinopathy, which is seen following traumatic chest compression asphyxia,
is characterized by large white patches on the retina in the macular and peripupillary areas (24, 36). It has been
reported in association with battered child syndrome (300).
SKELETAL EVIDENCE OF PHYSICAL ABUSE

As stated earlier, a skeletal survey should be conducted on victims of suspected physical abuse who are under
the age of 2 years (212). In living children, it is recommended that a skeletal survey be repeated 2 weeks later,
or, if this is unlikely to occur, a bone scan may be performed on the same day as the initial skeletal survey.
Obviously, these are not options in deceased children. When abnormalities are suspected on skeletal survey,
the bony area in question may be excised. Excision makes it possible to document associated soft tissue injuries,
obtain more detailed radiographic images, and perform a direct visual and histologic examination. It is wise to
review radiographs with a pediatric radiologist, who will recognize subtle but specific signs of inflicted injury.
Kleinman has grouped skeletal injuries according to their relative specificity for abuse (162). Those with a high
specificity for abuse, particularly in infants, include classic metaphyseal lesions, posterior rib fractures, scapular
fractures, spinous process fractures, and sternal fractures.
Skull fractures in infancy may be associated with falls and also with inflicted blunt trauma. Most accidental skull
fractures sustained in minor household falls are single, linear, nondisplaced fractures of unilateral parietal bones,
with no associated underlying intracranial injury (137, 270). Fractures that cross suture lines, multiple fractures,
and bilateral fractures are associated with inflicted injury more than with minor accidents (213). Kleinman
describes linear fractures as fractures with a low specificity for abuse, whereas complex skull fractures are
regarded as moderately specific (162).
Classic metaphyseal lesions are distinctive injuries of infancy and are highly specific for inflicted injury—perhaps
more so than any other skeletal or visceral abnormality. The lesion consists of a planar disruption through the
primary spongiosa. Hemorrhage is usually inconspicuous. The distal femur and proximal tibia are the most
common sites of these fractures, which are often bilateral. The lesions are thought to be produced by torsional
and fractional forces generated when the infant is twisted or pulled by an extremity, or when the extremities are
subjected to shear strains during violent shaking. Although extremely telling of abuse, these lesions are difficult
to date because the usual markers, such as subperiosteal formation of new bone, are often lacking (162, 163).
Because of the subtlety and specificity of these lesions, some have suggested that even metaphyses that appear
normal by radiographic examination be removed in postmortem cases in which abuse is strongly suspected
(164). Fractures of the shaft of a long bone are four times more frequent than epiphyseal-metaphyseal injuries
and are the most common fracture seen in child abuse (159, 189, 190, 195). However, these fractures are of low
specificity in ambulatory children (25, 212). Indeed, nondisplaced oblique or spiral fractures of the tibia are so
frequently caused by accidental falls in ambulating youngsters that they are referred to as “toddler's fractures”
(90, 162). The specificity for abuse increases as the age of the child decreases—spiral diaphyseal fractures of
nonambulatory infants are highly suspect. However, a recent
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report of two unintentional spiral-oblique humeral fractures in preambulatory infants illustrates the importance of
a investigating the case history thoroughly before formulating a final opinion (142). Although abusive fractures
can occur anywhere along the ribs, they are often distributed in a posterior location. Anteroposterior compression
of the thorax, as in squeezing by an adult, produces excess leverage over the fulcrum of the transverse
processes at the costovertebral junction (169). This in turn creates tension along the inner aspects of the rib
head and neck that leads to fractures. Abusive rib fractures may also be located laterally because the lateral
aspects of the ribs represent areas of outbending and tension during squeezing (Figure 7-41). Acute rib fractures
may not be visible radiographically. During healing, as calluses form, the fracture becomes visible
radiographically (162) (Figure 7-42). Attempts at cardiopulmonary resuscitation may be offered as an explanation
for rib fractures. However, in previous studies, routine skeletal survey or autopsy was unable to demonstrate rib
fractures in infants following cardiopulmonary resuscitation (82, 96, 124, 139, 280, 319, 321).
Like the dating of soft tissue trauma, the dating of skeletal trauma is imprecise (328). In an attempt to age or date
a skeletal injury, three separate factors must be taken into consideration. First, the age of the infant affects the
healing process. Callus develops in neonates sooner than in older infants. Dynamic changes occur in the
skeleton itself as an infant matures, and compact bone increases. Second, the history of the injury must be
considered, although many times in abusive injuries accurate histories are not forthcoming. Third, many abusive
skeletal fractures are subjected to repetitive trauma. Either the abusive act is repeated in the same location and
affects the same bone, or a failure to seek medical attention results in lack of treatment and immobilization, so
that the fracture is constantly disturbed (232). Histologic studies of the timing of abusive injuries are difficult to
undertake. Multiple episodes of trauma have often occurred, accurate histories are rarely provided, and histology
provides only a single window of time in a dynamic process. Consultation with a pediatric radiologist and
correlation of the histopathologic findings with the radiologic appearance are strongly encouraged.
FIGURE 7-41▪Schematic representation of the production of abusive rib fractures. Anteroposterior squeezing
results in posterior or lateral fractures.
FIGURE 7-42▪Multiple posterior rib fractures with callus formation.
On occasion, caregivers attempt to conceal fatal child abuse by discarding the child's body and claiming that the
child is missing. If the child is later discovered, the remains may be skeletonized. Ascertaining the cause and
manner of death becomes more difficult when soft tissue is absent. Consultation with a forensic anthropologist
may be extremely helpful in such cases. The forensic anthropologist can assist in determining the age, sex, and
postmortem interval. The sex of a prepubertal child is extremely difficult to assess (157). The anthropologist may
be able to detect subperiosteal new bone formation in association with healed fractures (305).
BLUNT TRAUMA OF THE ABDOMEN AND THORAX

Blunt abdominal trauma is the second leading cause of death in fatal child abuse (53). The victims are generally
toddlers. The inflicted injury may represent the delivery of a large force, such as a punch or a kick, to a small
surface area. The involved organs are trapped between the incoming force over the soft anterior abdominal wall
and the rigid spine or thoracic cage along the posterior aspect of the body (Figure 7-43). Evidence of blunt
trauma to the anterior abdominal wall is not a reliable marker for inflicted blunt abdominal trauma; in a study of
184 abusive child homicides in New York City, 43% of the children dying of blunt abdominal trauma displayed no
external evidence of trauma along the anterior abdominal wall (75).
Several studies indicate that hollow viscera in the midline upper abdomen, such as the duodenum and jejunum,
are the organs most commonly injured in abusive blunt abdominal trauma (184, 245). The two most common
causes of duodenal injury in children are abuse and motor vehicle collisions in which the lap belt has been
placed over the abdomen rather
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than the pelvic region and iliac crests (28). The duodenum and jejunum are thought to be more vulnerable to
blunt force injury in young children because of the unique characteristics of a toddler's abdomen—wide and
comparatively flared costal margins and a short anteroposterior distance (31, 245). Injuries to these organs
include duodenal hematomas, serosal avulsions, and full-thickness perforations with resultant peritonitis.
Because of its viscoelastic nature, human tissue responds in a rigid fashion when forces are rapidly applied. In
contrast, the tissue is more deformable when forces are slowly applied (284). When subjected to a rapid force,
such as a punch or kick, a hollow organ may perforate.
FIGURE 7-43▪Schematic representation of abusive abdominal trauma.
The mortality rate in victims with abusive hollow viscus perforations is very high [71% in a study by Ledbetter
(184)], and they often present for medical care in extremis or are dead at the scene. Historical information usually
reveals that the child has been ill, with nausea and vomiting, for a time period ranging from hours to days. At
autopsy, the prosecting pathologist must examine the bowel carefully to identify the perforation—a task made
more challenging by the accompanying peritonitis. The perforation itself is usually quite small. Such an injury is
highly amenable to surgical intervention if medical treatment is obtained in a timely fashion (Figures 7-44 and 7-
45). Injuries to the mesenteric root may be associated with the bowel perforation. All evidence of abdominal injury
—abdominal wall contusion, small-bowel perforation, mesenteric root injury—should be histologically sampled to
estimate the interval from “injury to death” and document any evidence of old injury. Vitreous electrolyte studies
may reveal dehydration in these cases, which coincides with postinjury nausea and vomiting (59, 62, 171).
FIGURE 7-44▪Small-bowel perforation with surrounding submucosal hemorrhage that resulted in death.
FIGURE 7-45 ▪ Acute peritonitis resulting from the small-bowel perforation shown in Figure 7-44.
The solid organ most commonly injured in child abuse is the liver (75, 184). Lacerations of the liver result in
hemoperitoneum; the mechanism of death in these cases is internal exsanguination. In contrast to small-bowel
perforation, injury to the liver is relatively quickly followed by profound symptoms of shock. The liver may display
massive, stellate lacerations—more severe than those generally seen in highvelocity motor vehicle collisions
(Figures 7-46 and 7-47). In cases of blunt force trauma, cardiopulmonary resuscitation may be offered as an
explanation. However, studies have shown that injuries caused by cardiopulmonary resuscitation
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are rare in children and not as severe as abusive injuries (34, 68, 96, 172, 222, 251, 257, 280, 285, 304). When
such an explanation of massive abdominal injuries is offered, the question arises: “Why did the child require
cardiopulmonary resuscitation in the first place?”
FIGURE 7-46▪Extensive stellate liver lacerations resulting from fatal abusive abdominal trauma in a 3-year-old
child.
FIGURE 7-47▪Transection of the liver resulting from fatal abusive abdominal trauma in a 20-monfh-old child.
Another frequently affected solid organ is the pancreas. Injury to the pancreas may be associated with a small-
bowel perforation or liver laceration which is the cause of death, but the pancreatic injury is further evidence of
blunt force trauma (46). If the child survives the initial insult, pancreatitis and pseudocysts are the most common
complications (9). As previously discussed (see “Skeletal Evidence of Physical Abuse”), rib fractures are often
caused by anteroposterior squeezing. This represents a force applied slowly over a large surface area, rather
than a large force delivered rapidly to a small surface area, as in a direct blow. The typical pattern is bilateral in
approximately the same location (posterior or lateral). Abusive blunt force injury of the thoracic visceral organs
from blows is relatively rare in comparison with head injuries and abdominal trauma. Homicidal cardiac
lacerations often consist of rupture of the right atrium at its junction with the great veins. This rupture is caused
by either of two possible mechanisms. In direct trauma to the precordial chest, the heart is compressed between
the incoming sternum anteriorly and the rigid thoracic cage and vertebral column posteriorly (58). In trauma via a
blow to the epigastric region of the abdomen, the force may be transmitted to the atrium via the inferior vena
cava (67). In both scenarios, the forces are applied rapidly, so that energy cannot be absorbed through
deformation of the viscoelastic tissue (302).
MIMICRY
Although some disorders can be mistaken for pediatric inflicted injury, courtroom claims are much more frequent
than actual occurrences. Impostors of inflicted childhood injury may be divided into three main categories:
cutaneous findings, bony abnormalities, and metabolic conditions.
Cutaneous Findings
Causes of cutaneous findings that can be mistaken for signs of abuse include natural diseases, congenital
markings, cultural folk medicine practices, and decompositional changes.
Impetigo contagiosa is one of the more common cutaneous childhood infectious diseases that can be mistaken
for child abuse. Impetigo contagiosa is most often a disease of preschoolers and may occur in epidemics. The
two most common etiologic agents are Staphylococcus aureus and group A streptococci. The lesions, which
usually occur in exposed areas, begin as relatively circular vesicopustules that rupture quickly and can be
mistaken for cigarette burns (Figure 7-48). After rupture, the lesions become covered with a thick yellow crust.
Histologically, the vesicopustule is located in the upper layers of the epidermis and contains numerous
neutrophils. It also may contain Gram-positive cocci (191) (Figure 7-49).
Staphylococcal scalded skin syndrome, as the name implies, may be mistaken for scald burns. It is characterized
by large, flaccid bullae that rupture almost immediately. In this syndrome, the staphylococcal infection is usually
extracutaneous, such as pharyngitis or conjunctivitis. The bullae are caused by a toxin produced by the
staphylococci (191). Streptococcal toxic shock syndrome may present in much the same way (229).
Cutaneous contact with calcium chloride may cause skin necrosis to an extent requiring debridement. Such a
lesion may raise concerns about child abuse (329).
Ehlers-Danlos syndrome is an inherited connective tissue disorder with ten different subtypes. Some subtypes
are characterized by poor wound healing with extremely thin skin, prolonged bleeding, and subsequent scarring
(306). Because a severe injury accompanies a history of minor trauma, such lesions have been confused with
abusive injuries.
Some hematologic disorders may cause a cutaneous manifestation of “unexplained bruising.” This may lead to a
suspicion of abuse. Disorders that have presented in this fashion include acute lymphoblastic leukemia and von
Willebrand disease (205, 306).
FIGURE 7-48▪Impetigo contagiosa, initially alleged to be a cigarette burn. Note additional lesion at the inferior
margin of the photograph. (Courtesy of Bill Smock, M.D., Department of Emergency Medicine, University of
Louisville, Kentucky.)
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FIGURE 7-49▪Impetigo contagiosa. Vesicle in the upper layers of the epidermis.
A common nonpathologic finding that may be mistaken for a contusion is a Mongolian spot. Mongolian spots are
generally located over the lumbosacral region and are present at birth (Figure 7-50). They usually fade during
the early years but may be retained into adulthood. They are seen in a high percentage of African-American,
Hispanic, and Asian babies. They also occur in Caucasian babies. Mongolian spots occasionally develop outside
the lumbosacral region (192). A congenital hemangioma known as nevus flammeus may be mistaken for a red
contusion. Two types exist. The medially located nevus flammeus is commonly located in the occipital region or
the center of the face. The laterally located nevus flammeus is found on the face or extremities. The laterally
located lesion may become darker and raised with age, whereas the medially located lesion remains flat and may
fade with age (193). These congenital hemangiomas are colloquially known as “stork bites” (Figure 7-51).
FIGURE 7-50▪Mongolian spot over the midline buttocks of an infant.
FIGURE 7-51▪Congenital hemangioma over the midline occipital region. An additional hemangioma is visible at
the bottom of the picture in the midline high thoracic region.
Various cultural folk medicine customs cause cutaneous lesions that can be confused with abuse if the examiner
is unaware of them. One of the most common is a practice of Vietnamese immigrants. Cao gio (“coin rubbing”) is
used to alleviate common illnesses. The lesions are produced by rubbing the skin with a coin. Common sites
include the back, neck, head, shoulders, and chest. Other practices include bat gio (“skin”).
Postmortem changes that may be confused with traumatic injuries by nonpathologists include lividity and
maceration. Victims of SIDS often are transported to the hospital with well-developed rigor mortis. Such infants
show a welldeveloped lividity pattern. Those unfamiliar with lividity may confuse it with antemortem bruising.
Lividity can be differentiated from contusion as follows: lividity occurs in dependent areas of the body as it rests
in the postmortem state, and pressure points are spared. Prominent lividity is seen in association with well-
developed rigor mortis. Livor mortis blanches under pressure for about the first 24 hours. It should be
remembered that both rigor and livor are greatly influenced by environmental factors (including temperature and
wind current) and by individual factors such as clothing and size of the decedent. Death in utero is followed by
macerative changes in relatively short order (274). Skin slipping, separation of the epidermis from the underlying
dermis, may be present as soon as 6 hours after death and is expected if the infant has been dead for 12 hours
or longer (311). Because the epidermis easily peels away and the underlying dermis displays a generalized red
color, skin slipping may be mistaken for scald burns.
Other Findings
Soon after death, the infant brain, with its very high water content, begins to liquefy. The bones of the cranium
then override one another, so that deformity of the cranium results. Such deformity may be mistaken for head
trauma.
Inherited metabolic conditions may cause signs and symptoms that can be confused with abuse (59, 171, 200).
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Methylmalonic aciduria may present as failure to thrive (306). Glutaric aciduria type I is an inherited metabolic
disorder that can be confused initially with head trauma. Children with glutaric aciduria type I may present at age
6 to 18 months with encephalopathic crisis following a minor illness. This encephalopathic crisis may lead to
destruction of the basal ganglia. Children with glutaric aciduria type I characteristically display a head
circumference above the 95th percentile at birth. Continued rapid growth of the head circumference after birth
leads to macrocephaly with frontal bossing (12). Glutaric aciduria type I may be diagnosed by a metabolic screen
of blood (see Chapter 5).
Osteogenesis imperfecta is a rare disorder of type I collagen that results in abnormal bone fragility. Type I
collagen is the major structural protein of the extracellular matrix of bone, skin, and tendon (201). Four main
types of osteogenesis imperfecta are known. Type I is the most common. It is characterized by abnormal fragility
with osteoporosis, blue sclerae, defective dentition (dentinogenesis imperfecta), and presenile hearing
impairment. Other features common in osteogenesis imperfecta type I include wormian bones of the skull and
short stature. Osteogenesis imperfecta type I accounts for approximately 80% of all cases of osteogenesis
imperfecta. It is inherited in an autosomal dominant fashion. The family history is extremely useful in evaluating
children for osteogenesis imperfecta type I.
Osteogenesis imperfecta type II is known as the fetal or perinatal form. Severe osteoporosis is generally
apparent at birth, and intrauterine growth retardation is present. The majority of children with osteogenesis
imperfecta type II succumb within the first few weeks of life. These infants display deep blue-black sclerae, a
characteristic facies, severe skeletal deformities, and multiple fractures and osteopenia at birth. Because of the
obvious bony deformities, this form is unlikely to be mistaken for child abuse (107). Type III is thought to be
caused by a sporadic mutation. The majority of patients with type III display characteristic triangular faces. These
infants may have fractures at birth. The color of the sclerae may appear normal. Children with type III often
display shortening, bowing, and angulation of the long bones in addition to growth retardation. Type IV, the
rarest form, is most often confused with abuse. Osteoporosis and deformity are present but may be mild. Type IV
children usually have wormian bones, and abnormal dentition is common. Metaphyseal fractures in osteogenesis
imperfecta are different from the metaphyseal corner fractures and bucket handle fractures seen classically in
child abuse (1). Although the potential for misdiagnosis exists, the probability is very small given the relative
prevalences of type IV osteogenesis imperfecta and abusive fractures (167). Microscopically, the osseous tissue
of a child with osteogenesis imperfecta demonstrates a relative abundance of osteocytes. The extracellular
matrix is reduced, and so the cells are much closer together than is normal (32). The diagnosis of osteogenesis
imperfecta remains a clinical one, based on the patient and family history and on the findings of diagnostic
imaging and physical examination. A skin biopsy may be used as a confirmatory test (see Chapter 27).
CARDIOPULMONARY RESUSCITATION INJURIES

Often, a child is brought to the attention of a health care professional or death investigator and the etiology of
injuries, in particular injuries secondary to cardiopulmonary resuscitation (CPR) versus inflicted blunt force
trauma, becomes a crucial issue (20, 124, 132, 134, 149, 172, 183, 222, 248, 251, 297, 319, 321). Perpetrators
will claim that a child's injuries were caused by CPR, either by the child him/herself or by a first responder.
Injuries secondary to CPR may be external and internal, usually involving the head/neck and rarely trunk
(abdomen/thorax). These injuries are due to the compressions and to ventilation/intubation. Several studies
report no injuries to children secondary to CPR. Others report that, if present, these CPR-related injuries are not
significant or life threatening. Investigators should be aware of the resuscitative technique used on children, if
abdominal compressions were performed, and note if the resuscitator is experienced in the technique(s) (222,
304). The EMS personnel or emergency department physician can easily demonstrate how he/she performs
CPR. The investigator can correlate injuries with points of contact during compressions and ventilation, type of
mask, and type of airway. The mask should be retained for comparison to the facial injuries. A doll can be used
as well as the type/size of mask. Note any adhesive used on the face to aid in ventilation/intubation.
Conventional CPR with chest compressions produces blood flow that is approximately 30% of normal. Interposed
abdominal compressions (IAC) augmenting resuscitation may be performed resulting in improved hemodynamics
without intra-abdominal injury (10, 304). IAC can increase the blood flow twofold, and when performed by trained
individuals, the midabdominal compressions increase organ perfusion without organ injury. Studies in canines
show that IAC improves arterial pressure, central venous pressure, oxygen consumption, and cardiac output
without causing trauma.
In children, unlike adults, rib and sternal fractures rarely if ever occur; this has even proven true in children with
an underlying bone disease (124, 134, 139, 172, 199, 268, 319, 321). Ribs in children are flexible and more
resilient against fracture. In the absence of radiographic evidence of bone disease, unexplained rib fractures are
indicative of abuse. Often, such rib fractures are associated with other signs of abuse and/or different stages of
rib healing. Recent radiographical studies have examined subtle CPR-related rib fractures (82). When present,
these rib fractures are not usually associated with hemorrhage and are extremely difficult to notice without
removal of the parietal pleura.
Abdominal injury is uncommon (14, 68, 178, 197, 248, 277, 285, 297). Rarely, gastric perforation (lesser
curvature), epicardial hematoma, pulmonary interstitial hemorrhage, hepatic/splenic contusion or laceration, and
pancreatic
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injury have been reported as secondary to CPR. Abdominal compressions in children may result in pancreatic
hemorrhage as well as injury to the liver and spleen. Such abdominal compressions should be documented
(304). However, it must be noted that some studies of many resuscitated children report no injuries. In one study,
injuries were noted, but none were abdominal.
More of the CPR-related injuries in children are soft tissue injuries of the head and neck from ventilatory efforts
(108, 121, 132, 152). These include facial abrasions (nasal bridge, undersurface of the nose, anterior chin) from
the airbag-valve mask that are usually symmetrical. As the resuscitator positions his/her hand on the child,
fingertip contusions beneath the chin and on the side of the head may be produced. If a mask is not used but
instead mouth-to-mouth breathing is performed, one may see scrapes/fingernail scratches over the perinasal
area. If intubated, adhesive tape marks may be present on the lateral aspects of the mouth and cheeks. The
child's oropharynx is more susceptible to damage by forceful digital clearing and suction as well as by
endoscopic instruments. Traumatic mucosal tears and hypopharyngeal perforation have occurred with digital
clearing of the airway (108). The adult-type oral injuries secondary to teeth are not seen in the edentulous child
(172). With regard to CPR causing retinal hemorrhages, studies and collaborative research conclude that CPR
does not result in retinal hemorrhages.
Various forms of barotrauma have been described in adults and children (259). These include tympanic
membrane injury, pneumothorax, pneumoperitoneum, pneumoscrotum, and air embolism (especially in the
premature newborn). With ventilation, injuries such as pneumothorax (due to positive pressure ventilation) and
gastric rupture (due to overdistension during ventilation; usually the lesser curvature) may result (257). Also,
though rare, complications of mechanical positive pressure ventilation can occur related to faulty bag ventilation
devices and the valve.
Findings secondary to CPR other than from compressions and ventilation include defibrillator marks over the
thorax, venipuncture and intraosseous line access marks, bruising about the neck from attempted vascular
access, adhesive marks from taping the endotracheal tube, and vomitus in the airway secondary to agonal
regurgitation and subsequent compressions. One can also see cardiac contraction band necrosis and focal
hemorrhage with the administration of catecholamines during prolonged resuscitation. Defibrillation can produce
subepicardial myofibril disintegration. Gastric rupture secondary to nasogastric tube placement has also
occurred.
GUNSHOT WOUNDS
Gunshot wounds are relatively uncommon in young children but account for significant morbidity and mortality in
adolescents. It has been shown that the availability of guns in the home increases the risk for suicide among
adolescents (18, 30). Furthermore, many adolescent students report easy access to handguns (47% of boys and
22% of girls in one study) (18, 45).
Information that may be derived from the examination of a gunshot wound includes an estimate of the range of
fire and path of the projectile. Soot and gunpowder particles emerge from the muzzle of a fired gun along with the
bullet. Depending on the distance of the target surface from the muzzle of the gun, these substances may be
deposited in or on the target surface and may be used to estimate the range of fire. In contact wounds, all gas,
soot, and gunpowder particles are blown into the wound bed along with the bullet. In wounds of the head, where
relatively thin tissue is stretched over a rigid bony skull, gas is trapped between the outer table of the skull and
the soft tissue. This causes a marked expansion of the soft tissue, which may exceed the elastic capability of the
skin. When the elastic capability of the skin is exceeded, stellate lacerations radiate from the margins of the
gunshot wound of entrance. Furthermore, as the skin is forced outward from the body by entrapped gas, a
“muzzle stamp abrasion” may be produced (Figure 7-52). In wounds produced at close range, soot and burning
gunpowder particles are deposited on the skin surrounding the gunshot wound of entrance. Soot is transient
evidence because it may be wiped away during medical treatment. Therefore, it is imperative that this evidence
be documented early or preserved in some fashion. As the distance between the muzzle and skin surface
increases, soot can no longer reach the body surface. However, the burned and burning gunpowder particles
continue to travel and become embedded in the skin. These are represented by small, reddish brown, punctate
lesions surrounding the gunshot wound of entrance. The pattern is referred to as “stippling” or “tattooing”
(Figure 7-53). The examiner should measure the dimensions of the stippling and describe
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its overall pattern. Also helpful is a notation concerning the location of the gunshot wound of entrance within the
stippling (eccentric or central). In general, stippling is seen in handgun wounds inflicted within approximately 2 ft
(76). However, actual numbers should be provided to investigators with caution.
FIGURE 7-52▪Contact gunshot wound with muzzle stamp.
FIGURE 7-53▪Intermediate-range gunshot wound with surrounding tattooing.
It is better to use descriptive terms, such as “close range” or “intermediate range.” When wounds have been
inflicted from distances greater than about 2 ft (with standard handguns), the skin surrounding the gunshot of
entrance does not display soot or stippling. The gunshot wound of entrance is represented by a defect with a
small “abrasion collar.” The abrasion collar is produced when the bullet initially encounters the skin and indents
it, thereby stretching and rubbing the skin around the gunshot wound of entrance.
Gunshot wounds of exit are often irregular and stellate, and abrasions are absent (Figure 7-54). Contrary to a
popular myth, the gunshot wound of exit is not always larger than the wound of entrance.
The path of the projectile through the body may be of forensic importance in proving or disproving a witness's
account of the event. The path of the bullet should be described in three planes: anteroposterior, superoinferior,
and lateral. Gunshot wounds should be classified as either penetrating or perforating. In a penetrating gunshot
wound, the bullet is retained within the body. In a perforating gunshot wound, the bullet enters the body,
proceeds through, and exits (77).
FIGURE 7-54▪Gunshot exit wound.
CONCLUSION
The diagnosis of physical abuse should be approached as a team effort because many disciplines are
involved, including radiology, ophthalmology, surgery, pathology, social services, and law enforcement. It is
useful to establish a child fatality review team within a community to review sudden unexpected and
traumatic deaths of children. Such a team facilitates close working relationships and the exchange of
information among multiple agencies. Furthermore, it allows for the discovery of trends and hazards
concerning childhood deaths. The identification of common causes of accidental death may facilitate public
awareness campaigns to lower the incidence of such tragedies in the future. Increased collaboration
between pediatric pathologists, forensic pathologists, and pediatricians should be aggressively pursued.
Collaborative efforts should involve research, scientific discussions, and publications, so that each specialty
can benefit from the knowledge base of the others.
Of utmost importance in the evaluation of childhood injury is the correlation of the history with the physical
findings. The pathologist must be familiar with various developmental milestones of infants and young
children in order to properly evaluate the provided history. The severity, site, and distribution of the injury
must also be correlated. Pattern injuries should be evaluated and properly documented. Ancillary studies
should be utilized. The history should be adequately documented as well. Persons evaluating childhood
injuries for forensic purposes are encouraged to keep an open mind when forming a differential diagnosis
and employ common sense at all times.
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Chapter 8
Transplant Pathology
Rish Pai
Theodore J. Pysher
Aliya N. Husain
Solid organ transplantation has become an accepted mode of therapy for a variety of end-stage diseases, with somewhat variable
long-term outcome depending on the organ, as is discussed in this chapter (small bowel, liver, pancreas, kidney, heart, and lung).
Kidney and liver transplant are relatively more common; thus, these are presented in greater detail. Although there are many organ-
specific features in posttransplantation pathology, there are many similarities also. Postsurgical complications have markedly
decreased due to better techniques and donor and recipient management. Immunosuppressive regimens, including multiple drug
combinations, are standard of care. Antibody-mediated rejection is uncommon, while acute cellular rejection occurs in a majority of
recipients and can usually be treated effectively. Chronic rejection is a fibrosing process that continues to be the major limiting factor
to long-term survival, being more frequent in lung than in kidney, heart, and liver recipients. These immunocompromised patients are
susceptible to both the usual bacterial as well as opportunistic infections, which often involve the lung. Posttransplant
lymphoproliferative disease (PTLD), reported to occur in 3% to 5%, appears to be decreasing even further. It can involve the
transplanted organ (rare in heart) as well as extranodal sites such as the gastrointestinal tract.
TRANSPLANT IMMUNOLOGY
Overview
The success of transplantation depends, in large part, on the immune response of the recipient to the donor tissue. The
phenomenon of graft rejection was first identified by Peter Medawar in the early 1940s (71, 111). Medawar and others demonstrated
that allogeneic skin grafts (graft from agenetically distinct individual of the same species) would undergo rapid necrosis; however,
syngeneic skin grafts (graft from a genetically identical individual) would survive. As almost all solid organ transplants occur between
two genetically different individuals (allogeneic graft), many potential foreign or nonself molecules (alloantigens) are available to
elicit an immune response and lead to graft failure. Most of these alloantigens are derived from polymorphic genes inherited from
both parents and expressed codominantly. One of the most important alloantigens responsible for rejection is encoded by the major
histocompatibility complex (MHC). There are three different histopathologic categories of rejection: hyperacute rejection, acute
rejection, and chronic rejection, each of which can also be characterized by immunologic effector mechanisms (humoral versus cell-
mediated). As transplant immunology is a complex field, only a limited discussion of this broad topic is presented here, and
interested readers are referred to many excellent reviews for further reading (90, 110, 118).
Hyperacute Allograft Rejection

Hyperacute rejection is characterized by thrombotic occlusion of the graft vasculature that begins within minutes to hours after blood
vessel anastomosis. The mechanism involves preformed antibodies present in the recipient that bind donor endothelial cells and
elicit an immune response characterized by complement activation. Complement proteins are powerful serum proteins that are able
to damage cells through either cell lysis or recruitment of inflammatory cells such as neutrophils and macrophages. Classical
complement activation occurs when an antibody of the IgM or IgG subclass binds to its cognate antigen and activates C1q. The
activation of complement leads to the destruction of donor endothelial cells, resulting in thrombosis. The IgM antibodies responsible
for hyperacute rejection are mainly those against the carbohydrate ABO blood group antigen expressed primarily on red blood cells
but also on vascular endothelial cells. As most donors and recipients are matched with respect to their ABO subtypes, hyperacute
rejection due to anti-ABO antibodies is rare (118).
Acute Allograft Rejection

Acute allograft rejection is commonly encountered in solid organ transplants and has been an area of extensive research.
Classically, acute rejection is characterized by the presence
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of infiltrating lymphocytes that mediate direct killing, macrophage activation, and tissue damage. The lymphocytes involved in this
process include CD4+ T-cells, CD8+ T-cells, natural killer (NK) cells, and B-cells. Much of transplant immunology has been focused
on the role of T-cells in rejection, as they are the principal mediators of acute rejection. Indeed, much of the immunosuppressive
therapies in use today are directed toward interfering with T-cell function. The mechanism underlying T-cell activation is complex
and involves two mechanisms: direct presentation of alloantigens (nonself MHC molecules) to recipient T-cells by donor-derived
leukocytes and indirect presentation of alloantigens to recipient T-cells by recipient leukocytes (90). The process by which recipient
T-cells can be directly activated by nonself MHC molecules on donor cells is still a mystery to most immunologists. During T-cell
development in the thymus, those cells, with T-cell receptors, with high affinity for self-MHC molecules are deleted and only those
with low affinity for self-MHC survive, thus preventing nonspecific T-cell activation (negative selection) (108). However, in the
transplant setting, recipient peripheral T-cells are exposed to nonself MHC. Immunologists hypothesize that since T-cells with high
affinity for these MHC-molecules were not deleted by negative selection, there will be a significant proportion (up to 1%) of
circulating recipient T-cells with high affinity for nonself MHC (80). These T-cells could become activated and mediate allograft
rejection. Both CD8+ and CD4+ T-cells become activated by allorecognition in response to class I and class II MHC molecules,
respectively. The subsequent secretion of cytokines leads to macrophage, neutrophil, and natural killer cell recruitment (through
chemokine and adhesion molecule expression) and tissue destruction (through reactive oxygen species, arachidonic acid
metabolites, thrombosis, etc.). In addition, through direct allorecognition, donor CD8+ T-cells can mediate killing. Direct
allorecognition is thought to be the principle mechanism by which cellular rejection is mediated.
Indirect recognition of alloantigens is much better understood immunologically as it mirrors what occurs during infections. In this
pathway, recipient antigen-presenting cells (dendritic cells and macrophages) phagocytose donor antigens and process them into
peptides for presentation on class I and class II MHC. T-cells specific for these peptide:MHC complexes then can become activated
and mediate rejection. The proportion of T-cells that would be activated in such a manner is much smaller than in direct
allorecognition, and for many years, the significance of this pathway of T-cell activation has been unclear. Recently, indirect
presentation has gained the interest of transplant immunologists as it can on its own mediate rejection (30). Moreover, indirect
presentation is essential in producing highly specific antidonor antibodies (16). The donor-specific antibodies are mainly directed
against donor MHC, both class I and class II. Once formed, these antibodies can bind to donor leukocytes and activated endothelial
cells (anti-MHC class II) or all donor cells (anti-MHC class I) resulting in tissue damage through activation of complement and
recruitment of inflammatory cells. Indeed, the use of C4d, a product of the complement cascade, as a surrogate of antibody-
mediated complement activation has helped pathologists recognize acute humoral rejection (14, 66).
Chronic Allograft Rejection

Histologically, chronic rejection in most organs is characterized by fibrosis and vascular damage, and immunologically this process
most likely represents repeated bouts of acute rejection (sometimes subclinical). Thus, both cell-mediated and humoral mechanisms
most likely contribute to chronic rejection. Upon activation, some T-cells can differentiate into effector cells that produce fibrosing
cytokines (65) resulting in collagen deposition and parenchymal extinction. In addition, other cytokines such as platelet-derived
growth factor and basic fibroblast growth factor can induce proliferation of smooth muscle cells leading to narrowing of the graft
vessels. Moreover, antidonor antibodies have been shown to activate endothelial proliferation and vascular remodeling in animal
models (16). The resulting ischemia further leads to parenchymal loss and graft dysfunction. Other causes of late graft dysfunction
might not necessarily be related to immune-mediated rejection. Indeed, systemic disease such as diabetes, hyperlipidemia, viral
infections, etc. can all contribute to late graft dysfunction and should be differentiated from chronic rejection.
Allograft Tolerance
Understanding the immunologic mechanisms of allograft rejection has been essential in developing new therapies as well as
defining new histopathologic entities (acute humoral rejection); however, many questions remain. One of the most exciting fields in
transplant immunology is uncovering the mechanisms behind allograft tolerance. The goal of such research is to determine which
patients can be removed from immunosuppressive therapy due to tolerance toward the donor allograft. This is particularly important
in the pediatric population as immunosuppressive therapy (particularly corticosteroids) is a major cause of morbidity and mortality.
To date, no serologic or histopathologic data can accurately predict graft survival upon withdrawal of medications; however,
evidence points to arole of donor-derived leukocytes in mediating allograft tolerance (110, 111). It is hypothesized that patients who
become microchimeras are more likely to become tolerant to their allografts (124). This finding is supported by the early
observations that solid organ allografts are accepted to a great extent in individuals who are also receiving partial bone marrow
transplants (63). In addition, the greater acceptance of liver allografts is thought to be due to the large number of donor-derived
leukocytes present in this organ, some of which may be pluripotent stem cells that can migrate to recipient bone marrow and persist.
The recent
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appreciation of regulatory T-cells has also shed light on allograft tolerance. Regulatory T-cells have been shown to suppress the
function of effector T-cells, and active research is underway to enhance the activity of regulatory T-cells in order to achieve allograft
tolerance (120).
TRANSPLANT PATHOLOGY OF THE INTESTINE
Overview
The introduction of improved immunosuppression (notably FK506) has led to a rise in small intestinal transplantation that is of
particular importance to the pediatric pathologist as many of the disorders requiring transplantation occur in the pediatric population:
necrotizing enterocolitis, intestinal volvulus, gastroschisis, massive resections, Hirschsprung disease, neuronal intestinal dysplasia,
neuropathic and myopathic pseudo-obstruction, protein-losing enteropathy, and microvillous inclusion disease (29, 51, 89). The
most frequent indication for intestinal transplantation in these patients is total parenteral nutrition-associated liver disease (52).
When the liver disease is mild, the intestine can be transplanted in isolation. Signs of portal hypertension and cirrhosis mandate
intestinal transplantation in combination with the liver, or as part of a multivisceral organ transplant. Indeed, patients receiving
combined intestinal/liver transplantation or a multivisceral organ transplant experience fewer episodes of acute rejection and
improved overall survival at 5 years (48). Currently, the major obstacle to intestinal transplantation is the availability of appropriate
grafts. In particular, size matching is of extreme importance as many pediatric patients have contracted abdominal cavities as a result
of previous surgeries.
The pathologist’s role in intestinal transplantation is to evaluate mucosal biopsies in patients with graft dysfunction or as part of a
surveillance program. Most institutions routinely take protocol biopsies for the first four to six weeks and when clinically indicated
thereafter. In evaluating mucosal biopsies, the pathologist must correlate histologic findings with the clinical and endoscopic findings.
As with most transplant specimens, a systematic approach evaluating the overall architecture, surface and crypt epithelium,
inflammatory infiltrate, and vasculature can prevent pitfalls in diagnosis.
Preservation Injury and Hyperacute Rejection

Due to the intestinal villous circulation, the regenerative compartment of the epithelium is protected from ischemia; thus, preservation
injury is less worrisome than in other solid organs. Biopsies taken prior to transplantation demonstrate lamina propria edema and
separation of the epithelium from the basement membrane. Shortly after reperfusion, numerous mitoses can be seen within the
regenerative compartment along with capillary congestion, villous blunting, and a mild neutrophilic infiltrate (58). Biopsies taken a
week after transplantation usually show normal histology even when epithelial damage was quite severe. Hyperacute rejection in
small bowel transplants has recently been described, and there is some overlap with preservation injury; however, distinction
between the two is usually not difficult. In instances of hyperacute rejection, there is a positive cross-match indicating preformed
donor-specific antibodies. These antibodies damage the endothelium leading to fibrin thrombi within the lamina propria vasculature
resulting in severe congestion and focal hemorrhage. Neutrophils can be seen marginating within the congested vessels. The
presence of fibrin thrombi and severe congestion distinguishes hyperacute rejection from preservation injury (125).
Acute Rejection
Unlike liver allografts, acute rejection is extremely common (up to 90% of patient’s will experience at least one episode) and remains
a major cause of intestinal graft failure (up to 50%). Acute rejection is clinically characterized by nonspecific symptoms such as
fever, nausea, vomiting, increased stomal output, abdominal pain, and distention. In severe acute rejection, hemodynamic instability
may occur leading to shock. Endoscopically, acute rejection is characterized by granularity, diminished peristalsis and, in some
cases, mucosal ulceration. Acute rejection can occur at any time in the posttransplant period; however, the first episode of rejection
usually occurs within 100 days (51, 89). The landmark paper by Lee et al. (58) analyzed the first 62 intestinal transplants performed
at the University of Pittsburgh and was the first study to develop histologic criteria for the diagnosis of acute rejection. Subsequent
modifications have led to a well-developed histologic grading system for acute rejection that provides a reliable assessment of
severity (126).
The histologic manifestations of acute rejection include crypt apoptosis, lamina propria inflammatory cell infiltrate, and crypt
architectural distortion. During most episodes of acute rejection, all biopsies taken from multiple sites will show histologic features of
rejection; however, in approximately 20% of cases, only the ileum will be involved. Crypt apoptosis is the earliest histologic sign of
rejection, and apoptotic counts should be routinely performed on mucosal biopsy specimens. Rejection is characterized by greater
than six apoptotic bodies per ten crypts, and in mild acute rejection, crypt apoptosis is the dominant histologic feature (Figure 8-1). In
addition, mild localized collections of inflammatory cells (predominately activated/blastic lymphocytes with lesser numbers of
eosinophils and neutrophils) are present around small venules and capillaries in the deep mucosa. Peyer patches become enlarged
and contain large numbers of activated lymphocytes. The crypt epithelium commonly shows features of regeneration including mucin
depletion, nuclear enlargement, and hyperchromasia. A mild increase in intraepithelial lymphocytes and occasional neutrophils is
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typically seen. The villi are shortened, and the crypts tend to be distorted due to lamina propria expansion.
FIGURE 8-1 ▪ Mild acute rejection of small bowel allografts. A: The villous architecture is usually preserved and there is only a mild
increase in lamina propria inflammation. B: Prominent apoptotic bodies are the most prominent feature. (Photos courtesy of Dr.
Reetesh Pai, Stanford University.)
Moderate rejection is characterized by increased crypt apoptotic bodies and a diffuse inflammatory cell infiltrate characterized by
activated lymphocytes. Crypt apoptotic bodies are increased and begin to appear in the midportions of the crypt. The villi are
flattened to a greater extent; however, extensive ulceration is not common. In severe acute rejection, crypt apoptotic body counts are
further increased (up to 20) and become confluent (Figure 8-2). Mucosal ulcerations are common and, in its place, are fibrinous
neutrophilic exudates mimicking pseudomembranous colitis. Care should be taken when evaluating biopsies for acute rejection 100
days posttransplant as the inflammatory infiltrate is generally mild and crypt apoptosis is the only dominant histologic feature (58).
FIGURE 8-2 ▪ Severe acute rejection of small bowel allografts. A: Surface ulceration with a prominent lymphocytic infiltrate is
common. B: Crypts are typically lost, and the surviving crypts are severely damaged. This differential diagnosis includes ischemia
and infection. (Photos courtesy of Dr. Reetesh Pai, Stanford University.)
Chronic Rejection
Chronic rejection in the intestine is less common than in heart, kidney, and lung; however, 8% of allografts at 5 years
posttransplantation develop chronic rejection (79). Patients with chronic rejection have persistent diarrhea despite increased
immunosuppressive therapy. Endoscopic and radiographic findings of chronic rejection include loss of mucosal folds, mural
thickening, focal ulcers, and decreased arborization of the mesenteric vasculature. Clinically, chronic rejection is encountered late in
the posttransplant period, with most cases occurring months after transplantation. There are many factors associated with the
development of chronic rejection. Those individuals with acute rejection within 30 days of transplantation and those with severe
acute rejection are more likely to develop chronic rejection. Other risk factors
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include prolonged cold ischemic time, old donor age, and episodes of CMV infection (79). Simultaneous liver transplantation greatly
protects from chronic rejection most likely by decreasing the number of acute rejection episodes. The pathologic process that results
in chronic rejection involves arterial obliteration; however, arteries are rarely sampled in endoscopic biopsies. Thus, on mucosal
biopsies, one can only suggest possible chronic rejection based on downstream features of chronic ischemia. Early histologic
changes that suggest possible chronic rejection include patchy mild fibrosis and focal crypt loss. These nonspecific changes can
persist for months. With worsening ischemia due to progression of chronic rejection, there is extensive loss of the intestinal crypts,
villous atrophy, mucosal ulceration, and increased lamina propria inflammation and fibrosis. The surviving crypts show evidence of
chronic damage including pyloric gland metaplasia and regenerative features (58, 79). Once chronic rejection proceeds to the
severe stage, the graft is very likely to fail. At resection, the vasculature should be adequately sampled to find the characteristic
changes of chronic rejection. In addition, extensive neural hyperplasia is a common finding at resection (76).
Complications of Transplantation
Infection remains a very common complication of transplantation, whether in the postoperative setting or due to immunosuppressive
therapies. The vast majority of infections are bacterial infections although fungal infections are also routinely encountered (89). Of
more importance to the pathologist is recognizing viral infections, in particular cytomegalovirus (CMV), Epstein-Barr virus (EBV), and
adenovirus. CMV infection is encountered in 5% to 29% of intestinal allograft specimens and can clinically mimic acute rejection (31,
89). Negative CMV serology in the pediatric recipient is associated with increased CMV infection when transplanted with a serologic
positive donor (60% will develop CMV enteritis) (64). In the majority of specimens, a moderate neutrophilic and mononuclear cell
infiltrate is seen in the lamina propria as well as in the crypts. Ulceration with abundant granulation tissue can be seen in severe
cases. In severely immunocompromised individuals, inflammation may be mild. In addition, crypt atrophy, cell drop out, and apoptotic
bodies may be present, mimicking rejection. The characteristic CMV inclusions are mainly confined to the endothelial and stromal
cells (Figure 8-3); however, epithelial cells can be infected in severe cases.
Adenovirus is a very common cause of pediatric gastroenteritis; however, until recently, infection of allografts has not been routinely
recognized. Pinchoff et al. (81) found a high prevalence of adenoviral infection in pediatric small bowel allografts. Adenoviral enteritis
most commonly affects the ileum and is characterized by smudgy epithelial cell nuclear inclusions, epithelial hyperplasia with
disarray, and a prominent lymphoplasmacytic infiltrate. While adenoviral infection limited to the intestinal allograft is not in itself a
matter of concern, disseminated adenoviral infection can be fatal. Moreover, those patients with a liver allograft are at risk of
developing adenoviral hepatitis and fulminant hepatic failure.
FIGURE 8-3▪Cytomegalovirus infection of small bowel allografts. In CMV infection, an inflammatory infiltrate with ulceration, crypt
atrophy, and apoptotic bodies can be seen; however, the characteristic cytoplasmic and nuclear inclusions are key in differentiating
CMV infection from rejection. (Photo courtesy of Dr. Reetesh Pai, Stanford University.)
Epstein-Barr virus (EBV) infection is another serious complication in the posttransplant period as it can lead to PTLD. Biopsy-proven
EBV infection is fairly common and occurs in up to 50% of intestinal allograft specimens, higher than in many solid organs (27). EBV
infection is associated with a wide histologic spectrum, from simple lymphoid hyperplasia to non-Hodgkin lymphoma. When
evaluating a specimen, particular attention should be paid to the type of lymphoid infiltrate (Figure 8-4). If small lymphocytes
predominate, one can be reassured; however, the presence of large atypical lymphoid cells should prompt concern for PTLD and in
situ hybridization for EBV early RNA (EBER)
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should be performed. A large number of EBER positive cells (>15 per high power field) with a heterogeneous population of lymphoid
cells, including immunoblasts, plasma cells, and large cleaved cells, are characteristic of polymorphous PTLD (Figure 8-4) (27). If
the lymphoid population is homogenous, the designation of monomorphic PTLD is made and further classification is made according
to established criteria (27). The vast majority of monomorphic PTLDs are B-cell in origin; however, T-cell PTLDs have been
described.
FIGURE 8-4 ▪ Posttransplant lymphoproliferative disorder of small bowel allografts. PTLD is commonly characterized by an atypical
inflammatory infiltrate, which can be mixed (polymorphous) as in this case or monomorphic. In situ hybridization for EBER can be
helpful in confirming the diagnosis. (Photo courtesy of Dr. Reetesh Pai, Stanford University.)
The Gastrointestinal Tract in Graft-Versus-Host Disease

The intestinal tract is one of the three major target organs in graft-versus-host disease (GVHD) (69, 70). The skin and the liver are
the other two organs affected when donor lymphoid cells are transfused into immunosuppressed host. GVHD usually occurs in the
setting of bone marrow transplantation but may also rarely occur following the transfusion of nonirradiated blood into patients with
primary or secondary immunodeficiency disorders (75). Conceptually, GVHD mirrors allograft rejection as donor leukocytes
recognize recipient tissues as “foreign” and attempt to “reject” them. Thus, the immunologic effector mechanisms are similar. GVHD
develops in two phases: acute, which begins 1 week to 4 months after transplantation, and chronic, which begins approximately 4
months or more after transplantation. The clinical and pathologic features of the two phases are distinctly different.
The gastrointestinal tract is affected in at least half of patients with acute GVHD (69). Intestinal GVHD is usually heralded by profuse
watery diarrhea, which indicates involvement of the small intestine and colon. Occasionally, the upper gastrointestinal tract will be
involved first or exclusively; the symptoms are nausea, vomiting, and anorexia. Acute intestinal GVHD is usually diagnosed by
colonoscopic biopsy or endoscopic biopsy of the upper gastrointestinal tract. The earliest histologic changes occur deep in the
crypts (the regenerative compartment) with epithelial infiltration by lymphocytes and subsequent apoptosis of individual glandular
cells, vacuolization of cytoplasm, and nuclear karyorrhexis (105, 122), mimicking the changes seen in acute rejection (Figure 8-5). If
appropriate therapy is not instituted, neutrophilic inflammation, glandular destruction, crypt abscesses, and ulceration are seen.
Complete crypt loss, villous atrophy, and extensive mucosal denudation occur in advanced acute GVHD. In the esophagus,
vacuolization and inflammation of the epithelial basal layer and eventual desquamation and ulceration are seen (103).
Chronic GVHD is a more insidious process that primarily affects the skin and liver. The intestinal tract is largely spared; however,
features of chronic injury can be seen (2). In the esophagus, a scleroderma-like fibrosis and dysmotility may develop (72). In the
evaluation of all the phases of intestinal GVHD, opportunistic infections must be ruled out (103) Interestingly, mycophenolate mofetil,
a commonly used immunosuppressive drug in solid organ transplantation, can give rise to histologic findings similar to acute GVHD
(78).
FIGURE 8-5 ▪ Graft-versus-host disease of the small bowel. GVHD is characterized by apoptosis of individual epithelial cells lining
the crypts similar to acute rejection seen in small bowel transplants. If severe, complete villous loss and surface ulceration can be
seen.
TRANSPLANT PATHOLOGY OF THE LIVER

Overview
In the United States, between 1998 and 2007, 1,589 liver transplants were performed in patients under the age of 17 accounting for
approximately 7% of the total number of liver transplants. Currently, there are approximately 800 pediatric patients on the transplant
list (77). The indications for liver transplant are diverse (Table 8-1) (77); the most common continues to be extrahepatic biliary
atresia.
Early in pediatric transplantation, the survival rates were dismal as only 30% of patients survived greater than 1 year (29). With
improved surgical techniques, patient screening, and immunosuppression, the current 1-year patient survival is 90% and the 5-year
survival is 80%. Graft survival is 85% at 1 year and 67% at 5 years (77). The early days of pediatric liver transplantation were also
complicated by a shortage of appropriate-sized liver allografts. With the advent of reducedsized liver transplantation, living-related
transplantation and, most importantly, split-liver transplantation, the shortage of pediatric liver allografts has been somewhat
alleviated (29, 38, 55). In split-liver transplants, the whole adult cadaveric liver is divided into two functional segments: one for adults
(right trisegment) and one for children (left lateral segment). Recent studies have shown that split-liver recipients have comparable
survival to whole liver recipients (4, 49). Despite these improvements, surgical complications continue to be more common when
compared with adults (29, 38, 68). In particular, the use of partial liver allografts predisposes to biliary complications (98). In addition,
hepatic artery thrombosis is more common in pediatric patients owing to the technically difficult surgery. However, the improvement
in surgical techniques and postoperative management has improved, allowing many of these grafts to be saved. Portal vein
thrombosis is occasionally encountered, which, in most cases, resolves without need for intervention (68). Hepatic vein thrombosis
is rarely encountered except in patients
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undergoing liver transplantation for Budd-Chiari syndrome. Bowel perforation is common in the pediatric population as most of these
patients have had previous abdominal surgery and suffer from poor nutrition. Other complications of liver transplant can be roughly
grouped into the time periods in which they are most likely to occur (Table 8-2) (121).
Table 8-1 ▪ INDICATIONS FOR PEDIATRIC LIVERTRANSPLANTATION
Noncholestatic cirrhosis
Autoimmune hepatitis
Chronic viral hepatitis
Cholestatic liver disease/cirrhosis
Caroli disease
Choledochol cyst
Primary sclerosing cholangitis
Biliary atresia
Extrahepatic
Alagille syndrome
Hypoplasia
Acute hepatic necrosis
Acute viral hepatitis
Drugs
Metabolic diseases
Alpha-1-antitrypsin deficiency
Wilson disease
Hemochromatosis
Tyrosinemia
Primary oxalosis
Glycogen storage disease types la, lb, III and IV
Hyperlipidemia
Urea cycle disorders
Crigler-Najjar syndrome
Malignant neoplasms
Hepatoblastoma
Hepatocellular carcinoma
Other
Cystic fibrosis
Budd-Chiari syndrome
Congenital hepatic fibrosis
TPN/hyperalimentation
Familial cholestasis
Hepatic adenomatosis
Table 8-2 ▪ APPROXIMATE TIMELINE OF BIOPSY FINDINGS IN LIVER TRANSPLANTATION
Posttransplant Interval Complications Histologic Features
Early (0-7 days) Preservation/harvesting injury Centrilobular pallor, ballooning

degeneration, cholestasis
Humoral rejection Extensive necrosis and perivenular

hemorrhage; positive C4d
Early hepatic artery thrombosis Zonal hepatocyte and bile duct necrosis
Middle (7-30 days) Acute cellular rejection Mixed portal infiltrate, bile duct damage,
and endothelialitis
CMV hepatitis Microabscesses and viral inclusions

Recurrent disease Features of original disease
Late (>30 days)
Chronic rejection Bile duct and arteriolar loss, foam cell
arteriopathy
Late-onset acute rejection Perivenular inflammation; interface and

lobular activity; less endothelialitis and
portal inflammation than classic acute
rejection
PTLD Atypical Iymphoid infiltrate, EBER+

Late hepatic artery thrombosis Centrilobular hepatocyte dropout; biliary
obstruction
De novo autoimmune hepatitis Lymphoplasmacytic infiltrate with

interface activity
The initial outcome of the liver allograft depends on the health of the donor liver, the amount of ischemic time the allograft suffered,
the presence of preformed antiallograft antibodies, and complications encountered during surgery and the perioperative period.
Acute rejection and viral infections tend to occur between 1 week and 2 months posttransplantation, whereas chronic rejection and
recurrent disease are late manifestations. However, the timing can vary significantly (e.g., late-onset acute rejection) and biopsy
interpretation remains essential.
Preservation Injury
Preservation (harvesting) injury results from donor and tissue procurement factors that contribute to poor allograft function in the
perioperative period. In order to diagnose preservation injury, one must exclude injury due to surgical complications, immunologic
reactions, and drug toxicity. Warm and cold ischemia preferentially damage hepatocytes and endothelial cells, respectively.
Endothelial cell damage leads to interference with vascular blood flow and subsequent allograft injury. Many donor factors can
increase the susceptibility of the allograft to ischemic time. One of the most studied is the presence of donor macrovesicular
steatosis. Transplantation of liver allografts with greater than 50% macrovesicular steatosis, on frozen section analysis results in
poor graft function as steatotic hepatocytes are
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sensitive to ischemic damage. Other donor factors that influence graft function include fibrosis, chronic liver disease, hemodynamic
instability, infections, donor atherosclerosis, and donor age (82, 91).
FIGURE 8-6▪Preservation injury of liver allografts. Pallor in the centrilobular areas with hepatocyte ballooning occurring shortly after
transplantation is characteristic of mild preservation injury. Hepatocyte and canalicular cholestasis can also be quite prominent in
some cases.
Clinically, preservation injury is characterized by poor bile production and persistent elevations of serum ALT and AST. The
histologic features of preservation injury are usually apparent within 1 to 2 days after revascularization. In mild preservation injury,
mild centrilobular hepatocyte ballooning and canalicular cholestasis are commonly seen. Occasionally, neutrophils may be present.
On low-power microscopic evaluation, preservation injury can be suggested by pallor in the centrilobular areas. The hepatocyte
injury is rapidly reversible; however, the cholestasis may persist for several weeks (Figure 8-6). In more severe injury, zonal necrosis
and severe neutrophilia may be seen. In these biopsies, bile ductular proliferation as well as cholestasis may be prominent. In
patients receiving a steatotic liver, reperfusion results in lysis of the steatotic hepatocytes with formation of sinusoidal fat droplets
that disrupt hepatic blood flow. The extracellular fat may persist for weeks after initial injury. Resolution of hepatic injury is the
hallmark of preservation injury, but if severe, the allograft may fail resulting in primary nonfunction. If hepatocyte injury persists
beyond one week, other diagnoses such as rejection and obstructive cholangitis should be considered.
It is our practice to report the percentage of macrovesicular steatotic hepatocytes, the amount of fibrosis, the presence of perivenular
necrosis, and neutrophilic infiltration (excluding surgical hepatitis) (28) to our transplant surgeons who ultimately determine allograft
use.
Hepatic Artery Thrombosis

As previously mentioned, hepatic artery thrombosis (HAT) remains a significant problem in pediatric liver transplantation and is a
complication in 5% to 10% of pediatric liver allografts (68). The incidence of HAT increases with decreasing age due to the smaller
size of the arterial anastomosis. As the hepatic artery is the sole blood supply to the biliary tree, HAT should be sought whenever a
biliary leak is found. HAT can occur early in the posttransplant period or late (occurring >30 days posttransplant). Early HAT is
associated with severe graft dysfunction and high mortality rate. In early HAT, rapid diagnosis and repair of the vascular tree are
essential in reversing biliary damage and prevention of allograft failure. Even with aggressive treatment, retransplantation may be
necessary; however, in one study, 40% of children with HAT survived without retransplantation (114).
In late HAT, the allograft is less susceptible to damage as collaterals have formed. Indeed, many patients are asymptomatic.
Symptomatic patients commonly present with recurrent cholangitis, biliary tract strictures (due to prolonged ischemic damage),
abscess, and fever. Biopsy findings in HAT are nonspecific, variable, and irregularly distributed within the graft (121). In early HAT,
coagulative necrosis of the centrilobular hepatocytes is frequently encountered along with bile duct necrosis. In late HAT, features of
biliary obstruction are encountered, including canalicular cholestasis, cholate stasis, and bile ductular proliferation. In addition,
centrilobular hepatocyte ballooning and dropout are commonly seen. Although these findings suggest HAT, definitive diagnosis
requires clinical correlation.
Biliary Complications
In children, biliary tract complications are more numerous due to surgical difficulties and the use of split-liver allografts (38, 67, 68).
Clinically, biliary complications should be suspected when preferential increases in alkaline phosphatase and gamma-glutamyl
transferase occur. Minor strictures may be asymptomatic with only minor elevations in biliary enzymes, whereas complete
obstruction, cholangitic abscess, and ascending cholangitis result in fever, jaundice, right upper quadrant pain, and bacteremia.
Liver biopsies typically show features of biliary obstruction. In the acute phase, portal edema, canalicular cholestasis, and portal
inflammation (mostly neutrophils) are commonly seen. Chronic obstruction leads to cholate stasis, chronic portal inflammation, focal
bile duct loss, and portal fibrosis. Progression to biliary cirrhosis can occur if the obstruction is not relieved. Biliary-vascular fistula is
a serious complication that warrants prompt surgical correction. Histologically, bile is found in blood vessels often with a giant cell
reaction, and red blood cells are found within bile ducts.
Hyperacute (Humoral) Rejection

Humoral rejection is a rare cause of early liver allograft failure and should be distinguished from primary nonfunction. Hyperacute
rejection occurs in the setting of preformed cytotoxic antibodies directed mainly against ABO blood group antigens, but also against
class I and II MHC antigens. The liver is relatively resistant to injury by these antibodies for multiple reasons including clearance of
antibodies by resident Kupffer cells, dual blood supply, and absence of conventional basement membrane (which is prothrombotic)
(21).
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Hyperacute rejection is suspected first in the operating room when the liver becomes swollen and hard and bile is not produced,
soon after revascularization. Hemostasis may be difficult to achieve in these patients. Histologically, hyperacute rejection may be
difficult to distinguish from primary nonfunction. In severe cases (mostly those due to ABO-incompatibility), there tend to be large
areas of infarction, portal vein thrombi, and necrotizing arteritis. In mild cases (ABOcompatible), centrilobular hepatocyte ballooning,
canalicular cholestasis, acidophil bodies, and bile ductular proliferation may be seen (features almost indistinguishable from
preservation injury). With appropriate clinical history, such as positive cross-match and short ischemic time, hyperacute rejection
may be suggested. Detection of C4d (by immunohistochemistry or immunofluorescence) may be helpful as a recent study found 91%
(10 of 11) of hyperacute rejection cases had positive staining for C4d in the hepatic vasculature (35).
Acute Rejection
Acute rejection is fairly common in pediatric liver allografts, affecting up to 60% of transplant recipients (67). Most episodes occur
within the first few months after transplantation and can easily be controlled by traditional immunosuppressive therapy. However, a
somewhat distinct form of acute rejection can occur late in the post transplant period, aptly termed late acute rejection. These
rejection episodes tend to be more resistant to standard immunosuppressive therapy and have unique histologic features. Most
cases of late acute rejection in children are due to inadequate immunosuppression (18). Clinically, acute rejection can be
asymptomatic when mild. More severe cases present with fever, decreased bile flow, and elevations in liver chemistry tests. The
gold standard for confirming the diagnosis remains liver biopsy; however, communication between the pathologists and clinician is
essential in determining which patients with rejection require increased immunosuppression.
In 1997, the Banff working group convened to develop histologic criteria outlining three core histological features: (a) portal
inflammation, (b) subendothelial inflammation, and (c) bile duct damage (5) (Figure 8-7). The portal inflammation is mixed. Activated
(blastic) lymphocytes and small mononuclear cells tend to predominate; however, eosinophils, macrophages, and neutrophils can be
prominent. Posttransplant lymphoproliferative disorder should be kept in mind when a monotonous portal infiltrate consisting of
blastic lymphocytes is present without other features of rejection. The presence of mononuclear inflammatory cells between the
endothelial cells of the portal or central vein and the underlying basement membrane, referred to as endothelialitis, is another
common feature of rejection. Occasionally, central vein endothelialitis may be the only prominent feature of acute rejection. Bile duct
damage is manifested by the presence of mononuclear cells inside the basement membrane and between cholangiocytes. In
addition, the bile duct epithelium shows loss of apical cytoplasm (increased nuclear/cytoplasmic ratio), paranuclear vacuolization,
nucleoli, nuclear overlap, mitosis, apoptotic bodies, and cytoplasmic eosinophilia. To make a diagnosis of acute rejection, two of
three of the above histologic features must be present. The diagnosis is further strengthened if greater than 50% of bile ducts are
damaged or if unequivocal endothelialitis is present. Other findings such as necrotizing arteritis (rarely seen in needle biopsies),
interface hepatitis, lobular inflammation, and eosinophilia are also seen in acute rejection but are not necessary for the diagnosis.
Early in the postoperative period, acute rejection may resemble preservation injury; however, the presence of portal inflammation
should distinguish between these two entities.
FIGURE 8-7▪Acute rejection of liver allografts. A: The portal tracts in acute rejection are expanded by a dense mixed inflammatory
infiltrate. B: Definitive evidence of endothelialitis along with bile duct damage confirms the diagnosis.
Once the diagnosis of acute rejection is made based on the above criteria, an indication of the global severity should be given. In
mild acute rejection, portal inflammation is mild. In moderate rejection, most or all of the portal tracts are expanded by an
inflammatory infiltrate. In severe rejection, there is spillover into the hepatic parenchyma with hepatocyte necrosis, both periportal
and perivenular. At our institution, only a global assessment of rejection is given;
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however, a rejection activity index has been developed to further characterize the severity of rejection and is routinely reported at
some institutions (5, 59).
As mentioned, late-onset acute rejection has some unique morphologic features when compared with acute rejection occurring early
in the posttransplant period (18, 20). Late acute rejection tends to have less portal inflammation, increased interface activity, less
endothelialitis, and more lobular activity; however, traditional features of acute rejection should still be present. In some cases, only
centrilobular pathology exists with perivenular inflammation and zone 3 hepatocyte dropout.
Chronic Rejection
Chronic rejection has become relatively rare with current immunosuppressive therapy and affects only 3% to 5% of total liver
allografts (67). Some studies report almost no cases of chronic rejection in pediatric patients (46); however, chronic rejection does
occur and is an important cause of late graft failure. Factors associated with chronic rejection include a primary diagnosis of
autoimmune liver disease, late-onset acute rejection, nonwhite race, baseline immunosuppression, certain tumor necrosis factor-2
alleles, and CMV infection (controversial) (24, 34, 117). Despite the name, many cases of chronic rejection occur within months of
transplant (2 to 6 months) and lead to graft failure within 2 years. Indeed, unlike other solid organ allografts, chronic rejection in the
liver decreases with time, except for a small group of patients with late-onset chronic rejection. The classic presentation of chronic
rejection is that of a patient with multiple episodes of acute rejection who develops progressive cholestasis and elevations in alkaline
phosphatase, bilirubin, and gammaglutamyl transferase, and is unresponsive to immunosuppressive therapy. Rarely patients
present with chronic rejection in the absence of any documented history of acute rejection.
FIGURE 8-8▪Chronic rejection of liver allografts. A: In early chronic rejection, the biliary nuclear/cytoplasmic ratio is increased, and
the cytoplasm shows prominent eosinophilia. B: In late chronic rejection, the bile ducts are lost and only portal veins and, to a lesser
extent, terminal hepatic arterioles remain to identify portal tracts. There is an “empty appearance to the often diminutive portal tracts.
As chronic rejection most commonly results from repeated bouts of acute rejection, there will be a period of overlap. Conceptually,
acute rejection refers to reversible and active lesions in which there is hepatocyte apoptosis and blastic portal inflammation,
whereas chronic rejection is generally nonreversible and refers to loss of key structures. If both features are present, both acute and
chronic rejection should be diagnosed based on their respective criteria. Late clinical findings of chronic rejection include hepatic
infarction and loss of synthetic function. Clinically, chronic rejection can resemble biliary obstruction, and cholangiography is
sometimes necessary to distinguish them.
Like acute rejection, there are three histopathologic features of chronic rejection: (a) bile duct atrophy, (b) foam cell arteriopathy, and
(c) bile duct loss, at least one of which should be present (19, 59). The diagnosis of chronic rejection mainly depends upon bile duct
features as the characteristic foam cell arterial changes are rarely encountered on routine liver biopsies. Thus, it is important to
exclude other causes of duct injury or loss such as hepatic artery thrombosis, obstructive biliary disease, recurrent chronic hepatitis,
drug reactions, and cytomegalovirus infections. The bile duct damage is thought to be ischemic in nature due to damage to the
peribiliary arterial plexus. The earliest manifestations of bile duct injury include eosinophilic transformation of the biliary cytoplasm,
uneven nuclear spacing, syncytia formation, nuclear enlargement and hyperchromasia, and ducts with focal epithelial cell loss. At
this early stage of chronic rejection, it is thought that these changes might be reversible with immunosuppression. In late chronic
rejection, bile ducts and, to a lesser extent, terminal hepatic arterioles are lost. When quantifying bile duct and arterial loss, it is
essential to remember that in a normal liver, not all portal tracts contain these structures. In fact, between 5% and 10% of portal
tracts do not contain bile ducts or hepatic artery branches (17). Thus, bile duct loss is only significant if greater than
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20% of the portal tracts do not have bile ducts. However, quantification of bile duct and arterial loss can be complicated in late
chronic rejection as portal tracts can be difficult to visualize. In these cases, portal tracts should be inferred from location within the
lobule, presence of connective tissue, and shape. Additionally, inflammatory cells may obscure bile ducts. In such cases,
immunohistochemistry for cytokeratin 7 may be useful in determining bile duct number; however, care must be taken to count only
true bile ducts and not ductules (36).
FIGURE 8-9▪De novo autoimmune hepatitis. The portal tract is expanded by a dense lymphoplasmacytic infiltrate with prominent
interface and lobular activity. Along with elevated ANA titers, these findings are consistent with a de novo autoimmune hepatitis.
Foam cell arteriopathy is another hallmark of chronic rejection; however, it is best appreciated in large-sized and medium-sized
hepatic artery branches that can only be sampled on hepatectomy specimens. Early chronic rejection is characterized by
accumulation of foam cells within the intima without luminal compromise. In late rejection, foam cell accumulation with luminal
compromise predominates. Changes in large bile ducts can also be appreciated in hepatectomy specimens, including fibrosis of the
wall, epithelial sloughing, and papillary hyperplasia. In most cases of chronic rejection, both bile duct loss and foam cell arterial
changes co-exist; however, up to 15% of cases may have only one feature.
Centrilobular changes can also be seen in chronic rejection and may be a prominent feature. In early chronic rejection, perivenular
mononuclear inflammation, hepatocyte dropout, acidophil bodies, pigmented macrophages, and mild fibrosis are commonly seen.
Late chronic rejection is characterized by perivenular fibrosis that can be extensive, resulting in bridging fibrosis. Vascular damage
due to chronic rejection may be a cause of these centrilobular changes; however, immunologic factors might also contribute to these
findings. Centrilobular cholestasis can also be prominent, especially when bile duct loss becomes severe. Many factors not related
to chronic rejection may also lead to similar centrilobular changes such as viral hepatitis, venous outflow obstruction, and hepatic
artery thrombosis. Thus, definitive diagnosis of chronic rejection must rely on bile duct and arterial changes.
De novo and Recurrent Autoimmune Hepatitis

Patients who are transplanted due to autoimmune hepatitis (AIH) can develop recurrent disease (30% by 5 years); however, some
patients without any prior history develop a syndrome remarkably similar to classic AIH termed de novo AIH. The diagnosis of de
novo or recurrent AIH requires the presence of autoantibodies, lymphoplasmacytic portal inflammation with prominent interface and
lobular activity, serologic evidence of liver injury, hypergammaglobulinemia, and no evidence of viral hepatitis, drug-related hepatitis,
or rejection (Figure 8-9) (20).
Other Recurrent Diseases

In children, recurrent hepatitis C or B is generally not routinely encountered as chronic viral hepatitis is an uncommon indication of
liver transplantation. In addition, intrinsic metabolic or synthetic liver disease does not recur in the allograft. However, metabolic
diseases that secondarily affect the liver can recur in the allograft. These diseases include Niemann-Pick disease, Gaucher disease,
cystinosis, and erythropoietic protoporphyria (45).
Recent evidence has confirmed that primary sclerosing cholangitis (PSC) can recur in approximately 5% to 20% of patients with
most recurrences diagnosed more than 1 year posttransplant (32). Moreover, PSC patients are at a higher risk of developing
rejection and worsening inflammatory bowel disease after transplantation. Because in the posttransplant setting there are many
causes of biliary disease, the diagnosis of recurrent PSC is often difficult, and no gold standard exists. Thus, close clinical,
radiologic, and histopathologic correlation is required to make this diagnosis. The presence of nonanastomotic biliary strictures is
suggestive of recurrent PSC but only if occurring late in the posttransplant period. In addition, other causes of late-onset biliary
strictures, such as chronic rejection and biliary infections must be excluded. Early stricturing is more likely due to complications of
preservation injury and hepatic artery thrombosis. Biopsies showing characteristic “onion-skinning” cholangitis or fibro-obliterative
changes have been shown to occur only in allografts from PSC patients, but these features are seen only in a small percentage of
patients. Thus while specific, absence of these features does not rule out recurrent PSC. Features of biliary obstruction are more
commonly seen in recurrent PSC; however, these features are nonspecific. Currently, guidelines suggest that recurrent PSC should
be suggested in cases with a confirmed diagnosis of PSC before transplant; if there is cholangiographic evidence of extrahepatic
biliary obstruction, beading, and irregularities at least greater then 90 days after transplantation; or if there is histologic evidence of
fibrous cholangitis and/or fibro-obliterative lesions with or without ductopenia, biliary fibrosis, or biliary cirrhosis.
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Idiopathic Posttransplantation Chronic Hepatitis
Recently, a group of patients without any previous history or serologic evidence of viral hepatitis or drug reaction has been shown to
develop a picture resembling chronic hepatitis late in the posttransplant period (23). Remarkably, in one pediatric study, 64% of
allograft biopsies at 10 years posttransplant had histopathologic features of chronic hepatitis including portal inflammation,
necroinflammatory activity, and fibrosis. Less than 5% of these cases met the criteria of de novo autoimmune hepatitis; only 2 of 113
were positive for hepatitis C, whereas no cause was found in the vast majority of cases. Although only a subset met the criteria for
de novo autoimmune hepatitis, many patients had increased ANA and SMA titers, some above 1:100. It is possible that these clinical
and histopathologic abnormalities represent a subclinical form of de novo autoimmune hepatitis or atypical chronic rejection. These
authors suggest that reinstitution of steroid therapy may be beneficial to these patients. As most institutions do not routinely obtain
protocol liver biopsies late in the posttransplant course, these findings have yet to be corroborated at other institutions.
Posttransplant Opportunistic Infections

As the pediatric transplant recipient may be naïve for many viral infections and many would not have completed their vaccinations,
viral infections of the allograft tend to be more severe. Indeed, even live attenuated viral vaccines are contraindicated in transplant
patients due to the possibility of graft infections. Viral infections occur most commonly between 1 week to 2 months after
transplantation and tend to follow episodes of acute rejection due to increased immunosuppression (121). Thus, distinction between
ongoing acute rejection and new-onset viral infection may be difficult clinically and histologically. The most common viral infections
leading to graft dysfunction are CMV and EBV. However, other, more rare viral infections such as adenovirus, varicella virus, and
herpes simplex virus can lead to graft failure.
CMV hepatitis is fairly common in pediatric liver transplantation, affecting up to 10% of allografts. However, with the advent of
prophylactic CMV therapy, the incidence and severity of disease have been reduced. Close monitoring of patients receiving an
allograft from a CMV positive donor, is essential as CMV hepatitis tends to be more common and more severe. CMV infection of the
allograft can lead to a variety of histologic manifestations, some of which overlap with acute rejection. As classic eosinophilic nuclear
inclusions are rare and may be absent, reliance on multiple histologic features is necessary. Mild-to-moderate lymphocytic portal
inflammation is found in almost all cases of CMV hepatitis. In addition, one of the most sensitive, but not specific, findings in CMV
hepatitis is the presence of scattered clusters of necrotic hepatocytes surrounded by a neutrophilic infiltrate forming microabscesses
(Figure 8-10A). However, microabscesses can be found in a wide variety of conditions such as biliary obstruction, ischemia, other
infections, and sepsis (57), and, therefore, CMV immunohistochemistry is indicated when microabscesses are present. Kupffer cell
hyperplasia, hepatocyte ballooning, and parenchymal inflammation are other common findings. Slight lymphocytic cholangitis may be
seen and should not be mistaken for acute rejection.
Pediatric transplant recipients commonly develop manifestations of EBV infection that can ultimately lead to PTLD (67). Naïve
recipients of EBV-positive allografts are at a much higher risk of developing infection in the posttransplant period. Interestingly,
transplantation for Langerhans cell histiocytosis also predisposes to EBV-associated PTLD (98). Clinically, EBV infection first
manifests as fever, pharyngitis, lymphadenitis, and jaundice. Liver enzymes are typically elevated. EBV infection results in a wide
variety of histologic manifestations in the liver allograft. Nonspecific findings such as portal inflammation and sinusoidal mononuclear
infiltrates, often forming linear aggregates, are common. Scattered acidophil bodies, hepatocyte ballooning, and mild lobular disarray
with pseudoacinar formation and plate hypertrophy are commonly seen. If EBV infection is not controlled, progression to PTLD may
occur. Early lesions consist of atypical lymphocytes in the portal inflammatory cell infiltrate. Endothelialitis may be present, closely
mimicking acute rejection. Differentiation relies on the presence of a monotonous portal infiltrate with few atypical lymphocytes rather
than the mixed infiltrate seen in acute rejection (Figure 8-10C). Frank PTLD can range from diffuse large B-cell lymphoma to
Hodgkin-like lymphoma. Extrahepatic involvement is common. EBER as well as immunohistochemistry for CD20, kappa, and lambda
is useful to confirm the diagnosis (Figure 8-10D).
Adenovirus, though rare, is more common in pediatric transplant recipients than in adults who are more likely to have protective
immunity (42). Clinically, patients present with fever, difficulty breathing, diarrhea, and liver dysfunction. Usually infection occurs in
the first 3 months, and serotype 5 is the most common. Large granulomas along with random areas of necrosis are characteristic of
adenoviral hepatitis (Figure 8-10B). Smudgy and granular viral nuclear inclusions can be found at the edge of the necrotic zones
and can be documented by immunohistochemistry. HSV and VZ are other rare causes of posttransplant viral hepatitis and can lead
to fulminant hepatic failure if infection goes unrecognized. Once again, infection is more common and more severe in pediatric
patients as they are more likely to lack protective immunity. HSV and VZ cause similar histopathologic findings, and because it is
difficult to separate these two infections based on H&E alone, immunohistochemistry is required. As in adenoviral hepatitis, random
confluent areas of coagulative-type necrosis are common. In the center of the necrotic areas, ghost hepatocytes and neutrophils are
seen. At the edge of these zones, virally infected hepatocytes with characteristic ground-glass intranuclear inclusions may be
present. Multinucleated infected hepatocytes are sometime seen.
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FIGURE 8-10▪Posttransplant infections of the liver allograft. A: Cytomegalovirus is a common infection in the posttransplant setting.
Characteristic findings include microabscesses near infected cells. B: Adenovirus infection, although rare, is a serious complication
in the pediatric setting. Zonal necrosis along with smudgy nuclear inclusions is characteristic. C: The histologic findings in PTLD are
varied. In severe cases, the portal tracts are greatly expanded by a dense, atypical lymphoid infiltrate. D: In situ hybridization for
EBER may be essential in confirming the diagnosis of PTLD.
If VZ or HSV is suspected, rapid communication to the clinicians is essential. Immediate antiviral therapy and cessation of
immunosuppression may reduce graft dysfunction and prevent graft failure.
The Liver in Bone Marrow Transplantation

Bone marrow transplantation is used in the management of a wide variety of diseases, including aplastic anemia, leukemia, and
immune disorders (70). Hepatic changes seen in these patients may be related to chemotherapy and total-body irradiation in
preparation for transplantation, graft-versus-host disease (GVHD), or the infections to which these immunocompromised individuals
are susceptible. Veno-occlusive disease (VOD) or sinusoidal occlusive syndrome (SOS) is a complication of chemotherapy
(particularly cyclophosphamide-based regimens) and may be mild, with full recovery, or severe and lead to death (119). VOD usually
occurs within the first 30 days after transplantation; however, lateonset VOD has been reported with newer chemotherapeutic
regimens. Chemotherapeutic agents damage the sinusoidal endothelial cell layer, resulting in deposition of extracellular matrix in the
sinusoids and terminal hepatic veins occluding blood flow. This results in ascites, weight gain, painful hepatomegaly, and jaundice.
In the early phase, centrilobular hemorrhage is associated with damaged venules and sinusoids (Figure 8-11). There is narrowing of
the lumina and widening of the subendothelial zone, which may contain collagen fibers, siderophages, and cell fragments.
Progression is manifested by partial to complete occlusion of the venules and sinusoids and centrizonal hepatocellular atrophy. The
reticulin stain is particularly helpful in identifying the occlusive sinusoidal and venular lesions (Figure 8-11). Cholestasis and
distortion of the lobular architecture may occur (97).
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Mild disease is followed by recovery by day 20, but death from fulminant hepatic failure occurs in some cases.
FIGURE 8-11▪Veno-occlusive syndrome/sinusoidal obstructive syndrome. A: VOD/SOS is characterized by centrilobular

hemorrhage and necrosis. B: A reticulin stain is helpful in highlighting the sinusoidal and venular deposition of extracellular matrix.
Immunologic mechanisms of rejection in liver allograft recipients and GVHD in bone marrow transplant patients are similar. They
manifest many of the same histopathologic changes in the liver. Both are characterized by portal inflammation and bile duct injury in
the acute phase, and severe duct damage and duct loss in the chronic stages (109). The acute form of GVHD manifests 3 to 4
weeks after transplantation with a skin rash, diarrhea, and jaundice. The early changes consist of mild, nonspecific lobular hepatitis.
Liver biopsy specimens evaluated 1 to 2 weeks after the onset of the disease, show characteristic bile duct abnormalities. The bile
ducts show epithelial degeneration and necrosis and lymphocytic infiltration (Figure 8-12). Destruction of ducts and ductular
proliferation occurs with progressive disease. There is a lymphocytic portal inflammation, but spillover is usually minimal. Mild
hepatocellular changes, occasional acidophil bodies, and cholestasis are seen in the lobule. Endothelialitis of the portal and central
veins may be seen (104).
FIGURE 8-12▪Graft-versus-host disease of the liver. A: GVHD is characterized by destruction of the bile ducts with lymphocytic
infiltration. Extensive iron deposition is commonly seen in these bone marrow transplant patients. B: In chronic GVHD, bile ducts are
lost and cholestasis is evident. (Photos courtesy of Dr. John Hart, University of Chicago.)
Chronic GVHD, seen 100 to 400 days after bone marrow transplantation, affects 30% of long-term survivors. It may be preceded by
acute GVHD or develop in patients without prior episodes of disease. Chronic liver disease is seen in most patients with chronic
GVHD with multisystem involvement or as a limited disorder with cutaneous and hepatic involvement, which has a more favorable
prognosis. Although cirrhosis and its complications are unusual, micronodular cirrhosis leading to death from hepatic failure has
been reported (69, 70). The liver in chronic GVHD may show a histologic appearance of chronic hepatitis with portal infiltration by
mononuclear cells. Long-standing GVHD results in bile duct loss (Figure 8-12). Additional findings include portal infiltration by
plasma cells and cholestasis
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with pseudoxanthomatous changes. Endothelialitis is not a feature of chronic GVHD (104).
TRANSPLANT PATHOLOGY OF THE PANCREAS

Overview and the Role of Histopathology
Pancreatic transplantation is becoming increasingly utilized for type I diabetes mellitus and is most commonly performed in
conjunction with a kidney allograft due to end-stage renal failure. As end-stage renal failure in type I diabetes occurs mainly in
adulthood, the pediatric pathologist is rarely asked to evaluate pancreatic biopsies in this setting. Moreover, histopathologic
evaluation of pancreatic graft biopsies is rarely indicated in current practice for a variety of reasons. First, the pancreas is a very
active exocrine and endocrine organ; thus, levels of synthetic products produced by the pancreas can accurately gauge graft
function. Second, biopsies from the kidney have proven to be a fairly accurate surrogate in evaluating pancreatic rejection in
patients who receive kidney/pancreas transplants (87). Third, in some instances, biopsies can be difficult to obtain and could cause
severe injury to the pancreatic duct and vessels. Despite these caveats, biopsies obtained from the pancreatic grafts can
occasionally provide useful information to the clinician, and at some large centers, biopsies are routinely performed (54).
Histopathology of Acute and Chronic Rejection

In 1997, a histologic grading scheme was proposed for acute rejection that divided acute rejection into six grades based on the
degree of septal, acinar, ductal, and vascular inflammation (22). Inflammation in the islets of Langerhans is rarely encountered and is
not a feature of acute rejection. This grading scheme showed good reproducibility, prognostic significance, correlation with
laboratory data, and response to immunosuppressive treatment. Chronic rejection is characterized by fibrous expansion of the septal
areas and loss of acinar parenchyma. Islets initially are not affected; however, extensive fibrosis can lead to loss of glycemic control.
The vascular changes are similar to those seen in other solid organs with intimal and medial fibrosis and narrowing of the lumen.
Critical to the evaluation of allograft pancreatic biopsies is assessment for other causes of graft dysfunction such as bacterial,
fungal, or viral infections, especially CMV infection (53). Clinically and histologically, CMV pancreatitis can mimic acute rejection;
however, the presence of the characteristic nuclear inclusions in endothelial or stromal cells is diagnostic.
RENAL TRANSPLANT PATHOLOGY

Overview
Approximately 600 renal transplants are performed on patients less than 18 years old each year in the United States. Based upon
the 9,837 renal transplants performed in 8,990 patients from 1987 through 2005 in the North American Pediatric Renal Trials and
Collaborative Studies (NAPRTCS) database, the primary diagnoses leading to renal transplantation in children are developmental
abnormalities (renal aplasia/hypoplasia/dysplasia (15.9% of total), obstructive uropathy (15.8%), reflux nephropathy (5.2%),
polycystic kidney disease (2.9%), medullary cystic disease (2.8%), and agenesis of abdominal musculature (2.7%), followed by focal
segmental glomerulosclerosis (FSGS) (11.7%), specific glomerulonephritis (9.9%) in aggregate, chronic glomerulonephritis (3.4%),
and hereditary nephritis (2.2%). Over this same period, the proportion of living donor (LD) renal allografts has increased from 43% to
60%, 81% of which were from a parent, and the proportion of cadaveric donor (CD) allografts has declined from 57% to 40% (101).
From 1987 to 2000, the proportion of deceased donors less than 10 years of age declined from 35% to 10% due to the inferior
outcomes of small CD kidneys given to small recipients (8). Approximately 25% of pediatric patients require removal of their native
kidneys to alleviate problems related to polyuria, proteinuria (including hyperlipidemia and thrombophilia), recurrent pyelonephritis,
and hypertension; and a similar percentage of pediatric renal transplants are performed preemptively, before reaching dialysis-
dependent ESRD (7). One-year allograft survival improved from 91% in 1987-1995 to 94% in 1996-2000 for LD and 81% to 93% for
CD, and the projected allograft half-life (the time at which one-half of allografts will be lost) improved from 15.4 (LD) and 9.5 (CD)
years in the 1987-1989 cohort to 25.4 and 16.4 years for the 1996-1998 cohort (8). The causes of graft failure in pediatric renal
transplants are chronic rejection (41.3%), vascular thrombosis (8.1%), recurrence of the original disease (7.9%), and acute rejection
(6.3%) (101). Important morbidities in pediatric renal transplant recipients are growth failure, cardiovascular disease, and infectious
diseases (7), and infectious diseases now exceed rejection as the most common reason for hospitalization of pediatric renal
transplant recipients (84).
The pathologist may encounter autopsy, nephrectomy, biopsy, or cytologic specimens from pediatric renal transplant patients.
Approximately 20% of our pediatric renal biopsy specimens are from renal allografts, all of which were performed to determine the
cause of graft dysfunction. The differential diagnosis in that situation includes acute or chronic rejection, infection, drug toxicity,
ischemic injury, urinary obstruction, recurrence of the original disease, de novo primary renal disease, posttransplant
lymphoproliferative disorder, and chronic allograft nephropathy. The widely used Banff Classification of Renal Allograft Pathology
defines a “minimal” sample for interpretation as seven glomeruli and one artery and an “adequate” sample as at least ten glomeruli
and two arteries, and recommends that there be two cores or at least two separate cortical areas for examination (86, 106). Multiple
levels should be examined with hematoxylin and eosin (H&E), trichrome, periodic acid-Schiff (PAS), and Jones silver stains; and
immunofluorescent or immunohistochemical stains for C4d and BK polyomavirus
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should be performed. Immunofluorescent stains for other immunoglobulin and complement components to look for recurrent or de
novo disease or immunohistochemical stains for T-and B-lymphocytes and in situ hybridization for Epstein Barr virus to rule out
posttransplant lymphoproliferative disease are performed without hesitation when these conditions are suspected clinically or
pathologically.
Frozen section prior to implantation of kidneys from older donors has been recommended as a means of eliminating organs likely to
experience delayed graft function or decreased survival, but glomerulosclerosis, interstitial fibrosis, and vascular disease may be
difficult to recognize in frozen sections, and no absolute threshold has been established beyond which a donor kidney should not be
used (15). Similarly, although Sarwal et al. (92) noted that the presence of acute tubular necrosis in the graft largely obviated the
advantage of transplanting adult-sized kidneys to infants and small children, it is difficult to diagnose acute tubular necrosis in a
frozen section. Permanent sections of biopsies obtained at the time of reperfusion (0-hour) or 1 hour later may show lesions
predictive of hyperacute (neutrophils in glomeruli and peritubular capillaries) or acute rejection (neutrophils, macrophages or
platelets in peritubular capillaries) and can provide a baseline for the interpretation of subsequent biopsies (15).
Protocol or surveillance biopsies taken at predetermined times irrespective of graft function may show evidence of acute rejection in
the absence of clinical signs of graft dysfunction, allow earlier recognition of chronic lesions, and reveal unsuspected (and
potentially treatable) infectious or inflammatory conditions. Postulating that the “renal reserve” created by transplantation of an adult-
sized kidney to a small child may delay the clinical recognition of acute or chronic injury, Birk et al. (11) performed regular protocol
biopsies on 21 pediatric renal allograft recipients and found subclinical acute rejection in four patients (19%). This group also
performed routine follow-up biopsies 1 month after the diagnosis of acute rej ection and showed that improvement of serum
creatinine did not reliably predict resolution of rejection (10). In another descriptive study of protocol biopsies in children, Shishido et
al. (96) found that subclinical acute rejection superimposed on chronic allograft nephropathy (CAN) had more graft dysfunction and
diminished graft survival compared to patients with CAN alone. Protocol biopsies have also been used in studies of steroid-free
immunosuppressive drug regimens in pediatric renal allograft recipients (1, 93).
Hyperacute and Accelerated Acute Rejection and Delayed Graft Function

These early events represent variations on the theme of acute humoral rejection due to preformed antidonor antibodies and
ischemic acute tubular necrosis. Hyperacute rejection is characterized by graft swelling and tenderness, and anuria almost
immediately or within the first few days after transplantation. Glomerular capillaries are distended with platelets, erythrocytes, and
fibrin thrombi, and there is necrosis of glomerular endothelium and tubular epithelium. Neutrophils marginate in small vessels and
peritubular capillaries (an important finding that helps to distinguish humoral rejection from perfusional injury, arterial or venous
thrombosis, or cyclosporine toxicity). Interstitial edema and hemorrhage progress to coagulative necrosis. Although the process is
mediated by antibodies, immunofluorescent staining of vessels may be negative. C4d staining along peritubular capillaries similar to
that seen in acute and chronic humoral rejection, as discussed below, is often seen in hyperacute rejection (15). The disease
formerly known as accelerated acute rejection is also caused by preformed antibodies that may not be detectable until the plasma
cell clone has been stimulated by the graft. It typically occurs 1 to 12 weeks after transplantation and is manifested by necrotizing
arteritis or a thrombotic microangiopathy. Delayed graft function may be caused by a variety of donor factors, peritransplant ischemic
injury, or drug toxicity. Typical histologic findings are reminiscent of acute tubular necrosis and include dilation of tubular lumens,
loss of the brush border in proximal tubules, epithelial cell necrosis and apoptosis, and cellular casts (102).
Acute Rejection
Historically, renal allografts in children had higher rates and earlier and more refractory episodes of acute rejection than allografts in
adults (7), but, with improved immunosuppressive therapy, the 12-month probability of acute rejection in children in the NAPRTCS
series decreased from 54% to 13% in LD recipients and 69% to 16% in CD recipients between 1987-1990 and 2003-2005. 53% of
LD recipients and 47% of CD recipients achieved complete reversal of rejection (return to baseline creatinine values), and only 4%
and 6%, respectively, lost their grafts or died as a result of acute rejection (101). For LD recipients, the relative risk of developing
acute rejection is increased in African-Americans, history of prior transplant or more than five blood transfusions, HLA mismatch, lack
of induction therapy, and female gender; and for CD recipients, additional risk factors include recipient age less than 1 year, prior
dialysis, and cold ischemic time greater than 24 hours (101). Tables 8-3 and 8-4 summarize the criteria for the scoring of lesions and
classification of patterns of rejection in the Banff Classification of Renal Allograft Pathology.
Acute T-cell—mediated rejection: The minimum criteria for acute T-cell-mediated rejection in the Banff Classification (Type IA) are
the following: mononuclear cell infiltrates involving more than 25% of the parenchyma (≥i2) and at least two foci with five to ten
intraepithelial mononuclear cells in a tubular cross section or five to ten mononuclear cells per ten tubular epithelial cells in a
longitudinal section (≥t2) (Figure 8-13A). Interstitial inflammation without tubulitis is not diagnostic of rejection, and lesser degrees of
interstitial inflammation and tubulitis are considered borderline or “suspicious” for acute rejection, although immunosuppressive
therapy
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prior to biopsy may have reduced the interstitial inflammatory response, and in that context, i1t2 lesions may indicate rejection (86).
Interstitial inflammation is not graded in areas of fibrosis, the immediate subcapsular cortex, and the adventitia around large veins,
but this is being reevaluated. The interstitial infiltrate in acute rejection is often mixed, but if there are more than 5% to 10%
eosinophils, neutrophils, or plasma cells, an asterisk is added to the “i” score and other diagnoses should be considered
(hypersensitivity reaction, acute bacterial infection or infarction, and infection or posttransplant lymphoproliferative disorder,
respectively). Similarly, while tubulitis should be assessed in the most severely involved area, there should be more than one focus
with the highest grade of involvement, and since tubulitis in atrophic tubules may be seen in the absence of rejection, it should not
be graded in tubules that show a 50% or greater reduction in caliber. Most of the infiltrating lymphocytes in acute rejection will be T-
cells, and a predominantly B-cell infiltrate raises the question of a posttransplant lymphoproliferative disorder (86), while nodular
aggregates of B-cell aggregates in a predominantly T-cell infiltrate may identify allografts that will be refractory to standard
antirejection therapy but responsive to anti-B-cell immunotherapy (60).
Table 8-3 ▪ BANFF CLASSIFICATION OF RENAL ALLOGRAFT PATHOLOGY (86, 106)- I. LESION SCORINGa
Lesion Code Grade 0 Grade 1 Grade 2 Grade 3 Comment
Findings associated with cell-mediated rejection
Tubulitis t None ≥2foci with 1-4 ≥2 foci with 5- ≥2foci with Do not count atrophic
intraepithelial 10 >10 tubules <50% normal
lymphocytes intraepithelial intraepithelial size
per tubular lymphocytes lymphocytes
cross section per tubular per tubular
or per 10 cross section cross section
epithelial cells or per 10 or per 10
epithelial cells epithelial cells
Interstitial i <10% of 10%-25% of 26%-50% of >50% of Indicate >5%-10%

inflammation unscarred unscarred unscarred unscarred eosinophils,
parenchyma parenchyma parenchyma parenchyma neutrophils, or plasma
cells and B-cell
nodules with an
Total ti <10% of total 10%-25% of 26%-50% of >50% of total

interstitial parenchyma total total parenchyma
inflammationb parenchyma parenchyma
Arteritis v No intimal ≥1 artery with ≥1 artery with ≥1 artery with Indicate infarction
arteritis intimal arteritis intimal arteritis fibrinoid and/or interstitial
and <25% and > 25% change and hemorrhage with an
luminal luminal transmural
occlusion occlusion arteritis with
medial smooth
muscle
necrosis
Findings associated with antibody-mediated rejection
C4d staining C4d Negative Minimal (1%- Focal positive Diffuse Immunohistochemistry
(0%) <10% of area (10%-50% of positive (> may be one grade
with ≥2+ area with >2+ 50% of area less sensitive than
linear staining linear staining with ≥2+ linear immunofluorescence
along along staining along
peritubular peritubular peritubular
capillaries) capillaries) capillaries)
Glomerulitis g None Glomerulitis in Segmental or Glomerulitis Specify types of

a minority of global (mostly global) inflammatory cells
glomeruli glomerulitis in in all or almost
25%-75% of all glomeruli
glomeruli
Peritubular ptc No luminal Cortical Cortical Cortical Do not score if <10%

capillaritis inflammatory peritubular peritubular peritubular of cortical capillaries
cells in capillary with capillary with capillary with involved. Specify
cortical 3-4 luminal 5-10 luminal >10 luminal type(s) of luminal
peritubular inflammatory inflammatory inflammatory cells.
capillaries cells cells cells
Glomerular cg Double Double Double Double

double contours in contours in contours in contours in
contour <10% of 10%-25% of 26%-50% of >50% of
peripheral peripheral capillary loops peripheral
capillary capillary loops in the most capillary loops
loops in most in the most severely in the most
severely severely affected severely
affected affected nonsclerotic affected
nonsclerotic nonsclerotic glomerulus nonsclerotic
glomerulus glomerulus glomerulus
Chronic changes
Tubular ct No tubular Tubular Tubular Tubular
atrophy atrophy atrophy in atrophy in atrophy in
≥25% of the 26%-50% of >50% of the
area of the area of area of cortical
cortical cortical tubules
tubules tubules
Interstitial ci Fibrosis of Fibrosis of Fibrosis of Fibrosis of Do not score

fibrosis ≥5%of 6%-25% of 26%-50% of >50% of subcapsular fibrosis
cortical area cortical area cortical area cortical area
Subintimal cv No chronic ≥25% luminal 26%-50% >50% luminal Elastica breaks,

fibrosis in vascular narrowing by luminal narrowing inflammatory cells in
arteries changes fibrointimal narrowing fibrosis suggest
thickening chronic rejection
Findings associated with calcineurin-inhibitor toxicity
Arteriolar ah No PAS- Mild-to- Moderate-to- Severe

hyalinization positive moderate severe PAS- PASpositive
hyaline PAS-positive positive hyaline
thickening hyaline hyaline thickening in
thickening in thickening in many
at least one more than one arterioles
arteriole arteriole
Alternate aah No typical Nodular Nodular Circumferential

arteriolar lesions of hyaline hyaline hyaline
hyalinizationb CNI deposits in deposits in involvement;
arteriolopathy only one more than one independent of
arteriole and arteriole, but the number of
no no arterioles
circumferential circumferential involved
involvement involvement
aNote the number of glomeruli and arteries present and number of sclerotic glomeruli
bTotal interstitial inflammation and alternate arteriolar hyalinization are undergoing evaluation
Table 8-4 ▪ BANFF CLASSIFICATION OF RENAL ALLOGRAFT PATHOLOGY (106)-II. DIAGNOSTIC

CATEGORIES
T-cell-mediated rejection
Suspicious (v = 0 and (i = 0 or 1 and t = 1, 2 or 3))

OR (v = 0 and (i = 2 or 3 and t = 1))
IA V = 0 and (i = 2 or 3 and t = 2)
IB V = 0 and (i = 2 or 3 and t = 3)
IIA v1
IIB v2
III v3
Chronic active cv > 0 with mononuclear cells in subintimal fibrosis
Antibody-mediated rejection
C4d+ C4d = 3, antidonor antibody detected,

and no morphologic evidence of
rejection
I C4d = 3, antidonor antibody detected, and

ATN-like change
II C4d = 3, antidonor antibody detected,

and (ptc > 0 and/or g > 0 and/or
thromboses)
III C4d = 3, antidonor antibody detected, and

v=3
Chronic active C4d = 3, antidonor antibody detected,

(cg > 0 and/or ct > 0 and/or ci > 0 and
or cv > 0)
Interstitial fibrosis and tubular atrophy without evidence of any specific etiology
I ct = 1 and ci = 0 or 1
II ct = 2 and ci = 2
III ct = 3 and ci = 3
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Intimal arteritis, seen as lymphocytic infiltration beneath the endothelium of arteries (Figure 8-13B), is the criterion by which Type II
acute T-cell-mediated rejection is defined. Type IIA shows mild-to-moderate endarteritis in at least one arterial cross section (v1),
and Type IIB shows severe intimal arteritis with at least a 25% reduction of the luminal area in at least one arterial cross-section (v2)
(86). Because of the potential for sampling error, the most severely involved
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artery should be graded, and neither lymphocytes attached to (but not beneath) arterial endothelium nor lymphocytes in venous
walls should be graded. Transmural arteritis or fibrinoid mural necrosis with lymphocytic inflammation is the criterion by which Type
III acute rejection is defined. Interstitial hemorrhage and infarction are not sufficient for a diagnosis of Type III rejection but are
designated with an asterisk after the “v” score (86).
FIGURES 8-13▪ Acute and chronic rejection. A:Tubulitis is a feature of acute T-cell-mediated rejection, and the >10 intraepithelial
lymphocytes per 10 epithelial cells in nonatrophic or only partially atrophic tubules seen here is a t3 lesion (PAS 40×). B: Intimal
arteritis is indicative of grade II acute rejection, and the 25% to 30% luminal narrowing seen here is a borderline v2 lesion (H&E
40×). C: Bright ribbon-like staining for C4d along peritubular capillaries between tubules is the hallmark of antibody-mediated
rejection (Fluorescein-conjugated anti-C4d 40×). D: Double contours along glomerular capillary loops are a sign of chronic antibody-
mediated rejection (Jones methenamine silver 40×).
Acute antibody-mediated rejection (AMR): Demonstration of diffuse linear staining for C4d along peritubular capillaries (C4d3)
(Figure 8-13C) has become the hallmark of AMR (86) and is seen in 20% to 30% of biopsies for acute rejection. C4d is an inactive
fragment of complement component C4 that binds covalently to adjacent structures, thereby avoiding the modulation that makes the
immunoglobulins responsible for initiating the attack undetectable. In normal kidneys, immunofluorescent staining for C4d is found in
the mesangium and at the vascular pole of glomeruli, presumably a consequence of the physiologic turnover of immune complexes,
and may be seen along glomerular capillaries in immune complex disorders, but peritubular capillary staining is characteristic of AMR
(26). Other histopathologic features of AMR include neutrophils in peritubular and glomerular capillaries and neutrophilic tubulitis,
but these lesions are seen infrequently in some series, and the Banff Classification has categories of AMR in which there is C4d
staining without these features, alone and with changes consistent with acute tubular necrosis (Table 8-4). AMR typically has its
onset one to three weeks after transplantation but may arise after several months or years, especially if immunosuppression is
decreased. There is no correlation with HLA match, ischemic time, or donor age. During episodes of rejection,
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C4d-positive cases show higher serum creatinine levels and are less responsive to steroid and anti-T-cell immunotherapy compared
to C4d-negative cases (66), and long-term graft survival is significantly reduced (40). Most patients with positive C4d staining have
HLA class I or II donor-specific antibodies, but ABO and non-HLA antiendothelial antibodies have been demonstrated in a few
patients (66).
Chronic Allograft Nephropathy (CAN)

Chronic Allograft Nephropathy (CAN), the leading cause of renal allograft failure in children, is due to a combination of immunologic
and nonimmunologic mechanisms, ischemia, hypertension, infection, immunosuppressive drug toxicity and noncompliance, and
recurrent disease. In the original Banff 97 classification, CAN was graded on the extent of tubular atrophy and interstitial fibrosis, but
recently there has been an increased emphasis on identifying the underlying cause. Chronic transplant glomerulopathy,
characterized by the presence of double contours along glomerular capillary walls on silver stains (Figure 8-13D), fibrous intimal
thickening without duplication of the internal elastica in arteries, and diffuse C4d staining along peritubular capillaries, are
suggestive of late or chronic AMR; and mononuclear cells in peritubular capillaries or glomeruli and interstitial plasma cell infiltrates
may also be seen in this condition (107). In a pediatric series, 50% of biopsies that showed features of CAN were C4d-positive;
these biopsies showed more transplant glomerulopathy and increased mesangial matrix, and patients with C4d-positive CAN had a
higher rate of graft loss (39). Immunofluorescent microscopy of chronic transplant glomerulopathy may show nonspecific segmental
granular deposits of IgG or IgM and C3 in the capillary wall and mesangium, and electron microscopy shows widening of the
subendothelial space due to accumulation of electron lucent flocculent material, but no electron-dense deposits. Reduplication of the
external lamina of peritubular capillaries is associated with positive C4d staining (44, 66, 74). Fibrointimal thickening with
reduplication of the internal elastica (cv) and arteriolar hyaline change (ah) may be related to hypertension; peripheral hyaline
nodules in arterioles suggest chronic calcineurin-inhibitor (cyclosporine, tacrolimus) toxicity as discussed below; marked tubular
ectasia with Tamm-Horsfall casts raises the question of chronic obstruction; and intratubular neutrophils, lymphoid follicles, and viral
inclusions are seen in infections (107).
Vascular Thrombosis
Vascular thrombosis accounts for 8.1% of graft failures and is the second leading cause of allograft loss in the NAPRTCS series
(101). Risk factors for vascular thrombosis include peritoneal dialysis prior to transplantation, cadaver kidneys from donors less than
6 years old or with more than 24 hours cold ischemic time, recipients less than 2 years old, and a history of prior transplant (8).
Recurrent Disease
Recurrence of the original disease that necessitated renal transplantation accounts for 7.9% of allograft loss in children (101). In
addition, any acquired renal disease may develop de novo in renal allografts. The most common recurrent disease in pediatric renal
allograft recipients is focal segmental glomerulosclerosis (FSGS), which accounts for 12% of ESRD leading to transplantation in all
children and adolescents in the United States and 23% among African-American patients, and recurs in the allograft in 20% to 50%
of patients. Rapid progression from onset to ESRD, younger age, white race, mesangial proliferation on biopsy, and recurrent
disease in one allograft are associated with a higher risk of recurrence (7). Autosomal recessive FSGS due to NPHS2 mutations
appear to have a much lower risk of recurrence after transplantation (95). Recurrences occur early, 78% within the first
posttransplant month (95), and patients with primary FSGS also experience twice the rate of early graft nonfunction/acute tubular
necrosis, requiring dialysis compared to all other groups, raising the question of subclinical recurrence (7).
Membranoproliferative glomerulonephritis (MPGN) Type I accounts for 2.1% of ESRD leading to transplantation in children (101)
and recurs in 30% to 77% of allografts, resulting in loss of the graft in approximately one-fourth to onethird of patients with recurrent
disease (95). MPGN Type II (dense deposit disease) accounts for 0.9% of pediatric renal transplants (101), and recurs in nearly all
allografts, but this results in graft loss in only 10% to 20% of patients (95). Lupus nephritis accounts for 1.6% of ESRD leading to
transplantation in children (101) and may recur in 30% of allografts, but the incidence of graft failure due to recurrent disease is low
(95). IgA nephropathy accounts for 1.3% of pediatric renal transplants (101) and has been reported to recur in 65% of adults who
had a graft biopsy for any reason (83). However, graft loss from recurrence was only 7% in one pediatric series and 3% in adults
(95). Henoch-Shoenlein purpura nephritis accounts for 1.4% of ESRD leading to transplantation in children (101), and recurrence
has been reported in 53% of allografts, all from living related donors, and 22% of grafts were lost (95). Congenital nephrotic
syndrome accounts for 2.6% of pediatric renal transplants (101), and though proteinuria recurs in 25% of patients with the Finnish
type of congenital nephrotic syndrome (CNF) who receive transplants, these patients do not appear to have recurrent CNF (56).
Membranous glomerulonephritis (MGN) accounts for 0.5% of ESRD leading to transplantation in children (101), and recurrence has
been reported in adults but not in children. However, de novo MGN was reported in initial allografts of seven children, four of whom
developed MGN in subsequent allografts (37). Familial nephritis accounts for 2.2% of pediatric renal transplants (101), but half of
males with X-linked Alport syndrome will require transplantation by age 25. This genetic disease does not recur in the allograft, but
crescentic glomerulonephritis due to antiglomerular
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basement membrane antibodies develops in 3% to 5% of transplanted Alport males, and nearly 90% of these grafts will fail (50).
Hemolytic uremic syndrome (HUS) is the most common cause of acute renal failure in children in developed countries and accounts
for 2.7% of pediatric renal transplants (101). HUS recurred in only 1 of 118 (0.8%) transplanted children with classic postdiarrheal
HUS, but in 13 of 63 (21%) of those with atypical HUS, and 5 of 11 (45%) of those with HUS due to Factor H deficiency (62). The
rate of graft failure in a smaller series of recurrent HUS was 83% (85), and one would suspect that the rate of recurrence in
subsequent grafts would also be high. A thrombotic microangiopathy indistinguishable from HUS may develop in transplants as a
result of humoral rejection, drug toxicity (oral contraceptives, cyclosporine and, rarely, OKT3), pregnancy, or other infection.
Cystinosis accounts for 2.1% of ESRD leading to transplantation in children (101), and cystine deposits commonly occur in renal
allografts of patients with cystinosis. This does not appear to affect graft function, but neither does the renal allograft prevent the
systemic complications of cystinosis. In contrast, recurrence of oxalate deposits in oxalosis, which accounts for 0.5% of pediatric
renal transplants (101), does impair graft function, and combined liver and kidney transplantation is the preferred treatment. Graft
survival in children transplanted for ESRD due to urologic abnormalities is comparable to children with normal urinary tracts if the
abnormalities can be corrected and careful attention is paid to possible sources of infection (9). Wilms tumor (WT) and Denys-Drash
syndrome (DDS) each account for 0.5% of pediatric renal transplants (101), and transplantation should be delayed for 1 to 2 years
after completion of chemotherapy.
Immunosuppressive Drug Toxicity

Of the immunosuppressive agents currently used in solid organ transplantation, the calcineurin inhibitors, cyclosporine and
tacrolimus, have the most significant renal toxicity. The monoclonal and polyclonal antibodies that deplete the T-cell pool (OKT3 and
antilymphocyte and antithymocyte globulins) or inhibit interleukin-2 (Basiliximab and Daclizumab) rarely cause renal disease (100).
However, there have been case reports of glomerular and larger renal vessel thrombosis with OKT3 (15). Steroids and
antiproliferative agents have numerous adverse effects but generally do not cause renal lesions. However, sirolimus has been
reported to delay recovery from acute renal failure in cultured mouse proximal tubular epithelium (61) and to result in delayed graft
function, which resulted in a myeloma-like cast nephropathy when sirolimus was used in combination with tacrolimus, possibly
because these two drugs are metabolized by the same pathway (102). Sirolimus has also been reported to rarely cause reversible
proteinuria and thrombotic microangiopathy (15).
FIGURE 8-14▪The PAS-positive nodules in the wall of the arteriole at the lower left are seen in calcineurin-inhibitor toxicity (PAS
40×).
Calcineurin inhibitors (CNI) can cause characteristic lesions in glomeruli, tubules, the interstitium, and vessels. Glomerular
thrombotic microangiopathy (TMA) and isometric vacuolization of proximal tubular epithelial cells indicate acute or ongoing toxic
injury, while chronic toxicity results in hyaline changes in arterioles and striped interstitial fibrosis (107). The differential diagnosis of
glomerular TMA includes antibody-mediated rejection, but CNI-induced TMA does not show C4d staining along peritubular
capillaries. The cytoplasmic vacuoles in CNI tubulopathy are small and uniform, in contrast to the large irregular vacuoles seen with
ischemic tubular injury, and they do not stain with H&E or PAS stains. The differential diagnosis includes an osmotic nephrosis due
to agents such as mannitol and intravenous immunoglobulin. Arteriolar lesions include ballooning of smooth muscle cells, probably
an early and reversible lesion like isometric vacuolization in tubules, and PAS-positive mural hyaline nodules along the adventitial
aspect of the vessel (Figure 8-14). The differential diagnosis of the hyalinosis includes diabetes mellitus and hypertension, but the
subadventitial nodules are relatively specific for CNI toxicity (15).
Polyomavirus Type BK (BKV) Infection
Cytomegalovirus and adenovirus are important causes of infection in renal transplant patients and can be diagnosed in biopsies on
the basis of characteristic inclusions or positive immunohistochemical stains. Epstein-Barr virus infection may lead to posttransplant
lymphoproliferative disorders and is best diagnosed by in situ hybridization. However, over the past decade, polyomavirus type BK
(BKV) has become the most important infection in kidney transplant patients. BKV infection develops in 1% to 5% of renal transplant
recipients, and one-half of these patients lose graft function. Up to 90% of the population worldwide is BKV-seropositive, and the
virus is known to persist in renal allografts (41). Histologically confirmed BKV nephropathy developed in six of 173 (3.5%) of
pediatric renal transplant recipients 4 to 47 months
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after transplant (median 15 months), which led to reduced long-term graft function, and BKV nephropathy was significantly
associated with recipient seronegativity (99). Allograft biopsies with BKV nephropathy characteristically show large basophilic
nuclear inclusions in tubular epithelial cells (Figure 8-15) that stain strongly with antibodies to the SV-40 T antigen, but central pale
inclusions surrounded by dark chromatin and vesicular nuclei have been described. In early stages of viral replication, the
immunohistochemical stain may be positive in normal-appearing nuclei and in late stages inclusion-bearing cells may be negative
(15). Interstitial inflammation and tubulitis may be present in more advanced BKV nephropathy, and biopsies may show both acute
rejection and BKV infection. Inclusion-bearing cells, known as “decoy” cells, can be identified in the urine, and in a prospective study
of 78 adult renal allograft recipients, decoy cell shedding was seen in 30%, viremia assessed by nested PCR in 13%, and biopsy-
proven nephropathy in 8%. With biopsy as the diagnostic standard, decoy cells had a sensitivity of 100% and specificity of 71%, and
BKV viremia had a sensitivity of 100% and specificity of 88%, but the viral load in patients with BKV nephropathy was significantly
higher than in those without nephropathy (41).
FIGURE 8-15▪ BK virus infection is evidenced by the staining of enlarged nuclei in these tubular epithelial cells. Mouse anti-BK virus
large T antibody 40×.
Posttransplant Lymphoproliferative Disorders

Posttransplant lymphoproliferative disorders (PTLDs) occur in 1% or less of pediatric renal transplant recipients, although there may
be considerable variations in case definition between centers since EBV infection may present along a continuum of clinical features
(33). There is also a continuum of pathologic lesions ranging from benign lymphoid hyperplasia to polymorphic PTLD to
monomorphic PTLD, and tissue from suspected cases should be handled in the same manner as a suspected non-Hodgkin
lymphoma, with samples sequestered for possible flow cytometry, immunohistochemical studies for T-cells and B-cells, in situ
hybridization for EBV, and B-cell gene rearrangement studies.
While the native kidney may be involved in PTLD arising in the setting of transplantation of other organs, the differentiation of PTLD
from acute cellular rejection is a problem unique to the renal allograft. Typically, the interstitial infiltrate is very dense and is either
monomorphic or contains a range of lymphoid cells, but no neutrophils or eosinophils, and tubulitis and endarteritis are usually
absent. Since most PTLDs are B-cell proliferations, in contrast to the predominantly T-cell proliferation seen in acute cellular
rejection, and more than 90% harbor the EBV genome, a combination of immunohistochemistry for T-cells and B-cells and in situ
hybridization for EBV-encoded nuclear RNAs (EBERs) can sort out most cases (116).
PATHOLOGY OF HEART TRANSPLANTATION

Overview
In 1967, the first successful human heart transplant was achieved but survival was limited by infection, graft failure/hemodynamic
collapse, and rejection. Important milestones include introduction of biopsy forceps for percutaneous endomyocardial biopsies in
1973, the development of calcineurin inhibitors and the introduction of Cyclosporine A in the heart transplant population in 1980, and
the introduction of antithymocyte globulin (ATG) in 1990 for both induction and treatment of rejection.
Volumes and Indications

The total number of pediatric heart transplant procedures reported to the Registry of the International Society of Heart Lung
Transplantation (ISHLT) has remained stable for the past 15 years at approximately 400 procedures per year (12). The 1st year of
life is the single most common year for a heart transplant procedure in patients aged 18 years and younger. The indications for
transplantation include congenital cardiac malformations in the infant [most often after surgery(ies)], and in the older child
cardiomyopathy (dilated), congenital malformation [also after surgery(ies)], endocardial fibroelastosis, adriamycin toxicity, and
retransplantation for chronic rejection. Dilated cardiomyopathy does not recur in the transplanted heart; however, the patient is at
higher risk for developing chronic rejection again after retransplantation for it.
Surgical Complications
In the current setting, surgical complications are exceedingly rare and include hemorrhage and wound infections.
Rejection
Endomyocardial biopsy (EMB) remains the gold standard for rejection surveillance. It has a high sensitivity and specificity for the
diagnosis of acute cellular rejection. There are currently no cardiac imaging modalities or serum markers that can replace it.
Typically, surveillance biopsies are performed
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once weekly for the 1st month, every 2 weeks for the 2nd month, and every 6 to 8 weeks between the 3rd and 12th months. After
the 1st year, the frequency can be decreased to quarterly, biannually, or annually. The current working formulation suggests a
minimum of three step levels for microscopic examination. No special stains are routinely required. Unstained slides can be saved
for immunohistochemical staining if needed. One to two pieces of biopsy should be obtained in addition and frozen for
immunofluorescence staining, if clinically indicated (115).
Table 8-5 ▪ OLD AND REVISED GRADING SYSTEMS OF THE ISHLT FOR ACUTE CELLULAR REJECTION
1990 2005
No rejection (grade 0) No rejection (grade 0R)

Focal, mild acute rejection Mild, low-grade, rejection: interstitial and/or perivascular cellular infiltrate with up to one
(grade 1A) focus of myocyte damage (grade 1R)
Diffuse, mild acute rejection
(grade 1B)
Focal, moderate acute
rejection (grade 2)
Multifocal moderate Moderate, intermediate-grade, rejection: two or more foci of cellular infiltrate with associated
rejection (grade 3A) myocyte damage (grade 2R)
Diffuse, moderate rejection Severe, high-grade rejection: diffuse cellular infiltrate with multifocal myocyte damage ±
(grade 3B) edema, ± hemorrhage ± vasculitis (grade 3R)
Severe acute rejection
(grade 4)
Hyperacute rejection is graft injury triggered by preformed antibodies and occurs rapidly after implantation of the graft, usually within
minutes to hours. This type of rejection is now extremely rare.
Acute cellular rejection consists of an inflammatory infiltrate that is predominantly a T-cell-mediated response directed against the
cardiac allograft. A substantial increase in activated B-lymphocytes and natural killer cells is seen in moderate rejection, suggesting
their important role as promoters and effectors of cellular rejection. Eosinophils and neutrophils are also present in severe rejection.
The grading system for acute cellular rejection has been revised by the ISHLT such that the old system can easily be translated into
the new one, which is simpler and more reproducible (Table 8-5; Figures 8-16,8-17,8-18,8-19,8-20 and 8-21; eFigures 8-1 and 8-2)
(113). In most transplant centers, mild as well as focal moderate rejection (grades 1A, 1B, 2/1R) is not treated if patient is
asymptomatic and there is no clinical indication of rejection.
FIGURE 8-16▪Negative for acute cellular rejection (grade 0/0R). Pediatric heart biopsies appear more cellular than adults do since
the myocytes are smaller. Also, capillary endothelium can be quite prominent in posttransplant biopsies (H&E, 200×).
FIGURE 8-17▪Focal mild acute cellular rejection (grade 1A/1R). There is a focal infiltrate of lymphocytes between the myocytes and
involving fat, which is often present in posttransplant biopsies (H&E, 200×).
Antibody-mediated rejection (AMR): AMR is an immunopathologic process associated with the production of antidonor-reactive
antibodies in which injury to the graft is, in part, the result of activation of the complement system. It is poorly responsive to
conventional immunosuppression, which targets the cellular arm of the immune response. Risk
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factors for developing AMR include blood transfusions, previous transplantation, use of ventricular assist devices, presence of
positive B-cell flow cytometry cross-match, and elevated panel-reactive antibodies. AMR has been associated with the development
of cardiac allograft vasculopathy (CAV) and decreased survival (88).
FIGURE 8-18 ▪ Mild acute cellular rejection (grade 1B/1R). There is a sparse but diffuse lymphocytic infiltrate between myocytes,
without any myocyte damage (H&E, 200×).
FIGURE 8-19 ▪ Focal moderate acute cellular rejection (grade 2/1R). There is one focus of activated lymphocytes associated with
myocyte damage (arrow) (H&E, 200×).
FIGURE 8-20▪Multifocal moderate acute cellular rejection (grade 3A/2R). The biopsy has two separate foci of moderate rejection
seen in A and B (H&E, 200×).
FIGURE 8-21▪Diffuse moderate acute cellular rejection (grade 3B/3R). There is a marked infiltrate associated with myocyte damage
and few eosinophils and neutrophils (H&E, 200×).
Histological features are capillary endothelial changes (swelling or denudation with congestion), macrophages and neutrophils in
capillaries, interstitial edema, and/or hemorrhage and fibrin in vessels. Immunopathologic evidence of AMR includes
Immunoglobulin (IgG, IgM, and/or IgA) plus complement deposition (C3d, C4d, and/or C1q) in capillaries by immunofluorescence
on frozen sections (Figure 8-22); and/or
CD68 staining of macrophages within capillaries (CD31-positive or CD34-positive) by immunohistochemistry; and
C4d staining of capillaries by paraffin immunohistochemistry (Figure 8-23) (25).
Chronic rejection (CAV) involves both epicardial and intramural coronary arteries. The whole length of the coronary vessels is
usually affected. There is diffuse concentric
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narrowing with luminal stenosis due to intimal fibrosis (eFigures 8-3 and 8-4) with long-term lesions resembling conventional
atherosclerosis (Figure 8-24). The incidence of CAV in children is 2.5% at 1 year, 11% at 5 years, and 16.7% at 10 years after
transplantation, which is much lower than that reported for adults. Infant recipients had the lowest risk of CAV, likely due to their
lower incidence of acute cellular rejection.
FIGURE 8-22▪Positive C4d staining is seen in all the vessels in this heart biopsy (Courtesy of Dr. Anthony Chang, University of
Chicago Medical Center, indirect immunofluorescence, 40×).
FIGURE 8-23▪Positive C4d staining is seen in all the vessels in this heart biopsy with strong endothelial staining
(immunohistochemical stain, 200×).
Infection
These chronically immunosuppressed patients are prone to bacterial and opportunistic infections mostly in the lungs, GI tract, skin,
and nervous system. Infection of the heart itself is rare; toxoplasmosis and CMV are seen most often.
The incidence of PTLD seems to be decreasing from the 3% to 5% reported in the past, perhaps due to better immunosuppressive
regimens. The proliferation is EBV driven and can be polyclonal lymphoplasmacytoid or monoclonal. It most often involves
extracardiac sites such as lymph nodes, gastrointestinal tract, lung, and skin.
FIGURE 8-24▪Chronic rejection (cardiac allograft vasculopathy) is seen in this epicardial coronary artery, which has eccentric intimal
fibrosis (H&E, 20×).
Other Complications
Hypertension is reported in 47% at 1 year, 63% at 5 years, and 72% of pediatric recipients at 10 years after transplantation. Renal
dysfunction occurs in 6% at 1 year, 9% at 5 years, and 17% at 10 years. Hyperlipidemia also increases steadily to 38% at 10 years
after pediatric transplantation (12).
Other Biopsy Findings

Quilty lesion: This is an endocardial lymphocytic lesion composed of mature lymphocytes with a central dendritic cell network upon
which the B- and T-cells are organized (neolymphogenesis) (94). The infiltrate often extends into the underlying myocardium where
it may be associated with myocyte damage and fibrosis (Figure 8-25). It is not known to be related to acute or chronic rejection,
infection, ischemic time, or poor outcome. The main issue is to differentiate it from cellular rejection and avoid over-treatment
(eFigures 8-5 to 8-8)
Adipose tissue: Over time, more and more fat accumulates in the transplanted heart and can be seen in the endomyocardial
biopsies (eFigure 8-9). Only when epicardial mesothelium is identified, should one alert the cardiologist as to the possibility of
perforation.
Site of previous biopsy: Due to the structure of the heart, the bioptome tends to be guided to the same location for each biopsy.
Thus, it is very common to see organizing biopsy site with fibrin, mild inflammation, granulation tissue, and fibrosis (eFigure 8-10).
Calcifications: Occasionally, dystrophic microcalcifications are seen on biopsy. These can be located in the myocyte or in areas of
scarring.
Fibrosis: Focal fibrosis is often seen on biopsy, especially after the 1st year posttransplantation. It may represent old biopsy site,
healed infarct, or drug-induced fibrosis.
FIGURE 8-25▪Quilty lesion. This endomyocardial biopsy shows a large infiltrate between myocytes, composed of mature
lymphocytes with multiple capillaries (H&E, 100×).
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Outcome
Overall survival is approximately 40% for patients up to 20 years after transplantation. The median survival is 15.8 years for infant
recipients, 14.2 years for childhood-age recipients, and 11.4 years for adolescents. Late survivors have continued to show excellent
rehabilitation in terms of functional status. The two main posttransplant morbidities that have steadily increased are CAV and renal
failure. Malignancy has remained an important but low-frequency event (12).
PATHOLOGY OF LUNG TRANSPLANTATION

Lung transplantation, single, bilateral, or, less often, heartlung, has been an accepted mode of therapy for a variety of end-stage
lung diseases for about 20 years. Methods of evaluation of allograft dysfunction are variable and, depending on the clinical
differential diagnosis, can include transbronchial biopsy (TBB), bronchoalveolar lavage (BAL) with culture, endobronchial biopsy
(EBB), and, least often, wedge biopsy and fine needle aspiration biopsy (FNAB).
Volumes and Indications

Since 1995, the numbers of procedures worldwide that are reported to the ISHLT have been fairly constant, with 76 lung transplants
in 2006. The majority of recipients are between 12 and 17 years of age with less than five procedures per year in infants (3). The
most common indications for children are cystic fibrosis (CF) and primary pulmonary arterial hypertension (PPAH), in contrast to
adults who are transplanted for emphysema, CF, idiopathic pulmonary fibrosis, and PPAH. Heritable surfactant deficiency and
alveolar capillary dysplasia are indications in neonates. There is no recurrence of original disease in children except for the rare
patient who gets retransplanted for chronic rejection and is at higher risk for developing chronic rejection again.
Vascular Complications
Postsurgical obstruction/thrombosis of the arterial or venous anastomosis, although rare, is a surgical emergency. Inflammatory
cells, endothelial disruption, and recent thrombus are seen in the early posttransplant period, while organizing/organized thrombus,
stenosis, and fibrosis with foreign body giant cells are present in the intermediate to late period.
Airway Anastomotic Complications

The anastomosis heals by formation of granulation tissue, the surface of which reepithelializes in a few days. Occasionally,
exuberant polypoid granulation tissue forms, which may need to be removed. Varying degrees of ischemic injury, manifested as
coagulative necrosis of airway wall components, are commonly present. Superimposed infection may interrupt and complicate the
healing process. Common organisms found on culture, endobronchial biopsy, and special stains include fungi (Candida and
Aspergillus sp.) and bacteria. Fungi tend to invade necrotic cartilage. Dehiscence of the anastomosis can allow infection to spread
into the mediastinum. Healing may result in fibrosis and stenosis of the airway, treatment for which includes stent placement.
Primary Graft Dysfunction

Primary graft dysfunction occurs in 22% of pediatric lung recipients (similar to adults) in the first 30 days after transplantation due to
some combination of ischemia, reperfusion, surgical trauma, denervation, and interruption of lymphatics, resulting in endothelial
injury and pulmonary edema with or without diffuse alveolar damage. Recovery occurs in the majority of patients in a few days to
weeks with supportive therapy, although the mortality and morbidity rates are high (73).
Rejection
Definite diagnosis and grading of rejection (especially acute rejection) are based on light microscopic examination of tissue obtained
by TBB, which may be performed based on the clinical symptoms or based on a surveillance protocol. Since rejection is a patchy
process, it is recommended that five fragments of alveolated lung tissue be examined at three different levels stained with
hematoxylin and eosin. A working formulation for the grading of pulmonary allograft rejection, initially developed in 1990 and revised
in 1996 (Table 8-6) and 2007, is widely used (112).
Hyperacute Rejection
Only a few well-documented cases of hyperacute (humoral or antibody-mediated) rejection of the lung (all in adults) have been
reported in the literature (47). Preformed antibodies bind to the endothelium and epithelium of the donor lung and activate
inflammatory, complement, and coagulation
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cascades. Within minutes to hours after transplantation, there is progressive respiratory failure, pulmonary edema, and pleural
effusion, with complete opacification of the allograft seen on radiologic examination. The histologic features of hyperacute rejection
include diffuse alveolar damage (DAD), alveolar hemorrhage, interstitial neutrophilia, fibrin thrombi and vasculitis.
Table 8-6 ▪ WORKING FORMULATION FOR THE CLASSIFICATION AND GRADING OF PULMONARY
ALLOGRAFT REJECTION
A. Acute rejection (perivascular)

Grade 0—none
Grade 1—minimal
Grade 2—mild
Grade 3—moderate
Grade 4—severe
B. Airway inflammation—lymphocytic bronchitis/bronchiolitis

Grade 0—none
Grade 1—minimal
Grade 2—mild
Grade 3—moderate
Grade 4—severe
C. Chronic airway rejection—bronchiolitis obliterans

Ca. Active
Cb. Inactive
D. Chronic vascular rejection—accelerated graft vascular sclerosis
Modified from Yousem SA, Berry GJ, Cagle PT, et al. Revision of the 1990 working formulation for the classification of
pulmonary allograft rejection, Lung Rejection Study Group. J Heart Lung Transplant 1996;15:1-15.
There is deposition of IgG and complement in the alveolar septa. Complement fragments C3d and C4d may also be detected. If
fresh frozen tissue is not available for immunofluorescence studies, C4d deposition can be demonstrated in the vascular
endothelium and/or the interstitium by IHC. Only strong staining without background should be interpreted as positive. However,
staining is patchy and the sensitivity and specificity of C4d staining are low.
Acute Rejection
Acute rejection is a cell-mediated process during which there is progressive infiltration of the graft by host mononuclear cells.
Immune cell activation causes release of inflammatory chemokines and upregulation of adhesion molecules. Major cellular targets
include endothelial and epithelial cells. With current immunosuppressive therapy, it is rare for a lung transplant recipient to die of
acute cellular rejection.
Although acute rejection can develop as early as 3 days to many years posttransplant, most patients experience some rejection
commonly around 3 months, with most episodes occurring between 2 and 9 months (43). Noncompliance with immunosuppressive
medications is a significant cause of late episodes of acute rejection.
Acute rejection is characterized by a predominantly lymphocytic infiltrate with scattered eosinophils, neutrophils, and plasma cells.
The infiltrate begins in the perivascular areas and variably extends into the airways and lung parenchyma. In minimal acute rejection
(grade A1/B1), there are scattered infrequent perivascular and airway mononuclear infiltrates forming two to three layers that are not
obvious at low magnification (Figure 8-26). Mild acute rejection (grade A2/B2) consists of greater than three layers of activated
lymphocytes, eosinophils, and neutrophils around small blood vessels (Figure 8-27) or a band-like infiltrate in the airway submucosa
(eFigure 8-11). Moderate acute rejection (A3/B3) is characterized by an extension of the inflammation into alveolar septa with or
without vasculitis or a band-like infiltrate in the submucosa extending into the airway epithelium with focal epithelial necrosis. In
severe acute rejection (grade A4/B4), diffuse perivascular, interstitial, and air space infiltrates associated with pneumocyte damage,
macrophages, hyaline membranes, hemorrhage and neutrophils or epithelial ulceration with fibrinopurulent exudates are seen.
FIGURE 8-26▪Minimal acute cellular rejection of lung (A1). There is an incomplete perivascular cuff of lymphocytes (H&E, 100×).
FIGURE 8-27 ▪ Mild acute cellular rejection of lung (A2). There is a complete perivascular cuff, more than three layers thick, which is
readily apparent at low power (H&E, 40×).
In the 2007 revision, the grading of perivascular and interstitial infiltrates remains the same (i.e., A0 to A4); however, the airway
inflammation is changed to B0 (none), B1R (lowgrade, 1996 B1 and B2), Grade B2R (high-grade, 1996 B3 and B4) (112). The latter
poses a problem for those centers that treat grade B2 rejection in a manner similar to grade A2. Asymptomatic minimal rejection is
clinically insignificant and not treated. Mild (grade A2/B2) and higher grades are treated irrespective of symptoms.
The main differential diagnosis is infection, and microbiologic cultures and TBB are most useful to distinguish this (eFigure 8-11).
Aspiration is a common event, which is gaining more significance since it may trigger episodes of acute rejection and increase the
risk of patients to develop chronic rejection (eFigures 8-13 to 8-15) Bronchial-associated lymphoid tissue (BACT) is often prominent
to lung transplant recipients, and care should be taken not to overcall it as rejection (eFigure 8-16).
Chronic Rejection
In the lung, chronic rejection is primarily manifested as bronchiolitis obliterans (BO). Despite improved baseline immunosuppression
and treatment of acute rejection, BO remains the most important cause of late graft failure. Although its etiology and pathogenesis
are still not completely understood, acute rejection is certainly one of the most important risk factors. In general, the process of
chronic rejection is
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believed to occur in stages. The initial wave of antibodymediated response is paralleled by a cellular infiltrate in which the
monocyte/macrophage compartment plays a central role as the critical effector cells. The high antigenicity of airway epithelial cells
through the upregulated expression of MHC, adhesion and co-stimulatory molecules, together with the abundance of antigen-
presenting cells and circulating lymphocytes, provide an increased propensity to damage of these structures, similar to epithelial-
lined conduits in other solid allografts (e.g., bile ducts, pancreatic ducts, and renal tubules). The production of inflammatory
mediators and growth factors contribute to the fibroproliferative response of the damaged graft leading to BO.
Although the term chronic implies a late temporal process, BO can be seen as early as three to six weeks after transplantation, but
primarily occurs 1 or more years later. The onset of chronic rejection is insidious with vague general symptoms and nonproductive
cough. There is progressive dyspnea on exertion and irreversible decline in pulmonary function tests, not explained by other causes
such as infection. When the decline is greater than 10% of baseline, a clinical diagnosis of bronchiolitis obliterans syndrome (BOS)
is made, which does not need pathologic confirmation. BOS is graded from 1 to 3 based on the degree of loss of lung function (13).
When the clinical diagnosis is not clear, a wedge biopsy is often needed since BO is a patchy process and diagnostic yield of TBB is
low.
BO is patchy both in distribution and severity in individual lobes and in the same airway. There is submucosal fibrosis, which either
bulges asymmetrically into the lumen and causes partial obstruction or is concentric and causes total obstruction (Figure 8-28;
eFigure 8-17). Chronic vascular rejection occurs much less frequently and is histologically similar to the transplant vasculopathy
seen in other solid organ allografts (intimal fibrosis and vascular thickening); however, in the lung, it does not usually cause
significant allograft dysfunction.
The main histologic differential diagnosis is organizing pneumonia (formerly known as bronchiolitis obliterans organizing pneumonia
or BOOP), which is a healing response to various forms of lung injury and manifests as loose fibromyxoid plugs of connective tissue
within alveoli and bronchioles. On the other hand, BO is a dense scar tissue (mature collagen) within small airways.
FIGURE 8-28▪Chronic rejection (bronchiolitis obliterans). Eccentric submucosal fibrosis partially occludes the lumen of this
bronchiole (H&E, 100×).
Once there has been a decrease in lung function due to BO, it cannot be reversed, but aggressive immunosuppression can stabilize
the disease for variable periods of time. Some patients can live with BO for a few years, but others have progressive dysfunction
and complications and die unless retransplanted.
Infections
Like any immunocompromised patient, lung transplant recipients are at high risk of developing infections, which can be bacterial,
viral, or fungal, and may cause tracheobronchitis, localized infection of the airway anastomosis or pneumonia. Most bacterial
infections occur in the first posttransplant month, whereas viral and fungal infections tend to be seen in the 3- to 6-month period
since they are on immunosuppressive drugs. Lung transplant patients remain susceptible to infections for the rest of their lives
especially in that substantial population of children transplanted because of cystic fibrosis (50% of cases in most pediatric lung
transplant programs).
Microscopic findings depend on the etiology of the infection and the host response, which may be minimal. Bacterial infections
usually elicit neutrophilic infiltration of airway, interstitium, and alveolar spaces. Occasionally, there is only bacterial growth and
infarction with no inflammation. The most common viral infection is caused by CMV, which often infects endothelial cells. This may
lead to bleeding complications after diagnostic TBB. CMV is diagnosed by finding the classical single intranuclear and multiple small
cytoplasmic inclusions in an enlarged cell (eFigure 8-21). Treated patients often have smudged, eosinophilic inclusions, which may
be difficult to identify as CMV (Figure 8-29). Adenovirus infection is more common in children, and scattered adult and pediatric
patients develop serious pneumonias due to the other
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respiratory viruses (respiratory syncytial virus, parainfluenza, influenza). Fungal infections are often caused by Aspergillus or
Candida sp. especially in children with cystic fibrosis. Pneumocystis pneumonia is rare due to routine prophylaxis.
FIGURE 8-29▪Treated CMV. Soon after treatment, CMV inclusions become eosinophilic and smudged as seen in this
photomicrograph (H&E, 200 ×).
FIGURE 8-30▪ Early CMV pneumonitis. In the lung transplant recipient, detection of any nuclear stain even without classic
intranuclear inclusions is indicative of CMV infection (immunohistochemical stain, 200×).
In the very early stage of CMV infection, IHC staining against immediate-early antigen may demonstrate nuclear positivity in cells
lacking diagnostic cytopathic changes (Figure 8-30). IHC is also very useful for confirming the diagnosis in patients already on
treatment for CMV.
The main differential diagnosis is from acute rejection, since the symptoms are similar. Infection may precipitate rejection and vice
versa. Infections can be very difficult to treat, with new resistant strains emerging in some patients. Prophylaxis plays an important
role in preventing PCP and CMV pneumonia.
OTHER FORMS OF LUNG INJURY

DAD, organizing pneumonia, acute interstitial pneumonia, and interstitial fibrosis may all be seen as nonspecific responses to lung
injury in the posttransplant patient. The etiology of these responses is diverse and is not specifically related to either acute or
chronic rejection.
Posttransplant Lymphoproliferative Disorder

PTLD occurs in 3% to 5% of lung transplant recipients with frequent involvement of the allograft, often as one or multiple nodules. A
high index of suspicion should be maintained, and the diagnosis can be suggested on FNAB and TBB. Particularly with low-grade
lesions, the need to obtain adequate tissue for complete workup may require a wedge biopsy. The histologic and molecular features
are similar to those seen in any other transplant patient (Chapter 22).
Outcome
Advances in donor management, surgical techniques, and immunosuppressive drugs have led to improvement in the short-term
survival of patients. However, in contrast to other solid organ transplants, over half of the lung transplant recipients (pediatric and
adult) continue to suffer and die of chronic rejection (bronchiolitis obliterans) 3 to 10 years posttransplantation. Although surveillance
biopsies can detect infection in asymptomatic children, the early detection of AR (seen in 4%) is unlikely to have a major impact on
longterm survival (6).
Pulmonary Complications After Hematopoietic Stem Cell Transplant

With the use of effective infection prophylaxis, noninfectious causes of pulmonary dysfunction after stem cell transplant are the major
pulmonary causes of morbidity and mortality. These include acute and chronic graft-versus-host-disease, idiopathic pneumonia
syndrome, diffuse alveolar hemorrhage, pulmonary veno-occlusive disease, and organizing pneumonia (123).
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Chapter 9
The Placenta
Raymond W. Redline
INTRODUCTION
Perinatal pathology, the subdiscipline of pediatric pathology devoted to the study of abnormal pregnancy
outcomes, is a rapidly developing field interfacing with obstetrician-gynecologists, neonatologists, and clinical
geneticists. A central tenet of this field is that analysis of adverse pregnancy outcome begins with study of the
placenta and its adnexa. The fetus is entirely dependent on the placenta for sustenance and protection
throughout gestation. Indeed the placenta has been called a “diary of intrauterine life.” Artificial barriers are often
placed between the study of so-called products of conception resulting from early pregnancy loss and placentas
submitted to pathology following complications of later pregnancy. Such a separation has no anatomic or
functional basis and has probably hindered a complete and holistic understanding of the underlying biologic
factors responsible for adverse outcomes in couples with sporadic or recurrent pregnancy loss. The first part of
this chapter briefly summarizes key stages of placental development as a basis for understanding the problems
of the first and early second trimester. The second part outlines the structure of the mature placenta to provide
an anatomic framework for disease processes occurring in the late second and third trimester of pregnancy.
EARLY PREGNANCY
Development
The fertilized zygote undergoes a series of cleavage divisions to form a solid 16-cell morula by 5 days following
ovulation (123). Between 5 and 8 days, a number of important events occur: the lose aggregate of cells becomes
compacted, cells at the periphery develop tight junctions and begin transporting fluid into the center of the morula
(blastocyst formation), and the surrounding zona pellucida is shed as the blastocyst attaches to, crosses, and
invades the endometrium (Figure 9-1). The formation of tight cell-cell junctions at the periphery of the blastocyst
marks the emergence of the trophectoderm lineage (trophoblast), which is the principal component of the
placenta. Transport of fluid into the blastocyst and invasion of the gestational endometrium (decidua)
foreshadow the two most important functions of trophoblast throughout gestation: transport of maternal
substrates to the fetus and tissue remodeling of the maternal uterus to ensure adequate delivery of these
substrates. Cells within the blastocyst (inner cell mass) separate into two lineages: epiblast, which gives rise to
the epithelium surrounding the amnionic cavity (day 8) and the embryonic germ layers (days 15 to 28), and
hypoblast, which forms the connective tissue of the placenta (extraembryonic mesoderm) and the primary yolk
sac (23).
Development of the maternal and fetal placental circulations occurs in parallel. The maternal circulation begins
when capillaries are eroded by an outer layer of primitive syncytial trophoblasts (131). Blood subsequently flows
into lacunar spaces within the syncytium. These lacunae gradually enlarge eventually forming the intervillous
space. Trophoblasts also migrate centripetally within the arterial circulation, forming cellular plugs that retard
blood flow into the intervillous space until approximately 10 weeks of gestation (Figure 9-2A) (69). The basis for
arterial versus venous invasion is unknown but may involve differential expression of angiogenic signaling
molecules (190). During this period of retarded blood flow, the walls of the spiral arteries are remodeled in a
series of events that includes dissolution of the muscular media, dilatation of
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the lumen, and reconstitution of the vessel wall by extracellular matrix secreted by endovascular trophoblasts. By
the time that the plugs disappear, the cells of the developing placenta and the underlying structural integrity of
the intervillous space are sufficient to withstand the oxygen tension and pressure of arterial blood flow.
Subsequent remodeling of deeper arteries in the inner third of the myometrium continues until 18 weeks of
pregnancy (the so-called secondary wave of implantation) (127). During this process, the placenta also expands
laterally by attachment to and cooptation of large veins at the margins of the conceptus (so-called marginal sinus
formation) (39). By the end of pregnancy, approximately 80 to 100 spiral arteries open into the mature intervillous
space (28).
FIGURE 9-1▪Early implanting gestational sac (cytokeratin stain): Portions of embryo and unattached amnionic
sac are surrounded by circumferential primary villi anchored in the peripheral cytotrophoblast shell with early
intermediate trophoblasts infiltrating the adjacent endometrium.
The fetal circulation of the placenta develops in two distinct phases (44). Extraembryonic mesoderm from the
hypoblasts migrate peripherally into the primitive biphasic trophoblasts (cytotrophoblast stem cells and syncytial
trophoblast) between the developing lacuna to form the so-called primary villi. A villous capillary circulation
subsequently forms via local inductive interactions between cytotrophoblasts and extraembryonic mesoderm
(Figure 9-2B). Later, this villous capillary net becomes connected to the embryonic circulation via anastomoses
with large vessels growing out into extraembryonic connective tissue covering the trophoblastic portion of the
placenta (chorionic plate). These large vessels reach the placenta via the body stalk, later to become the
umbilical cord. Paired arteries develop along the allantoic duct and a vein develops along the
omphalomesenteric duct. The vascularized extraembryonic mesoderm undergoes branching morphogenesis to
form villous trees. As these trees increase in complexity and the placenta enlarges, the more proximal
intraplacental vessels develop a muscular media and form the so-called stem villous arteries and veins.
FIGURE 9-2▪Early pregnancy vascular development: A: Spiral arteries surrounded by intermediate trophoblasts
with luminal plugs of endovascular trophoblasts and remodeling of the vessel wall. B: Fetal capillaries arising
from pluripotent villous stromal cells induced by villous trophoblasts.
The final stage of early placental development is formation of the membranes (185). The initial phase occurring
at about 9 to 11 weeks of gestation is disappearance of the extraembryonic coelom and primitive yolk sac,
resulting in attachment of the amnionic cavity surrounding the fetus to the chorionic connective tissue covering
the trophoblastic portion of the placenta. This is followed at 11 to 17 weeks by the gradual atrophy and collapse
of the intervillous space in all portions of the placenta not directly overlying the implantation site. It has been
suggested that higher oxygen tension due to the lack of endovascular trophoblasts plugging away from the
implantation site is responsible for this pattern of peripheral collapse (70). Finally, at about 17 to 20 weeks, the
enlarging chorionic sac makes contact with and fuses to opposite side of the uterus, forming the mature
multilayered placental membrane composed of vascularized decidua vera, avascular decidua capsularis,
chorionic trophoblasts (chorion laeve), chorionic connective tissue, amnionic connective tissue, and amnionic
epithelium.
Multiple Pregnancy
Background
Twinning (and higher order multiple pregnancies) can occur from either fertilization of multiple eggs (dizygotic) or
fission of a single fertilized egg (monozygotic) (136).
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Monozygotic twinning occurs at a constant rate in most populations and can be associated with a variety of
different placental types (Figure 9-3). Separation prior to blastocyst formation leads to separate placentas
(dichorionic diamnionic). Separation between blastocyst formation and amniogenesis leads to a single placenta
but separate amnionic sacs (monochorionic diamnionic) while separation after amniogenesis results in a single
placenta and amnionic sac (monochorionic monoamnionic). The incidence of dizygotic twinning is variable in
different populations and depends on the frequency of polyovulation, either natural related to endogenous FSH
levels or artificial related to ovulation-inducing drugs used in association with assisted reproductive technology It
had been thought that dizygotic twins always have separate (dichorionic) placentas, but a recent report has
confirmed that on rare occasions, probably at the late morula stage, dizygotic twins can fuse to form a
monochorionic placenta (172).
FIGURE 9-3▪Diagrammatic representation of placentation in monochorionic twinning. (From Gersell DJ, Kraus
FT. Diseases of the placenta. In: Kurman RJ, ed. Blausteins pathology of the female genital tract. New York:
Springer Verlag, 1998:986, with permission.)
More important than zygosity per se from a clinical standpoint are connections in the placental vasculature (17,
93). Surface anastomoses between chorionic arteries or veins (artery to artery and vein to vein) are common in
monochorionic twins. These connections can lead to sharing of blood (chimerism) but do not generally cause
circulatory imbalance. An exception may occur when major arteries immediately adjacent to their umbilical cord
insertion sites are connected. In this situation, an artery from one twin may develop sufficient pressure to reverse
the circulatory flow in the second twin, leading to secondary atrophy of the heart and other rostral structures
(acardiac fetus) (21). Aberrant connections in the period when the villous circulation anastomoses with the
embryonic circulation, on the other hand, may lead to areas of the placenta that are perfused by the arterial
circulation of one twin and drained by the venous system of the other. The resulting twin-twin transfusion
syndrome is discussed below.
Pathology
The most important role of the pathologist in multiple pregnancy is to determine the number of chorions and
amnions in each placenta, usually by direct inspection followed by confirmatory histologic sections from the
dividing membrane between the two placentas. Such examination is not required when the placental discs are
completely separate. If a single placenta is noted and the dividing membrane is opaque, two amnions flanking a
fused central chorion are most likely (dichorionic diamnionic). This can be confirmed at gross examination by
peeling the three layers, and the placentas can then be separated for weighing and processing. If the dividing
membrane is translucent, only two amnions are expected (monochorionic diamnionic). The chorion is absent
because it surrounds but does not divide the two fetal sacs. This is again confirmed by peeling the two layers. A
monochorionic placenta is, as the name indicates, one placenta and should be weighed without division.
Inspection of the chorionic plate for surface anastomoses should be performed. Air injection studies are a quick
and easy method for detecting clinically significant deep arteriovenous anastomoses (87). More complete
injections with colored or radiopaque dyes are usually conducted only in a research context. Pathologic lesions,
most frequently found with discordant twin growth (see below) in both monochorionic and dichorionic twins,
include peripheral cord insertion, avascular villi, and indicators of maternal vascular under perfusion (60, 154).
Clinical Correlation
Twin gestations of all types are at an increased risk for premature delivery, fetal growth restriction, preeclampsia,
and cerebral palsy (129). Many of these complications are increased in the presence of discordant twin growth,
usually defined as a greater than 25% difference in body weights. Adverse outcome is generally more frequent in
the smaller and/or the nonpresenting (second) twin. Chronic twin-twin transfusion syndrome is a specific form of
discordant growth related to deep arteriovenous anastomoses in monochorionic twins (183). The syndrome is
characterized by marked growth restriction and anemia in the donor twin and macrosomia, polycythemia, and
congestive heart failure in the recipient. Acute twin-twin transfusion without growth discordance can occasionally
occur when previously balanced anastomoses become unbalanced due to either changes
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in fetal blood pressures or secondary occlusion of bridging fetal vessels. The most dramatic example of acute
transfusion syndrome occurs after fetal demise of one twin. In this case, sudden blood shifts from the survivor to
the decedent are associated with a very high risk of perinatal brain damage (126).
Gestational Trophoblastic Disease

Background
Trophoblast has two major functions: tissue invasion and substrate transfer. As described above, invasion
occurs early in gestation as the primitive mononuclear and syncytial trophoblasts invade the superficial
endometrium, and later in gestation, when intermediate trophoblasts implant more deeply in the myometrium.
Neoplastic transformation of primitive mononuclear and syncytial trophoblasts result in choriocarcinoma while
that of intermediate trophoblasts result in placental site trophoblastic tumor (PSTT). A third more recently
described subtype of trophoblastic neoplasm, the epithelioid trophoblastic tumor (ETT), is derived from cells
phenotypically similar to the trophoblasts of the membranes (chorion laeve trophoblasts) (166). While
choriocarcinoma can develop de novo from apparently normal early and late placentas, more than half of cases
are preceded by molar pregnancies. Trophoblastic proliferation is largely dependent on growth-promoting
factors, transcribed only from paternally inherited genes (14). Control of proliferation and differentiation, on the
other hand, depends on antiproliferative genes of maternal inheritance. Molar pregnancies have an
overrepresentarion of paternal chromosomes (141). Complete moles are derived from fertilization of an empty
ovum and contain only paternal chromosomes, while partial moles are triploid gestations resulting from dispermy
and have a 2:1 ratio of paternal to maternal chromosomes. The increased frequency of molar pregnancies
accounts for the relatively high incidence of choriocarcinoma in Asian populations. PSTT and ETT, most
commonly, develop after term pregnancies, often years after delivery. It is well known that some intermediate
trophoblasts are left behind after delivery and can persist for many years. Although not proven, these persistent
rests of trophoblasts, known as placental site nodules, may well be precursors for these rare tumors (189).
FIGURE 9-4▪Molar pregnancy: A: Complete hydatidiform mole— uniformly hydropic villi with central cisterns and
circumferential trophoblastic hyperplasia. B: Partial hydatidiform mole—molar villi (left) and irregularly shaped
fibrotic villi (right) showing mild circumferential syncytiotrophoblastic hyperplasia. C: Early complete hydatidiform
mole—bulbous villous branching, densely cellular myxoid stroma, and trophoblastic hyperplasia.
Pathology
Molar pregnancies are characterized by villous edema and trophoblastic hyperplasia. Edema is often extreme
leading to cavitation of the villous stroma. In complete moles, the edema and hyperplasia affect the entire
conceptus, while in partial moles, they affect only a subgroup of villi (Figure 9-4A). Partial moles also show
irregularly shaped villi, as may be seen in
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other chromosomal abnormalities (Figure 9-4B) (147). Classic complete moles are easily diagnosed based on
clusters of fluid-filled vesicles (hydatidiform or “grape-like” change) and marked trophoblastic hyperplasia. With
current use of early ultrasound, approximately one-third of complete moles are curetted at a stage before
development of edema or diffuse trophoblastic hyperplasia. These early complete moles can be difficult to
recognize but manifest a number of helpful diagnostic features including a cauliflower-like growth pattern,
densely cellular myxoid villous stroma, focal trophoblastic hyperplasia, and atypia of implantation site
trophoblasts (Figure 9-4C) (80). Occasional early pregnancy specimens show nonspecific trophoblastic
hyperplasia without edematous or molar villi. Cytogenetic study of these specimens has shown a high
prevalence of the two relatively uncommon trisomies, 7 and 15 (146). Whether these cases have an increased
risk of later choriocarcinoma is not known.
Choriocarcinomas are often characterized grossly by large areas of hemorrhage and necrosis. They are
composed of two cellular populations—clusters of 10 to 50 mononuclear cytotrophoblasts surrounded by a
wreath-like garland of syncytial trophoblasts (Figure 9-5A). The mononuclear trophoblast shows mild-moderate
nuclear atypia and watery clear cytoplasm. Syncytial trophoblast contains multiple enlarged hyperchromatic
nuclei and deep eosinophilic cytoplasm. The latter stains intensely for human chorionic gonadotropin and human
placental lactogen, while the former lack both hormones. Both cell types are cytokeratin positive. Unlike normal
trophoblasts, individual clusters of malignant cells in choriocarcinoma perpendicularly invade smooth muscle
fascicles in the myometrium. PSTT is composed of larger mononuclear cells with more nuclear atypia and an
intensely eosinophilic cytoplasm (Figure 9-5B) (167). Binucleation is occasionally seen, but greater numbers of
nuclei are rare. Unlike normal intermediate trophoblasts, tumor cells invade in large cohesive sheets and are
associated with tissue necrosis. Some large arteries show remodeling by tumor cells, but large nontransformed
arteries are also seen and are diagnostically helpful. Tumor cells stain positively for cytokeratin and human
placental lactogen but are usually only weakly and focally positive for human chorionic gonadotropin (hCG). ETT
contains vacuolated cells often in a hyaline matrix, bearing a striking resemblance, by both light microscopy and
immunostaining, to the cells of the membranous chorion laeve (Figure 9-5C). They tend to grow in a nodular
expansile pattern in the lower uterine segment or cervix where
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they can mimic squamous carcinomas. Their antigen profile includes diffuse expression of keratin, alpha inhibin
and p63 plus focal/variable hCG, hPL, MelCAM (CD148), and placental alkaline phosphatase (168).
FIGURE 9-5▪Trophoblastic tumors: A: Choriocarcinoma—clusters of mononuclear cytotrophoblasts surrounded

by a wreath-like garland of poorly differentiated syncytial trophoblasts. B: Placental site trophoblastic tumor—
loosely cohesive sheets of atypical intermediate trophoblasts with strongly eosinophilic cytoplasm. C: Epithelioid
trophoblastic tumor—sheets of vacuolated extravillous trophoblasts invading myometrium.
All preneoplastic and neoplastic lesions of trophoblasts are combined under the rubric gestational trophoblastic
disease (92). While molar pregnancies are usually confirmed by tissue diagnosis, subsequently developing
choriocarcinomas are generally not. Clinical management relies on serum monitoring of the tumor marker, hCG,
and radiographic imaging. Persistence or elevation of hCG levels, after evacuation of a molar pregnancy, is
treated empirically with single agent chemotherapy. Tumors with extremely high hCG levels, metastasis to
organs other than the lung, and other high-risk factors are treated with multiple agent chemotherapy. On rare
occasions, chemotherapeutically resistant tumors manifest a distinct pathologic phenotype known as atypical
choriocarcinoma (100). PSTT and ETT usually present with vaginal bleeding. Curettage is suspicious for a
neoplasm and hCG levels are usually positive, but often at low levels. Radiographic studies confirm a mass
lesion, and hysterectomy is performed. Unlike choriocarcinoma, PSTT and ETT are relatively indolent and only
rarely metastasize. However, they respond poorly to chemotherapy so local control is paramount. Clinical
management of the occasional early pregnancy specimens with nonspecific or unclassifiable trophoblastic
hyperplasia referred to above should include a single hCG titer to ensure return to baseline.
Anembryonic Pregnancy
Background
Missed abortion refers to a pregnancy in which a nonviable chorionic sac is retained in the uterus requiring
curettage for evacuation. An early gestational sac lacking sonographic and histologic evidence of embryonic
development (anembryonic pregnancy) is the most common form of missed abortion. A large percentage of these
specimens have embryonic lethal chromosomal abnormalities (158). The remaining chromosomally normal
specimens most likely represent random major disruptions of early embryogenesis resulting in complete or partial
resorption of the inner cell mass derivatives. The proportion with sporadic mutations in major developmental
genes is unknown (see Chapters 2 and 3).
Pathology
Anembryonic pregnancies show a typical pathologic profile. They consist of a relatively thin chorionic membrane,
uniformly edematous (hydropic) villi, and well-preserved gestational endometrium and implantation site (Figure 9-
6A). Amnion, yolk sac, umbilical cord, and embryonic tissue are usually absent, and no fetal blood vessels are
apparent. The uniformly hydropic nature of the villi is caused by continuing trophoblastic transport function
leading to fluid buildup in villi with no egress to the fetal circulation. With prolonged retention, the hydropic villi
can undergo secondary fibrosis (hyalinization). Gestational endometrium and an implantation site in these cases
are usually unremarkable.
The management of women with first trimester losses, particularly when recurrent, is highly dependent on the
nature of the loss. A careful pathologic examination can often guide clinical management in cases where
cytogenetic analysis has either not been obtained or is unsuccessful (158). Recognition of an anembryonic
gestation (also referred to as blighted ovum or hydropic abortus) by early ultrasound or pathologic examination is
clinically useful in that it identifies a cohort with a low recurrence rate. Early and late miscarriages with
thromboinflammatory lesions or endometrial pathology and no apparent defects in fetal development are much
more likely to recur in subsequent pregnancies.
Miscarriage
Background
The term miscarriage refers to early pregnancy specimens in the process of being expelled from the mother
(threatened incomplete, and complete abortions). Most common are specimens with evidence of remote fetal
death (hyalinized villi, obliterated fetal vasculature) (Figure 9-6B). These cases are a heterogeneous mixture of
chromosomally normal and abnormal gestations with intrauterine fetal demise due to malformation, deformation,
or disruption. A second group is characterized by well-preserved and normally vascularized chorionic villi with
copious intervillous hemorrhage (Breus mole) (Figure 9-6C). Specimens in this second group are often
chromosomally normal. Several underlying maternal abnormalities may contribute to miscarriage.
Antiphospholipid syndrome leads to maternal vascular maldevelopment, thrombosis, and hemorrhage via
antibody plus complement-mediated interactions with maternal endothelial cells and trophoblasts (30, 164).
Other less specific maternal vascular abnormalities may interfere with the endovascular trophoblastic plugs that
normally retard blood flow into the intervillous space in early pregnancy. This can lead to oxidative damage to the
developing placenta and high-pressure flow into the early intervillous space (73). Finally, there is a group of
poorly understood thromboinflammatory processes (see below) characterized by evidence of maternal immune
responses in fetal tissues. Some evidence links these uncommon lesions to abnormal maternal immune
responses to foreign fetal antigen in the placenta (158).
Pathology
The general phenotype of most miscarriages is a well-developed chorionic sac with adherent amnion,
collagenized (hyalinized) villi, and hemorrhagic necrosis of the implantation site and gestational endometrium.
More specific findings are sometimes
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identified in specimens from patients with multiple consecutive miscarriages. Massive perivillous fibrin deposition
(“maternal floor infarction”) can present at any gestational age and is discussed later. Chronic histiocytic
intervillositis is characterized by extensive infiltration of the intervillous space by a monomorphic infiltrate of
monocytes-macrophages (Figure 9-6D) (29). This lesion can also present at later stages but is most frequently
observed in the first trimester. Breus mole, in addition to marked hemorrhage in the intervillous space, may show
pathologic thrombosis or congestion of spiral arteries and an absence of endovascular trophoblastic plugs
(Figure 9-6E). Finally, the spiral arteries of some patients with antiphospholipid syndrome or other autoimmune
diseases may show vasculitis, mural hypertrophy, perivascular decidual fibrin deposition, and/or plasma cell
infiltration (Figure 9-6F) (116).
FIGURE 9-6▪Spontaneous abortion: A: Anembryonic pregnancy—uniformly hydropic villi adjacent to chorion
without an adherent amnion. B: Hyalinized villi with adjacent fused chorioamnion, consistent with remote fetal
death. C: Breus mole—well-preserved villi with marked intervillous hemorrhage. D: Chronic histiocytic
intervillositis—early chorionic villi surrounded by sheets of immature monocytes-macrophages. E: Pathologic
congested spiral arteries lacking endovascular trophoblast plugs. F: Spiral arterioles with marked chronic
perivasculitis in maternal autoimmune disease.
Patients with antiphospholipid syndrome are currently treated with low-dose heparin therapy often accompanied
by lowdose aspirin. Approximately 70% to 80% of women treated with this regimen achieve successful
pregnancy outcome in subsequent pregnancies (30). Those failing this regimen may be treated with full
anticoagulation, intravenous gamma globulin, or corticosteroids with unclear efficacy. Patients with vascular
pathology lacking antiphospholipid antibodies are often treated similarly. Chronic histiocytic intervillositis is more
frequent in women with underlying immunologic abnormalities and abnormal alloimmune responses to fetal
(paternal) antigens (29, 47). They frequently have an abnormal cytokine response to pregnancy, as manifest by
increased TNF-a (embryotoxic factor) (63). Women with abnormal alloimmune responses have been extensively
studied often without any pathologic correlation. Although no randomized controlled trials demonstrating efficacy
have been published, these patients are sometimes treated empirically with progesterone, immunosuppressive
drugs, intravenous immunoglobulin, and immunization with paternal leukocytes (37).
FIGURE 9-7▪TORCH infections: A: Syphilis—histiocytic villitis with villous edema. B: CMV-plasma cell villitis
with villous fibrosis.
Congenital Infection
Background
Although ascending infections caused by organisms from the cervicovaginal tract can occur in the second
trimester, most congenital infections in the first half of pregnancy are acquired hematogenously (24, 87). The
majority are the result of primary infection, since previous exposure usually elicits protective antibodies in the
mother. Bacterial and fungal infections are rare. Spirochetes (T. pallidum, B. burgdorfei), parasites (T. gondii, T.
cruzi, P. falciparum, S. hematobium), and viruses (cytomegalovirus, varicella zoster virus, herpes simplex virus,
rubellavirus, poxviruses, parvovirus B19, enteroviruses, HIV, hepatitis B and C) are the major causative agents.
Organisms may localize to and elicit inflammation exclusively in the intervillous space (P. falciparum, B.
burgdorfei, S. hematobium) or they may cross the placenta without eliciting an inflammatory response
(parvovirus B19, HIV, hepatitis B and C, most enteroviruses), but more commonly they infect both placenta and
fetus. Most fetal infections occur in the second trimester. Spread to the fetus in the first trimester is less common,
but the infections are generally more severe. Maternal infections in very early pregnancy often spare the
products of conception (see Chapter 6).
Pathology
Organisms limited to the intervillous space lead to accumulations of fibrin and chronic inflammatory cells at that
location. The remaining infections lead to a diffuse chronic placentitis with chronic inflammatory cells in the
chorion, decidua, and villous stroma (6). Unlike villitis of unknown etiology (also discussed below), infectious
villitis tends to involve most or all villi. Two overlapping patterns are seen. The first, edematous villi with
increased Hofbauer cells, is typical of syphilis (Figure 9-7A). The second, fibrotic villi with evidence of remote
hemorrhage and, occasionally, villous plasma cells is typical of CMV (Figure 9-7B). Many infections have unique
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features allowing a specific histopathologic diagnosis. These include the presence of organisms or viral
inclusions in the villous stroma (CMV, herpes simplex virus, varicella zoster virus, parvovirus B19, T. cruzi),
umbilical cord (T. pallidum, T. gondii), or intervillous space (P. falciparum, S. hematobium).
A common clinical mnemonic for congenital infection is the acronym TORCH standing for toxoplasmosis,
(others), rubella virus, cytomegalovirus, and herpes simplex (58). In the United States, two infections, CMV and
syphilis, account for more than 90% of congenital infections. Infants with any of the TORCH infections have a
number of common features including intrauterine growth restriction (IUGR), pancytopenia, hepatosplenomegaly,
and coagulopathy. Each infection also has specific features, a description of which is beyond the scope of this
chapter. A standard serologic screen known as the “TORCH titer” tests for maternal IgG specific for the common
TORCH agents and is part of the routine workup for IUGR or suspected antenatal maternal infection. Specific
testing for IgM is required to distinguish recent from remote infection. Many infections can also be diagnosed by
PCR testing of fetal blood or amniotic cells obtained by amniocentesis.
LATE PREGNANCY
Anatomy
The mature placenta is composed of four distinct units of structure-function:
1. Chorionic plate and its contiguous vascularized fetal connective tissue

2. Interhemal villous trophoblasts and the adjacent intervillous space
3. Basal plate and underlying maternal uterine vasculature
4. Tripartite placental membranes, consisting of amnion, chorion, and decidua.
The chorionic plate (or fetal surface) consists of the fibrous connective tissue supporting the large muscular
arteries and veins that distribute fetal blood flow from the umbilical cord to the family of 20 to 30 large villous
trees (20, 28). The umbilical cord is a squamous epithelial-lined conduit normally measuring between 40 to 80
cm in length at term that conducts fetal blood from the umbilicus to some location on the chorionic plate (or
occasionally the adjacent placental membranes). It contains paired arteries that spiral around a central vein, all
surrounded by a hyaluronate-rich matrix (Wharton jelly), which provides considerable protection from external
compression. The two arteries are connected, at or just before their insertion site, into the chorionic plate by an
anastomosis (Hyrtyl anastomosis). Villous trees, emanating from the underside of the chorionic plate, branch
multiple times as they conduct fetal blood through a succession of smaller arteries and veins until they reach
capillaries that abut the trophoblastic interhemal membrane in the terminal villi. These conducting villi are
referred to as stem and intermediate villi, with the latter representing the arteriolar level at which blood flow to the
gas-exchanging terminal villi is ultimately regulated (54). The two critical anatomic features that must be
maintained in this compartment are patency of the large villous vessels and short diffusion distance between
fetal capillaries and interhemal villous trophoblasts. Maturation of the villous tree with advancing gestational age
in the third trimester is characterized by an increase in the number of terminal villi relative to intermediate villi and
a decrease in the amount of villous connective tissue associated with the peripheralization of capillaries and the
formation of specialized vasculosyncytial membranes. However, there is considerable regional variation in villous
maturity. Well-perfused villi, overlying the opening of the maternal spiral artery (central cotyledon), appear
considerably less mature than those at the “watershed” between arteries (peripheral cotyledon) (Figure 9-8A and
B).
Interhemal villous trophoblasts consist of a single multinucleated layer of differentiated syncytiotrophoblasts with
underlying basement membrane and occasional basally located cytotrophoblastic stem cells. Each stem cell is
the progenitor for 80 to 100 fused syncytiotrophoblastic cells, and these large syncytial sheets form a mosaic
covering the entirety of the villous tree (169). Turnover of syncytiotrophoblasts occur via clustering of nuclei in
syncytial knots followed by apoptosis and shedding into the maternal circulation (98). Critical features for the
interhemal membrane are cellular viability, appropriate maturation in terms of endocrine, transport, anticoagulant,
and immunoprotective functions, and accessibility to maternal blood flow—meaning absence of adherent fibrin or
inflammatory exudate.
The basal plate consists of 80 to 100 anchoring villi inserted into the endometrium plus a similar number of
perpendicularly oriented perforating maternal arteries and tangentially oriented draining maternal veins.
Intermediate trophoblasts, arising from cytotrophoblasts on the underside of the anchoring villi, diffusely infiltrate
the basal plate and elaborate large amounts of a fibronectin-rich extracellular matrix that provides structural
integrity and unites these elements into a coherent anatomic structure (Nitabuch layer). Closely related
endovascular trophoblasts are normally present in the wall of basal plate arteries. Important features of the
normal basal plate are adequate remodeling of maternal arteries to ensure adequate blood flow into the
intervillous space and sufficient depth and extent of trophoblastic implantation to prevent premature separation.
The margins of the placenta require additional consideration. The process of continuing lateral placental growth
involves growth of villous trees into maternal veins at the periphery of the disc (39, 115). This process results in
the formation of distinct large sinusoidally dilated veins surrounding the placenta, structures previously
misinterpreted as a discrete marginal venous sinus (Figure 9-8C).
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FIGURE 9-8▪Normal placental anatomy at term: A: Central lobule—distal villi are enlarged with abundant stroma,
numerous capillaries, and uniform layer of villous trophoblasts. B: Peripheral lobule—distal villi are much smaller
with scant peripheral capillaries and prominent syncytial knots. C: Margin/membrane—peripheral villi extend into
a large venous space within the basal plate that is covered by fused amniochorion and decidua.
The placental membranes, at first glance, appear distinct from the first three compartments. While this is certainly
true in terms of function, the anatomic differences are minor. The membranes form by involution of the placenta
and retain all of its layers. The fetal surface of the membranes is covered by amnion and consists of chorionic
connective tissue and occasional chorionic villi, albeit without fetal blood vessels. The villous trophoblasts
coalesce as the intervillous space is obliterated to form a third distinct morphologic variant of trophoblast known
as chorion laeve or epithelioid trophoblast. This noninvasive trophoblastic layer is supported by underlying
maternal decidua. Critical requirements for this compartment include the integrity and contiguity of all layers. In
particular, chorionic prostaglandin dehydrogenase in chorion laeve trophoblasts must be functionally active and
spatially positioned to inactivate labor-inducing prostaglandins elaborated by the amnion, decidua, and
myometrium (34, 179).
Chronic Disease Processes

Disease processes affecting the placenta can be classified in a variety of ways including anatomic location,
mechanism of injury, or clinical outcome. Another way that is particularly useful for understanding the causal
sequence of events leading to adverse pregnancy outcome is time of onset (149). The rationale for such a
separation is that earlier events and placental lesions may significantly decrease placental reserve lowering the
threshold for fetal injury and resulting in an enhanced effect of comparatively minor stresses at the time of
parturition. In the following scheme the term chronic refers to lesions evolving over weeks, subacute to those
evolving over many hours to days, and acute to those evolving over just a few hours.
Maternal Vascular Under Perfusion
Background
Chronic maternal underperfusion of the intervillous space can result from a variety of causes including underlying
cardiac insufficiency, failure to expand intravascular volume during pregnancy, or structural abnormalities in
arteries supplying the uterus. It is currently believed that the major process leading to underperfusion is failure of
trophoblasts to appropriately invade and remodel the uterine spiral arteries. While the exact sequence or
sequences of events leading to this outcome have not yet been worked out, a number of
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contributing factors have been identified. These include initial exposure to fetoplacental antigens in first
pregnancies, inherited polymorphisms in genes of the renin-angiotensin system, antiendothelial cell antibodies,
and underlying uterine small vessel disease (15, 105, 161, 181). The common denominator for all of these
factors seems to be decreased oxygen delivery to the implantation site resulting in dysregulation of growth factor
and protease expression, impaired trophoblastic differentiation, and inadequate placentation (32). In the absence
of arterial remodeling, the placenta is chronically underperfused leading to decreased fetoplacental growth and,
in some cases, the release of vasoactive mediators in late pregnancy leading to the clinical syndrome of
preeclampsia. Several of these mediators have been identified in the last few years including soluble form of
vascular endothelial growth factor (VEGF) receptor 1 (sflt-1), soluble endoglin, and circulating AT1 receptor
antibodies (67, 90, 99, 165).
Pathology
Placentas affected by maternal underperfusion generally show two or more of a constellation of features that
together allow a specific diagnosis to be rendered (144). One important and often overlooked feature is
decreased weight for gestational age and decreased placental weight relative to that of the infant (increased
fetoplacental weight ratio) (109, 187). In severe cases, this correlates with late impairment of placental growth
(distal villous hypoplasia) as the fetus sacrifices placental perfusion in order to supply critical vascular beds such
as the central nervous and cardiovascular systems (Figure 9-9A) (68, 88). Also, common in severe cases is a
thin umbilical cord resulting from extracellular volume depletion and decreased hydration of Wharton jelly.
Complete maternal vascular obstruction secondary to spiral artery thrombi leads to villous infarcts (Figure 9-9B)
(31, 180). Partial maternal vascular obstruction can lead to stasis with intervillous fibrin deposition (Figure 9-9C),
hypoxia with accelerated syncytiotrophoblast turnover and increased syncytial knots (Figure 9-9D), and localized
ischemia with villous agglutination (Figure 9-9E) (7, 65, 132). Two other types of placental abnormalities may
also be seen. First, persistent muscularization of basal plate arteries and aggregates of placental site giant cells
or epithelioid (chorion laeve type) trophoblasts in the basal plate) correlate with superficial implantation (153).
Second, maternal arteriopathies, medial hypertrophy (Figure 9-9F) and fibrinoid necrosis (acute atherosis)
(Figure 9-9G), may be linked to inheritance of a variant
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angiotensinogen allele and/or the vasoactive mediators discussed above (82, 90, 105, 182).
FIGURE 9-9▪Maternal underperfusion: A: Distal villous hypoplasia— decreased number of long thin poorly
branching distal villi. B: Villous infarction—large aggregate of nonviable villi with collapse of the intervillous
space and remote ischemic necrosis of the villous trophoblast. C: Increased intervillous fibrin—irregular
aggregates of fibrin coating large proximal villi and protruding from denuded portions of the distal villous tree.
FIGURE 9-9▪D: (continued) Increased syncytial knots—numerous aggregates of large numbers of
syncytiotrophoblastic nuclei within the villous trophoblast layer. E: Villous agglutination—small areas of
aggregated villi with syncytial knots and intervillous fibrin (microinfarct). F: Mural hypertrophy of decidual
arterioles-hypertrophy of the vascular smooth muscle wall (arteriolosclerosis). G: Acute atherosis of decidual
arterioles—fibrinoid necrosis of the vascular smooth muscle wall with scattered aggregates of embedded lipid-
laden macrophages.
Chronically underperfused placentas are associated with fetal growth restriction, premature birth owing to either
premature labor or premature rupture of membranes, premature placental separation (abruptio placenta), and an
increased risk for the development of preeclampsia (10, 43, 110, 186). Clinical conditions predisposing to
maternal underperfusion include type I diabetes mellitus, connective tissue disease, chronic renal insufficiency,
essential hypertension, and underlying maternal coagulopathies including thrombophilic mutations and
antiphospholipid syndrome (119). Familial aggregation of preeclampsia and underlying maternal vascular
disease may at least in part be due to inheritance of the so-called metabolic syndrome characterized by
abnormal serum lipid levels, enhanced production of acute phase inflammatory mediators, and a predisposition
to vascular damage related to oxidative stress. These patients are often overweight and predisposed to
developing cardiovascular disease, type II diabetes, and sleep-disordered breathing in later life.
Chronic Abruption
Background
As discussed above, lateral growth of the placenta involves remodeling of large uterine veins (39). These large
obliquely oriented structures may rupture prematurely if poorly supported by the surrounding endometrium or
subjected to elevated intramural pressure due to obstruction of larger upstream maternal veins (38, 130). Unlike
arterial rupture resulting in abruptio placenta, venous hemorrhages tend to occur at the placental margins and at
relatively lower pressure (61). For these reasons, marginal separation may not cause immediate delivery but may
instead present as threatened
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abortion in early pregnancy or vaginal bleeding in later pregnancy. Factors that have been associated with
chronic abruption include multiparity, smoking, oligohydramnios, and excessively deep uterine implantation (114,
177).
FIGURE 9-10▪Chronic abruption: A: Circumvallation—nonperipheral membrane insertion with underlying

organizing blood clot. B: Membrane hemosiderin—cytoplasmic golden-brown refractile pigment within
macrophages in the chorioamnion.
Pathology
Chronic abruption, like maternal underperfusion, is associated with a constellation of placental findings. These
include old marginal blood clot, circumvallate membrane insertion, chorioamnionic hemosiderin deposition, and
green (biliverdin) staining of the fetal surface (Figure 9-10) (155). Circumvallation develops as a consequence of
blood accumulating in the space between the decidua and chorion, leading to undermining or folding of the
marginal chorionic plate. When circumvallation is attributable to chronic marginal separation, old blood clot and
local hemosiderin deposition are seen on histologic sections. Hemosiderin stains blue by iron stain, but other
hemoglobin-related pigments do not. Any pigment seen in a premature placenta favors chronic abruption rather
than meconium release, which is uncommon before 37 weeks.
Chronic abruption is often clinically associated with oligohydramnios, a syndrome known as the chronic
abruptionoligohydramnios sequence (48). Chronic marginal hemorrhages may be detected by ultrasound as so-
called subchorionic hemorrhages (74). Serial ultrasound studies have documented the development of
circumvallation following repeated subchorionic hemorrhages (22). Chronic abruption is an important cause of
preterm delivery and may be associated with an atypical form of neonatal lung disease (188). It is also a
significant risk factor for cerebral palsy and other forms of neurologic impairment in term infants (118, 149).
Finally, acute marginal hemorrhages that result in immediate delivery (marginal abruptions) are important causes
of preterm delivery and should be distinguished from abruptio placenta (see below) by pathologic examination.
Villitis of Unknown Etiology

Background
Diffuse chronic villous inflammation with fibrosis and mineralization is typical of relatively rare TORCH-type
congenital infections (see above). Localized lymphohistiocytic villous inflammation is far more common and is
seen in approximately 5% to 10% of term pregnancies (84). While these localized infiltrates could reflect
unrecognized infections, extensive investigation over many years has failed to reveal organisms and neither the
mothers nor the infants of these pregnancies have shown any consistent clinical or laboratory evidence of an
infectious process. It has been shown that the villous infiltrates are largely composed of maternal T-lymphocytes
(151). It is currently believed that this lesion, known by convention as villitis of unknown etiology (VUE), is the
result of maternofetal cell trafficking with a localized host-versus-graft reaction in the villous tree. Maternofetal
cell trafficking is a well-known phenomena in animal and human pregnancies and can rarely result in neonatal
graft-versus-host disease and connective tissue diseases of childhood (16, 117).
Pathology
The majority of cases of VUE are characterized by small groups of less than 10 affected villi in either a random or
predominantly basal distribution (Figure 9-11A,B). Less commonly, larger groups of villi are involved (patchy or
diffuse VUE) (Figure 9-11C). Stem villous vasculitis and perivasculitis are a special subcategory of VUE, where
lymphocytic infiltration is not confined to the distal villous tree (Figure 9-11D). This pattern is often associated
with extensive downstream avascular villi and has been termed obliterative fetal vasculopathy (139, 143). All
types of VUE are commonly accompanied by a lymphoplasmacytic infiltrate in the basal plate (chronic deciduitis).
Diffuse perivillous fibrin deposition and intervillositis with a polymorphous inflammatory infiltrate including
neutrophils (active chronic
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villitis) are other variations most commonly seen with patchy or diffuse VUE. The presence of neutrophils, plasma
cells, or eosinophils increases the possibility of an underlying infection, which can be further evaluated by
special stains and clinical correlation. Histiocytic giant cells, on the other hand, are common and do not suggest
an infectious etiology.
FIGURE 9-11▪Chronic villitis: A: Focal—clusters of less than ten villi with a nonuniform lymphohistiocytic infiltrate
in the villous stroma. B: Basal—lymphohistiocytic infiltrate involving anchoring stem and adjacent villi
accompanied by a lymphoplasmacytic infiltrate in the decidua basalis. C: Patchy/diffuse—aggregates of ten or
more chronically inflamed villi. D: Obliterative fetal vasculopathy—marked perivascular chronic inflammation
involving stem villi leading to vascular occlusion.
Focal and basal villitis are generally not associated with adverse outcomes (149). Basal villitis is more common
with underlying uterine abnormalities such as malformations, leiomyomas, previous curettage, chronic
endometritis, low implantation, and adherent placenta (142). Patchy and diffuse villitis is associated with IUGR,
particularly when it occurs at term in the absence of hypertension (152). Stem villous vasculitis and perivasculitis
with avascular villi (VUE with obliterative fetal vasculopathy) are associated with an increased risk of cerebral
palsy and other forms of neurologic impairment. Recurrence of VUE occurs in approximately 10% to 25% of
cases (142, 160). This is particularly common with more severe involvement. A small subgroup of women
experience recurrent fetal losses at all gestational ages secondary to diffuse chronic villitis. Also of interest is the
association of VUE with ovum donation pregnancies where the fetus shares no antigens with the mother (125,
175).
Fetal Vascular Thrombo-occlusive Disease
Background
Thrombo-occlusive lesions of large fetal vessels in the placenta and umbilical cord occur in the context of one or
more of the classic triad of risk factors: vascular stasis, loss of endothelial resistance to coagulation, and
circulatory hypercoagulability (143). Possible causes of fetal vascular stasis include prolonged umbilical cord
obstruction, increased central venous pressure, and elevated hematocrit. Loss of
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endothelial resistance to coagulation may occur with severe fetal inflammation, antiphospholipid syndrome, and
other forms of vessel wall damage. Circulatory hypercoagulability may be present with platelet disorders,
maternal diabetes, or thrombophilic mutations involving protein C, protein S, antithrombin II, factor V, prothrombin
2010, and methyltetrahydrofolate reductase. Other causes of inherited and acquired thrombophilia are emerging
with increasing recognition.
Pathology
Sustained proximal vascular occlusion leads to degenerative changes in the distal villous tree. Because of the
extensive branching of the villous tree, changes in distal villi are a more sensitive indicator of disease than the
obstructive lesions themselves. Long-standing occlusion of large fetal vessels leads to hyalinized avascular villi
(Figure 9-12A) (150). Earlier stages lead to circulatory stasis with degeneration of red blood cells, endothelial
cells, and villous stromal fibroblasts (Figure 9-12B). This pattern of change occurs diffusely in the placentas of
stillbirths (52). When seen in a focal distribution in either livebirths or stillborns, it has been termed hemorrhagic
endovasculitis (162) or more recently villous stromal-vascular karyorrhexis (143). Both types of degenerative
villous change can affect large or small groups of villi and can either be localized or distributed throughout the
placental parenchyma. When the number of affected villi exceeds an average of >15 villi/slide, the term fetal
thrombotic vasculopathy is used. Thrombi in large fetal vessels are identified in approximately one-third of such
cases (Figure 9-12C). Other lesions associated with fetal thrombo-occlusive disease include intimal fibrin
cushions and fibromuscular sclerosis of stem arteries (45). Intimal fibrin cushions are intramural aggregates of
fibrin or fibrinoid in proximal fetal veins that may be attributable to increased intramural pressure (Figure 9-12D).
At late stages, they may undergo mineralization. Fibromuscular sclerosis represents concentric narrowing of the
vascular lumen by proliferating smooth muscle cells and subendothelial fibroblasts, typically occurring in
placental vessels lying between the point of occlusion and the distal villi secondary to lack of flow (Figure 9-12E).
FIGURE 9-12▪Fetal thrombo-occlusive lesions: A: Extensive hyalinized avascular distal villi. B: Distal villi with
degenerative stromal-vascular karyorrhexis. C: Occlusive stem villous thrombus.
Fetal thrombotic vasculopathy is a significant risk factor for cerebral palsy and other forms of neurologic
impairment in term infants (86, 137, 149). It may also be associated with other manifestations of thromboembolic
disease in the fetus
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including renal vein thrombosis, perinatal liver disease, and limb infarction (41, 122). Avascular villi are also
associated with IUGR, chronic monitoring abnormalities, and discordant twin growth (134, 152). Nonocclusive
thrombi in severely inflamed chorionic vessels are occasionally seen with severe acute chorioamnionitis in very
low-birth-weight infants and represent a risk factor for neurologic impairment in this subgroup (156).
FIGURE 9-12▪(continued) D: Recent intimal fibrin cushions—layered subendothelial eosinophilic fibrin/matrix
deposits in the walls of large stem villous or chorionic veins. E: Fibromuscular sclerosis—concentric fibrosis of
large fetal vessels with entrapment of degenerating red blood cells.
Massive Perivillous Fibrin Deposition (“Maternal Floor Infarction”)
Background
Massive perivillous fibrin deposition is characterized by the accumulation of excessive amounts of fibrin and
extracellular matrix-rich fibrinoid around gas-exchanging distal villi in the lower two-thirds of the placenta (9, 35,
108). It should be distinguished from increased intervillous fibrin owing to maternal underperfusion, which usually
begins around proximal villi in the upper portions of the placenta (7). Deposition of fibrin and/or other matrix
components could be the primary abnormality providing a substrate that may promote differentiation of villous to
intermediate trophoblasts followed by migration into the intervillous space. Alternatively, the lesion may represent
an aberrant response to trophoblastic injury in which cytotrophoblasts generate intermediate trophoblasts, rather
than syncytiotrophoblasts (trophoblastic metaplasia). These intermediate trophoblasts would then, in turn,
secrete large quantities of extracellular matrix proteins that surround the distal villous tree (42, 50). Massive
perivillous fibrin deposition is idiopathic and often recurrent in subsequent pregnancies. Maternal autoimmune
disease, preeclampsia, and thrombophilic states have all been implicated in its pathogenesis (18, 78, 163). Case
reports of discordancy in twins and an association with fetal long-chain acyl CoA dehydrogenase deficiency
suggest a fetal genetic component as well (97, 148).
Pathology
Massive perivillous fibrin deposition occurs in two distinct patterns: basal-predominant with a rind-like gross
thickening of the basal plate and diffuse with fine lacy strands of firm with fibrin marbling the entire cut surface of
the placenta. Microscopically, distal villi are surrounded by a matrix of fibrin and fibrinoid elements intermixed
with large numbers of intermediate trophoblasts (Figure 9-13). In some cases, degenerative changes such as
eosinophilia or karyorrhexis of villous trophoblasts and stroma may be seen. The lesion is distinguished from
villous infarction by lack of villous agglutination and the absence of degenerating cellular debris in the intervillous
space. Localized plaques of perivillous fibrin and increased intervillous fibrin in areas of marginal placental
atrophy may be seen at all gestational ages (51). These localized lesions should not be mistaken for massive
perivillous fibrin deposition.
FIGURE 9-13▪Massive perivillous fibrin deposition/maternal floor infarction: eosinophilic fibrin/matrix material with
embedded intermediate trophoblasts completely surrounding large portions of the distal villous tree.
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Massive perivillous fibrin deposition (“maternal floor infarction”) is a rare but important placental lesion
associated with spontaneous abortion, stillbirth, severe IUGR, and neurologic impairment (2, 9, 108). It is a
recognized cause of recurrent reproductive failure (13). It most commonly begins in the late second and early
third trimester and can develop quite rapidly. It has been associated with a typical sonographic picture, which
some have termed a “jelly-like” placenta (72). Severe IUGR, decreased pulsed flow Doppler studies, and
abnormal biophysical profile are common, and delivery at the earliest possible opportunity is recommended (95).
No controlled trials of therapy have been conducted. Empiric use of heparin, aspirin, or immunomodulatory
agents has been attempted in some cases.
Villous Capillary Proliferations (Chorangioma/Chorangiomatosis/Chorangiosis)
Background
As described above, early vascularization of the first trimester placenta occurs by vasculogenesis. Mesenchymal
precursor cells form vessels de novo under the inductive influence of adjacent villous trophoblasts. At later
stages of pregnancy, new vessels form by angiogenesis in which new vessels arise via budding and sprouting
from existing vessels. Angiogenic growth factors such as VEGF released under the influence of hypoxia, growth
factors, or inflammatory cytokines can stimulate reactive villous capillary proliferative lesions at several sites in
the mature placenta (121).
Pathology
Chorangiomas are spherical expansile lesions usually found arising from major stem villi under the chorionic
plate or at the placental margins (Figure 9-14A). Histologically, they resemble capillary hemangiomas and are
composed of a mixture of endothelial cells, pericytes, and myofibroblastic stromal cells. Associated nonspecific
surface trophoblastic proliferation is seen in up to 40% of cases and is benign (81). Chorangiomatosis can arise
in the loose reticular connective tissue of either stem or intermediate villi. The lesion is composed of small
vessels with endothelial cells and pericytes surrounding an intact central villous core (Figure 9-14B). Rather than
expanding eccentrically to form a mass as in chorangioma, the vessels in localized chorangiomatosis extend
proximally, distally, and around their site of origin. Diffuse multifocal involvement of immature intermediate villi
appears to be a distinct pattern. Chorangiosis is confined to distal villi, and the vessels are lined by endothelium
alone (Figure 9-14C). The threshold for making a diagnosis of chorangiosis is the presence often or more
capillary cross sections in ten or more villi in several areas of the placenta (3). Occasional villi with 15 to 20 or
more capillaries are usually identified (174).
Chorangiomas are most frequent at sites such as the placental margin and with scenarios such as preeclampsia
and multiple gestations that are associated with relative hypoxia (19, 121). They may be multifocal in rare cases
and are occasionally associated with hemangiomas in the fetus. When large, they can serve as niduses for fetal
consumptive coagulopathy or may act as arteriovenous shunts leading to heart failure and hydrops fetalis (75,
176). Localized chorangiomatosis and chorangioma have similar associations, while diffuse multifocal
chorangiomatosis is more common in preterm placentas and has been associated with IUGR (121). Chorangiosis
is most common in large diabetic placentas but often accompanies placentas with other chronic and subacute
pathologic processes (3, 170). It is also seen in placentas delivered at high altitude and may be a compensatory
physiologic adaptation to reduced oxygen tension without maternal underperfusion of the intervillous space.
Villous capillary vascular lesions of all three types are increased in Beckwith-Wiedemann syndrome as is
mesenchymal dysplasia, a more pervasive abnormality presenting with abnormal large and small fetal vessels,
increased villous connective tissue, and villous cavitation (Figure 9-14D) (71). Mesenchymal dysplasia has also
been associated with several types of confined placental mosaicism (11, 64) (see Chapter 3).
Subacute Disease Processes

Amniotic Fluid Infection/Chorioamnionitis
Background
The products of conception develop in the normally sterile uterine cavity. Parturition, however, requires an outlet
to the external environment. This outlet, the cervicovaginal tract, like most other body orifices has a rich and
complex microbial flora that can include aerobic and anaerobic bacteria, mycoplasma, and fungi. This
environment can also transiently harbor organisms with a particular capacity to infect the products of conception.
These include group B streptococci, Listeria monocytogenes, and the predominantly anaerobic flora associated
with bacterial vaginosis. A connection between the gestational sac and the cervicovaginal tract does not occur
until about 18 to 19 weeks of gestation (55). After that time, the secretory immune system, structural integrity of
the cervix, and the placental membranes serve to protect the fetoplacental unit from ascending infection. Failure
of one or more of these mechanisms may allow organisms to enter the endometrium or amniotic fluid. Local
immunosuppressive mechanisms, fetal immunologic immaturity, and the anatomic enclosure of the fetoplacental
unit all inhibit effective immune responses at these sites. The early inflammatory response to ascending infection
is composed of maternal neutrophils emanating from the intervillous circulation and small venules in the
membranous decidua (26, 27). Later this maternal response may be supplemented by a fetal response
composed of neutrophils emanating from large vessels of the umbilical cord and chorionic plate. The localization
of the inflammatory response reflects the site of infection in the amniotic cavity and the placental layers through
which maternal and fetal neutrophils migrate (chorion and amnion).
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FIGURE 9-14▪Villous capillary lesions: A: Chorangioma—nodular vascular tumor composed of capillaries,

surrounding pericytes, and adjacent fibrous stroma. B: Chorangiomatosis—proliferating capillaries with
surrounding pericytes in the outer reticular zone of an immature intermediate villus. C: Chorangiosis—increased
(>10) number of capillary cross sections in the terminal villi. D: Mesenchymal dysplasia—varying combinations of
increased small and large fetal vessels, excessive villous stroma, and cavitated edematous cisterns affecting
large segments of the villous tree.
While the majority of amniotic fluid infections occur via the ascending route, other mechanisms including
hematogenous spread from distant sites, contiguous spread from other pelvic organs, and direct inoculation of
organisms during diagnostic procedures such as amniocentesis also occur.
Pathology
The pathologic description of chorioamnionitis must be separated into its two components: the maternal and fetal
responses. Each of these in turn should be subcategorized in terms of spatiotemporal progression (stage) and
severity (grade) (145). The stages of maternal infection are (a) acute subchorionitis (neutrophils restricted to
subchorionic fibrin and the membranous decidual-chorionic interface) (Figure 9-15A), (b) acute chorioamnionitis
(neutrophils in chorion and amnion), and (c) necrotizing chorioamnionitis (signs of amnion necrosis) (Figure 9-
15B). These signs include karyorrhexis of neutrophils, desquamation of amnionic epithelial cells, and intense
eosinophilia of the amnionic basement membrane. The stages of fetal infection are (a) neutrophils in chorionic
vessels (chorionic vasculitis) and/or umbilical vein (umbilical phlebitis), (b) neutrophils in one or both umbilical
arteries (umbilical arteritis), and (c) neutrophils and neutrophilic debris forming arcs around umbilical vessels in
the Wharton jelly (necrotizing funisitis) (Figure 9-15C). Severe maternal responses are characterized by large
accumulations of neutrophils (microabscesses) under the chorion (79). Severe fetal responses are characterized
by near confluent neutrophilic infiltrates in the amnionic side of chorionic vessels with attenuation and
degenerative changes of the vessel wall (intense chorionic vasculitis) (Figure 9-15D). Severe fetal responses
may in some cases lead to the formation of mural thrombi in affected vessels.
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FIGURE 9-15▪Chorioamnionitis: A:Early subchorionitis—diffuse neutrophilic infiltration of the subchorionic fibrin.

B: Necrotizing chorioamnionitis—necrosis and sloughage of amniocytes combined with a thickened eosinophilic
basement membrane and karyorrhexis of adjacent neutrophils. C: Necrotizing funisitis—loosely organized
perivascular arcs of eosinophilic precipitate and degenerating neutrophilic debris in the umbilical Wharton jelly.
D: Intense chorionic vasculitis—near confluent neutrophilic infiltration of the amnionic side of major chorionic
vessels accompanied by myocyte disarray and/or endothelial activation.
The prevalence of histologic chorioamnionitis is inversely proportional to gestational age reaching over 50%
below 28 weeks (33, 106). It is believed that placental infection is the leading cause of premature delivery at less
than 32 weeks. In some cases, chorioamnionitis may be preceded by premature membrane rupture. Bacterial
vaginosis is another risk factor for infection (53). In general, the ability to effectively eradicate intrauterine
infections with antibiotics is limited and preterm delivery is inevitable. Spread of organisms from the infected
placenta to the fetus (early onset sepsis) is much less common, and chorioamnionitis is rarely the direct cause of
intrauterine fetal death. An exception is untreated group B streptococcal infection, which is associated with a
higher but still limited risk of fetal infection. Recently, the fetal response to amniotic fluid infection has received
special attention (fetal inflammatory response syndrome).
It is currently believed that various aspects of this response including circulating cytokines, bacterial toxins, and
activation of the coagulation cascade predispose to cerebral palsy and other forms of neurologic impairment (56,
91, 135). A role for fetal inflammatory response syndrome in the development of chronic lung disease has also
been proposed with conflicting evidence (57, 157, 178).
Amniotic Fluid Meconium
Background
Acute episodes of in utero hypoxia, regardless of duration, can trigger redistribution of blood flow resulting in the
release of fetal stool (meconium) into the amniotic fluid (101). This vagally mediated reflex is believed to
represent an adaptation preserving adequate perfusion to more critical vascular beds such as the central
nervous and cardiovascular systems.
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In most cases, the hypoxic episodes are brief and caused by transient umbilical cord occlusion, which is common
after 39 weeks as the fetus continues to grow and move in the face of decrease in the amount of protective
amniotic fluid and umbilical cord Wharton jelly. Meconium is composed of large amounts of bile acid and
phospholipases that have direct caustic effects on fetal and placental tissues. Particularly important are effects
on umbilical and chorionic blood vessels (5). The amount of meconium passed and the volume of fluid available
to suspend it are important variables in determining its effects on the placenta and fetal lungs. Since meconium
diffuses relatively slowly through fetoplacental tissues, duration of exposure is a critical factor in terms of toxic
effects on fetal blood vessels. Longer duration of exposure is also significant insofar as it is an indicator of
hypoxia occurring more remote from the time of labor and delivery. Meconium increases the risk for
chorioamnionitis by several mechanisms including neutralization of bacterial inhibitory factors in amniotic fluid
and direct chemotactic properties (128). In some cases, prolonged meconium exposure and severe fetal
chorioamnionitis may synergize to cause chorionic vessel injury.
FIGURE 9-16▪Meconium: A: Numerous vacuolated pigment-laden macrophages and amnionic edema with
necrosis of amniocytes. B: Numerous vacuolated pigment-laden macrophages deep in the connective tissue of
the chorionic plate. C: Meconium-associated vascular necrosis—diffuse eosinophilic cytoplasmic degeneration
and nuclear pyknosis (apoptosis) of peripheral vascular smooth muscle cells in the vessels of the chorionic plate
and/or umbilical cord.
Pathology
The pathologist’s role is to determine the chronicity and secondary effects of meconium exposure. Meconium is a
fine particulate red-brown pigment generally found within the vacuolated cytoplasm of tissue macrophages.
Other membrane pigments such as hemosiderin and lipofuscin are morphologically distinct, are not associated
with a clinical history of meconium-stained fluid, and do not cause degenerative changes in the amnion such as
dehiscence from the chorion, necrosis of amniocytes, and connective tissue edema (Figure 9-16A). Estimating
the duration of meconium exposure is inexact (102). It is believed that meconium pigmentladen macrophages
appear in amnion approximately 1 hour after release. Spread to the membranous decidua takes at least 3 hours.
Significant accumulations of pigment-laden macrophages in the deeper layers of the chorionic plate and Wharton
jelly, and green staining of the umbilical cord probably take at least 6 to 12 hours (Figure 9-16B). A rare and
clinically significant lesion associated with prolonged meconium exposure is meconium-associated vascular
necrosis (4). This lesion is characterized by apoptotic cell death of peripheral myocytes in the umbilical and
chorionic vessels (Figure 9-16C).
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Meconium passage occurs in approximately 14% of all deliveries but rarely occurs before 34 weeks of gestation.
While statistically associated with obstetric and neonatal complications, it is neither a specific nor a sensitive
indicator for them. Meconium-associated vascular necrosis has been strongly associated with cerebral palsy and
other adverse neurologic outcomes (149). The presence of abundant pigment-laden macrophages in the
chorionic plate also has a borderline significant association with adverse outcome (138). The meconium
aspiration syndrome is denned as respiratory distress requiring oxygen therapy associated with meconium
release and an abnormal chest x-ray (159). It is associated with serious respiratory and neurologic complications
and a significant mortality rate. It occurs in 11% of meconium-stained infants and has been correlated with the
presence of meconium below the vocal cords. However, prompt suctioning of meconium from the airways after
delivery has not made a major impact on morbidity and mortality (77). Current thinking suggests that meconium
aspiration syndrome is largely due to significant perinatal stresses that lead to the deep aspiration of the
meconium prior to birth.
Fetomaternal Hemorrhage
Background
One of the consequences of the close proximity of maternal and fetal circulations in the placenta is that small
disruptions in the integrity of the villous tree can lead to fetal hemorrhage into the intervillous space. Some
degree of fetomaternal hemorrhage has been estimated to occur in at least 50% of all pregnancies, and fetal
cells may persist in the mother for many years leading to modulation of the immune response and in some cases
maternal autoimmune diseases such as scleroderma (117). More substantial hemorrhages of 0.5 to 40 mL occur
in 8% of pregnancies and hemorrhages of greater than 40mL in 0.3% to 1% of pregnancies (49). Diagnosis of
fetomaternal hemorrhages depends on either flow cytometry or the Kleihauer-Betke test. These tests are
performed on a peripheral blood sample from the mother, and the volume of hemorrhage is calculated from the
percentage of fetal cells relative to the maternal blood volume.
FIGURE 9-17▪Fetomaternal hemorrhage/increased NRBC. A: Intervillous thrombus—fresh laminated hematoma

completely surrounded by distal villi. B: Marked increase in circulating NRBC—clusters of immature nucleated
red blood cells including erythroblasts in villous capillaries.
Pathology
Definitive diagnosis of massive fetomaternal hemorrhage can be confirmed only by direct measurement of fetal
red blood cells in the maternal circulation. Placental findings, which suggest the diagnosis in the proper clinical
context are intervillous thrombi (Figure 9-17A), markedly increased circulating nucleated red blood cells (NRBC)
(Figure 9-17B), or signs of developing hydrops fetalis (placentomegaly, villous immaturity, diffuse villous edema)
(Figure 9-17C). NRBCs are discussed below. Intervillous thrombi are spherical collections of clotted blood that
are completely surrounded by villous tissue. They have been shown to represent sites of fetomaternal
hemorrhage (76). However, they are extremely common and are not, in most cases, associated with large
volume bleeds. The finding of multiple or large intervillous thrombi increases the probability of a clinically
significant hemorrhage.
Predisposing factors for massive fetomaternal hemorrhage include severe maternal underperfusion of the
placenta; large edematous placentas associated with fetal congestive heart failure; and traumatic insults
including abruptio placenta, amniocentesis, maternal trauma, or external cephalic version. Most cases have none
of these predisposing factors (49). Cases may present in utero with decreased fetal movements, nonreactive
fetal monitoring, or a distinct sinusoidal fetal heart rate pattern. Affected fetuses and neonates can develop
circulatory collapse, CNS damage, hydrops fetalis, or stillbirth due to a combination of hypovolemia and chronic
high output congestive heart failure due to profound fetal anemia (89).
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FIGURE 9-17▪ (continued) C: Villous hydrops-marked stromal edema with artifactual dehiscence of the villous
trophoblastic layer. D: Mild-to-moderate increase in circulating NRBC—scattered normoblasts in terminal villous
capillaries
Prolonged/Repetitive Antenatal Hypoxia
Background
Prolonged or repetitive shorter periods of antenatal fetal hypoxia are well-documented causes of CNS damage in
experimental pregnancy models and selected clinical cases (107). While the underlying cause of hypoxia is
sometimes indicated by one or more of the pathologic lesions discussed above, in other cases, the insults are
not accompanied by recognizable tissue changes. One useful indicator of sustained significant hypoxia is the
finding of an increased number of circulating NRBC in the placental circulation (113, 171). This physiologic
response is the result of both premature release of red blood cell precursors into the systemic circulation and,
later, increased fetal erythropoiesis. It is, at least in part, mediated by hypoxia-inducible elements in the promoter
regions of erythropoietin.
Pathology
The identification of increased NRBCs in the placental circulation is most important in cases such as stillbirths
where early neonatal blood counts are not available. While erythroblastosis is readily identified (Figure 9-17B),
the recognition of lesser numbers of circulating normoblasts requires a conscious effort to inspect several fields
of distal villi at 40x magnification in every case (Figure 9-17D). A relatively simple semiquantitative method for the
estimation of increased NRBCs in the placenta has been described (140). Others have actually counted NRBCs
in cross sections of large umbilical or chorionic vessels (40).
Increased NRBCs reflect decreased oxygen availability in the fetal hematopoietic microenvironment. This can
occur secondary to maternal hypoxemia, decreased placental oxygen transfer, or insufficient fetal oxygen-
carrying capacity (anemia). Accumulation of red blood cell precursors in hematopoietic tissues and their
subsequent release in large numbers in the fetal circulation require a time interval measured in hours. Variable
estimates of the time required vary from 2 to 24 hours and are controversial (62, 112). Our patient data and the
available animal studies suggest that a marked significant elevation in a previously normal host probably requires
at least 6 to 12 hours to develop (25, 103, 140). Persistence of elevated NRBCs for several days postnatally may
indicate a longer period of antenatal hypoxia associated with markedly increased fetal erythropoiesis.
Acute Disease Processes

Abruptio Placenta
Background
Abruptio placenta (placental abruption), the sudden separation of a significant portion of the placenta from its
underlying maternal blood supply prior to delivery, is one important cause of acute hypoxic injury. While
separation can occur at any location, clinically significant abruptions tend to occur in the central part of the disc
and tend to involve the rupture of maternal spiral arteries rather than veins. Three major factors are associated
with arterial rupture: (a) an abnormal vessel wall (e.g., acute atherosis in preeclampsia), (b) physical force (e.g.,
increased luminal pressure secondary to severe hypertension or shear force associated with maternal trauma),
and (c) ischemia-reperfusion injury (e.g., vasospasm associated with substance abuse involving cocaine or
nicotine) (1, 120, 186). Other processes leading to sudden catastrophic uteroplacental separation include
cervical dilatation with placenta previa and uterine rupture with attempted vaginal delivery following a previous C-
section.
Pathology
It is often stated that the correlation between pathologic and clinical abruption is poor (59). Indeed vaginal
bleeding followed by immediate delivery can occur in the absence of placental lesions. Likewise, clinical signs
and symptoms of
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abruption may also prove unreliable. The gold standard for diagnosis of abruptio placenta is direct visualization
of retroplacental hemorrhage at the time of C-section. Nevertheless, most placentas in cases of abruptio
placenta show one or more of a constellation of findings that allow a diagnosis of findings consistent with
abruption to be made with some confidence. The best pathologic evidence is a finding of a retroplacental
hematoma with either placental indentation or intraplacental extension (Figure 9-18A). In the absence of these
findings, microscopic evidence of interstitial hemorrhage in the basal plate or diffuse retromembranous
hemorrhage is helpful. Ischemic changes in the overlying placenta such as recent villous infarction (Figure 9-
18B) or villous stromal hemorrhage (Figure 9-18C) are also highly suggestive of abruption (104). Finally, lesions
associated with chronic maternal underperfusion, as listed above, are often associated with abruption and can
help strengthen a strong clinical suspicion of the diagnosis (46).
The classical clinical signs of abruptio placenta include vaginal bleeding, abdominal pain, and uterine rigidity.
Abruption is associated with a number of adverse outcomes including preterm delivery, fetal growth restriction,
stillbirth, and hypoxic ischemic encephalopathy (8, 111). Hypertension, maternal substance abuse, advanced
maternal age, low pregnancy weight gain, grand multiparity, and strenuous physical labor are known
predisposing risk factors. A subgroup of patients have repetitive abruptions and both inherited and acquired
maternal coagulation disorders may play a role in some of these patients (66, 83, 184).
FIGURE 9-18 ▪ Recent abruption: A: Laminated blood clot spreading within, indenting, and focally perforating
the basal plate. B: Recent villous infarction—eosinophilic degeneration and karyorrhexis of villous trophoblast
with partial collapse of the intervillous space. C: Villous stromal hemorrhage— diffuse fresh hemorrhage filling
the stroma of immature distal villi.
Umbilical Cord Occlusion
Background
A second common cause of acute hypoxic injury is complete obstruction to umbilical blood flow (cord occlusion).
Obstruction to flow can occur via a variety of mechanisms including occlusive umbilical venous thrombi, tight true
knots, compression of the cord between the fetus and the bony pelvis, hypercoiling, torsion of bridging vessels
associated with anomalous cord insertions (marginal, membranous, furcate) and cord entanglements around
fetal body parts (12, 36, 85, 94, 173). Several scenarios increase the risk of cord occlusion including decreased
Wharton jelly, increased cord length, decreased amniotic fluid volume, sudden changes in
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fetal position, and fetal thrombophilia states. The umbilical vein is the more easily compressed structure by virtue
of its thin wall and its nonduplicated status compared to the umbilical arteries. Cord occlusion prevents
oxygenated placental venous blood from returning to the fetus and may be associated with dramatic differences
between umbilical arterial and venous pH and base excess values (96).
Pathology
The umbilical cord itself may show a distinct abnormality such as a tight overhand knot (Figure 9-19A).
Sometimes, the only gross clue is a difference in diameter and/or color on opposite sides of a putative site of
obstruction (Figure 9-19B). In other cases, obstruction can be inferred by changes within the placenta such as
intimal fibrin cushions (increased venous pressure) or villous stromal karyorrhexis (circulatory stasis). More
recently, the degree of dilatation in chorionic stem villous veins has been used to help make the diagnosis of
cord obstruction as a cause of stillbirth (Figure 9-19C) (124). Also important is documentation of pathologic
abnormalities that may predispose to cord obstruction such as long, thin, hypercoiled, and/or marginally inserted
umbilical cords (Figure 9-19D).
FIGURE 9-19 ▪ Umbilical cord obstruction: A: Tight overhand umbilical cord knot with marked morphologic
changes in vessels on one side of the obstruction. B: Acute cord prolapse—transverse indentation of the
umbilical cord with congestion on the fetal side. C: Markedly dilated chorionic plate veins. D: Excessive long,
diffusely hypercoiled, and macerated umbilical cord associated with an intrauterine fetal demise.
A recent study found either clinical or pathologic abnormalities of the umbilical cord in 63% of term infants with
cerebral palsy (133). Cord occlusion is also a well-recognized, although occasionally controversial, cause of
intrauterine fetal demise (124). Prolapse of the cord with compression between the fetus and pelvic brim is most
commonly seen in premature or breech deliveries and can be a cause of intrapartum death. Transient umbilical
cord occlusion during labor is believed to be responsible for the fetal heart rate abnormality known as variable
decelerations. Variable decelerations can develop a “late” component, a pattern indicative of acidosis and
suggestive of more prolonged and severe occlusion. The correlation between clinical cord entanglements and
outcome is weak and controversial. This reflects the fact that the severity and duration of cord occlusion are
poorly estimated by the observed state of the cord at the time of delivery.
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Fetal Hemorrhage
Background
Finally, the least common mechanism of acute hypoxic injury is fetal hemorrhage. One cause of acute fetal
hemorrhage is massive fetomaternal hemorrhage (discussed above) occurring during labor. Other causes
include transection of umbilical vessels in the placental membranes at the time of membrane rupture; perforation
of umbilical or chorionic vessels at the time of amniocentesis; and sequestration of extravasated blood in the
placenta (subamnionic or subchorionic hemorrhage), umbilical cord (umbilical hematoma), or fetus (liver, lung, GI
tract, central nervous system, caput succedaneum).
Pathology and Clinical Correlation

The relationship between intervillous thrombi and fetomaternal hemorrhage is discussed above and the
pathology of hemorrhages in the fetus is outside of the scope of this chapter. Other placental hemorrhages must
be carefully considered in terms of the clinical history. Fetal vessels traveling in the fetal membranes associated
with peripheral cord insertion are quite common, and these vessels are frequently torn after delivery of the infant
in the third stage of labor. Only tears showing significant amounts of adjacent hemorrhage, distortion of
neighboring tissues, or organization of the hematoma in the setting of fetal distress should be diagnosed.
Likewise, subamnionic and intraumbilical hemorrhages commonly occur with traction on the umbilical cord after
delivery of the infant. Hemorrhages at these sites should be diagnosed only in the presence of predisposing
events such as in utero instrumentation or cord traction (e.g., external version, short umbilical cord) followed by
fetal distress with neonatal anemia and/or hypovolemia.
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Chapter 10
The Nervous System
Christopher Dunham
Arie Perry
GENERAL NEUROPATHOLOGIC PROCESSES AND PRINCIPLES

The practice of neuropathology demands an extensive knowledge of normal central nervous system (CNS)
cytology and architecture, in addition to common artifacts. During development, the CNS (from fetal life and
beyond) changes dramatically, especially in terms of histology, making pathologic assessments even more
challenging. A full discussion of normal CNS histology and common artifacts is beyond the scope of this text.
Some of the more common neuropathologic changes and pathophysiologic processes are introduced below.
The CNS contains a variety of neurons, which vary in size from the small neocortical granular (stellate) neurons
(<15 jim) to the large neocortical pyramidal neurons (measuring from 10 to 100 jim for the Betz cells of the
primary motor cortex). Pyramidal neurons are often considered the morphologic prototype that bears ample
lightly basophilic cytoplasm, darker clumpy Nissl substance, a large central nucleus, a prominent nucleolus (the
“Owl's eye”), and coarse cytoplasmic processes. Neocortical neurons exhibit a prominent apical dendrite
oriented perpendicular to the cortical surface. Neurons may display several different cytologic abnormalities,
some of which are specific, but many of which are nonspecific and must thus be interpreted in the correct clinico-
pathologic context. Acutely necrotic (or “dead”) neurons are a form of nonspecific change that contain two
essential components: (a) shrunken pyknotic angular nuclei and (b) cytoplasmic eosinophilia (“red is dead”)
(Figure 10-1A). Although commonly caused by ischemia (and hence often referred to as ischemic neurons), any
process that causes acute neuronal death may lead to the formation of red neurons (e.g., hypoxia,
hypoglycemia, carbon monoxide, epilepsy, HSV encephalitis). Acute neuronal death is much more difficult to
appreciate in fetal brains that contain a predominance of primitive neurons (small dark nuclei with little
cytoplasm); in such cases, nuclear fragmentation or karyorrhexis (at times in keeping with apoptosis) can be
appreciated on high magnification (Figure 10-1B). A large variety of neuronal inclusions may be seen, including
both intranuclear and intracytoplasmic types. These inclusions vary tremendously in color, size, and shape, from
essentially rounded and eosinophilic/basophilic to more fibrillar (e.g., neurofibrillary tangle). These inclusions are
commonly seen in viral and neurodegenerative/metabolic diseases. Abnormal vacuolization of the cytoplasm can
be seen and may correlate with swelling of ultrastructural elements (e.g., mitochondria). Vacuolization that
seemingly occurs within the neuropil (the meshwork of neuronal processes among which all the cell bodies of the
neocortex reside) may be a result of neuronal loss or the expansion of neuronal processes (which occurs with
the prion diseases or “spongiform” encephalopathies). Some damaged neurons, for instance those near
infarction, may undergo mineralization (i.e., ferruginization). Damage to the axon can lead to several cytologic
alterations. Disruption of the axon may result in chromatolytic changes that include swelling of the soma,
dispersion of the Nissl substance, eccentric displacement of the nucleus, and accumulation of cytoskeletal
filaments. This latter event results in axonal spheroids (i.e., swellings) that can be highlighted with
immunohistochemical (IHC) stains [e.g., P-amyloid precursor protein (β-APP)] (Figure 10-2). The distally
transected portion of the axon degenerates and initially forms ovoids that are later taken up by macrophages,
which serve to localize areas of degeneration (26). Somewhat more fusiform axonal spheroids known as
“torpedoes” are commonly found in the upper cerebellar cortex and reflect Purkinje cell damage.
Normal astrocytes are situated throughout the gray (protoplasmic astrocytes) and white (fibrillary astrocytes)
matter. For the most part, these astrocytes partake in their physiologic duties rather inconspicuously. However,
in response to almost any insult, these cells undergo proliferation (i.e., hyperplasia) and enlargement (i.e.,
hypertrophy) termed reactive astrocytosis or gliosis, which has been equated to the “scar tissue” of the CNS.
Their normally inapparent cytoplasmic processes (by routine H&E staining) accumulate intermediate GFAP
filaments after stimulation, and thus take on a starburst-like pattern (Figure 10-3). In contrast to astrocytic
neoplasms, these reactive astrocytes are
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typically distributed evenly throughout the parenchyma. Myelination glia are a notable pitfall to the determination
of gliosis in the newborn; these glia also display relatively abundant eosinophilic cytoplasm, but they are a
normal finding in the young myelinating nervous system. Although their distinction from gliosis is often difficult,
myelination glia often display hyperchromatic small round nuclei associated with cytoplasm that is shaped like a
comet. In the more chronic stages of gliosis, the cytoplasmic processes of astrocytes retract and become less
obvious on routine staining, although their nuclei remain in increased number and continue to mark areas of prior
damage. Gliosis occurring in the cerebellar cortex in response to Purkinje cell loss is termed Bergmann gliosis,
which is characterized by parallel fibrillary processes radiating through the molecular layer toward the pial
surface and the accumulation of astrocytic nuclei within the Purkinje layer. The somewhat nonspecific form of
gliosis that occurs immediately under the pia mater in the neocortex is called Chaslin gliosis, a reaction that is
often attributed to previous seizure activity. Occasionally found within these areas of longstanding gliosis are the
brightly eosinophilic structures termed Rosenthal fibers (RFs) and less often eosinophilic granular bodies
(EGBs); however, these structures are also seen in a variety of neoplastic [e.g., pilocytic astrocytoma (PA)] and
nonneoplastic (i.e., Alexander disease) conditions (Figure 10-4). RFs have a characteristic EM appearance,
manifesting as an electron-dense core surrounded by fibrillary material. An entirely nonspecific but characteristic
astrocytic reaction occurs in abnormal physiologic states often associated with hyperammonemia; under these
conditions, Alzheimer type II astrocytes (of no relation to Alzheimer disease) accumulate, particularly in the basal
ganglia and deep layers of the neocortex. These astrocytes,
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often seen in pairs, exhibit inconspicuous cytoplasm, enlarged pale nuclei, and often a prominent nucleolus.
Astrocytes may bear inclusions, both cytoplasmic and nuclear, although these are often difficult to appreciate on
routine stains, necessitating the use of special stains and immunohistochemistry for their detection.
FIGURE 10-1 ▪ Acutely “necrotic” or dead neurons. A: Adult. B: Premature infant. Note the nuclear fragmentation
(i.e., karyorrhexis) and eosinophilic cytoplasm in two shrunken subicular neurons.
FIGURE 10-2 ▪ Axonal spheroids from a case of infantile neuroaxonal dystrophy (i.e., Seitelberger disease).
FIGURE 10-3 ▪ Reactive gliosis (Glial fibrillary acidic protein immunohistochemistry (IHC)).
FIGURE 10-4 ▪ Rosenthal fibers and eosinophilic granular bodies are nonspecific eosinophilic structures that are
most commonly seen in the context of long-standing gliosis or within low-grade primary brain neoplasms. A:
Perivascular accumulation of RFs in this case of Alexander disease. B: EGBs within the microcystic component
of a pilocytic astrocytoma.
Normal oligodendroglia fulfill their metabolic roles (most importantly myelination) rather inconspicuously from a
histologic point of view. As opposed to astrocytes, the spectrum of pathology occurring in oligodendroglia is
much more restricted. By routine staining, normal oligodendroglia exhibit inconspicuous cytoplasmic processes
and hyperchromatic, round regular nuclei. As may be predicted, insults affecting oligodendroglia result in
demyelination (i.e., myelin destruction), which can be elucidated with myelin special stains [e.g., Luxol fast blue
(LFB)]. Usually, there is concomitant oligodendroglial dropout and astrocytic gliosis. Some pathologic processes
cause myelin to separate between its layers, resulting in intramyelinic splitting, which manifests as vacuolar
change in the white matter on light microscopy. Like astrocytes, oligodendroglia may bear abnormal nuclear
[e.g., progressive multifocal leukoencephalopathy (PML)] or cytoplasmic inclusions [e.g., multiple systems
atrophy (MSA)], the latter of which usually require special/IHC stains for their detection. The normally
inconspicuous cytoplasm of oligodendroglia may become slightly more conspicuous when they suffer cytotoxic
insults (e.g., ischemia).
Pathologic reactions of the ventricular lining cells, or ependyma, are generally very limited and nonspecific.
These normally columnar to cuboidal cells form a simple (i.e., single layered) epithelium. With hydrocephalus
(HCP) or cerebral atrophy, the epithelium stretches and becomes atrophic, or even discontinuous. Soon after
acute injury, subependymal astrocytes proliferate and produce nodular excrescences, which protrude into the
ventricular cavity. Although previously termed granular ependymitis, this nonspecific pathologic reaction is not
always related to an underlying inflammatory process; as such, the terms subventricular gliosis or ependymal
granulations are preferable. If exuberant, this gliosis may entrap portions of ependyma resulting in subependymal
rosettes/tubules. As with the other cellular elements of the CNS, residual ependymal cells may bear inclusions,
usually of viral etiology.
Microglial reactions are unique to the CNS. Microglia are inflammatory and antigen-presenting cells derived from
bone marrow monocytes. The nuclei and cytoplasmic processes of these parenchymal cells are very
inconspicuous within normal CNS tissue. Generally, they are of two types: (a) resident microglia are those that
reside within the neuropil (and also the perivascular space) and do not undergo significant turnover with
hematogenous monocytes and (b) perivascular microglia, whose population is continually renewed via
hematogenous monocytes (26). Microglia have also been termed rod cells since, after parenchymal insult, their
presence is heralded by a proliferation of small elongate naked nuclei. After CNS damage, perivascular microglia
phagocytose necrotic debris and accordingly accumulate lipid material, which distends their cytoplasm yielding a
foamy appearance. When resident microglia are stimulated, two basic pathologic patterns may be seen. First,
there may be a diffuse microglial activation, wherein rod cells are evenly distributed throughout the diseased
tissue; some have termed this uniquely CNS reaction “neuroinflammation” (21) (Figure 10-5A). Second, and
often associated with viral encephalitides, are microglial nodules, which are roughly spherical aggregates of
microglia (Figure 10-5B). Microglia may also surround and digest dying neurons, a process termed
neuronophagia.
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FIGURE 10-5 ▪ Activated microglia. A: Neuronophagia is seen within this diffuse microgliosis. B: In addition to
some perivascular lymphocytes, a microglial nodule is seen toward the left side of the figure.
Increased Intracranial Pressure, Edema, and Hydrocephalus (HCP)

Once the cranial sutures fuse early on in postnatal life, the skull essentially acts as a rigid closed box, the
contents of which include brain parenchyma, blood, and cerebrospinal fluid (CSF). A mature- sized brain (-1,400
g) contains 75 mL each of blood and CSF Note: a term brain weighs ˜300 to 350 g). This CSF results in a normal
intracranial pressure (ICP) of 15 mm Hg. The cerebral perfusion pressure (CPP) equals the mean arterial
pressure minus the ICP. Cerebral blood flow (CBF), which for the brain as a whole is 50 mL/100 g/minute, is
calculated by dividing CPP by resistance (i.e., the vasculature). Through autoregulation, the CBF is kept
constant despite changes in the systemic blood pressure. Notably, the autoregulatory capabilities of the prenatal
cerebral vasculature are poor, making the brain vulnerable to fluctuations in blood pressure. When a mass-
forming disease process increases the intracranial contents and elevates ICP (e.g., brain tumor), the brain
compensates by expelling contents from the “closed box” so as to maintain the CBF at near normal levels. CSF
leaves the cranial cavity first, and once autoregulatory mechanisms fail, blood is expelled (i.e., global ischemia),
and then finally brain tissue (i.e., cerebral herniation). Several dural folds exist in the cranial cavity (e.g., cerebral
falx, tentorium) and effectively serve to compartmentalize the brain. However, these extremely tough pieces of
connective tissue are unyielding in the setting of increased ICP. In response to a mass lesion, brain tissue will
shift or herniate from one compartment to the next, resulting in tissue damage (including contusion). Subfalcine,
transtentorial (i.e., uncal), and tonsillar herniations are the most important forms, with the latter often being fatal
due to compression of nearby cardiorespiratory centers in the medulla (26).
Cerebral edema is a local or generalized accumulation of fluid within the brain parenchyma that can result in
increased ICP. If severe, cerebral edema may result in herniation. There are three main types of cerebral edema:
(a) vasogenic, (b) cytotoxic, and (c) hydrocephalic. The blood-brain-barrier (BBB) results from the specialized
properties of the endothelial cells, their intercellular junctions, and a relative lack of vesicular transport (51).
Breakdown of the BBB results in vasogenic cerebral edema. This type of cerebral edema is often seen in the
context of CNS neoplasia and is responsive to steroid therapy. Cytotoxic cerebral edema refers to the
intracellular swelling that occurs in neurons, glia, and endothelial cells. This results from failure of the ATP-
dependent Na+7K pump, and subsequent osmotic accumulation of intracellular fluids. Cytotoxic cerebral edema
occurs after hypoxia, or more commonly with global ischemia due to cardiac arrest. Hydrocephalic cerebral
edema is the result of transependymal CSF accumulation. Both vasogenic and hydrocephalic cerebral edema
are elucidated by hyperintense signals seen on T2-weighted MRI and fluid-attenuated inversion recovery
(FLAIR) sequences. Notably, these forms of cerebral edema are not mutually exclusive, and often occur
simultaneously. Grossly, the edematous brain exhibits congestion, with flattening of gyri and narrowing of sulci.
There may be evidence of cerebral herniation (see above) manifesting as areas of necrosis and hemorrhage.
Certain types of herniation routinely cause compression of large arteries and hence infarction (e.g., uncal
herniation with posterior cerebral artery compression and primary occipital lobe infarction). On coronal
sectioning, the ventricles are collapsed from surrounding pressure and appear slit-like. By histology, the
parenchyma is pale and vacuolated.
HCP refers to the accumulation of excess CSF and concurrent expansion of the cerebral ventricles (Figure 10-6).
CSF can accumulate under normal ICP, usually in the context of cerebral atrophy; this is called HCP ex vacuo.
Conventional HCP occurs under the pressure of excess CSF, usually resulting from a paucity of CSF absorption
but also more
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uncommonly from abnormal CSF production [e.g., from a choroid plexus papilloma (CPP)]. HCP due to impaired
resorption can be communicating or noncommunicating, with the former resulting from a lack of arachnoid
granulation-mediated CSF uptake, and the latter being caused by an obstruction in the ventricular system.
Communicating HCP is often a result of meningitis or subarachnoid hemorrhage (SAH), while more rare causes
include arachnoid villi aplasia or dural venous sinus obstruction. Noncommunicating HCP may be primary (i.e.,
congenital) or secondary (i.e., acquired). Primary causes include the enigmatic aqueductal obstruction (e.g.,
related to gliosis, stenosis, atresia/forking, or an obstructing septum) and X-linked HCP (caused by mutations in
the L1CAM gene on Xq28). Secondary causes include tumor, hemorrhage, or infection. Several structural CNS
abnormalities may exhibit concurrent HCP (e.g., holoprosencephaly) of unknown pathogenesis, and some
suggest that the nonspecific term ventriculomegaly may be more appropriate in these cases.
FIGURE 10-6 ▪ Hydrocephalus. There is marked dilatation proximal to and including the fourth ventricle in this
sagittally sectioned autopsy brain, which also exhibited evidence of meningitis.
TRAUMA
Birth
Various craniospinal injuries may be mechanically incurred at birth. A number of extracranial hemorrhages may
occur within the scalp whose layers can be remembered via the mnemonic “scalp” (Skin, Connective tissue,
Aponeurosis epicranialis or galea, Loose connective tissue, Periosteum). Hemorrhage into the subcutaneous
connective tissue is called caput succedaneum. There may be subgaleal bleeding and subperiosteal
hemorrhage (i.e., cephalohematoma) that often occur over the parietal bone, which may be attributable to
forceps delivery. Usually these hemorrhages resolve after a few weeks or months. Perinatal skull fractures are
also frequently parietal in location, and often linear in quality. Depressed skull fractures tend to be more common
in children over 2 years of age. Separation of the squamous and lateral aspects of the occipital bone is called
occipital osteodiastasis, and this may result in contusion of the cerebellum and posterior fossa subdural
hemorrhage (SDH). Epidural hemorrhage is less common than SDH and SAH. A cerebral contusion with
subsequent evolution to intracerebral/intraventricular hemorrhage (IVH) is rare. However, white matter tears, that
are potentially hemorrhagic (i.e., gliding or internal contusions), may be seen in young infants and are thought to
arise from shearing forces between the gray and white matter.
FIGURE 10-7 ▪ Laceration and intraparenchymal hemorrhage within the spinal cord secondary to a complicated
breech delivery seen here in cross sections of the spinal cord.
The spinal cord may absorb fractional or rotational forces at the time of birth. Breech and cephalic deliveries
typically result in upper thoracic/low cervical and midcervical damage, respectively. Large forces can result in
laceration (i.e., tearing) of the parenchyma (Figure 10-7). Petechial hemorrhages and axonal spheroids may be
seen microscopically. Clinical outcome is variable; there may be acute respiratory failure and death or, in those
survivors who are initially hypotonic, spasticity. The brachial plexus may be injured via fractional forces at the
time of delivery. Damage to the C5-6 roots results in shoulder deficits (i.e., Erb paralysis), whereas the wrist and
digits are affected with C8-T1 insult (Klumpke paralysis). Simplistically, surgical repair involves resection of the
resultant traumatic neuroma with anastomosis of more normal proximal and distal nerve stumps; frozen section
assessment of the degree of nerve stump viability may be requested intraoperatively.
Infancy and Childhood

Pediatric patients may suffer from both accidental and nonaccidental (i.e., inflicted or abusive) injury. Motor
vehicle accidents (MVAs), falls, and assaults are the leading causes of pediatric neurottauma. The
neuropathology of severe or fatal head injury in children older than 1 year is very similar to that seen in adults
(26). Infants less than 1 year old suffer a different pattern of injuries, which is thought to be related to the unique
anatomic features of this age (39). These include a highly deformable skull, unfused cranial sutures, a high
head:body ratio, an elastic spinal column with immature joints, reduced neck muscle tone, and a relatively
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unmyelinated brain. Accordingly, the cause of death in fatal infant craniospinal injury is often related to cerebral
edema, secondary to hypoxia-ischemia. The craniocervical junction is particularly vulnerable and damage to vital
brainstem cardiorespiratory centers may account for the frequent clinical presentation of apnea (39). SDH may
be seen and is typically thin and bilateral (see below).
Inflicted Injury in Infants

The pathogenesis of fatally inflicted CNS injury among infants is controversial. Several terms have been used to
describe the classic pattern injuries and circumstances, the most common of which is likely “shaken baby
syndrome.” Many disfavor the use of this term since it implies knowledge of the mechanisms surrounding injury;
hence we use the term “inflicted injury.” Infants suffering inflicted injury often present clinically in a moribund
state with respiratory distress or apnea. There may be lethargy, irritation, poor feeding, vomiting, and seizures.
Fundoscopy may reveal retinal hemorrhages and CT/MRI often reveals cerebral swelling and a diffuse thin layer
of subarachnoid blood. In addition to inflicted mechanisms, this clinical feature may be mimicked by other
etiologic entities, including MVAs, vasculopathies/coagulopathies, infection, dehydration, and metabolic
abnormalities; hence a thorough forensic-based investigation of these cases may be required (see Chapter 7).
Autopsy of an infant with inflicted injury needs to be meticulous since many findings may be subtle. Extracranial
injuries may include rib and long bone fractures, which may be more conspicuous in older children. A variety of
cranial fractures and hemorrhages is somewhat characteristic but nevertheless nonspecific in isolation. Skull
fractures are relatively common in infants despite the inherent deformability of these bones at this age. Fractures
tend to be linear and may result in dural tears and hence CSF leaks (e.g., rhinorrhea, otorrhea). Growing
fractures occur when a portion of the leptomeninges herniate through the dural defect and intercede between the
two sides of a bony interruption; with time, CSF accumulates in a cyst-like space and erodes bone, thus
preventing proper healing. In general, skull fractures are most often parietooccipital in location. Several different
types of hemorrhage may be incurred. Epidural hemorrhage occurs between the outer surface of the dura and
the adjacent skull. It is uncommon in infants, possibly because the dura is tightly adherent to the skull, and since
the middle meningeal artery is more easily displaced than torn (39). SDHs are common yet different than the
space-occupying variety seen in older children and adults. They are thin and bilateral and are often described as
“trivial.” Accordingly, infant SDHs are not thought to be due to the tearing of bridging veins, and they tend to be
accompanied by retinal hemorrhages (which are somewhat nonspecific). SAH and IVHs are generally negligible.
Cerebral contusions are essentially “brain bruises,” which manifest as areas of parenchymal hemorrhage and
necrosis among the crests of gyri. Acutely, the hemorrhage of contusions is perivascular and oriented
perpendicular to the cortical surface. Although they may be seen beneath skull fractures, contusions are
generally uncommon. Cerebral edema is often the immediate cause of death, and is related to global hypoxic-
ischemic injury. As mentioned above, the craniocervical junction is particularly susceptible to injury, especially
that which is related to stretch. Careful dissection of the cervical paraspinal muscles may reveal soft-tissue
hemorrhage. Spinal epidural hemorrhage may be seen but must be cautiously interpreted since this can be
artifactually induced. When the craniocervical region suffers from severe hyperextension injury, pontomedullary
rents (or tears) may be identified. If the injured infant survives but dies later on, signs of cerebral atrophy in
keeping with hypoxic-ischemic encephalopathy (HIE) may be seen.
Although previously claimed to be indicative of diffuse axonal injury (DAI) by some, the microscopic feature of
infant-inflicted injury is not in keeping with such. DAI is due to profound acceleration/deceleration and rotational
forces placed upon the head, and results in widespread axonal damage. As opposed to the axonal damage of
DAI, infant-inflicted injury is localized often to the lower pons and upper medulla, and particularly to descending
corticospinal tracts (32). In addition, axonal damage may be apparent in the cervical and other spinal roots (95).
Axonal spheroids can be seen on routine stains about 24 hours after injury, but IHC staining for P-APP can
highlight microscopic axonal damage after as little as 2 hours. The lysosomal marker, CD68, is often used to
label microglia, and may be used to highlight the acute and more remote cellular reactions to axonal damage.
HIE is frequent and is seen more diffusely throughout the brain. Early features include acute neuronal necrosis
(i.e., red neurons), parenchymal vacuolation, microglial activation, and myelin pallor, whereas in chronic stages,
parenchymal rarefaction predominates with neuronal loss and gliosis.
SUDDEN INFANT DEATH SYNDROME

Sudden infant death syndrome (SIDS) is a complex multifactorial disorder characterized by the sudden clinico-
pathologically unexplained death of an infant (<1 year old). Numerous factors are associated with SIDS, but the
role to which each impacts this disorder remains elusive. Many of these factors are hypothesized in terms of their
effect on the infant's innate autonomic physiologic response to normal homeostatic stressors (e.g., apnea,
hypercarbia/blood pH, hypotension, etc.). These infant responses are largely mediated by brainstem nuclei.
Maternal factors increasing the risk of SIDS include low socioeconomic status, low maternal education, cigarette
smoking, and alcohol consumption. These maternal factors may play a role in predisposing the embryo/infant to
hypoxicischemic damage [e.g., periventricular leukomalacia (PVL)] or brainstem abnormalities, which can be
seen pathologically in a subset of SIDS cases (67); notably, the histopathologic changes seen in SIDS cases are
often inconspicuous.
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Cerebellar and more so medullary abnormalities have been hypothesized. Derivatives of the rhombic lip [e.g.,
external granule layer (EGL) of the cerebellum, inferior olive, and the arcuate nucleus] have been implicated in
SIDS. Abnormalities in medullary serotonergic (5HT) nuclei have been postulated (74). These nuclei have been
suggested to normally modulate and integrate autonomic, respiratory, and somatomotor responses to
homeostatic stressors; in particular, lowered 5HT receptor binding has been demonstrated in the arcuate
nucleus (56). Susceptibility genes have been proposed, as evidenced by the detection of polymorphisms in the
promoter regions/coding sequences; candidate genes include the 5HT transporter, IL-10, and heat shock protein
60 (39). Rare SIDS-like cases have even been associated with cardiac sodium channel mutations (causing
arrhythmias) and inborn errors of metabolism (especially medium chain acetylCoA dehydrogenase deficiency).
Death often occurs during sleep and is associated with the prone position. Integration and synthesis of these
factors have led to the triple risk model of SIDS (27). This model proposes that there are three key factors
leading to infant death when present simultaneously: (a) a vulnerable infant (i.e., those with pathophysiologic
abnormalities), (b) a critical period of development (the peak incidence of SIDS cases occurs at 2 to 4 months
and may be related to brain maturation), and (c) an exogenous stressor (e.g., prone sleeping). As researchers
continue to unravel the mysteries of this disorder, management and minimization of these recognizable risk
factors are the best means of preventing this devastating syndrome (see Chapter 7).
STRUCTURAL MALFORMATIONS OF THE CNS

Neural Tube Defects, Axial Mesodermal Defects, and Tail Bud Defects
Classic embryologic describes three primitive germ layers: endoderm, mesoderm, and ectoderm. Near 16 days
postovulation, the mesodermally derived notochord induces the development of CNS tissue from the overlying
ectoderm; the signaling molecule sonic hedgehog (Shh) is important to this process. This newly formed
neuroectoderm first thickens into the neural plate. A longitudinal neural groove then develops, and subsequently
at 18 to 20 days postovulation, neural folds arise from the lateral aspects of the plate. The neural crest (the
forerunner of the spinal, cranial nerve and autonomic ganglia, leptomeninges, Schwann cells, melanocytes, and
other tissues) originates from the apices of these folds, which eventually meet at distinct closure sites in the
midline to form the neural tube. In humans, two initial closure sites are well recognized, one (Site 1) at the
cervicaloccipital boundary (on day 22 postfertilization), and a second (Site 2) at the extreme rostral end of the
neural plate. A third closure site at the forebrain-midbrain boundary may also exist. Fusion of the neural fold
proceeds bidirectionally from Site 1 and caudally from Site 2. Fusion of the cranial portion of the neural tube is
completed at the anterior neuropore (24 days postfertilization), which subsequently develops into the lamina
terminalis. The caudal aspect of the neural tube finishes closure at the posterior neuropore (28 days
postfertilization). In general, the process of neural tube formation is called neurulation and is divided into two
aspects: (a) primary neurulation describes the fusion of neural folds, which form the rostral aspects of the CNS
and (b) secondary neurulation describes the formation of the caudal-most neural tube (i.e., lumbosacral spinal
cord) that occurs through canalization of a solid mass of cells. Primary and secondary neurulated tissues
eventually join to form the complete neural tube. After neural tube formation, the axial skeleton begins its
development and eventually encases the maturing CNS. The skull has a dual origin: the cranial vault and occiput
develop from axial mesoderm (endochondral bone formation), while the skull base and facial bones arise from
cranial neural crest (membranous bone). The vertebrae also arise from axial mesoderm.
Neural Tube Defects

Neural tube defects (NTDs) are the result of defective neural tube closure during the third to fourth week of
gestational age (GA). The true incidence of NTD is difficult to determine since severe forms lead to spontaneous
abortion. Birth prevalence has been estimated to be between 3 and 7/1,000 depending on several factors,
including geography (e.g., high prevalence in Northern Ireland) (30). There is a spectrum of CNS involvement,
from widespread (e.g., complete craniorachischisis) to more focal (e.g., lumbosacral myelomeningocele).
Patients with focal spinal forms may survive with motor and sensory deficits below the level of nonclosure, which
include rectal and urinary sphincter involvement (e.g., incontinence, urinary tract infections). Additional problems
may include HCP, Chiari II malformation, and kyphosis. Although the use of folic acid supplements has reduced
the frequency of NTDs, the mechanism by which they act is unclear. Craniorachischisis is the most severe form
of NTD wherein there is complete failure of neural tube closure such that the brain and spinal cord are exposed
to the amniotic fluid. There may be some forebrain development rostrally, but neural tube closure is usually
deficient distal to the midbrain. In anencephaly, the NTD is generally limited to the cranial and cervical regions
(Figure 10-8). The skull vault is absent, the skull base is malformed (thick and flat anomalous sphenoid bones),
and the orbits are shallow. The majority of the brain (minus portions of the anterior pituitary, cranial nerves,
medulla, and some cerebellar folia) is replaced by the area cerebrovasculosa. This CSF-filled cystic
angiomatous area contains neuroepithelial remnants (including ependyma, neurons, neuroblasts, and choroid
plexus) and numerous thin-walled blood vessels. Keratinizing squamous epithelium, that is continuous with
normal skin, covers the defect. Due to the deficiency of cerebral tissue, there is a paucity of descending spinal
cord tracts.
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Overlying spinal leptomeninges are vascular and may contain glioneuronal heterotopias (39). Associated
abnormalities include hypoplastic adrenals and lungs, plus an enlarged thymus. Myelomeningoceles can occur
throughout the spinal cord, but lumbosacral lesions are the most frequent (Figure 10-9). An association with the
Chiari II malformation may be noted. The spinal cord may be “closed” (i.e., no NTD per se) or “open” posteriorly
as a flattened lesion. Closed lesions are cystic and covered by a delicate membrane/skin that contains a
hydromyelic cord (Figure 10-9A,B). Open lesions contain a vascularized mass of disorganized neuroepithelial
tissue, the area medullovasculosa that is covered by atrophic cutaneous tissue. In either case, the spinal cord
and meninges herniate through an associated vertebral defect. There may be spinal cord abnormalities above
the NTD (e.g., hydromyelia, syringomyelia, and diplomyelia).
FIGURE 10-8 ▪ Anencephaly Because of the absent calvarium (or “skull cap”), malformed vascularized neuroglial
tissue (i.e., area cerebrovasculosa) can be directly visualized (see arrow).
FIGURE 10-9 ▪ Cervical myelomeningocele (A and B). Gross dissection revealed contiguity of the midline
cervical mass and the cervical spinal cord. (Images courtesy of Dr. Beth Levy Department of Pathology, St. Louis
University School of Medicine, St. Louis, MO.).
Herniation Through Axial Mesodermal Defects

Portions of CNS tissue (with proper neural tube closure) may herniate through axial mesodermal (i.e., bony)
defects. These include encephaloceles and meningoceles. Encephaloceles may be anteriorly located (fronto-
ethmoidal cases are common in Southeast Asia), but occipital cases are the most frequent (Figure 10-10).
Occipital encephaloceles can involve the foramen magnum and include portions of cerebellum, brainstem, and
occipital lobe (e.g., Chiari III malformation). Meckel-Gruber syndrome is a lethal autosomal recessive disorder
that is characterized by the triad of CNS malformations (especially occipital encephalocele), cystic dysplasia of
the kidneys, and ductal plate malformations of the liver (3). It can be detected by ultrasound prior to 14 weeks
gestation. There is genetic linkage to three loci: 17q21-24 (MKS1), 11q13 (MKS2), and 8q24 (MKS3).
Meningoceles are typically lumbosacral in location. All three layers of meninges herniate through the bony
vertebral defect, while the spinal cord remains in a normal position. Accompanying
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spinal cord defects may be seen and include hydromyelia, syringomyelia, diastematomyelia, and cord tethering.
FIGURE 10-10 ▪ Encephalocele. A: Atrophic cutaneous tissue overlies brain parenchyma in this surgical
specimen. (Image courtesy of Dr. Beth Levy, St. Louis University.) B: Histologically, neuroglial tissue (arrow) is
embedded within the deep subcutaneous connective tissue from a more subtly involved example.
Tailbud Defects
Tailbud defects are thought to involve abnormalities of secondary neurulation. Cord abnormalities are
lumbosacral and include hydromyelia (dilatation of the central canal), diastematomyelia (splitting of the cord into
hemisections and often due to a bony spur), diplomyelia (duplication), and cord tethering. The tethered cord
syndrome per se involves lower limb motor and sensory deficits, pain, and neuropathic bladder, all of which
presumably result from traction on distal cord elements. There may be a thickened filum terminate, low or dilated
conus medullaris, spinal lipoma, or other abnormalities in the lumbosacral cord or sacral region in general (39).
Detethering frequently leads to clinical improvement, although surgical specimens are not common (61).
Disorders of Forebrain Development

The early development of the forebrain and midline structures, as it pertains to the neuropathology of structural
malformations, is described in greater detail by Ellison (26). Three primary brain vesicles are present by the
fourth week of gestational age (GA): (a) prosencephalon (forebrain), (b) mesencephalon (midbrain), and (c)
rhombencephalon (hindbrain). Forebrain induction is thought to be governed by the prechordal plate, the
ventralizing molecule Shh, and the dorsalizing molecule bone morphogenic protein 7. By constraining growth in
the ventral midline of the forebrain primordium, the relatively rapid dorsolateral growth leads to the formation of
paired telencephalic secondary vesicles by the sixth week GA. There are five secondary brain vesicles: (a) the
telencephalon (cerebral hemispheres and basal ganglia) and (b) diencephalon (thalamic substructures), both
arise from the prosencephalon; (c) mesencephalon; (d) the metencephalon (pons and cerebellum) and (e)
myelencephalon (medulla), both arise from the rhombencephalon. Shh also induces the optic primordium to
divide and grow out from the diencephalon at 4 to 5 weeks GA. The paired olfactory vesicles are induced by the
olfactory placodes and their ingrowing olfactory nerves at 6 weeks GA. The anterior commissure begins its
development at 10 weeks GA, arising from or adjacent to the lamina terminalis. At this same time, the fornices
and hippocampal primordia arise nearby and grow in a reverse C-shaped manner en route to their destination in
the temporal lobe. The corpus callosum arises at 12 weeks GA from the massa commissuralis, which is slightly
rostral and superior to the anterior commissure. It grows in a rostro-caudal manner, and in doing so results in the
formation of the septum pellucidum at 20 weeks GA.
Holoprosencephaly and Agenesis of the Corpus Callosum

Holoprosencephaly is a disorder of induction and patterning of the rostral neural tube occurring at 4 to 5 weeks
GA. This may be the result of a faulty prechordal plate (17). The majority of cases are sporadic, and the
incidence is approximately 5 to 9/100,000 live births. Risk factors for holoprosencephaly include maternal
diabetes and possibly alcohol consumption. Chromosomal abnormalities are commonly seen, the most frequent
of which is trisomy 13 (Patau syndrome). Molecular genetic investigations have revealed seven genes that are
associated with holoprosencephaly, including SIX3 (HPE2), SHH (HPE3), TGIF (HPE4), ZIC2 (HPE5), and
PTCH (HPE7). Mutation of the sonic hedgehog gene (SHH) is especially intriguing in light of its role in
neuroectodermal induction. Holoprosencephaly may be seen in the context of a well-recognized syndrome, such
as Smith-Lemli-Opitz syndrome (due to a defect in cholesterol biosynthesis). Notably, the Shh molecule must
undergo autoproteolytic cleavage for
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proper functioning, which in turn is dependent on cholesterol attachment to its carboxy-terminus.
Holoprosencephaly has a variable clinical picture; severe forms result in death early in life, while more mild forms
allow survival into adulthood. Craniofacial and ocular abnormalities that can be present have been hypothesized
to be a result of defective mesencephalic neural crest (94) (Figure 10-11A). Microcephaly (i.e., small head) is
common; brains are correspondingly micrencephalic (i.e., low weight) and often less than 100 g at term. There
may be hypotonia, seizures, developmental delay, and mental retardation. Hypofunctioning of the pituitary gland
results in pan-endocrinopathies.
There are three main clinico-pathologic categories of holoprosencephaly that together likely represent a
spectrum of disease severity. Lobar, semilobar, and alobar forms correspond to increasingly severe structural
and clinical diseases, which is generally defined by the extent of the midline longitudinal fissure. In less severe
forms, the fissure can be seen “cleaving” the cerebrum into two cerebral hemispheres more caudally. In alobar
holoprosencephaly, this midline fissure is absent, resulting in a single cerebral mass or holosphere. The Sylvian
fissure, gyrus rectus, and the olfactory structures are also absent. This anomalous gyral pattern prohibits the
delineation of cerebral lobes. The holosphere is horseshoe shaped and contains a single ventricle that opens
postero-dorsally (Figure 10-11B). The opening of this ventricle is covered by a delicate membranous roof that
attaches to the tentorium; this membrane may balloon to form a dorsal cyst. The lateral aspects of this membrane
are bounded by a single arch-shaped hippocampus. The floor of the single ventricle is formed by the fused deep
gray nuclei. Although the corpus callosum is absent, there is no bundle of Probst (see below). The anterior
commissure and the septum pellucidum are also absent. Both the brainstem and the cerebellum are grossly
normal (with the exception of hypoplastic corticospinal tracts). The skull base is malformed. The anterior aspects
of the circle of Willis are anomalous, and both the anterior and middle cerebral arteries are replaced by a
disorganized collection of vessels called the rete mirable. Microscopically, the cortical gray matter is dysplastic
(93). It is excessively thick and dyslaminated, and may demonstrate a progressively abnormal latero-medial
gradient of architectural disturbance (94). The sparsely cellular external layer is segmented and arranged into
irregular clusters, which may form thick cords of neurons that can traverse the entire pallium (i.e., developing
cortical gray matter). There may be acellular deep zones or “glomeruli.” The deeper neocortical neurons are
often maloriented. The leptomeninges of the holosphere can be laden with glioneuronal rests and form a
superficial “crust” over the brain. The architecture of the hippocampi, deep gray nuclei, and cerebellum is also
often abnormal, with the latter exhibiting dysplasia, heterotopia, and an association with trisomy 13.
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The least severe lobar form of holoprosencephaly, despite its resemblance to normal brain, still contains cerebral
cortex that is continuous across the midline (at the frontal pole, in the orbital region or above the corpus callosum
causing cingulosynapsis). Portions of the olfactory structures and posterior corpus callosum may be present.
Semilobar holoprosencephaly is intermediate in appearance. In the recently described middle hemispheric
variant of holoprosencephaly, portions of the deep gray nuclei and fronto-parietal lobe are fused across the
midline, with relative rostral, caudal, and ventral brain sparing.
FIGURE 10-11 ▪ Alobar holoprosencephaly. A: Examination of the face reveals a proboscis (arrow), which is
superior to a single orbit bearing two fused globes. B: Superior and caudal views of the brain reveal the
horseshoe-shaped holosphere containing a single ventricle that opens postero-dorsally. (Images courtesy of Dr.
Robert Schmidt, Department of Pathology and Immunology, Washington University School of Medicine, St.
Louis, MO.)
Agenesis of the corpus callosum (ACC) may be isolated or seen in combination with other CNS abnormalities.
These associations (e.g., a neuronal migration disorder) make it difficult to assess the clinical impact of agenesis
of the corpus callosum per se. However, isolated agenesis of the corpus callosum is most often asymptomatic
and found incidentally on imaging. Potential signs and symptoms may include seizures, mental retardation, subtle
perceptual deficits, or a disconnection-like syndrome. ACC may be associated with a well-recognized syndrome
(e.g., Aicardi) or an inborn error of metabolism (e.g., nonketotic hyperglycinemia). Pathologically, the
characteristic findings of ACC are seen grossly on coronal sectioning of the brain. Agenesis may be complete or
partial, with latter forms being found more caudally (i.e., splenium) in keeping with the corpus callosum's rostro-
caudal embryologic development. Laterally situated and longitudinally directed bundles of white matter are
usually identified immediately superior to the lateral ventricles; these are called the bundles of Probst, and are
thought to represent misdirected callosal fibers (Figure 10-12). The normal dorso-lateral angles of the lateral
ventricles take on an abnormal superior orientation (i.e., “bat-wing ventricles”). The distended membranous roof
of the 3rd ventricle displaces the fornices and leaves of the septum pellucidum laterally. The cingulate gyrus is
replaced by several short radiating gyri, and the anterior commissure may also be absent. Other structural
abnormalities that may accompany agenesis of the corpus callosum include HCP, olfactory hypoplasia and
neuronal migration deficits (see below). A subset of ACC may be the result of mechanically impeding mass lesion
(e.g., lipoma), but the pathogenesis of most other cases is unclear. Some have speculated that abnormalities in
the “glial sling,” which normally guides commissural fibers across the midline, may be a potential cause of ACC
(20).
FIGURE 10-12 ▪ Agenesis of the corpus callosum. Coronal sections of the brain do not reveal a normal corpus
callosum; in its absence, dorso-laterally directed “bundles of Probst” (arrows)are noted. (Image courtesy of Dr.
Barry Rewcastle.)
Other disorders of forebrain induction include olfactory aplasia, atelencephaly, aprosencephaly, and
abnormalities involving the septum pellucidum (26). Just as the development of the cingulate gyrus and that of
corpus callosum are linked, so are those of the olfactory bulbs and the gyrus rectus; true olfactory aplasia is
usually accompanied by absence of the gyrus rectus.
Cell Migration and Specification Disorders

The embryology of early neocortical development is reviewed in greater detail by Golden (39). The wall of the
early neural tube is composed of a pseudostratified neuroepithelial layer. By 4 weeks GA, the outer preplate
zone has emerged from the inner ventricular zone of neuroepithelium. The preplate is composed of two layers:
an outer layer of Cajal Retzius cells (CRCs) (i.e., neurons) and an inner layer of subplate neurons. These
transient subplate neurons play an important role in the early organization of neocortical connectivity. CRCs
secrete reelin, an important extracellular matrix protein that assists migrating neuroblasts in finding their correct
neocortical laminar destination. By 6 weeks GA, radial glia processes have essentially spanned the cortical
mantle and aligned themselves perpendicular to the brain surface. These radial glia processes serve as physical
guides for the migrating neuroblasts, which will form the cortical plate and eventual neocortex. The process of
radial migration is complex and involves numerous molecules, including neuregulin, ErbB4, cell adhesion
molecules, astrotactin, extracellular matrix molecules, and their receptors (39). A two-part physical barrier to
overmigration lies in the marginal zone (i.e., laminae I) of the pallium and is composed of (a) the glia limitans,
which is formed by the expanded end feet of the radial glia and the basal lamina of pial blood vessels, and (b)
the horizontal processes and synapses of CRCs. Neuroblasts leave the ventricular zone to populate the cortical
plate “split the preplate” and form the future neocortical laminae in an inside-to-outside sequence, with the
deepest layers forming prior to more superficial layers (i.e., laminae VI prior to V, etc.). These neuroblasts
migrate in waves between 6 and 20 weeks GA. There is also tangential migration in the developing neocortex,
wherein migrating neuroblasts (future inhibitory neurons) are guided by neuronal processes (rather
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than radial glia) to their destination. The primitive neocortex is initially overpopulated by neurons, and their
numbers are normally culled by apoptosis. The remaining neurons terminally differentiate and establish the
connectivity pattern indicative of the developed neocortex.
Lissencephaly, Types I and II

Lissencephaly type I (i.e., classical type) is a diffuse and abnormally “smooth” (i.e., agyric) cerebral surface,
whereas pachygyria represents a more focal agyric abnormality among more normally gyrated cortex.
Lissencephaly type I is caused by disrupted neocortical cell migration. A number of additional CNS malformations
may be associated with lissencephaly type I, and imaging that reveals such features can guide genetic testing.
Four main genes have been linked to lissencephaly type I and some appear associated with distinct
histopathology (39). LIS1 (17p13.3) encodes the LIS1 protein [aka platelet-activating factor acetyl hydrolase 1
subunit bi (PAFAH1β1)] that is involved in a complex cellular cascade, which influences dynein (and hence cell
movement) and possibly cell proliferation. LIS1 is associated with Miller-Dieker syndrome. XLIS (Xq22.3-23) (or
DCX) encodes doublecortin, which is a microtubule-associated protein; while affected males have lissencephaly
type I, females exhibit subcortical band heterotopia (SBH; see “Cerebral Heterotopia” below). The reelin gene's
(RELN; 7q22) product (i.e., reelin) is the extracellular ligand that influences intracellular downstream targets
(e.g., LIS1 protein). Clinically, mutation in RELN causes lissencephaly type I associated with cerebellar
malformations. Finally, ARX (Xp21) encodes a transcription factor important in CNS/PNS development; mutations
result in lissencephaly type I and ambiguous genitalia. The general clinical features of lissencephaly type I
include developmental delay, mental retardation, seizures (including infantile spasms), and microcephaly.
Pathologically, lissencephaly type I is characterized by thickened neocortical gray matter and a paucity of white
matter (Figure 10-13). The aygric cortex may be preferentially seen more rostrally (XLIS or RELN mutation) or
caudally (LIS1 mutation). Foci of pachygyria tend to have an ill-defined border with more normal brain.
Heterotopic gray matter may be seen in the periventricular and deep white matter, and if associated with an XLIS
mutation, a subcortical band of gray matter may be seen. There may be HCP. The cerebellum is typically
hypoplastic in cases associated with RELN mutations. The inferior olives may be dysplastic, and the cortico-
spinal tracts may be abnormal. Classically, the histology of lissencephaly type I is described as a malformed four-
layer cortex: (a) the outer first layer (i.e., molecular layer) is fairly normal and contains CRCs, (b) the second
layer contains large maloriented pyramidal neuron, (c) the cell poor third layer may be myelinated in older
children, and (d) the fourth layer is a thick and contains disorganized small to medium pyramidal and granular
neurons. This classic four-layer pattern corresponds to the LIS1 mutation and is more prominent in the posterior
cerebral aspects. Recent investigations have suggested additional histologic variants, including four-layer
anteriorly predominant (DCX mutation), three-layer (ARX mutation), and two-layer forms (29).
FIGURE 10-13 ▪ Lissencephaly type I. The markedly thickened cerebral gray matter displays an absence of
gyration. There is a concomitant paucity of cerebral white matter (coronal section). (Image courtesy of Dr. Beth
Levy, St. Louis University.)
The cell migratory defect in lissencephaly type II (i.e., cobblestone type) appears to be one of overmigration. A
defective glia limitans allows radial glial processes to extend beyond the normal limits of the neocortex,
facilitating the excessive migration of neuroglial precursors. Lissencephaly type II shares a thickened neocortical
gray ribbon and at times an agyric surface with lissencephaly type I. However, the five main autosomal recessive
syndromes associated with lissencephaly type II exhibit a characteristic triad of cerebral, ocular, and muscle
diseases that are not seen in type I disease. These syndromes include WalkerWarburg syndrome, Fukuyama
congenital muscular dystrophy (FCMD), Muscle-Eye-Brain disease, congenital muscular dystrophy type ID
(MDC1D), and, MDC1C, a disorder associated with fukutin-related protein (FKRP). FCMD is the most common
(incidence of 3/100,000/year) and characteristically occurs in Japan. The abnormalities in these syndromes (and
hence lissencephaly type II) are thought to be due to defective glycosylation, in particular O-mannosylation.
Glycosylation is a common posttranslational protein modification and, in general, is important to normal
development. O-glycosylation of α-dystroglycan
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appears particularly important to the etiology of these conditions (65). Pathologically, these micrencephalic
brains have a lissencephalic cortex that may have a “bumpy” quality (i.e., cobblestone). The gray-white junction
tends to be distinct below the thickened gray matter. White matter is deficient, and there may be HCP. The
brainstem is small, in part due to hypoplastic corticospinal tracts. The cerebellum in these cases is
characteristically small, especially in the vermal region, and cases of Walker-Warburg syndrome may exhibit
features of a Dandy-Walker malformation (DWM) and an occipital encephalocele. Microscopically, the cortex is
very disorganized and unlaminated. Superficial aspects tend to be more abnormal and may resemble
polymicrogyria. The gray-white junction may exhibit a nodular appearance. The deep and superficial areas are
separated by large internalized and hyalinized blood vessels that likely represent the original overrun
leptomeningeal vasculature. Less severely affected areas may contain a leptomeningeal “crust” of glioneuronal
ectopia. The cerebellum is disorganized, and although the internal granular and Purkinje neurons retain their
somewhat normal relations, the normal architecture is disrupted. Bands of white matter are seen over the
cerebellar surface. The overall appearance of the cerebellum may also resemble polymicrogyria.
Polymicrogyria
Polymicrogyria is a cortical malformation where the neocortical gray matter ribbon is microscopically thin,
excessively folded, and fused. Intrinsic and acquired origins for this lesion have been proposed (39). The risk
factors for polymicrogyria include (a) intrauterine infection (e.g., “TORCH”), (b) intrauterine ischemia, (c)
metabolic diseases (e.g., Zellweger syndrome), and (d) a family history. Polymicrogyria may also be associated
with well-recognized syndromes. Karyotypic abnormalities have been noted (e.g., -Ip36, -22q11), but with the
exception of FGRR3 mutations in thanatophoric dwarfism, specific mutational information is limited (44).
Clinically, localized polymicrogyria may be asymptomatic, but more often it is associated with developmental
delay, psychomotor retardation, spastic diplegia, pseudobulbar palsy, and seizures. MRI highlights this abnormal
cortex and may reveal additional structural abnormalities (e.g., decreased white matter or other white matter
changes, calcification, schizencephaly, porencephaly, etc.). Grossly, the cerebral surface in polymicrogyria is
irregular and bumpy. Coronal sections reveal thickened neocortical gray matter composed of serpiginous,
heaped-up thin layers. Polymicrogyria may be widespread and symmetric, or focal and asymmetric. Cingulate
and striate cortices are often spared. Polymicrogyria may be seen in the relatively spared temporal lobe of
hydranencephaly, or adjacent to porencephalic defects. Microscopically, the cortex is composed of numerous
attenuated, excessively folded, and fused layers (Figure 10-14). Fusion of adjacent molecular layers results in a
branching pattern of paucicellular tissue, which often bears a central blood vessel. The cortex is usually
unlayered but may be four layered (similar to lissencephaly type I). Leptomeningeal glioneuronal and nodular
heterotopias may also be seen.
FIGURE 10-14 ▪ Polymicrogyria. Transverse sectioning of this surgical brain specimen reveals abnormal
undulation of the cortical ribbon.
Cerebral Heterotopia
Cerebral heterotopia refers to malformative lesions wherein groups of cytologically normal brain cells (i.e.,
neurons and glia) do not reach their neocortical destination. Three main categories are discussed here:
leptomeningeal heterotopia (LH), periventricular heterotopia (PH), and SBH. LH and PH are often associated
with other CNS malformations, while SBH is usually seen in isolation. LH is likely the most common of these
three forms and is usually focal. Genetic and epigenetic risk factors are associated with each form of cerebral
heterotopia. Genetic syndromes linked to LH include trisomy 13, holoprosencephaly, and lissencephaly type II.
The genetics of PH are complex, but one X-linked form involves mutations of the FLNA gene (Xq28) (99). SBH is
usually due to mutations in the X-LIS gene (i.e., doublecortin, DCX) (see above). Epigenetic risk factors likely
represent the most common mechanisms underlying LH/PH and include HIE, PVL, and germinal matrix/subpial
hemorrhage. Damage to the glia limitans and radial glia likely underlies the pathogenesis of LH and PH,
respectively. It is difficult to assess the clinical impact of these heterotopias since LH and PH are frequently
associated with other CNS malformations. However, mental retardation and seizures often accompany all three
forms of cerebral heterotopia. Grossly, LH is often inapparent unless seen in the context of lissencephaly type II.
SBH appears as a band of gray matter (outside of the intragyral white matter) that is flanked on either side by
white matter. On close inspection, the gray matter of the SBH may be broken up into nodules, which are split by
white matter bundles. PH may also be confluent (i.e., band-like) or nodular. Like LH, PH is associated with
abnormal adjacent neocortex (cortical dysplasia with LH, and polymicrogyria with PH). Microscopically, all three
forms of cerebral heterotopia appear similar.
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Pyramidal and granular neurons are associated with glia and other normal neocortical elements, but there is no
lamination and the neurons are maloriented. In LH, there is usually some connection to the underlying cortex.
Malformations of Cortical Development, Including Focal Cortical Dysplasia

This group of epileptogenic CNS malformations regrettably has been plagued by a plethora of confusing
terminology. A recent multidisciplinary consensus has been achieved regarding the terminology of these cortical
lesions (72). Malformations of cortical development (MCD) is the umbrella term and encompasses several
entities such as the neuronal migration disorders (NMD; see above), focal cortical dysplasia (FCD), and
microdysgenesis. Previously, the term “cortical dysplasia” was often used nonspecifically to describe many
different abnormal cortical histologies. Microdysgenesis has similarly been utilized in the past to describe more
mild abnormalities in cortical architecture (e.g., excess white matter neurons, excess perivascular oligodendroglia
in the white matter, “glioneuronal hamartia,” abnormal neuronal clustering, dyslamination, cortical
columnarization) (52); however, the term mild MCD is now favored over microdysgenesis (see below).
FCDs are a relatively common surgical specimen. The seizures associated with FCD usually manifest in the first
decade. A variety of genetic predispositions and environmental insults have been hypothesized to play a role in
the pathogenesis of FCD. At what time FCD arise during embryology is unclear (i.e., insult occurring before,
during, or after neuroglial migration). Genetic studies have suggested general roles for the PDK/mTOR and
reelin pathways in FCD. In addition, polymorphisms of the TSC1 gene have been associated with FCD type lib,
while polymorphisms of the TSC2 gene have been seen with ganglioglioma (GG) and FCD type Ha (5).
Identification of these lesions has been facilitated by MRI (90). Pathologically, lesions may be grossly inapparent
or seen as a focal thickening of gray matter with blurring of the underlying gray-white matter junction. The main
histologic features of FCD were recently reviewed and used to devise a new classification scheme (72).
Dyslamination and columnar disorganization are common findings among most FCDs, and are usually associated
with other “mild” abnormalities (in particular, ectopic neurons in the subarachnoid space or within layer I of the
cortex); these findings have been termed mild MCD. FCD has been divided into types I and II (i.e., Taylor type),
with only the latter containing overtly dysmorphic neurons and balloon cells (Figure 10-15). Dysmorphic neurons
are neurofilament (NFP)-rich, maloriented, and/or abnormally large neurons with atypical coarse Nissl substance
and thick dendritic processes. Balloon cells are abnormal cells with abundant glassy eosinophilic cytoplasm and
eccentrically placed vesicular nuclei, often with prominent nucleoli; larger than gemistocytes, these cells may
demonstrate neuronal, glial, or hybrid features by routine staining and by immunohistochemistry (e.g.,
coexpressing GFAP and neuronal markers). Neuronal abnormalities are limited to immature (round/oval cells with
an enlarged nucleus and a thin rim of cytoplasm) and or giant (normal but enlarged, 50 to 80 jim pyramidal)
neurons in FCD type I (see Table 10-1). Other changes may be numerous but often include gliosis,
hypomyelinated zones, an abnormally myelinated layer I, and calcification (39). FCD type lib is often
indistinguishable from the tubers of tuberous sclerosis (TS), and as such may represent a forme fruste of this
condition, a hypothesis which is somewhat strengthened by genetic studies (see above). The histologic changes
of FCD
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may also be similar to those seen in hemimegalencephaly, an epileptogenic disorder that describes the
syndromic or isolated occurrence of an enlarged abnormal hemisphere associated with hemiparesis and
developmental delay (39).
Table 10-1 ▪ HISTOLOGIC CLASSIFICATION OF FCD
Histologic FCDTypelA FCD Type FCDType MA (Taylor FCD Type MB (Taylor

Change IB A) B)
Dyslamination ±
MILD MCD
Immature ±
giant
neurons
Dysmorphic neurons
Balloon “Cells”
FCD, focal cortical dysplasia; MCD, malformation of cortical development (see text for details)
Adapted from Palmini A, Najm I, Avanzini G, et al. Terminology and classification of the FCDs.
Neurology 2004;62(Suppl. 3):S2-S8
FIGURE 10-15 ▪ FCD type lib. Balloon cells are seen within the gliotic and calcified white matter immediately
subjacent to malformed cortical gray matter.
Antenatal Disruptive Lesions
Although in a sense this group of lesions could be considered malformative, they are felt to be largely due to the
impact of a hypoxic-ischemic insult on the developing brain. These insults are acquired in utero. Intrauterine
infections may play a role in the etiology of some of these lesions. Hydranencephaly is the severe and diffuse
necrosis of the cerebral mantle and deep gray (and concomitant HCP ex vacuo) due to perfusion failure of the
internal carotid territory at 15 to 16 weeks gestation. The residual mantle is markedly thinned, and there is
evidence of secondary brainstem and spinal cord atrophy. There may be sparing of parenchyma supplied by the
posterior cerebral artery. Porencephaly describes the focal transmantle necrosis of cerebrum (most often in the
middle cerebral artery territory) wherein the ventricle communicates with the subarachnoid space.
Polymicrogyria, gliosis, and calcification often rim the porencephalic defect. If there is bilateral MCA damage that
spares the cingulate gyri (i.e., leaving a “handle”), the resulting defect is called basket brain. Schizencephaly
describes a nontransmantle cleft in the cerebrum. Multicystic encephalopathy is the result of a diffuse white/gray
matter insult to the cerebrum, causing widespread necrosis and cystic change. The insult in MCE is presumed to
occur late in gestation.
Microcephaly and Micrencephaly

Microcephaly refers to a small head, whereas micrencephaly refers to a small brain. Some authors have used
the term microcephaly interchangeably to refer to both of these abnormalities.
Hindbrain Malformations
As described above, by the sixth week of GA, the secondary brain vesicles that give rise to the cerebellum and
pons (metencephalon), in addition to the medulla (myelencephalon), have begun their development. There are
similarities in the development of the hindbrain and the spinal cord. The alar and basal plates give rise to the
dorsal sensory and ventral motor spinal cord horns, respectively. With respect to hindbrain, its dorsal aspect is
essentially splayed out such that the motor basal plates lie medial, while the sensory alar plates lie lateral. The
metencephalic alar plates fuse and give rise to the cerebellum. The Purkinje and deep gray neurons of the
cerebellum arise from the ventricular zone of the alar plate, while the eventual internal granule neurons arise
from the upper aspect of an alar plate derivative called the rhombic lip (109). A lateral to medial outward
migration of granule neuron precursors has populated the EGL by 14 weeks, and persists until 1 year of age.
Neurons from the EGL migrate inward to their eventual destination in the internal granule layer. The
flocculonodular, anterior and posterior lobes are already identifiable by 12 weeks GA. Cerebellar folia can be
seen by 20 weeks. Precerebellar nuclei (i.e., the pontine and inferior olivary nuclei) arise from the lower rhombic
lip.
Chiari and Dandy-Walker Malformations

Although the Chiari malformations are discussed in the context of the hindbrain, their actual pathogenesis is
unclear (see below). Three forms are well recognized. Chiari I malformations are characterized by the caudal
displacement (not true herniation) of the cerebellar tonsils through the foramen magnum and into the upper
cervical spinal canal. Chiari I malformations are often associated with syringomyelia. The clinical picture includes
neck pain and signs/symptoms associated with syringomyelia (i.e., “cape-like” or “hanging” dissociated sensory
loss in the shoulders and arms) that develops in older teenagers and young adults. Pathologically, surgical
resection specimens reveal sclerotic and gliotic degenerated tonsillar tissue. Pathogenesis of the Chiari I
malformation is unclear. Chiari II malformations (previously known as the Arnold-Chiari malformation) occur in
young children and describe the caudal displacement of cerebellar vermis into the upper cervical spinal canal
plus added hindbrain abnormalities. Ninety-five percent of Chiari II malformations are associated with a
lumbosacral myelomeninocele (39). Maternal vitamin A deficiency is a risk factor for Chiari II. The clinical picture
of Chiari II is dominated by HCP and the myelomeningocele. Pathologically, the fourth ventricle, midbrain, pons,
and medulla are all elongated and caudally displaced. There may be “tectal beaking” and an S-shaped “kinking”
of the medulla onto the dorsal spinal cord (Figure 10-16). The posterior fossa is small, and there may be
abnormalities of the cranial nerves. Additional CNS malformations may include PH, polymicrogyria, and
pachygyria. Pathogenesis of the Chiari II malformation is unknown. Hypotheses include (a) hydrodynamic
(related to mechanic pressure from HCP or excess CSF egress from the myelomeningocele), (b) cord tethering,
(c) a defect in neurulation, or (d) a defect in the posterior fossa mesenchyme leading to restricted cerebellar
growth. Chiari III malformations are rare and are defined by an occipito-cervical encephalocele (that includes
cerebellar tissue) that is accompanied by a distorted brainstem and local anatomy. The clinical picture is similar
to Chiari II but is more severe and the prognosis is poor. Pathologic changes may include cerebellar dysplasia.
Pathogenesis is also unclear but is likely related to defective neurulation.
The DWM (or syndrome) typically presents sporadically in infancy as an isolated finding or in association with
other CNS malformations. It contains five main features. First, there is cystic dilatation of the fourth ventricle.
Second, the cerebellar vermis is hypoplastic or absent. Third, as opposed to the Chiari II malformation, DWM is
characterized by a large posterior fossa. Fourth, and likely related to the large posterior fossa, is the elevation of
the tentorium and related
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dural sinuses. Finally, there is HCP. The Dandy-Walker variant has only some of the features of the DWM, which
include an anteriorly rotated vermis ± fourth ventricular dilatation. The pathogenesis of the DWM is unknown, but
the development of the fourth ventricular roof and its outlet foramina (especially Magendie) is thought to be
important. Maternal isoretinoin use is a risk factor. Clinically, features related to HCP and increased ICP are
common. Surprisingly, cerebellar signs/symptoms are less common. There may be concomitant mental
retardation. Pathologically, the main findings are those seen grossly (see above). The fourth ventricular cyst wall
is composed of an outer pial and inner ependymal layers, with residual cerebellar parenchymal in between.
FIGURE 10-16 ▪ Chiari II malformation. A: This posterior dissection reveals cerebellar vermis that has herniated
through the foramen magnum and an associated lumbar myelomeningocele. (Image courtesy of Dr. Barry
Rewcastle.) B: Sagittal section of the brain highlights herniated and discolored cerebellar vermis along the
posterior aspect of the spinal cord. (Image courtesy of Dr. Robert Schmidt, Washington University School of
Medicine, St. Louis, MO.)
A variety of additional cerebellar malformations has been characterized. Cerebellar heterotopia and dysplasia are
reviewed by Golden (39). Rarer malformations include cerebellar agenesis, Joubert syndrome,
pontoneocerebellar hypoplasia, and granular cell aplasia.
Malformations of the brainstem are numerous but rare. Some are described elsewhere in this chapter (i.e.,
Moebius syndrome, X-linked HCP with congenital absence of the pyramids). Olivary heterotopia and
dentate/olivary dysplasias may occur in association with a number of different CNS malformations or syndromes,
and in light of their origin from the metencephalic alar plate, it is not surprising that these may occur in
conjunction with cerebellar abnormalities.
Cystic Lesions of the CNS

“Cysts” within the CNS are biologically benign and nonneoplastic. Their characteristic sites and pathology are
summarized in Table 10-2. During embryologic development, ectopic placement of germ layer tissue may
account for the formation of many of these lesions.
METABOLIC, NEURODEGENERATIVE, AND MISCELLANEOUS DISORDERS

Lysosomal Storage Disorders
Lysosomes are the digestive organelles of the cell. They contain numerous hydrolytic enzymes (e.g.,
phosphatases, nucleases, glycosidases, proteases, sulfatases, phospholipases, etc.) that assist in normal
cellular metabolism. These enzymes are often directed to cleave off the sugar chains from larger
macromolecules. A deficiency in one or more of the glycoprotein enzymes results in a lysosomal storage disorder
(LSD). The LSDs are relatively uncommon disorders that often result in progressive and multisystemic disease
that is fatal in childhood. LSD can be subdivided into categories, which are roughly based on the class of
macromolecule that is not correctly metabolized; these include (a) sphingolipidoses (lipids), (b)
mucopolysaccharidoses (MPS)
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[disaccharide molecules of glycosaminoglycans (GAGs)],(c) glycoproteinoses (glycoproteins), and (d) a number
of miscellaneous LSDs, which include the neuronal ceroid lipofuscinoses (NCL) and Pompe disease (type II
glycogenosis).
Table 10-2 ▪ CNS CYSTS
Cyst Type Common Sites Pathology
Neurenteric Intradural, Cuboidal to columnar respiratory-type or Gl-type epithelium

(i.e., extramedullary, and covering a connective tissue stroma. Possible goblet cells and
endodermal) ventral to the cervical cilia. Immunohistochemistry3 (IHC)
spinal cord
Colloid Antero-superior third Simple columnar epithelium. Possible cilia. Cyst contents
ventricle near the PAS-positive. IHC3
Foramen of Monroe
Rathke cleft Sella Similar to neurenteric. Degenerate forms with atrophic

epithelium and xanthogranulomatous inflammation. IHC3
Dermoid Midline: fontanelle, Stratified squamous epithelium with dermal adnexal

fourth ventricle, appendages. Cyst contents (grossly “cheesy”): degenerate
cauda equina. keratinocytes, sebaceous material, hair, etc.
Epidermoid Cerebello-pontine Similar to dermoid epithelium but without dermal appendages.

angle (CPA), Keratinizing epithelium. Cyst contents: “dry” keratin. Gross
parasellar, diploe of “pearly” appearance
skull
Ependymal Intraventricular, Columnar epithelium similar to ependyma. Possible cilia. No

leptomeningeal, goblet cells. IHC: GFAP-positive and S-100 protein-positive.
intraparenchymal
Choroid Lateral ventricles Simple cuboidal to columnar epithelium. Cytokeratin-positive

plexus and S-100-positive.
Pineal Pineal parenchyma Three layers: (a) internal fibrillar layer with RFs. IHC: GFAP-
positive. (b) Pineal parenchymal “middle” layer. IHC:
Synaptophysin-positive. (c) Outer connective tissue layer.
Arachnoid CPA, Sylvian fissure CSF filled. Inner arachnoid (EMA-positive) and outer
connective tissue layers.
3Cytokeratin and EMA-positive,with collagen IV immunoreactive subepithelial basement membrane.

Usually CK7-positive, CK20-negative.
The LSDs are generally autosomal recessive disorders that are usually the result of a mutation in the gene that
encodes a particular lysosomal enzyme. There is extensive clinicopathologic overlap among the LSDs as a
whole, many of which are individually indistinguishable without ancillary biochemical and genetic testing.
Conceptually, the LSDs can be divided into four basic clinico-pathologic phenotypes: (a) neuronal lipidoses, (b)
leukodystrophies, (c) storage histiocytoses, and (d) MPS (or the Hurler phenotype) (81). Most LSD storage
products are water soluble and thus are washed out during routine histologic processing, leaving behind only the
clear, vacuolated, and distended cytoplasm of the cells they have affected. This accumulated material
mechanically disrupts cellular processes and eventually leads to cell death. The neuronal lipidoses are
characterized by substrate storage in cytoplasm of neurons, leading to the gross finding of megalencephaly early
in the disease course (Figure 10-17). Subsequent neuronal death and gliosis eventually result in cerebral
atrophy. Involvement of the retina may lead to the characteristic ”cherry red spot,” while other clinical
manifestations of the neuronal lipidosis (NL) phenotype include psychomotor retardation and dementia, loss of
acquired motor and perceptual skills, epilepsy, and myoclonus. The leukodystrophies, which are part of the
LSDs [e.g., metachromatic leukodystrophy (MLD) and Krabbe leukodystrophy (KLD)], are the result of substrate
accumulation in oligodendrocytes and Schwann cells, causing a loss of myelin and myelinating cells. Clinical
manifestations include psychomotor retardation, spasticity, ataxia, visual abnormalities, and a demyelinative
peripheral neuropathy. Substrate accumulation in mesenchymal and epithelial cells, in addition to the
extracellular matrix, results in the Hurler phenotype. Clinically, these patients have core features, which include
coarse facies, skeletal and joint abnormalities (i.e., dysostosis multiplex and arthropathies), organomegaly,
cloudy corneas, cardiovascular disease, and CNS disease (entrapment neuropathies, HCP, and NL). Finally,
substrate storage in monocytes/macrophages causes
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the storage histiocytosis (SH) phenotype, which clinically manifests in hepatosplenomegaly, hematologic, and
skeletal abnormalities. Table 10-3 lists a subset of the LSD, their specific enzymatic defects, and some of their
characteristic clinico-pathologic features (note: MLD and KLD are further described below) (see Chapter 5).
FIGURE 10-17 ▪ Neuronal lipidosis (NL). The cytoplasm of these neurons is markedly distended by lipofuscin-
like storage products in this example of neuronal ceroid lipofuscinosis (NCL).
Leukodystrophies
The leukodystrophies are a group of genetically based progressive disorders that share common abnormalities
in myelin formation and metabolism. These disorders have hence been referred to as dysmyelinating, to
distinguish them from demyelinating disorders (e.g., multiple sclerosis) where myelin is thought to form normally
but is later destroyed. Pathogenetically, the leukodystrophies are a heterogeneous group of disorders, which, for
example, include some of the lysosomal and peroxisomal storage disorders. These disorders often have onset
during childhood, but adults can also be affected. Clinically, these disorders can affect numerous neurologic
modalities and hence result in a myriad of signs and symptoms, which may include psychomotor retardation and
dementia, pyramidal and extrapyramidal manifestations including spastic paraparesis, ataxia, visual and hearing
abnormalities, as well as signs of bulbar involvement. Characteristic clinical manifestations (i.e., age of onset,
signs/symptoms) may accompany specific forms of leukodystrophy. The white matter of not only the CNS but
also the PNS [e.g., MLD, KLD, and less so adrenoleukodystrophy (ALD)] may be affected. Some
leukodystrophies are more systemic in nature and thus bear extra-CNS/PNS disease manifestations (e.g.,
adrenal and testicular involvement with ALD; biliary and renal involvement with MLD). Genetically, many of these
disorders are inherited in
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an autosomal recessive manner; however, some follow an X-linked or sporadic pattern.
Table 10-3 ▪ LYSOSOMAL STORAGE DISEASES
Enzymatic/Protein Characteristic Clinico-

LSD Deficiency Stored Material Pathologic Features
GM1 β-Galactosidase GM1 ganglioside, NL, MPS, cherry red spota

gangliosidosis Keratan sulfate
GM2 Hexosaminidase A GM2 gangliosides NL, SH (Sandhoff), cherry red

gangliosidosis and/or B spota
(Tay-Sachs
and
Sandhoffs)
Niemann-Pick Sphingomyelinase Sphingomyelin NL, SH (Niemann-Pick cells),

A/B cherry red spota
Niemann-Pick ER membrane Phospholipids and NL, SH (Niemann-Pick cells),

C protein with role in glycolipids axonal swellings, and
intracellular neurofibrillary tanglesa
cholesterol
transport
Gaucher Glucocerebrosidase Glucocerebroside SH with Gaucher cells

disease (“wrinkled tissue paper”)a
Fabry disease agr;3- Trihexosylceramide Painful peripheral neuropathy;

Galactosidase ischemic CNS and heart
disease; bathing trunk
telangectasias, renal, and eye
diseasea
Farber Ceramidase Ceramide NL, LD, cherry red spot; painful

granulomatosis arthropathy, subcutaneous
nodules, and hoarseness
related to lipid granulomasa
MPS type I: α-L-lduronidase Dermatan and heparan MPS, mental retardation,

Hurler disease sulfate dysostosis multiplex, cloudy
corneas, heart disease. EM:
reticulogranular inclusions
NCL 1-4 Palmitoyl protein Saposin A&D (NCL1), NL. EM: granular osmiophilic
thioesterase SCMAS (subunit C of deposit (NCL1), curvilinear
(NCL1), Tripeptidyl mitochondrial ATPase bodies (NCL2), fingerprint
pedtidase (NCL2) synthase), (NCL2-4) bodies (NCL3), or “mixed” with
peptidase (NCL2) lipofuscin-like (NCL4)
Pompe α-Glucosidase (acid Glycogen (membrane Vacuolar myopathy,

disease maltase) bound and free by EM) cardiomegaly, macroglossia
(glycogenosis
type 2)
EM: in general, many of these disorders contain membranous cytoplasmic or zebra body inclusions
within lysosomes; GM1 may contain added reticulogranular material; Gaucher disease exhibits tubular
inclusions; Farber granulomatosis features “banana bodies.”
aSphingolipidoses.
LSD, lysosomal storage disease; NL, neuronal lipidosis; MPS, mucopolysaccharidosis; SH, storage
histiocytosis; ER, endoplasmic reticulum; NCL, neuronal ceroid lipofuscinosis.
Pathologically, the leukodystrophies characteristically cause bilaterally symmetric white matter-predominant

disease that can involve the cerebrum, brainstem, cerebellum, and even the spinal cord. Usually, subcortical U-
fibers are spared from myelin destruction (Figure 10-18A). There may be a rostral (Alexander disease, MLD) or
caudal (ALD; KLD) predominance of cerebral white matter disease. In general, early stages of disease are
characterized by widespread myelin destruction with relative axonal preservation; macrophages are often
present and distended by bubbly PASpositive cytoplasmic material. Later stages often demonstrate axonal and
oligodendrocyte destruction plus reactive gliosis. Characteristic pathologic changes often accompany individual
leukodystrophies and hence assist in diagnosis (Table 10-4; Figure 10-18B). A subset of leukodystrophies are
considered “sudanophilic” since macrophages and other cells that accumulate indigestible substrates stain
positive with Sudan B or Oil Red O fat stains. Included in these sudanophilic leukodystrophies are ALD,
Pelizaeus-Merzbacher disease (PMD), and a host of less well-described entities that may contain characteristic
histopathology including calcification, pigmentation, meningeal angiomatosis, and cavitation with oligodendrocyte
proliferation or vanishing white matter disease, which recently has been linked to mutations in any of the genes
encoding subunits of the eukaryotic translation factor eIF2B (87, 115).
Peroxisomal Disorders
Peroxisomes are cellular organelles that have a single membrane, which encloses a matrix wherein numerous
important biochemical reactions take place. Peroxisomes generate hydrogen peroxide (H2O2), a molecule that
assists in oxidizing several cellular toxins. However, H202 can itself be toxic; hence peroxisomes contain
catalase, an enzyme that serves to break down H2O2 into water and oxygen. Peroxisomes also play an important
role in the oxidation of very long chain fatty acids (VLCFAs), plasmalogen biosynthesis (an important cell
membrane and myelin component), cholesterol biosynthesis, and the metabolism of amino acids, bile acids, and
purine nucleotides. Knowledge of these basic biologic functions is clinically useful since the routine laboratory
workup of the peroxisomal disorders often involves initial assessment of VLCFAs, hepatic peroxisomes, and RBC
plasmalogens.
There are three main categories of peroxisomal disorders:
(a) the peroxisomal biogenesis disorders (e.g., Zellweger spectrum, and rhizomelic chondrodysplasia punctata
type 1),
(b) the single enzyme deficiencies (e.g., D-bifunctional protein deficiency and adult Refsum disease), and (c) X-
linked ALD. In general, these disorders are neuropathologically characterized by neuronal migration defects,
leukodystrophy-like white matter abnormalities, CNS lipid deposition, and systemic abnormalities (including the
adrenal cortex and liver). Both the biogenesis disorders and the single enzymes deficiencies include autosomal
recessive inheritance. The former involve mutations in the PEX genes; these encode the peroxin proteins that
are important to peroxisomal functioning. Zellweger spectrum includes three disorders, which are considered to
form a spectrum of diseases related to mutations in PEX1. These include (from most to least severe) the
following: Zellweger syndrome, neonatal ALD, and infantile Refsum disease. Zellweger syndrome (aka,
cerebrohepatorenal syndrome) is a systemic disorder primarily affecting the liver (cirrhosis) and brain. Patients
have dysmorphic facies and neurologic manifestations that include psychomotor retardation, hypotonia,
depressed deep tendon and Moro reflexes, seizures and nystagmus. These infants die within the 1st year of life.
Neuropathologic findings include NMD
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(pachygyria, polymicrogyria), leukodystrophy-like white matter abnormalities, abnormalities of rhombic lip-derived
structures (dentate nucleus/inferior olivary dysplasias, cerebellar heterotopias), and prominent deposition of
sudanophilic lipid in the CNS (primarily within macrophages and showing trilaminar appearance on EM)
(Chapters 5 and 15).
FIGURE 10-18 ▪ Leukodystrophy. A: Coronally sectioned case of Krabbe disease demonstrates symmetric
dysmyelination of cerebral white matter with relative sparing of the subcortical U-fibers. (Image courtesy of Dr.
Barry Rewcastle.) B: LFB-PAS-stained case of ALD demonstrates pale white matter and characteristic
perivascular lymphocytic cuffing.
Table 10-4 ▪ THE LEUKODYSTROPHIES
Biochemical/Genetic
Abnormality
(Chromosomal
Leukodystrophy Locus) Characteristic Pathology
Adrenoleukodystrophy Deficiency of a Perivascular lymphocytic inflammation. EM:

(ALD) peroxisomal ATP- trilaminar inclusions. Striated lamellar cytoplasm
binding cassette inclusions in CNS and select systemic organs
transporter, resulting
in accumulation of
VLCFAs. X-linked
(Xq28)
Metachromatic Aryl-sulfatase A Metachromatic material (using acidic cresyl violet

leukodystrophy (MLD) deficiency. or toluidine blue) in the brain (macrophages), PNS
Accumulate sulfatide (Schwann cells), and viscera (biliary epithelium
(22q13) and renal tubules). EM: herringbone, prismatic,
and tuftstone inclusions.
Krabbe Galactocerebroside P- Globoid cells, often perivascular. PNS:

leukodystrophy (KLD) galactosidase hypertrophic neuropathy with fibrosis and “onion-
deficiency. bulbs.” EM: tubular inclusions
Accumulate
psychosine (14q25-
31)
Alexander disease Sporadic mutation of RF accumulation, especially in perivascular and

the GFAP gene subpial locations. Grossly cavitated white matter.
(17q21) EM: amorphous electron-dense material
surrounded by 10-nm intermediate filaments
Canavan disease Aspartoacylase More central aspects of central myelin lost with
deficiency. relative oligodendroglial and axonal sparing.
Accumulate A/- Vacuolation at neocortical gray-white junction. No
acetylaspartate macrophages and little gliosis (versus other LSDs).
(17p13-ter) EM: myelin splitting at intraperiod line plus
elongate mitochondrial with “ladder-like” cristae
Pelizaeus-Merzbacher Deficiency of normal Perivascular patchy dysmyelination (i.e., tigroid).

disease (PMD) proteolipid protein
(PLP). Accumulate
abnormally folded
PLPintheER.X-
linked(Xq22)
Mitochondrial Disorders
The mitochondria are the “powerhouse” of the cell. They produce the energy needed for life in the form of ATP
via aerobic respiration. The final stages of aerobic respiration are mediated by the electron transport chain,
which comprises five protein complexes that are embedded within the inner mitochondrial membrane. Each of
these five complexes is composed of multiple protein subunits (86 in total), most of which are encoded by nuclear
DNA. The mtDNA genome is circular, double stranded, and includes 16,569 base pairs. Up to ten copies of the
mtDNA genome may be seen within a cell. This genome encodes for 22 tRNAs, 2 rRNAs, and 13 subunits of the
electron transport chain. Genetically induced defects in the assembly or formation of the electron transport chain,
or in the maintenance of the mitochondrial DNA, result in mitochondrial disorders. Dysfunction of the electron
transport chain presumably results in cell death via numerous mechanisms, including energy deprivation, free
radical toxicity, and apoptosis. Since electron transport chain functioning relies on proteins encoded by both
mitochondrial and nuclear DNA, mitochondrial disorders may be inherited via either maternal or classic
Mendelian patterns.
Mitochondrial disorders, such as the LSDs, may display significant clinico-pathologic overlap. Many of these
disorders, when viewed in isolation, may be caused by more than one mutation, and in turn, any given mutation
may lead to more than one mitochondrial disorder. This biologic complexity makes the diagnosis of mitochondrial
disorders challenging. These disorders are often described as encephalomyopathies, since muscle (cardiac and
skeletal) and brain tissues are usually affected due to their heavy reliance on mitochondrial energy production.
Clinically, the presence of a mitochondrial disorder may be suspected via characteristic lab abnormalities, which
often include an elevation in blood/CSF lactate and the lactate-to-pyruvate ratio. Ragged red fibers (RRF) are a
common manifestation of muscle disease and represent a localized proliferation of abnormal mitochondria. RRFs
are detected histochemically on frozen sections via modified Gomori trichrome (dark red) or succinic
dehydrogenase (dark blue) stains (Figure 10-19A). The cytochrome oxidase C (COX) stain often fails to stain
such affected fibers (i.e., “pale fibers”). By EM, the abnormal mitochondria of RRFs may take several unusual
configurations including concentric spirals and rectangular paracrystalline arrays that resemble “parking lots”
(Figure 10-19B). It is important to note that RRFs are not entirely specific for the mitochondrial disorders, and
that not all mitochondrial disorders bear RRFs. Other common neuropathologic findings seen among the
mitochondrial disorders include hypoxic-ischemic-like and infarct-like changes, intramyelinic edema/spongy
myelinopathy, tract/system
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degenerations, and vascular mineralization (deep gray and adjacent white matter, dentate nucleus and the brain
stem). Only a few of the more common mitochondrial disorders are briefly discussed below.
FIGURE 10-19 ▪ Mitochondrial myopathy. A: RRF. (Modified Gomori trichrome.) B: EM reveals abnormal
mitochondria with paracrystalline (“parking lot”) inclusions.
Mitochondrial encephalopathy with lactic acidosis and strokes (MELAS) is a maternally inherited disorder that
most often results from an adenine to guanine point mutation at nucleotide 3,243 of mtDNA. This mutation is
within the gene that encodes the tRNA for leucine. Those afflicted are usually young, although both the age of
onset and initial presentation may be quite variable. Sudden focal neurologic signs (e.g., hemiplegia,
hemianopsia, seizures, etc.), migraine-like attacks, or more nonspecific symptoms (such as vomiting or a change
in mental status) may be seen. Myopathic features include proximal limb weakness, fatigability, and deficits in
eye movements. Episodes of such neurologic dysfunction tend to be recurrent. Pathologically, foci of necrotic
brain damage resemble true infarcts; however, these lesions do not follow standard vascular distributions. The
occipital lobes, deep gray matter (which also may demonstrate vascular mineralization), and cerebellum are often
affected. RRFs are present.
Myoclonic epilepsy with ragged red fibers (MERRF) is another maternally inherited disorder. MERRF most often
results from an adenine to guanine point mutation at nucleotide 8,344 of mtDNA. This mutation is within the gene
that encodes the tRNA for lysine. Like MELAS, those afflicted are often young. Clinical features include a
proximal myopathy, myoclonic epilepsy, sensorineural hearing loss, cognitive deficits, short stature, and ataxia.
Pathologic changes involve neuronal loss and gliosis among the dentato-rubro-olivary system, substantia nigra,
dorsal column nuclei (gracile and cuneate), and Clarke column. Vascular mineralization may be noted in the deep
gray matter, and muscle pathology includes RRFs.
Leigh disease (subacute necrotizing encephalopathy) is most frequently caused by nuclear DNA mutations, and
hence usually inherited in an autosomal recessive pattern. Genes encoding subunits of the electron transport
chain complexes I, II, IV, and V may be mutated, or alternatively there may be a deficiency of pyruvate
dehydrogenase. Disease onset often manifests prior to 2 years of age and includes weight loss, vomiting,
psychomotor retardation, and weakness. Movement disorders, ataxia, eye abnormalities (optic atrophy,
ophthalmoplegia, nystagmus), respiratory difficulties, hypotonia, and epilepsy are also often characteristic.
Pathologically, the deep gray and brainstem are primarily affected by vasculo-necrotic lesions. The brainstem
tegmentum, inferior colliculi, and substantia nigra are characteristically affected. Grossly, these lesions are
atrophic, soft, and symmetrically distributed. Microscopically, the findings resemble those seen in Wernicke-
Korsakoff syndrome (although hemorrhagic features are absent and the mamillary bodies are normal). Typical
lesions bear rarefaction of the neuropil with spongiosis and relative neuron preservation, foamy macrophages,
and gliosis and an increased density of capillaries that is thought to result from vascular proliferation and/or
neuropil collapse.
Kearns-Sayre syndrome (KSS) is a sporadic disorder that is most frequently due to a deletion in the mtDNA
genome (˜5 kb). KSS is usually of pediatric onset and is neurologically characterized by eye findings
(ophthalmoplegia, ptosis, retinitis pigmentosa, and vision loss), hearing deficits, weakness, ataxia, proximal
myopathy, cognitive impairment, and seizures. Extra-CNS abnormalities include short stature, often fatal cardiac
pathology (cardiomyopathy and
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conduction problems), plus additional GI, renal and endocrine perturbations. Chronic progressive external
ophthalmoplegia (CPEO) may be seen as a component of KSS, or alternatively can be the sole manifestation of
a mitochondrial disorder. Neuropathologic abnormalities classically include RRFs on muscle biopsy and a diffuse
spongy myelinopathy. This white matter pathology is not accompanied by prominent gliosis or macrophagic
infiltrates. As can be seen in many of the disorders characterized by spongy myelinopathy, splitting of myelin
lamellae at the intraperiod line results in vacuole formation. Like many mitochondrial diseases, deep gray matter
may bear vascular mineralization. Correlating with clinical ataxia, cerebellar pathology includes Purkinje neuron
dendritic deformities plus eventual neuronal dropout.
Amino Acid Disorders

Amino acid disorders are inherited (mostly autosomal recessive) deficits in the enzymatic degradation of amino
acids. This group includes the urea cycle disorders, phenylketonuria (PKU), nonketotic hyperglycinemia,
homocystinuria, maple syrup urine disease (MSUD), and some of the organic acidemias (e.g., proprionic and
methylmalonic acidemia). Although some of these diseases have an insidious onset and pursue a chronic
course, most follow a severe and fatal clinical picture in early childhood. Encephalopathy/psychomotor
retardation, seizures and motor findings (spasticity, tetraplegia) are seen. Neurologic dysfunction is thought to be
related to a combination of toxic biochemical accumulations (e.g., amino acid intermediaries, hyperammonemia),
deficits in the biosynthesis of key metabolic compounds, and energy dysfunction. Neuropathologic alterations
commonly include a spongy myelinopathy (like that seen in KSS) that tends to affect infratentorial structures,
Alzheimer type II astrocytes (see below), and neocortical/deep gray hypoxic-ischemic lesions. Vascular pathology
(i.e., infarction) is characteristic of homocystinuria.
Congenital Disorders of Glycosylation

The congenital disorders of glycosylation (CDGs) are an uncommon but evolving group of relatively recently
described inborn errors of metabolism. N and O-linked glycan synthesis/processing are affected and ultimately
result in hypoglycosylated glycoproteins; hence, these disorders are often multisystemic in nature. These
disorders are inherited in an autosomal recessive manner, and the genetic basis underlying many of the CDGs is
a missense mutation. Isoelectric focusing of transferritin is the common test used in the diagnosis of CDGs. CDG
type la is the most common and best described entity. The R141H missense mutation in the PMM2 gene on
16p13.3-13.2 is the most frequent mutation in CDG la. Infants are affected, in the early stages, by prominent
psychomotor retardation, ataxia, and alternating strabismus. This early phase of disease is often fatal. If the
patient survives, later neurologic manifestations include retinitis pigmentosa, seizures, and stroke-like episodes.
Other clinical aspects of CDG la may include hypotonia, feeding problems, liver disease (fatty infiltration and
cirrhosis, leading to coagulopathies), pericardial effusions, dysmorphic features (inverted nipples, subcutaneous
buttock fat pads, and contractures), and musculoskeletal abnormalities. Neuropathologic features are dominated
by olivopontocerebellar atrophy (OPCA); EM may reveal myelin-like lysosomal inclusions.
Acquired Metabolic Disorders and Vitamin Deficiencies
Acquired metabolic diseases affecting the nervous system are numerous. They are encountered more frequently
in adults, although pediatric examples are clinically and pathologically similar. Some of these include
hypoglycemia, electrolyte disorders [e.g., central pontine myelinolysis (CPM), disorders of calcium hemostasis],
hepatic encephalopathy, porphyria, uremic, and dialysis-related encephalopathy. Several vitamin deficiencies
may also characteristically lead to neurologic disease. These include thiamine (Bl) and Wernicke-Korsakoff
syndrome, pyridoxine (B6), B12 (subacute combined degeneration), nicotinic acid (pellagra), folic acid, vitamin A
(including intoxication), and vitamin E.
Neurodegenerative and Miscellaneous Disorders

There are a number of neurodegenerative and more nondescript neurologic disorders that characteristically
affect the pediatric population. A subset of these entities are discussed below.
Alpers-Huttenlocher syndrome [progressive neurodegeneration of childhood (PNDC)] is now considered by
many to reside among the mitochondrial disorders (40). This disease manifests in infancy and is characterized by
the acute onset of intractable seizures, developmental delay, hypotonia, ataxia, cortical blindness, failure to
thrive, and liver disease (which pathologically reveals bile duct proliferation and cirrhosis). Death frequently
occurs by 3 years of age. Molecular genetic studies have revealed mtDNA depletion and mutations in the
polymerase gamma gene (POLG). Genetic transmission has been suggested to be autosomal recessive. Gross
pathologic findings reveal a patchy neocortical atrophy that has a predilection for the visual cortex. Along with
thalamic and focal hippocampal atrophy, there may be concomitant HCP ex vacuo. Microscopically, the
neocortex is especially affected by a spongy rarefaction, with superimposed neuronal loss and gliosis; severe
cases are transcortical, while less involved cortex shows these abnormalities more superficially. These spongy
changes are reminiscent of those seen in CreutzfeldJacob disease (CJD). Neuronal loss is often prominent in the
inferior olives. Neutral fat deposition in diseased parenchyma is highlighted with oil red O staining.
Axonal spheroids are a microscopic accompaniment of many diseases that affect the nervous system. When
numerous, these axonal spheroids may signify the presence of a neuroaxonal dystrophy (NAD). Axonal
spheroids are
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rounded eosinophilic structures ranging in size from 20 to 120 jim. They are highlighted with silver stains or by
various IHC stains (e.g., NFP and ubiquitin). EM reveals mitochondria, membrane-bound electron-dense
granules, tubulomembranous structures all among an amorphous matrix; NFPs are surprising sparse but if seen
are often displaced toward the periphery. Two NADs are discussed further: infantile neuroaxonal dystrophy
(INAD, or Seitelberger disease) and neurodegeneration with brain iron accumulation type 1 [NBIA; pantothenate
kinase-associated neurodegeneration (PANK), or Hallervorden-Spatz disease (HSD)]. Both are rare progressive
neurologic diseases.
INAD typically has an onset just after 1 year of age. Sporadic and familial forms (with an autosomal recessive
inheritance pattern) may be seen. A definitive pathogenesis has not been defined. Normal development may be
seen early on, but psychomotor retardation eventually develops. Weakness, hypotonia, depressed deep tendon
reflexes, visual difficulties, and cerebellar deficits (ataxia, pendular nystagmus) may also be seen clinically.
Terminal stages of disease (between 6 and 15 years of age) include tetraplegia/spasticity with decerebrate
posturing, bulbar palsies, and bowel/bladder incontinence. Gross pathologic changes reveal cerebral and
cerebellar atrophy with HCP ex vacuo. Optic nerves may be atrophic as well. The globus pallidus is large and
pale early on but later takes on a rusty discoloration. Microscopically, the deep gray, brainstem (including the
substantia nigra), cerebellar cortex, and spinal cord are especially affected; abnormalities include axonal
spheroids (central, peripheral, and in the autonomic nervous systems), spongy degeneration, gliosis, and
lipid/iron pigment accumulation (especially within the globus pallidus and the substantia nigra reticulata). These
regions as well as a few white matter tracts (e.g., corticospinal, spinobulbar, optic, olfactory) may display
demyelinating-like pathology.
NBIA occurs in both sporadic and familial (autosomal recessive) forms and may have an onset at any age.
Infantile (<1 years old), late infantile (2 to 5 years old), juvenile (“classic;” 7 to 15 years old), and rarely adult
onset cases have been described. Infantile/late infantile cases are often fatal before 10 years of age and are
more often associated with mutations in the PANK2 gene. An insidious gait disorder with hypotonia often heralds
the onset of disease, although psychomotor deficits may predate such manifestations. Hyperkinetic movement
disorders are a key clinical feature and are seen in approximately 50%; these include choreoathetosis, dystonia,
and tremors, which may result in dysarthria, dysphagia, and abnormal extraocular movements. Additional
neurologic deficits include ataxia and nystagmus, visual abnormalities, hyper-reflexia with a Babinski response,
and leg amyotrophy. Cognitive impairment leads to dementia. HARP syndrome (hypo-P-lipoproteinemia,
acanthocytosis, retinitis pigmentosa, and pallidal degeneration) is considered to reside within the NBIA clinico-
pathologic spectrum (15). MPJ findings characteristically reveal the “eye of the tiger” sign where a ring-like
region of T2 hypointensity is seen in the globus pallidus externa and the substantia nigra, while T2
hyperintensity is seen in the globus pallidus interna. Gross pathology reveals atrophy within the cerebrum and
cerebellum; this atrophy has a rusty hue in the globus pallidus interna and substantia nigra (Figure 10-20).
Microscopically, the pallidonigral system is characteristically affected by iron pigment deposition, neuronal loss,
gliosis, and spheroid formation. Granular pigment containing iron, lipofuscin, and neuromelanin may be
intracellular (neuronal somata and axons, astrocytes, and microglia) or extracellular. Spheroids may also be seen
in other deep gray nuclei, as well as the neocortex, brainstem tegmentum, and the spinal cord. Notably, the
peripheral nervous system is not affected.
FIGURE 10-20 ▪ NIBA (PANK or HSD). This coronal brain section reveals a rusty discoloration of the substantia
nigra.
Epilepsy may be a manifestation of a number of different disorders that cause dysfunctioning of the neocortex.
Vascular, infectious, traumatic, autoimmune, neoplastic, metabolic, malformative, and neurodegenerative are
some of the etiologic classes that may be involved in the pathogenesis of epilepsy. Pasquier et al. (73)
discussed their surgical pathology experience with 327 cases of drug-resistant epilepsy and highlighted the
spectrum of disease that may be seen in this context. Included within these specimens was a large number
displaying a common form of idiopathic pathology to which we will limit our discussion: mesial temporal sclerosis
(MTS). MTS (also known as Ammon horn sclerosis or hippocampal sclerosis) is not usually associated with a
clear genetic abnormality and does not have a clear pathogenesis. However, some cases are associated with a
history of prolonged febrile seizures during infancy, and moreover, the pathology is similar if not identical to that
seen within the context of hypoxic-ischemic injury. MTS may be seen in isolation or in conjunction with a second
form of temporal lobe pathology (e.g., neoplasm, vascular formation, cortical dysplasia, etc.). Gross pathology
reveals atrophy of the hippocampal formation with concomitant dilatation of the adjacent inferior temporal horn of
the lateral ventricle (Figure 10-21A). Microscopically, neuronal loss and gliosis are most striking in
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the CA1 and CA4 (i.e., endfolium) hippocampal subregions (Figure 10-21B). Neuronal loss, dispersion, and/or
duplication may also be seen within the dentate granular layer. Dysmorphic neurons may occasionally be seen in
the endfolium and, like some of the dentate granule neuron alterations, may be a reactive, rather than primary
component of MTS.
FIGURE 10-21 ▪ Mesial temporal sclerosis (MTS). A: Using NeuN immunohistochemistry, this low-power
magnification image reveals a dropout of neurons in the hippocampal CA1 subregion (black arrow), as well as
neuronal dispersion within the dentate granule layer of the hippocampal formation (white arrow). B: GFAP-
stained section reveals marked gliosis of CA1. Note: unstained neurons (left) taper off into a region of more
intense gliosis (arrow).
Neurodegenerative disorders that prominently affect the cerebellum are numerous but uncommon. Secondary
forms of cerebellar disease (i.e., paraneoplastic, toxic/nutritional, vascular, infectious/inflammatory, prion related,
and metabolic) will not be discussed here. Primary forms of cerebellar disease may be inherited or sporadic (e.g.,
multiple system atrophy, idiopathic degeneration) in nature. Familial cerebellar ataxia may follow an autosomal
recessive or dominant pattern of inheritance. Some of the more common forms of autosomal recessive
[Friedreich ataxia (FA); ataxia telangectasia (AT)] and autosomal dominant diseases [the “spinocerebellar
ataxias”; dentatorubropallidoluysial atrophy (DRPLA); and the episodic ataxias (EA1 and EA2)] are discussed
below.
FA is a progressive multisystem disorder that typically has an onset prior to 15 years of age and results in death
by the end of the fourth decade. This disorder involves an abnormally expanded intronic GAA trinucleotide repeat
within the frataxin gene located on 9q 13-21.1. The gene product frataxin encodes for a mitochondrial protein
involved in iron transport; dysfunction of this protein with disease is thought to lead to iron accumulation and
oxidative neuronal damage. Ataxia (of gait, limb and voice, or dysarthria) is the result of cerebellar and sensory
degeneration. There is a loss of position and vibratory sense, along with areflexia. A pyramidal pattern of leg
weakness is accompanied by a Babinski response. Extra-CNS manifestations include pes cavus, scoliosis,
cardiomyopathy, and diabetes mellitus. Gross pathologic CNS findings are generally limited to atrophic dorsal
roots of the spinal cord; ischemic CNS disease may be seen and may be attributed to cardiac disease.
Microscopic changes are prominent within the spinal cord and include tract degenerations (spinocerebellar,
corticospinal, and dorsal columns) plus degeneration of Clarke columns (Figure 10-22). The dorsal root ganglion
shows neuronal depletion and concomitant nodule of Nageotte formation (a proliferation of satellite cells that
normally rim sensory neurons of the dorsal root ganglia). Large myelinated sensory fibers are lost in peripheral
nerves. Neuronal loss in the accessory cuneate and gracile nuclei reflects transsynaptic degeneration. Neuronal
loss and gliosis are seen in the vestibular, cochlear, and superior olivary nuclei. Cerebellar abnormalities include
white matter gliosis plus neuronal loss in the dentate nuclei with concomitant superior cerebellar peduncle
atrophy. Hypoxic-ischemic changes may be seen in the cerebellum and neocortex.
FIGURE 10-22 ▪ Friedreich ataxia (FA). In this myelin-stained histologic preparation of the spinal cord in
transverse section, there is a symmetric lack of staining in the dorsal columns, corticospinal tracts, and less so in
the spinocerebellar tracts.
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Strictly speaking, spinocerebellar atrophy (SCA) is a heterogeneous group of autosomal dominant
neurodegenerative disorders affecting the cerebellum and additional CNS structures. The reciprocal circuitry
between the cerebellar cortex, dentate nucleus, and the inferior olive (the “cerebellar module”) is thought to be
particularly important to the pathogenesis of ataxia (57). The number of disorders included under the rubric of
SCA continues to grow at a rapid pace, and types 1 to 25 have recently been described (note: there is no SCA 9)
(114). Many of the SCAs are trinucleotide repeat disorders, of which six forms (SCA 1 to 3, 6, 7, and 17) bear
expanded CAG coding repeats along with clinical evidence of “anticipation,” wherein the repeat becomes
progressively longer, and disease onset is progressively earlier with increasing disease severity for each
subsequent generation of patients. SCA 10 is exceptional in that it exhibits an ATTCT pentanucleotide
noncoding repeat. Despite their recognition, the mechanism by which these repeats cause disease is unclear.
Although the atypical age of onset is usually after the fifth decade, pediatric forms of SCA may be seen. The
spectrum of neurologic deficits includes cerebellar dysfunction (truncal and limb ataxia, dysarthria, nystagmus),
abnormal gait, spasticity, weakness, parkinsonism and other extrapyramidal movement disorders, pyramidal
signs, autonomic dysfunction, sensory abnormalities (including visual difficulties), and cognitive impairment.
Some SCAs cause a multitude of neurologic deficits, while others are considered “purely” cerebellar. SCA 3 (or
Machado Joseph disease) is the most common form among this group of diseases. Pathologic descriptions are
available for only a subset of these disorders. SCA 2 affects many neurologic systems. The gross brain weight is
reduced, and there is OPCA. Although there may be gross cerebellar atrophy in SCA 6, considered one of the
“purely” cerebellar forms, such atrophy is not conspicuous in SCA 3. Microscopically, both SCA 2 and SCA 6
demonstrate cerebellar cortical atrophy with Purkinje cell dropout, while SCA 3 cerebellar disease is centered
upon the dentate nucleus (with neuron loss and “grumose degeneration”). SCA 2 demonstrates dropout of
neurons from the basis pontis and inferior olive. The spinal cord is abnormal in both SCA 2 and 3; while both
demonstrate fiber loss in the posterior columns and neuronal loss in Clarkes nucleus, SCA 3 exhibits additional
lateral column degeneration reminiscent of FA (but without dorsal spinal root involvement),
rmmunohistochemistry may reveal diagnostically useful intranuclear inclusions or more diffuse staining in the
SCAs with expanded CAG repeats (SCA 6 bears abnormal cytoplasmic staining only). These inclusions may
stain with antibodies targeted against the abnormal gene product involved, ubiquitin, expanded polyglutamine
residues (e.g., IC2), or against other “chaperone” proteins.
Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder resulting from the
homozygous mutation or deletion of the SMN1 gene on 5q13. Three forms are generally recognized. SMA 1 (or
Werdnig-Hoffmann disease) has an onset early in infancy. Proximal muscle weakness in the limbs progresses to
involve the axial and diaphragmatic muscles; there may be bulbar involvement with respiratory insufficiency
related to intercurrent infection and aspiration. Death is often seen prior to 1 year of age. In SMA 2, early motor
development may be normal, but weakness prevails by 3 months of age. There may be tongue atrophy and hand
tremor. Fasciculations and depressed deep tendon reflexes are also seen. Eventually, contractures and
kyphoscoliosis develop. Most die by 25 years of age. SMA 3 is a more chronic form of disease but still may have
a young onset. A functional motor deficit is appreciated and includes clinical weakness. Knee jerk reflexes are
depressed and there may also be hand tremor. This gradual form of disease does not affect respiratory
musculature or lead to a shortened life span. Pathologically, SMA 3 exhibits an adult pattern of denervation
within muscles. In contrast, SMA 1 and 2 show large groups/fascicles of small rounded (not angular like that
seen in adults) type I and II fibers intermixed with hypertrophic type I fibers, the latter possibly reflecting a
compensatory response (Figure 10-23). In the end stage, endomysial
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connective tissue and fat replacement may mimic a muscular dystrophy. Within the CNS, all forms of disease
demonstrate anterior spinal root atrophy grossly, with anterior horn cell loss and concomitant gliosis
microscopically. Earlier stages may feature neuronophagia and ballooned (NFP) neurons. A useful diagnostic
feature in the thalamus is the presence of chromatolytic neurons. Bulbar motor neurons may also be affected.
FIGURE 10-23 ▪ Spinal muscular atrophy (SMA). A: H&E-stained frozen section of skeletal muscle reveals the
typical distribution of rounded atrophic and enlarged fibers. B: Many of the latter prove to be type I (ATPase pH
10.4).
Autism is an enigmatic neurologic disorder characterized by three key features: impaired social interaction,
communication deficits (both verbal and nonverbal), and restricted/stereotyped behavior. Onset is before 3 years
of age, and there is a 4:1 male-to-female sex distribution. Prevalence has most recently been estimated at 1/150
live births, making autism a rather common disorder. Genetic factors are clearly critical, but rather than a simple
Mendelian pattern of inheritance, multiple genes are likely to be involved in the predisposition to this disease. In
particular, duplication of chromosome 15q1 1-q13 is observed in a subset, and there appears to be a strong
linkage between this GABA (53 subunit-encoding locus and the clinical feature of “insistence on sameness” (96).
Neurotransmitter studies have suggested deficits in GABA-A receptors (hippocampal formation), ACh receptors
(frontal and parietal lobes plus the cerebellar cortex), and decreased 5-HT synthesis (dentothalamocortical
pathway) (7, 16, 78). Although the prevalence of autism is clearly higher in monozygotic (60% to 90%
concordance) versus dizygotic (5% to 10% concordance) twins, environmental factors likely play a significant
etiologic role as well. Neuropathologic descriptions remain limited, although the most common gross finding,
especially in young patients, is a nonspecific megalencephaly. Purkinje cell dropout is the most common
histologic finding. Limbic structures (including the amygdala, hippocampus, and entorrhinal cortex) exhibit small
and closely packed neurons. Neocortical malformations may be seen and include a thickened neocortex, focal
increased neuronal density, dyslamination, pyramidal neuronal malorientation, an increase in white matter, and
molecular layer neurons (4). Cortical microcolumns, thought to be the smallest functional unit of the neocortex,
have also been studied and found to be abnormally developed (13). Several brain regions, including the vertical
limb of the nucleus of the diagonal band of Broca, the dentate nucleus, and the inferior olive, demonstrate
neuronomegaly in younger patients with autism, followed by atrophy in older patients; there may be
superimposed neuronal loss in some of these regions.
Neoplasia
Primary CNS tumors are common in pediatrics and only superseded by lymphoid-hematopoietic disorders in
terms of frequency (86). Although adults and children may incur similar tumors, their individual incidence varies
significantly with age. Prominent in adults are the following: diffusely infiltrating astrocytomas (DAs), metastases
(primarily carcinoma), meningioma, and nerve sheath tumors (especially schwannoma). In pediatrics, PAs are the
most frequent. Other common pediatric CNS tumors include DA, medulloblastoma, ependymoma, and
craniopharyngioma (CPG) (Table 10-5). The current epidemiologic data are due in part to the ongoing
refinements of our classification schemes, of which the WHO Classification of Tumors of the Nervous System
(2007) is considered the standard (Table 10-6) (63). Despite these advances, several pediatric neoplasms
remain difficult to classify.
Clinical Considerations
The presenting signs and symptoms of pediatric brain tumors are largely similar to those described in adults
(Table 10-7). However, tumors occurring in infancy often display more insidious features. The myriad of focal
neurologic abnormalities are more easily appreciated in older children who are better able to articulate their
deficits. For example, tumors of the pineal gland characteristically result in Parinaud syndrome, typified by
upgaze paralysis and convergence
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nystagmus due to compression of dorsal midbrain visual centers. Seizure activity is an especially frequent
presenting feature; not only do they suggest cortical involvement, but they also commonly accompany temporal
lobe tumors. Posterior fossa tumors are anatomically speaking in a “hightraffic area” and result in a number of
cerebellar, brainstem, and “long tract” abnormalities.
Table 10-5 ▪ THE MAIN HISTOLOGIC TYPES OF PRIMARY CNS TUMORS IN CHILDREN AND
THEIR RELATIVE FREQUENCIES
Tumor Type, WHO Grade Percentage
Pilocytic astrocytoma (PA), I 23.5
Diffuse astrocytoma, II 5
Anaplastic astrocytoma, III 7.2
Glioblastoma (GBM), IV 7.2
Pleomorphic xanthoastrocytoma (PXA), II-IIIa 1.9
Subependymal giant cell astrocytoma (SEGA), I 2.5
Medulloblastoma, IV 16.3
Ependymoma, II-IIIa 10.1
Craniopharyngioma (CPG), I 5.6
Germ cell tumors 2.5
Ganglioglioma (GG), I-IIIa 2.5
Meningioma, I-IIIa 2.5

Supratentorial primitive neuroectodermal tumor (sPNET), IV 1.9
Pineal parenchymal tumors (PPTs) (pineocytoma; pineoblastoma), II-IV 1.9
Atypical teratoid rhabdoid tumor (ATRT), IV 1.3
Choroid plexus tumors (CPTs) (papilloma; carcinoma), I and III 0.9
Desmoplastic infantile ganglioglioma (DIG)/astrocytoma (DIA), I 0.6
Dysembryoplastic neuroepithelial tumor (DNT), I 0.6
Pituitary adenoma 0.9
Schwannoma 1.3
Neurofibroma 0.3
Langerhans' cell histiocytosis 0.6
aTumors where a range of grades are listed, the highest grade is generally called “anaplastic.” Data
from Rickert CH, PaulusW. Epidemiology of central nervous system tumors in childhood and
adolescence based on the new WHO classification. ChildsNervSystem 2001;17:503-511.
Table 10-6 ▪ WHO CLASSIFICATION OFTUMORS OFTHE NERVOUS SYSTEM
Tumors of Neuroepithelial Tissue
Astrocytic tumors Large cell medulloblastoma
Pilocytic astrocytoma (PA)

Pilomyxoid astrocytoma
Subependymal giant cell astrocytoma (SEGA)
Pleomorphic xanthoastrocytoma (PXA)
Diffuse astrocytoma
Fibrillary astrocytoma
Protoplasmic astrocytoma
Gemistocytic astrocytoma
Anaplastic astrocytoma
Glioblastoma (GBM)
Giant cell GBM
Gliosarcoma
Gliomatosis cerebri
Oligodendroglial tumors
Oligodendroglioma
Anaplastic oligodendroglioma
Oligoastrocytic tumors
Oligoastrocytoma
Anaplastic oligoastrocytoma
Ependymal tumors
Subependymoma
Myxopapillary ependymoma
Ependymoma
Cellular
Papillary
Clear cell
Tanycytic
Anaplastic ependymoma
Choroid plexus tumors (CPTs)
Choroid plexus papilloma (CPP)

Atypical CPP
Choroid plexus carcinoma (CPC)
Other neuroepithelial tumors
Astroblastoma
Chordoid glioma of the third ventricle
Angiocentric glioma
Neuronal and mixed glioneuronal tumors
Dysplastic gangliocytoma of cerebellum (Lhermitte-Duclos)

Desmoplastic infantile astrocytoma/ganglioglioma
Dysembryoplastic neuroepithelial tumor (DNT)
Gangliocytoma
Ganglioglioma (GG)
Anaplastic ganglioglioma
Papillary glioneuronal tumor
Rosette-forming glioneuronal tumor of the fourth ventricle
Central neurocytoma
Extraventricular neurocytoma
Cerebellar liponeurocytoma
Paraganglioma of the filum terminale
Tumors of the pineal region
Pineal parenchymal tumors (PPTs)

Pineocytoma
PPT of intermediate differentiation
Pineoblastoma
Papillary tumor of the pineal region
Embryonal tumors
Medulloblastoma
Desmoplastic/nodular medulloblastoma
Medulloblastoma with extensive nodularity
Anaplastic medulloblastoma
Large cell medulloblastoma
CNS primitive neuroectodermal tumors (PNETs)
CNS PNET NOS
CNS ganglioneuroblastoma
CNS neuroblastoma
Medulloepithelioma
Ependymoblastoma
Atypical teratoid/rhabdoid tumor
Tumors of Cranial and Paraspinal Nerves

Schwannoma (Neurilemmoma, Neurinoma)
Cellular
Plexiform
Melanotic
Neurofibroma
Plexiform
Perineurioma
Intraneural perineurioma
Soft-tissue perineurioma
Malignant peripheral nerve sheath tumor (MPNST)
Epitheliodi
MPNST with divergent mesenchymal and/or epithelial differentation
Melanotic
Tumors of the Meninges

Tumors of meningothelial cells
Meningioma
Meningothelial
Fibrous (fibroblastic)
Transitional (mixed)
Psammomatous
Angiomatous
Microcystic
Secretory
Lymphoplasmacyte-rich
Metaplastic
Chordoid
Clear cell
Atypical
Papillary
Rhabdoid
Anaplastic (malignant)
Mesenchymal tumors
Lipoma
Angiolipoma
Hibernoma
Liposarcoma (intracranial)
Solitary fibrous tumor
Fibrosarcoma
Malignant fibrous histiocytoma
Leiomyoma
Leiomyosarcoma
Rhabdomyoma
Rhabdomyosarcoma
Chondroma
Chondrosarcoma
Osteoma
Osteosarcoma
Osteochondroma
Hemangioma
Epithelioid hemangioendothelioma
Hemangiopericytoma
Angiosarcoma
Kaposi sarcoma
Primary melanocytic lesions
Diffuse melanocytosis
Melanocytoma
Malignant melanoma
Meningeal melanomatosis
Other neoplasms related to the meninges
Hemangioblastoma
Lymphomas and Hematopoietic Neoplasms
Malignant lymphomas
Plasmacytoma
Granulocytic sarcoma
Germ Cell Tumors
Germinoma
Embryonal carcinoma
Yolk sac tumor
Choriocarcinoma
Teratoma
Mature
Immature
Teratoma with malignant transformation
Mixed germ cell tumors
Tumors of the Sellar Region
Craniopharyngioma (CPG)
Adamantinomatous
Papillary
Granular cell tumor
Pituicytoma
Spindle cell oncocytoma of the adenohypophysis
MetastaticTumors
Modified from theWHO 2007 classification scheme (63).
Neuroradiologic studies are an important tool of the neuropathologist. In particular, T1-weighted MR images with
gadolinium enhancement and T2-weighted/FLAIR (fluidattenuated inversion recovery) studies allow (somewhat
simplistically) assessment of the vascularity and edema associated with a tumor, respectively. These radiologic
studies help tremendously in the formation of differential diagnoses. Cystic lesions bearing a mural nodule [e.g.,
PA, pleomorphic xanthoastrocytoma (PXA) and GG], intracortical lesions [e.g., dysembryoplastic neuroepithelial
tumor (DNT)], and other tumors that remodel the inner table of the skull (presumably through mechanical
compression) all suggest a slowly growing low-grade lesion. An exophytic lesion of the dorsal brainstem is
usually PA, while an intrinsic pontine or white matter-based cerebral lesion (especially enhancing cases) points
to a more ominous tumor, such as a DA (28). Newer imaging modalities may also prove useful in the future. For
example, using magnetic resonance spectroscopy (MRS), Tamiya et al. (106) found positive correlations
between choline to creatinine ratios and the proliferative IHC marker Ki-67, suggesting that this modality may
assist in tumor grading.
Table 10-7 ▪ PRESENTING SIGNS AND SYMPTOMS OF PEDIATRIC BRAIN TUMORS
Specific Feature Symptom/Sign

Category
General illness Irritability, listlessness, failure to thrive, loss of developmental

milestones, behavioral disturbance, poor feeding
Increased ICP or HCP Headache, nausea, vomiting, macrocephaly, “sun-setting eyes,”

papilledema
Focal neurological Seizures

disturbances (“focality”) Motor deficits (e.g., weakness)
Visual field loss/deficit (e.g., Parinauds')
Neuroendocrine dysfunction
Cranial neuropathies
Cerebellar dysfunction (nystagmus, ataxia, Romberg sign, abnormal
tone, tremor, vertigo, etc.)
Long tract signs (paraparesis, hyperreflexia, Babinski sign)
Treatment strategies generally involve three modalities: surgery, radiotherapy, and chemotherapy. Low-grade
tumors are generally treated with surgery alone since this circumvents potential negative sequelae of
chemotherapy and radiotherapy (113). High-grade neoplasms (i.e., WHO grade III-IV) usually receive adjuvant
radiation and chemotherapy. Stereotactic biopsy may be performed in cases where the tumor lies within delicate
anatomy (e.g., brainstem, pineal, and spinal cord); however, this yields very small biopsies, and therefore
sampling adequacy is often a concern. A more thorough assessment of the current state of therapeutic neuro-
oncology is available elsewhere (113).
General Pathologic Considerations

The workup of pediatric CNS tumors often employs both routine and adjuvant pathologic studies. As
classification schemes are regularly updated, it is important to review prior surgical specimens at the time of
recurrence or progression, as previous diagnoses occasionally need revision.
The frozen section provides a preliminary diagnosis and enables tissue allocation algorithms, the latter of which
are increasingly being utilized for tumor banking, local research, ancillary molecular testing, and participation in
clinical protocols. However, diagnostic accuracy ultimately remains
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most critical, and ensuring histologically superior permanent sections [i.e., formalin-fixed and paraffin-embedded
(FFPE) sections] should always be the first priority. Retention and processing of the cavitronic ultrasound
aspirator (CUSA) material may be somewhat less preserved than resected tissue due to partial autolysis and
other artifacts but can nonetheless be essential to the final diagnosis, especially in the context of small tumors.
Fixing a small portion of the tumor (i.e., 1 mm3) in glutaraldehyde for ultrastructural analysis is recommended
when the initial diagnosis is in question. If sufficient tissue is provided, a fragment should be snap frozen and
stored for future studies. If only a small portion of tumor remains for FFPE, requesting a number of unstained
sections upfront will avoid wasting any tissue at the time of slide preparation.
Evaluation of the FFPE material allows the first precise characterization of tumor. Primary CNS tumors may
simplistically be considered as either “well circumscribed” or “diffusely infiltrating,” and this dichotomization often
assists in narrowing the differential diagnosis. Glial, neuronal, embryonal, and a number of other cytologic
features can usually be appreciated on routine stains. Detail should be directed at the nuclear features since
these are often key to many diagnoses (especially gliomas). Degenerative-type atypia (large hyperchromatic
nuclei, often bearing pseudoinclusions of cytoplasm) is a common feature to many low-grade primary brain
tumors and should not be over-interpreted as a concerning finding in the absence of other malignant features.
Mitotic activity is critical to the grading of many CNS primary tumors, and specific cutoff numbers [generally
expressed as #/10 high-powered fields (HPF)] exist for some tumor types. Grading is generally based not on the
entirety of the specimen but on the most malignant portion identified (i.e., one rotten apple spoils the bunch).
Microvascular proliferation (MVP), also referred to as endothelial proliferation or endothelial hyperplasia,
represents foci of multilayering in blood vessel; several cellular elements (including smooth muscle cells,
pericytes, and endothelial cells) are identified in these hyperplastic walls despite the focus on endothelia in the
name. It is an important finding in the diffuse gliomas, as is necrosis (which may be pseudopalisading), where
both of these features raise the WHO grade; notably both of these features may be seen in PA but do not impact
prognosis and, thus, do not elevate the tumor grade. EGBs and RFs, while nonspecific, usually imply the
presence of a slow-growing neoplasm, as does calcification to a lesser extent.
IHC analysis plays an important ancillary role in the diagnosis of brain tumors. The more commonly utilized stains
are listed in Table 10-8. Special histochemical stains and electron microscopy (EM) modalities have been
supplanted by IHC; but the former still have great utility in some scenarios. Reticulin is frequently used to identify
extracellular matrix deposition in several tumors (e.g., PXA, gliosarcoma, GG, desmoplastic medulloblastoma,
etc.) and in some tumors may stain in a pericellular pattern (e.g., schwannoma), indicative of basal lamina (The
immunostain collagen IV is a more specific marker of such.). Periodic acid Schiff (PAS) and PASwith-diastase
confirm the presence of glycogen within the
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cytoplasm of tumor cells (diastase sensitive), while the latter also highlights EGBs. Trichrome stains highlight
RFs in addition to collagen. Bielschowsky silver stain labels axons (like NFP by IHC) and often the ganglion cell
component of glio-neuronal tumors. LFB, counterstained with H&E or PAS, can be used to stain myelin and,
hence, highlight tumor infiltration of white matter, or active demyelination (note: myelin breakdown products
within macrophages is first blue, then changes to magenta or PAS-positive with time and concomitant
degradation.), as seen in tumefactive (or tumor-like) multiple sclerosis. Ultrastructural analysis remains the gold
standard for a few tumor types, such as ependymoma. The cilia, basal bodies, and intercellular “zipper-like”
junctions of ependymoma are often well preserved and may even be identified in tissue that was previously
subject to FFPE. EM can also help support the presence of a neuronal differentiation when dense-core granules,
clear vesicles, microtubulefilled processes, and synapse formation are seen.
Table 10-8 ▪ IHC STAINS COMMONLY USED IN THE INVESTIGATION OF PEDIATRIC CNS
TUMORS
Stain Utility
GFAP Glial differentiation, primarily in gliomas, reactive gliosis
S-100 protein Nonspecific neuroectodermal marker, gliomas, DNT (OLCs), CPT, nerve sheath
tumors, melanocytic
Neuronal Facilitate identification of a “neuronal component” in a tumor. Synaptophysin and

markersa chromogranin also for neuroendocrine differentiation; NFP labels normal axons and
hence infiltration
Cytokeratinb Epithelial differentiation in CPTs, ATRT metastatic carcinoma
EMA Ependymomac, meningioma, ATRT, metastatic carcinoma
CD34 Epileptogenic tumors: GG and PXA
INI1/BAF47 ATRT” d
c-kit Germinomatous differentiation in germ cell tumors
Ki-67 Proliferative marker
CD68 Lysosomal marker; used to identify reactive elements, including macrophages and
microglia; histiocytic tumors
p53 Labels many tumors including astrocytic tumors, high-grade CPTs and MPNSTs.
Particularly useful to identify “naked nuclei” of an infiltrating astrocytoma when
strongly positive
HMB-45and Melanocytic markers

Melan-A
CD45, Markers of white blood cells (CD45 is general, CD20 and 79a for B-cells, and CD3
CD20and79a, forT-cells) in reactive conditions and lymphoma
CD3
Muscle Muscle type differentiation in rhabdomyosarcoma, ATRT, medullomyoblastoma, etc.

markerse
aNeuronal markers: synaptophysin, Neu-N, NFP, MAP-2, chromogranin
bCAM 5.2 (low molecular weight cytokeratin most commonly used)
cEpendymomas also stain with CD99 in a membranous and dot-like pattern

dFor ATRT, a triad of vimentin, EMA and SMA positivity also useful.
eIncludes SMA, MSA, desmin, myogenin, myoglobin, and caldesmon

DNT, dysembryoplastic neuroepithelial tumor; OLC, oligodendroglial-like cells; CPT, choroid plexus
tumors; NFP, neurofilament; ATRT, atypical teratoid rhabdoid tumor; GG, ganglioglioma; PXA,
pleomorphic xanthoastrocytoma; MPNST, malignant peripheral nerve sheath tumor.
Genetic studies are becoming more frequent in the daily practice of neuropathology. Karyotyping remains an
excellent method of globally screening rare pediatric brain tumors for cytogenetic abnormalities. Several pediatric
brain tumors are amenable to testing, and some may exhibit signature molecular alterations. Fluorescence in situ
hybridization (FISH) is occasionally used in the workup of several tumor types, including astrocytomas,
oligodendrogliomas, medulloblastomas, atypical teratoid rhabdoid tumors (ATRTs), and meningiomas.
Polymerase chain reaction (PCR)-based loss of heterozygosity (LOH) analysis is also used by some, as is
chromogenic in situ hybridization (CISH). As newer genetic techniques continue to elucidate the molecular
underpinnings of these tumors [e.g., gene expression profiling, array comparative genomic hybridization (aCGH),
and single nucleotide polymorphism (SNP) arrays or “chips”], it is anticipated that additional routine genetic
testing will be employed for diagnostic, prognostic, and predictive purposes.
What follows here is a brief account of the pertinent pathologic and molecular genetics of the most common
pediatric brain neoplasms. A more exhaustive description exists elsewhere (10, 63, 68).
Gliomas
Pilocytic Astrocytomas
PAs, WHO grade I, are slowly growing tumors that most commonly occur in the cerebellum, hypothalamus, and in
relation to the optic pathway (especially in relation to NF1), although cerebral, brainstem, and spinal cord cases
also occur. Imaging often reveals a cystic lesion bearing an enhancing mural nodule.
Histologically, PAs are fairly discrete GFAP-positive tumors that exhibit only limited infiltration of adjacent native
parenchyma. They are classically described as biphasic with (a) compact areas that contain spindled cells with
long thin fibrillary processes (i.e., “piloid” or hair-like) emanating from opposite ends of the cell (i.e., bipolar); and
(b) more loosely textured microcystic areas populated by small cells with round-oval nuclei bearing short
cytoplasmic processes (Figure 10-24); RFs are seen in the former areas, while EGBs are seen in the latter.
Degenerative atypia (see above) and vascular hyalinization are both common. Several histologic features, taken
out of this typical context, can raise suspicion of a more ominous neoplasm, in particular a diffusely infiltrating
type glioma. Areas of a PA may closely resemble DA or oligodendroglioma. MVP, often termed “glomeruloidtype,”
can closely mimic that found in high-grade gliomas, and may be accompanied by a bland “infarct-like” necrosis.
Mitotic activity can be seen but is generally low. Extension involving the local subarachnoid space is fairly
common, but does not adversely impact prognosis. Cases of anaplastic PA (WHO grade III), which secondarily
develop the typical features of a high-grade DA (see below), have been reported but are exceedingly rare and
remain poorly characterized to date. Pilomyxoid astrocytomas (PMA), considered by some to be an infantile-
variant of PA, typically occur in the hypothalamus and are characterized by a monotonous population of small
oval-to-elongate cells; these cells are embedded in mucoid background and form occasional perivascular
pseudorosettes similar to those seen in ependymoma along with mitoses, necrosis, and variable infiltration. This
proposed variant has been suggested to recur and seed the CSF spaces more frequently than typical PA (111).
As such, it has been designated as WHO grade II in the 2007 WHO classification scheme. With exception of
these latter two variants, WHO grade I PAs behave in a prognostically favorable manner.
FIGURE 10-24 ▪ Pilocytic astrocytoma (PA). Low-power magnification demonstrating a sharp tumor-brain
interface (left), and a typical biphasic solid/microcystic architecture.
Genetic studies of PA have failed to identify a consistent signature abnormality. Cytogenetic abnormalities are
often limited to gains of only a single chromosome (92). Gene expression profiling of 21 PAs suggested
deregulation of several groups of genes in PA, including those involved in neurogenesis, cell adhesion, synaptic
transmission, and CNS development in cellular differentiation (119). In addition to
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demonstrating that PAs could be separated into two groups based upon differences in gene expression, this
latter study also showed that PAs immunonegative for myelin basic protein were more likely to progress.
Although loss of the tumor suppressor gene, neurofibromin, appears to play a role in NF1 related PAs, its role in
sporadic PAs is less clear (112). More recent studies suggest roles for Sox-10-regulated overexpression of
ErbB3 (as part of a tyrosine kinase receptor-mediated pathway) (2), and overexpression of mRNA related to
matrilin-2 (an extracellular matrix protein) in sporadic PA (98).
Diffusely Infiltrating Astrocytomas

As a group, DAs, WHO grade II-IV, represent the second most common pediatric tumor type. These grossly
graytan-to-gelatinous tumors obscure the native gray-white boundaries, with higher grade examples often
containing additional hemorrhage and necrosis. Microscopically, tumor cells invade adjacent brain structures in a
single cell manner and have a tendency to aggregate around preexisting structural elements (neurons, blood
vessels, underneath pial, and ependymal surfaces); these are called secondary structures of Scherer. Irregularly
arranged infiltrating astrocytic tumor cells' morphology range from uniform, and minimally atypical, to highly
pleomorphic in terms of both their cytoplasmic and nuclear features (Figure 10-25). Eosinophilic cytoplasm is
fibrillary to gemistocytic (“belly-like”), and processes are often few and coarse; this cytoplasm is often
immunoreactive for GFAP. In contrast, reactive astrocytes are evenly spaced and have a “starburst” appearance
with several thin processes. “Naked nuclei” (i.e., nuclei without discernable cytoplasm on routine staining) may
be common or predominant in fibrillary astrocytomas, making their identification difficult in cases with only mild
hypercellularity and atypia. Nuclear features often reflect tumor grade. In general, the lowest grade tumors (i.e.,
WHO II) display less pleomorphism; they are moderately hyperchromatic, oval to slightly angulated, and bear
indistinct nuclear membranes/nucleoli. In higher grade examples (i.e., WHO III-IV), nuclei often become
increasingly pleomorphic and hyperchromatic.
FIGURE 10-25 ▪ Anaplastic astrocytoma, WHO grade III. Enlarged, hyperchromatic, and irregular tumor nuclei
are associated with little cytoplasm and are seen diffusely invading neocortical gray matter.
DA grading criteria are currently fairly well defined. The four key features are encompassed by the mnemonic
“AMEN”: nuclear atypia, mitoses, endothelial proliferation, and necrosis. WHO grade II tumors show nuclear
atypia alone. One mitotic figure is generally not considered sufficient to warrant an anaplastic designation,
especially in large resections (35). WHO grade III tumors (i.e., anaplastic astrocytoma) generally exhibit mitotic
activity (at least 2 to 3 mitoses within the entire surgical material). Suspicion of a higher grade neoplasm is also
often deduced from radiologic enhancement, which often (but not always) correlates with the presence of MVP.
Either MVP or necrosis (in particular pseudopalisading necrosis, wherein tumor cells cluster or palisade around
an area of central necrosis) elevates the grade to IV [i.e., glioblastoma (GBM)].
The inherently infiltrative nature of diffuse astrocytomas precludes surgical resection, and recurrences are
inevitable despite current adjuvant therapy. Although some studies suggest better outcomes for childhood versus
adult DAs, the prognosis remains poor (9). A recent epidemiological study of 987 children and adults estimated
the median survival times for grade II, III and IV DAs at 5.6, 1.6, and 0.4 years, respectively (71).
The traditional EGFR (seen in primary or de novo GBM) versus p53 (secondary GBMs developing from a lower
grade precursor) molecular pathogenic dichotomy, which characterizes adult DAs does not appear to be entirely
applicable to pediatric DAs: EGFR and p53 mutations are relatively infrequent (83, 112). However, Phosphatase
and Tensin homolog (PTEN) mutations, which characterize both primary and secondary adult GBMs, may be
seen in children and appear to portend a poor prognosis (83); therefore, pediatric DAs may involve similar but
also potentially different components of these complicated pathways. In general, mouse modeling of gliomas has
suggested molecular abnormalities in three basic cellular processes: (a) external signaling (e.g., involving
receptor tyrosine kinases EGFR and PDGFR), (b) signal transduction (SRC, AKT, PTEN, RAS, RHO), and (c)
cell cycling (INK4a/CDK4/RB/E2F and ARF/MDM2/p53 pathways) (68). Notably, one recent pediatric microarray
study found differential expression of the EGFR/FKBP12/HIF2-alpha growth and angiogenesis-promoting
pathway in higher grade DAs (54). In addition, microsatellite instability, considered a marker of defective DNA
repair, has been noted by some researchers to be more common in pediatric versus adult high-grade DAs (103),
although a recent study has failed to find such an association (25).
Ependymomas
Ependymomas, WHO grade II—III, are discrete radiologically enhancing gliomas, which in children most often
occur
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in relation to the fourth ventricle. Supratentorial and spinal cord cases also occur, the latter of which are more
common in adults. Of several microscopic variants (classic, cellular, papillary, clear cell, tanycytic,
andmyxopapillary), the classic and cellular types are the most common. The interface with adjacent brain is
sharp. Tumor cells are more often fibrillar, but epithelial morphologies can be seen; the former result in
characteristic perivascular pseudorosettes and the less common true rosettes, while the latter form epithelial
canals and surfaces (Figure 10-26A). Long GFAP-positive fibrillary processes radiate toward a central blood
vessel, creating perivascular nuclear free zones and hence pseudorosettes. The epithelial quality of many
ependymomas is reflected in IHC cytoplasmic dot-like positivity seen for epithelial membrane antigen (EMA) and
CD99. EM can facilitate the diagnosis by demonstrating long zipper-like intercellular junctions, microvilli, cilia,
and intracytoplasmic lumina (Figure 10-26B).
FIGURE 10-26 ▪ Ependymoma. A: Perivascular pseudorosettes. B: EM demonstrating “zipper-like” intercellular

junctions ({long arrow) and microvilli {(short arrow).
Numerous grading systems have been proposed for ependymomas. Regrettably, no consensus has been
achieved as to the criteria that best typify anaplastic (WHO grade III) examples. Ho et al. (45) suggested that two
of the following four criteria were indicative of anaplasia: mitoses & sup3;4/10 hpf, hypercellularity, MVP, and
necrosis. Other studies have found atypia, hypercellularity, and MVP to be reliable prognosticators (58, 70). One
recent study suggested that indicators of cellular proliferation, in particular cell densityadjusted mitotic rate and
ki-67, are especially important (60). Another new study claims that telomerase activity is reflective of anaplasia,
with IHC positivity for h-TERT seen in those cases (104).
Ependymomas are usually treated with surgery and, in many cases, radiation. Gross total resection has been
proven a key prognostic indicator. Radiotherapy is often withheld in children under 3 years of age because of the
heightened risk of CNS damage in this cohort. Five-year progression free survival (PFS) and overall survival
(OS) for grade II and grade III ependymomas were 90 and 93 months versus 27 and 61 months, respectively
(58).
To date, no single genetic feature characterizes the majority of pediatric ependymomas. While overall, loss of
chromosome 22q is the most commonly seen abnormality, this usually occurs in the context of adult spinal cases
and in NF2 patients. Loss of the tumor suppressor gene 4.1B (DAL-1 on 18pl 1.3) has been noted to be more
common in intracranial examples (especially in the ”clear cell variant”), and abnormalities of the 4.1R gene (on
lp32-33) may also be important (85, 100). Various CGH studies on pediatric intracranial ependymoma have
revealed gain on lq (spinal cases may show gain of chromosome 7) and losses on chromosomes 6q, 9, 13, and
17p in subsets of tumors (79). Poorer clinical outcomes have been suggested in cases with (a) partial
chromosomal losses (or structural alterations) and gain of lq (23) and (b) elevated ErbB2/4 receptor
coexpression levels, which is especially predictive when combined with the IHC Ki67 index and the extent of
resection (37). More recently, microarray and Q-PCR data have suggested several potential genes of interest in
the pathogenesis of pediatric ependymoma (102), with patterns supporting a possible histogenetic link to radial
glia (108).
Less Common Gliomas

Additional relatively discrete and generally low-grade gliomas include PXAs, SEGAs, and DIAs. These
neoplasms are usually associated with a favorable prognosis post resection and are briefly discussed below.
Both astroblastoma (with lowgrade and high-grade forms proposed) and gliomatosis cerebri (generally
considered WHO grade III) are rare tumors that are essentially considered forms of glioma (the former sharing
some ependymal features, while the latter demonstrates astrocytic or rarely oligodendrocytic cytology).
Astroblastomas are discrete and contain epithelioid-to-nbrillary GFAP positive cells arranged in distinctive
astroblastomatous-type
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rosettes (i.e., perivascular pseudorosettes containing cellular processes with broad-based vascular
attachments). Vascular hyalinization is characteristic. Gliomatosis cerebri is defined as a widely infiltrative glioma
involving more than two lobes and potentially infratentorial structures. Types I and II are recognized with the
latter being associated with a distinct mass. Prognosis is poor for these patients; the majority of whom die within
12 months (63). Angiocentric glioma is a recently described epileptogenic primary neoplasm that displays
ependymal-like differentiation, characteristic nuclear cytology, and a perivascular/subpial infiltrative growth
pattern (118).
In comparison with adults, oligodendrogliomas (WHO grade II and III) are much less common. These tumors are
generally white matter-based lesions of the cerebrum that have a tropism for the neocortical gray matter.
Secondary structures of Scherer and calcification tend to be more prominent in oligodendrogliomas as opposed
to DAs. The typical “fried-egg” appearance of tumor cells is a helpful, although not entirely consistent, tissue-
processing artifact wherein round/regular tumor nuclei are surrounded by a clear halo of cytoplasm. Delicate
chicken-wire-type vasculature courses between the tumor cells. Scattered mitotic activity is tolerated within
grade II forms. In general, mitotic activity greater than 6/10 HPF and/or MVP is necessary for a designation of
anaplastic oligodendroglioma (WHO grade III) (36). Oligodendrogliomas are probably best known for their
favorable prognosis and chemotherapeutic responsiveness when accompanied by codeletion of the lp and 19q
chromosomal arms (12). Unfortunately, this favorable genetic signature is more commonly encountered in adult
oligodendrogliomas and is uncommon in pediatric cases (59, 84). Moreover, when present in children, the
prognostic significance of this deletional pattern is not as clearly established. Mixed oligoastrocytomas (MO A)
contain both oligodendroglial and astrocytic tumor components, manifesting either as geographically separate
forms or more often intermingled forms.
PXAs, WHO grade II and III, are epileptogenic and usually occur as cortically superficial lesions of the temporal
lobe. Histologic features are quite characteristic, but prior to its recognition as a distinct entity, PXA was
commonly misdiagnosed as GBM. Large pleomorphic cells are variably GFAP-positive, and often contain
substantial eosinophilic to lipidized clear cytoplasm. Spindle-shaped cells are arranged in interweaving fascicles
that often engender a mesenchymal quality, which is accompanied by pericellular reticulin deposition. EGBs and
perivascular lymphocytes are typical. Both subarachnoid space involvement and a limited infiltrative component
can be seen and are not indicative of a poor outcome. Grading criteria have been proposed to mark a subset of
PXAs that are associated with a poor prognosis; five mitoses/10 HPF has been suggested as a criterion for
anaplastic PXA (WHO grade III) (34). Genetic studies are limited and initially suggested alterations that differ
from DAs (53). More recently, some of the abnormalities previously described in DA have also been identified in
PXAs, namely changes in chromosome 9p (53) and the MDM2 gene (66).
SEGA, WHO grade I, is almost entirely restricted to patients with TS (see later discussion). They usually occur
near the foramen of Monro, and accordingly result in obstructive HCP. Whether this entity is neoplastic or
hamartomatous remains unclear. Imaging reveals contrast enhancement and often calcification. Tumor cells
contain abundant glassy eosinophilic cytoplasm and, despite their name, are more aptly considered larger than
“giant” (giant cells of a giant cell GBM are much larger, bizarre, and are often multinucleate.). Both spindled cells
and epithelioid to gemistocyte-like forms are seen, typically forming sweeping fascicles and occasional
perivascular pseudorosettes. Nuclei often bear prominent nucleoli, resulting in comparisons to ganglion-like cells.
These hybrid astrocytic/neuronal features are reiterated in the IHC results in these cases that sometimes reveals
both GFAP and neuronal marker positivity. Accordingly, some experts have favored the alternative term
subependymal giant cell tumor. Mitoses, MVP, and necrosis are usually absent.
Embryonal Tumors
This group of tumors comprises approximately 20% of pediatric tumors. Histologically, they are united by their
small round blue cell cytology: primitive appearing cells exhibiting a high nuclear-to-cytoplasmic ratio and
hyperchromatic nuclei. All are WHO grade IV.
Medulloblastoma
Medulloblastomas are tumors of the cerebellum, and generally originate from the vermis. They are contrast-
enhancing tumors that may contain necrosis, although MVP is somewhat uncommon. These tumors have a
tendency to seed the CSF pathways and may result in “drop metastases” to the spinal cord. Distant metastases
may also rarely occur (most requently bone and lymph nodes).
Numerous histologic subtypes of medulloblastoma exist, the most common of which include classic (i.e.,
undifferentiated); desmoplastic-nodular (D-N); medulloblastomas with extensive nodularity (MBEN); and large
cell-anaplastic (LC-A). Less commonly, medulloblastomas may exhibit glial, skeletal muscle, and/or melanotic
types of differentiation. Classic medulloblastomas contain patternless sheets of “small round blue cells,” with or
without Homer Wright rosettes, wherein primitive tumor cells surround a central island of delicate fibrillary
material (i.e., neuropil) (Figure 10-27A). These Homer Wright rosettes are also sometimes referred to as
neuroblastic rosettes since they are identical to those encountered in neuroblastomas of the peripheral nervous
system. Ganglioid (intermediate in size between neurocytes and ganglion cells) and ganglion cells are less
common manifestations of neuronal differentiation and maturation. When significant nuclear atypia accumulates,
this variant essentially blends into the more aggressive LC-A variant.
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FIGURE 10-27 ▪ Medulloblastoma, classic subtype. A: H&E 1,000× magnification. B: Synaptophysin

immunohistochemistry
LC-As medulloblastomas are characterized by two types of tumor cells, either of which may predominate: (a)
large cells are rounded and contain enlarged vesicular nuclei, prominent nucleoli, and variable amounts of
cytoplasm and (b) anaplastic cells are similarly enlarged, but show significant nuclear atypia and
hyperchromasia. These anaplastic regions often display “nuclear molding” and “cell wrapping” (Figure 10-28).
D-N medulloblastomas have a characteristic low-power appearance of rounded pale islands of tumor, separated
by darker internodular tumor. The pale islands are less cell dense and composed of uniform round-oval, less
mitotically active and cells embedded within a fine fibrillary/neuropil-like reticulin-poor background. These slightly
more mature neuronal-appearing cells sometimes resemble neurocytes. The internodular tumor is more cell
dense and primitive appearing, with mitotically active cells embedded within reticulin-rich tissue. Sometimes
parallel rows of single tumor cells are identified (cellular streaming). D-N medulloblastomas comprise a
genetically distinct subset of medulloblastomas that behave in a prognostically favorable manner (69). Tumors
bearing a predominance of large pale islands (which are often grossly or radiologically visible) and minimal
internodular areas have been termed MBENs; these rare medulloblastomas seen in very young children are also
considered to form a prognostically more favorable subgroup. Such tumors have been referred to as cerebellar
neuroblastoma in the past.
IHC staining of tumor cells in medulloblastoma is most reliably done with synaptophysin, consistent with at least
a limited degree of neuronal differentiation in the vast majority of medulloblastomas (Figure 10-27B). More
variable and often limited degrees of GFAP positivity may also be seen. In the D-N medulloblastomas, the
greatest degree of synaptophysin and GFAP positivity is usually seen in the intranodular regions, consistent with
the notion that these represent islands of maturation. Occasionally, Neu-N nuclear positivity may be seen within
the neurocytic-like tumor cells of these intranodular areas. As expected, ultrastructural evidence of neuronal
differentiation is also common.
Recent attempts at grading medulloblastomas have concluded that greater degrees of anaplasia (as defined by
nuclear enlargement, mitoses, apoptosis, large cells, angulation/pleomorphism, cell crowding, and cell wrapping)
are associated with worse clinical outcomes (Fig. 10-28) (24, 33).
Several biologic pathways have been implicated in medulloblastoma pathogenesis: (a) sonic hedgehog (SHH),
(b) wingless (WNT), and (c) ERBB receptor tyrosine kinase I family. SHH is important to cerebellar granular cell
development and mutations in the SHH pathway (most notably the PTCH gene associated with Gorlin syndrome)
have been linked to D-N medulloblastoma (80). Approximately, 15% of sporadic medulloblastomas involve
mutations in the WNT pathway, which includes contributions from APC (related to Turcot syndrome), axin, GSK-
3beta, beta-catenin, and the
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transcription factor complex TCF/LEF (105). Overexpression of the ERBB2 receptor has been associated with
poor clinical outcome, while elevated Trk-C expression has been linked to a more favorable behavior and the D-
N medulloblastoma variant (42, 112). The most common cytogenetic alteration in medulloblastomas involves loss
of chromosome 17p, most often resulting from the formation of an isochromosome 17q [i(17q)] with an associated
duplication of the long arm; i17q is encountered in about 30% of cases (79). Isolated losses of 17p have been
associated with aggressive behavior, as have amplifications in the MYC oncogenes, either c-MYC or N-MYC;
such amplifications are seen in approximately 10% of cases (105, 112). More recent data have drawn attention
to epigenetic phenomena, in particular hypermethylation of key DNA segments involved in transcriptional
regulation, including the tumor suppressor genes RASSF1A and HIC-1 (64, 117).
FIGURE 10-28 ▪ Large cell—anaplastic medulloblastoma. “Cell wrapping” is prominent in this example.
Five-year survival rates for medulloblastomas have continually improved over the last 25 years, rising from 36%
in 1980 to approximately 70% to 80% now. However, this has come with a significant price in terms of long-term
side effects, since craniospinal radiation is particularly toxic to the developing CNS, especially in those children
less than 5 years old.
Supratentorial Primitive Neuroectodermal Tumor

A previously held conceptualization suggested that all CNS embryonal neoplasms were of a similar origin. For
example, supratentorial primitive neuroectodermal tumor (sPNET) was considered to simply represent the
supratentorial form of medulloblastoma. However, the prognosis for sPNET has been demonstrated to be
significantly worse than for its postulated cerebellar counterpart; moreover, recent studies have revealed
separate genetic alterations (see below). sPNETs are contrast enhancing and may exhibit calcification,
hemorrhage, and/or necrosis. Histologically, these densely cellular tumors are reminiscent of classic
medulloblastomas, but sometimes with greater evidence of divergent differentiation, both on routine stains
(Homer-Wright rosettes, perivascular pseudorosettes, ependymal canals, pigmented cells, neurons) and
immunohistochemically (positivity with neuronal markers including synaptophysin, GFAP, muscle markers,
epithelial markers). Tumors with evidence of extensive neuronal differentiation have been alternately termed
cerebral neuroblastoma.
Gene expression profiling (80) and CGH data (46) support the separation of medulloblastoma from sPNETs. In
particular, CGH has revealed that sPNET, as compared to medulloblastoma, do not demonstrate i(17q) or -10q
but do exhibit+lq, -16p and -19p. This latter study also revealed a significantly worse prognosis for sPNET. One
recent review suggested a role for the pRB/Ink4/p53 and DNA repair pathways in the development of sPNETs
(62). With such a paucity of data accumulated to date, additional molecular genetic investigations of sPNET are
clearly needed.
Atypical Teratoid/Rhabdoid Tumor (ATRT)

ATRT is an uncommon, but distinctive tumor of infants and young children (generally <5 years old). These often
large, cystic, hemorrhagic, and enhancing tumors may be seen supratentorially, in the posterior fossa or rarely in
the spinal cord. Routine and IHC stains yield a polyphenotypic pattern (i.e., presence of multiple lineage-
associated markers that are usually not coexpressed). Characteristic to ATRT are rhabdoid cells, which exhibit
eccentrically placed vesicular nuclei, prominent nucleoli, and a globular or fibrillar eosinophilic paranuclear
inclusion corresponding to whorled bundles of intermediate filaments ultrastructurally (Figure 10-29A). Areas of
both mesenchymal and epithelial differentiation may be noted. Primitive appearing cells may predominate in
some, causing diagnostic confusion with sPNET or medulloblastoma (43). The IHC profile is highly variable but
typically includes a triad of positivity for EMA, smooth muscle actin (SMA), and vimentin. ATRTs result from
biallelic inactivation of the INI1/BAF47/hSNF5 gene tumor suppressor gene (located at 22q11) via either large-
scale deletion (which may be identified with FISH) or smaller single base pair mutation (detected through gene
sequencing). A highly sensitive and specific IHC stain for this gene's protein product (called INI1/BAF47) has
recently become commercially available; nonneoplastic nuclei retain nuclear staining of this ubiquitously
expressed protein, whereas there is loss of expression in rumor nuclei (43) (Figure 10-29B). These extremely
aggressive tumors often cause death within 1 year.
Other rare embryonal neoplasms are listed in Table 10-9. Pineoblastomas are also a form of embryonal tumor
but are discussed under the pineal parenchymal tumors (PPTs) section below.
Tumors Related to the Third Ventricle/Suprasellar Space

Craniopharyngioma (CPGs), WHO grade I, are squamous epithelial neoplasms that are thought to be derived
from remnants of Rathke pouch. They are typically suprasellar and result in dysfunction of the hypothalamic-
pituitary axis, visual difficulties, obstructive HCP, and increased ICP. These contrast-enhancing, cystic and
calcified tumors contain a characteristic dark sparkled fluid similar to “machinery oil” (when spilled in vivo may
result in chemical meningitis). Papillary and adamantinomatous are the two main subtypes.
Adamantinomatous CPGs are typically present in children, although there is also a second smaller peak in
adults. Epithelial cells are arranged in sheets, whorls, and trabeculae, and may line cyst spaces. Solid foci bear
orderly islands of epithelia with (a) peripheral or basal palisades, (b) adjacent polygonal cells, and (c) a loose
meshwork of epithelial cells termed stellate reticulum resulting from intercellular fluid accumulation (Figure 10-
30). Cellular outlines (or“ghosts”) of squamoid tumor cells with brightly eosinophilic cytoplasm and indistinct
nuclei constitute wet-keratin, a diagnostic feature even in the absence of viable epithelium. A
xanthogranulomatous inflammatory
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reaction is typical and accompanied by needle-shaped clear cholesterol clefts and necrosis. The ragged
interface with adjacent brain is typified by dense piloid gliosis (including RFs), which may resemble PA in the
absence of adjacent epithelium. Immunohistochemistry is positive for cytokeratins and EMA. The outcome is
dependent on the extent of surgical resection and tumor size, with 10-year survivals ranging from 64% to 96%
(63). Recent mutational and IHC analyses have suggested a role for abnormal WNT pathway signaling in
adamantinomatous CPGs; exon 3 of beta-catenin was mutated in 77% of these tumors, with corresponding
nuclear accumulation of beta-catenin in 94% (11).
FIGURE 10-29 ▪ Atypical teratoid rhabdoid tumor (ATRT). A: Rhabdoi cells. B: INI-1/BAF-47
immunohistochemistry demonstrating a lack of staining in tumor nuclei, while nonneoplastic lymphocytes and
endothelial cells retain nuclear positivity
Papillary CPGs are relatively discrete papillary tumors that primarily affect adults. These tumors are composed of
stratified, nonkeratinizing squamous epithelium situated on a fibrovascular stroma that lacks the characteristic
histology of the adamantinomatous variant. Scattered goblet cells may be highlighted with mucin stains. Papillary
CPGs are more commonly intraventricular (third ventricular), and although some studies have suggested that this
variant displays a better prognosis than the adamantinomatous variety (107), other studies have failed to
demonstrate such an association (22).
Germ cell tumors are thought to be derived from ectopically placed germ cells during gestation. Included in this
group are germinoma, yolk sac tumor, choriocarcinoma, embryonal carcinoma, teratoma (mature and immature
variants), and mixed neoplasms (comprised of two or more of the preceding types). Pineal and suprasellar
regions are especially favored; at times, synchronous (and separate) lesions may be detected in each of these
two areas. Suprasellar lesions typically result in dysfunction of the hypothalamic-pituitary axis and abnormal
vision, whereas pineal lesions result in Parinaud syndrome and HCP. Clinical outcomes correlate with certain
subtypes and segregate into favorable (e.g., germinoma, teratoma) and unfavorable groups (e.g., yolk sac tumor,
choriocarcinoma, embryonal carcinoma), the latter of which are often suspected via imaging/gross features of
necrosis and hemorrhage. Histologic features of CNS germ cell tumors are essentially identical to their extra-
CNS counterparts (nongerminomatous examples are discussed in Chapters 18 and 19).
CNS germinomas have a characteristic histology composed of two cell types. The neoplastic component has
large round-to-oval epithelioid cells that are glycogen rich and have a clear-to-eosinophilic cytoplasm; the
associated
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nucleus is large, vesicular and bears a prominent nucleus (Figure 10-31A). The second cell type comprises a
variably prominent reactive lymphocytic infiltrate, which is dispersed within an architectural lobularity created by
delicate fibrovascular septae. This inflammation can also be granulomatous and may overshadow the tumor
cells, occasionally leading to a misdiagnosis of inflammatory disorders; this pitfall is particularly important to
consider when dealing with small biopsy specimens. Immunohistochemically, the large tumor cells stain positively
for placental alkaline phosphatase (PLAP) and c-kit (CD117) in a membranous pattern, of which the latter is now
preferred (49) (Figure 10-31B). Recent genetic studies have yielded similar cytogenetic alterations within CNS
and extra-CNS germinomas: isochromosome 12p [i(12p)] which should not come as a surprise. Germinomas are
extremely radiosensitive and chemosensi-tive and therefore are among the prognostically favorable group of
CNS germ cell tumors, with 5-year survival varying from 80% to 96% (10).
Table 10-9 ▪ ORARE EMBRYONAL TUMORS
Tumor Type (ref) Key Histologic Features IHC/EM
Medulloepithelioma Neoplastic epithelium bearing an external Vimentin and nestin

(Molloy et al., 1996)* limiting membrane; divergent
differentiation.
Ependymoblastoma Multilayered true rosettes Vimentin, S-100; abortive

(Cruz-Sanchez et al., (ependymoblastic) ependymoma-like
1988)** ultrastructure
ETANTR (Eberhart et Ependymoblastoma-like rosettes, neuropil Synaptophysin, NFPGFAP;

al., 2000)*** and varibable neuronal differentiation EM as above
ETANTR, embryonal tumor with abundant neuropil and true rosettes.
*J Neurosurg 1996;84:430-436.
**Histopathology 1988;12:17-27.
***Pediatr Dev Pathol 2000;3:346-352.

FIGURE 10-30 ▪ Adamantinomatous CPG. In addition to its characteristic epithelium, “wet keratin” (arrow) can be
seen and often bears “ghosts” of degenerate tumor cells.
FIGURE 10-31 ▪ Intracranial germinoma. A: Typical biphasic histology including large mitotically active cells and
reactive lymphocytes. B: CD117 (c-kit) immunohistochemistry demonstrating membranous positivity in the large
tumor cells.
PPTs are thought to be derived from the native pineocyte, a neuron-like cell with photoreceptor and
neuroendocrine characteristics. These contrast-enhancing tumors obstruct CSF flow and compress adjacent
structures with Parinaud syndrome being typical. The WHO 2000 classification system recognizes three main
PPTs: Pineoblastoma, WHO grade IV, primarily affects children, while pineocytoma, WHO grade II, usually
occurs in adults. PPT of intermediate grade is “intermediate” in terms of grade and clinical features and is
considered WHO grade III. Jouvet et al. (48) have proposed an alternative four-tier grading scheme for PPTs,
where pineocytomas are grade I, PPT of intermediate grade are grade II and III, and pineoblastomas are grade
IV; the degrees of mitotic activity and NFP staining in tandem serve to differentiate these groups into
prognostically meaningful categories. Pineoblastomas are somewhat poorly demarcated and may contain
hemorrhage and or necrosis. Histologically, these embryonal tumors are populated by primitive mitotically active
cells. Hypercellular sheets of tumor cells may contain Homer-Wright rosettes or Flexner-Wintersteiner rosettes,
but none of the pineocytic rosettes are characteristic of pineocytoma. Pineocytomas bear uniform small mature
cells with round-oval bland nuclei and moderate amounts of eosinophilic cytoplasm. These cells closely resemble
the neurocytes encountered in central neurocytoma. Mitoses and necrosis are infrequent. Pineocytic rosettes
resemble HomerWright rosettes but are larger and are not formed by primitive cells (Figure 10-32). Degenerative
atypia and ganglionic differentiation may also be seen. Immunohistochemistry reveals staining for neuronal
markers, especially synaptophysin, but also for the more nonspecific neural/neuroendocrine marker neuron
specific enolase (NSE). In general, the grading of PPTs utilizes cytology, mitotic activity, necrosis,
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the present or absence of pineocytic/Homer-Wright/FlexnerWintersteiner rosettes, and a decrease in NFP
staining (10) (see also above). Five-year survival rates for pineocytoma and pineoblastoma have been estimated
at 86% and 58%, respectively (63). Genetic information on PPTs is very limited and reviewed elsewhere (62).
FIGURE 10-32 ▪ Pineocytoma. Pineocytic rosettes (arrows), which are larger than Homer Wright rosettes, are
scattered throughout this example.
Neuronal and Mixed Glioneuronal Tumors
Ganglioglioma (GG)
GGs (usually WHO grade I) are epileptogenic tumors that preferentially occur in the temporal lobe. The defining
feature of GGs is dysmorphic neurons. Their morphology deviates from normal neurons (large vesicular nucleus,
prominent nucleolus, basophilic cytoplasm bearing Nissl substance) in exhibiting binucleation or multinucleation,
vacuolated cytoplasm, and clumpy irregularly formed Nissl substance (Figure 10-33A). Coarse irregular
processes and Alzheimer type degenerative changes (including neurofibrillary tangles and granulovacuolar
degeneration) may be seen. Architecturally, the ganglion cells are often clumped or haphazardly arranged in
comparison to the laminar, well-ordered arrangement of normal cortex. However, in areas where the glial
component predominates, GGs may resemble DA, oligodendroglioma, and even PA. Gangliocytomas are
essentially GGs without the glial component; in cases where a glial component is more equivocal, a diagnosis of
“ganglion cell tumor” may be more appropriate. Connective tissue-rich areas and calcification may also be seen.
Although generally considered noninfiltrating and discrete neoplasms, neuropil-like areas (including axons)
indistinguishable from native parenchyma are frequent and make the designation of infiltration versus neoplastic
neuropil (i.e., tumor cell process constitute the meshwork of process, which to some degree mimics the
appearance of normal neuropil) difficult. EGBs are very common, as are perivascular lymphocytes. Features
characteristic of high-grade gliomas (mitoses and necrosis) are usually absent, although MVP is fairly common.
High-grade glial transformation (i.e., anaplastic GG, WHO grade IE, and rarely IV) is exceedingly rare and difficult
to define (10). The glial and neuronal components can be highlighted immunohistochemically with GFAP and
neuronal markers (most commonly synaptophysin), respectively. More recently, scattered CD34 positivity has
been suggested to be characteristic of GG, both within tumor cells and in the adjacent dysplastic cortex (8)
(Figure 10-33B). EM can also be used to support the finding of neuronal differentiation. Prognosis is favorable
with surgical resection.
Dysembryoplastic Neuroepithelial Tumor (DNT)

DNT, WHO grade I, is a controversial lesion that was first described in 1988 (18). Although currently considered
a mixed glioneuronal tumor by the WHO, many consider it
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a hamartomatous mass. These epileptogenic lesions are cortically based, with a marked predilection for the
temporal lobe. Imaging may reveal calcifications, cyst formation, and enhancement. The histologic hallmark of
classic DNT is the specific glioneuronal element, which is composed of columns of bundled axons/capillaries
(arranged perpendicular to the cortical surface) that are lined by oligodendroglial-like cells (OLCs). The exact
histogenesis of OLCs is debated; they stain for S-100 and are negative for both GFAP and neuronal markers,
suggesting a nondescript neuroepithelial origin. The columns of the specific glioneuronal element are separated
by pale basophilic mucin, within which are nondysmorphic floating neurons (Figure 10-34 A, B). Stellate
astrocytes may be seen among the specific glioneuronal element. Complex and simple forms of DNT are the
most commonly recognized. Complex DNTs contain patterned glial nodules (mucin ±) and a multinodular
architecture associated with the specific glioneuronal element and/or foci of cortical dysplasia. The glial
component, although typically nodular, may resemble conventional diffuse glioma or low-grade glioma. In
addition, this glial component may exhibit rare mitoses, nuclear atypia, necrosis, and even MVP, but these
features are not common. Areas of cortical dysplasia, characterized mainly by disorganized and dyslaminated
cortex, may lie adjacent to DNTs. Even more controversial is the proposed nonspecific variant of DNT; this
variant is not widely accepted (19). Chief in differential diagnosis of DNT is oligodendroglioma; features used to
differentiate these tumors are described by Burger et al. (10). DNTs have a favorable prognosis, even after
subtotal resection. Genetic studies of DNT are rare, but unlike oligodendrogliomas, they lack lp and 19q
codeletions (31, 76, 82).
FIGURE 10-33 ▪ Ganglioglioma (GG). A: H&E section reveals numerous neoplastic neurons, including
vacuolated and binucleate (arrow) forms. B: CD34 immunohistochemistry highlights tumor cells with highly
ramifying cytoplasmic processes.
FIGURE 10-34 ▪ Dysembryoplastic neuroepithelial tumor (DNT). A: Low-power microscopy reveals acortically
based neoplasm. B: High-power microscopy demonstrates “floating neurons” and “oligodendroglial-like cells.”
Other Neuronal/Glioneuronal Tumors

Desmoplastic infantile ganglioglioma (DIG), WHO grade I, is a distinctive tumor usually occurring in the 1st year
of life. These large superficial lesions often have dural attachment, cyst formation, and contrast enhancement.
Macrocephaly and increased ICP herald its presence. Histologically, reticulin-rich desmoplastic areas contain
spindled cells with a fascicular or storiform arrangement; these areas often obscure the astrocytic component
that often requires GFAP IHC to fully appreciate. The latter resemble slender fibrillary astrocytes embedded
within the densely desmoplastic stroma; scattered small gemistocytes may be seen. The neuronal component is
often equally subtle since they are often considerably smaller than the neurons of conventional GG. Less
common are more classic ganglioglioma-like foci complete with EGBs. As with the glial element, IHC or EM may
be needed to detect this neuronal component. If neuronal elements are still lacking after special studies, the term
“dyplastic infantile astrocytomas” (DIA) is appropriate. However, both DIA and DIG are now thought to represent
opposite ends of a single entity (i.e., dysplastic infantile tumors). Worrisome and mitotically active primitive
neuroectodermal tumor (PNET)-like foci bearing MVP and necrosis may be seen but fortunately do not impact
prognosis since these patients have favorable outcomes after surgery.
Dysplastic gangliocytoma of the cerebellum (Lhermitte Duclos disease) (DGCC), WHO grade I, is a unique
cerebellar neoplasm often associated with Cowden syndrome. Predictably, these patients present with signs and
symptoms of cerebellar dysfunction and CSF obstruction. These solid tumors are characteristically striped on
T2-weighted MRI due to in part to thickened folia. Microscopically, a distinctive abnormal architecture is seen
and has been likened to cortex “flipped inside-out.” More normal cerebellar cortex is progressively replaced by
two layers. The outer layer consists of parallel arrays of myelinated axons, whereas the inner layer is composed
of abnormal smaller and larger neurons (ganglioid and ganglion-like, respectively), which replace the internal
granule cells. These ganglion cells may resemble Purkinje cells but are far too numerous, disordered, and
pleomorphic. Abnormal
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vascular proliferation may be noted in the subarachnoid space and white matter (which may be vacuolated).
Since patients with Cowden syndrome bear germline mutations in the PTEN tumor suppressor gene on
chromosome 10q23, genetic studies (including sporadic cases) have investigated the PTEN/Akt/mTOR pathway.
IHC and mutational analysis have confirmed frequent PTEN mutations (15/18, 83%) and secondary activation of
mTOR (1, 120). A workup for other features of Cowden syndrome may also be warranted since such patients are
at risk for numerous systemic manifestations, including breast and gastrointestinal (GI) carcinomas.
Choroid Plexus Tumors

Choroid plexus tumors (CPTs) are intraventricular papillary epithelial tumors that are derived from the choroid
plexus. These are intensely enhancing lesions that often present with increased ICP and HCP. Although CPTs
may affect any site where native choroid plexus resides, the lateral ventricle is the most frequent location in
children.
CPP, WHO grade I, closely resembles normal choroid plexus in that papillae contain a fibrovascular core, a
simple to cuboidal epithelium with minimal to mild atypia, and little mitotic activity. However, this neoplastic
epithelium differs from normal choroid plexus in being more cell dense and lacking the normal surface hobnailing
(i.e., bumpy) architecture (Figure 10-35A). Mesenchymal type metaplasia, pigmented epithelium, and focal
ependymal differentiation may all be seen rarely, the latter manifesting GFAP immunoreactivity.
Choroid plexus carcinoma (CPC), WHO grade III, usually presents before 3 years of age. Papillary architecture is
variably replaced by areas of solid tumor growth (Figure 10-35B). Epithelium is clearly anaplastic in most
examples, although transitions with better-differentiated areas are occasionally seen and may suggest a role for
progressive malignant transformation in some. Pseudostratified epithelium is markedly cell dense and tumor cells
exhibit a high nuclear-to-cytoplasmic ratio. Nuclei are hyperchromatic and mitotic activity is generally prominent
(>5 per 10 HPF). Foci of necrosis are characteristic, and MVP may be seen. CPCs often invade adjacent brain
parenchyma.
Atypical CPP is considered an intermediate WHO grade II tumor. Atypical CPP has recently been defined by a
minimum of two mitoses per 10 HPF, while often containing at least two of the following as well: hypercellularity,
pleomorphism, foci of solid growth, and necrosis (47).
In general, CPT immunohistochemistry reveals positivity for cytokeratins and S-100 protein, with the latter often
being more limited in CPCs. Unlike most carcinomas, EMA is usually negative and focal GFAP expression is
relatively common. Transthyretin and synaptophysin staining have been touted as markers of CPTs, but these
are generally unreliable due to poor specificities. The genetics of CPTs are reviewed by Kamaly-Asl et al. (50).
TP53 mutations may be frequent in CPC (especially those related to Li-Fraumeni syndrome) but are rare in CPP.
Kamaly et al. further suggest that rare cases of CPC with INI1 mutation are truly ATRTs, an important differential
diagnostic consideration, since this tumor afflicts the same age group, can be intraventricular, and may show
papillary features. CPPs are often cured with surgery (5-year survival 100%), while CPCs often grow rapidly and
have an unfavorable prognosis (5-year survival 40%) (63).
Miscellaneous CNS Tumors

A variety of less common tumors arise in the CNS of pediatric patients. As opposed to adult patients, Meningeal-
based tumors, in particular meningioma, are uncommon (75). Pituitary adenoma is also much more common in
adults. Nerve sheath tumors (including schwannoma and neurofibroma) are covered in the soft tissue chapter
(Chapter 24), whereas bony skull-based tumors (including chordoma, Langerhans cell histiocytosis) are covered
in the chapter on the skeletal system (Chapter 27). Primary melanocytic lesions (melanomas,
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melanocytoma) are rare and thought to be derived from leptomeningeal melanocytes, hence their typical extra-
axial location. Vascular tumors are similarly rare, with hemangioblastomas being common only in adults. Their
presence in children strongly raises the possibility of von Hippel-Lindau (VHL) disease.
FIGURE 10-35 ▪ Choroid plexus tumors (CPTs). A: Choroid plexus papilloma (CPP). B: CPC; note the better
differentiated area left versus the more poorly differentiated tumor right.
Table 10-10 ▪ CANCER PREDISPOSITION (NEUROCUTANEOUS) SYNDROMES
Syndrome Gene (Locus) Nervous System Extraneural Manifestations

Pathology
Neurofibromatosis NF1 (17q11) Neurofibromas (diffuse, Skin (café au lait spots, axillary
type 1 nodular, plexiform); freckling); Lisch nodules;
MPNSTs; pheochromocytoma; carcinoid
optic/hypothalamic gliomas; tumors; rhabdomyosarcoma;
diffuse astrocytomas; CML; bone lesions
“UBOs”
Neurofibromatosis NF2(22q12) Bilateral vestibular Minimal skin stigmata (rare

type 2 schwannomas; plexiform schwannomas);
schwannosis; multiple cataracts.
meningiomas; MA; spinal
cord ependymomas; glial
microhamartoma
Ataxia ATM (11q22- Cerebellar degeneration; Mucocutaneous and

telangectasia 23) intracranial hemorrhage; conjunctival telangectasias;
(AT) cytomegaly and nuclear immunodeficiency and related
atypia (CNS and extraCNS respiratory infections; tumor
tissues) predilection and radiation
sensitivity
Neurocutaneous sporadic Diffuse melanocytosis, Cutaneous nevi (giant and or

melanosis melanocytoma, or primary multiple, including the
syndrome malignant melanoma of the congenital nevus of Ota)
leptomeninges
Nevoid basal cell PTCH Desmoplastic Odontogenic keratocysts;

carcinoma (9q22.3) medulloblastomas; palmar/plantar dyskeratoses;
(Gorlin) syndrome meningioma; CNS skeletal malformations; ovarian
malformations (agenesis of fibromas; melanoma;
the corpus callosum, leukemia/lymphoma;
cerebral falcine breast/lung carcinoma
calcifications; HCP)
Von Hippel- VHL(3p25-26) Hemangioblastomas Renal cell carcinoma;

Lindau (VHL) (cerebellum, retina); pheochromocytoma; pancreatic
papillary endolymphatic sac tumors; polycythemia
tumor (PELST)
Cowden PTEN/MMAC1 DGCC (Lhermitte Duclos Verrucous skin; cobblestons

(10q23) disease) oral papules; trichilemmomas;
colonic polyps; thyroid
nodules; breast carcinoma
Li-Fraumeni TP53 Gliomas (astrocytoma, Bone and soft-tissue

(17p13.3) ependymoma; ± sarcomas; leukemia;
multicentric); cerebral adrenocortical/breast
PNETs; CPTs; carcinoma; visceral epithelial
meningioma; schwannoma malignancies
Turcot Type 1: Type 1: GBM (younger Type 1: ± hereditary

hMLH1 (3p21) onset versus sporadic) nonpolyposis colorectal
hMSH2 (2p22- Type 2: medulloblastoma carcinoma Type 2: familial
21); hPMS2 adenomatous polyposis
(7p22)Type
2:APC(5q21)
Familial RB(13q14) Retinoblastoma; Osteosarcoma
retinoblastoma pineoblastoma
MPNST, malignant peripheral nerve sheath tumor; UBOs, unidentified bright object onT2-weighted or
FLAIR MR images; MA, Meningioangiomatosis
Cancer Predispostion (Neurocutaneous) Syndromes

These syndromes are summarized in Table 10-10. Patients with one of these syndromes may incur both
neoplastic and nonneoplastic forms of pathology and hence are often multisystemic in nature. Onset of these
syndromes is often during the pediatric years. While many of these syndromes are inherited in an AD pattern,
some (e.g., AT) are autosomal recessive in nature, while others (e.g., neurocutaneous melanosis syndrome)
appear sporadically. The specific clinical diagnostic criteria for each of these syndromes are not given here; for
such, the reader is directed to other texts (81).
INFECTIOUS DISEASE
Bacterial Infections
Acute Meningitis
Neonatal acute bacterial meningitis is most frequently due to group B streptococcus (Streptococcus agalactiae)
and Escherichia coli. Several other Gram-positive
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(Listeria monocytogenes and Staphylococcus aureus) and Gram-negative (Citrobacter, Klebsiella,
Enterobacter, Proteus, and Salmonella species, as well as Pseudomonas aeruginosa) bacteria may also be
causative. Infants and young children are affected primarily by S. pneumoniae, Neiserria meningitides, and
Haemophilus influenzae type b (Hib), while children older than five (like adults) are infected most frequently with
the former two pathogens. Notably, immunization with the Haemophilus, pneumococcal, and meningococcal
conjugate vaccines has significantly reduced the incidence of previously devastating infections (14).
FIGURE 10-36 ▪ Acute bacterial meningitis. A: Coronal section of the cerebrum reveals abundant purulent
material within the leptomeninges. B: Microscopy highlights a neutrophil-rich leptomeningeal infiltrate.
Bacteria reach the CNS via hematogenous spread, often from an upper respiratory focus, or direct spread from a
contiguous site of disease (i.e., mastoids, inner ear, nasal sinus, mouth). Neonates often acquire organisms via
passage through an infected birth canal. Once arriving at the CNS, breech of the blood-brain barrier is facilitated
by bacterial surface proteins. Although host immune defenses and antimicrobial therapy may effectively
neutralize the pathogen at hand, bacterial products can persist in stimulating the inflammatory response (14).
Focal neurologic deficits, changes in mental status, fever, rash, seizures, and signs of meningeal irritation herald
the presence of acute meningitis. Nonspecific changes such as irritability, lethargy, poor feeding, apneic spells,
and a bulging fontanelle may be seen in infants. CSF examination is key to the diagnosis, and findings include
granulocytic pleocytosis, elevated protein, decreased CSF to serum glucose ratio, and identification of organisms
on Gram stain. Definitive diagnosis is made with culture, organism specific PCR, and latex agglutination tests.
Gross examination of the brain reveals diffuse edema and possibly cerebral herniation. Surface vasculature is
congested. A light-colored thick leptomeningeal exudate may be seen grossly but may be less prominent in
partially treated cases. Focal areas of parenchymal softening are suggestive of infarction (Figure 10-36A).
Microscopic sections reveal a neutrophil-predominant exudate (lymphocytes and macrophages are seen in later
stages.) that often extends down the Virchow-Robin spaces (Figure 10-36B). This inflammation can result in a
vasculitis with secondary thrombosis (and hence infarction). Inflammation may also be seen in the choroid plexus
and along the ventricular lining. Organisms are highlighted with the Gram stain. Complications among survivors
predictably follow the areas of pathologic damage. Cortical infarcts lead to focal neurologic deficits (e.g.,
spasticity, dysphasia) and seizures and, when widespread, may result in mental retardation (or when less
pronounced, learning disabilities and behavioral disturbances). Resolution of leptomeningeal inflammation with
concomitant fibrosis overlying cranial nerves may result in cranial nerve palsies (e.g., hearing loss). Scarring
(i.e., gliosis and fibrosis) of the ependyma and leptomeninges can obstruct the flow of CSF causing HCP (see
Chapter 6).
Cerebral Abscess
Bacteria, which cause abscesses, like those causing meningitis, also arrive at the CNS via hematogenous or
direct contiguous spread. Children with congenital heart disease are particularly predisposed to hematogenous
dissemination of bacteria, resulting in abscesses within brain regions
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receiving a high blood flow (e.g., middle cerebral artery territory). Dental infections (i.e., abscesses), mastoiditis,
paranasal sinusitis, or otitis media may traverse local anatomic boundaries and lead to abscess formation in
adjacent brain parenchyma.
The spectrum of microorganisms causing abscesses has changed with time (91). While the incidence of S.
aureus has been decreasing, the identification of anaerobes has increased. Streptococcus milleri and S.
viridans are frequently associated with direct brain inoculation and hematogenous spread, respectively, and as a
group, aerobic and anaerobic forms of streptococcus cause 60% to 70% of cerebral abscesses (26). Other
causative aerobic and microaerophilic bacteria may include Haemophilus, Gram-negative enteric bacilli, and
Pseudomonas aeruginosa. Common anaerobes include Bacteroides, Peptostreptococcus, Fusobacterium,
Propionbacterium, Prevotella, and Actinomyces. Penetrating head injuries, neurosurgical procedures, and
immunocompromised states all predispose to abscess formation, with the latter invoking less common bacterial
(e.g., Nocardia, Listeria, Mycobacterial species), fungal (e.g., Candida, Aspergillus, Cryptococcus,
Histoplasma, Coccidioides, and Mucor), and parasitic (e.g., Toxoplasmosis)pathogens. MR imaging reveals a
mass with a thin smooth rim of enhancement. The resulting gross and microscopic appearance of cerebral
abscesses evolves through stages. Early stages (<4 days) begin with a cerebritis that grossly appears as an ill-
defined area of hyperemia and edema; microscopically endothelial swelling is accompanied by perivascular and
parenchymal neutrophils. After 4 days, areas of confluent necrosis emerge, as both macrophage and
mononuclear infiltrates become more conspicuous. An early granulation tissue reaction at the margin of necrosis
heralds the initiation of capsule formation at approximately 10 days; chronic inflammatory cells are noted within
the capsule, which is in turn surrounded by edema and reactive astrocytosis in the adjacent brain. A well-formed
reticulin-rich capsule is noted at 2 weeks, at which time the classic multilayered abscess wall is best appreciated.
Organisms (most highlighted with the Gram stain) are seen at all stages of evolution, particularly at the capsule-
necrosis boundary.
Subdural empyema and epidural abscesses are uncommon and not discussed further here.
Chronic Bacterial Infections

Mycobacteria uncommonly cause infection in North American children. Most cases are seen in the
immunocompromised (in particular, AIDS patients) and importantly in the developing world. Mycobacterium
tuberculosis (TB) causes meningitis (the most common form of disease), tuberculous masses (i.e.,
tuberculomas), and spinal epidural abscesses. Initial infection results from inhalation of bacteria-laden droplets;
subsequent CNS spread is related to hematogenous dissemination. Reactivation of a focus of latent CNS
infection (i.e., a “tubercle” or “Rich focus”) is also a source of active disease. Symptoms in children are subacute
(occur over 2 to 3 weeks) and may include fever, meningismus, signs of increased ICP (headache, nausea, and
vomiting), seizures, cranial nerve palsies, and epilepsy, with changes in mental status seen later on. Examination
may reveal a sixth cranial nerve palsy. Diagnosis may be made on CSF specimens via culture (often slow
growing) and PCR or via direct visualization using Ziehl-Nielsen or auramine rhodamine fluorescence staining
(see below).
Gross pathologic findings include a gelatinous and, at times, nodular leptomeningeal exudate that is often most
prominent along the Sylvian fissure and base of the brain. The choroid plexus and ventricular lining may be
similarly affected, and result in HCP. Areas of parenchymal softening are suggestive of superimposed infarcts
related to endarteritis obliterans with vascular thrombosis. Necrotizing granulomas are typical. Chronic type
inflammatory cells, variable fibrosis, and multinucleated (Langhans type) giant cells may be accompanied, albeit
rarely in most cases, by acid-fast bacilli on Ziehl-Nelsen staining (i.e., “red snappers”). Inflammation may spill
over into the adjacent brain, causing microglial activation and gliosis. Tubercles are the nodular macroscopic
confluence of these granulomas, while the histologically similar tuberculomas are grossly “mass forming.”
Other bacteria causing chronic CNS disease are uncommon but include Lyme disease (Borrelia burgdorferi) and
syphilis (Treponemapallidum).
Viral Infections
Viral meningitis is defined as a febrile illness associated with clinical signs of meningeal irritation but lacking
neurologic dysfunction and positive cultures. These often banal cases occur with seasonal and geographic
variations; rarely do they come to the attention of the pathologist. The most common causative entities include
the enteroviruses (echoviruses, Coxsackie A and B, and enterovirus per se) and HSV-2 (i.e., Mollaret
meningitis). Histology reveals, at best, a scanty lymphocytic predominant perivascular and leptomeningeal
infiltrate, which may affect the choroid plexus and creep into the superficial aspects of the brain parenchyma.
Viral encephalitis also causes fever but is additionally characterized by brain parenchymal dysfunction
manifesting as an altered state of consciousness and/or objective signs of neurologic dysfunction (i.e., seizures,
focal neurologic deficits); the equivalent pathologic process occurring in the spinal cord is called myelitis. Mixed
forms (i.e., meningoencephalitis or encephalomyelitis) also occur. Each of these main pathologic processes can
result in either acute or chronic disease depending on the particular type of viral pathogen involved. The clinical
severity of disease induced by these viruses ranges from minimal to fatal. Antivirals exist for some pathogens
(e.g., HSV and acyclovir), but care is limited to supportive therapy for others. Diagnoses are made by serology,
culture (in the past from CNS biopsy specimens), and more recently via PCR-based assays (especially of CSF),
which uncover specific viral nucleic acids.
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Herpes simplex virus (HSV) is one of the most common members of the Herpesviridae family, a group of
generally necrotizing double-stranded DNA viruses that also include varicella-zoster (VZV), cytomegalovirus
(CMV), and Epstein-Barr virus (EBV). Viral DNA is enclosed in a nucleocapsid and surrounded by a viral
envelope. Neonatal disease is most often caused by the HSV type 2, while the less frequent childhood form of
infection is caused by HSV type 1. Neonatal disease is usually acquired in the perinatal period from an infected
mother bearing recurrent but often asymptomatic genital disease (55). Neonates present within the first 4 weeks
of life with one of three main forms of disease: (a) localized skin, eyes, or mouth (SEM) disease including
vesicles and/or keratoconjunctivitis; (b) encephalitis ± SEM; or (3) diffuse or disseminated HSV with SEM,
encephalitis, and multiple visceral organ disease. These affected infants may be lethargic, irritable, feed poorly,
and suffer from seizures. Pathology reveals a diffusely swollen and congested brain. Hemorrhagic and necrotic
lesions of the gray and white matter are accompanied by macrophages and lymphocytes. Intranuclear viral
inclusions may be seen within neurons, glia, and/or endothelia. Survivors are left with a parenchymal loss and
gliosis (i.e., cystic encephalomalacia).
Childhood disease is much less common and presents in a similar fashion to that in adults. Primary HSV infection
is often asymptomatic, although some may develop oropharyngeal ulcers. Once the virus is absorbed, it
replicates and subsequently travels in a retrograde fashion along sensory axons (e.g., olfactory or trigeminal)
toward the respective ganglion where a latent infection ensues. Reactivation of viral disease is accompanied by
replication and anterograde travel down sensory axons toward the periphery whereupon mucocutaneous
vesicles erupt. HSV encephalitis is thought to arise either with primary infection or after reactivation of latent
trigeminal ganglia disease. Common clinical presenting features include fever, headache, altered mental status,
and seizures. The classic distribution of disease (see below) may be seen via imaging, and definitive diagnosis
using PCR to find viral DNA in the CSF has largely supplanted brain biopsy. Swelling, congestion, hemorrhage,
and necrosis are typically localized initially (often asymmetrically) to the posterior orbitofrontal, temporal lobes,
cingulate gyrus, and insulae (Figure 10-37A). Acutely necrotic (i.e., “red”) neurons are accompanied by
parenchymal/perivascular lymphocytes and macrophages, plus the nonspecific but characteristic viral
encephalitic features of perivascular lymphocytes, microglial activation, microglial nodule formation, and
neuronophagia. Neuronal, glial, and/or endothelial intranuclear viral inclusions (Figure 10-37B) may be difficult to
appreciate in some cases, wherein IHC staining for HSV can be very helpful. Endothelial and hence vascular
involvement may result in thrombosis and infarction. A necrotizing myelopathy may be seen but is rare. In
survivors, the extensive residual damage usually manifests in the form of cystic encephalomalacia.
CMV is the most common intrauterine viral infection. Congenital CMV is usually acquired transplacentally from a
newly infected mother, and while acquisition is most successful in third trimester gestations, first trimester
infections lead to the most severe (often systemic and fatal) disease. Survivors are left with sequelae that include
hearing loss, language disorders, microcephaly (the most common neurologic presentation), mental retardation,
seizures, chorioretinitis, and motor deficits. Imaging reveals micrencephaly, cerebral microcalcifications (often
periventricular), HCP, and gyral abnormalities. Diagnosis of fetal infection can be made via viral culture or PCR
of amniotic fluid, or by fetal IgM serology. Gross pathology confirms the imaging impressions, and
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may reveal porencephaly and polymicrogyria. Microscopy reveals a necrotizing ventriculoencephalits, with areas
of calcification and gliosis. Perivascular lymphocytes are accompanied by macrophages and activated microglia
(± nodules). Cytomegalic cells bear a single haloed intranuclear inclusion whose abundant cytoplasm also
contains multiple small inclusions (Figure 10-38); immunohistochemistry often highlights more widespread
involvement than is appreciated by routine stains. Subependymal gliosis may result in HCP. CMV infections are
less common in older children and are largely restricted to immunosuppressed patients (e.g., those with HIV) with
systemic disease, which may be related to reactivation of latent bone marrow virus. Symptoms may include
changes in mental status, nystagmus, and cranial nerve palsies, all of which are often indicative of a poor
prognosis. Pathologically, several forms of disease may be seen including encephalitis of varying severity,
ventriculitis, and lumbosacral myeloradiculitis (see Chapter 6).
FIGURE 10-37 ▪ Herpes simplex encephalitis. A: Ventral view of the brain demonstrating marked hemorrhagic
necrosis in a congenital case caused by HSV 2 (image courtesy of Dr. Barry Rewcastle). B: High-power
histology showing an eosinophilic intranuclear inclusion, likely within a glial cell.
FIGURE 10-38 ▪ CMV encephalitis. Cytomegalic cell containing a large intranuclear inclusion.
Primary infection with VZV results in chicken pox (varicella), whereas reactivation of latent sensory ganglia
disease causes shingles (zoster). Either form of VZV may result in CNS disease, which is typically necrotizing
and accompanied by intranuclear inclusions. Varicella may cause an embryopathy, transient cerebellitis,
meningoencephalitis (that can resemble Acute Disseminated Encephalomyelitis (ADEM), and has been
associated with Reye syndrome (an encephalopathic illness that has been correlated with salicylate ingestion).
Zoster has been associated with encephalitis, myeloradiculitis, and a vasculopathy/vasculitis. Varicella
embryopathy is acquired transplacentally and results in the most severe disease when acquired in the first half of
gestation. Cutaneous scarring, limb hypoplasia, chorioretinitis, cataracts, and mental retardation are seen.
Pathologically, scarring and gliosis are seen within the meninges and parenchyma, respectively, with the latter
showing evidence of degeneration but rarely an active necrotizing infection with demonstrable virus. A chronic
inflammatory infiltrate is accompanied by microglial activation. There may be neuronal loss and degeneration
within the dorsal root ganglia, anterior horns, and posterior/lateral funiculi, along with denervation muscular
atrophy. Vasculitis (± granulomatous) with infarction is seen in AIDS patients. The recent development of a live
attenuated vaccine will likely decrease the future incidence of VZV-related CNS disease.
The arboviruses are a group of mostly single-stranded RNA viruses that are usually transmitted to humans via
mosquitos. Infections are seasonally distributed and generally occur in the summer and fall. While West Nile
virus has garnered much of recent spotlight, it only rarely results in symptomatic CNS disease in pediatric
patients. More common in children, yet still rare, are Western and Eastern equine encephalitides and La Crosse
encephalitis (88). Incubation periods are less than 3 weeks and presenting symptoms include fever, malaise, and
myalgias. Neurologic disease is diverse and includes aseptic meningitis, increased ICP, altered level of
consciousness (which can lead to coma), motor deficits, and seizures. Diagnosis is made via serology or via
PCR-specific RNA assay of the CSF. Gross pathology may reveal swelling, congestion, hemorrhage, and, if
severe, necrosis. Some arboviruses tend to affect certain areas of the brain and spinal cord (26), but despite
these predilections, the microscopic features are nonspecific. Chronic leptomeningeal inflammation is
accompanied by the typical features of encephalitis (microglial activation, microglial nodules, perivascular
lymphocytes) and occasionally perivascular hemorrhage/myelin destruction. Vessels may be thrombosed, but
only rare and severe cases demonstrate significant necrosis. Notably, although viral inclusions are absent on
routine staining, IHC staining (available for some arboviruses) can help to highlight neuronal and glial infection.
Although more commonly associated with meningitis, the enteroviruses (see above) may all rarely cause a
poliomyelitis. These small single-stranded RNA viruses (including the formerly more common Poliovirus) cause a
lytic infection of motor neurons in the anterior horn of the spinal cord and in the brainstem. Initial infection is via
the fecal-oral route, and after hematogenous dissemination, the virus enters the CNS. Roughly 10 days after the
resolution of a nonspecific flu-like illness, a prodrome of fever, headache, vomiting, meningismus, irritability, and
myalgia ensues. Paralytic encephalomyelitis follows this prodrome and is often asymmetric and lower extremity
predominant. Gross pathologic findings are uncommon, but severe cases include congestion, hemorrhage, and
necrosis of motor nuclei within the brainstem and spinal cord anterior gray matter. Microscopically, affected
areas are intensely inflamed. Parenchyma and leptomeninges first contain neutrophils and later lymphocytes
plus activated microglia (with microglial nodules and neuronophagia). Chronic forms of disease manifest as
areas of neuronal loss, gliosis, and scanty inflammatory infiltrates.
The measles virus is a single-stranded RNA pathogen from the paramyxoviridae family. Measles is highly
contagious virus that is acquired through inhalation. Primary infection is systemic and results in fever, a
maculopapular rash, and
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rarely CNS disease, which can include aseptic meningitis or ADEM. Measles mediates two less common chronic
CNS diseases that are now rare since the institution of the MMR vaccine: measles inclusion body encephalitis
(MIBE) (which occurs in the immunocompromised a few months after primary infection) and the more acclaimed
subacute sclerosing panencephalitis (SSPE). SSPE results in CNS disease approximately 5 to 10 years after
primary infection, with some occurring after vaccination (which normally reduces the risk of disease dramatically).
In SSPE, the viral genome is mutated such that the virus is unable to assemble or bud from infected cells.
Clinical disease progresses through the early stages of cognitive and behavioral dysfunction; through motor
deficits, seizures, and ataxia; and finally autonomic dysfunction, altered mental status, and finally death. Median
survival is less than 2 years. Diagnosis can be made via antibody titers or PCR of fresh frozen brain.
Pathologically, the gross brain may show signs of atrophy and leukodystrophylike changes. A
meningoencephalitis is seen microscopically, with lymphocytic infiltrates (including perivascular) and
parenchymal microglial activation. The neocortex, deep cerebral gray, and white matter regions are especially
involved. There may be neuronal loss, and Alzheimer-like neurofibrillary tangles may be identified in residual
neurons. Extensive white matter gliosis (i.e., “sclerosing”) may be accompanied by demyelinated patches.
Intranuclear eosinophilic and haloed viral inclusions may be seen in neurons and oligodendroglia, but these are
often sparse, necessitating immunohistochemistry for their detection.
Human immunodeficiency virus (HIV) is a single-stranded RNA retrovirus that causes AIDS. HIV infection is
acquired by numerous routes, including sexual, hematologic, iatrogenic (e.g., contaminated instruments), and
perinatal. This latter mode of infection is the most common in children. Primary infection may result in aseptic
meningitis, after which a reservoir of virus is established in CD4-positive T-cells, macrophages, and microglia.
With viral-mediated destruction, CD4-positive T-cells plummet to numbers less than 200/JIL; thereafter, the
systemic and CNS features of AIDS ensue. CNS disease related to AIDS includes (a) direct HIV infection, (b)
opportunistic infections, (c) nonspecific CNS damage (related to ischemia, metabolic insults, etc.), and (d)
treatment-related disease (e.g., AZT myopathy). Although pathologic reports are early, highly active antiretroviral
therapy (HAART) appears to have significantly impacted the patterns (i.e., incidence, prevalence) of AIDS-
related disease in developed nations, especially in terms of reducing opportunistic infections (41). However,
these opportunistic infections are much less common in children as compared to adults regardless. Moreover,
socioeconomic barriers have impeded the implementation of HAART therapy in many developing nations. HIV
encephalitis/encephalopathy (HIVE) and vacuolar myelopathy are disorders that are thought to be directly related
to CNS HIV infection. Almost 40% of HIV-positive children develop HIVE, which is the most frequent HIV-specific
disease and tends to occur in the later stages of immune suppression (6). Clinically, HIVE is characterized by
developmental delay, apathy, seizures, and spastic quadriparesis (39). Grossly, HIVE brains may be atrophic.
Microscopically, the multinucleated giant cell is characteristic; it is thought to be of phagocytic lineage, expresses
HIV antigens, and harbors virus (97). Loosely aggregated microglia and glial cells (similar to microglial nodules)
and perivascular (at times vasculitic) inflammatory infiltrates may be seen and predominate in the deep cerebral
white, basal ganglia, and brainstem. Leukoencephalopathic features can be present and include white matter
myelin pallor and gliosis; there also may be degeneration of the corticospinal tracts. Somewhat characteristic of
pediatric AIDS brains are the angiocentric calcifications seen within basal ganglia and frontal white matter.
Fungal Infections
Fungal infections are largely restricted to those pediatric patients who are immunocompromised. Typically, these
pathogens gain access to the CNS through hematogenous dissemination, often via lung infection. CNS invasion
may be accompanied by only a sparse inflammatory reaction, which in part may be related to the patient's
immunosupression. The clinico-pathologic features of the most commonly encountered fungal pathogens are
summarized in Table 10-11. Other CNS fungal infections include Mucormycosis, Coccidiomycosis,
Blastomycosis, Histoplasmosis, and Chromoblastomycosis.
Parasitic Infections
Parasitic infections of the pediatric CNS are uncommon. Two of the most common, toxoplasmosis and
neurocysticercosis (NEC) are briefly described below. Other parasitic infections include cerebral malaria,
amoebic infections (e.g., Entamoeba histolytica, Nagleriafowleri, Acanthamoeba species),
neuroschistosomiasis, trypanosomiasis, and helminthic infections (e.g., Ecchinococcus granulosis).
Toxoplasmosis is caused by Toxoplasma gondii, an obligate intracellular protozoan. Cats are the definitive host,
and human infection is acquired via inadvertent ingestion of parasitic oocysts passed through feline feces.
Primary infection is essentially asymptomatic in the immunocompetent; although the immune system may prevent
the development of disease, the parasite is not eradicated, and lies dormant in muscle/brain cysts. Of particular
interest is the CNS toxoplasmal disease, which is congenital or occurs in the immunosuppressed. Congenital
toxoplasmosis results from transplacental spread of organisms primarily during initial maternal infection and
parasitemia. Like CMV, transmission of disease is most efficient during late gestation but more severe earlier on
(highest risk for severe disease is 10 to 24 weeks). Following birth, affected infants classically present with Sabin
tetrad, which includes seizures, chorioretinitis, cerebral calcifications, and HCP. The pathology of congenital
toxoplasmosis differs from the disease seen in
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older immunosuppressed individuals. Parasites proliferate in ependymal and periventricular regions,
disseminating widely from there. Ependymal destruction and gliosis result in obstructive HCP. There is
leptomeningeal, parenchymal, and perivascular inflammation, in addition to vascular thrombosis with secondary
coagulative necrosis with mineralization. Inflammation is chronic, and there is often microglial activation.
Encysted bradyzoites are more easily appreciated, whereas extracellular tachyzoites may be difficult to
distinguish from karyorrhectic nuclear debris; in these cases, IHC stains and EM help to highlight the parasites
(Figure 10-39). Toxoplasmosis disease related to immunosupression is associated with reactivation of a dormant
infection. Clinical presentation is variable but can include changes in mental status, features of increased ICP,
and focal neurologic deficits. Imaging usually reveals multiple ring enhancing lesions. Pathologically, areas of
hemorrhage and necrosis are often centered upon the basal ganglia. Microscopically, foci of coagulative
necrosis are surrounded by mononuclear and neutrophilic inflammation plus granulation tissue and
gliosis/microgliosis. Inflammation may also be perivascular. These pathologic changes depend in part on the
immune status of the host: greater degrees of suppression are associated with less inflammation and scarring.
Vascular damage with superimposed thrombosis is often present. Older lesions can become cystic.
Table 10-11 ▪ SUMMARY OF COMMON FUNGAL INFECTIONS
Fungal Clinical
Organism Morphology Source Presentation Pathology
Cryptococcus Narrow Pigeon Subacute to Thick gelatinous meninges and

neoformans budding yeast; excreta chronic soap bubble' deep gray matter
polysaccharide Inhaled meningitis lesions. Perivascular yeast
capsule accumulation. PAS-positive and
mucicarminepositive
Candida Pseudohyphae Endogenous Low-grade Cerebritis and microabscesses

albicans and yeast (e.g., Gl, meningitis in an ACA/MCA distribution.
GU, skin, PAS and Grocott methenamine
etc.; Silver (GMS)-positive
Aspergillus Acutely Soil Inhaled Hemorrhagic Hyphal angioinvasion ±

species branching infarction and granulomatous reaction. PAS-
(fumigatus septated abscess positive and GMS-positive
and flavus) hyphae formation
NEC is likely the most common parasitic CNS infection worldwide. Humans are the definitive host in the non-
CNS form of disease wherein larval forms residing in poorly cooked pork are ingested; thereafter, the larvae
mature into the adult tapeworms (Taenia solium), which reside in the GI tract. CNS disease occurs when
humans become the intermediate host after eating food contaminated with tapeworm eggs (or oocytes). Once
ingested, the eggs develop into larvae, which burrow through the GI tract wall and disseminate hematogenously
throughout the body (including the CNS
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and muscle). Numerous larval cysts may develop within the brain and often remain asymptomatic for years.
However, if the larvae are in eloquent brain or die, an intense inflammatory reaction may occur and herald the
parasites' presence via a myriad of often location-dependent specific neurologic signs and symptoms. Imaging
reveals 1 to 2 cm ring enhancing cystic lesions that may bear a calcified scolex. Microscopically, the larval cyst
wall contains three layers: outer/cuticular, middle/cellular, and inner/reticular/fibrillary components. Favorable
sections of the scolex may reveal parts of the four muscular suckers and or the double row of 22 to 32 hooklets
(i.e., teeth) (Figure 10-40). Once the larvae dies and begins to degenerate, a chronic inflammatory response
(including multinucleate giant cells, eosinophils, and neutrophils) ensues and a zone of granulation tissue may
eventually wall off the deceased larva, which in turn undergoes fibrosis and mineralization.
FIGURE 10-39 ▪ Toxoplasmosis. A: An encysted organism (bradyzoites) is accompanied by foamy macrophages
and coagulative necrosis. B: Immunohistochemistry against toxoplasmosis helps to highlight free-living tachyzoite
forms that mimic karyorrhectic debris on routine staining.
FIGURE 10-40 ▪ Neurocysticercosis (NEC). This viable larval form has been favorably sectioned and reveals the
characteristic hooklets of the scolex (arrow).
VASCULAR DISORDERS
Several pediatric CNS disorders are best considered within the vascular category. These include both congenital
and acquired disorders, and they may affect all pediatric age groups. In general, these cause an interruption of
blood supply (either global or localized), which results in ischemia and/or hemorrhage. The incidence of pediatric
CNS vascular disorders varies tremendously; likely the most commonly encountered entities are those affecting
premature infants [e.g., HIE and germinal matrix hemorrhage (GMH)]. The gray or white matter may be
preferential targets of damage.
Hypoxic-ischemic encephalopathy (HIE) is a common form of injury, especially in premature infants. HIE causes
a global insult (e.g., with septic shock or cardiac arrest). The clinical impact may be minimal or may lead to
profound neurologic impairment and even death. The areas of the brain that are susceptible to damage are age
dependent; premature infants suffer damage primarily in the deep gray matter (i.e., basal ganglia and thalamus),
while term infants and older children exhibit hippocampal and neocortical damage preferentially. However, these
general patterns are only guidelines and damage can be more widespread in all ages. Regional factors
associated with increased vulnerability include (a) high metabolic activity; (b) vascular watershed zones; and (c)
specific neurotransmitter receptor distributions (especially glutaminergic). These factors contribute to the general
concept of selective vulnerability, which dictates that certain areas of the CNS are preferentially susceptible to
certain injurious processes, such as hypoxia and ischemia. Grossly, there may be cerebral swelling, dusky gray
matter, and loss of normal gray-white junctions that eventually result in cerebral atrophy.
The microscopic CNS pathologic changes of HIE are age and region dependent. Neocortical damage may occur
in a variety of patterns. As with any HIE, the initial phase is one of cerebral swelling and edema that manifests as
parenchymal pallor and vacuolation. Acute neuronal death occurs within the first 24 hours. If mature neurons
contain ample cytoplasm (i.e., large pyramidal neurons), the latter will become brightly eosinophilic (Figure 10-
1A). The neuronal nucleus becomes pyknotic and angulated. However, if cells are small and immature (i.e., little
cytoplasm), evidence of HIE will be limited to nuclear fragmentation (i.e., karyorrhexis) (Figure 10-1B). If the
nucleus fragments into multiple rounded bodies, cell death may be considered apoptotic rather than necrotic.
Microglial activation is a common early occurrence, while foamy macrophages appear after a few days. Vascular
changes include early swelling of endothelia, while capillary proliferation occurs after a week. Reactive gliosis is
generally not apparent until 1 week after injury. Some have suggested that the premature brain cannot
demonstrate gliosis in the first half of gestational development; as such, this would facilitate the rough dating of
an in utero insult; however, exceptions to this rule clearly exist (101). Mineralization within neurons and
macrophages may be seen after 10 to 14 days. Notably, these changes are very similar to those of frank
infarction, although the latter typically involves all cell types within a vascular region, rather than individual
neurons. For example, vascular watershed zones are particularly susceptible to HIE, resulting in selective
neuronal necrosis, but if injury is more severe, complete parenchymal involvement occurs (i.e., watershed
infarction). Near-term infants may display a peri-Rolandic or rarely a columnar distribution of cortical damage.
The sulcal cortical depths are particularly susceptible to HIE, leading over time to deep sulcal atrophy and
superficial sparing; grossly, this pathology is termed ulegyria because of the “mushroomlike” appearance of the
affected gyrus.
The hippocampus is somewhat more resistant to the effects of HIE in premature infants, as compared to its more
classic involvement in older patients. If involved, however, acute neuronal cell death (i.e., red neurons) is most
often seen in Sommer sector (CA1) and the end folium (i.e., CA4), while CA2
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(the “dorsal resistant zone”) is largely spared. Later stages are characterized by neuronal loss and gliosis (i.e.,
hippocampal sclerosis). Infants less than 8 to 9 months may display a microglial reaction beneath the dentate
gyrus as a marker of HIE. The hippocampal subiculum is more commonly affected in premature infants with HIE;
although initially called pontosubicular necrosis (PSN) when seen in association with pontine damage, it has
been shown that the neuronal cell death is actually apoptotic and not necrotic (89).
HIE may preferentially affect the deep gray nuclei (including the basal ganglia and thalami), primarily in preterm
but also term infants. These nuclei display a high metabolic activity near term, which might underlie their
susceptibility to insult (110). Histologic changes are similar to those in the neocortex. As a response to injury, the
deep gray nuclei may display abnormal myelination, wherein oligodendroglia mistakenly invest reactive
astrocytic, rather than axonal processes. Grossly, these deep gray nuclei adopt a marbled appearance called
status marmoratus. Survivors with this type of damage may suffer from cerebral palsy.
The cerebellum is also commonly affected by HIE. Both the cortex and dentate nuclei are often damaged. While
Purkinje and dentate neurons die via necrosis, internal granule neurons are lost by apoptosis (39).
Overall, the brainstem is uncommonly affected by HIE (with the exception of the aforementioned PSN). While the
inferior olives may incur neuronal death, other changes are generally rare. Severe HIE may result in bilaterally
symmetric dorsal lower brainstem necrosis, which may explain the pathogenesis behind a subset of Moebius
syndrome cases (facial diplegia and bilateral abducens palsies). Only severe cases of HIE tend to affect the
entirety of the brainstem, and for that matter the spinal cord.
If HIE occurs during prenatal life, the pathways and cytoarchitecture of the developing brain may be significantly
disturbed. Accordingly, this may lead to secondary malformations (i.e., acquired and not congenital). The type of
malformation that results is GA dependent. It would be predicted that earlier insults result in more profound
malformations. Polymicrogyria and schizencephaly are thought to be the result of early damage, while some
forms of cortical dysplasia and hippocampal sclerosis are considered to be of late onset (39).
The white matter may be preferentially damaged in premature (and less so term) infants. The most acclaimed
member of this group of lesions is periventricular leukomalacia (PVL).
PVL is less frequently encountered than it was in the past, likely because of advances in prenatal care.
Premature infants between GAs of 24 and 35 weeks (peak age is 28 weeks) are most frequently affected.
Besides age, two of the most important risk factors include feto-maternal cardiorespiratory instability (e.g., fetal
cerebral hypoperfusion and immature cerebral autoregulatory mechanisms) and intrauterine infection (e.g.,
chorioamnionitis). These two factors may act synergistically to trigger an inflammatory response (in part
mediated by reactive astrocytes and activated microglia) that primarily targets the premyelinating
oligodendrocytes of the fetal brain via excitotoxic amino acid-based mechanisms, oxidative stress, and cytokine
cascades (116). Clinically these premature infants are generally “sick” (i.e., septic with unstable cardiac and
respiratory function), and neurologically, they may have weak legs and seizures. The more uncommonly affected
term infant often suffers from congestive heart disease or a congenital diaphragmatic hernia. Long-term sequelae
include cerebral palsy, cognitive deficits (mental retardation, learning deficits), behavioral abnormalities, and
epilepsy. Twenty-five percent of SIDS cases display evidence of PVL at autopsy. Genetic features of PVL are
not well understood.
Pathologically, PVL is defined by two features: (a) periventricular necrosis that may be cystic and mineralized
and (b) evidence for a more diffuse white matter gliosis (Figure 10-41). Some have conceptualized these two
lesional components as the vascular “core” and the “penumbra,” respectively. Adding further to the vascular
hypothesis is the suggestion that the periventricular areas of predilection likely represent a region of vascular
watershed during this age (26). Microscopically, periventricular white matter damage begins with the
development of coagulative necrosis of all cell types within the first 24 hours of the insult (all cells develop
nuclear pyknosis and eosinophilic cytoplasm). Axonal spheroids are seen on routine staining but can be further
highlighted with b-APP IHC. Activated microglia, elucidated with CD68 immunohistochemistry, are prominent in
early stages. Within the first week, macrophages infiltrate the areas of necrosis and are surrounded by early
reactive gliosis. Gross cavitation and mineralization (of axons) can be seen after a few weeks. Cystic spaces
collapse to form glial scars or areas of encephalomalacia. Destruction of these axonal processes undoubtedly
affects the development of the overlying neocortical gray matter and likely accounts for subsequent
cytoarchitectural abnormalities. Surrounding these areas of periventricular
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necrosis is a more subtle diffuse white matter gliosis that is relatively devoid of axonal pathology. However,
prominent loss of premyelinating oligodendrocytes from these areas leads to delayed and impaired myelination.
Since premature infants are mainly at risk, it is not uncommon to see coexistent germinal matrix hemorrhage
(GMH) (see below) and HIE.
FIGURE 10-41 ▪ Periventricular leukomalacia (PVL). Coronal section of the brain demonstrating cystic
abnormalities in the white matter; note the markedly thinned corpus callosum.
GMH is also characteristic of premature infants. Given its subependymal location, IVH is present in most
examples. Although in rare examples term infant may also experience GMH and IVH, the latter is more often a
result of choroid plexus hemorrhage, possibly related to congenital vascular malformation (see below). Young GA
is the most important risk factor; the incidence of GMH is inversely proportional to GA. Those infants less than 28
weeks are at the greatest risk for severe GMH (38). Other risk factor may include respiratory compromise (which
is interrelated with age), intrauterine growth retardation (IUGR), feto-maternal sepsis (e.g., related to
chorioamnionitis), hypothermia, intubation, and transportation between hospitals. Although the large veins of the
germinal matrix are the likely source of the hemorrhage, the exact pathogenesis of GMH remains unclear. The
germinal matrix is a major source of neuroglial precursors and persists until 34 weeks gestation (involution
occurs by 38 to 40 weeks gestation). Enhanced fibrinolytic activity is characteristic of the involuting matrix
making it susceptible to hemorrhage, in part due to the lack of sufficient parenchymal support of the matrix
vasculature and poor hemostasis. Hypoxic-ischemic injury of the germinal matrix may further impair the already
primitive autoregulatory capabilities of these vessels, making them vulnerable to fluctuations in CPP.
Clinically, GMH usually presents within the first 24 to 48 hours after birth. There may be a decreased level of
consciousness or irritability, a tense fontanelle, and seizure activity. Severe GMH is often fatal (38). Occult cases
of GMH are presumably of less clinical severity. Grading of the extent of GMH has been widely applied to
ultrasonography. Grade I is confined to the germinal matrix; grade II additionally includes IVH that in grade III
causes HCP; and finally, grade IV adds intraparenchymal extension of hemorrhage beyond the germinal matrix.
Higher grades of GMH correlate with greater degrees of long-term neurological disability (39). Grossly, the
appearance of GMH is in keeping with the aforementioned grading scheme (Figure 10-42). Extension of
ventricular blood out of the foramina of the fourth ventricle into the basal cisterns yields a subarachnoid
component that likely plays a role in chronic HCP. Areas of parenchyma adjacent to GMH may be necrotic (with
microscopic mineralization) and often show concomitant PVL. Microscopically, there are relatively few reactive
changes in the parenchyma.
A variety of acquired vascular disorders may be seen in children, many of which are rarely encountered by the
neuropathologist. Essentially, all of these disorders result in “stroke” (i.e., infarction). Risk factors for pediatric
stroke include diabetes, cardiac abnormalities (e.g., congenital and rheumatic heart disease, arrhythmias),
thrombophilias, hyperhomocysteinemia, hematologic conditions, trauma, drug use (e.g., smoking, amphetamines,
etc.), hypertension, obesity, and oral contraceptive use. Meningitis often leads to infarction via inflammation,
damage, and thrombosis of leptomeningeal vessels (i.e., secondary vasculitis). Clinically, pediatric and adult
stroke may present similarly (i.e., focal signs/symptoms and or more global neurologic impairment). Angiography
and diffusion/perfusion weight MRI are frequently used in the patient's workup. Microscopically, edema and
congestion precede acute neuronal cell death that is most readily visible by 24 hours. At 1 to 2 days, there is
infiltration of neutrophils (PMNs) and endothelial swelling. Within the first week, PMNs make way for
macrophages. Angiogenesis and reactive gliosis are seen by 2 weeks. Later stages are characterized by
neuronal loss, gliosis, and cystic degeneration. Well-recognized clinico-pathologic entities that have a relative
predilection for the CNS vasculature include Moyamoya disease, fibromuscular dysplasia, venous sinus
thrombosis, arterial dissection, vasculitides (e.g., Takayasu arteritis, primary angiitis of the CNS), and
vasculopathies (e.g., HIV vasculopathy). Many systemic disorders characteristically affect the large and small
blood vessels of the CNS including systemic lupus erythematosis, other collagen vascular disorders, sickle cell
disease, antiphospholipid antibody syndrome, fat emboli, thrombotic thrombocytopenic purpura, and hemolytic
uremic syndrome.
FIGURE 10-42 ▪ Bilateral GMH. Hemorrhage on left side has extended into the adjacent ventricular system and
out into the subarachnoid space (cisterna magna) overlying the cerebellum through the foramina of the fourth
ventricle (coronal section). (Image courtesy of Dr. Barry Rewcastle.)
Many of the congenital CNS vascular anomalies are also uncommonly seen. Berry (i.e., saccular) aneurysms are
extremely rare in young children. A defective internal elastic lamina may predispose to their formation over time
and thus accounts for the low prevalence in this population. These often present with massive and fatal SAH.
The key to their discovery is a careful dissection of blood and the circle of Willis in the fresh state when
structures are more manipulatable. Microscopically, the aneurysm wall is focally attenuated; the internal elastic
lamina and media are replaced by fibrous connective tissue, and possibly atherosclerosis plus hemosiderin.
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Vascular malformations include arterio-venous malformations (AVMs), cavernous hemangiomas (i.e., cavernous
angiomas or “cavernomas”), venous angiomas, and capillary telangectasias, the former two of which are more
commonly symptomatic. AVMs may present with hemorrhage or with ischemic signs and symptoms that relate to
arterio-venous shunting and vascular steal. Arterial feeders and draining veins are usually well appreciated
angiographically. Microscopically, there are arteries, veins, and “arterialized veins” of varying mural thickness
and caliber, often with entrapped fragments of gliotic brain between these abnormal vessels. Hybrid vessels
appear partly arterial and partly venous in favorable histologic sections. Staining of the internal elastic lamina
(e.g., Musto Moat, Verhoff van Gieson) assists in highlighting the arterial components. Recent and remote
hemorrhage may be seen in the abnormal vessels, as well as gliotic brain. Evidence of embolization may be seen
in the form of foreign material within the vascular lumina of the malformation. Cavernous angiomas are
essentially venous structures that present as mass lesions, which may cause hemorrhage, focal neurologic
deficits, or seizures. Gradient echo MRI sequences highlight these malformations. Microscopically, hyalinized
veins of various caliber are packed together in a back-to-back fashion, generally excluding intervening
parenchyma in most examples. Gliosis and signs of prior hemorrhage surround these abnormal blood vessels.
Vein of Galen aneurysms are actually arterio-venous fistulas that are associated with aneurysmal dilatation of
the vein of Galen. These are thought to arise early in gestation (between 6 and 11 weeks) (39). The posterior
cerebral artery is a frequent “feeder artery.” The most common clinical presentation is high output congestive
heart failure in a young child. Vascular steal may lead to atrophy and parenchymal necrosis (with dystrophic
calcification). Microscopically, feeder vessels are dilated and hypertrophic, while the “aneurysmal” vein similarly
displays a thickened wall. Vessels in the adjacent brain parenchyma may also be hyperplastic in response to
high pressure shunting.
FIGURE 10-43 ▪ MA characterized by a variably hyalinized, fibroblastlike perivascular spindle cell proliferation,
adjacent to normal-appearing or mildly dysmorphic cortical neurons.
Meningioangiomatosis (MA) is a form of meningovascular malformation or hamartoma occurring either in the

setting of NF2 or sporadically (77). The former is typically asymptomatic, whereas the latter usually presents
before adulthood with seizures and/or headache. This often plaque-like proliferation of meningothelial, smooth
muscle, and fibroblast-like spindled cells appears to extend down the Virchow-Robin spaces into the superficial
brain and invest blood vessels (Figure 10-43). The surrounding brain is gliotic and often displays dysmorphic
neurons, dystrophic calcification, and fibrosis. In contrast to pure MA, cases associated with an overlying
meningioma likely represent an unusual mimic with perivascular tumoral spread, rather than a true malformation.
Other CNS vascular anomalies include Fowler syndrome, meningocerebral angiodysplasia/renal agenesis, and
Sturge-Weber-Dimitri disease (i.e., encephalotrigeminal angiomatosis) (39).
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Chapter 11
Pediatric Ophthalmic Pathology
J. Douglas Cameron
INTRODUCTION
The observations and opinions of surgical pathologists are critical in managing many pediatric ocular conditions
including potentially fatal entities such as retinoblastoma and suspected nonaccidental trauma.
Because pediatric ophthalmic surgical specimens tend to be infrequent, this chapter includes a discussion of
pertinent ocular anatomy and pivotal events in embryologic development of the eye; the intention is to provide a
context for pathologic features. In addition, the type of surgical procedure used to obtain the tissue specimen is
described to assist in understanding the origin of the specimen and orientation of gross specimens.
This chapter is organized by the types of tissue most frequently received in the laboratory; eyelid tissue,
conjunctiva, cornea, vitreous, orbital soft tissues, whole globes removed surgically, and globes removed at
autopsy. Crystalline lens tissue removed because of congenital cataracts and extraocular muscle tissues
removed during some types of strabismus procedures are infrequently processed because histological
observations of this type of specimen are not relevant to management of the ocular abnormality.
THE NORMAL EYELID

Structure of the Eyelid
The eyelid is covered by stratified squamous epithelium associated with a thin keratin layer. The surface merges
with the mucous membrane at the mucocutaneous junction located on the eyelid margin (Figure 11-1). The
epidermis is associated with pilosebaceous units, eccrine glands, and apocrine glands. The apocrine glands
have no recognized function in the eyelid tissues. The cilia are the product of a modified pilosebaceous unit in
that there are no associated piloerector muscles and there is a prominent sebaceous component (glands of
Zeis). The tarsal plate is a dense collagenous structure that supports the delicate eyelid and houses a large
volume of sebaceous glands (the Meibomion glands). No cartilage is present in the eyelid. The holocrine
secretion of the Meibomion gland is applied to the tear film surface from pores located along the eyelid margin
anterior to the mucocutaneous junction and posterior to the eyelid cilia (38, 61).
SURGICAL PROCEDURES OF THE EYELID

An eyelid biopsy may be a simple removal of an ellipse of skin because of the suspicion of cutaneous neoplasm.
These specimens are handled as are cutaneous biopsies elsewhere. When a lesion involves the eyelid margin,
particularly near the punctum, the surgery becomes more complicated because scarring in the region of punctum
may cause lacrimal drainage abnormalities (chronic tearing, epiphora) and because scarring of the eyelid margin
may damage the cornea. Some type of superficial lamellar dissection in the region of the punctum may be done
to spare punctal function. For lid margin lesions a full-thickness wedge of eyelid is removed because restoration
of lid margin function is facilitated. A lid-splitting procedure removes the tissue either anterior or posterior to the
anterior border of the tarsal plate. Full-thickness and partial-thickness lid margin specimens are generally
oriented perpendicular to the lid margin (or row of cilia) with nasal and lateral surgical margins.
VASCULAR ABNORMALITIES OF THE EYELID

Capillary hemangioma is a benign proliferation of blood vessels of the soft tissue of the face that may involve
both eyelids and the orbit (57). The proliferation does not usually involve the contents of the globe. The
cutaneous lesions are red and lobulated, and may markedly distort the contours of the face (Figure 11-2). The
lesions are rarely biopsied but occasionally may be surgically debulked. Initially, there is capillary lesion in a
lobular pattern characterized by proliferation of endothelial cells around a small caliber vascular channel. The
lesion is not encapsulated and lobules of the hemangioma extend into the surrounding soft tissue. With time the
endothelial profile flattens and the lumen becomes
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more prominent as interstitial tissue develops. Intralesional steroids are sometimes injected to shrink the mass
and may be seen as amorphous material in the vascular lumen or in the interstitial space (128). The lesions
appear clinically at birth or 2 to 4 weeks after birth and may progress rapidly to produce soft-tissue enlargement
and mechanical ptosis of the eyelid. Obstruction of vision by mechanical ptosis may interfere with the
development of visual function (amblyopia) (77). The hemangioma may enlarge over the following several years.
The lesions tend to spontaneously involute generally by age 7 years. There may be vascular abnormalities
elsewhere in the body.
FIGURE 11-1▪Structure of the eyelid: The anterior surface of the eyelid is composed of stratified squamous
epithelium with a thin keratin layer. The keratinized surface merges with mucous membrane lining the posterior
surface of the eyelid at the mucocutaneous junction (arrow). Large pilosebaceous units represent the eyelashes
that lack a piloerector muscle. Meibomion gland secretion covers the surface of the tear film at the
mucocutaneous junction. (Hematoxylin-eosin stain, original magnification ×40).
Nevus flammeus is congenital vascular lesion in the distribution of the first and second divisions of the trigeminal
nerve (42). The vascular abnormality is present at birth and does not progress or regress. The cutaneous lesion
may be treated with laser but is generally not biopsied (117). It is clinically important because this vascular
malformation is associated with ipsilateral glaucoma and ipsilateral choroidal hemangioma. Glaucoma, when
present, is treated as are cases of glaucoma from other causes. The choroidal hemangioma is very difficult to
treat and may progress to serious retinal detachment, a potential cause of loss of vision (98, 133).
FIGURE 11-2▪Capillary hemangiomas in the periorbital soft tissue of the face cause dysfunction of the eyelid
because of mechanical ptosis. If the eye is not stimulated by formed images the retinal function will not develop
(amblyopia).
INFLAMMATORY ABNORMALITIES OF THE EYELID

Pyogenic granuloma refers to a polypoid lobular capillary hemangioma of the skin in the discipline of
dermatopathology. This term is used by clinical ophthalmologists to describe a granulation tissue reaction usually
located in the tarsal conjunctiva, which is a response to mechanical trauma or to the presence of a chalazion
(Figure 11-3) (37). A reddish lobulated mass develops on the conjunctival surface that may be large enough to
protrude through the interpalpebral fissure. The area is excised if the mass interferes with the surface lubrication
of the eye from malpositioning of the eyelid margin. The overlying mucous membrane may show effects of drying
and reactive proliferation. In the subepithelial tissue there are acute and chronic inflammatory reactions
associated with multiple delicate vascular channels and an edematous stroma (granulation tissue). Conjunctival
pyogenic granuloma usually will spontaneously involute over days to weeks.
Chalazion is a granulomatous reaction to sebaceous products of the Meibomion gland of the eyelid. The gland
becomes occluded and ruptures into the surrounding soft tissue. The affected area is initially tender but evolves
into a firm nontender nodule. In cases of exceptional size, an incision and curettage is performed. The tissue is
usually friable
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amorphous gray tissue composed of a lipogranulomatous reaction with foreign body and occasional Langhans-
type giant cells (Figure 11-4). The surrounding normal tissue is usually not represented in the specimen,
however, infiltration among the fibers of the orbicularis muscle may be observed occasionally.
FIGURE 11-3▪Pyogenic granuloma is a clinical term used by ophthalmologists to refer to a transient fibrovascular
response in a mucous membrane (the conjunctiva). With remodeling normally found in the repair process the
lesion will spontaneously diminish over time. The lesion is occasionally removed if it causes symptoms related to
eyelid dysfunction.
FIGURE 11-4▪Chalazion is a lipogranulomatous reaction to a lipid globule (white arrow) representing sebaceous
secretion from a ruptured Meibomion gland of the eyelid (black arrow). The rupture is thought to be the result of
blockage of the outlet mechanism of the gland. The tissue submitted will often be limited to the contents of the
chalazion (Hematoxylin-eosin stain, original magnification ×100).
Generally, the nodule resolves over weeks or months. In some individuals, there may be multiple episodes in
different locations in the eyelid. In children, chalazion may cause refractive error and pose a risk of amblyopia if
persistent (4). In adults, a coexisting sebaceous carcinoma may be present.
Juvenile xanthogranuloma (nevoxanthoendothelioma) is a non-Langerhans’ cell histiocytosis. Well-defined
purple to red nodules appear on the skin and the anterior surface of the eye (24, 135). The reaction may also
appear in the orbit and in the uveal tract within the eye. Lesions in the iris may cause spontaneous hyphema,
which may be bilateral (22). The cutaneous lesions are characterized by a mononuclear lipidized or non-lipidized
cells and Touton giant cells. The lesions tend to involute spontaneously. Occasionally, intraocular lesions cause
intractable glaucoma requiring enucleation. The mononuclear cells are usually limited to the uveal tract but may
involve adjacent structures as well. This type of proliferation may occur in adults as well as in children.
Molluscum contageosum is a poxvirus infection of the epithelium of the skin. Multiple, well-demarcated,
elevated, umbilicated, nontender nodules develop on the skin. Shedding of viral particles from infected epithelial
cells near the lid margin may produce a localized, persistent, follicular conjunctivitis, which brings the patient to
medical attention (113). This infection is often found associated with immune deficiency (23). The lesion consists
of acanthotic stratified squamous epithelial cells with prominent intracytoplasmic inclusions (Figure 11-5). The
contents of infected cells desquamate into the environment from the umbilicated region. Conjunctival follicular
reaction from Molluscum contageosum is difficult to treat medically. Lid margin lesions often require surgical
excision.
FIGURE 11-5▪Molluscum contageosum consists of virus-infected cells (white arrow) of the eyelid margin that
may cause regional conjunctivitis because of exfoliation of infected cells and debris (black arrow) (Hematoxylin-
eosin stain, original magnification ×20).
A sty or hordeolum is an abscess of one of the adnexal units of the eyelid skin. This condition is very infrequent
and is generally not biopsied. An external hordeolum is superficial and an internal hordeolum is located deeper in
the eyelid skin. In most cases, a lesion described clinically as a sty may actually be a chalazion (see above)
(108).
Preseptal cellulitis is a bacterial infection of the subcutaneous tissue of the eyelid anterior to the orbital septum.
The orbital septum is a fibrous diaphragm extending from the periosteum of the orbital rim to the eyelid margin.
Its major function is to compartmentalize and protect orbital fat from external influences. Preseptal cellulitis may
present with a marked increase in soft-tissue volume. If the inflammatory reaction or infection extends posterior to
the orbital septum (orbital cellulitis), there is swelling of intraorbial tissue forcing the globe to move anteriorly
(proptosis or exophthalmoses). The malposition of the globe as well as direct inflammation of the extraocular
muscle may cause limitation of excursion of the globe (ophthalmoparesis) resulting in double vision (diplopia).
The infected tissue is rarely biopsied, although fine needle aspirations may be used for culturing
microorganisms. The most common organisms found are Haemophilus influenza and Streptococcus species (31,
76). Treatment is with systemic antibiotics.
NEOPLASTIC LESIONS OF THE EYELID SKIN

Melanocytic nevus is a proliferation of abnormal melanocytes at the dermal-epidermal junction. Clinically, the
lesions appear as hyperpigmented areas of the skin that vary in degree and extent of pigmentation (see Chapter
25).
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Spindle cell and epithelioid nevus (Spitz nevus) is a proliferation of melanocytes that has many histological
features of melanoma even though the clinical course is usually benign. The lesion may present as a rapidly
enlarging, welldemarcated nodule of the eyelid skin (60). Histological patterns include spindle and epithelioid cell
as well as mixed types. The mitotic rate is usually low, and actively dividing cells are generally located near the
superficial dermis. The size of the melanocytes usually diminishes toward the base of the lesion. Regional lymph
node metastasis has been reported; however, even in those cases the long-term course remains favorable.
Cutaneous malignant melanoma of the skin may arise in childhood (18).
Oculodermal melanocytosis is congenital hyperpigmentation of the skin associated with hyperpigmentation of
the ipsilateral episcleral tissue located deep to the conjunctiva. Hyperpigmentation is present at birth and
generally remains stationary in intensity and extent throughout the life of the individual. The lesions may become
more prominent during puberty (120). An increased concentration of typical melanocytes is located at the dermal-
epidermal junction and in the episcleral tissue. There is minimal risk of malignant transformation of cutaneous
and episcleral melanocytes. There is an increased risk for ipsilateral uveal melanoma, particularly for
Caucasians affected with the problem. Malignant transformation may also occur in the nevus itself as well as
orbit, optic nerve, and brain (97).
Xeroderma pigmentosa is an autosomal recessive defect in the DNA repair systems of the body. Exposure to
ultraviolet light induces the formation of basal cell carcinoma, squamous cell carcinoma, and malignant
melanoma in the facial skin of even young children. The number of lesions is characteristically large. Children
may also develop squamous cell carcinoma of the conjunctiva as well as pterygia. Ocular surface scarring from
these lesions may significantly affect visual function (51, 85). There is no treatment to replace the deficient
DNAase that normally repairs ultraviolet-damaged DNA. Treatment of individual lesions is accomplished by
standard surgical therapy. Prevention is attempted by limiting exposure of the facial skin to sun light.
Basal cell nevus syndrome (Gorlin-Goltz) is an autosomal dominant condition associated with the development
of basal cell carcinoma at multiple sites. Basal cell carcinoma of the eyelid has been observed in a 16-year old.
The eyelid lesions tend to be aggressive and may involve the orbit (63). In addition to cutaneous malignancies
there are associated skeletal abnormalities such as odontogenic cysts of the jaw and bifid ribs. Palmar and
plantar pits as well as mental retardation and intracranial calcifications may be present. As with other ectodermal
dysplasia syndromes, the ocular surface may be abnormal due to meibomion gland dysfunction. Degenerative
pannus may be associated with loss of vision (68).
Neurofibromatosis type I (NF-1) is an autosomal dominant condition in which various types of abnormally
produced cytokines lead to the development of neoplasia of various types. The syndrome is recognized by the
presence of hyperpigmented cutaneous regions with a smooth contour (Café-au-lait) spots and proliferation of
elements of peripheral nerve (neurofibroma) within the eyelid skin. The neurofibromas are acquired and consist
of nodular or plexiform patterns. The nodular form generally does not affect eyelid function. The plexiform variety
may produce massive enlargement of the soft tissues of the face including the eyelid, causing major
deformations of the eyelid margin (ectropion or entropion). The cornea depends on normal eyelid function to
maintain corneal clarity. Corneal scarring can result from eyelid distortions. Surgical debulking of lesions is
occasionally performed to improve eyelid function, which is often only partially successful (34). The lesion
consists of proliferation of all cellular elements of the peripheral nerve including axons, Schwann cells, and
perineural cells. Occasionally, the native peripheral nerve trunk can be identified. The lesions of plexiform
deformity are often progressive. In this tissue as well as elsewhere, there is a risk of developing malignant
peripheral nerve sheath tumors.
Optic pathway glioma may be found in 15% to 20% of individuals with NF-1 and may account for significant
morbidity in young children. Symptoms include vision loss, proptosis, and precocious puberty (34, 79). Globe
enlargement and glaucoma have been reported on the ipsilateral side oforbito-facialNF-1 (95). The association
of NF-1 and uveal melanoma appears to be coincidental (64) (see Chapter 10).
THE PEDIATRIC CONJUNCTIVA

Structure of the Conjunctiva
The conjunctiva extends from the eyelid margin to the junction of the cornea and sclera (the limbus). The
conjunctiva is a mucous membrane with many specialized regions. Along the internal lining of the eyelid (the
tarsal conjunctiva) the surface is tightly adherent to the tarsal plate. There is redundant conjunctiva at the
forniceal regions of the eyelid to allow full mobility of the globe. The conjunctiva over the globe (bulbar
conjunctiva) is loosely applied. The associated accessory lacrimal tissue is regional and clinically inconspicuous.
There is associated nonnodal lymphoid tissue in the subepithelial space, particularly in the region of the fornix.
The conjunctival surface is composed of stratified, nonkeratinizing epithelium containing a variable number of
intraepithelial goblet cells found most prominently in the bulbar conjunctiva (Figure 11-6A,B). Dendritic
melanocytes and antigen-processing cells (Langerhans cells) are present throughout the surface epithelium. The
underlying tissue is nonspecific, delicate fibrovascular tissue (substancia propria). The substancia propria of the
conjunctiva fuses with the fibrovascular tissue of the globe (episcleral tissue and Tenon capsule) at the limbus
but is otherwise distinct and separate. Lymphatic channels are present throughout the substancia propria of the
conjunctiva to the limbus where they form arcades. The limbus is one of the locations of stem cells
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and is a common site for the development of both squamous cell carcinoma and malignant melanoma. Lymphatic
channels of the conjunctiva drain to the preauricular, parotid, and submental nodes (38, 61).
FIGURE 11-6▪A: The normal conjunctiva is composed of nonkeratinizing squamous epithelium containing goblet
cells (arrow). Goblet cells are concentrated in the bulbar conjunctiva (Hematoxylin-eosin stain, original
magnification ×100). B: There is normally a nonnodal collection of lymphocytes within the stroma of the
conjunctiva, particularly in the far periphery (conjunctival fornix) (Hematoxylin-eosin stain, original magnification
×100).
SURGICAL PROCEDURES OF THE CONJUNCTIVA

The repair process of the conjunctiva results in scarring that may restrict movement of the globe and may result
in a cosmetically unacceptable appearance. Therefore, biopsies of the conjunctiva are generally limited in extent
even in the presence of a suspected malignancy. Most biopsy sites of the conjunctiva are described in reference
to the limbus (e.g., the specimen is from the 3 o’clock position of the right eye and extended nasally). Biopsies for
the diagnosis of systemic disease (e.g., sarcoidosis) are often performed in the inferior fornix where there is a
normal high density of resting lymphocytes. Surgical access to the eye (cataract incision, orbital biopsy) and for
strabismus procedures is through the conjunctiva. Inclusion cysts may arise at a suture line if the wound margins
are not precisely apposed. Inclusion cysts may be removed by a second procedure to improve cosmetic
appearance. Conjunctival tissue is essentially a nonrenewable resource, making the surgeon hesitant to remove
any more tissue than is absolutely necessary. Histopathologic interpretation of biopsy is often difficult because
this tissue, which may harbor potentially serious disease (melanoma, lymphoma, rhabdomyosarcoma), is delicate
and can easily be crushed with the forceps; furthermore the samples are usually small. Adding to the processing
problem is the tendency of the conjunctival tissue to curl or deform, if immersion fixed in formalin. The optimal
method of submission is to fix the conjunctiva after the tissue has been flattened on support media such as filter
paper. Many tumors of significance arise at the limbus (the junction of cornea and sclera). Therefore, the tissue
sections are usually oriented perpendicular to the limbus. Close communication with the surgeon is necessary to
establish tissue margins of significance.
INFLAMMATORY CONDITIONS OF THE CONJUNCTIVA

Microbial infections of the conjunctiva due to bacteria, viruses, and fungi occur commonly but are rarely
biopsied. Trachoma remains a worldwide cause of significant blindness that results from infection with
Chlamydia trachomatis. Early stages of the disease are characterized by an indolent follicular conjunctivitis. Late
in the evolution of the disease, superficial scarring of the tarsal conjunctiva deforms the eyelid orientation
(entropion) to allow eyelashes (cilia) to come in contact with and damage the superficial structure of the cornea.
Blindness from trachoma results from the corneal scarring and not from the conjunctival infection. Treatment of
established cases is surgical (132).
Ligneous conjunctivitis is an accumulation of fibrin in the subepithelial space of mucous membranes throughout
the body, caused by a systemic reduction in the levels of plasminogen (90, 136). The condition usually presents
in young females due to conjunctival symptoms (itching, burning, decreased vision) because of the presence of
subconjunctival nodules composed of a woody-like accumulation of fibrin (114). The overlying epithelium is
usually unremarkable, although signs of drying (epithelial thinning, reactive keratinization) may be present.
Amorphous fibrin sometimes associated with an acute or chronic nongranulomatous inflammatory infiltrate may
be present. Topical plasminogen concentrate has been used for treatment (90). Ligneous conjunctivitis may be
associated with congenital occlusive hydrocephalus and juvenile colloid milium (114).
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DEVELOPMENTAL ABNORMALITIES OF THE CONJUNCTIVA
Developmental abnormalities of the conjunctiva are infrequent. Occasionally, redundant, tortuous, dilated
lymphatic vessels (lymphangiectasia) are present that may lead to symptoms because of dryness of elevated
portions of the tissue or because of hemorrhage into the lymphatic spaces.
Episcleral osseous choristoma is due to embryonic rests of bone in the episcleral tissue, which may present as a
stationary nodule often in the upper temporal quadrant of the conjunctiva or of the lower eyelid (50). The lesion
generally consists of mature bone surrounded by mature fibrous tissue.
Limbal dermoid of the conjunctiva is a choristomatous nodule of dermal tissue, usually located at the limbus.
The nodule may also be situated on the central corneal surface with only a minimal connection with the vascular
system of the conjunctiva. The mass is an obstruction to vision light and alters the contour of the cornea. Without
treatment, the lack of symmetric vision in a child can lead to amblyopia (failure of physiologic development of
vision, “lazy eye”) (12). The surface is nonkeratinizing squamous epithelium overlying dermal elements including
mature fat. The lesion may involve the full thickness of the cornea and sclera but does not involve intraocular
structures (93) (Figure 11-7). The lesion is usually solid without cystic elements as are found in cystic dermoid of
the orbit (see below). Surgical removal may not result in normalization of corneal curvature.
Neuronal ceroid lipofuscinosis is a group of neurodegenerative diseases inherited in an autosomal recessive
pattern that results in accumulation of lipopigments within cells and consequently with disruption of cellular
function (94). The conjunctiva is considered a convenient site for diagnostic biopsy. Intracellular “curvilinear
bodies and fingerprint bodies” are found by examination with transmission electron microscopy (126) (Figure 11-
8A,B) (see Chapters 5 and 10).
FIGURE 11-7▪Limbal dermoid, light micrograph. A: Solid dermoid is present at the limbus (junction of cornea and
sclera) involving the eye of a child, which was enucleated for other reasons. The conjunctiva is markedly
thickened (between two gray arrows) (Hematoxylin-eosin stain, original magnification ×20). B: Mature
pilosebaceous units as well as eccrine and apocrine glands (black arrow) are present. The lesion tends to
remain stationary in size and location. If the opacity involves the central visual axis retinal function may not
develop in a normal manner (amblyopia) (Hematoxylin-eosin stain, ×40 original magnification).
MELANOCYTIC ABNORMALITIES OF THE CONJUNCTIVA

Melanosis of the conjunctiva is recognized clinically as hyperpigmentation of the conjunctiva without alteration of
the surface contour of the conjunctiva. The lesion is present at birth or develops in early childhood as a yellow-
brown to brownish black discoloration. There is a larger than average number of typical melanocytes and a
higher than average accumulation of melanin in the conjunctival epithelium basal layers. This lesion is not a
precursor for melanoma.
Melanosis of the episclera and scleral tissue is visible melanosis of the tissues deep to the conjunctiva,
although the normal, transparent conjunctiva is visible as an area of slate gray discoloration. Heterochromia iridis
and hyperpigmentation of the uveal tract may be present. Hyperpigmentation may extend to the meninges of the
optic nerve and the brain. The contour of the overlying conjunctiva is not altered. The lesion may also present at
birth and is generally stationary. A larger than average number of melanocytes is present as individual cells or
small clusters interspersed in connective tissue. There is an increase in the number of melanocytes in the uveal
tract; these melanocytes are larger than the indigenous melanocytes. The melanocytes are relatively
hyperpigmented (Figure 11-9A,B). This lesion is a risk factor for melanoma, but the melanoma arises in the
ipsilateral uveal tract or deep orbital tissues but not in the conjunctiva.
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FIGURE 11-8▪ A: Neuronal lipofuscinosis is a neurodegenerative disease resulting in pigment degeneration of
the retina (arrow and arrow head). B: The conjunctiva may be used as a biopsy site to confirm the diagnosis by
transmission electron microscopy. Within affected cells curvilinear and fingerprint bodies are recognized
(arrows).
Nevus of Ota is a risk factor for ipsilateral uveal and orbital melanoma, particularly if it occurs in Caucasians. In
addition to the hyperpigmentation of the eye (melanosis oculi) and orbit, there is hyperpigmentation of the skin of
the eyelids and periorbital facial skin. Meningeal melanocytoma has also been associated with the nevus of Ota
(104).
Acquired melanosis of the conjunctiva may be a characteristic of aging in races with high-density melanin
pigmentation of the skin. Onset is well beyond the pediatric age and is generally bilateral and indolent. Primary
acquired melanosis (PAM) of the conjunctiva primarily involves women. PAM in races with low-density melanin
pigmentation is a risk factor for conjunctival melanoma, if the condition is unilateral with the onset in middle age.
The risk is greatest if atypia of the abnormal melanocytes is present. This condition has not been reported to
involve the pediatric age group (39).
FIGURE 11-9▪ A: Melanosis oculi may be associated with hyperpigmentation of the uveal tract (black arrow) as
well the episcleral surface (gray arrow). The uveal pigmentation is a clinical risk factor for the development of
uveal melanoma (Hematoxylin-eosin A ×40 original magnification). B: The hyperpigmentation of the episcleral
fibrous tissue is clearly visible clinically but subtle histopathologically (gray arrow) (Hematoxylin-eosin stain,
original magnification ×200).
Melanocytic nevus of the conjunctiva is an accumulation of abnormal nevus cells in the region of the conjunctival
epithelium. Early in life, in the junctional nevus stage, the nevus is a relatively well-demarcated area of the
conjunctiva, which may not alter the surface contour and may be amelanotic or lightly pigmented. Conjunctival
nevi are often
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associated with an anomalous development of the conjunctival epithelium, where the conjunctival epithelium is
drawn into the substancia propria of the conjunctiva to form solid nests of squamous epithelium or cysts lined by
squamous epithelium (Figure 11-10). The cysts may alter the surface contour of the conjunctiva, particularly if
the cystic lining contains goblet cells or accessory lacrimal tissue that secretes into the lumen of the cysts. The
melanocytes may be extensively pleomorphic, ranging from spindle-shaped cells to epithelioid cells. These
atypical melanocytes may occur in the epithelium of the inclusion cysts giving the false impression of lymphatic
spread of a melanoma. Clusters of melanocytic nevus cells may indent the lining of lymphatic channels also
giving the appearance of possible distant spread (41).
FIGURE 11-10▪ A: Conjunctival nevus. The surface stratified squamous cells are somewhat flattened. Multiple
cysts lined by squamous epithelial cells are located among nests of nevus cells in the subepithelial tissue.
Material accumulating in the cystic spaces may simulate growth by clinical appearance (Hematoxylin-eosin stain,
original magnification ×100). B: Aggregates of melanocytic nevus cells may indent the lining of lymphocytic
channels in the conjunctival stroma suggesting lymphatic invasion and possible metastasis. The appearance of
the cells is bland and the aggregates are covered by lymphatic endothelial cells (Hematoxylin-eosin, original
magnification ×400).
Later in the natural history of a conjunctival melanocytic nevus, a dermal component develops (compound
nevus). At puberty, there may be proliferation of melanocytes and increased density of pigmentation creating
concern about the presence of a conjunctival melanoma. The nevus may appear to enlarge because of
simultaneous proliferation of the squamous epithelial component of the inclusion cysts and increasing volume of
the contents of the cyst (137). Irritation from drying of the elevated surface of the conjunctiva may also add to the
impression of growth due to reactive inflammation and vascularization.
Melanoma of the conjunctiva is the possibility of melanoma arising in a preexisting nevus or de novo even
though PAM with atypia does not generally occur in the pediatric age group. A review of the international
literature by Taban found 28 reported cases in individuals under the age of 15 years (125, 137).
A conjunctival melanoma is an atypical proliferation of conjunctival melanocytes that has the potential of wide
spread metastasis and death. In the presence of a preexisting nevus or in the absence of histological signs of a
preexisting nevus there is invasion of the substancia propria of the conjunctiva by atypical melanocytes.
Features of malignancy include the presence of mitotic figures, atypical melanocytes in clusters, lack of the
expected maturation with depth, and infiltrative growth at the deep margin. Cytological features such as a
spindle-shaped appearance of the cells is of no prognostic significance (87).
Squamous carcinoma of the conjunctiva is usually associated with exposure to ultraviolet light in middle-aged or
elderly persons (99). The lesion may present as a papilloma, a gelatinous lesion, or as a leukoplakic mass of the
conjunctiva. The initial histopathologic findings are atypia progressing to carcinoma in situ. The lesions are
invasive if the underlying basement membrane is breached. Squamous cell carcinoma of the conjunctiva is
usually indolent but may spread to regional nodes. Spindle cell and mucoepidermoid variants tend to be more
aggressive and may invade the eye itself. Squamous cell carcinoma of the conjunctiva rarely involves the
pediatric age group, and is seen in xeroderma pigmentosum.
THE PEDIATRIC CORNEA

Structure of the Cornea
The cornea is the dominant element of optical system of the eye, providing up to 80% of its refracting power. The
power of the cornea is determined by its curvature, a feature that is highly conserved throughout life. The cornea
is composed primarily of extracellular matrix (the corneal stroma). The principal component of the corneal stroma
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is uniform type I collagen bundles separated at a precise distance by highly specialized and uniform
proteoglycans. The interfiber distance is determined by the degree of hydration of the proteoglycans (Figure 11-
11). The anterior surface of the cornea is composed of extraordinarily homogeneous nonkeratinizing squamous
epithelium. The corneal epithelium does not contain goblet cells or antigen-processing cells. The normal
basement membrane of the corneal epithelium is not visible by light microcopy. It rests on an acellular band of
dense type I collagen (Bowman membrane) that is found only in primates and birds. Bowman membrane
probably functions in maintaining corneal curvature, does not thicken with age, and is not restored if damaged by
pathologic processes (Figure 11-12). The corneal endothelium is derived from neural crest and not mesoderm
and therefore, is not stained by vascular endothelial markers (e.g., factor VIII). The corneal endothelium
produces a thick basement membrane (Descemet membrane) that is not physically attached to the corneal
stroma and thickens continuously throughout life (38, 61).
FIGURE 11-11▪Normal cornea. The surface epithelium is stratified, nonkeratinized squamous epithelium that
does not contain goblet cells. The epithelial basement membrane is not visible by light microscopy when the
epithelium is normal. The corneal stroma is composed of uniform collagenous fibers regularly separated by
proteoglycans. The splitting artifact of the stroma between collagenous lamellae is a normal finding. Lack of the
splitting artifact correlates with clinical corneal edema. Descemet membrane continues to thicken throughout life.
There are no firm architectural attachments between Descemet membrane and corneal stroma and Descemet
membrane and corneal endothelial cells. Corneal endothelial cells are similar to mesothelial cells of the pleura
and maintain corneal hydration (Periodic acid/Schiff stain; original magnifications ×20).
FIGURE 11-12▪Normal corneal anterior surface. The corneal surface is nonkeratinized and is composed of
extremely uniform cells. The epithelial basement membrane is not visible by light microscopy when the epithelium
is normal black arrow). Bowman layer is acellular type I collagen that is not replaced if damaged (white arrow)
(Periodic acid/Schiff stain, original magnification ×200).
SURGICAL PROCEDURES OF THE CORNEA

Biopsy of the cornea is performed infrequently except in the case of infection (fungus and acanthamoeba) that is
resistant to therapy. Repair processes of the cornea result in loss of transparency and therefore, biopsies are
extremely small and originate as far away from the visual axis (center of the cornea) as possible. Most of the
biopsy specimens, in the case of suspected fungus, will be submitted for culture; however, histopathologic
evaluation in cases of suspected acanthamoeba is more likely to be diagnostic than microbiologic studies.
Classically full-thickness penetrating keratoplasty has been the most common method of treating disease-
damaged corneas. A trephine, usually 7.5 mm in diameter, is used to create an incision through 90% of the
thickness of the cornea. The incision is completed with scissors. The entire specimen is submitted only after the
donor graft is in place and secured. Drying artifacts of the host cornea may accumulate during the interval. The
most common indications for penetrating keratoplasty in the pediatric age group are keratoconus and opacity
from corneal trauma. More recently surgical techniques have been developed in which only the specific layer of
the cornea, which has been altered by disease, is removed [deep lamellar keratoplasty (DLK)]. Currently, the
most common use of this procedure is for the removal of the diseased posterior corneal stroma (e.g., Descemet
membrane and endothelium in Fuchs endothelial dystrophy). The diseased host tissue is replaced by a similar
donor graft tissue. Surgical treatment of Fuchs endothelial dystrophy is not usually performed in the pediatric age
group. Surface damage of the cornea at any age may be treated with anterior lamellar keratoplasty. The surgical
procedure and problems
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associated with visual recovery limit the use of this procedure in children.
The object of refractive corneal surgery is to change the optical qualities of the cornea. Initially, the strategy was
to flatten the central cornea by weakening the peripheral cornea (radial keratotomy). Currently various methods
are used to decrease corneal thickness [photorefractive keratectomy (PRK) and laser in situ keratomileusis
(LASIK)] and thus, reduce the refractive error of myopia (nearsightedness, the eye is too long). Because the
optical system of the eye is not stable until age 18 to 22 years, these procedures are generally not performed in
children.
DEVELOPMENTAL ABNORMALITIES OF THE CORNEA

There is a small range of tolerance of corneal diameter in which the normal optical properties of the cornea
function. Similarly, the radius of curvature must be precise in order to focus light on the retina.
Microcornea is a condition in which the cornea is less than 9 mm in diameter (horizontal limbus to limbus) at 1
year of age. Other anatomic abnormalities such as microphthalmos with cyst or persistent hyperplastic primary
vitreous (PHPV) often coexist. Even nonsyndromic-associated abnormalities of the trabecular meshwork are
likely to cause glaucoma. The cornea tissue generally has a normal histological appearance except in the case
of Peters syndrome (see following).
Megalocornea is a condition in which the cornea is greater than 11.5 mm in diameter (horizontal limbus to
limbus) at 1 year of age. If the megalocornea is acquired and unilateral, then distortion by intraocular pressure
(buphthalmos of congenital glaucoma) may be the cause of enlargement rather than developmental process.
Developmental abnormalities are generally stationary and tend to be bilateral. Megalocornea may be associated
with ectopia lentis and other abnormalities of the anterior segment. The structure of the cornea in megalocornea
is generally normal.
Cornea plana refers to a flattened corneal contour (decreased radius of curvature). The associated decrease in
axial length often results in severe degrees of hyperopia (farsightedness) (44). The corneal structure may have
characteristics of nonuniform sclera rather than uniform cornea and be opaque (129).
Sclerocornea is the result of failure in the development of the unique homogeneous architecture of the cornea.
The corneal tissue resembles sclera both in radius of curvature (cornea plana) and in characteristics of
extracellular matrix.
Peters anomaly most likely results from failure of separation of the cornea from the crystalline lens during
embryonic development. In most cases the central posterior corneal stroma, Descemet membrane and central
corneal endothelium are absent. Rupture of Descemet membrane (Haab stria), during forceps delivery or with
corneal enlargement from glaucoma (buphthalmos), may have a similar histological appearance.
INFLAMMATORY CONDITIONS OF THE CORNEA

Herpes Simplex Keratitis
The herpes simplex virus initially affects the body as a systemic infectious disease with the cutaneous
expression being a vesicular dermatitis. Live virus is retained in the Gasserian ganglion and, for undetermined
reasons, will periodically travel via sensory peripheral nerve to infect the corneal epithelium. Cytopathologic
effects of the infected cells are seen as a linear, branching ulcer of the corneal epithelium (dendritic figure). With
repeated episodes of infection, the corneal stroma becomes involved, not with direct viral infection but with
lymphocytic infiltration, peripheral vascularization, and proteolysis of the extracellular matrix (discoid herpes
keratitis, herpes metaherpetica). The cornea can thin to the point of rupture. There may or may not be an intact
epithelial covering. Bowman membrane ulcerates and Descemet membrane ruptures resulting in perforation of
the cornea. A foreign body granulomatous response to the severed ends of either or both Bowman membrane
and Descemet membrane is a unique response in herpes simplex keratitis. There may be an extensive
lymphocytic infiltrate in the keratoplasty specimen that may not be appreciated clinically. The degree of
vascularization of the cornea is a risk factor for immunologic corneal rejection (119) (see Chapter 6).
Acanthamoeba Keratitis
Acanthamoeba is a protozoa commonly found in soil and water. The organism can gain access to the cornea via
microabrasions often associated with wearing contact lenses (17). The organism is neurotropic accounting for
the extreme pain associated with infection. Organisms can be identified clinically by using confocal microscopy
(35). The trophozoite form is motile and is able to spread extensively throughout the corneal stroma creating
necrotizing keratitis and scarring. The encysted form can be identified throughout the cornea with most
commonly used stains (Figure 11-13). There
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may be a limited inflammatory response because of topical treatment. Acanthamoeba is resistant to most forms of
therapy (29). Occasionally, biopsy for diagnosis or penetrating keratoplasty for advanced stages of the infection
is performed.
FIGURE 11-13▪Acanthamoeba keratitis. Multiple encysted acanthamoeba organisms are present throughout the
corneal stroma (Periodic acid/Schiff, original magnification ×200).
DYSTROPHIC CONDITIONS OF THE CORNEA

Corneal dystrophies are metabolic abnormalities of the cornea that cause clinically detectable opacities. The
prevalence of corneal dystrophy is extremely low. Dystrophies are mostly inherited (usually in an autosomal
dominant pattern, except for macular corneal dystrophy), bilateral, usually symmetric, progressive (at markedly
variable rates), and recur in corneal grafted tissue. Recently, multiple clinical entities that were thought to be
distinct from each other have been found to have a common genetic defect located at 5q31 (62, 71, 74, 124).
The discovery has totally changed the classification of corneal stromal dystrophies. The dystrophies reclassified
include Reis-Bücklers dystrophy, lattice corneal dystrophy type I, granular corneal dystrophy, and Avellino
corneal dystrophy (103). Even though the conditions are inherited, they generally do not progress sufficiently to
be treated with penetrating keratoplasty in the pediatric age group except for congenital hereditary corneal
dystrophy (CHED). (See cornea 2008;27(Suppl 2:S1-S42)).
CHED is a congenital structural abnormality of the corneal stroma and endothelium that is inherited both in
autosomal recessive (2, 116) and autosomal dominant forms (70). The two forms are genetically distinct but both
involve a region of chromosome 20 (13) with the recessive form mapping to 20p13 (92). At birth both corneas of
an affected individual are thickened and opaque. The corneal collagen fiber diameter is nearly twice the normal
diameter and is haphazardly arranged in a manner that limits transmission of light. Descemet membrane is often
thin and the endothelium is abnormal. There is associated secondary bullous keratopathy and degeneration of
Bowman membrane. Subepithelial amyloid accumulation has been observed in some cases (81). Treatment is
penetrating keratoplasty.
Map-dot-fingerprint dystrophy, also known as anterior basement membrane or Logan-Guerry dystrophy, is
characterized by excessive production of basement membrane material by the corneal epithelial cells (105). The
epithelium is loosely adherent because of abnormal adhesive properties of the redundant basement membrane.
Corneal abrasions tend to occur more frequently (recurrent erosion) (Figure 11-14). Secondary reactive
degeneration of the Bowman membrane and anterior corneal stroma may occur if the abrasions are extensive,
leading to superficial corneal opacification that is permanent. The condition rarely affects the pediatric age group
and is treated topically.
Meesmann corneal dystrophy is a degeneration of the corneal epithelial cell cytoskeleton. Because the corneal
epithelial cells are replaced in a 10-day cycle this condition is rarely symptomatic and does not require treatment.
FIGURE 11-14▪Map-dot-fingerprint dystrophy. Epithelial cells have produced a defective basement membrane
with abnormal adhesive characteristics. The epithelium has separated from Bowman membrane to form a
subepithelial bulla. The bullae are fragile and may rupture causing exposure of nerve endings and pain. The
absence of epithelial cover is a risk factor for corneal infection (Periodic acid/Schiff stain, original magnification
×100).
Dystrophies of Bowman membrane (Reis-Bücklers and Thiel-Behnke dystrophies) occur extremely rarely. There
is destruction of Bowman membrane possibly due to a protease produced in the corneal epithelium. Recent
evidence suggests a relationship to a mutation of the TGFBI gene (21).
Macular corneal dystrophy is an abnormality of mucopolysaccharide production by corneal keratocytes (1). The
condition is inherited in an autosomal recessive pattern (16q22). Unlike the other corneal dystrophies, there is a
systemic abnormality in a subset of persons with macular corneal dystrophy.
Granular corneal dystrophy is an abnormality of protein metabolism of the corneal epithelial cells associated with
the genetic defect at 5q31. Well-demarcated deposits occur initially in the anterior corneal stroma and progress
to accumulate in deeper stromal layers. The intervening collagen is normal.
Lattice corneal dystrophy type I is an accumulation of amyloid in the corneal stroma often in a linear pattern
associated with the genetic abnormality at 5q31. Other subgroups of lattice corneal dystrophy involve other
processes leading to amyloid deposition and are extremely rare (123).
Avellino corneal dystrophy is caused by the genetic defect at 5q31 that presents initially with features of
granular corneal dystrophy and then progresses to develop features of lattice corneal dystrophy in addition to the
features of granular corneal dystrophy. Persons living in Avellino, Italy were the initial group studied that led to
the discovery of the common genetic defect at 5q31 being associated with multiple phenotypic expressions (40).
Fuchs endothelial dystrophy is a common corneal dystrophy that is expressed generally in the older age groups.
The corneal endothelium is not able to dehydrate the cornea and the corneal stroma becomes thickened and
opaque. Descemet membrane becomes thickened focally (corneal guttata) or generally (multilaminar Descemet
membrane). There is a significant loss of corneal endothelial cells far beyond what
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is observed during normal age-related attrition. There are secondary degenerative changes of the corneal
epithelium, bullous keratopathy, including intraepithelial basement membrane formation, subepithelial bullae,
degenerative pannus, and reactive destruction of Bowman membrane. This condition is one of the most common
indications for penetrating keratoplasty particularly following cataract extraction in older age groups (131).
FIGURE 11-15▪Keratoconus. There is a break in Bowman membrane (arrows) that is associated with alteration
of the anterior contour of the cornea (formation of a “cone”) (Periodic acid/Schiff stain, original magnification
×200).
Posterior polymorphous dystrophy consists of endothelial cells with epithelial cell characteristics (59). The
epithelial cell metaplasia can be detected clinically but is generally stationary and does not affect visual function.
Generally, no treatment is required.
Keratoconus is an acquired localized stromal thinning of the cornea, usually located in the inferior nasal quadrant
(Figure 11-15). The thin area is displaced anteriorly by normal levels of intraocular pressure altering the anterior
corneal curvature. Keratoconus is not considered to be a corneal dystrophy. Its etiology has not been
established but appears to relate to abnormal activity of the matrix metalloproteases normally produced by
corneal keratinocytes. The natural history is one of progressive myopia and irregular astigmatism that can be
corrected initially with contact lenses. In time, some cases progress to corneal stromal scarring in the region of
the cone (the area of maximal distortion). The stroma becomes thin to the point where corneal rupture is
possible. Rupture of Descemet membrane in the region of the cone may allow aqueous from the anterior
chamber to instantaneously hydrate the normally dehydrated corneal stroma (corneal hydrops). There is sudden
appearance of corneal opacity that may slowly clear over weeks or months as the corneal endothelium repairs.
Complete clarity is rarely accomplished. Distinct, focal breaks of Bowman membrane characterize keratoconus.
Scarring of variable degrees is associated with the breaks in Bowman membrane. In the event of corneal
hydrops, there is rupture of Descemet membrane. The severed ends of Descemet membrane generally curl
inward. Endothelial cells may migrate over exposed posterior corneal stroma to establish a new, but considerably
thinner Descemet membrane. Keratoconus is one of the most common indications for penetrating keratoplasty in
children (36) (Figure 11-16).
FIGURE 11-16▪Corneal hydrops. The lack of tensile strength of the cornea has progressed to the point of
rupture of Descemet membrane, exposing the relatively dehydrated corneal stroma to be exposed to aqueous
humor (arrows). Hydration of the corneal stroma results in opacity in the region of rupture. With time, the
posterior cornea may repair causing at least partial clearing of the stroma and improved vision. (Periodic
acid/Schiff, original magnification ×40).
DEGENERATIONS OF THE CORNEA

Band keratopathy is a degeneration of the anterior cornea often associated with chronic anterior uveitis or
chronic keratitis. It appears as superficial opacification of the anterior cornea with focal oval well-demarcated
areas of translucency that causes marked loss of vision. Calcium is deposited in Bowman membrane and corneal
stroma by dystrophic calcification (Figure 11-17). The calcified Bowman membrane is as fragile as an egg shell
and may fracture and extrude onto the anterior corneal surface. There is no effective treatment for band
keratopathy (20).
THE CRYSTALLINE LENS

Structure of the Crystalline Lens
The crystalline biconvex lens is located in the visual axis posterior to the iris diaphragm and contributes about
10% of the refractive power of the eye. Until approximately age 40 years, the lens is pliable enough to allow
variable focus from distance to near. After age 40, the lens loses its pliability and bifocals or reading spectacles
are necessary for near
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tasks. After age 70 years, the lens loses its transparency to the point where cataract surgery may be necessary.
FIGURE 11-17▪Band keratopathy is dystrophic calcification of Bowman membrane (large arrow) and corneal
stroma (small arrows) following chronic keratitis or uveitis. In advanced cases, calcified Bowman membrane may
fracture and be displaced onto the corneal surface causing a foreign body sensation (Hematoxylin-eosin stain,
The crystalline lens is surrounded by a dense type IV collagen capsule that is variable in thickness. The thickest
portion of the capsule is at the point of insertion of the supportive lens zonule system of fibers and is thinnest at
the posterior pole, adjacent to the vitreous in the visual axis. The lens cortex and nucleus are initially entirely
cellular. In the anterior hemisphere there is a single layer of cuboidal “epithelial cells” that terminate at the lens
equator by forming a curvilinear “lens bow” (the stem cells of the lens). The remainder of the lens cells lose their
nuclei and become anucleate lens fibers. The lens fibers have a very regular structure associated with few
organelles but have an intricate system of balland-socket connections between lens fibers. Lens fibers are
continuously added to the surface of the cortex beneath the lens. The older fibers are compacted in central lens
and tend to become opaque (nuclear cataract). Lens zonules originate from the surface cells of the pars plana,
anterior to the vitreous base, and extend through the posterior chamber to the equator of the lens. Zonules are
composed of fibrillin, maintain lens position, and change lens shape (and optical power) during accommodation
(38, 61).
SURGICAL PROCEDURES OF THE CRYSTALLINE LENS

Cataract extraction is one of the most common surgical procedures performed on the elderly in the United States.
Cataract surgery is infrequently performed in the pediatric age group except for congenital cataract or
inflammation-related cataract (e.g., uveitis associated with juvenile rheumatoid arthritis and trauma). Surgery is
usually performed through a small corneal incision. The opaque material of the lens is removed by mechanical
aspiration through a sophisticated auger-like device. The lens is replaced by a synthetic (usually plastic) lens
constructed of various types of biostable polymers. In children, glasses for near tasks will be necessary because
the current intraocular lenses correct only for distance. The intact lens is usually not removed, and the aspirated
lens cortical material is usually not submitted for histopathologic examination. The lens capsule remains to
support the intraocular lens. The posterior lens capsule often becomes opaque due to fibrous metaplasia of the
remaining lens epithelial cells. A YAG laser is used as a postoperative office procedure to create a clear axial
opening through this type of reactive membrane.
DEVELOPMENTAL ABNORMALITIES OF THE CRYSTALLINE LENS

The crystalline lens develops from an invagination of the surface ectoderm to form the lens vesicle. The
developing lens interacts with the retina derived from the neuroectoderm to form the functional aspects of the
eye. Neural crest cells form the supportive tissue of the eye and orbit. Fibers of the lens cortex form a visible
suture in the shape of an inverted Y anteriorly and an inverted Y posteriorly. Subtle changes in lens biochemistry
may lead to opacification of lens fibers as punctate or diffuse opacities or opacification of the Y sutures. A rich,
temporary, vascular plexus (tunica vasculosa lentis) supports the lens during embryonic development, which will
undergo apoptosis approximately at birth. At that time, nutritional support for the lens changes to the aqueous
produced by the cilia epithelium. The tunica vasculosa lentis may be retained in various degrees to form
fibrovascular membranes in the papillary space (persistent fetal vasculature.) The lens is supported by a system
of zonules composed primarily of fibrillin. Variations in zonular structure may allow various degrees of lens
dislocation (15).
Primary aphakia is an exceedingly rare event because of the contribution of the lens to ocular development; if
the lens is not present the remainder of the globe is unable to develop.
Microphakia may occur in generalized ocular abnormalities such as PHPV. In this situation abnormalities of the
vitreous will limit development of the lens. Eyes affected by PHPV are usually small with limited visual potential.
Many complex factors determine lens size, most of which are not yet fully characterized. The microphakic lens is
usually also spherical. The abnormal shape may be caused by deficiencies in the quality or quantity of lens
zonules (19, 56).
In Lowe syndrome the lens is small and has a discoid shape with no clear demarcation between fetal nucleus
and cortex. There is apparent lack of formation or, alternatively, a degeneration of primary lens fibers at the lens
equator. The profile of the equatorial lens is sharply angled and the anterior-posterior dimension of the lens is
reduced. The lens is densely opaque.
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A pyramidal cataract is a dense elevation on the anterior surface of the lens usually in the visual axis. In addition
there may be absence of axial posterior corneal stroma and Descemet membrane and endothelium (Peters
syndrome).
A lens notch may be observed in an inferior nasal hiatus of the iris diaphragm of an iris coloboma. Faulty closure
of the fetal fissure during development leads to failure of formation of lens zonules in the area of the coloboma.
The absence of lens zonules allows the natural pliability of the lens to form a notch at the lens equator. A lens
notch is usually not associated with opacification of the lens.
Phakomatous choristoma is the presence of ectopic lens tissue in the cutaneous structures of the anterior orbital
soft tissue, usually in the inferior nasal quadrant. The abnormal lens is characterized by basement membrane-
producing epithelial cells and primitive lens fibers. The condition is usually stationary and not associated with
other ocular abnormalities (88, 140).
In Alports syndrome, most often inherited in an X-lined pattern, abnormal basement membrane may lead to
alteration in lens contours; increase in anterior lens curvature, anterior lenticonus, or increase in posterior lens
curvature, posterior lenticonus. The anterior lens capsule is thin and is characterized ultrastructurally by
numerous full-thickness cracks associated with degeneration of regional epithelial cells. The capsule appears
thin enough and weak enough to explain reported spontaneous lenticular ruptures. Posterior lenticonus,
independent of Alport syndrome, has also been reported (102). A dot-and-fleck retinopathy also occurs in a high
percentage of affected individuals (28).
CONGENITAL LENS OPACITIES

Congenital cataracts are the final expression of many types of developmental and metabolic defects and are
relatively rare (52). Autosomal dominant transmission with high penetrance is most common, but autosomal
recessive and X-linked transmissions also occur. It is not surprising that several cataract types have been linked
to regions that regulate crystallin genes, the major proteins of the lens fibers (100). The pathologic features of
congenital cataracts are classified by geographic localization of the clinical opacity.
Anterior polar cataracts arise in the papillary space as an elevated plaque that may be the result of faulty
separation of the lens vesicle from the surface ectoderm or may be the result of a momentary toxic environment
(e.g., inflammation) during gestation. Posterior polar cataracts are more likely related to failure of involution of
components of the tunica vasculosa lentis (Mittendorf dot). Opacification in the region of the Y sutures is
common and is estimated to occur in at least 20% of the population. Congenital cataract may affect any region of
the lens with markedly variable degrees and patterns of relative opacity. A cerulean cataract is a club-shaped
opacity with a blue tinge. A zonular congenital cataract has opacification in zones around a clear nucleus. In
contrast, the fetal nucleus may be translucent or totally opaque as found in the rubella syndrome. There is
potential risk indicated by cataract as there is intracellular sequestration of live virus within the central fiber cells
of the lens, which may persist long after birth and may cause viral endophthalmitis if released during cataract
surgery.
LENS OPACITIES IN DISEASES OF GENETIC ORIGIN

There are dozens of other syndromes of known or suspected genetic cause in which some form of cataract has
been described; however, seldom are the clinical characteristics of the lens opacity specific for that syndrome.
Extensive bibliographies of these associations are available (45), and new examples are reported yearly. The
most common lens opacity in these often autosomal dominant diseases is the posterior subcapsular cataract
(PSC). What may be difficult to determine is whether the cataract is directly due to the genetic defect or is a
secondary effect of the disease process. Discoveries such as expression of crystallins in nonlenticular cells
promise new avenues for understanding these associations (100).
In galactosemia, polysaccharides may accumulate in the lens, changing the state of hydration and the clarity of
the lens. If the biochemical abnormality is corrected through dietary measures early in the course of the disease,
the lens may return to its normal state of transparency. If the condition persists, secondary structural changes in
the lens will cause permanent opacity (see Chapter 5).
Lysosomal storage diseases result from deficiency of the lysosomal enzymes necessary for cellular metabolic
functions and are transmitted as autosomal or X-linked recessive traits. They show lysosomal inclusions that
occur in lens epithelial cells, causing subtle anterior lens opacification.
In Fabry disease, there is an intracellular accumulation of neutral glycosphingolipids, especially
trihexosylceramide, in many ocular and systemic epithelial and endothelial cells (43). Niemann-Pick disease is
characterized by accumulation of laminated membranous inclusions in many cells including the lens epithelial
cells although causing minimal opacification (110).
Cataract formation is an important feature in myotonic dystrophy, the clinical characteristics of which include
muscle weakness, cardiac muscle conduction defects, and a slowness of contracted muscles to relax. Cataracts
occur in almost all adults, with myotonic dystrophy, as iridescent polychromatic crystals in a zone deep to the
anterior and posterior capsules, exhibiting especially green and red colors. A posterior subcapsular stellate
cataract may then develop, followed by cortical vacuoles and clefts of a nonspecific type that are not
distinguishable from an aging cataract.
THE ZONULAR APPARATUS AND LENS DISLOCATION (ECTOPIA LENTIS)

The lens zonules are composed of a cystine-rich glycoprotein fibrillin, which is encoded on chromosome 15q21.1
(80).
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A lens is luxated when it is completely dislocated from its normal position and the zonular support is nearly or
completely absent. Subluxated lenses are partially removed from their normal position with variable degrees of
zonular support remaining (Figure 11-18).
FIGURE 11-18▪Crystalline lens dislocation. The center of the lens is displaced (subluxated) from the pupillary
margin (white arrow) but is not completely dislocated (luxated). Stretched zonules can be seen extending from
the margin of the lens posteriorly to their insertion. The majority of lens zonules attached to the elevated portions
of the lens margin.
The most common cause of lens subluxation-luxation in most large series has been trauma (96). It usually follows
penetrating injury or severe contusive injury and is often associated with cataract and rhegmatogenous retinal
detachment.
Marfan syndrome is the most common heritable cause of crystalline lens dislocation; it is caused by mutations in
the fibrillin-1 gene (FBN1) on chromosome 15q21.1 (30). The most important systemic abnormality of Marfan
syndrome is the high risk of dissecting aneurysm of the aorta. Lens subluxation may be present at birth or may
appear after birth, and may be stationary or progressive (84). The zonules can be easily seen stretching from the
periphery of the lens across the peripheral pupillary space. The subluxated lens may be normal in size or small,
with a flatter curvature of the lower half and a posterior bulge as a result of weakness or absence of the inferior
zonules. The zonular bundles may be thin, thick, or of normal caliber but in most cases show thin and poorly
aggregated zonules (106) (see Chapter 13).
Homocystinuria is an autosomal recessive disease based on a virtual absence of cystathionine β-synthase (58).
Lens dislocation is not present at birth but is usually present by age 30 years. The lens is often spherical to the
point where it may dislocate into the anterior chamber producing pupillary block glaucoma. The globe tends to be
elongated, increasing the risk of retinal detachment and the ciliary musculature tends to be hypodeveloped. The
zonular bundles inserting on the lens show an abnormal porous sponge-like appearance (107), probably as a
result of the short, disoriented fibrils of which they are composed. The zonules tend to rupture midway between
origin and insertion.
The biochemical defects of Weill-Marchesani syndrome and sulfite oxidase deficiency are also associated with
abnormalities of zonular structure that may lead to ectopia lentis.
CONGENITAL CATARACT FROM ENVIRONMENTAL FACTORS

The developing lens is extremely sensitive to changes in its biochemical microenvironment. Even transient
changes may lead to localized opacities in specific regions of the lens cortex. The closer the opacity to the
epicenter of the lens the more likely the event was early in development. Lens opacities formed in this manner
generally do not progress after birth and may not affect visual function.
Rubella: The rubella virus may gain access to the developing lens and infect the cells of the lens cortex during
pregnancy. Infection in this manner produces a dense white (pearl-like) cataract that may be limited to the
embryonic nucleus. The cataract is distinguished by retention of lens cells with nuclei in the center of the lens,
which, in normal development, would have involuted and disappeared except at the equatorial lens bow (Figure
11-19). Rubella virus remains viable in the lens for years after birth (130, 139). Early surgical procedures
designed to remove congenital cataracts piecemeal may have contributed to virus release into the eye and
subsequent viral panophthalmitis.
FIGURE 11-19▪Rubella cataract. Normally, there are no nucleated crystalline lens cells in the center of the lens,
the lens nucleus. With rubella infection early in gestation the central lens cells are infected with the rubella virus,
retain their nuclei (small arrows), and are densely opaque. There is also some degeneration of the lens cortex
(large arrow) which also causes peripheral translucent opacity (Hematoxylin-eosin stain, original magnification
×20).
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FIGURE 11-20▪Anterior subcapsular cataract. A: The cornea is opaque from long-standing anterior uveitis and
keratitis creating a toxic environment in the anterior chamber. B: Crystalline lens repair processes have caused a
dense anterior subcapsular cataract (arrow) by fibrous metaplasia of the crystalline lens epithelium. The lens
capsule undulates because of contracture of the fibrous scar (Periodic acid/Schiff stain, original magnification
×40).
Toxic cataract: With chronic damage from anterior uveitis, the anterior lens epithelial cells will be stimulated to
undergo fibrous metaplasia resulting in dense anterior subcapsular cataract (Figure 11-20). Following trauma,
intraocular inflammation, or vitrectomy, crystalline lens cells may migrate from the lens equator to the posterior
pole of the lens to create a “ground-glass” opacification of the posterior lens cortex. The migrating cells retain
their nuclei but are very polymorphic. The posterior subcapsular cells are said to resemble urothelial cells of the
bladder and have been called “bladder cells (15) (Figure 11-21).
Traumatic cataract: The crystalline lens will instantly become opaque if the lens capsule is disrupted allowing
fluid to disturb the homogeneity of the lens cortex as in a penetrating injury of the cornea or sclera. A shock wave
associated with blunt trauma may also cause the formation of a cataract; however, the clinical onset of the
opacity may be days or years following the injury. This type of cataract may be characterized by posterior
migration of the lens epithelium from the equator along the internal surface of the posterior capsule to the
posterior pole of the lens. This type of PSC is also associated with advanced diabetes mellitus, chronic treatment
with corticosteroids and with inflammation (14).
FIGURE 11-21▪Posterior subcapsular cataract (PSC). Lens epithelial cells have migrated from the lens equator
to the posterior cortex of the lens in the visual axis. The cells have retained their nuclei and are irregular in shape
and size manifesting as a “ground glass” appearance of the posterior lens cortex clinically (Periodic acid/Schiff
stain, original magnification ×40).
THE VITREOUS
Structure of the Vitreous
The vitreous is composed of a type II collagen matrix containing hyaluronic acid. The majority of the vitreous is
composed of water and may attain a volume of 4 cc weighing 4 g. The vitreous is formed near the junction
adherent to the internal surface of the retina at the optic disc, in the region of the peripheral macula, along the
course of retinal blood vessels and at the posterior surface of the crystalline lens. The matrix of the vitreous
degenerates over time (usually beyond the pediatric age) and separates from the surface of the posterior retina
forming “floaters,” which cast a symptomatic shadow on the retina. The vitreous is a biochemical sink and also
functions in maintaining retinal attachment (38, 61).
Surgical Procedures of the Vitreous

The entire vitreous can be surgically removed (vitrectomy) without immediate effect on the structure of the eye. In
most cases, a cataract will develop due to subtle biochemical alteration. Vitrectomy is most often used to remove
acquired mechanical factors affecting the retina
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(e.g., epiretinal membrane formation associated with macular hole and subretinal neovascularization from a
variety of causes). Vitrectomy is also used to correct fibrovascular membranes (traction retinal detachment) that
are found in advanced stages of diabetic retinopathy. Occasionally, vitrectomy is used in pediatric cases for
diagnostic purposes (diffuse infiltrating retinoblastoma, medulloepithelioma). The specimen often contains only a
small number of cells, as is found in fine needle aspirations used at other sites. Usually, the histopathologic
diagnosis is established by examining a cellblock. Fine needle aspiration is rarely done intraocularly because the
mechanical forces generated during aspiration are much more difficult to control than the aspiration forces
generated by the highly sophisticated vitrectomy instrument.
Intermediate uveitis or pars planitis is a localized inflammation probably of autoimmune origin that involves the
vitreous base and peripheral retina (8). Children and young adults are primarily affected. The majority of affected
patients are asymptomatic and become symptomatic only with secondary changes such as cystoid macular
edema (reactive swelling of the retina), cataract, or glaucoma. In the region of the peripheral retina there is
phlebitis and retinal edema. The vitreous structure collapses and becomes opaque over the area of proliferation
of the nonpigmented epithelium of the ciliary body. Granulomatous inflammation is present in the region of
reactive proliferation (53). Medulloepithelioma of the ciliary body and diffuse infiltrating retinoblastoma may
present in a similar manner (122).
Familial exudative vitreoretinopathy is a congenital abnormality of the peripheral retinal circulation resulting in
secondary extracellular matrix abnormalities throughout the vitreous in the form of organized membranes and
focal opacities (snowflake-like) (7). Autosomal dominant, recessive, and X-lined inheritance patterns have been
identified. At least five mutations associated with familial exudative vitreoretinopathy have been identified on the
long arm of chromosome 11 (67). Along with the basic vascular abnormality the membranes are responsible for
causing retinal detachment, displacement of the macula, cataract, and anterior chamber angle closure (6).
THE OPTIC NERVE

Structure of the Optic Nerve
The optic nerve is the aggregation of retinal ganglion cell axons that exit the eye through the scleral canal in the
posterior medial portion of the sclera. The hydraulic integrity of the globe is maintained by a sieve-like structure
(the lamina cribrosa) at the plane of the sclera through which the optic nerve axons pass. Just beyond the lamina
cribrosa, oligodendroglia form a myelin sheath around each axon. This addition increases the diameter of the
optic nerve from 1.5 mm at the scleral canal to 3.0 mm in the myelinated portion. The axons of the optic nerve
are supplied by branches of the ophthalmic artery in the arachnoid, extending across the subarachnoid space to
the pia. The central retinal artery crosses the dura and arachnoid 12 mm posterior to the lamina to assume a
central position in the proximal optic nerve but does not supply the optic nerve itself. The dura of the optic nerve
is contiguous with the periostium of the orbital apex and with the sclera. The subdural space is truncated or
closed through the optic canal of the sphenoid bone. The subarachnoid space of the optic nerve is contiguous
with the subarachnoid space of the central nervous system (38, 61).
Surgical Procedures of the Optic Nerve

The optic nerve is a sensitive structure that is in a relatively surgically inaccessible position. The anterior
approach usually requires removal of a portion of the orbital rim. The best exposure is via a frontal craniotomy.
On occasion biopsy of the optic nerve may be necessary in cases where a diagnosis cannot be established by
other means and usually where there is no or limited visual potential for the eye. Fistualization of the meninges
(optic nerve fenestration) has been attempted in certain limited medical conditions that do not usually affect the
pediatric age group.
Optic nerve gliomas are juvenile pilocytic astrocytomas that cause fusiform enlargement of the optic nerve. In
many cases the tumor limits visual potential but is not a threat to the life of the child. Approximately 15% of
persons with NF-1 will have optic nerve gliomas. In this setting, the clinical course may be more aggressive but
the tumor rarely, if ever, undergoes malignant transformation (78, 112). Only rarely will an optic nerve glioma
extend into the eye. Two cytological patterns are found in pilocytic astrocytomas: areas of fibrillar matrix with oval
to round nuclei and a more mucoid matrix that may contain microcysts. Rosenthal fibers (intracellular electron-
dense material surrounded by glial elements), eosinophilic granular bodies (membrane-bound intracellular
osmophilic material), and microcalcifications also characterize the lesion. Neither Rosenthal fibers nor granular
bodies are unique to pilocytic astrocytoma. Surgical resection of the optic nerve or enucleation may be
necessary if there is sufficient proptosis to cause degeneration of the cornea or abnormal development of the
orbit. Risk factors for a poor therapeutic outcome include the association with NF1 or involvement of sensitive
structures such as the optic chiasm (3, 65) (see Chapter 10).
Meningioma of the optic nerve may arise in the arachnoid sheath of the optic nerve (primary optic nerve
meningioma) or may involve the orbit from an intracranial meningioma, usually one situated along the sphenoid
ridge. Optic nerve meningioma is uncommon. Most are of the transitional and meningotheliomatous types. They
usually do not invade the eye itself but may cause proptosis and ophthalmoplegia due to mass effect of the
tumor. Treatment is usually surgical. Treatment with external beam radiation is being evaluated (75).
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THE ORBIT
Structure of the Orbit
The globe is housed in a 30 cc orbit bordered by bone that provides physical protection for the globe, primarily
by the presence of the orbital rim. The interior orbit is separated from the soft tissues of the face by the orbital
septum, a fibrous membrane that originates from the periostium of the facial bones at the orbital rim. The rectus
and oblique muscles have their origin at the periosteum of the orbital apex and function to align the two globes,
allowing the brain to receive two slightly dissimilar images providing for perception of depth. The veins traversing
the orbit have no valves. Direction of blood flow is determined by differential pressure gradients between the
internal and external carotid systems. The only epithelial structure in the orbit is the lacrimal gland, a portion of
which is located anterior to the orbital septum (the palpebral lobe) and a portion is posterior to the septum (the
orbital lobe). The ducts from the orbital lobe extend through the palpebral lobe to reach ostia in forniceal region
of the conjunctiva. There are no lymphatic channels or lymph nodes in the orbit except the lymphoid tissue
associated directly with the lacrimal gland. The only cartilaginous structure in the orbit is the trochlea in the
superior nasal orbit that serves as a pulley to direct orientation of the superior oblique tendon. Orbital soft tissue
is divided into multiple intercommunicating compartments by delicate fibrous septa. The area between the rectus
muscles has been referred to as the intraconal space, but that space has no unique functional or prognostic
significance for tumors. Preseptal soft tissue forms the eyelids, the conjunctiva, and the lacrimal drainage
apparatus. The eyelids protect the eye from the external environment and close when stimulated by visual
threats, movement of the eyelashes, or disturbance of corneal sensation. The orbicularis oculi closes the eyelids,
and the levator palpebrae open the eyelids. The eyelid is also responsible for forming and maintaining the tear
film. The tear film has at least three functional layers: an aqueous portion that contains oxygen and other
nutrients, a mucous portion that allows smooth layering of the aqueous portion, and a lipid portion that retards
evaporation of the aqueous portion. The major volume of aqueous is not formed by the lacrimal gland in the orbit
but is formed by accessory lacrimal gland acini located in the upper eyelid, the upper conjunctival fold (the
fornix), and the conjunctiva directly covering the globe (bulbar conjunctiva). The lacrimal gland itself plays only a
minor reflex role of tear film function. Tear film components exit via puncta located at the superior and inferior
nasal eyelid margins. Tears and surface debris flow through the canaliculus to the nasal lacrimal duct and finally
to the lateral wall of the nasal mucosa under the inferior turbinate. A pumping mechanism for aqueous is thought
to function via contraction of the orbicularis oculi. The caruncle is a sequestered portion of the lower eyelid
margin and contains all of the dermal elements of the eyelid but is covered by mucous membrane contiguous
with the conjunctiva (38, 61).
Surgical Procedures of the Orbit

As indicated with the optic nerve, surgical approaches to the orbit are technically difficult and undertaken only
with strong clinical indications. Lacrimal gland tissue is usually removed anteriorly through the skin and orbital
septum. Particularly when adenoid cystic carcinoma is encountered, bone in the region of the lacrimal fossa is
often removed. Because of the evolution of treatment of rhabdomyosarcoma only a biopsy sampling of the tumor
is required prior to chemotherapy and radiation (although debulking procedures may be performed). In the past
the entire contents of the orbit including all eyelid tissue to the orbital rim (exenteration) were required. Tumors
located in the posterior orbit are best approached via a frontal craniotomy. Occasionally expanded orbital
contents are decompressed into an adjacent sinus cavity such as in aggressive Graves disease. Release of
trapped orbital contents in an orbital floor fracture is indicated only if the entrapped tissue causes major
abnormalities of movement of the globe.
A dermoid cyst of orbital tissue is a cystic choristoma usually containing benign dermal elements that tend to
progressively enlarge because of internal desquamation of the surface epithelium and adnexal units. The cyst
arises from embryonic rests of mesenchyme that tend to be adjacent to membranous bones particularly in the
region of facial fusion lines (46). A classification by location has been proposed. Juxtasutural cysts are often
found along the orbital rim and are attached to bone by fibrous septa that do not distort the bone. Sutural
dermoid cysts (including giant dermoid cysts) originate in the synostosis of orbital bones and may extend within
cancellous bone or may extend either into the orbital cavity or into the intracranial cavity. This type of cyst is
associated with defects of bone and may develop the appearance of a draining sinus. A soft-tissue dermoid cyst
develops within soft tissue and is not associated with bone.
Orbital dermoid cysts may develop either anterior or posterior to the orbital septum. Cysts present anterior to the
orbital septum tend to occur at an earlier age (usually before age 5 years) and are often found at orbital rim
suture lines. The cyst is usually a smooth, firm, nontender, oval mass along the superior orbital rim and is less
than 2 cm in diameter. Cysts posterior to the orbital septum tend to be associated with intraorbital suture lines
and present at a later age. Anteriorly positioned cysts may herniate through the orbital septum. Posterior lesions
may extend through the superior orbital fissure or into the temporal fossa. Medial lesions generally do not extend
into the ethmoid air cells, but superior cysts may extend into the frontal sinus. Most lesions present in the first
two decades, although presentation in advanced age is also possible. There is no gender specificity.
There has been a distinction between epidermoid cysts that do not contain adnexal elements and dermoid cysts
that do contain adnexal elements. However, the histological distinction does not guide management and the
general term dermoid cyst is most often used. The lining of the cyst is
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stratified, keratinizing squamous epithelium with or without adnexal units. When adnexal units are present, the
cyst wall is usually more robust than in those lesions without adnexal units. The cyst may contain desquamated
squamous epithelium, cholesterol, hemorrhage (hemosiderin), hair, or calcium. The contents may subdivide into
various fluid levels. In regions were the cyst wall has ruptured, there is a granulomatous foreign body reaction
and fibrous reaction that may be extensive (Figure 11-22).
FIGURE 11-22▪Dermoid cyst. This cyst was removed from a 3-year old who developed superior temporal orbital
rim pain and tenderness. The symptoms may have been due to rupture of the cyst wall (arrow), allowing a
foreign body granulomatous reaction to the keratin contents of the cyst.
Generally there is progressive enlargement of the lesion because of expanding volume of the intraluminal
contents. Continuous pressure may cause erosion through bone into contiguous tissues and spaces. There is no
malignant transformation. Treatment is surgical excision.
Langerhans cell histiocytosis (LCH) (formerly known as histiocytosis X) is a proliferative disorder with multiple
clinically distinct forms (Hand-Schuller-Christian disease, Letterer-Siwe disease, and eosinophilic granuloma).
The pathophysiology of LCH is not understood; however, there is no evidence of metabolic abnormality or
infection (83). The Langerhans cell is an immune-processing cell of the monocyte-macrophage system found
among the squamous epithelial cells in the skin, in bone marrow, and in the paracortical region of lymph nodes
as well as multiple other sites. Despite the clinical dissimilarity, all these diseases have a histological pattern that
suggests a granulomatous inflammatory infiltrate containing pathologic Langerhans cells. The normal
Langerhans cell has dendritic processes, an eccentric folded nucleus, small nucleolus, and a cytoplasmic
structure, the Birbeck granules, that has a central striation and a “tennis racket” profile. The function of the
Birbeck granule is unknown, but it appears to be composed of plasma membrane components. Pathologic
Langerhans cells lack dendritic processes but retain Birbeck granules. CD1a positivity differentiates Langerhans
cells from other macrophages.
The prevalence of LCH in children under age 15 years ranges between 4.6 (55) and 8.9 (121) per 100,000.
There have been no reported instances of familial, time, or geographic location clustering (83). There appears to
be no gender specificity.
LCH may present as a single system disease with a lesion in a single tissue type or a multisystem disease
including disseminated forms. Orbital involvement most often presents as a single system disease of orbital
bone. The onset of proptosis is acute, and there are associated signs of inflammation. The degree of
involvement is highly variable. When the lesion is located in orbital bones there has been concern that there
would be progression to central nervous system involvement and such lesions have been treated with
chemotherapy with conflicting opinions about long-term benefit. Extraocular lesions of sufficient size may cause
intraocular findings of compression (choroidal folds, optic disc swelling) and compressive optic neuropathy.
Rarely, LCH may present as uveitis where a vitreous biopsy could be interpreted as containing only
macrophages and other benign inflammatory cells (unless stained for CD1a) (127). Secondary glaucoma may
develop if the trabecular meshwork is affected. The eyelid skin is unusually not involved. Late recurrences have
been reported (134) (see Chapters 22 and 27).
The light microscopic pattern is that of chronic granulomatous inflammation characterized by an infiltrate of
histiocytes, lymphocytes, giant cells, and eosinophils. The presence of eosinophils is not essential for the
diagnosis of LCH. Birbeck granules can be detected only by transmission electron microscopy and are found in
only 20% of the cases studied (91). Langerhans cells can be identified by the CD1a stain. The number of CD1a-
stained cells generally decreases as the lesion matures or regresses.
Single system disease survival is nearly 100%. Multisystem disease survival is associated with an 80% survival.
Age at presentation of less than 1 year is a risk factor for a poor prognosis. LCH itself is a risk factor for
secondary malignancy including Hodgkin lymphoma and acute leukemia. Treatment includes surgical debulking,
chemotherapy, and simple observation.
Lymphangioma is a developmental abnormality of lymphatic vessels and their precursor cells and lymphoid
tissue in the soft issue of the orbit. Normally no lymphatic channels or populations of lymphocytes are found in
the orbit, thus this lesion is a choristoma and presents at birth, although the condition may not present clinically
until advanced age. One clinical classification is by the character of hemodynamics in the lesion (no flow, venous
flow, or arterial flow) that guides surgical and other means of therapy (49). There is no gender specificity with the
majority of lesions presenting in the first decade.
Lesions of various sizes and degrees of functional significance may be found in the eyelid, the conjunctiva,
anterior (preseptal) orbital soft tissue, and posterior (postseptal) orbital soft tissue. The eye itself is not involved.
Noncontinuous vascular lesions may be present in patients with intracranial vascular lesions (69). The size of the
lymphangioma may vary with posture, straining, or inflammation of the upper respiratory tract. There is usually
less effect during indolent periods on the optical system than that found with hemangioma of similar volume.
There is minimal pain unless acute hemorrhage suddenly expands the volume of the
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lesion. In this circumstance, there may be various degrees of loss of vision, development of an afferent pupillary
defect, choroidal folds, optic disc swelling, and compressive optic neuropathy (potentially to the point of complete
and permanent loss of vision). Hemorrhage may be spontaneous or associated with trauma and is more likely to
occur in a child or adolescent than in older persons. This event is also more likely in the postoperative period
after debulking procedures. Long-standing lesions beginning in childhood may result in expansion of the orbital
contours.
The gross appearance is that of diffuse lesion with no external capsule. The cut surface is composed of vascular
channels of various sizes that may contain translucent fluid or hemorrhage or both. The vascular channels are
separated by fibrous septa that also may contain areas of fresh to old brownish hemorrhage. The vascular
channels are lined by low-profile vascular endothelial cells with little apparent support by pericytes or
extracellular matrix. Within the fibrous septa there are variable amounts of lymphoid tissue, some of which may
contain germinal centers. By transmission electron microscopy endothelial cell gaps and fragmented basement
membrane may be seen.
Treatment is limited to embolization and surgical debulking (27). Multiple procedures are often necessary. In
extreme cases, due to corneal exposure and ulceration, enucleation of the eye may be necessary.
Idiopathic inflammatory disease of the orbit (also known as inflammatory pseudotumor of the orbit) is a
syndrome of inflammation of the soft tissues of the orbit of undetermined cause. The condition may arise at any
age (range 2 to 89 years) with no gender predilection (47). Approximately, 5% of cases arise in the 2 to 18 years
age-group. The usual presentation is orbital pain. The onset is often explosive and may be either unilateral or
bilateral. The bilateral cases may be simultaneous or sequential. The presentation tends to be bilateral in
children (44%) (47). Other common findings are ophthalmoparesis, proptosis, and a palpable mass.
Cerebrospinal fluid pleocytosis may be present in cases of extraobital inflammation (82). Imaging findings include
thickening of extraocular muscles including the tendon insertion to the sclera (in contrast with thyroid
ophthalmopathy where the tendon is spared), lacrimal gland enlargement, contrast enhancement of the sclera,
and inflammation of orbital fat. Histological findings include pleomorphic inflammation, fibrovascular tissue
proliferation, and fat necrosis (granulomatous inflammation to fat necrosis). There is no clonal restriction. The
plasma cells may be IgG4-positive (89). Early in the course of the disease, there is a fine collagenous stroma
and a rich cellular infiltrate consisting of plasma cells, eosinophils, and lymphocytes. Later in the course of
disease there is often a dense deposition of extracellular matrix and a granulomatous pattern in the region of fat
necrosis. The rate of progression is variable from case to case and within a given case. The response to
treatment is variable. Bilaterality is a risk factor for poor therapeutic response. The histological character of the
lesion may not be predictive of therapeutic success.
Rhabdomyosarcoma is the most common sarcoma in children as a proliferation of primitive rhabdomyoblasts.
There are two distinct clinical presentations. The most characteristic is the sudden, unexplained onset of signs of
inflammation in a child suggestive of preseptal or orbital cellulitis, except that there is no response to
conventional treatment, indicating that a biopsy is necessary. In the embryonal variant, there may be marked
pleomorphism of cells, which are often spindled, with prominent nucleoli and a variable degree of cytoplasmic
eosinophila. Rarely, actin and myosin filaments may be identified by PTAH staining. Myosin filaments and
sarcomeric units with Z-banding are evident in the tumor cells by transmission electron microscopy. The
immunohistochemical profile is positive for desmin, smooth muscle actin, and focally for myogenin. A rare
subtype is the botryoid rhabdomyosarcoma that may present in the subconjunctival space or anterior orbital soft
tissues, suggestive of a lymphoma. It is most often seen in older children and its prognosis is more favorable
than with other types of rhabdomyosarcoma (10, 101).
In older children, the alveolar variant may present in paraorbital sinuses and secondarily may involve the tissues
of the orbit. Clinical signs are those of a soft-tissue mass in the orbit or more likely in the ethmoid sinuses with
temporal displacement of the globe. The tumor is composed of aggregation of primitive round cells with an
acellular center vaguely suggestive of alveoli of a normal lung. Positive immunohistochemical markers include
those for muscle with strong reactivity for myogenin.
Treatment of orbital rhabdomyosarcoma is no longer surgical but a combination of chemotherapy and radiation
(25).
Tumors of the lacrimal gland, which is the only epithelial structure of the orbit, most often tend to be the result of
inflammation or lymphoma, generally in the adult age groups. The most common epithelial tumor of the lacrimal
gland is pleomorphic adenoma (benign mixed tumor), which is generally found in adults. The most common
malignant tumor of the lacrimal gland is adenoid cystic carcinoma, which can occur in the pediatric age group.
Early clinical signs and tumification of this neoplasm may be subtle. The most common histological pattern is that
of proliferation of small cells with hyperchromatic nuclei in a “Swiss cheese” pattern. Solid, basaloid, and
sclerosis patterns are also possible. The tumor tends to spread early due to its propensity to involve perineural
spaces, to adjacent orbital bone. Evaluation of surgical margins in this situation is at best problematic. The long-
term outcome for all cases is generally poor (48).
THE EYE
Structure of the Eye
The eye (globus oculi) is an extension of the brain that collects and transmits images gathered from the
environment. Two elements are essential: a method of focusing light with tissue
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lenses (the anterior segment) and a method of converting energy from a restricted portion of the electromagnetic
spectrum via a photochemical process into impulses that can be integrated with the remaining processes of the
brain (the posterior segment) (38, 61).
FIGURE 11-23▪The normal eye. The anterior segment is composed of the cornea, anterior chamber and the
posteior chamber, and cyrstalline lens. The posterior segment is composed of the vitreous, retina, choroid, and
optic nerve.
The anterior segment is composed of the cornea, the anterior chamber, the iris, the posterior chamber, and the
crystalline lens (Figure 11-23). The main function of the anterior segment is to transmit and refract light (reorient
parallel rays of light to a focal point). The anterior chamber is bordered anteriorly by the cornea, peripherally by
the anterior chamber aqueous filtering apparatus (the trabecular meshwork), and posteriorly by the iris stroma
and the anterior crystalline lens capsule at the pupil. Aqueous is produced by the nonpigmented epithelium of the
ciliary processes and flows through the pupil into the anterior chamber. The aqueous nourishes the anterior
hemisphere of the crystalline lens and all of the tissues bordering the anterior chamber. Spent aqueous is filtered
into the systemic vascular system, at the periphery of the anterior chamber initially, through a porous trabecular
meshwork and then through a protein membrane [the juxtacanalicular connective tissue (JXT)]. Beyond the JXT
the aqueous is discharged through the canal of Schlemm, through emissary veins, and finally into veins of the
general circulatory system. Abnormalities of drainage (glaucoma) usually occur in the JXT. Tumor cells in the
anterior chamber may exit the eye via the aqueous drainage channels.
The trabecular meshwork collagen cores are covered by an endothelium contiguous with the corneal epithelium.
There is no epithelial or endothelial lining of the anterior surface of the iris. The iris stroma is variably pigmented
by dendritic melanocytes allowing for iris “color.” The degree of iris epithelial pigmentation is uniformly dense
despite the degree of iris stromal pigmentation. The vessels of the iris stroma are unique because of a very thick
adventitial lining. The blood column cannot be seen during clinical evaluation. The thick adventitia is probably
necessary because of the continuous movement of the iris associated with changes in pupil diameter. The
sphincter muscle at the pupil is located in the iris stroma. The dilator muscle fibers are located in the cytoplasm
of the anterior iris pigment epithelium.
The posterior chamber is bordered by the posterior surface of the iris, the equatorial crystalline lens, the ciliary
body, and the anterior border of the vitreous (the vitreous face). The posterior chamber contains aqueous. The
lens zonules extend through the posterior chamber from posterior ciliary body to the lens equator and are freely
mobile in that space.
The posterior segment is composed of the vitreous, the retina, the optic disc, the uveal tract, and the sclera. The
main function of the posterior segment is to detect and transfer images from the external environment to the
brain.
The main architectural structure of the retina is provided by the Müller cells. Incident light travels through the
fullthickness of the retina before it is absorbed by visual pigments in the photoreceptor outer segments and is
converted into electrical signals for the brain. The outer limiting membrane of the retina is not a true retina but a
series of attachments between Müller cells and photoreceptors. The visual pigments of the rods are embedded
in the plasma membrane of separate disc-shaped structures of the photoreceptor outer segments. The visual
pigments of the cones are located in a folded but continuous plasma membrane of the outer segments. In both
the rods and cones, the signal is transported from the photoreceptor outer segments via cilia to the
photoreceptor inner segments. The photoreceptor inner segments contain abundant mitochondria. The visual
signal is then passed to the horizontal, bipolar, and amacrine signal-processing cells in the middle retina across
connections in the outer plexiform layer. A series of synapses in this layer forms the middle limiting membrane.
The modified signal is then passed to the ganglion cells across connections of the inner plexiform layer and
passed to the lateral geniculate via long axonal processes that make up the optic nerve (Figure 11-24).
Muller cells, modified astrocytes, support the retina by extending from the internal limiting membrane (a true
basement membrane that it produces) across the full-thickness of the neurosensory retina to the external limiting
membrane that is actually a series of connections between the apical portion of the Muller cell and the
photoreceptor cells. The nuclei of the Muller cells are located in the same region as the nuclei of the
photoreceptors. The retina also contains microglia and oligodendroglia. The central retinal artery supplies the
internal retina to the level of the middle limiting membrane. There are three layers of capillaries posteriorly
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and two layers in the equatorial retina. Beyond the equator there is a single capillary layer and at the periphery
the retina is solely supplied by external sources in the uveal tract.
FIGURE 11-24▪The normal retina: The retina contains photoreception system, an image processing system, and
image transmission system. The retina is rarely biopsied (Hematoxylin-eosin stain, original magnification ×20).
The retinal pigment epithelium (RPE) is derived from neuroectodermal cells of the outer layer of the optic cup.
The RPE cell is among the first in the body to produce melanin a molecule that is necessary for the development
of the neurosensory retina. The retina does not fully develop in ocular albinism. The melanin granules, which are
large and oval, are easily distinguished as individual granules by light microcopy. Collections of extracellular
RPE melanin in the vitreous may be mistaken for bacteria. In contrast, individual melanin granules in the dendritic
melanocytes of the uveal tract have a small diameter that is not easily resolvable by light microscopy. The RPE
is a monolayer of cells residing on its basement membrane with an undulating basal surface and long apical
processes. The apical processes interdigitate among the photoreceptor outer segments and help to physically
isolate individual photoreceptor outer segments. Interphotoreceptor mucoid substance is also present among the
photoreceptor outer segments. Among the functions of the RPE is metabolism of spent photoreceptor outer
segment lipoproteins and visual pigment molecules. The RPE has no physical connection with the overlying
neurosensory retina but is held in place by physiologic forces generated between the vitreous and the choroid. If
these factors are altered, or if the physical integrity of the neurosensory retina is violated by the formation of a
physical hole, the retina will detach from the RPE (retinal detachment).
The fovea centralis is the most highly specialized region of the retina. It is a thin area of the retina located directly
in the visual axis in the center of a portion of the retina designated as the macula. The macula is a region of the
retina generally lying between the temporal inferior and superior vascular arcades. All factors that might interfere
with the transmission of light are eliminated. The internal limiting membrane is thin; the internal retina including
ganglion cells and nerve fiber layer are absent; the internal retinal circulation is absent; and the external
plexiform layer is oriented obliquely to reach peripherally located ganglion cells. Only cones are present and are
in such a high concentration that their profiles are cylindrical rather than cone-shaped. The RPE is thicker in this
region and there is a higher concentration of melanin granules. In the center there is a avascular zone of
diameter 500 μm where nutrition is solely supplied by the uveal tract vessels (choriocapillaris). There is
compensatory thickening of the ganglion cell and nerve fiber layer in the retina immediately peripheral to the
fovea centralis.
The uveal tract receives its blood supply from the short and long posterior ciliary system. The larger vessels are
located external and progressively diminish in caliber to finally form the choriocapillaris, which is a large volume
chamber lined by fenestrated vascular endothelial cells. The basement membrane of the vascular endothelium, a
layer of extracellular matrix containing elastin and the basement membrane of the RPE cells together make up
Bruch membrane. In the region between lumens of the choriocapillaris vessels, Bruch membrane is composed of
only two layers. Venous blood is drained via vortex veins located in each quadrant through a long intrascleral
channel to mix with systemic venous blood on the episcleral surface. Among the vascular channels there is a
dense concentration of dendritic melanocytes characterized by retention of intracellular small caliber melanin
granules. The uveal tract also contains the long posterior ciliary nerve, a branch of the trigeminal, and may
contain collections of peripheral nervous system ganglion cells.
The retina is protected from vascular insults by a bloodretinal barrier similar in function and form to the blood-
brain barrier. The vessels of the choriocapillaris are porous but any extravascular fluid is blocked from retinal
penetration by intercellular tight junctions near the apical portions of the RPE cell. Similarly, the vascular
endothelial cells of the intraretinal vascular system are connected by tight junctions to form the intraretinal
portion of the blood-retinal barrier. This barrier may be breached by either inflammation or degeneration.
The optic disc is formed by the confluence of ganglion cell axons exiting through the scleral canal to form the
optic nerve. The hydraulic integrity of the globe is maintained at the level of a specialized relatively porous zone
of the sclera, the lamina cribrosa. The majority of the fibers exit via large pores in the superior and inferior
lamina. The region is not supplied by the central retinal artery but by end arteries of the short posterior ciliary
system, which is a branch of the ophthalmic artery. The exiting fibers form a central concavity, the optic cup,
which is not covered by the internal limiting membrane of the retina but does contain glial tissue.
The sclera is formed by randomly oriented collagenous fibers that are more hydrated than the cornea and are
therefore opaque. The sclera contains elastin fibers to accommodate changes in intravascular blood volume
during systole and diastole. The sclera is relatively thin at the insertions
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of the four rectus muscles and the insertion of the superior oblique. The insertion of the inferior oblique is
muscular rather than tendinous, and therefore, there is no compensatory thinning. The sclera is breached by
multiple ostia for the short and long posterior ciliary arteries posteriorly, the vortex veins near the equator,
sensory nerves anteriorly, and multiple emissary channels carrying aqueous to the venous system near the
limbus.
Surgical Procedures of the Eye

In addition to cataract extraction and penetrating keratoplasty, glaucoma-filtering procedures are performed on
the anterior segment. In order to reduce intraocular pressure a fistula is created at the limbus (trabeculectomy).
In certain difficult cases (neovascular glaucoma) a silicone tube is placed in the anterior chamber to route
aqueous posteriorly to a filtration chamber placed at the equator of the eye. Strabismus procedures consist of
surgically transposing the insertion of a rectus muscle or oblique muscle or surgically shortening a muscle by
excising a portion of the muscle. At the site of insertion the sclera is reduced to half thickness to allow attachment
of the rectus muscle. Penetration of the globe is possible when resuturing rectus muscles. In cases of intraocular
tumor or advanced degeneration of the eye from trauma or other insults (atrophia bulbae, phthisis bulbi), the
entire eye is removed (enucleation). The volume of the eye is replaced with a plastic sphere and covered with
conjunctiva. A contact lens-like prosthesis (“glass eye”) is then placed behind the eyelids but anterior to the
conjunctiva. The prosthesis should be removed and cleaned similar to cleaning a contact lens. In some
circumstances, particularly with advanced endophthalmitis, and when there is no evidence of intraocular
malignancy, the cornea and ocular contents are removed leaving the rectus muscles attached to the sclera,
which is left in place (evisceration). Processed coral material (hydroxyl apatite) is placed in the scleral shell.
Fibrous tissue will grow into and stabilize the coral relative to the sclera. A prosthesis is attached to the coral by
a peg, which extends thorough a fistula in the conjunctiva. Increased mobility of the prosthesis is the clinical
advantage to evisceration versus enucleation.
Abnormalities of the Eye

Retinoblastoma is a malignant tumor of the retina, resulting from an uncontrolled proliferation of retinoblasts. The
malignancy is capable of widespread metastasis leading to death. The retinoblast is a pleuripotential
neuroectodermal cell that will differentiate into the various components of mature retina. This tumor has become
a model for heritable malignant tumors based on a genetic deletion of the Rb (retinoblastoma) gene. The tumor
initially proliferates in the plane of the retina but is capable of involving all structures within the eye. The tumor
may spread to the central nervous system via the optic nerve and through lymphatics and blood vessels to
tissues at distant sites. In the past 10 years retinoblastoma has changed from an almost uniformly fatal disease
to one in which 95% of the patients are stabilized using methods that preserve the eye and vision.
The genetic defect at chromosome 13q14 is associated with retinoblastoma in early childhood and is also
associated with malignant tumors in other tissues (e.g., pinealoblastoma, osteosarcoma) at later stages of life.
Thus, there is a risk of death not only from the initial retinoblastoma but from other second primary malignancies
as well.
The Knudson two-hit hypothesis states that retinoblastoma arises as a result of two mutational events (72, 73). If
both chromosomal 13q14 regions are normal, no retinoblastoma develops; if one of the two 13q14 regions is
abnormal, no retinoblastoma results. If both chromosomes 13 have a 13q14 deletion or functional abnormality,
retinoblastoma results. When both mutations occur in the same somatic postzygotic cell, a single unilateral
retinoblastoma results. Because the mutations occur in a somatic cell, this condition is, therefore, not inherited. In
the hereditary form, the first mutation occurs in a germinal prezygotic cell, which means that this mutation is
present in all resulting somatic cells and a second mutation occurs in the somatic postzygotic cells, resulting in
multiple retinal tumors as well as nonretinal tumors in other sites at different times of life, such as sarcomas. In
the inherited form with a germ-line mutation (i.e., a carrier of the retinoblastoma genetic abnormality), the
probability of developing the tumor is 95 in 100 (32).
The tumor arises when the retinoblastoma gene is absent (point deletion) or nonfunctional in affected cells. The
protein produced by the gene (RB1) is a phosphoprotein that inhibits progression through the cell cycle by
binding to DNA. The RB1 protein functions as a tumor suppressor in the retina by inhibiting proliferation and
promoting differentiation of retinal progenitor cells. In the absence of this protein there is an accumulation of
proliferating embryonic retinal cells (9, 138). In the eye the tumor arises in differentiated retina, may arise in
multiple sites of the same eye and may develop in both eyes.
Retinoblastoma is inherited in an autosomal dominant pattern with incomplete penetrance even though at a
cellular level the disease is autosomal recessive. In heritable retinoblastoma all of the 200 million cells of the
developing retina of an individual are susceptible to acquiring a second mutation. Even though the probability of
any one cell being damaged is low, the chance of one hit in 200 million is high.
Small subsets of cases of retinoblastoma arise in children with extensive deletions in chromosome 13. These
children in addition may exhibit holoprosencephaly, midface dysmorphism including cleft lip and palate, and
mental and growth retardation. This is the least frequent form of retinoblastoma (115).
Retinoblastoma is the most common intraocular neoplasm in children with a frequency of 1 in 16,000 to 1 in
20,000 live births in the United States (86, 111). The incidence of retinoblastoma decreases with age. There is
no significant sex or race predilection, and 20% to 35% of the cases are bilateral (resulting from germ-line
mutations), although only
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5% of all children with retinoblastomas have a family history of retinoblastoma (111). The tumor occurs in both
sexes and is found in all cultures and all geographic areas. The incidence of heritable retinoblastoma appears
stable. There is a recent suggestion that the incidence of nonheritable, unilateral retinoblastoma is increasing in
certain groups due to environmental influences (nutritional deficiencies and human papilloma virus infection).
The heritable form is likely when another family member has been identified with retinoblastoma. Approximately
40% of cases of retinoblastoma are heritable. This form is generally diagnosed before age 12 months, much
earlier than the nonheritable form. In 80% of cases of heritable retinoblastoma, retinal tumors are multiple in each
eye and are found in both eyes. An associated intracranial tumor (primitive neuroectodermal tumor or trilateral
retinoblastoma) is found in 2% to 3% of cases. The children who survive heritable retinoblastoma are at risk of
developing osteosarcoma, soft-tissue sarcoma, and other mesenchymal tumors during the first two decades of
life or later. In adulthood there is a significant risk of developing malignant melanoma and central nervous system
tumors. In the elderly there is an increased incidence of cancer of the bladder. The 30-year cumulative incidence
rate for second, nonocular, primary tumors is approximately 26%. The risk may be increased with radiation and
chemotherapy (109).
The nonheritable form arises spontaneously and comprises about 60% of cases. The average age at
presentation is 24 months. There is generally a single tumor in one eye. There is no detectable chromosomal
abnormality and, therefore, much less risk of developing retinoblastoma in succeeding generations. These
children have the same risk for second primary tumors, as does the general population.
Historically, retinoblastoma has presented as a fungating mass emanating from a ruptured globe. There was
often associated facial soft-tissue invasion and involvement of the central nervous system. Death usually
followed in the subsequent weeks or months.
Currently retinoblastoma is often discovered by parents or relatives who notice a difference between the quality
of the light reflex in one eye relative to the other either in person or on viewing family photographs (118) (Figure
11-25). At this relatively early stage, usually at age less than 3 years, the eye does not appear to be inflamed
and the child does not appear to be aware of loss of vision. The tumor, whether limited to the posterior retina, in
the vitreous or in the subretinal space associated with retinal detachment, will reflect the light that is normally
absorbed by blood pigments and melanin in the RPE and choroid. Children with retinoblastoma also may present
with strabismus (misalignment of the visual axes of the two eyes), iris neovascularization (response to retinal
ischemia leading to heterochromia, dilated fixed pupil, secondary glaucoma), or tumor accumulation in the
anterior chamber (neoplastic hypopyon). More advanced cases may present with signs of intraocular
inflammation (panophthalmitis), or ruptured globe with orbital extension. In some of cases of regressed
retinoblastoma, the sole clinical sign may be a small calcified tumor in the plane of the retina with surrounding
retinal pigment epithelial scarring.
FIGURE 11-25▪Retinoblastoma. The right pupil appears white (leukocoria) because of light reflecting off a large
intraocular tumor (retinoblastoma) through a clear lens and cornea. Cataract and corneal opacification are
features of only an advanced retinoblastoma that fills the entire eye.
Leukocoria (white pupil) is not an exclusive sign of retinoblastoma. Any condition that changes absorption of
ambient light to reflection of ambient light through the pupil may cause this sign. Some of the more common
nonretinoblastoma conditions presenting with leukocoria include PHPV (a developmental anomaly of the vitreous
resulting in intraocular fibrosis and retinal detachment), Coats disease (a developmental vascular malformation of
the retina leading to retinal detachment) and presumed ocular toxacariasis (a parasitic intraocular infection
leading to intraocular scarring and retinal detachment).
Clinical findings may be unilateral or bilateral. Bilateral cases are often asymmetric. Retinoblastoma appears
initially as an isolated or multicentric translucent-to-opaque thickening or globular expansion of the retina in any
quadrant of the eye (Figure 11-26). Larger tumors become vascularized with a feeding artery and a draining vein.
Focal opacities within larger masses correspond to areas of dystrophic calcification. The mass progressively
enlarges and expands into the vitreous where it may simulate vitreous inflammation or
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into the subretinal space causing a serous retinal detachment. The mass continues to enlarge to fill the entire
posterior compartment and displaces the lens-iris diaphragm anteriorly (Figure 11-27). Retinal ischemia
associated with tumor growth will promote neovascularization of the anterior iris surface. The anterior contour of
the iris flattens and the pupil may become distorted, enlarged, and nonreactive. The delicate neovascular
vessels may hemorrhage and deposit hemosiderin within the iris stroma that darkens the iris color
(heterochromia iridis). Neovascularization of the trabecular meshwork interferes with the egress of aqueous and
causes the intraocular pressure to rise (neovascular glaucoma). The sclera of a child is pliable and may
markedly expand in an anteriorposterior dimension resulting in a large eye (buphthalmos). Increased intraocular
pressure also causes corneal decompensation, opacification, and scarring. With additional tumor growth, the
tumor will seek sites of weakness in the eyewall (cornea and sclera). The largest opening is the scleral canal
containing the optic nerve, the most likely and earliest site of extraocular extension. The tumor may also extend
through any of the numerous scleral ostia; however, this stage in the evolution of the tumor is not visible
clinically. When extraocular, the tumor may extend through the lymphatics of the conjunctiva and infiltrate the soft
preseptal tissues of the orbit and facial lymph nodes. Direct expansion to the posterior septal orbital tissues is
usually across the posterior sclera, presenting as proptosis. The cornea is the most likely site of frank rupture of
the eye when the tumor completely fills the eye. Extension through the blood vessels of the choroid, orbit, and
face allows the tumor to spread to distant sites.
FIGURE 11-26▪Retinoblastoma. The intraocular tumor causing the leukocoria in Figure 11-25 extends into the
vitreous space from the retina. Areas of calcification and irregular vascularization are evident.
FIGURE 11-27▪Retinoblastoma. The tumor has filled the entire volume of the posterior segment and is displacing
the lens-iris diaphragm anteriorly. The cut surface of the tumor has a “brain-like” quality. Multiple calcific
highlights are present.
Fluorescein angiography of the retinoblastoma is characterized by early filling and late hyperfluorescense
associated with leakage of fluorescein into the vitreous. Echographic features of retinoblastoma include general
low internal reflectivity alternating with intense hyper-reflectivity in regions of dystrophic calcification. There is a
shadow effect posterior to thick areas of the tumor.
Standard radiography has been important in identifying intraocular opacities (dystrophic calcification) and
outlining signs of extraocular extension. Dystrophic calcification, however, can occur in nonneoplastic conditions,
particularly those associated with degeneration (e.g., following trauma). Computed tomography (CT) and
magnetic resonance imaging (MRI) allow more precise recognition of extraocular extension. These techniques
are particularly important in the detection of mass lesions in the pineal and suprasellar regions of the brain
(trilateral retinoblastoma). By CT imaging, retinoblastoma has approximately the same density as brain. By MRI
T1-weighted imaging, the tumor is hyperdense relative to the vitreous, and by T2-weighted imaging, the tumor is
hypodense relative to the vitreous. There is minimal-to-marked enhancement on contrast-enhanced T1-weighted
images with fat suppression techniques.
In most cases of retinoblastoma, the external dimensions of the eye are normal for the patient’s age. The
exceptions are rare and are associated with developmental abnormalities affecting the size of the globe (e.g.,
microphthalmos) and advanced cases with buphthalmos or frank rupture of the globe. On gross sectioning the
tumor has a brain-like consistency associated with focal areas of dystrophic calcification. The tumor may arise in
any region of the retina. The location of greatest clinical significance is near the optic disc.
There are several growth patterns. The tumor may remain confined to the plane of the retina usually at the
retinal periphery in a rare variant of retinoblastoma, diffuse infiltrating retinoblastoma. The usual tumor is densely
white or gray with an irregular outline that is sharply demarcated from surrounding differentiated, uninvolved
retina. In most cases the tumor invades the vitreous (endophytic growth pattern), into the subretinal space
(exophytic growth pattern) or both. Tumor within the vitreous is poorly supported by blood vessels and develops
extensive areas of necrosis giving it a friable character that appears similar to inflammation within the vitreous.
This form of tumor extension may also be associated with metastatic seeding to the surface of the retina or optic
disc making the distinction between multiple primary sites and multiple metastatic sites difficult. The posterior
chamber, the anterior chamber, and the surface of the optic disc may similarly be seeded by tumor from the
vitreous. Tumor in the subretinal space is associated with serous fluid accumulating in the subretinal space and
detaching the overlying retina. Advanced tumors may invade the choroidal tissues (26).
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The final growth pattern is regression. Retinoblastoma may progress in one eye and regress in the other. The
remaining tumor tissue is often extensively calcified and surrounded by retinal pigment epithelial reaction for a
variable distance from the main tumor.
The cross-sectional diameter of the optic disc in most patients is 1.5 mm. Immediately posterior to the lamina
cribrosa, the ganglion cell axons acquire a myelin coat increasing the cross-sectional diameter of the optic nerve
(dural sheaths and neural axis) to 3.0 mm. Any optic nerve cross-sectional diameter, greater than 3.0 mm,
harbors extraocular retinoblastoma until proven otherwise. The desired length of the optic nerve specimen is a
minimum of 10 mm. Removal of 20 mm of optic nerve is technically possible, even in a child. A short optic nerve
specimen is a prognostic risk factor.
It is unusual for a cataract to form, except in the most advanced tumors characterized by extensive necrosis. In
these cases iris neovascularization and anterior chamber hemorrhage (hyphema) may be a presenting clinical
feature.
The majority of the tumor cells are characterized by a large vesicular nucleus with homogeneously dispersed
chromatin of variable shape and size without a nucleolus. There is only a small amount of visible cytoplasm.
Retinoblastoma cells are positive with S100 but are usually negative with glial fibrillary acidic protein.
Ultrastructurally, the cells contain few internal organelles. In some regions there may be triplication of the nuclear
membrane. Numerous mitotic figures are present throughout the tumor. There may be some background cells
with features of glial cells; however, it is difficult to distinguish neoplastic glial cells from reactive glial cells
originating in surrounding normally differentiated retina. Outside the confines of the retina (e.g., in the subretinal
space) retinoblastoma cells tend to adhere to each other in small clusters. Multiple bizarre cells may be present.
Inflammatory cells and macrophages may be present in vitreous samples.
There are several types of more differentiated cells generally grouping in the form of rosettes. Rosettes are
composed of one or two layers of nuclei encircling a central space. Mitotic figures may be seen in the cells
making up the rosettes. Some rosettes are incompletely formed and blend with the surrounding undifferentiated
cells. Rosettes are usually found in random areas of greater differentiation rather than within areas of totally
undifferentiated retinoblasts.
The most primitive and least specific of the forms is the Homer-Wright rosette. It is composed of poorly
differentiated cells with definite epithelial characteristics. The central portion of the rosette does not form a
definitive lumen but contains neurofibrillary processes and is thought to be composed of cells with ganglion cell
characteristics. This type of configuration is found in neuroblastoma of the adrenal gland and cerebellar
medulloblastoma among others. The Homer-Wright rosette appears much less frequently in retinoblastoma than
the Flexner-Wintersteiner rosette.
The Flexner-Wintersteiner rosette is more differentiated and more specific for retinoblastoma as compared with
the Homer-Wright rosette (Figure 11-28). The layer of cuboidal cells is taller and has a more definite epithelial
character. The apical portion of the cell forms an inner limiting structure of terminal bars, delimiting the cells from
a central lumen. The central lumen contains acid mucopolysaccharide that is similar to the acid
mucopolysaccharide found in the interphotoreceptor mucoid substance. Some cells may have characteristics of
inner photoreceptor elements such as abundant nuclei, cytoplasmic microtubules, and 9 + 0 cilia. Occasionally
laminated membranous structures resembling the discs of rod outer segments have been identified.
FIGURE 11-28▪Retinoblastoma. Flexner-Wintersteiner rosettes are an indication of tumor differentiation.
The fluerette is the most differentiated and is the most specific for retinoblastoma but is identified in only 6% of
cases of retinoblastoma. This type of rosette is more linear than round and is composed of more differentiated
cells with small less basophilic nuclei and prominent eosinophilic cytoplasm. Cytoplasmic processes extend
through a fenestrated membrane in a cluster-like configuration suggesting a bouquet of flowers (i.e., a
“fleurette”). There may be associated areas of deposited calcium, but mitotic figures are rare in fleurettes and
there is no necrosis. The cells have ultrastructural characteristics of cone photoreceptors.
Retinoblasts spread initially in the plane of the retina. There does not seem to be any architectural resistance of
either the inner or outer retina to the advance of the tumor cells. The tumor spreads across the inner limiting
membrane into the substance of the vitreous. In the vitreous, proliferation of blood vessels appears to be limited.
Tumor cells are arranged in sleeves around dilated blood vessels originating in the retina. Approximately 50 to
200 cells are seen surrounding the lumen of blood vessels in contrast with the one to two cell layers that make
up a true rosette. The thickness of the sleeve depends on the metabolic activity of the cells of the tumor. If
twenty to one-hundred and ten micrometer from the vessel lumen there is ischemic necrosis and dystrophic
calcification but little or no inflammatory infiltrate. Extensive cellular necrosis leads to liberation of substantial
amounts of DNA that can deposit and be identified by light microscopy
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along the internal limiting membrane of the retina, along blood vessel walls, and along the posterior crystalline
lens capsule.
FIGURE 11-29▪Retinoblastoma. Both viable and necrotic tumor has extended into the anterior chamber in a case
of advanced retinoblastoma (Hematoxylin-eosin stain, original magnification ×40).
The mode of spread for retinoblastoma includes local extension, extension into the optic nerve and intracranial
spread, and distant metastases. Spherules of tumor cells separate from the primary tumor in the vitreous and
deposit on the inner limiting membrane and secondarily reinvade the retina at a site distant from the original
tumor. Spherules also gain access to the posterior chamber where aqueous convection currents carry the cells
to the iris surface and trabecular meshwork of the anterior chamber (Figure 11-29). Aqueous seeding may
simulate a hypopyon.
Tumor cells readily cross the external limiting membrane of the retina and enter into the subretinal space (Figure
11-30). Disturbance of retinal pigment epithelial function due to the presence of tumor cells breaks down the
blood retinal barrier and allows fluid to accumulate in the subretinal space (serous retinal detachment). There is
no secondary neovascularization to support tumor cells in the subretinal space. Nutrition appears to be derived
from the serous fluid itself. Tumor cells become configured into spherules as in the vitreous cavity; however, the
inner most cells of the spherules tend to be necrotic rather than the externally situated cells in the vitreous. Cells
may obtain access to the space under the RPE and across Bruch membrane and choriocapillaris to the vessel-
rich choroidal layer.
FIGURE 11-30▪Tumor cells (arrows) have extended from the plane of the retina into the subretinal space
(Hematoxylin-eosin, original magnification ×20).
FIGURE 11-31▪Retinoblastoma. The intraocular retinoblastoma has extended to the superficial tissues of the
optic disc but not through the lamina cribrosa (Hematoxylin-eosin stain, original magnification ×20).
Retinoblastoma spreads in the plane of the retina, to the optic disc, through the lamina cribrosa into the
substance of the optic nerve (Figure 11-31). Once beyond the lamina cribrosa, extraocular extension has
occurred (Figure 11-32). The tumor in the optic nerve axis has access to the subarachnoid space through which
it is able to spread throughout the central nervous system (Figure 11-33).
In the uveal tract, tumor cells have access to the intravascular compartment and may spread extensively to the
liver,
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bones, and lungs. In the anterior chamber, tumor cells may traverse the trabecular meshwork to gain access to
episcleral tissue including the lymphatics of the conjunctival stroma. The lymphatic channels collect at the
preauricular nodes and submental nodes of the soft tissues of the face.
FIGURE 11-32▪Retinoblastoma. The tumor has spread beyound the lamina cribrosa to the optic nerve
(Hematoxylin-eosin stain, original magnification ×20).
FIGURE 11-33▪Retinoblastoma tumor cells have completely replaced the axons of the optic nerve (Hematoxylin-
eosin stain, original magnification ×20).
Tumor cells may escape the eye along surgical wounds in those unfortunate cases where retinoblastoma has
been misinterpreted as a congenital cataract and the cataract has been surgically removed.
In terms of prognostic factors, besides tumor size and location, a differentiated tumor with abundant Flexner-
Wintersteiner rosettes has a better prognosis than one without rosettes. Similarly, a tumor composed entirely of
fleurettes (retinocytoma) has a much better prognosis (73). Although many factors affect the prognosis, the most
important is the extent of invasion by the retinoblastoma, with extension into the optic nerve and ocular coats
being the two most important predictors of outcome (77) and extraocular invasion being the most important
predictor of death. Massive choroidal invasion and extension into the sclera are associated with a high incidence
of systemic metastases. With respect to assessing extraocular extension, it is important to note that isolated
episcleral “free-floating” tumor cells may sometimes represent artifact of dislodged tumor cells during opening of
the globe. Full-thickness choroidal involvement is associated with 60% mortality. Subretinal pigment epithelial or
superficial choroidal extension is frequent and is not very significant. Uveal inflammation in the presence of
choroidal invasion is associated with a poor prognosis. Thus, massive choroidal involvement, deep choroidal
involvement with emissarial extension short of the surface of the eye, concomitant choroidal inflammation, and a
large tumor are associated with an adverse outcome (102). With respect to invasion of the optic nerve, invasion
up to but not beyond the lamina cribrosa has relatively little prognostic significance, but invasion up to the line of
transection carries a poor prognosis. Tumor beyond the lamina cribrosa and involving the pia arachnoid also is
associated with a poor prognosis (111). Presence of iris neovascularization is a poor prognostic sign and may
relate to the quantitative volume of tumor and to significant choroidal invasion (102). Besides local extension and
intracranial involvement, distant metastases may involve long bones and skull, viscera (most often the liver), and
lymph nodes.
Medulloepithelioma is a rare tumor originating from the medullary epithelial cells of the optic vesicle that have
differentiated toward the epithelium of the ciliary body. There is a bimodal distribution of tumors presenting as
congenital lesions in children and acquired lesions in adults. In both instances, the clinical and histopathologic
distinction between benign and malignant lesions may be difficult in the early stages of tumor progression. The
single best differentiating feature is invasion of adjacent tissues and even that finding can be found in tumors
with an indolent course.
Congenital medulloepitheliomas tend to arise in the first decade of life presenting with pain, decreased vision, a
sectoral cataract (leukocoria), or increased intraocular pressure (11). The tumors are not heritable. A ciliary body
mass is found by clinical examination. The tumors tend to be white or gray with an irregular, sometimes cystic
surface. The cystic components of the tumor may separate from the primary mass and may float freely in the
anterior chamber or even in the vitreous. Infrequently the tumor may arise in the plane of the RPE or along the
course of the optic nerve. The tumor is composed of primitive neuroblastic cells arranged in chords or sheets of
cells associated with an extracellular matrix containing hyaluronic acid. Flexner-Wintersteiner (photoreceptor
differentiation) and Homer-Wright rosettes (ganglion cell differentiation) lined by a single layer of cells may be
present. In addition, primitive rosettes (ciliary epithelial differentiation) may be present; however, this type of
rosette is lined by several layers of undifferentiated neuroepithelial cells. Reactive proliferation and formation of a
cellular membrane may also occur and extend across the vitreous face. Because there is often an inconsistent
degree of pleomorphism and variable mitotic activity, the natural history may be difficult to predict by cytological
features. Invasion of adjacent uveal structures, especially extension to and through the sclera, is a distinct risk
factor for additional local invasion, although the tumor only rarely produces distant metastasis (54). Tumors
significantly affecting ocular function are often treated with enucleation because of the uncertainty of the natural
history of any individual tumor and the difficulty in determining the significance of involvement of adjacent
structures, particularly the vitreous (33).
A subgroup of medulloepitheliomas (teratoid medulloepithelioma) contain heterotopic elements, particularly
cartilage, however brain and muscle tissue may also be present. Again, histological clues to a malignant course
are not distinctive
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enough for certain categorization. Tumors with heterotopic elements tend to have a more aggressive course.
Treatment criteria for the two groups are similar because the heterotopic elements cannot be distinguished
clinically with any degree of certainty. The overall mortality in one series was reported in the range of 10% (11).
Benign and malignant acquired medulloepitheliomas also occur, but arise most often in adults. Again, the
distinction between benign and malignant lesions may be difficult. Treatment is usually surgical depending on
symptoms and volume of tumor (33, 54).
Fuchs adenoma is a benign proliferation of fully differentiated ciliary epithelium of adults that is found in up to
30% of autopsy series (5, 66). The lesion is of no clinical significance.
PEDIATRIC GLAUCOMA
Glaucoma is an imbalance between production of aqueous and drainage of aqueous into the systemic
circulation. Any developmental abnormality of the anterior segment may lead to glaucoma; the more extensive
the architectural abnormality the more likely is glaucoma to develop. In the vast majority of cases, the imbalance
is caused by abnormalities of filtration rather than overproduction of aqueous. Increased intraocular pressure will
not decrease aqueous production until the intraocular pressure is in the range of the diastolic blood pressure. In
the pediatric age group the tissues of the eye remain pliable to the point where increased intraocular pressure
may actually expand the dimensions of the eye leading to apparent enlargement of the cornea and anterior-
posterior dimensions of the globe (buphthalmos). The expansion is not uniform enlargement of the globe, rather
it is stretching of the thinnest portion of the eyewall at the junction of the cornea and sclera (the intercalary
zone)). In advanced cases of glaucoma from any cause, the anterior chamber may collapse allowing the anterior
surface of the iris to come in contact with the posterior surface of the cornea (total anterior synechiae), further
limiting the ability of aqueous to exit the eye. The front of the eye may bulge forward (ectasia) and the ectatic
area may become lined by iris (anterior staphyloma).
In many cases of congenital glaucoma there is no histological sign of architectural abnormality of the anterior
chamber angle including the delicate trabecular meshwork. However, there is a range of anatomic abnormalities
in developmental disturbances from total immaturity of the draining structures to regional minimal structural
changes. Except in extreme cases, the intraocular pressure cannot be predicted from the nature of the
architectural changes.
In the pediatric age group, one of the most common forms of secondary glaucoma is neovascular glaucoma. In
this situation ischemia of the retina induces formation of local angiogenic factors [e.g., vascular endothelial
growth factor (VEGF)]. The process stimulates angiogenesis of the anterior surface of the iris. This fibrovascular
growth flattens the contour of the anterior iris (clearly seen by light microscopy) and also causes adhesions
between the anterior surface of the iris and the posterior surface of the peripheral cornea [peripheral anterior
synechia (PAS)]. The PAS limit aqueous access to the trabecular meshwork and cause increased intraocular
pressure. This mechanism is found in retinopathy of prematurity, advanced retinoblastoma, and uncontrolled
diabetic retinopathy, among others.
Sustained increased intraocular pressure causes degeneration of the ganglion cell and nerve fiber layer of the
retina. The exact cause for internal retinal atrophy has not been definitely determined for all types of glaucoma.
In addition, there is retrodisplacement of the structural support of the optic disc (the lamina cribrosa), which is a
finding unique to increased intraocular pressure. In other forms of atrophic optic neuropathy the position of the
lamina cribrosa relative to the surrounding sclera is not affected. Progressive loss of retinal ganglion cell axons
progressively increases the cup-to-disc ratio to the point of “total cupping” of the optic disc. This clinical finding
can be confirmed by anterior-posterior histological sections of the eye if the plane of section is through the optic
disc. Total optic cupping correlates with total loss of optic nerve axons, widening of pial septa, and enlargement
of the subretinal space. The character of the dura is not changed by increased intraocular pressure.
OCULAR TRAUMA
The most common indication for the surgical removal of an eye in the pediatric age group is trauma. Loss of
visual function of the eye is usually due to a combination of hemorrhage, inflammation, and ultimately glaucoma.
Accidental trauma to the eye is generally categorized into nonpenetrating or penetrating trauma. The distinction
is important in guiding the initial therapy of the injured eye. Generally, a nonpenetrating injury does not require
surgical repair, although the degree of injury in many cases exceeds that found in penetrating injury. Surgical
repair is usually necessary in cases of penetrating trauma (open globe injury).
In most cases of severe trauma treated with enucleation there is a rupture or laceration of the corneal-scleral
coat, the “eye wall”. Most ruptures are found in the region of the corneal sclera limbus, which is a normally thin
region of the eye wall. Lacerations are also most commonly found in the anterior eye wall but may also be
located posteriorly. By the time of enucleation there usually has been fibrovascular repair of the eye wall wound
that can be identified by discontinuity of the collagen pattern, rupture of Bowman or Descemet membrane, or
interruption of one of the pigmented coats of the eye. The anterior chamber is usually disorganized and PAS are
present. The lens may be totally absent, be represented by crystalline lens remnants (best identified by Periodic
acid/Schiff staining), or show changes of anterior or posterior fibrous metaplasia of the crystalline lens epithelial
cells. The retina is most often detached with blood or serous fluid in
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the subretinal space. The surface of the retina may undulate due to the formation of a contracted membrane on
its surface (epiretinal membrane). The vitreous may be contracted and filled with inflammatory cells or with
proliferating fibrovascular tissue (proliferative vitreoretinopathy). There may be hemorrhage within, superficial to,
or deep to any of the coats of the eye. The choroid may be infiltrated by nongranulomatous or granulomatous
inflammatory cells either diffusely (see below) or focally. The optic nerve frequently has signs of early or
advanced atrophy (14).
The eye may be collapsed, if there has been loss of intraocular contents, including the lens, vitreous, and retina.
Intraocular foreign material may be present, depending on the nature of the original injury. Identifying foreign
material is aided by the use of polarized light.
The eye is removed within 2 weeks, if there is no clinical indication of retention of useful vision (no light
perception) in order to reduce the risk of losing vision in the contralateral eye because of sympathetic ophthalmia
(14).
Sympathetic ophthalmia is a bilateral granulomatous inflammation of the uveal tract appearing 5 days to many
years following trauma to one of the two eyes. The inflammation is thought to be due to an autoimmune response
to an unknown type of antigen that is expressed during trauma. The first clinical indication of the presence of
sympathetic ophthalmia is a loss of the ability to focus at near objects (accommodation) followed by a
generalized uveitis. Untreated, the uveitis in the initially uninvolved eye may be more severe than in the injured
eye. The major histological sign is a granulomatous inflammatory response in any portion of the uveal tract
characterized by epithelioid histiocytes and giant cells. The giant cells may contain melanin pigment, but this
finding is not specific to sympathetic ophthalmia. There is generally an intense infiltrate of lymphocytes but not
plasma cells or eosinophils in the surrounding tissue. Epithelioid histiocytes also accumulate between the RPE
and Bruch membrane (Dahlen-Fuchs nodules) (Figure 11-34). Dahlen-Fuchs nodules may also be found in
sarcoid uveitis. There may be some sparing of the choriocapillaris in the noninjured (sympathizing) eye, but the
finding is also not specific for sympathetic ophthalmia. An inflammatory reaction to exposed lens protein (lens-
induced uveitis or phacoanaphylactic endophthalmitis) is also found in some cases. All of the histological findings
are nonspecific. To establish the diagnosis of sympathetic ophthalmia there must be a history of some type of
ocular trauma, which may include such surgical procedures such as cyclocryotherapy (freezing of the ciliary body
to treat intractable glaucoma) or pars plana vitrectomy (see above).
Eyes removed within the 2-week risk period for sympathetic ophthalmia may still have suture material at sites of
penetration of the sclera and may also have surgical appliances used in retinal detachment repair (scleral
buckles) and treatment of glaucoma (glaucoma filtration devices) on the episcleral surface. There may be
considerable fibrosis from the episcleral tissue at sites of injury and at surgical sites even a few days after the
original injury.
FIGURE 11-34▪Sympathetic ophthalmia. There is a massive chronic granulomatous inflammatory infiltration of
the uveal tract. Epithelioid histiocytes have accumulated between the RPE and Bruch membrane (arrow). The
features of the retina are distorted by trauma, inflammation, and sectioning artifacts (Hematoxylin-eosin stain,
Eyes removed months or years following trauma are generally small, shrunken, and have assumed a cuboidal
shape (phthisis) (Figure 11-35). The ocular degeneration may be so advanced as to make identification of
laterality difficult. The most reliable landmarks are the insertion of the superior and inferior oblique muscles. On
sectioning, there may be extreme resistance because of dystrophic calcification both in the remaining lens tissue
and in the plane of the RPE. Decalcification for several days is often necessary. Histologically, there is often
scarring and vascularization of the cornea, complete fibrosis of the anterior chamber, atrophy of the iris, cataract
(potions of which may be calcified), total retinal detachment with atrophy and gliosis, fibrosis of the vitreous, and,
most often, profound optic atrophy. The most important observations are those of the uveal tract to determine the
presence or absence of sympathetic ophthalmia.
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In adults, it is also important to determine if this “blind painful eye” has been harboring a neoplasm such as
malignant melanoma (14).
FIGURE 11-35▪Phthisis. This eye has degenerated following surgical repair for a detached retina. The eye is
small and cuboidal in shape.
AUTOPSY SPECIMEN
Removing the eyes of a child at autopsy is a very uncommon event, except when there is suspicion of child
abuse homicide (16). Globes may be removed via an anterior approach as with surgical enucleation or may be
removed through a window created in orbital roof. Whichever approach is used, it is important to obtain as much
optic nerve as possible and to obtain a sample of orbital fat.
In most cases of death due to shaking or simple blunt force injury in which death occurred shortly following the
injury there is little external sign of trauma. There is also usually no abnormality of the cornea, anterior chamber,
or external surface of the globe. It is important to note any signs of scleral thinning indicated by a blue tinge of
the sclera, which may be found in very young infants or in individuals affected by osteogenesis imperfecta.
Subdural and subarachnoid hemorrhage of the optic nerve is indicated also by a blue-togray discoloration of the
dura. Cross sections of the optic nerve itself are normal in diameter and character, however, there may be
marked expansion of the dural and subdural spaces by hemorrhage (Figure 11-36). On sectioning of the eye the
cornea, lens, and anterior chamber generally are normal. Retinal hemorrhages may be present. The location and
extent of the intraretinal hemorrhage, particularly if the hemorrhage extends to the ora serrata, is an important
observation (Figure 11-37). The retina in the region of the macula may also be elevated. Hemorrhage may
extend into the vitreous itself. Histologically, the hemorrhages may be located completely within the architecture
of the retina (intraretinal), between the neurosensory retina and the RPE (subretinal), between the retina and the
cortex of the vitreous (subhyaloid), or within the vitreous (intravitreal). There may also be signs of disruption of
the internal limiting membrane in the region of the macula. Hemorrhage may also be noted in the sclera at the
insertion of the dura of the optic nerve (the circle of Zinn-Haller). Hemorrhage may be found in the surrounding
orbital fat (see Chapter 7).
FIGURE 11-36▪Nonaccidental trauma. A: The cross section of the optic nerve is normal at 3.0 mm. There is
extensive hemorrhage in the subdural and subarachnoid spaces. B: Subdural and subretinal hemorrhages are
present in this low magnification view.
In cases where the child died after a longer interval from abuse, the hemorrhages may be less apparent. There
may be atrophy and gliosis in the region of resolved retinal hemorrhage. There may or may not be hemosiderin
staining in the area of suspected former hemorrhage. There may be considerable optic atrophy.
SUMMARY
The most common eyelid specimens in the pediatric age group usually consist of inflammatory lesions:
Molluscum contageosum and chalazion. Most important lesions would be those of metastatic neuroblastoma
in very young children and rhabdomyosarcoma in slightly older children. There is a variant of
rhabdomyosarcoma with a subconjunctival presentation, the botryoid variant. Basal cell carcinoma and
squamous cell carcinoma can occur with xeroderma pigmentosa but are otherwise uncommon. Sebaceous
cell carcinoma can occur but again is extremely rare.
Conjunctival nevus may be an important clinical problem in the pediatric age group. Malignant melanoma
can occur in very young children but most of the pigmented lesions will be nevi. Concern is generated
because of enlarging size due to expansion of subepithelial squamous cysts, increased pigmentation found
during adolescence, and inflammation of the nevus from a combination of factors. Pterygium and squamous
cell carcinoma are found generally in a much older age group.
Corneal tissue is most often evaluated because of surgical treatment of keratoconus. Confirmatory findings
include focal ruptures of Bowman membrane and occasionally rupture of Descemet membrane (corneal
hydrops). Other corneal
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specimens will be submitted following accidental trauma and will show evidence of repair with fibrous
proliferation. Corneal dystrophies, except for CHED in some restricted geographic areas, are not commonly
treated with surgery in this age group. An important biopsy evaluation would be for acanthamoeba keratitis,
particularly if there is a history of correction of refractive error with soft contact lenses.
FIGURE 11-37▪Nonaccidental trauma. A: Hemorrhages are found throughout the retina (blackarrows) and
extend as far anteriorly as the ora serrata (yellow arrow). B: Intraretinal hemorrhage is shown extending to
the ora serrata.
Cataracts in this age group are treated surgically, but the tissue is generally not examined histologically.
Retina, vitreous, and uveal tract are very infrequently evaluated by fine needle aspiration biopsy. The major
exception would be anterior chamber paracentesis for diffuse infiltrating retinoblastoma as a differential
diagnosis in the evaluation of protracted intermediate uveitis, both found in the older child-younger teenage
group. Congenital melanoma of the uveal tract has been reported but uveal melanoma generally occurs in
the fifth to sixth decade.
The most common orbital lesions in children include ruptured dermoid cyst and various vascular
developmental abnormalities such as lymphangioma. Idiopathic orbital inflammation (orbital inflammatory
pseudotumor) can occur in children where the presentation is often bilateral and the progression more
aggressive. Rhabdomyosarcoma has a predilection for orbital tissue in children. Surgical treatment for
rhabdomyosarcoma is now not as common as formerly, being replaced currently by combinations of
chemotherapy and occasionally radiation. The biopsy of orbital tumor tissue is often essential in managing
treatment strategies for rhabdomyosarcoma. Adenoid cystic carcinoma of the lacrimal gland can occur in
children. Its treatment at any age is difficult as the outcome tends to be poor.
The treatment of retinoblastoma is in rapid evolution. Enucleation, once the standard of care for all cases of
retinoblastoma, is now done selectively and often after prior treatment with chemotherapy, cryotherapy,
photocoagulation, transpupillary thermotherapy, and, occasionally, radiation. All therapeutic efforts will
change the histological appearance of the primary tumor. Definite histologically defined risk factors,
especially optic nerve involvement by the retinoblastoma tumor, continue to guide therapy when enucleation
is performed. The most important part of gross examination of a retinoblastoma eye is extremely careful
evaluation of the surgical margin at the site of transaction of the optic nerve. Sections of the retinoblastoma
eye must include levels through the optic disc.
Most eyes enucleated in children are the result of irreparable trauma to the eye. The most important
histological observations include those for diffuse granulomatous inflammation of the uveal tract with or
without signs of Dalen-Fuchs nodules (i.e., sympathetic ophthalmia). Sarcoidosis and other inflammatory
lesions may have exactly the same histological appearance as sympathetic ophthalmia. Correlation of
histopathologic findings of the pathologist with clinical findings of the ophthalmologist is essential in
establishing the diagnosis of sympathetic ophthalmia. Eyes removed many years following the original
trauma may even become small externally and distorted internally (phthisis bulbi). The evaluation for
sympathetic ophthalmia remains an important function of the pathologist even if the interval between the
injury and enucleation has been decades.
Autopsy eye specimens are usually collected for assessment of ocular developmental abnormalities as part
of evaluation for congenital syndromes (e.g., trisomy 13 or 18) or as a part of a homicide investigation for
child abuse. In cases of suspected nonaccidental injury, important observations include external signs of
ocular injury; the apparent thinness of the sclera (osteogenesis imperfecta); sign of cataract formation; the
presence, location, and extent of retinal hemorrhage; the presence and extent of vitreous hemorrhage; the
presence and extent of subretinal hemorrhage; signs of traction retinal detachment; signs of intrascleral
hemorrhage at the sclera insertion of the dura of the optic nerve (Circle of Zinn-Haller); the presence of
hemorrhage in the soft tissues of the orbit; presence and extent of subdural and subarachnoid hemorrhage;
and finally the presence and degree of optic atrophy.
P.438
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Chapter 12
The Respiratory Tract
J. Thomas Stocker
Haresh Mani
Aliya N. Husain
DEVELOPMENT OF THE LUNG

The lung begins as a pouch or groove originating from the primitive foregut in week 3 of embryologic
development, when the embryo is 3 mm long. As the groove enlarges caudally, a tubular lung bud is formed; the
upper portion develops into the epithelium of the larynx, and the caudal portion into the epithelium of the
tracheobronchial tree (1).
The embryonic period of lung development (Table 12-1) begins in week 4 of gestation as the single lung bud
from the foregut divides into two primary bronchial buds, the forerunners of the right and left lungs (Figure 12-1A
to C). During week 5 of gestation, the primary bronchi divide. Each forms three lobar buds that, by the end of
week 6 of gestation, divide again to form 10 segmental bronchi on the right and eight to nine on the left. These
potential airways consist of a central core of epithelial cells surrounded by loose primitive mesenchyme that
contains widely separated capillaries. The primitive pulmonary arteries begin to form from the sixth aortic arch,
near the end of the embryonic period (2). The pulmonary veins begin as evaginations of the left atrium during
week 4 of gestation and coalesce with the mesenchymal capillary plexus early in week 5 (3).
The pseudoglandular period (weeks 6 to 16 of gestation) begins with the completion of the proximal airways and
encompasses the development of the conducting airway system to the level of the terminal bronchioles (Figure
12-2A,B). The pseudostratified columnar epithelium of the proximal airway displays cilia at week 10 of gestation.
The appearance of cilia extends to the epithelial cells of the peripheral airways by week 13. Goblet cells appear
in the bronchial epithelium at weeks 13 to 14 of gestation, and submucosal glands begin as solid buds
originating from the basal layers of the epithelium by weeks 15 to 16. Smooth muscle cells develop around
airways by the end of gestational week 7 and organize to form an identifiable wall to the larger bronchi by week
12. Lymphatics appear first in the hilar region of the lung in gestational week 8 and in the lung itself by week 10
(4). Cartilage is first seen in week 4 of gestation and forms distinct rings along the trachea and main bronchi by
the end of week 10.
The acinar or canalicular period extends from weeks 17 to 28 of gestation and is characterized by the
development of the basic structure of the gas-exchanging portion of the lung (Figure 12-3A,B). Smooth-walled
respiratory bronchioles, lined by cuboidal epithelium, subdivide into multiple, irregular alveolar ducts. By week 20
of gestation, the cells lining the ducts develop into type II alveolar lining cells with lamellar and multivesicular
bodies associated with surfactant synthesis. Type I alveolar lining cells then differentiate from type II cells to form
the thin air-blood interface required for gas exchange. As the interstitium thins in the latter portion of the acinar
period, the capillaries of the interstitium proliferate and come to lie beneath the type I cells. Submucosal glands in
the trachea and bronchi progress from tubules to mucuscontaining acini. By week 24, the cartilage has extended
to the most distal bronchi.
Table 12-1 ▪ PHASES OF INTRAUTERINE LUNG DEVELOPMENT
Gestation
Phase Period Major Event
Embryonic 26 days to Development

6 weeks of major
airways
Pseudoglandular 6-16 Development

weeks of airways to
terminal
bronchioles
Acinar or canalicular 16-28 Development

weeks of acinus and
its
vascularization
Saccular 28-34 Subdivision of

weeks saccules by
secondary
crests to term
Alveolar 34 weeks Alveolar

(and acquisition
beyond)
Adapted from Langston C. Prenatal lung growth and pulmonary

hypoplasia. In: Stocker JT, ed. Pediatric pulmonary disease.
Washington, DC: Hemisphere, 1989:2, with permission.
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FIGURE 12-1 ▪ Embryonic periods in respiratory tract development. A: At 29 to 31 days gestation (stage 14), the
primary bronchial buds are surrounded by primitive mesenchyme. Note the small esophagus above and between
the bronchi. (Hematoxylin and eosin stain, original magnification ×75.) B: By 35 to 37 days (stage 16), the
primary bronchi have divided into secondary and early tertiary buds. Note the centrally located esophagus and
the large amount of hepatic parenchyma (lower half). (Hematoxylin and eosin stain, original magnification ×60.)
C: In a sagittal plane of a 37- to 40-day (stage 17) embryo, the relationship between the esophagus (nearest
vertebral column) and trachea (between esophagus and heart) can be seen. The heart and liver are ventral to
the foregut structures. (Hematoxylin and eosin stain, original magnification × 20.)
The saccular period begins at week 28 of gestation with the development of secondary crests, which are formed
as distal airspaces divide into smaller units (Figure 12-4A,B). With an accompanying marked decrease in the
interstitial tissue and further increase in the capillary bed, a complex, interwoven capillary network develops in
the wall of the saccules. This provides for effective gas exchange as alveoli begin to develop at the end of the
period (32 to 36 weeks of gestation).
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FIGURE 12-2 ▪ Pseudoglandular period. A: At 9 weeks gestation, the proximal airways are present throughout
the right and left lobes. (Hematoxylin and eosin stain, original magnification ×30.) B: By 13 weeks, bronchiolar
development is well under way and early division into lobules and clusters of acini is apparent. (Hematoxylin and
eosin stain, original magnification ×40.
The final period of development, the alveolar period, begins in utero at 32 to 36 weeks of gestation and extends
until 18 to 24 months after birth. Alveoli develop as flask-shaped structures with thin walls whose double capillary
network meshes to appear as a single capillary bed (Figure 12-5). At term, type I alveolar cells are extremely thin,
resulting in an air-blood barrier of only 0.2 μm including the type I cell, the underlying basement membrane, and
the cytoplasm of the capillary endothelial cell. Lymphatic channels are distributed around pulmonary arteries,
bronchi, and bronchioles
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and extend along interlobular septa to anastomose with a plexus beneath the pleura. Lymphatic spaces do not
exist between alveoli (4).
FIGURE 12-3 ▪ Acinar period. A: In a 370-g fetus, acinar development is characterized by pulmonary arteries
and proximal bronchioles surrounded by alveolar ducts still widely separated by mesenchymal tissue.
(Hematoxylin and eosin stain, original magnification ×40.) B: The alveolar duct structures are lined by cuboidal
epithelium (early type II cells), but blood-filled capillaries are present just beneath the cells. (Hematoxylin and
eosin stain, original magnification × 425.)
FIGURE 12-4 ▪ Saccular period. A: In a 650-g fetus, discrete acini are identifiable within a lobule. (Hematoxylin
and eosin stain, original magnification × 50.) B: Secondary crests are covered by thinning type I cells, which
expose capillary beds immediately beneath the cells. (Hematoxylin and eosin stain, original magnification × 350.)
The vascular supply of the lung changes appreciably in late gestation and infancy. The bronchial arterial
circulation, originating from the aortic arch, supplies the bronchi, bronchioles, and interlobular septa in older
children and adults; however, the bronchial artery contributes substantially to the circulation of the alveolar ducts
and alveoli in the central portions of the lungs through bronchopulmonary artery anastomoses in utero and in
early infancy (5).
FIGURE 12-5 ▪ Alveolar period. At 2 months of age, a respiratory bronchiole (left) gives rise to alveolar ducts,
alveolar saccules, and thin-walled alveoli. (Hematoxylin and eosin stain, original magnification × 50.)
At birth, the surface area of the lung is about 4 m2, with the number of alveoli ranging from 10 to 150 million
(mean of 53 million) (6). Alveoli increase in number after birth, reaching the adult range of 300 to 600 million
alveoli by 2 years of age. Thereafter, lung growth occurs in terms of volume and alveolar size, with no further
increase in alveolar numbers (7).
NASOPHARYNX
Choanal Atresia
Choanal atresia occurs in about 1 in 5 to 8,000 livebirths and consists of unilateral or bilateral occlusion of the
airway between the posterior nasal passage and the nasopharynx (8, 9). The entity has been seen in
monozygotic twins (10) and has also been noted following radiotherapy for nasopharyngeal carcinoma (11, 12).
The septum blocking the airway is usually composed of bone or cartilage, but in as many as 20% to 50% of
cases,
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it may be composed of mucous membrane alone (13). Choanal atresia may exist as an isolated sporadic lesion,
in an autosomal dominant form, or possibly in an autosomal recessive form. It has been associated with palatal
defects, tracheoesophageal fistula (TEF), congenital heart malformations (14), trisomy 6 (15), Pfeiffer syndrome
(16), Treacher Collins syndrome (17), the fetal carbimazole syndrome (18), and the CHARGE (Coloboma, Heart
defect, choanal Atresia, Retardation, Genital, Ear anomaly) association (CHD7 mutation on 8q12.2) (19, 20), of
which it is a major component.
Cleft Lip and/or Palate

Cleft lip, with or without unilateral and bilateral involvement of the hard palate, the soft palate, or both, is the most
common malformation of the respiratory tract (21, 22). It occurs once in 750 live births as an isolated anomaly or
as part of a wide variety of chromosomal, inherited, and noninherited syndromes, of which over 250 have been
described (23) (Table 12-2). Cleft palate is associated with other anomalies in 47% of cases, cleft lip and palate
in 37%, and cleft lip alone in 14%. Anomalies most frequently seen are those in the central nervous system
(CNS) and skeletal system, followed by urogenital and cardiovascular anomalies. Maternal cigarette smoking
and alcohol use are associated with a 1.6- to 2.0-fold increase in isolated cleft lip, cleft palate, or both (24). The
incidence of cleft lip and palate is dose-related, increasing with increased cigarette smoking (25).
Table 12-2 ▪ EXAMPLES OF SYNDROMES ASSOCIATED WITH ISOLATED CLEFT PALATE-

PRESUMED INHERITANCE
Autosomal Dominant Autosomal Recessive X-Linked Sporadic

Occurrence
Stickler syndrome Diastrophic dwarfism Oculopalatodigital Hanhart or aglossia-

Apert syndrome Smith-Lemli-Opitz syndrome adactyl complex
Marfan syndrome syndrome Bruan-type Congenital oral
Mandibulofacial dysotosis Multiple pterygium nephrosis teratoma
Spondyloepiphyseal syndrome Gorlin Buccopharyngeal
dysplasia congenita Stapes fixation and skeletovascular membrane
Camptodactyly and oligodontia syndrome Oral duplication
clubfoot Cerebro-costo- Bilateral renal Caudal regression
Larsen syndrome mandibular syndrome agenesis anomaly
Beckwith-Wiedemann Chondrodystrophia Klippel-Feil anomaly
syndrome calcificans congenital Oligohydramnios
Wildervanck syndrome Dubowitz syndrome sequence
Chotzen syndrome Campomelic syndrome Bilateral renal
CPLS syndrome Tel Hasomer agenesis
Achondroplasia camptodactyly syndrome Various chromosomal
Cleidocranial dysplasia Juberg-Haywood syndromes
Brachmann-de Lange syndrome
syndrome
Hereditary renal
adysplasia
Maternally transmitted
myotonic dystrophy
Ectodactly, ectodermal
dysplasia syndrome
Popliteal pterygium
syndrome
Rapp-Hodgkin syndrome
Shprintzen syndrome
Treacher Collins
syndrome
CPLS, cleft palate-lateral synechia

Adapted from Gilbert EF. Respiratory system. In: Gilbert-Barness E, ed. Potter pathology of the fetus
and infant. St. Louis: Mosby, 1997:721, with permission.
Laryngocele
Laryngoceles occur rarely in childhood but may present as airway obstruction or as a neck mass in a neonate or
an older child (26, 27). The lesion, seen predominantly in boys and containing air or fluid, or both, may be within
the larynx behind the thyroid cartilage (33%), external to the larynx (25%), or involving both locations (28).
Infection of the lesion may occur (pyolaryngocele), leading to acute respiratory distress (29). Laryngoceles have
been described in association with laryngeal papillomatosis (30) and in later life with laryngeal carcinoma (31).
Laryngomalacia
Stridor and feeding difficulties in the newborn may be caused by laryngomalacia due to flaccidity of a long
epiglottis, short arytenoepiglottic folds, or bulky arytenoid swellings, resulting in partial obstruction of the larynx.
Kay and Goldsmith (32) have developed a classification based on the underlying pathophysiologic processes
with type 1 characterized by a
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foreshortened or tight aryepiglottic fold, type 2 defined by the presence of redundant soft tissue in the
supraglottis, and type 3 applying to cases caused by other etiologies. Potentially serious complications of the
obstruction include pulmonary hypertension and cor pulmonale, sudden death during respiratory tract infections,
failure to thrive, and possible impaired intellectual development secondary to episodes of hypoxia and
hypercapnia. Twenty percent of these infants have severe neurologic compromise or multiple congenital
anomalies (33). Surgical procedures including supraglottoplasty have been used in severe cases (about 10% to
15% of cases) and have been successful in relieving respiratory symptoms in 80% of those cases (33, 34).
Laryngomalaciainduced stridor has been reported in patients with Pierre Robin (35), acrocallosal, Marshall-Smith
(36), cri du chat, fetal warfarin (37), Down (38), Freeman-Sheldon (39), and Mohr syndromes. Chen et al. (40)
have described a familial form of laryngomalacia.
Laryngeal Stenosis and Atresia

Supraglottic, glottic, and subglottic developmental webs may produce varying degrees of laryngeal stenosis and
have been described in families with an autosomal dominant inheritance pattern.
FIGURE 12-6 ▪ Laryngeal atresia. A: The larynx reveals a patent upper opening (upper piece) and a patent
trachea (lower two cross sections). B: A histologic section from the area in the region of the cricoid cartilage
reveals only a pinpoint lumen (bottom center). (Hematoxylin and eosin stain, original magnification ×4.)
Subglottic stenosis, as an acquired lesion, has been seen secondary to short-term and long-term intubation in
the neonatal intensive care nursery with increased incidence when the infant is intubated for longer periods (41).
With acquired stenosis, dense submucosal fibrous connective tissue is present circumferentially in the subglottic
area and may narrow the lumen significantly. Submucosal glands are usually absent, and the cricoid cartilage
may display evidence of erosion.
Congenital laryngeal atresia occurs in three patterns:
1. Type 1, atresia of both supraglottic and infraglottic portions of the larynx

2. Type 2, atresia of the infraglottic region (Figure 12-6A,B)
3. Type 3, glottic atresia (42)
Associated conditions include esophageal atresia (EA), TEF, “total sequestration” of the lungs in the absence of
TEF, anal anomalies, urinary tract malformations, skeletal anomalies, and heart malformations (43). Many of the
conditions are part of the vertebral, anal atresia, cardiac, TEF, renal, and limb (VATER or VACTERL)
association. Other associations include partial diaphragmatic obliteration (44), Frasier
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syndrome, DiGeorge developmental field defect, and partial trisomy 9 (45, 46 and 47). Pulmonary hyperplasia
has been noted in infants who have laryngeal atresia with lung weights ranging from 150% to 300% of normal
(48).
FIGURE 12-7 ▪ Types of laryngotracheoesophageal cleft. A: Supraglottic interarytenoid cleft. B: Partial cricoids
cleft. C: Total cricoid cleft. D: Complete cleft to level of carina.
Laryngotracheoesophageal Cleft
Failure in formation of the tracheoesophageal septum, normally complete by day 35 of gestation, leads to the
development of one of four forms of laryngotracheoesophageal cleft (Figure 12-7A to D):
1. Supraglottic interarytenoid cleft (50% of cases)
2. Partial cricoid cleft
3. Total cricoid cleft
4. Complete cleft of the trachea to the level of the carina (42, 116)
Maternal polyhydramnios is seen in many cases, and a familial occurrence has been reported with relative
frequency. Associated conditions include TEF and other elements of the VATER association—pulmonary
hypoplasia, exstrophy of the bladder, polysplenia, double outlet right ventricle, and the G syndrome (49).
TRACHEA
Tracheal Agenesis
Tracheal agenesis is a rarely occurring, uniformly fatal malformation that is usually associated with
tracheoesophageal or bronchoesophageal fistula. Various classifications divide the entity into three to seven
types (Figure 12-8A to G); however, nearly 70% of cases consist of agenesis of the entire trachea with a small
fistulous connection between the esophagus and the carina (Figure 12-8C to E) (50, 51). The lungs may be
normally developed or totally absent (pulmonary agenesis). In the rare cases of tracheal agenesis with no
fistulous connection to the esophagus (i.e., total sequestration of the lungs), the lungs are uniformly distended,
histologically resembling extralobar sequestration (48). There is a male predominance of approximately 2:1 and
an association with maternal polyhydramnios in tracheal agenesis. In addition to the anomalies of the VATER
association, tracheal agenesis has been seen in association with duodenal atresia, annular pancreas,
syndactyly, and CNS malformations (52). Evans et al. (51) describe four groups based on the type of anomalies
associated with the tracheal agenesis: group 1, anomalies restricted to the trachea, larynx, and cardiovascular
system; group 2, severe cardiovascular anomalies and abnormal lung lobulation; group 3, a caudal component in
addition to thoracic abnormalities, with anal and renal anomalies being common; and group 4, multisystem
involvement with a high incidence of aberrant vessels, complex cardiac malformations, lung lobation defects, and
anomalies of other foregut derivatives.
Tracheal Stenosis
Although laryngeal, or tracheal, stenosis is usually seen as an acquired lesion related to intubation or to the
presence of a foreign body, congenital stenosis of the trachea is rare (53, 54 and 55). Congenital stenosis may
be diffuse, funnel-like, or segmental. Diffuse, generalized hypoplasia accounts for about 30%) of cases, funnel-
shaped or “carrot-shaped” stenosis for 20%, and segmental stenosis for the remaining 50%. Segmental stenosis
may be due to complete tracheal cartilage
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rings, “napkin-ring” stenosis, or too small but normally shaped rings with a narrow pars membranosa (Figure 12-
9) (56). Associated anomalies include anomalous bronchi, TEF, unilateral pulmonary agenesis, Crouzon
syndrome, Larsen syndrome, Down syndrome (57), Alagille syndrome, and ventricular septal defect (58). Wong
et al. (59) describe tracheobronchial stenosis in monozygotic twins.
FIGURE 12-8 ▪ Types of tracheal agenesis. A: Total pulmonary agenesis (8% of cases). B: Tracheal agenesis
with main bronchi arising directly from esophagus (10%). C: Tracheal agenesis with fused main bronchi and
bronchoesophageal fistula (56%). D: Tracheal agenesis with larynx joined by atretic strand to distal trachea,
which has a fistulous connection with the esophagus (10%). E: Upper tracheal agenesis with large direct
tracheoesophageal communication (5%). F: Tracheal agenesis with no communication with esophagus (5%). G:
Short-segment tracheal agenesis (5%).
FIGURE 12-9 ▪ Tracheal stenosis. A cross section from the mid trachea shows a complete cartilage ring beneath
the mucosa, significantly narrowing the tracheal lumen. (H&E, ×2.)
Tracheal stenosis, or narrowing, may also be produced by extrinsic pressure, most commonly by abnormally
placed or abnormally large blood vessels including
Vascular ring due to double aortic arch

Vascular ring due to right aortic arch
Aberrant right subclavian artery
Anomalous innominate artery
Anomalous left carotid artery—aneurysmal left and right pulmonary arteries
“Sling” retrotracheal left pulmonary artery
Advances in surgical management of congenital tracheal stenosis have improved survival, especially since the
advent of extracorporeal membrane oxygenation (ECMO) (60, 61).
Tracheomalacia
Congenital tracheomalacia (i.e., soft or collapsing trachea) is exceedingly rare and overlaps with tracheal
stenosis secondary to cartilage plate deficiency (62). Isolated cases have, however, been reported in association
with Down syndrome (63), EA, CHARGE association, Larsen syndrome (64), pulmonary vascular sling (65),
polychondritis, and various chondrodystrophies including Ellis-van Creveld syndrome, Langer-type mesomelic
dwarfism, and diastrophic dwarfism (66). Aortopexy has been successfully employed in the treatment of
tracheomalacia in infants (67). Acquired tracheomalacia may be seen in infants and young children who have
been intubated for prolonged periods or as a result of trauma, radiation, or a neoplasm (68, 69).
Tracheobronchiomegaly
Tracheobronchiomegaly, or the Mounier-Kuhn syndrome, usually involves men 20 to 40 years of age but has
been reported in children of both sexes and has a familial occurrence, suggesting an autosomal recessive type
of inheritance (70). The tracheal diameter exceeds the normal by three standard deviations. Saccular bulging of
the intercartilaginous membranes is frequent. The disorder has been noted in a child with cutis laxa and in an
adult with Ehlers-Danlos syndrome (71).
Tracheoesophageal Fistula and Esophageal Atresia

EA, with or without TEF, occurs sporadically with an incidence of 1 in 3,500 live births (72, 73). Maternal
polyhydramnios is present in more than 30% of cases, and nearly 35% of the infants are premature (56). The
anomaly can be divided into five (or more) types (Figure 12-10A to E). More than 95% of the patients have EA
with the clinical findings of excessive oral and pharyngeal secretions or choking, cyanosis, or coughing during
first attempts at feeding. A least three separate genetic factors have been identified for esophageal atresia (74).
TEF and EA can be most easily demonstrated at autopsy by removing the esophagus and trachea en bloc (see
Chapter 1), and then opening the esophagus lengthwise along its posterior or dorsal margin. EA is readily
apparent as a blind pouch (Figure 12-11A,B), but a small fistula between the anterior or ventral portion of the
esophagus and the trachea can also be visualized, as can the rare esophagobronchial fistula. Histologically,
squamous metaplasia of the trachea and bronchi may be seen in 80% of patients, primarily along the posterior
wall of the trachea but frequently extending around the entire internal surface of the trachea and into the bronchi.
The segment of esophagus may show tracheobronchial remnants in the form of abnormal mucous glands and
ducts, abnormal mucin secretion, the presence of cartilage, and a disorganized muscle coat (75). Aspiration of
gastric contents may be present in the lung, producing pneumonia with foreign body giant cell reaction.
Associated anomalies are seen in 49% to 72% of infants with EA and TEF, with multiple anomalies frequently
present (Table 12-3) (76). A nonrandom association of TEF with other malformations has been recognized in
about 45% of cases and given the acronyms of VATER, VACTER, or VACTERL (vertebral, anal, cardiac,
tracheoesophageal, renal, or radial and limb anomalies) (77, 78 and 79).
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Other less frequently associated anomalies include congenital pulmonary airway malformation (CPAM) (80),
diaphragmatic hernia, duodenal atresia, biliary atresia, sirenomelia, trisomy 18, and intracardiac epithelial cyst
(81).
FIGURE 12-10 ▪ Types of TEF and esophageal atresia (EA). A: EA with TEF to the distal esophageal segment
(>85% of cases in various series). B: EA without TEF (8%). C: TEF without EA (4%). D: EA with TEF to the
proximal esophageal segment (1 %). E: EA with TEF to both proximal and distal esophageal segments (1%).
Postsurgical survival of patients with EA and TEF has increased steadily over the last 50 years, presently
ranging from 75% to over 90% (43, 77). The highest mortality rate occurs in infants with low birth weight or with
coexisting cardiac malformations. TEF may recur after surgical repair in nearly 10% of cases (82). Tracheal
narrowing may persist for years in nearly one-third of the patients, along with respiratory infections and
gastroesophageal reflux. Histologically, esophageal inflammation may be seen in 51% of cases, Barrett
esophagus in 6%, and Helicobacter pylori infection in 21%) of cases (83). TEFs may develop in burn patients,
with foreign body impaction, such as a disc battery (84), and following radiation and chemotherapy for
mediastinal malignancies, including lymphoma (85). An increased incidence of esophageal adenocarcinoma in
adulthood in patients with TEF has been suggested (86).
BRONCHUS
Bronchial Atresia
Bronchial atresia is an entity seen almost exclusively in infants and is most frequently associated with infantile
(congenital) lobar emphysema (87). Cases of bronchial atresia with mild emphysema, however, have been
reported in children from 1 day to 13 years (median, 4 years) with
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symptoms of chronic cough and fever in nearly all of the cases, often related to the recurrent pneumonia noted in
more than 90% of cases. The atretic bronchus is connected to the right lower lobe, left upper lobe, and right
upper lobe in decreasing order of frequency. Histologically, the affected bronchus may be obstructed by
circumferential or eccentric luminal fibrosis with or without abnormalities of the cartilage plates. The fibrosis may
be the result of in utero inflammation in the neonate or possibly postpartum inflammation in the case of children
and adults. The similarity between the lungs of congenital bronchial atresia and infantile lobar emphysema (ILE),
both radiographically and pathologically, is striking, and suggests that they may be a single entity.
FIGURE 12-11 ▪ TEF and esophageal atresia. A: In a posterior view of the tongue (top), trachea, and lung, the
esophagus is seen to end in a blind pouch (center). B: With the trachea and esophagus open posteriorly, a
fistula can be seen connecting the carina with the distal end of the esophagus. (Courtesy of David Kelly, M.D.,
University of Alabama, Birmingham, Alabama.)
Table 12-3 ▪ ANOMALIES ASSOCIATED WITH ESOPHAGEAL ATRESIA ANDTEF

Organ System Incidence Most Frequent Examples
(%)
Musculoskeletal 14.7-24 Vertebral defects, rib defects, radial amelia, caudal

dysgenesis
Cardiovascular 11.0-49 Ventricular septal defect, patent ductus arteriosus, right

aortic arch
Gastrointestinal 20.3 Imperforate anus, malrotation, duodenal atresia
Genitourinary 12.2-50 Renal malposition, renal cysts or hypospadias, horseshoe

agenesis kidney
Craniofacial 9.7 Choanal stenosis, ear malformations, micrognathia
Central nervous 7.2 Hydrocephalus

system
Pulmonary 2.1 CPAM pulmonary hypoplasia
Adapted from Stocker JT. Congenital and development diseases. In: Dail DH, Hammer SP, eds.
Pulmonary pathology. 2nd ed. Heidelberg: Springer-Verlag, 1994:163, with permission.
Bronchial stenosis may also be associated with ILE. The lumen of the bronchus may be intrinsically narrowed by
postinflammatory fibrosis or by an intraluminal mass such as aspirated meconium or other foreign material,
bronchial adenoma, ectopic thyroid tissue, or bronchial mucosal web. Extrinsic causes of bronchial stenosis
include parabronchial masses such as teratoma and bronchogenic cyst, enlarged or abnormally located
pulmonary arteries, and cardiac or
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left atrial enlargement (88, 89). Bronchial stenosis has also been associated with EA and TEF. More recently,
studies have also suggested that bronchial stenosis and/or atresia are common features of CPAM, extralobar
sequestration, and intralobar sequestration (ILS) as well as ILE (90, 91).
Bronchomalacia
Bronchomalacia and tracheobronchomalacia are seen most frequently in premature infants treated for prolonged
periods with mechanical ventilation (92). Congenital bronchomalacia, however, is a rarely occurring disorder in
which there is abnormal development of bronchial cartilage, leading to collapse of the lumen and possible
development of secondary pneumonia. Bronchomalacia has also been suggested as a cause of sudden death,
especially in those infants with respiratory distress (93). Deficiency of subsegmental bronchial cartilage with
bronchial collapse is also a feature of Williams-Campbell syndrome and has been noted in children with Larsen
syndrome (64, 94). Children with Down syndrome have a high incidence (to 50%) of laryngomalacia,
tracheomalacia, and bronchomalacia (63).
Histologically, the affected bronchus is decreased in size, with the usual cartilage plates replaced by scattered
small islands of immature-appearing cartilage. The lung, distal to the collapsed bronchus, may show pneumonia
or is distended in a pattern typical of ILE. Bronchial stents are used in the treatment of this abnormality but have
been associated with complications including an aortobronchial fistula (95).
Bronchial Isomerism Syndromes

Bronchial isomerism results in “mirror-image” lungs (i.e., bilateral right or left lung), and is associated with five
types of “polysplenia/asplenia” orheterotaxy syndromes (96).
FIGURE 12-12 ▪ Anatomic variations of right upper lobe bronchus. (From McLaughlin FJ, Strieder DJ, Harris GB,
et al. Tracheal bronchus: association with respiratory morbidity in childhood. J Pediatr 1985;106:751, with
permission.)
Type 1, Ivemark asplenia syndrome, is a nonfamilial malformation complex involving bilateral right-sidedness,
including absence of the spleen, intestinal malrotation, symmetric liver, and bilateral three-lobed “right” lungs
with bronchi for both lungs. A variety of cardiac malformations are also associated with this type, including right
aortic arch, symmetric venae cavae, transposition of the great vessels, and total anomalous pulmonary venous
return.
Type 2, M-anisosplenia, involves boys who have one or more larger and one or more smaller spleens, along with
congenital heart malformations, bilateral three-lobed “right” lungs, and relatively normal visceral situs.
Type 3, the polysplenia syndrome, is characterized by a bilateral two-lobed “left” lung bronchial pattern with
intestinal malrotation, symmetric liver, congenital heart malformations, and 4 to 14 uniform small spleens.
Type 4, F-anisosplenia, involves females who have bilateral two-lobed “left” lungs, congenital heart malformation
(usually double-outlet right ventricle), and anisosplenia.
Type 5, O-anisosplenia, is characterized by bilateral twolobed “left” lungs, an approximately 50% incidence of
intestinal malrotation, multiple spleens, an equal sex ratio, and congenital heart malformations, particularly
double-outlet right ventricle, ostium atrioventriculare commune, or both (see Chapter 13).
Abnormal Bronchial Branching and Origin

Abnormal branching patterns, mostly minor anomalies such as double stem superior segments of lower lobe
bronchi and trifurcation of the left upper lobe bronchus, are seen in nearly 10% of bronchograms (97). However,
major anomalies are also seen, including double right lobe bronchus, accessory cardiac bronchus, tracheal
origin of the right upper lobe bronchus (also called pre-eparterial bronchus), and bridging bronchus (Figure 12-
12) (98, 99).
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McLaughlin et al. (100) noted a tracheal origin of a bronchus in 2% of 412 symptomatic patients younger than 5
years of age who were undergoing bronchoscopy. The various forms of tracheal bronchus (Figure 12-12) may
lead to recurrent episodes of pneumonia requiring resection of the bronchus and lobe. Other anomalies are
noted in more than 75% of patients with tracheal bronchus. Wells et al. suggest that in patients with sling left
pulmonary artery, the tracheal bronchus often associated with the right upper lobe may represent the “normal”
origin of the bronchus, and the bronchi supplying the right middle and lower lobes are branches of the left main
bronchus that are crossing or “bridging” the mediastinum (Figure 12-13). They note that the origin of the tracheal
bronchus is at the normal level of tracheal bifurcation (T4-5) and the bifurcation of the bronchi supplying the left
lung and right middle and lower lobes is at the T6-7 level.
Bronchobiliary and Bronchoesophageal Fistulae

Bronchoesophageal fistula probably represents a variation of TEF (101) but may also be seen with infectious
diseases such as tuberculosis (102) and has been reported in association with Crohn disease (103).
Congenital bronchobiliary fistula rarely occurs (104); however, when it does, it is usually located between the
right mainstem bronchus and the left hepatic duct (105). The bronchobiliary fistula is thought to represent a
duplication of the upper gastrointestinal tract from its junction with the airway to the level of the ampulla of Vater.
The fistula arises from the proximal portion of the right main bronchus, accompanies the esophagus through the
diaphragm, and joins the biliary tree at the left hepatic duct. In its proximal portion, the tract resembles a
bronchus with cartilage rings and respiratory epithelium, and in its distal portion, the tract resembles a bile duct
or esophagus. Bronchobiliary fistulas may be seen in older children and adults in association with biliary
obstruction or infections (such as echinococcosis) involving the liver (106). It has also been noted as a
postsurgical complication in a child with undifferentiated embryonal sarcoma of the liver (107).
FIGURE 12-13 ▪ A bronchus “bridges” the mediastinum in the case of tracheal agenesis.
Bronchiectasis
Bronchiectasis was once a common acquired disorder seen in a variety of infectious diseases associated with
chronic inflammation of the bronchi (e.g., tuberculosis and pertussis). It is now primarily associated with a
number of congenital and familial conditions including immunodeficiency states (e.g., IgG, IgA, a-1-antitrypsin,
neutrophil, or complement deficiency), the immotile cilia syndrome, cystic fibrosis (CF), and the Williams-
Campbell syndrome.
Primary ciliary dyskinesis or the immotile cilia syndrome, also called Kartagener syndrome when associated with
situs inversus (50% of cases), is characterized by immobility of the cilia of mucosal cells in the upper and lower
airways, in the ependymal lining of the ventricular system, and in the various cells of the reproductive tract
(including tails of spermatozoa) (108). The incidence of the disease is 1 in 20,000 to 30,000. The abnormalities
of these cells lead to the clinical manifestations of chronic rhinitis, sinusitis, otitis, bronchitis, diffuse or localized
bronchiectasis, headaches, and male subfertility. A variety of ultrastructural abnormalities, many nonspecific,
have been described. These abnormalities include the absence of both inner and outer dynein arms, radial
spoke defects, missing nexin links, microtubular transpositions, and compound cilia (109). An autosomal
recessive mode of inheritance is suspected with extensive locus heterogeneity primarily on chromosome 19q and
10% of patients having a mutation in DNAIlor DNAH5 (110). An association with rheumatoid arthritis has recently
been noticed (111). Treatment may require bilateral lung transplantation.
The Williams-Campbell syndrome, or familial congenital bronchiectasis, is a disorder characterized by a
deficiency of bronchial cartilage distal to the main segmental bronchi, usually of the fourth to sixth order (112).
Cartilage is absent, markedly diminished, or soft. The syndrome usually is seen in the neonatal period or early
infancy, and familial cases have been reported. The disease may proceed rapidly or have a more benign course
compatible with prolonged survival. Lung transplantation may be unsuccessful because of cartilage problems in
the recipients' residual right and left mainstem bronchi (94).
CF (see below) is probably the most common cause of bronchiectasis, accounting for about 50% of all cases. No
airway lesion specific for CF has been discovered, although mucus stasis in bronchi and pseudomonas
pneumonia are frequently seen.
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Bronchogenic Cyst
The bronchogenic cyst is a discrete, extrapulmonary mass filled with fluid and composed of a wall lined by
respiratory epithelium overlying fibromuscular connective tissue that contains seromucinous glands and cartilage
plates. It is noted most frequently in the hilar or middle-mediastinal area, but it may be present in a midline
location from the subcutaneous region of the suprasternal area to beneath the diaphragm (113, 114).
Esophageal and enteric duplication cysts and pericardial cysts may also be present in the mediastinal region.
Bronchogenic cysts are rarely connected to the tracheobronchial tree or involve the pulmonary parenchyma.
Case reports of “intrapulmonary bronchogenic cysts” probably represent instances of type 1 CPAM [formerly
congenital cystic adenomatoid malformation (CCAM)] (115).
Bronchogenic cysts are seen most frequently in children and young adults as incidental findings on chest
radiographs, at surgery, or at autopsy, but they may present with symptoms related to secondary infection of the
cyst, including fever, hemorrhage, or perforation. In infants, bronchogenic cysts located near the trachea,
especially the carina, may produce obstruction and respiratory distress (116).
FIGURE 12-14 ▪ Bronchogenic cyst. A: A CT of the chest displays a large mass in the middle mediastinum. B: A
resected bronchogenic cyst, which was separate from the lung, is covered by connective tissue. C: Ciliated
pseudostratified columnar epithelium overlies a wall composed of fibrous connective tissue, glands, and a
cartilage plate in a bronchogenic cyst. (H&E, × 100.)
In infants, the gross appearance of the cysts consists of a 1- to 4-cm, smooth-to-irregular, spheroid mass
attached to, but not in communication with the tracheobronchial tree (Figure 12-14A-C). The cysts may contain
clear serous fluid, but if they are infected, the fluid may be turbid or hemorrhagic. In older patients, the cysts may
reach a diameter of 8 to 10 cm and may be found throughout the mediastinum as well as in or beneath the
diaphragm. Extrathoracic cysts are usually confined to the subcutaneous region in the suprasternal area (117).
Microscopically, the lining of the cyst is composed of ciliated, cuboidal to pseudostratified columnar epithelium.
Cartilage plates and seromucinous glands are present in the wall, as is fibromuscular connective tissue (Figure
12-14C). The presence of striated muscle and stratified squamous or columnar epithelium is consistent with an
esophageal cyst (Figure 12-15). Enteric cysts are lined by mucus-secreting columnar epithelium and contain
gastric glands with parietal cells in the wall. All three types of cysts may display squamous metaplasia, mucosal
ulceration, inflammation, extensive necrosis, or a combination of these, making an exact diagnosis difficult.
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FIGURE 12-15 ▪ Esophageal cyst. A: A cystic structure was resected from the middle mediastinum adjacent to
the esophagus. B: Columnar epithelium overlies a wall composed of thick muscular bands in this esophageal
cyst from the mediastinum. (Hematoxylin and eosin stain, original magnification × 75.)
Bronchogenic cysts have been noted between the sequestration and the midline in association with extralobar
sequestrations in older children. This suggests that the cysts have arisen from “rests” of bronchogenic cells
along the abortive foregut tract that gave rise to the sequestration (118).
Plastic Bronchitis
Children with cardiac defects (119) or an underlying pulmonary disease (asthma or allergic disease) may
develop obstructive bronchial casts (120). There are two types of casts: type I, cellular cast made up of
inflammatory cells with fibrin, and type II, acellular casts composed mainly of mucin (121). Other underlying
causes include CF, neoplasia (122), thalassemia a (123), and acute chest syndrome of sickle-cell disease (124).
Grossly the cast may display a partial or complete outline of the bronchial tree with either tube-like features or
partial or completely solid cores. Those composed of acellular mucin may be partially clear to opaque.
Microscopically, the structures, as indicated by the types, are composed of an inflammatory cellular infiltrate
embedded in fibrin (type I) or mucin-like material with scattered cellular debris (type II) (120).
LUNG
Pulmonary Agenesis
Complete absence of both lungs is extremely unusual and incompatible with life. However, unilateral agenesis,
involving one or more lobes, has been seen in 1 in 10,000 to 20,000 autopsies and, in the absence of other
severe anomalies, is compatible with long-term survival (113, 125). There is a 1.3:1 female predominance with
unilateral agenesis; the right and left lungs are absent with equal frequency. Associated anomalies are noted in
about 75% of cases and include, in decreasing order of frequency, cardiovascular, gastrointestinal, skeletal, and
urogenital systems (126). Cardiovascular malformations include dextrocardia, septal defects, patent ductus
arteriosus, and total anomalous pulmonary venous return (127). Skeletal anomalies include hemivertebrae and a
high frequency of thumb malformations, especially triphalangeal thumb (128). Along with the radial and vertebral
anomalies, imperforate anus and TEF have been described, suggesting an association of pulmonary agenesis
with the VATER or VACTERL association (129). Osborne et al. (126) propose that a neural crest injury may
account for both the skeletal and pulmonary abnormalities because both are supplied by the second, third, and
fourth thoracic nerves.
The larynx and upper trachea are usually well formed in unilateral pulmonary agenesis, although with bilateral
agenesis the total trachea may be absent. The lower trachea in unilateral agenesis may continue directly into the
existing lung as a tracheobronchus or bifurcate at the carina, giving rise to a rudimentary, blind-ending bronchus
on the side of the agenesis. The pulmonary artery and vein to the side of the agenesis are absent or hypoplastic
and may have an unusual course to the lung, often forming a pulmonary sling (130). Shift of the mediastinum to
the side of the agenesis is usually present, often giving the appearance of dextrocardia in right-sided agenesis.
Studies in older infants have demonstrated an absolute increase in the number of alveoli in the existing lung
despite a reduced number of bronchial generations and pulmonary artery branches.
Abnormal Lobation, Location, and Shape

Abnormalities of lobation of the lung are usually of little clinical significance unless they are associated with other
anomalies such as the asplenia or polysplenia syndrome (discussed earlier in chapter). Lobes may be fused to
give the appearance of a single lobe on the right or left, or pleural fissures may produce the appearance of
multiple lobes.
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The appearance of multiple lobes may be seen in infants with long-standing healed bronchopulmonary dysplasia
(LSHBPD) (131). Fusion of the lungs in the midline behind the heart produces a conjoined, or “horseshoe,” lung
analogous to the horseshoe kidney (132). Additional anomalies are often present, including those of the VATER
association and the PAGOD syndrome (Pulmonary hypoplasia, Agonadism, Omphalocele, Dextrocardia and
Diaphragmatic defect), among others (133, 134). Bronchial supply to both lungs may be anatomically normal, but
there is usually an anomalous pulmonary artery supply and venous drainage resembling that seen in scimitar
syndrome (135).
Herniation of the lung across the mediastinum into the opposite hemithorax, associated with ILE, CPAM,
extralobar sequestration, and other conditions, occurs relatively frequently (1). The lung can also herniate
outside the thoracic cavity, usually into the neck. Cervical herniation or “protrusion” is most frequently reported
as a “normal variant” in some infants and children (136). It may also be seen, however, as a result of trauma or
surgery, and in association with iniencephalus, the Klippel-Feil syndrome, and the cri du chat syndrome (137). A
familial occurrence has been noted, and the condition is thought to be an autosomal dominant hereditary disease
(138). Herniation through the diaphragm and intercostal spaces may also occur (139).
Sequestrations
Extralobar
Extralobar sequestrations of the lung are discrete masses of pulmonary parenchyma outside the normal pleural
investment of the lung and are not connected to the tracheobronchial tree. They apparently originate from an
outpouching of the foregut, separate from the normally developing lung (Figure 12-16A). This outpouching then
loses its connection with the foregut, isolating the parenchyma from the tracheobronchial tree (118). Extralobar
sequestrations are diagnosed prenatally in about 25% of cases, and about 60% of patients present with it by 3
months of age (140). Presenting symptoms, often noted on the first day of life, include cyanosis, dyspnea, and
difficulty in feeding. Approximately 10% of patients are asymptomatic. Fetal nonimmune hydrops, anasarca,
pleural effusion, or localized edema may be present along with maternal polyhydramnios. Extralobar
sequestrations may be seen in older children, occasionally in association with a bronchogenic cyst, and they
have been reported in adults as old as 81 years of age (141). There is a slight female predominance.
Associated anomalies are present in more than 65% of cases of extralobar sequestration, with 50% of lesions
containing CPAM type 2 within the sequestration or, less frequently, in a lobe of the “normal” lung. The
ELS/CPAM cases are seen more frequently in the first 3 months of life and on the left side (140). Other
anomalies include bronchogenic cyst, cardiovascular malformations, bronchopulmonary-foregut connection,
pectus excavation, absence of pericardium, and diaphragmatic hernia with concomitant pulmonary hypoplasia
(142, 143). High levels of CA19-19 have been reported in a few cases of extralobar sequestration (142, 144).
Extralobar sequestration is usually a single round to ovoid lesionranging from 0.5 to 15 cm in diameter (Figure
12-16B). In a report of 50 cases, 48% of the lesions were located in the left hemithorax, 20% in the right
hemithorax, 8% in the anterior mediastinum, 6% in the posterior mediastinum, and 18% beneath the diaphragm
(140). The blood supply to the extralobar sequestration is through a direct branch of the thoracic or abdominal
aorta in over 75% of cases. The remaining receive their blood supply from smaller systemic arteries, the
pulmonary artery, or rarely, from the pulmonary artery and a systemic artery (48). Venous drainage is through the
systemic circulation in over 80% of cases; the remaining 20% of cases are drained either partially or completely
by the pulmonary veins, or rarely, by the portal vein (145).
Grossly, the lesion is covered by a smooth to wrinkled pleura overlying a fine, reticular network of lymphatics.
These lymphatics may be prominent in 30% or more of cases. Cut sections of the lesion display homogenous,
pink-to-tan tissue resembling normal pulmonary parenchyma, or clusters of small cysts. Prominent subpleural
lymphatics may also be seen.
Microscopically, extralobar sequestrations consist of uniformly dilated bronchioles, alveolar ducts, and alveoli in
a normal acinar pattern (Figure 12-16C). Bronchioles are usually tortuous with undulating, cuboidal to columnar
epithelium. In 50% of cases, the lesion may consist partially or entirely of back-to-back, dilated, bronchiole-like
structures typical of CPAM type 2 (Figure 12-16D). Lymphatics are unremarkable in the majority of cases but
may be dilated and increased in number beneath the pleura and around bronchovascular bundles, occasionally
resembling congenital pulmonary lymphangiectasia (CPL) (Figure 12-16C). Although they are rare, infarction,
arteritis, and inflammation may be present in an extralobar sequestration. In the absence of severe anomalies,
survival is good, although with large intrathoracic lesions, the associated pulmonary hypoplasia may be severe
enough to cause death. Rhabdomyomatous dysplasia is seen 25%-30% of cases (Figure 12-16D).
“Total” Sequestration with Pulmonary Hyperplasia

Infants with laryngeal or tracheal atresia without TEF have, in effect, “total” sequestration of the lungs and
display a histologic appearance virtually identical with that seen in extralobar sequestration. The lungs are often
two to four times the expected weight and crowd the chest cavity, flattening the diaphragm and leaving an
impression of the ribs on the visceral pleural surface (Figure 12-17) (48). Similar pulmonary changes may be
seen with in utero hyperextension of the neck that appears to compress and obstruct the larynx. Scurry et al.
(146) have noted normal sized or hyperplastic lungs in infants with varying degrees of upper airway obstruction
despite the presence of renal dysgenesis
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and oligohydramnios, conditions more frequently associated with pulmonary hypoplasia. Lymphatics are
unremarkable. Atresia or obstruction of a single bronchus to a lobe may lead to hyperplasia of that lobe and the
development of one form of ILE (see below) (147).
FIGURE 12-16 ▪ Extralobar sequestration. A: The normal lung develops as an evagination from the foregut (top
half). A second evagination (bottom) from the foregut gives rise to lung tissue not attached to the normally
developing lung. B: A large right-sided thoracic mass is attached to the mediastinum by a thin vascular pedicle.
Note the hypoplasia of the right lung. C: The pulmonary parenchyma is uniformly dilated from the bronchioles to
the most distal alveoli. (Hematoxylin and eosin stain, original magnification × 25.) D: Back-to-back bronchiole-like
structures typical of CPAM type 2 are seen in 50% of extralobar sequestrations. Note also the rhabdomyomatous
dysplasia. (Hematoxylin and eosin stain, original magnification ×75.)
FIGURE 12-17 ▪ Hyperplastic lungs in the case of laryngeal atresia are massively enlarged, displaying the
markings of the ribs on their surface.
FIGURE 12-18 ▪ Sequence of events in the formation of intralobar pulmonary sequestration. A: Occlusion of a
bronchial branch by means such as aspirated material or inflammatory debris can lead to the development of
pneumonia distal to the occlusion. B: As the pneumonia persists or progresses, the lung seeks oxygenated
blood to aid in resolution and repair. If pulmonary artery flow is inadequate, systemic blood supplying pleural
granulation tissue through the pulmonary ligament arteries may be “parasitized.” C: As the pneumonia resolves
(or progresses or recurs), the major arterial supply to the sequestered portion of lung is derived from the
hypertrophied pulmonary ligament artery (or arteries). (From Stocker JT, Malczak HT. A study of pulmonary
ligament arteries: relationship to intralobar pulmonary sequestration. Chest 1984;86:611, with permission.)
Intralobar
ILS, by definition, consists of a portion of lung within the normal pleural investment that is isolated (sequestered)
from the tracheobronchial tree and is supplied by a systemic artery (Figure 12-18) (148). Although a small
percentage of ILSs are clearly congenital in origin and might more correctly be called arteriovenous
malformations (149, 150), the vast majority of ILSs are probably acquired lesions formed through repeated
episodes of pneumonia. During the course of these episodes, normal pulmonary ligament arteries become
hypertrophic to provide the systemic artery supply (Figure 12-18A-C) (48, 151, 152). Some examples of ILS may
develop from a previously existing malformation (e.g., CPAM) (142, 153). The following evidence suggests the
acquired nature of ILS:
ILS is rarely seen in the newborn (<15 cases described in children younger than 5 years of
age).
ILS is infrequently associated with other congenital malformations.
ILS is limited to the lower lobes in 98% of cases, allowing access to normally occurring
pulmonary ligament arteries.
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ILS-affected patients have a frequent history of repeated pulmonary infections (154, 155).
ILS presents with a clinical picture of chronic or recurrent pneumonia (e.g., cough, sputum
production) in over 85% of cases (148).
ILSs involve the lower lobe in 98% of cases with this probably reflecting the availability, within pleural granulation
tissues, of branches of normally occurring pulmonary ligament arteries or arteries within the diaphragm that are
parasitized for access to oxygen-rich systemic blood. The pulmonary ligament arteries originate from the thoracic
aorta and extend through the pulmonary ligament between the mediastinum and the lower lobes of the lung
(152). No comparable arteries except the bronchial arteries are present for potential use by the upper lobes in
cases of chronic or recurrent pneumonia.
Radiographic findings include cystic areas, some with fluid levels, along with homogenous and inhomogeneous
shadows. Lack of communication with the tracheobronchial tree is demonstrable by bronchography in about 85%
of cases; the other 15% of cases show some communication between the bronchial tree and the sequestration.
Arteriography demonstrates single (84%) or multiple (10%) systemic arteries (Figure 12-19A,B). The majority of
the arteries (73%) originate from the thoracic aorta, but about 21% originate from the abdominal aorta or celiac
axis and another 4% from the intercostal arteries (156). In rare instances, arteries may originate from the
coronary, subclavian, innominate, internal thoracic, or pericardiophrenic arteries (156). Venous drainage occurs
through the pulmonary veins in 95% of cases, and the remaining 5% of cases drain into the systemic circulation.
Increased serum levels of CA19-19 and CA125 have been noted in patients with ILS (157).
ILS is located on the left side in 55% of cases and on the right in 45% of cases; bilateral involvement is rare (48).
Grossly, the sequestered segment of lung displays variable pleural thickening with adhesions between
mediastinal structures, the diaphragm, and the parietal pleura. Variably sized (1 mm to 5.0 cm) cysts filled with
thin to viscid fluid are noted amid a dense fibrous parenchyma on cut section (Figure 12-19). Microscopically, the
pulmonary parenchyma is distorted by chronic inflammation and fibrosis (Figure 12-19C). The cysts are lined
with cuboidal or columnar epithelium and are filled with amorphous eosinophilic material, foamy macrophages, or
both (Figure 12-19D). Elastic and muscular arteries are present within the interstitium and may show medial
hypertrophy, thrombosis, and arteritis.
Hypoplasia
Pulmonary hypoplasia is the incomplete or defective development of the lung resulting in overall reduced size
due to reduced numbers or size of acini (Figure 12-20A). Lung weight and lung weight-to-body weight ratio are
the simplest means of determining whether hypoplasia exists. The normal lung weight-to-body weight ratio for
term and near-term infants is 0.222 ± 0.002 (158). Emery and Methal (159) describe a radial alveolar count using
a line intersect method in which a line is drawn from a terminal bronchiole perpendicular to the nearest septal
division or pleura surface (Figure 12-20B). The number of alveoli intersected by the line determines the count
with the mean for term infants of 4.4 ± 0.9 (160). Alveolar counting and lung volume measurements may also be
used (161). MRI, and twodimensional or three-dimensional ultrasound have also been used in determining
whether the lungs of an in utero fetus or newborn infant may be hypoplastic (162, 163).
Pulmonary hypoplasia is noted in more than 10% of neonatal autopsies and occurs in association with another
malformation (or malformations) in more than 85% of cases (164). The most frequently occurring anomalies are
diaphragmatic defects and renal malformations (Table 12-4), but a wide variety of anomalies have been
described (1). The common feature of most of these anomalies is that they directly or indirectly compromise the
thoracic space available for lung growth. The cause of the decreased thoracic space may be intrathoracic (e.g.,
abdominal contents herniated through a defect in the diaphragm) or extrathoracic (e.g., oligohydramnios with
uterine fetal compression). The thorax itself may be abnormal as in Jeune asphyxiating thoracic dystrophy,
spondyloepiphyseal dysplasia congenita, and achondroplasia (165). In utero accumulation of fluid within the
thorax as pleural effusion or chylothorax has also been implicated in the production of pulmonary hypoplasia.
Pulmonary hypoplasia may also occur in the absence of other anomalies or in cases of preterm premature
rupture of amniotic membranes (162, 166, 167, 168 and 169). As with infants with hypoplasia secondary to other
anomalies, these infants present with respiratory distress, are difficult to ventilate, and frequently have episodes
of pneumothorax (PT) and interstitial pulmonary emphysema (IPE). Potter sequence with sloping forehead,
flattened face and nose, receding chin, large ears, broad spade-like hands, and deformations of the limbs
secondary to compression by the uterus in the absence of adequate amniotic fluid is a consistent finding in
cases associated with oligohydramnios from any cause. Pulmonary hypoplasia has been noted in children with
Down syndrome, but it is thought to result from failure of the lung to develop properly in the postnatal period
(170).
At autopsy, the lungs may be either uniformly reduced in size or markedly asymmetric (e.g., with diaphragmatic
hernia). In cases in which the pulmonary hypoplasia is the direct cause of death, the lung weight usually is less
than 40% of expected and is often as low as 20% to 30%. Histologically, the acini are small for the infant's
gestational age, but alveolar and capillary development is usually consistent with the gestational age.
Infantile (Congenital) Lobar Emphysema

ILE is the overdistension or hyperplasia of a pulmonary lobe as the result of a partial or complete obstruction of
the bronchus to the lobe by intrinsic or extrinsic factors (171, 172 and 173) (Table 12-5). Boys are more
frequently affected than girls
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(1.5:1) (147). ILE presents in the first week of life in about 50% of cases (with about 40% presenting in the first
day of life) and in the first 6 months of life in over 80%, but ILE can occasionally be seen in children and young
adults from 7 months to 20 years of age. Wall et al. (174) described ILE in a mother and daughter each
presenting in their first month of life and Roberts et al. (175) noted the lesion in a father and son, both with
cartilage deficiency of the bronchus. Symptoms are those of mild respiratory distress increasing over a period of
hours to days to weeks; cyanosis, respiratory infections, vomiting, choking, and feeding difficulties may also be
seen. Rarely, the lesion may present as a sudden pneumothorax (147). Imaging studies reveal, in the classic
form (see below), a characteristic hyperlucent, overdistended lobe producing mediastinal shift and compression
of the uninvolved lobes (176) (Figure 12-21A). In the polyalveolar lobe form (see below), imaging may display a
lobe of normal lucency but one that occupies a disproportionate part of the hemithorax with mediastinal shift.
Less frequently, retained lung fluid may be seen in the involved hyperexpanded lobe (usually the polyalveolar
lobe type) on initial examination but which may clear over subsequent days. Associated anomalies are present in
from 5% to 40% of patients, and 70% of these anomalies are cardiovascular (177, 178 and 179). The upper
lobes are involved in over 95% of cases—the left slightly more often than the right. Multiple lobe involvement
occurs in about 15% of cases, usually with at least one lobe being an upper lobe. Bilateral involvement has been
reported in one case with the left upper and right middle lobes displaying ILE (180). Lower lobe involvement is
rarely seen except in the “acquired” form of ILE, as in premature infants receiving mechanical ventilation who
develop granulation tissue obstruction of a lower lobe bronchus, probably as a result of endotracheal tube
suctioning (181).
FIGURE 12-19 ▪ Intralobar sequestration. A: An arteriogram demonstrated arteries arising from the descending
aorta (mid right) supplying a portion of pulmonary parenchyma. B: A CT demonstrates a mass in the posterior
area of the right hemithorax. C: An artery arising from the descending aorta and passing through the pulmonary
ligament supplies a cystic portion of lung in the left lower lobe. D: Dense fibrous connective tissue containing
lymphoid aggregates surrounds irregular cysts filled with debris and macrophages. (Hematoxylin and eosin stain,
original magnification ×25.)
FIGURE 12-20 ▪ Pulmonary hypoplasia. A: The right lobes of the lung are markedly diminished in size,
secondary to herniated abdominal organs through a right-sided diaphragmatic hernia. By weight, the left lung is
also hypoplastic. B: At the periphery of an acinus in this hypoplastic lung, a radial alveolar count (RAC) is far
below the normal of 4 to 6 for a term infant, confirming the diagnosis of hypoplasia. (H&E, ×50.)
Table 12-4 ▪ ANOMALIES ASSOCIATED WITH PULMONARY HYPOPLASIA
Common
Renal agenesis, bilateral
Renal dysgenesis, bilateral
Obstructive uropathy
Polycystic renal disease (autosomal recessive)
Large abdominal wall defects

Infrequent
Diaphragmatic hypoplasia or eventration
Anophthalmia/microphthalmia—usually in association with diaphragmatic hernia
Hemolytic disease of the newborn
Pleural effusion, as with nonimmune fetal hydrops
Musculoskeletal abnormalities, such as thoracic dystrophies
Anencephaly
Scimitar syndrome
Chromosomal anomalies, including trisomy 13, 18, and 21
Rare
Abdominal pregnancy
Ascites secondary to congenital cytomegalovirus infection
Cloacal dysgenesis
Congenital hydropericardium
Down syndrome (probably postnatal “hypoplasia”)
Eagle-Barret syndrome
Giant cervical teratoma
Glutaric acidemia, type II
Homozygous α-thalassemia
Horseshoe lung
Hypoplasia of the arcuate nucleus
Laryngotracheoesophageal cleft
Neonatal hypophosphatasia
Pena-Shokeir syndrome, type I
Phrenic nerve agenesis
Right-sided cardiovascular malformation, as with hypoplastic right side of heart and pulmonary valve
or artery atresia
Rhabdomyoma in tuberous sclerosis
Thoracic neuroblastoma
Upper cervical spinal cord
Extralobar sequestration
Table 12-5 ▪ CAUSES OF ILE
Bronchial abnormality
Bronchial stenosis
Bronchial atresia
Abnormal origin of bronchus
Extrinsic obstruction of bronchus
Vascular anomaly
Pulmonary artery sling
Anomalous pulmonary venous return
Left-to-right shunting with dilated pulmonary arteries
External mass
Bronchogenic cyst
Intrinsic obstruction of bronchus
Aspirated meconium
Mucous plug
Granulation tissue
Bronchial mucosal folds
Torsion of bronchus
Foreign body
Adapted from Stocker JT. Congenital and developmental diseases. In: Dail DH, Hammer SP, eds.
Pulmonary pathology. Heidelberg: Springer-Verlag, 1989:55, with permission
FIGURE 12-21 ▪ Infantile lobar emphysema. A: A hyperinflated left lung shifts the mediastinum to the right. B: At
surgery, the hyperinflated lung bulges from the opening in the thorax.
FIGURE 12-21 (continued) C: “Classic” form of ILE. The alveolar duct and alveoli are dilated to 3 to 10 times the
normal size but are otherwise unremarkable. (Hematoxylin and eosin stain, original magnification ×60.) D:
“Hyperplastic” form of ILE. While not overinflated, this lung displays a complex acinar formation with a larger
number of alveoli (and consequently a large radial alveolar count) than would be expected at this age.
(Hematoxylin and eosin stain, original magnification × 25.)
Grossly, the lobe in vivo and after resection is hyperexpanded with individual alveoli, which may be readily
visualized (Figure 12-21B). Microscopically, two patterns (classic and polyalveolar) are identified. Nearly, 70%
(the classic pattern) display a uniform overdistension of apparently normally developed acini with alveolar
saccules and alveoli three to ten times the normal size but with radial alveolar counts (RAC) similar to those of
age-matched controls (Figure 12-21C) (147). There may be focal disruption of alveolar walls. The other 30% (the
polyalveolar pattern) show only little overdistension of what appear to be “complex” acini of
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the type seen in polyalveolar lobes and hyperplastic lungs (Figure 12-21D) (Munnell, 1973), and these have
RACs that are two standard deviations beyond the mean of age-matched controls. Seventy-five percent of these
infants with polyalveolar lobe present clinically within the first day or two of life and are likely to show radiologic
features of retained lung fluid (182, 183). Examination of the bronchus to the lobe may reveal stenosis, atresia,
or intrinsic obstruction (Table 12-5), or the bronchus may be unremarkable if extrinsic compression was present.
Cartilage abnormalities of the bronchial wall have been described, but special techniques must be employed to
demonstrate these changes convincingly. Surgical resection of the involved lobe is curative, although
nonsurgical management has been successful in unusual cases (176).
Congenital Pulmonary Lymphangiectasis

CPL is a rare, usually fatal disorder that presents in the first hours to days of life (184). It is characterized by the
presence of dilated thin-walled to thick-walled lymphatics within the interlobular septa and beneath the pleura of
the lung. CPL may be seen as a primary disorder or as secondary to obstructive cardiovascular lesions,
particularly total anomalous pulmonary venous return, but it may occur as part of a generalized lymphangiectasis
or as an isolated pulmonary lesion (185, 186). There is a distinct male predominance in occurrence of CPL of
over 2.5:1, and 5% to 10% of affected infants are stillborn. Symptoms include cyanosis and acute respiratory
distress. Fluid abnormalities including chylothorax, pleural effusion, fetal hydrops, and maternal polyhydramnios
have been described in utero and postpartum (187). In addition to the 60% of cases with cardiovascular
anomalies, CPL is associated with renal malformations, generalized lymphangiectasis, and other anomalies in
another 20% of cases (1). The confusion between CPL and interstitial pulmonary emphysema (IPE) has led to
the misdiagnosis of CPL in many cases, including the purported occurrence of CPL in two related female infants
(188). Illustrations of one of these
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cases strongly suggest that IPE was the pulmonary lesion. The diagnosis of CPL in the absence of
cardiovascular or other anomalies should be strongly suspect.
FIGURE 12-22 ▪ Congenital pulmonary lymphangiectasis. A: A fine network of dilated lymphatics is present
beneath the pleura, most notably where interlobular septa intersect the pleura. B: Cut section of the lung from an
infant with total anomalous pulmonary return reveals enormously dilated lymphatics.
FIGURE 12-22 ▪ (continued). C: Dilated lymphatics extend laterally beneath the pleura (top) and centrally along
an interlobular septum (center). Note the slight increase in connective tissue between the channels.
(Hematoxylin and eosin stain, original magnification × 60.) D: Numerous dilated lymphatics extend along
interlobular septa surrounding bronchovascular bundles. (Hematoxylin and eosin stain, original magnification ×
20.)
The lungs in CPL are bulky, firm, noncompressible, and covered by a milky network of dilated subpleural
lymphatics (Figure 12-22A). Rarely, a single lobe is involved by this process (189). On cut section, the
lymphatics are fluid filled and extend from the interconnecting subpleural network into the interlobular septa and
around the bronchovascular bundles (Figure 12-22B). Microscopically, the lymphatics are diffusely and uniformly
dilated, and may appear to be increased in number. Identification of lymphatics can be aided by the CD31 and
D2-40 immunohistochemical marking of endothelial cells (186). These small, irregular cysts are lined with a thin
layer of endothelial cells and surrounded by a loose myxoid to occasionally dense connective tissue that often
contains foci of extramedullary hematopoiesis. Clusters of lymphatics surround bronchovascular bundles within
the interlobular septa and may separate acini beneath the pleura (Figure 12-22C,D). This is in contrast to the air-
filled, larger, “unlined” cysts of IPE that are limited to the interlobular septa and do not extend laterally beneath
the pleura.
Congenital Pulmonary Airway Malformation (CPAM)

CPAM is a hamartomatous lesion of the lung, with an incidence of about 1 in 5,000 live births, that can be
separated into five major types based on clinical and pathologic features (Figure 12-23) (190). We proposed that
the former designation of this lesion as “CCAM” be changed to “CPAM” to reflect the fact that the lesions as
described below are “cystic” in only three of the five types and “adenomatoid” in only one type (type 3). CPAM
more accurately encompasses all five types in this classification.
CPAM, type 0, also known as acinar dysplasia or agenesis, is a rarely occurring, infrequently described
malformation that is largely incompatible with life (191). It is seen in term and premature infants who are cyanotic
at birth and survive only a few hours and is associated with cardiovascular abnormalities and dermal hypoplasia.
Grossly, the lungs are small and firm and have a diffusely granular surface (Figure 12-24A). Microscopically,
tissue consists of bronchus-like structures with muscle, glands, and numerous cartilage plates
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(Figure 12-24B). Prominent mesenchymal tissue separates these structures and contains extramedullary
hematopoiesis, large thin-walled vascular channels, and collections of amorphic basophilic debris. Rarely,
structures resembling proximal bronchioles are present, along with a few scattered acini at the periphery of the
lesion.
FIGURE 12-23 ▪ Classification of CPAM. The classification is based on the similarity in appearance of the
hamartomatous components of the lesion with the various areas of the normal tracheobronchial tree. Type 0,
composed of bronchus-like structures, appears to be a malformation of the most proximal tracheobronchial tree.
Type 1, containing bronchus-like and proximal bronchiole-like structures, mimics the distal bronchial tree and
proximal acinus. Type 2, composed of bronchiole-like structures, resembles the bronchiolar segment of the
acinus. Type 3, composed of bronchiolelike structures and alveolar ducts and saccules lined by cuboidal
epithelium, resembles the midacinar region. Type 4, with thin-walled structures lined by type 1 alveolar lining
cells, suggests a malformation of the distal acinar components. (From Stocker JT. Congenital and developmental
diseases. In: Dail HD, Hammer SP, eds. Pulmonary pathology. 2nd ed. New York: Springer-Verlag, 1994:182,
with permission.)
FIGURE 12-24 ▪ CPAM, type 0. A: A small nodular mass representing the right lung is largely devoid of air. A
similar lung was present on the left side. B: Bronchial-like structures are surrounded by irregular cartilage plates
and loose mesenchyme-containing thin-walled vascular structures. (Hematoxylin and eosin stain, original
magnification × 25.)
CPAM, type 1, the large or predominant cyst type, presents primarily within the first week to month of life but can
be seen in older children and even young adults (Figure 12-25A) (192). It accounts for nearly 65% of cases and
is usually readily amenable to surgery with a good prognosis. Grossly, the type 1 lesion is characterized by
single or multiple large cysts (3 to 10 cm in diameter) surrounded by smaller cysts and compressed normal
parenchyma (Figure 12-25B,C). Microscopically, the larger cysts are lined with ciliated, pseudostratified
columnar epithelium and the smaller ones by cuboidal to columnar epithelium (Figure 12-25D,E). More than 45%
of the cases display segments of mucus-producing cells among the epithelial
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lining of the larger cysts or in bronchioles and alveolar ductlike structures adjacent to the larger cysts (Figure 12-
25F). These mucous cells have similar characteristics to those of pyloric mucosa (193). Wang et al. (194, 195)
suggest that these mucous cells may have the potential for malignant transformation to bronchioloalveolar
carcinoma. Several reports of the occurrence of CPAM and bronchioloalveolar carcinoma have been published,
and a convincing argument can be made in establishing an association between the two diseases (196, 197,
198, 199 and 200). The walls of the CPAM, type 1 cysts are composed of elastic tissue overlying fibromuscular
connective tissue, and in 5% to 10% of cases, cartilage plates.
FIGURE 12-25 ▪ CPAM, type 1. A: A cystic mass is present in the lower right hemithorax in a newborn with
respiratory distress. B: Multiple large, fluid-filled cysts distend the left lobe from a fetus in the second trimester.
FIGURE 12-25 ▪ (continued) C: When opened, the mass consists of intercommunication cysts. D: Cysts of type
1 CPAM are chararacteristically lined by ciliated columnar epithelium in a sawtooth configuration with underlying
fibromuscular connective tissue. (H&E, × 200). E: A larger cyst wall (top) is covered by columnar epithelium in a
papillary configuration. Note the columnar epithelial lining of the smaller cysts as well. (Hematoxylin and eosin
stain, original magnification ×20.) F: Clusters of mucogenic cells line are present within the cyst lining. (H&E, ×
200.)
CPAM, type 2, the medium cyst type, accounts for 10% to 15% of cases, is seen exclusively within the first year
of life, and has a poorer outcome owing to its more frequent association with other anomalies (140), some of
which are incompatible with life (e.g., renal agenesis). The type 2 lesion is composed of cysts 0.5 to 2.0 cm in
diameter (rarely larger) that are evenly distributed and blend with the adjacent normal parenchyma (Figure 12-
26A). The cysts occasionally surround normal appearing bronchi. The typical back-to-back bronchiole-like
structures are lined by cuboidal to columnar epithelial cells with a thin, underlying, fibromuscular layer (Figure
12-26B). Mucous cells and cartilage plates are absent
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except as components of “entrapped” normal bronchi. A variant or subgroup of the type 2 lesion, termed
rhabdomyomatous dysplasia (201, 202 and 203), contains ribbons of striated muscle fibers throughout the
lesion, both in association with the cysts and between alveolar ducts and around blood vessels (Figure 12-26C).
The cysts of this rhabdomyomatous variant may be less prominent than other type 2 lesions.
Rhabdomyosarcoma has been reported to originate from CPAM, but this represents a pleuropulmonary blastoma
(PPB) rather than CPAM. CPAM, type 2-like features are present in 50% of extralobar sequestrations (140).
FIGURE 12-26 ▪ CPAM, type 2. A: Small cysts (0.2 to 0.5 cm) are scattered throughout the lobe and blend with
normal parenchyma. B: The backto-back bronchiole-like structures are separated by structures resembling
alveolar ducts. (Hematoxylin and eosin stain, original magnification ×40.) C: In a variant of type 2, striated
muscle fibers are present in the connective tissue between and around cysts. (H&E, ×200.)
CPAM, type 3, the small cystic or solid type, occurs infrequently (5% of cases), is seen exclusively in the first
days to month of life, has a notable male predominance, and owing to its large size and association with maternal
polyhydramnios and fetal anasarca, has a high mortality rate (204). Increased maternal levels of serum α-
fetoprotein have been noted in the second trimester of two cases of type 3 CPAM (205, 206). CPAM, type 3, the
original lesion described by Ch'in and Tang (207), consists of a large, bulky, parenchymal mass involving an
entire lobe or even an entire lung (Figure 12-27A,B). The mass effect of the lesion consistently produces
mediastinal shift and often results in hypoplasia of the uninvolved lung. Cysts are rarely larger than 0.2 cm in
diameter, with the exception of scattered, larger, bronchiole-like structures. Microscopically, the lesion resembles
an immature lung devoid of bronchi. Irregular, stellate-shaped, bronchiole-like structures lined with cuboidal
epithelial cells are surrounded by alveolar ductules and saccules that are also lined by cuboidal cells, imparting
the “adenomatoid” appearance for which this lesion was originally named (Figure 12-27C). Mucous cells,
cartilage, and rhabdomyomatous cells are not present, and there is a paucity of vessels within the lesion.
CPAM, type 4, the peripheral acinar cyst type, appears to be a hamartomatous malformation of the distal acinus.
This variant is seen equally in boys and girls, with an age range of newborn to 4 years and accounts for 10% to
15%
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of cases. Until recently, most of these cases were included in the type 1 category. Clinically, the type 4 lesions
may present with mild respiratory distress, sudden respiratory distress from tension PT, pneumonia, or on
occasion, as an incidental finding with no symptoms (190, 192). Radiographically, the lesion displays large air-
filled cysts with mediastinal shift and, occasionally, is associated with a PT. The lesion involves a single lobe in
about 80% of cases and rarely may be bilateral. Grossly, large thin-walled cysts are present at the “periphery” of
the lobe and appear to be lined by a smooth membrane (Figure 12-28A). Microscopically, the cysts are lined by
flattened epithelial cells (type I and II alveolar lining cells) over most of wall, with occasional low cuboidal
epithelium seen (Figure 12-28B to D). The wall of the cyst is composed of loose mesenchymal tissue with
prominent arteries and arterioles. Loose mesenchyme must not be confused with similar features seen in the
cystic type of PPB (see later). Dense connective tissue may be present in some cases in older patients. Survival
is excellent with resection.
FIGURE 12-27 ▪ CPAM, type 3. A: A large air-containing mass in the right hemithorax pushes the mediastinum to
the left. B: The resected lesion is nearly solid with only a few slit-like openings. C: Randomly distributed irregular
bronchiole-like structures are separated by dilated alveolus-like structures all of which are lined by cuboidal
epithelial cell imparting an adenomatoid (or gland-like) appearance. (H&E, ×100.)
Ultrasonography has been demonstrated to be a highly useful modality in the in utero diagnosis of CPAM (208).
In utero serial sonography has demonstrated the gradual reduction in the size of CPAM, type 1 and 2, with
subsequent normal development of the uninvolved lung (209).
There are several examples of anomalies seen in association with CPAM, mostly with type 2:
Bilateral renal agenesis/dysgenesis

Extralobar pulmonary sequestration
Cardiovascular malformation
Hydrocephalus and macrocephaly
Myelomeningocele
Jejunal atresia
Prune-belly syndrome
Sirenomelia
Bilateral nephromegaly
Pierre Robin syndrome
Pulmonary hypoplasia
Skeletal malformation
Bile duct hypoplasia
Left heart hypoplasia
Polycytosis of a solitary medial kidney
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FIGURE 12-28 ▪ CPAM, type 4. A: The lung is distended by thin, almost translucent cyst walls. B: The walls of
the cysts are composed of loose mesenchyme covered by an indistinct epithelial lining not apparent at this
magnification (Hematoxylin and eosin stain, original magnification ×25) C: The cyst walls are variously covered
by an attenuated epithelium of alveolar lining cells. (Hematoxylin and eosin stain, original magnification ×150). D:
The epithelium stains positively for cytokeratin (H&E, ×50).
Anomalies are noted in 15% to 20% of all cases of CPAM, particularly in association with the type 2 lesion (210).
CPAM is a unilateral lesion in about 95% of cases and involves a single lobe in 80% to 90% of cases. The right
and left sides of the lung are nearly equally involved, with the lower lobes affected in about 60% of cases. Type
2 CPAM has been noted in nearly 50% of cases of extralobar sequestrations. An association of CPAM with the
later development of a bronchioloalveolar carcinoma has been established (211, 212).
Variants of CPAM exist as unique entities or are the result of alteration by associated anomalies. Fisher et al.
(213) described a type 1 CPAM with large cysts filled with large papillary projections that consisted of delicately
branching fibrovascular stalks covered with cuboidal to columnar epithelial cells.
Congenital Alveolar Capillary Dysplasia

Congenital alveolar capillary dysplasia with or without misalignment of pulmonary veins is a rare entity that
presents as progressive hypoxemia in the newborn and is uniformly fatal (214, 215). Familial occurrence has
been noted (216). A number of cases have been shown to be associated with mutations in STRA6 on
chromosome 15q24.1 (217). Associated anomalies (Table 12-6) are seen in over 50% of cases and include
duodenal atresia, congenital heart disease, asplenia, phocomelia, and ureteric and urethral obstruction, among
others (218). It is characterized by the failure of formation and ingrowth of alveolar capillaries. Broad alveolar
septa with large alveolar capillaries within the septal wall are the hallmark of this disorder (Figure 12-29A to D)
(219). Capillaries
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are centrally placed well beneath the basement membrane of the alveolar lining cells and surrounded by loose
mesenchyme (Figure 12-29C). Ectatic veins are present within bronchovascular bundles, occasionally within the
adventitia of the pulmonary arteries, and may form an intermittent ring around the bronchiole (Figure 12-29B).
Small muscularized arteries are also present within the acini, extending to the precapillary area (Figure 12-29D).
Treatment with inhaled nitric oxide and ECMO has prolonged life but has been uniformly unsuccessful in
changing the fatal outcome of this disorder without lung transplantation (220).
Table 12-6 ▪ ANOMALIES ASSOCIATED WITH ALVEOLAR CAPILLARY DYSPLASIA
Anophthalmia and distinct eyebrows (217)
Familiary microphthalmia (460)
Degeneration of the anterior segment of the eye (461)
Atrioventricular septal defect and quadricuspid pulmonary valve (462)
Down syndrome (463)
Gastrointestinal (464)
Duodenal atresia and anorectal anomaly
Intestinal malrotation
Total colonic Hirschsprung disease.
Duodenal atresia (216, 465)
Left-right asymmetry (466)
Arteriovenous malformation of the liver
Bilateral ureteropelvic junction obstruction
Atrioseptal defect
Abnormally lobated lungs

Hydronephrosis
Urethral atresia (467)
Atrioventricular canal
Absence of gall bladder
Absence of left umbilical artery
Pulmonary lymphangiectasia
Hypoplastic left heart (468)
Sturge-Weber syndrome
Anomalous pulmonary veins (466)
Diaphragmatic hernia (469)
Bilateral tibial agenesis/ectrodactyly dysostosis (470)
FIGURE 12-29 ▪ Congenital alveolar capillary dysplasia. A: Bulky stiff lungs display focal hemorrhage and
prominent interlobular septa. B: Dilated veins are present adjacent to and within the adventita of a pulmonary
artery (center-right). (Hematoxylin and eosin stain, original magnification ×40.)
FIGURE 12-29 ▪ (continued) C: Broad alveolar septa contain many centrally located capillaries with only a few of
them approaching the alveolar epithelium. (Hematoxylin and eosin stain, original magnification ×75.) D:
Muscularized arteries are present within alveolar septa well away from bronchioles. (Hematoxylin and eosin
stain, original magnification ×75.)
Peripheral Cysts of the Lung

Peripheral, air-containing cysts of the lung can be seen in neonates, infants, and young children; it occurs in
association with Down syndrome as a result of pulmonary infarction, or in association with idiopathic
spontaneous PT (5). Occlusion of the pulmonary artery in infants can result in peripheral infarction of the lung,
which, with necrosis and organization, can produce subpleural cysts of varying size. Gonzalez et al. (221)
reported peripheral cysts in 18 of 98 patients with Down syndrome and suggested that the cysts are an intrinsic
feature of the disease that may result from reduced postnatal production of peripheral small air passages and
alveoli. The 0.2- to 1.0-cm air-filled cysts are located beneath the pleura and are formed of vascular fibrous
connective tissue walls lined by alveolar lining cells (Figure 12-30A,B). The cysts communicate with more
centrally located bronchioles and alveolar ducts. The cysts resemble those seen in the upper lobes of adult
males with idiopathic spontaneous PT and have also been noted in a case of ILE (147).
Hyaline Membrane Disease (HMD)

HMD is the pathologic counterpart of neonatal or idiopathic respiratory distress syndrome (RDS). It is
characterized by firm, atelectatic lungs with an uneven air-expansion pattern, focal hemorrhage, edema fluid in
alveoli, and hyaline membranes along terminal and respiratory bronchioles and alveolar ducts (222).
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Although infrequently seen in its “pure” form since the advent of surfactant replacement, sophisticated
mechanical ventilation and oxygen supplementation, HMD occurs primarily in premature infants with pulmonary
surfactant deficiency due to a variety of conditions. There is also, however, an increased incidence of HMD in
postterm infants (223). Infants present with tachypnea, intercostal retractions, and hypoxemia and display a
typical x-ray image of ground-glass alterations of the lungs with an air bronchogram and diffusely scattered
reticulogranular opacities (Figure 12-31A) (1).
FIGURE 12-30 ▪ Peripheral cysts of the lung. A: Small intercommunicating cysts lie between the pleura and
normal pulmonary parenchyma. B: The fibrovascular cyst walls are continuous with the interlobular septa of the
underlying lung. (Hematoxylin and eosin stain, original magnification ×15.)
Grossly, the lungs are firm and resemble liver more than lung. Microscopically, there is an uneven air-expansion
pattern with atelectatic acini and dilated bronchioles and alveolar ducts (Figure 12-31B). Scattered foci of
alveolar hemorrhage and edema are present, but most striking is the presence of smooth, homogeneous, pink
membranes lining
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terminal and respiratory bronchioles and alveolar ducts, particularly at points of division or branching (Figure 12-
31C). These hyaline membranes are composed of necrotic alveolar lining cells, plasma transudate, inhaled
amniotic fluid including squames, and fibrin, if hemorrhage is present. Hyaline membranes may be seen in infants
who die as early as 3 to 4 hours after birth and are uniformly present as well-formed structures by 12 to 24 hours
in infants with RDS. In the absence of severe disease requiring high oxygen tensions and ventilatory pressures,
at 36 to 48 hours the membranes begin to organize and separate from the underlying wall to be replaced by
alveolar lining cells or bronchiolar cuboidal or columnar epithelium (Figure 12-31D) (131).
FIGURE 12-31 ▪ Hyaline membrane disease. A: In this 24-hour-old, 1,050-g infant with respiratory distress, the
lungs display a classic “ground glass” opacity. B: The lungs in HMD are often atelectatic and display focal
hemorrhage. C: Dilated bronchioles and alveolar ducts are lined by thick hyaline membranes. (Hematoxylin and
eosin stain, original magnification ×25.) D: At 72 hours of age, the membranes are being covered by
regenerating alveolar lining cells. (Hematoxylin and eosin stain, original magnification ×100.)
Bacteria may alter the appearance of the membranes by producing fragmented, faintly basophilic structures, with
organisms often readily demonstrable by Gram stain on or within the membranes. Conditions associated with
hyperbilirubinemia (e.g., kernicterus, intraventricular hemorrhage,
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intrahepatic bile stasis, disseminated intravascular coagulation) may produce, in infants surviving 3 or more
days, yellow hyaline membranes as a result of the presence of unconjugated bilirubin (151).
Surfactant replacement therapy, although radically decreasing the incidence of HMD in premature infants and its
morbidity and mortality in these infants, does not appear to alter the pathologic features of HMD in infants dying
of RDS, although clinically there may be a slightly higher incidence of pulmonary hemorrhage and a lower
incidence of IPE, PT, and retinopathy of prematurity (224, 225). Surfactant therapy appears to accelerate the
rate of epithelial cell regeneration (226).
Bronchopulmonary Dysplasia (Chronic Lung Disease of Prematurity)

Bronchopulmonary dysplasia was first described in 1967 by Northway et al. (227). In a retrospective study, they
describe the clinical and pathologic features of 19 infants dying following mechanical ventilation with high
concentrations of oxygen for severe HMD (RDS). The pathology was correlated with clinical and radiographic
findings and included a 2- to 3-day period of acute RDS, followed by a weeklong period of “regeneration,”
another 10-day period of transition to chronic disease, and a final period of chronic disease extending beyond 1
month of life. The pathologic features in the first stage included the typical findings of HMD (e.g., atelectasis,
uneven air expansion pattern, hemorrhage, and hyaline membranes). During the second stage, there was
necrosis of bronchiolar and alveolar epithelium with persistence of hyaline membranes (Figure 12-32A to C). In
the transition to chronic disease, injury to alveolar epithelium continued, along with widespread bronchial and
bronchiolar mucosal metaplasia and marked mucus secretion. Clusters of hyperexpanded alveoli alternated with
areas of atelectasis. In the chronic stage, bronchioles displayed marked peribronchiolar smooth muscle
hypertrophy associated with clusters of “emphysematous alveoli.” The birth weights of the 19 infants dying of
bronchopulmonary dysplasia varied from 900 to 2,466 g, with two-thirds of them weighing more than 1,300 g.
Northway et al. (227) suggested that bronchopulmonary dysplasia was due to the toxic effects of oxygen, poor
bronchial drainage, and the effects of mechanical ventilation.
FIGURE 12-32 ▪ Bronchopulmonary dysplasia (BPD), acute and severe. A: The lungs are bulky and firm. Note
the tube perforating the upper lobe. B: Necrotizing bronchiolitis is a key feature of acute BPD and with occlusion
such as this precludes the acinus distal to it from being available for air exchange (H&E, × 125). C: In acini
whose bronchioles are not occluded by necrotizing bronchiolitis, the distal portion is exposed to the full
barotrauma and oxygen toxicity used in the treatment of HMD. As a result, there is alveolar cell
hyperplasia/dysplasia and alveolar septal fibroplasia (H&E, ×25).
In the 10 years following that initial brief description of the pathology of BPD, a number of other studies
described in more detail the pathologic features including the changes noted in alveoli, airways, lymphatics,
vessels, and connective tissue as criteria for the staging of bronchopulmonary dysplasia. In 1976, Bonikos et al.
(228) described a severe necrotizing bronchiolitis in the acute stages of BPD and
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implicated prolonged exposure to high levels of oxygen as a major feature in the cause of bronchiolitis. Since that
time, a number of additional factors, including infection, inflammation, poor nutrition, dehydration, and others,
have been implicated in the pathogenesis of BPD (229). In addition to the bronchiolitis, Bonikos et al. (230)
described a prominent alveolar septal fibrosis in the healed stages along with an increased incidence of cardiac
hypertrophy.
FIGURE 12-33 ▪ Long-standing healed bronchopulmonary dysplasia. A: Irregular clefting and fissuring of
pulmonary lobes probably represent the loss of acini during the acute phases of BPD. B: The acinus at top
represents the one “protected” by occluded bronchioles from the damage of barotrauma and high oxygen
pressures. At the bottom, this acinus displays the diffuse alveolar septal fibrosis caused by previous exposure to
barotrauma and high oxygen pressures (Masson trichrome, ×25).
The sequelae of this necrotizing bronchiolitis was described by Stocker in 1986 in a series of 28 patients with
long-standing “healed” bronchopulmonary dysplasia, who died at 3 to 40 months of age (131). Noting the
presence of deep pleural fissures and acini with varying degrees of alveolar septal fibrosis (Figure 12-33), It was
suggested that the necrotizing bronchiolitis seen in the acute phases, while prohibiting adequate ventilation,
often served to “protect” acini from damage by mechanical ventilation or high levels of oxygen (Figure 12-34A-C).
Stocker also suggested that the alveolar fissures might represent areas of complete loss of acini corresponding
to the marked decrease in internal surface area and number alveoli noted by Sobonya et al. (231). The 6- to 10-
fold reduction in number of alveoli suggested not only an absolute loss of some acini but a generalized reduction
in lung growth. In 1991, Margraf et al. (232) confirmed the reduction in lung volume and small airway density
noted by Sobonya.
FIGURE 12-34 ▪ Bronchopulmonary dysplasia (BPD) before the advent of surfactant replacement therapy. A:
Schematic representation of three uniformly distended acini (a to c) with associated bronchiole, alveolar ducts,
and alveoli. B: In the early stages of BPD, hyaline membranes or necrotic debris may totally occlude a
bronchiole (a) protecting the distal acinus. Bronchioles that remain partially or completely open (b, c) allow the
distal acinus to be exposed to varying degrees of injury from barotrauma and high oxygen tension. C: In the
healed stages of BPD with resolution of the bronchiolar obstruction in (A), the “protected” distal acinus expands
and continues to develop new alveoli. Depending on the degree of injury, acini may atrophy and disappear (c),
producing pleural fissures (see Figure 12-33A), or display varying degrees of alveolar septal fibrosis (b) and be
inhibited from further alveolar development. (From Stocker JT. Pathologic features of long-standing “healed”
bronchopulmonary dysplasia: a study of 28 3- to 40-month-old infants. Hum Pathol 1986; 17:943, with
permission.)
In recent years, with the advent of surfactant replacement therapy and increased sophistication in the use of
mechanical ventilation (including high frequency jet ventilation) and oxygen supplementation, another stage in
the evolution of
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the pathology of bronchopulmonary dysplasia has been seen. Although the occasional case of “classic” acute
bronchopulmonary dysplasia with necrotizing bronchiolitis, alveolar cell hyperplasia, and peribronchiolar and
alveolar septal fibroplasia is still seen along with focal alveolar septal fibrosis in the older patient (Figure 12-35A
to D), the few infants who now die from bronchopulmonary dysplasia display what might best be described as
“acinar simplification.” These simplified acini are characterized by uniformly dilated alveoli whose walls consist of
thin alveolar septa with little or no interstitial fibrosis (233).
FIGURE 12-35 ▪ Bronchopulmonary dysplasia or chronic lung disease of the premature since the advent of
surfactant replacement therapy. A: Schematic representation of three normally expanded and aerated pulmonary
acini in an immature infant. Note the appropriately thick septa of the developing lung. B: With normal growth and
development, the acini not only increase in size [relative to (A)] but also in complexity, with the appearance of
“new” alveolar saccules and alveoli. C and D: In infants receiving surfactant replacement therapy who develop
moderate-to-severe BPD, the acini increase in size [relative to (A)] but show little, if any, increase in the number
of alveolar saccules or alveoli. The alveolar septa in (C) appear normal in thickness compared with the less
injured or uninjured lung (B), or they may display a uniform mild alveolar septal fibrosis as in (D).
The bronchioles are similarly unremarkable, with only an occasional mild increase in peribronchiolar
musculature. These changes seem to represent an “arrest” of development of the acini, with a resulting markedly
decreased number of alveoli within each acinus (Figure 12-36A to C). As a result, the surface area of the lung is
significantly decreased even in the absence of significant pathology (e.g., alveolar septal fibrosis).
Although high concentrations of oxygen over prolonged periods of time are known to cause alveolar cell
hyperplasia and necrotizing bronchiolitis with resulting alveolar septal fibrosis, it is possible that low levels of
oxygen (25% to 35%), while not producing significant alteration in the epithelial lining of the lung or not causing
damage sufficient to cause septal fibrosis, may, in very immature infants, inhibit growth of the lung, that is, the
development of new alveolar ducts and alveoli. Although the lung appears to “mature” and alveolar septa appear
to thin and expand to resemble the septa of term infants, there is no accompanying significant increase in the
surface area of the lung through an increase in number of alveoli. Thus, although recent advances in mechanical
ventilation have limited the amount of injury to the bronchiole (i.e., no necrotizing bronchiolitis), the continued
patency of all bronchioles throughout the course of therapy allows equal injury or inhibition of growth to all acini
from even low levels of oxygen therapy.
Husain et al. (233) examined the lungs at autopsy of 22 patients with BPD, of whom 14 had received surfactant
therapy and compared them with 15 age-matched controls. Using readily available morphometric techniques
[RAC and mean linear intercept (MLI)], they displayed a virtual arrest of alveolar development in both the
surfactant-treated and nonsurfact ant-treated infants whether or not the typical feature of LSHBPD (i.e., alveolar
septal fibrosis) was present. Incidentally, septal fibrosis was infrequently seen in surfactant-treated BPD patients
even though their disease was severe enough to contribute significantly to their death. The RAC/MLI ratio (an
indicator of the number of alveoli) in the BPD patients who lived weeks to months was virtually unchanged from
that expected at the infant's birth weight. In other words, an infant born at 28 weeks' gestation, who developed
HMD and BPD and lived for 12 weeks, had the same number of alveoli as the one born at 28 weeks' gestation
who died in a few days.
As a result of surfactant replacement therapy and sophisticated methods of ventilation, we now see a much
smaller percentage of immature and premature infants with chronic lung disease, and when this chronic lung
disease does occur, it does not at all resemble the BPD described in the 1970s and 1980s. The classic features
of BPD (necrotizing bronchiolitis, epithelial cell hyperplasia, bronchiolar muscular hyperplasia, and alveolar
septal fibrosis) are, in fact, rarely seen today. The chronic lung disease of prematurity of today is an extremely
subtle disease (at least from a pathologic perspective) that manifests itself primarily as an inhibited or arrested
growth and development of the lung. The etiology of this type of failure of development is unclear. Long-term
sequelae of BPD include late sudden unexpected death, lobar overinflation, and right, left, or biventricular
myocardial hypertrophy (234).
Congenital Surfactant Deficiency

Inherited deficiency of one or more surfactant proteins (most frequently surfactant protein B) is often a fatal
autosomal recessive disorder of lung cell metabolism and is characterized by rapidly progressive respiratory
failure immediately after birth (235). The disease is caused by a deficiency of adenosine triphosphate-binding
cassette (ABC) protein (236), most frequently ABC A3 (237). Chorionic villous sampling can be used to identify
the homozygous state in utero (238). Less frequently, abnormalities of surfactant protein-A
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and surfactant protein-C may occur. Lung transplantation has been successful, although patients may develop
anti-surfactant protein-B antibody (239). Gene therapy utilizing adenoviral vectors has been studied for
surfactant protein B deficiency (240).
FIGURE 12-36 ▪ Chronic lung disease of the premature. A: The lung appears largely unremarkable (compare
with Figure 12-33B) with an evenly aerated parenchyma. B: In this section of lung from a 4-monthold infant born
at 26 week's gestation who developed moderate respiratory distress and clinical BPD, the acinus is simplified
with dilated alveolar ducts and saccules and with very few alveoli arising from them (see Figure 12-35C)
(Hematoxylin and eosin stain, original magnification ×60.). C: In this section of lung from a 2-month-old infant
born at 28 week's gestation who developed severe prolonged respiratory distress and clinical BPD, the alveolar
septa of all acini show mild though uniformly interstitial fibrous thickening. (Masson trichrome stain, ×40.)
Grossly, the lungs in congenital surfactant deficiency are heavy and appear consolidated. Microscopically, in the
early stages, alveoli are lined by a continuous layer of cuboidal alveolar lining cells (Figure 12-37A). As the
disease progresses, alveoli may be filled with eosinophilic granular material admixed with desquamated alveolar
cells and macrophages resembling congenital alveolar proteinosis (241) (Figure 12-37B). In the later stages,
alveolar septa are widened by fibroblasts producing alveolar septal fibrosis, although the alveolar cell
hyperplasia persists (Figure 12-37C). Immunohistochemical stains of the typical SPB-deficient lung demonstrate
decreased to absent SP-B and normal to increased amounts of A and C in alveolar lining cells (Figure 12-
37D,E). Electron microscopy displays alveolar type II cells with irregular electron-dense bodies, which also may
be present in alveolar spaces and macrophages (242).
Interstitial Pulmonary Emphysema

IPE is the dissection by air around bronchovascular bundles and along intralobular septa as the result of rupture
of alveoli, usually in association with mechanical ventilation (Figure 12-38). Dissection of air peripherally through
the pleura produces PT, whereas medial dissection can lead to pneumomediastinum, pneumopericardium, and,
rarely, pneumomyocardium (Figure 12-39A to C) (192). Although these air leaks can occasionally be observed in
normal infants and may spontaneously occur in about 5% of infants with RDS, the highest incidence is seen in
infants with RDS who are receiving mechanical ventilation. Although the incidence of IPE and its complications in
neonatal intensive care nurseries (NICU) was as high as 40% or more among all NICU patients 30 years ago,
early administration of surfactant and increasingly sophisticated means of ventilation have reduced the incidence
to 20% to 35% in only the sickest infants. Those particularly at risk include infants with lower 1 and 5 minute
APGAR scores, increased surfactant utilization, and higher inspired oxygen concentration (243). IPE has also
been reported in 20% of patients dying of acute asthma, as
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a result of cardiopulmonary resuscitation, and in association with a variety of infectious diseases (244).
FIGURE 12-37 ▪ A: Congenital surfactant deficiency. In the early stage, alveolar type II cells are hyperplastic,
lining up side by side along alveolar septa. (Hematoxylin and eosin stain, original magnification × 100.) B: As the
disease progresses, alveolar cells may be sloughed and undergo dissolution, producing the features of alveolar
proteinosis. (Hematoxylin and eosin stain, original magnification ×100.) C: With further progression, alveolar
septa become widened with fibrous connective tissue. Note the continued alveolar cell hyperplasia. (Masson
trichrome stain, original magnification ×100. D and E: Stains for surfactant in this surfactant-deficient lung are
positive for surfactant A (D) and negative for surfactant B (E). (Immunoperoxidase stains ×125.)
IPE can be acute (<7 days' duration) or persistent and may be localized to a single lobe or distributed diffusely
through all lobes (245). Acute IPE (AIPE) presents grossly as 0.1- to 0.5-cm air blebs located beneath the pleura
along junctions between the interlobular septa and the pleura (Figure 12-40A). On cut section, round to oval air
spaces may be seen around bronchovascular bundles and along the interlobular septa (Figure 12-40B). Only
rarely do the air-filled cysts dissect laterally from the septa beneath the pleura, which aids in the differentiation of
IPE from CPL. Microscopically, the cysts of AIPE are confined to the interlobular septal and peribronchial region,
compressing the adjacent blood vessels and acini (Figure 12-40C). The walls consist primarily of loose
connective tissue and compressed parenchyma. AIPE may incorporate some of the lymphatics of the interlobular
septa, but the vast majority of cysts appear to be formed from air-dissected connective tissue. Subpleural
lymphatics are rarely involved and appear unremarkable.
Persistent interstitial pulmonary emphysema (PIPE) occurs in infants with AIPE that lasts more than 1 week. The
cysts of
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PIPE may be localized to a single lobe or, when seen in association with BPD, diffusely radiate throughout most
or all of the lobes (245). PIPE is grossly characterized by multiple 0.1- to 0.3-cm cysts localized to the interlobular
septa and extending radially from the hilum to the pleura (Figure 12-41A,B). Cysts in the localized form of PIPE
tend to be larger than those in lungs that are diffusely involved, occasionally as large as 5 cm (246, 247). The
intercommunicating, irregularly shaped cysts are air-filled and lined with a smooth, glistening membrane. A
communication between the airway system and the interstitium may be demonstrable (245). Microscopically, the
cysts are composed of a thin to thick fibrous connective tissue wall intermittently “lined” with multinucleated
foreign body giant cells, the pathognomonic feature of PIPE
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(Figure 12-41C,D). The adjacent parenchyma is usually compressed and, in the diffuse form, frequently displays
the features of BPD. Treatment of localized PIPE consists of surgical resection or a variety of forms of selective
intubation, mechanical ventilation, or both (248).
FIGURE 12-38 ▪ Potential complications related to mechanical ventilation and pulmonary interstitial air. (From
Askin FB. Pulmonary interstitial air and pneumothorax in the neonate. In: Stocker JT, ed. Pediatric pulmonary
disease. Washington, DC: Hemisphere, 1989:166, with permission.)
FIGURE 12-39 ▪ Pneumopericardium, PT, and pneumomediastinum. A: In this chest x-ray, air can be seen within
the pericardial sac surrounding the heart (pneumopericardium), and in the right hemithorax (PT). B: At autopsy,
the air distended the pericardial sac. (Courtesy of Ralph E. Franciosi, M.D., Children's Hospital of Wisconsin,
Milwaukee, Wisconsin.)
FIGURE 12-39 ▪ (continued) C: Blebs of air dissect the tissues of the mediastinum (pneumomediastinum).
FIGURE 12-40 ▪ Acute PIPE. A: Air can be seen beneath the pleura at the junction of interlobular septa and
pleura. B: Round to oval, air-containing cysts are present within interlobular septa. The cysts extend radially
from the hilum to the pleura. C: The pulmonary artery (bottom) is surrounded and partially compressed by air-
filled spaces. (Masson trichrome, ×50.)
Aspiration
Aspiration of material into the tracheobronchial tree can occur in utero, during delivery, or in the neonate or
young child (Table 12-7). The material aspirated may obstruct the major airways and produce sudden respiratory
distress and even death (e.g., tracheal obstruction from aspiration of a peanut), or the distribution of the material
may be more diffuse, leading to a “chemical” pneumonitis (e.g., aspiration of meconium or gastric contents).
Aspiration of amniotic fluid in utero is a normal physiologic process, and a few sloughed squamous epithelial
cells (“squames”) can be seen in the lungs of virtually every term or near-term infant, but massive aspiration of
amniotic debris may be seen in postterm infants or in infants with oligohydramnios.
Grossly, the lungs are expanded and firm. Microscopically, squames distend alveolar ducts and alveoli. As noted
above, however, small number of squames can be seen in the lungs of virtually all infants born after 34 to 36
weeks of gestation (249).
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Meconium staining of amniotic fluid is seen in up to 29% of all pregnancies and was noted in 12.15% of 176,790
neonates reported by Wiswell et al. (250). Approximately, 5.5% of meconium-stained neonates (0.66% of all
neonates) develop the meconium aspiration syndrome (MAS). Boys are more frequently affected than girls. MAS
may cause death in approximately 4% of affected neonates.
MAS presents as respiratory distress in the meconium-stained neonate and requires mechanical ventilation in
about 30% of cases (251). Pneumothoraces are noted in more than 11% of MAS infants. ECMO and surfactant
lavage have contributed to the increasing survival of neonates with MAS (252).
FIGURE 12-41 ▪ Persistent PIPE. A: Air blebs are noted beneath a partially “clouded” pleura. B:
Intercommunicating, irregular, air-filled cysts lined by a smooth membrane compress the pulmonary parenchyma.
C: The irregular cysts extend along interlobular septa. The cyst walls are composed of fibrous connective tissue
of varying thickness, irregularly covered by foreign body giant cells. Note the bronchus (left). (Hematoxylin and
eosin stain, original magnification ×4.) D: The foreign body giant cells contain multiple, eccentrically placed
nuclei amid a smooth to granular cytoplasm. (Hematoxylin and eosin stain, original magnification ×210.)
Meconium, the residual of gastrointestinal secretions accumulated in the lower gastrointestinal tract of the fetus,
can, when aspirated, obstruct the trachea, bronchi, and bronchioles. The tenacious green-yellow material can
frequently be seen grossly as plugs within bronchi and bronchioles on cut section of the lung. Microscopically,
the material is composed of amorphous, acellular, faintly basophilic debris (Figure 12-42A). In infants surviving
more than a few hours, a chemical pneumonitis develops with alveoli filled with neutrophils and basophilic debris.
Chronic intrauterine meconium aspiration may cause pulmonary infarction, rupture, and meconium embolism.
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Table 12-7 ▪SOURCES OF ASPIRATED MATERIAL
Amniotic debris
Meconium
Blood
Milk
Gastric contents
Foreign bodies
Plants
Pins
Pieces of toys
Small batteries
Toxic fluids
Kerosene
Furniture polish
Mineral oil
Aspiration of maternal blood during delivery may produce clinical features that mimics pulmonary hemorrhage. In
infants, milk may be aspirated during feeding or regurgitation. Older infants with esophageal reflux or neurologic
disorders (e.g., cerebral palsy) are also prone to aspiration. Gastric contents may obstruct bronchi or bronchioles
and produce a chemical pneumonitis in which meat and vegetable fibers may be identified in association with
granulomata and foreign body giant cells. (Figure 12-42B to D)
Aspirated foreign bodies are responsible for about 2,000 deaths a year in children, usually lodge in the upper
airway or bronchi, and include virtually any object that will pass through the glottis into the larynx (253, 254).
Bronchial obstruction may lead to acute or chronic pneumonia including the development of an ILS (48).
Aspirated fluid such as kerosene, mineral oil, and furniture polish may result in severe pulmonary damage
including diffuse alveolar damage, lipoid pneumonia, and diffuse necrosis. ECMO has been helpful in treating
these patients, as well as patients with meconium aspiration (see below).
FIGURE 12-42 ▪ Aspiration. A: Meconium. Amorphous debris containing scattered neutrophils occludes a
bronchiole in this term infant who was intensely meconium stained. (H&E, ×50) B: Vegetable fibers are
accompanied by acute bronchiolitis (H&E, ×50).
FIGURE 12-42 ▪ (codntinued) C: Gastric contents—milk. (H&E, ×40) D: Amniotic debris. (H&E, ×50).
Extracorporeal Membrane Oxygenation

The development of ECMO and its use in the treatment of meconium aspiration, alveolar capillary dysplasia,
diaphragmatic hernia (among other causes of pulmonary hypoplasia), Listeria monocytogenes infection, and
congenital heart malformations have produced a variety of pathologic changes involving the lung and other
organs (220, 255).
Chou et al. (256) have described the autopsy findings in 23 infants receiving ECMO therapy and noted the
presence of interstitial and intra-alveolar hemorrhage along with hyaline membrane formation during the first few
days of therapy. They described the hyperplasia of type II alveolar cells and bronchial epithelial cells after 2 days
of ECMO therapy in some patients and, by 7 days, in all patients. Interstitial fibrosis was also noted beginning at
7 days. After 15 days of treatment, there was replacement of the terminal airways and alveoli by tall columnar
and mucin-producing epithelium (Figure 12-43). Squamous metaplasia of bronchial epithelium was also seen in
the majority of patients, and one patient developed mucinous metaplasia as well. Clusters of calcified material
were present in the alveoli of 7 of 23 cases (255). Long-term survivors of ECMO show a high frequency of
hyperinflation and airway obstruction (257).
Extrapulmonary changes include ischemic neuronal necrosis, focal cerebral infarcts, intracerebral hemorrhages,
periventricular leukomalacia, pericardial hemorrhage, carotid artery injury, and retinal vasculopathy (258, 259).
Pulmonary Hemorrhage
Hemorrhage into the alveoli or interstitium of the lung is a frequent finding in tissue removed at surgery at all
ages. In the neonatal period, however, pulmonary hemorrhage is frequently associated with HMD and BPD but
may also occur in association with patent ductus arteriosus (260), erythroblastosis, congestive heart failure,
disseminated intravascular coagulation, congenital malformations, acute pneumonia,
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systemic lupus erythematosus (261), Goodpasture syndrome (262), and rarely, as an isolated finding (263, 264).
The incidence of neonatal massive pulmonary hemorrhage, defined as hemorrhage involving more than two-
thirds of the lung, is seen in up to 40% of neonatal autopsies. The appearance of hemorrhage may also be
produced by the intrapartum aspiration of maternal blood, which may mimic both the clinical and pathologic
features of massive pulmonary hemorrhage. Identification of the type of alveolar red blood cells (maternal versus
fetal) allows for separation of the two entities.
Pulmonary Veno-occlusive Disease

Pulmonary venous obstruction may be secondary to congenital cardiac malformations such as mitral stenosis or
cor triatriatum, or it may be due to an intrinsic disease of the pulmonary veins, that is, pulmonary veno-occlusive
disease (PVOD). PVOD is a rare cause of pulmonary hypertension that mainly affects children and young adults
(265). Patients with PVOD present with symptoms of right-sided heart failure. Radiologic examination shows
prominent pulmonary arteries with Kerley B lines, interlobular septal thickening, pleural effusion, and mediastinal
adenopathy (266). Microscopically, pulmonary veins and venules show eccentric intimal fibrosis or are occluded
by thrombi, which may be partially recanalized and are best seen with an elastic or connective tissue stain.
Evidence of pulmonary hypertension is seen in arterialized veins and medial hypertrophy of arteries.
Its cause is unknown, although viral infections (including HIV), antiphospholipid antibody, and drugs have been
implicated (267, 268). PVOD has also been seen to develop in patients following bone marrow transplantation
(269) and as a component of pulmonary capillary hemangiomatosis (268, 270). No effective treatment is
available; lung transplantation has been tried. The prognosis associated with PVOD is poor.
Pulmonary Alveolar Microlithiasis

Pulmonary alveolar microlithiasis is a rare and unusual disorder reported worldwide as single cases or in siblings
or other family members (271). Patients often are asymptomatic with a diffuse miliary pattern concentrated along
bronchovascular bundles, interlobular septa, and beneath the pleura on routine chest x-ray study (272).
Diagnosis is made by demonstrating calcium phosphate microconcretions on bronchoalveolar lavage or lung
biopsy. An autosomal recessive inheritance pattern has been proposed, and one case has been associated with
the Waardenburg-anophthalmia syndrome (273). The disease progresses with eventual pulmonary hypertension,
cor pulmonale, and respiratory failure.
Pulmonary Hemosiderosis
Hemosiderin in the lung usually indicates previous hemorrhage or aspiration of blood and is thus relatively
nonspecific
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(Table 12-8). Macrophages containing hemosiderin can be found in alveolar or interstitial regions in association
with conditions such as infection, blood dyscrasia, chronic heart failure, pulmonary hypertension, and neoplasia.
There exists a group of rare disorders that are characterized by single or repeated episodes of bleeding that can
lead to massive hemorrhage or progress to chronic pulmonary disease. Based on the clinical, laboratory, and
immunopathologic findings, they are divided into two categories:
1. Idiopathic pulmonary hemosiderosis (IPH), with pulmonary hemorrhage as an isolated process
2. Secondary pulmonary hemorrhage associated with immunologically mediated renal or vascular disease.
FIGURE 12-43 ▪ Extracorporeal membrane oxygenation. This term infant with a large left-sided diaphragmatic
hernia and severe pulmonary hypoplasia was on ECMO for 16 days before dying. Note the alveolar septal
fibrosis and the replacement of the terminal airways and alveoli (right) by cuboidal to columnar epithelium.
(Hematoxylin and eosin stain, original magnification ×40.)
Table 12-8 ▪ DISORDERS ASSOCIATED WITH DIFFUSE PULMONARY HEMORRHAGE AND

HEMOSIDEROSIS IN INFANCY AND CHILDHOOD
Idiopathic pulmonary hemosiderosis (isolated)
Pulmonary hemosiderosis associated with sensitivity to cow's milk
Pulmonary hemosiderosis and glomerulonephritis
With antibodies to GBM (Goodpasture syndrome)
Without antibodies to GBM (usually immune-complex glomerulonephritis)

Pulmonary hemosiderosis associated with collagen-vascular or purpuric disease
Systemic lupus erythematosus
Wegener granulomatosis
Polyarteritis nodosa
Rheumatoid arthritis
Schönlein-Henoch purpura
Idiopathic thrombocytopenic purpura
Pulmonary hemosiderosis secondary to cardiac disease, Intrapulmonary vascular lesions, or

malformations Chronic left-sided or right-sided heart failure (e.g., mitral stenosis)
Pulmonary hypertension
Pulmonary veno-occlusive disease
Pulmonary lymphangiomyomatosis
Arteriovenous fistulas and other congenital vascular malformations
Vascular thrombosis with infarction
GBM, glomerular basement membrane
From Cutz E. Idiopathic pulmonary hemosiderosis and related disorders in infancy and childhood.
Perspect Pediatr Pathol 1987;11:49, with permission.
Idiopathic Pulmonary Hemosiderosis

IPH presents with symptoms including anemia, hypoxemia (85%), dyspnea, and hemoptysis (65%). It occurs
primarily in children 3 to 6 years of age but can be seen in children as young as 4 to 6 months of age.
Consanguinity and environmental factors may be involved in the development of IPH (274). Sex incidence is
equal, and 15% to 20% of cases occur in adolescents and young adults. Less specific non-pulmonary symptoms
include fever (in as many as 79% of cases), lymphadenopathy, hepatomegaly, and splenomegaly.
Radiographically, early stages are characterized by patchy or diffuse pulmonary infiltrates or massive confluent
shadows that may rapidly clear. In later stages of the disease, there is a perihilar reticulation or a pattern of
diffuse interstitial disease. The clinical triad of hemoptysis, iron deficiency anemia, and diffuse parenchymal
infiltrates is strongly suggestive of IPH (274). The presence of hemosiderin-laden macrophages on
bronchoalveolar lavage is also highly correlated with IPH, with the presence of 35% or more hemosiderin-laden
macrophages associated with a sensitivity of 1% and a specificity of 96% (275).
Hypochromic microcytic anemia is seen in virtually all cases of IPH, and eosinophilia is present in 12% to 15% of
patients (276). Bone marrow examination shows reactive erythroid hyperplasia and depleted iron stores. Levels
of serum iron are low, and total iron binding capacity is increased. Most patients with IPH have normal renal
function without circulating autoantibodies (compare with Goodpasture syndrome; see Chapter 17). An
association with celiac disease has also been reported (277, 278) and, rarely, juvenile dermatomyositis (279).
Although some children with IPH may die of massive hemorrhage shortly after presentation, other patients have a
history of progressive respiratory insufficiency leading to death 2 to 5 years after diagnosis, although a 5-year
survival of 86% was reported in 17 patients receiving corticosteroids (280) and other immunosuppressant agents
(281, 282).
Bronchoalveolar lavage demonstrates hemosiderin-laden alveolar macrophages in large numbers (283). Lung
biopsy and autopsy specimens show varied involvement. Focal areas of consolidation are common owing to
massive accumulations of hemosiderin-laden macrophages, which obliterate alveolar spaces and are associated
with interstitial fibrosis. Corrin et al. (284) describe capillary endothelial swelling and focal thickening of the
basement membrane. Stainable iron is present in alveolar and tissue macrophages, free in connective tissues,
and encrusting elastic fibers of small blood vessels and alveolar septa (Figure 12-44A to C). There is mild-to-
moderate alveolar cell hyperplasia, peribronchial lymphoid hyperplasia, and alveolar septal mastocytosis (75).
Immunofluorescence is negative for immunoglobulin, complement and antibasement membrane antibodies (285).
Infectious Diseases
Infectious diseases affecting the lungs (among other organs) are described in detail in Chapter 6. Organisms
specifically or primarily affecting the lungs bear special mention.
Respiratory Syncytial Virus

Respiratory syncytial virus (RSV) is the most important respiratory pathogen of infancy and childhood, and
creates sizable outbreaks of infection each year. An RNA virus, RSV occurs in regularly recurring epidemics in
midwinter and early spring. It has been identified as causing 5% to 40% of pneumonias (50% of viral
pneumonias), from 50% to 90% of cases of bronchiolitis, and from 10% to 30% of cases of persistant bronchitis
in young children (286). RSV presents clinically with fever, cough, rhinitis, pharyngitis, and dyspnea and can
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produce severe enough bronchiolitis to require hospitalization in 1% to 2% of cases, primarily in children 2 to 5
months of age, accounting for 90,000 hospital admissions annually in the United States (286). The mortality rate
in these hospitalized patients is 1 % to 3%, causing 4,500 deaths in infants and children in the United States
annually. RSV may, however, be associated with a higher mortality rate in children infected with pathogens such
as adenovirus, pneumococcus, cytomegalovirus, and Pneumocystis jiroveci (287). Of patients with RSV
requiring mechanical ventilation, the majority have at least one additional risk factor for a severe course of
infection (prematurity 50%, chronic lung disease 20%, congenital heart disease 35%, immunodeficiency 20%)
(288). Of those dying of RSV, Thorburn noted one of the following preexisting medical conditions in every
patient-chromosomal abnormalities 29%, cardiac lesions 27%, neuromuscular disorder 15%, chronic lung
disease 12%, large airway abnormality 9%, and immunodeficiency 9% (289).
FIGURE 12-44 ▪ Idiopathic pulmonary hemosiderosis. A: Hemosiderin is present in clusters of alveolar and
septal macrophages. B: An iron stain demonstrates masses of iron in alveolar macrophages and in connective
tissue. (Prussian iron stain, ×40.) C: Iron is also present in the media as well as encrusting elastic fibers adjacent
to a pulmonary artery. (Prussian iron stain, ×70.)
In fatal cases, RSV produces extensive alveolar and terminal bronchiolar plugging by granular eosinophilic debris
accompanied by peribronchiolar lymphocytic inflammation and edema (290). Eosinophils may be an integral part
of the inflammatory process (291). In less severely involved areas, bronchiolar epithelium displays uneven
proliferation with a polypoid appearance, squamous metaplasia, and desquamation. Syncytial giant cells may be
present along alveolar walls and may contain granular, mildly basophilic cytoplasmic inclusions, which may also
be seen in bronchial, bronchiolar, and alveolar epithelia (Figure 12-45A to C). Dense cytoplasmic inclusions can
be demonstrated by electron microscopy (286). RSV antigen can be demonstrated in formalin-fixed, paraffin-
embedded autopsy tissue by immunohistochemical techniques (Figure 12-45C).
Human Metapneumovirus
Human metapneumovirus (MNPV) accounts for nearly 10% of community-acquired alveolar pneumonia, but,
when compared with infants with RSV pneumonia, are older and have a more common history of acute otitis
media requiring tympanocentesis, wheezing and gastrointestinal symptoms, and a lower hospitalization rate
(292). MNVP is also seen more frequently
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than RSV in children with congenital abnormalities, particularly those with cardio-pulmonary problems and when
associated with an increased ventilatory requirement (293).
FIGURE 12-45 ▪ Respiratory syncytial virus. A: Amorphous inflammatory debris fills a bronchiole and
surrounding alveoli. Note the syncytial giant cells throughout the section. (H&E, ×75.) B: Hyperplastic alveolar
cells line the surface of this alveolus, which contains numerous syncytial giant cells. (H&E, ×200; courtesy of
Eduardo J. Yunis, M.D., Children's Hospital, Pittsburgh, Pennsylvania.). C: The cytoplasmic viral inclusions stain
intensely positive for RSV. (RSV immunoperoxidase stain, ×400.)
The pathology of HMPV has only recently been described (294). Bronchoalveolar lavage shows epithelial
degenerative changes and eosinophilic cytoplasmic inclusions within epithelial cells, multinucleate giant cells,
and histiocytes. Lung biopsy shows chronic airway inflammation and intra-alveolar foamy and hemosiderin-laden
macrophages.
Adenovirus
Adenovirus, a DNA virus, is frequently associated with gastroenteritis in infants and young children but also
accounts for 5% to 11% of cases of bronchitis, 2% to 10% of bronchiolitis, and 4% to 10% of pneumonia in
children (295). Adenovirus infections are seen most frequently in children younger than 5 years of age who
spend portions of their days in child care centers or other closed environments. Infections are also common in
grade and junior high school children during winter, spring, and early summer.
Severe cases of pneumonia are most common in children 3 to 18 months of age and are associated with
adenovirus types 3, 7, and 21. The onset is acute, and the child presents with high fever, persistent cough,
lethargy, diarrhea, vomiting, and pharyngitis. Adenovirus infection with or without interstitial pneumonia has been
implicated in up to 25% of sudden deaths in infants. Extrapulmonary complications (e.g., meningitis, myocarditis)
are common, and serious pulmonary sequelae (e.g., bronchiectasis, bronchiolitis obliterans (296), unilateral
hyperlucent lung) are seen in 14% to 60% of cases with documented lower respiratory tract disease (297).
The pneumonia is characterized by severe necrotizing bronchitis, bronchiolitis, and alveolitis (298). Adjacent to
areas of necrosis, the alveolar and bronchiolar epithelial cells are enlarged and contain small eosinophilic and
larger basophilic intranuclear inclusions. These inclusions have a characteristic amphophilic (smudged)
appearance. When viewed by electron microscopy, it can be seen that these inclusions contain viral particles
that measure 70 to 80 nm and are arrayed in a tight periodic pattern along diagonals, which create hexagonal
unit groups.
Legionella Pneumonia
Legionella pneumonia is seen infrequently in infants and children, but may be one of the many infections seen in
immunocompromised patients (299).
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Chlamydia Trachomatis
Chlamydia trachomatis, an obligate intracellular bacterial parasite, is a well-known oculogenital pathogen that
can also produce pneumonia in infants. Respiratory distress is noted in premature infants, and a progressive
staccato cough is seen in older infants. The disease is readily treatable with antibiotics, and the mortality rate is
low. Lung biopsy specimens display interstitial and intra-alveolar infiltrates of lymphocytes, plasma cells,
histiocytes, eosinophils, and neutrophils (300). Necrotizing bronchiolitis may be present, along with emphysema,
airway plugging, and atelectasis. Intracytoplasmic inclusions of Chlamydia trachomatis are only rarely seen in
the lung.
Eosinophilic Pneumonia
Acute eosinophilic pneumonia (AEP), while rare in the pediatric age group, can be seen in older children and is
characterized by acute onset, respiratory distress, eosinophilic infiltration in the lung, resolution of symptoms with
corticosteroids, and the absence of relapse (301, 302). An increase in peripheral blood hypersegmented
eosinophils may precede the onset of symptoms (303). Bronchoalveolar lavage also demonstrates the presence
of many eosinophils. Although the etiology of AEP is often unknown, it has been associated with a wide variety of
drugs, parasites, and other infectious agents (304). The lungs are consolidated with alveoli filled with eosinophils
and macrophages, accompanied in about 50% of the cases with an eosinophilic proteinaceous exudate. The
interstitium may be widened by a mixture of inflammatory cells rich in eosinophils but also containing plasma cells
and lymphocytes. Alveolar cells may be hyperplastic. Chronic eosinophilic pneumonia is rarely seen in children
(305).
Interstitial Lung Diseases

As the name implies, these are diseases, which primarily affect the interstitium (alveolar walls, interlobular
septae, and connective tissue surrounding bronchovascular bundles) of the lung. They are bilateral with
multilobar involvement. Terminology and definitions of acute and chronic interstitial lung diseases (ILDs) have
evolved over the last few years, both in children and adults. Use of uniform criteria has helped characterize
clinico-pathological entities whose course, response to treatment, and prognosis are better understood, although
there are still many patients whose diseases cannot be classified. It has also become evident that responses to
lung injuries occur in certain pathologic patterns that can be recognized on light microscopy, such as organizing
pneumonia, which point to a differential diagnosis but are not specific for a disease.
The spectrum of diseases seen in the pediatric population is almost completely different from that in adults.
Idiopathic pulmonary fibrosis/usual interstitial pneumonia and smoking-related disorders [desquamative interstitial
pneumonia (DIP) and respiratory bronchiolitis-interstitial lung disease] are never seen in children. Nonspecific
interstitial pneumonia (NSIP), which is usually associated with connective tissue diseases, can rarely be seen in
older children. The terms DIP and NSIP have been used in children, but these are not specific diagnoses, rather
the intent is to be descriptive. Thus, it is better to avoid terminology, which leads to confusion; rather one should
be as specific as possible.
A multidisciplinary working group reviewed 165 lung biopsies from children less than 2 years of age who had
diffuse lung disease and grouped them according to clinical and pathologic features (306). These groups are
given below, with the first four being more prevalent in infancy:
1. Diffuse developmental disorders
a. Acinar dysplasia
b. Congenital alveolar dysplasia
c. Alveolar capillary dysplasia with misalignment of pulmonary veins
2. Growth abnormalities reflecting deficient alveolarization
a. Pulmonary hypoplasia
b. Chronic neonatal lung disease
c. Related to chromosomal disorders
d. Related to congenital heart disease
3. Specific conditions of undefined etiology
a. Neuroendocrine cell hyperplasia of infancy
b. Pulmonary interstitial glycogenosis
4. Surfactant dysfunction disorders
a. Surfactant protein B mutations
b. Surfactant protein C mutations
c. ABCA3 mutations
d. Histology consistent with surfactant dysfunction disorder
i. Pulmonary alveolar proteinosis
ii. Chronic pneumonitis of infancy
iii. Desquamative interstitial pneumonia
iv. Nonspecific interstitial pneumonia
5. Disorders related to systemic disease processes
a. Immune-mediated collagen vascular disorders

b. Storage disease
c. Sarcoidosis
d. Langerhans cell histiocytosis
e. Malignant infiltrates
6. Disorders of the normal host-presumed immune intact
a. Infectious/post-infectious processes
b. Related to environmental agents
i. Hypersensitivity pneumonitis
ii. Toxic inhalation
c. Aspiration syndrome
d. Eosinophilic pneumonia
7. 7. Disorders of the immunocompromised host
a. Opportunistic infections
b. Related to therapeutic interventions
c. Related to transplantation and rejection
d. Diffuse alveolar damage, unknown etiology
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8. 8. Disorders masquerading as ILD
a. Arterial hypertensive vasculopathy

b. Congestive changes related to cardiac dysfunction
c. Veno-occlusive disease
d. Lymphatic disorders
As evident from the above groups, these include all diffuse lung diseases including developmental disorders, not
just interstitial diseases. However, this is workable framework in which to develop a differential diagnosis when
looking at a lung biopsy. Many of these conditions are discussed in other sections of this chapter.
Chronic Lung Disease of Infancy

After excluding known causes of ILDs, such as surfactant disorders and complications of prematurity
(bronchopulmonary dysplasia), there remains a group of infants who were born at term or near-term and
developed slowly progressive respiratory insufficiency several days or weeks after birth. On biopsy, there is a
mild chronic interstitial inflammation with minimal-to-mild fibrosis, reactive type 2 pneumocytes, and some
simplification of alveolar architecture. Possible etiologies are postinfectious changes, nutritional deficiencies, and
circulatory imbalances such as edema.
Neuroendocrine Cell Hyperplasia of Infancy

This is a relatively recently described rare disorder of unknown etiology seen in infants and young children. It is
characterized by significant tachypnea, hypoxia, and failure to thrive. Radiographs demonstrate hyperinflation,
interstitial markings, and ground glass densities. Lung biopsies show minimal changes on routine H&E stain. By
immunohistochemistry (bombesin and serotonin) significant increase in neuroendocrine cells is seen. All patients
in the initial series improved and survived for a mean of 5 years (307).
FIGURE 12-46 ▪ A: Pulmonary interstitial glycogenosis is illustrated in this lung biopsy from a young infant,
showing widened alveolar septa. B: These cells contain glycogen. (A: H&E stain, ×200, B: PAS stain, ×200.)
Pulmonary Interstitial Glycogenosis

This entity was first described in 2002 (308) based on seven infants who presented with tachypnea, hypoxemia,
and diffuse interstitial infiltrates with overinflated lungs on chest radiographs in the first month of life. Lung
biopsies from all cases showed expansion of the interstitium by spindle-shaped and polygonal cells containing
periodic acid-Schiff-positive, diastase-labile material consistent with glycogen (Figure 12-46A,B).
Immunohistochemical staining showed these cells to be vimentin-positive but negative for leukocyte common
antigen, lysozyme, and other macrophage markers. Electron microscopy revealed primitive interstitial
mesenchymal cells with few cytoplasmic organelles and abundant monoparticulate glycogen. Minimal or no
glycogen was seen in the alveolar lining cells. Since then a few more case reports have been reported. It
appears that this is most likely a relatively benign disease with resolution of signs and symptoms over the course
of a few months, with or without treatment (309).
LUNG TUMORS
Tumors of the lung, both benign and malignant, are decidedly unusual in the pediatric age group (310). In a
review of the files of the Armed Forces Institute of Pathology (AFIP) over a 40-year period, 166 pulmonary tumors
(including “pseudotumors”) were noted in patients 21 years of age and younger (Table 12-9). The ratio of benign
to malignant tumors in this series, 1:1.68, is similar to that appearing in the English literature, as described by
Hartman and Schochat, who identified 230 examples of primary neoplasms, 79 benign and 151 malignant (311),
whereas Hancock et al. (312) reported a ratio of 1:3.16 in a more recent literature review. However, metastatic
tumors are much more common than primary lung tumors in children (313). Fever, cough, and pneumonitis are
the most frequent presenting symptoms; respiratory distress
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and hemoptysis are more often seen with malignant tumors. About a quarter of children with benign neoplasms
are asymptomatic. These earlier series were compiled before the advent of pleuropulmonary blastoma (PPB).
Table 12-9 ▪ PRIMARY PULMONARYTUMORS IN CHILDREN
Benign 62 total
Inflammatory pseudotumor 52
Chondromatous hamartoma 3
Granular cell myoblastoma 3
Leiomyoma 2
Bronchial chondroma 1
Teratoma 1
Malignant 104 total
Bronchial adenoma 46
subtotal
Carcinoid 35
Mucoepidermoid 9
Adenoid cystic 2
Bronchogenic carcinoma 27
subtotal
Adenocarcinoma 14
Squamous cell carcinoma 7
Small-cell carcinoma 3
Large-cell carcinoma 3
Sarcoma 25
subtotal
Fibrosarcoma 8
Rhabdomyosarcoma 7
Leiomyosarcoma 6
Undifferentiated 4
Pulmonary blastoma 6
Compiled from cases seen at the Armed Forces Institute of Pathology from 1950 to 1989
(166 cases).
Benign Tumors
Inflammatory Pseudotumor/Inflammatory Myofibroblastic Tumor
Inflammatory pseudotumor of the lung (IPL) is by far the most common “benign tumor” of the lung in children,
accounting for up to 84% of cases; these tumors are more common in older children, and only anecdotal in
infancy (Table 12-10) (314). Unfortunately, the term “inflammatory pseudotumor” has been rather loosely used in
older literature to encompass different lesions including organizing pneumonia, so-called pseudolymphoma and
inflammatory myofibroblastic tumor (IMT). In the current literature, the term inflammatory pseudotumor is largely
restricted to IMT and its variant, the so-called plasma cell granuloma (315).
Table 12-10 ▪ INFLAMMATORY PSEUDOTUMOR IN CHILDREN (MYOFIBROBLASTICTUMOR)
Age (years) No. of Cases
<1 1
1-4 3
5-9 5
10-14 8
15-21 17
Male:female ratio, 15:19 (34 cases).
Compiled from cases seen at the Armed Forces Institute of Pathology.
Adapted from Stocker JT. Congenital and developmental diseases. In: Dail DH, Hammer SP, eds.
Pulmonary athology. Heidelberg: Springer-Verlag, 1988:53, with permission.
Children with IPL present with fever (22%), cough (20%), chest pain (11%), hemoptysis (9%), or pneumonia
(8%). Although IPL is thought by many to begin as a reactive process, a history of preceding pulmonary disease
is noted in only 20% to 33% of cases, and about 30% of cases (70% in some series) are asymptomatic when
discovered (314). The presence of clonal chromosomal aberrations also suggests that these lesions may be
neoplastic proliferations (316), and IMT is considered to be of intermediate biologic potential neoplasm. Although
the WHO classification of lung tumors (317) has defined IMT as being characterized by a molecular
rearrangement on chromosome 2p23 involving the tyrosine kinase receptor anaplastic lymphoma kinase (ALK)
(318), this genetic association is seen in less than 50% of all cases (319, 320). HHV-8 sequences with IL-6
overexpression have been described in pulmonary IMT/IPL (321), although this has not been substantiated by
other authors (322). Arber et al. (323) have reported frequent presence of Epstein-Barr virus (EBV) in IPL.
Pulmonary IMT may also represent metastasis from an extrapulmonary site (324, 325). In a study of 59 IMTs,
Coffin et al. (325) observed a mean age of 13.2 years, mean tumor size of 7.8 cm, and involvement of the lung in
22% of cases. Imaging studies usually show a single round, well-defined, peripheral mass with visible calcium
deposits in 25% to 35% of cases (Figure 12-47A) (314). Grossly, the lesion is usually seen as a firm,
circumscribed, 3- to 10-cm, grayish white mass, peripherally or centrally (Figure 12-47B), although they may also
involve the major bronchi and trachea (326, 327). Even peripheral lesions have been suggested to be closely
related to airways, as peribronchial, submucosal, or endobronchial nodules (328).
Microscopically, the tumor infiltrates adjacent lung, even though it may appear grossly well defined. There are
different histologic patterns, probably representing a morphologic continuum (329). The so-called plasma cell
granuloma pattern comprises of a fasciitis-like spindle cell proliferation with a vascular stroma rich in
lymphocytes, plasma cells, histiocytes, and mast cells (Figure 12-47C). Large lymphoid aggregates with or
without germinal centers may be seen, along with multinucleated giant cells, xanthoma cells, and/or abscess
formation. Some cases show a sclerosed hypocellular desmoid-like stroma, probably representing a burntout
stage. In other cases, the spindle cell proliferation may mimic a sarcoma but retains a prominent inflammatory
component; these cellular lesions may recur as inflammatory fibrosarcomas and have metastatic potential.
Atypical histologic features included hypercellularity, a prominent fascicular architecture, a focal herringbone
pattern, necrosis, abundant large ganglion-like cells, multinucleated or anaplastic giant cells, cellular and nuclear
pleomorphism,
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atypical mitoses, a round or polygonal cell component, and necrosis (325). Areas of organizing pneumonia may
be present at the margin of the lesion. Foci of calcification, osteoid metaplasia, and myxomatous changes may
be present (Figure 12-47D). Immunohistochemistry for smooth muscle actin and/or desmin may be helpful in
demonstrating the myofibroblastic nature of these cells. ALK positivity may or may not correlate with more
aggressive histology and behavior (330, 331). Electron microscopy indicates that the spindle cells are fibroblasts
or myofibroblasts. Treatment of IPL is by excision of the mass. The lesion tends to grow slowly and is locally
invasive and recurs in up to 24% of cases (332, 333). Recurrence correlates with local invasion and is rare
following complete excision but is more likely after simple enucleation (334). In Coffins series, three of 13
pulmonary IMTs metastasized. Metastasis was confined to ALK-negative lesions, although ALK reactivity was
associated with local recurrence (325). Secondary nephrotic syndrome has been reported in association with
pulmonary IMT (335). At present, there is no specific accepted adjuvant therapy for aggressive lesions.
FIGURE 12-47 ▪ Inflammatory pseudotumor. A: A discrete round mass is present in the right lower lobe of this 4-
year-old boy. B: A circumscribed nodule bulges from the cut surface of a resected section of lung. C: Interlacing
fascicles of myofibroblasts are separated by an infiltrate of lymphocytes and plasma cells. (Masson trichrome
stain, ×50.) D: A densely sclerotic area contains a focus of osteoid material. (H&E, ×75.)
Chondroma and Chondromatous Hamartoma

Although pulmonary chondroid hamartoma is the most common benign neoplasm of the lung in adulthood, they
are rare in children (312). They occur as isolated lesions and show a pathognomonic stippled, “popcorn”
calcification on chest radiographs, although this finding is present in less than 25% of cases. They are usually
single and variable in size; a giant (18 cm) cystic chondroid hamartoma has been reported in an asymptomatic
11-year-old boy (336). When discovered in adolescent girls, they may be associated with the Carney triad
(pulmonary chondroma, gastric epithelioid gastrointestinal stromal tumor, and functioning extra-adrenal
paraganglioma) (337). In this situation, the lung tumors, which may be multiple, are soft to firmbosselated masses
composed of a mixture of mature cartilage, bone, vascular adipose tissue, and stellate cells in a myxoid stroma
(Figure 12-48). Chondroid matrix may also be seen in pulmonary epithelioid hemangioendothelioma (338) and
pleomorphic adenoma (339); the latter tends to occur in association with large airways, whereas the former is
more likely to be peripheral and pleural based.
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FIGURE 12-48 ▪ Chondromatous hamartoma. Irregular lobules of cartilage are separated by vascular adipose
tissue and fibrous connective tissue. (H&E, ×30.)
Juvenile Laryngotracheal Papillomatosis

Juvenile squamous papillomas are benign neoplasms that occur most commonly in the larynx but in 5% of
patients extend into the trachea and, rarely, into the pulmonary parenchyma. The papillomas occur in 1,500 to
2,000 infants and children in the United States each year and are caused by a human papillomavirus (HPV) 6
and 11, which may be transmitted to the child from the mother during parturition (340, 341). The papillary
growths in the larynx produce hoarseness and inspiratory stridor, which may progress to acute respiratory
distress. Treatment includes standard surgical resection, cryosurgery, and laser therapy (342, 343). Within the
larynx and trachea, the lesion grows as papillary or sessile structures along the mucosal surface (Figure 12-49A)
and shows the morphology of a benign squamous papilloma with orderly stratified squamous epithelium covering
a vascular connective tissue core or stalk (340). Koilocytes are seen as evidence of HPV infection. Recurrences
are noted in many patients requiring multiple resections and even tracheostomy. However, spontaneous
regression may occur in older children. With repeated manipulation (e.g., surgery, intubation) fragments of the
papillomas may spread down the trachea into the bronchi and pulmonary parenchyma and produce solid and
cavitary lesions composed of sheets of squamous epithelial cells (Figure 12-49B to D) (344). The incidence of
lung involvement in recurrent papillomatosis has been estimated at 3.3% (345). Dissemination and subsequent
growth of the benign squamous epithelium may be extensive enough to produce respiratory insufficiency and,
rarely, death. Squamous cell carcinoma of the lung has been reported in patients with recurrent juvenile
laryngotracheal papillomatosis (346, 347), with a 16% incidence of cancer in cases with lung involvement (345).
HPV 11 transforms in these cases of malignancy.
Other Benign Tumors

Leiomyomas of the lung are seen in children, but leiomyosarcomas are more frequent (348, 349). In addition to
cartilage-containing hamartomata of the lung, a wide variety of benign lesions composed of varying amounts of
fibrous tissue, smooth muscle, adipose tissue, and vascular tissue are occasionally seen in children. Hull et al.
(350) described multiple pulmonary fibroleiomyomatous hamartomata in a 9-year-old girl. EBV-associated smooth
muscle tumors may occur in immunocompromised children (351).
Mesenchymal cystic hamartomas of the lung, first reported in 1986 (352), are rare tumors originating from
nodules of primitive mesenchymal cells, that gradually increase in size and then become cystic. They may occur
multifocally and should be distinguished from both pulmonary metastasis of endometrial stromal sarcoma and
lymphangioleiomyomatosis (353). Complications include hemorrhage from systemic arteries within the cyst wall,
PT, and hemothorax (354, 355). Mesenchymal cystic hamartoma is usually considered to carry a good
prognosis. However, malignant transformation has been reported (356). These are suspected PPBs.
Benign vascular tumors of the lung in children are rare and include lymphangiomatosis, lymphangiomyomatosis,
and pulmonary capillary hemangiomatosis. Diffuse pulmonary lymphangiomatosis is a rare disorder that
presents as wheezing or dyspnea over a period of months. Patients are from 1 month to 35 years of age at
presentation, with a mean age of 10 to 15 years. There is a male predominance (357). Chest images display
bilateral interstitial infiltrates, which are often greatest in the lower lobes associated with smooth thickening of the
interlobular septa and bronchovascular bundles (358). Microscopically, anastomosing endothelium-lined spaces
are present beneath the pleura and along interlobular septa, accompanied by irregular collections of spindle cells
containing hemosiderin and reactive for vimentin, desmin, actin, and progesterone receptor (357). The
endothelial lining cells are positive for factor VIII and ulex europaeus. The disease is progressive, especially in
younger children. Small pulmonary nodules, possibly representing early vascular malformations, may be seen in
patients with hereditary hemorrhagic telangiectasia (359).
The so-called sugar tumor of the lung is a tumor of the perivascular epithelioid cells (PEComa) and presents as
a circumscribed mass that is histologically composed of cells with clear cytoplasm rich in glycogen and
immunoreactivity for HMB45 and Melan-A, but negative for cytokeratin (360). These tumors may reach large
sizes, up to 12 cm (361). Lymp-hangioleiomyomatosis, a related lesion, has been reported in prepubertal girls in
association with renal and hepatic angiomyolipomas with or without tuberous sclerosis (362).
Sclerosing hemangioma (although not a vascular tumor), seen most frequently in adult women (363), also
occurs in the 15- to 21-year age group and is usually asymptomatic (364). The solitary, well-circumscribed
peripheral lesion measures 0.4 to 8 cm in diameter and shows a constellation of microscopic findings with two
cell types (surface and round cells) in a mixture of solid, hemorrhagic, papillary, and sclerotic patterns.. The
distinct round cells have abundant, pale, eosinophilic cytoplasm. Immunohistochemically, both surface and round
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cells stain with epithelial membrane antigen (EMA) and thyroid transcription factor-1 (TTF-1), while the round
cells are uniformly negative for pancytokeratin, surfactant proteins (A and B), and Clara cell antigens, suggesting
that they are derived from primitive respiratory epithelium (365). Rare cases are reported to have regional lymph
node metastases, which does not appear to affect the excellent prognosis.
FIGURE 12-49 ▪ Juvenile laryngotracheal papillomatosis. A: The larynx and trachea are covered by papillary
growths of firm, tan tissue. B: The lung contains cystic areas where fragments of tissue broken off from the
trachea and larynx have settled into the distal airways. C: Fragments of squamous papillomas that disseminated
to the lung have proliferated to fill alveoli (H&E, ×60). D: Fragments of papillomas from the larynx and trachea
can move down the trachea to lodge in alveolar ducts and alveoli (1, 2), where they can proliferate and extend
throughout adjacent acini (3, 4). Central cavitation of large lesions may take place (5 to 7) with further spread of
the tissue fragments. (From Kramer SS, Wehunt WD, Stocker JT, Kashema H. Pulmonary manifestations of
juvenile laryngotracheal papillomatosis. AJR Am J Roentgenol 1985;144:687, with permission.)
Although tumors of neural origin are relatively common in the mediastinum, they are rarely seen in the bronchi or
lung parenchyma. Neurofibromas and neurilemomas, however, have been reported and successfully treated
with conservative resection (311). Granular cell tumor is a disproportionately frequent endobronchial tumor in
children, considering its rarity in all sites (Figure 12-50A,B) (366). Multifocal intrapulmonary and hilar infantile
myofibromatosis has been described in a neonate (367). The lung may be secondarily involved in thoracic
fibromatosis. Similarly, teratomas are seen much more frequently in the mediastinum, and are exceedingly rare
as primary lesions of the lung (368).
Malignant Tumors
The most common malignant tumors in the lung are metastatic lesions (313). Although the lungs are frequently
involved in children dying from a variety of malignancies, in surgical pathology practice, the most frequently
encountered resections are metastatic osteosarcoma (348, 369, 370). Other common childhood tumors that
metastasize to the lung include Ewing sarcoma, rhabdomyosarcoma, Wilms tumor, hepatoblastoma, and germ
cell tumors. It must be remembered, however, that in patients with multiple lung lesions, all lesions may not
represent metastases and the possibility of a primary lung cancer following treatment of sarcoma must also be
considered in the differential diagnosis (371).
The most common malignant primary pediatric pulmonary tumors are the so-called “bronchial adenomas,” which
include carcinoid, mucoepidermoid carcinoma, adenoid cystic carcinoma, and the benign mucous gland
adenoma, in view of their intra-endobronchial nature. Carcinoids arise from bronchial neuroendocrine cells,
whereas the other two tumors arise from bronchial minor salivary glands.
FIGURE 12-50 ▪ Granular cell tumor. A: A densely cellular tumor nodule abuts a bronchus (H&E, ×100). B: The
tumor nodule is composed of large uniform cells with abundant granular cytoplasm. (H&E, ×200.)
Carcinoid
Carcinoid is the single most common primary malignant tumor of the lung in children and adolescents, accounting
for about 35% of all cases and nearly 50% of all malignant epithelial tumors. Presenting symptoms are cough
(80%), pneumonitis (60%), and hemoptysis (33%). Although the condition is occasionally seen in younger
children, more than 75% of pediatric carcinoids occur in patients older than 15 years of age (372). Carcinoid
syndrome is rarely seen in children although Cushing syndrome has been reported (372, 373). The tumors
usually arise in a bronchus (Figure 12-51A) as a fleshy, smooth, polypoid mass covered by intact mucosa but
may extend through the bronchial wall to invade the adjacent parenchyma. Peripheral carcinoids are rare in
children.
Microscopically, the lesion presents a mosaic pattern of solid ducts, cords, nests, trabeculae, and ribbons of
uniform cells with abundant clear or lightly eosinophilic cytoplasm and regular, centrally placed nuclei (Figure 12-
51B). Numerous capillaries are present in the delicate fibrous septa separating the cells. Rarely, spindle cell or
other atypical patterns may be seen. Argyrophilic granules may be demonstrated by Grimelius or Churukian
Schenk stains, but the tumors are usually argentaffin-negative, consistent with their foregut derivation.
Immunohistochemically, the tumors are positive for cytokeratin, synaptophysin, chromogranin, Leu-7 (CD56), and
variably positive for a variety of other neuroendocrine products including bombesin, serotonin, vasoactive
intestinal peptide, somatostatin, calcitonin, and/or adrenocorticotrophic hormone. Treatment of pulmonary
carcinoid is by conservative resection with removal of involved lymph nodes. Prognosis is excellent (374), with
about 90% survival.
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However, long-term follow-up is mandated since metastases occur in 10% to 25% of cases (366).
FIGURE 12-51 ▪ Bronchial carcinoid. A: A bronchus is occluded by a densely cellular tumor mass. (H&E, × 100.)
B: Uniform cells with round nuclei and finely granular cytoplasm are separated into discrete bundles by delicate
vascular septa. (H&E, ×200.)
Mucoepidermoid Carcinoma
Mucoepidermoid carcinoma accounts for up to 20% of bronchial adenomas and, like carcinoids, presents with
cough, fever, hemoptysis, recurrent pneumonia, or a combination of these symptoms (366). The lesion usually
occurs in a main bronchus as an obstructing, soft, polypoid mass. Microscopically, the tumor presents a solid
and cystic appearance with an admixture of mucus-secreting, intermediate, and epidermoid cells arranged in
sheets and glands (Figure 12-52), and may be associated with a dense lymphoplasmacytic infiltrate (375).
Tumors with higher proportions of squamoid cells tend to be of higher grade and higher stage, and have worse
outcomes (376). Translocation <(11;19) has been reported to be the primary chromosomal aberration for
pulmonary MEC in children, and the MECT1-MAML2 fusion transcript may be associated with better prognosis in
these tumors (377). Prognosis is favorable after conservative resection (374), although lymph node metastases
may rarely be present.
FIGURE 12-52 ▪ Bronchial mucoepidermoid carcinoma. Epidermoid and intermediate cells are admixed with
mucinous cells in the same tumor cluster (H&E, ×200.)
Adenoid Cystic Carcinoma

Adenoid cystic carcinoma accounts for less than 5% of bronchial adenomas in children, and presents in a
manner similar to other bronchial adenomas. The tumor resembles that seen in the salivary gland with groups of
cells arranged in cords or nests. Accumulation of mucin or hyaline material within the clusters imparts a
characteristic cribriform pattern. Treatment is by resection, and although metastases may occur, prognosis is
relatively favorable (311).
Other salivary-gland type malignancies are rare in children, with only anecdotal reports of acinic cell carcinoma
of the bronchus (378, 379) and epithelial-myoepithelial carcinoma (380).
Bronchogenic Carcinoma
Bronchogenic carcinoma accounts for up to 25% of primary malignant tumors of the lung and was the second
most common primary malignant pediatric pulmonary neoplasm until PPB. The majority of lesions are
adenocarcinomas or undifferentiated carcinomas (311, 312). Patients present with cough, pneumonitis, and
chest pain but may be asymptomatic if the tumor is peripheral. Over 60% of children and adolescents with
primary lung carcinoma experience a delay in diagnosis, leading to an advanced stage at diagnosis and poor
outcome. However, patients with localized resectable disease have a more favorable prognosis (312, 381, 382).
Over 60% of children and adolescents with primary lung carcinoma experience a delay in diagnosis, with
advanced stage at diagnosis and poor outcomes. However, patients with localized resectable disease have
favorable prognosis (312, 381, 382).
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Adenocarcinomas in children may be primary or may occur as a second malignancy following treatment for
Hodgkin lymphoma, Ewing sarcoma, and testicular germ cell tumor (383, 384). Adenocarcinoma may also arise
in a setting of type 1 CPAM and is thought to derive from the mucogenic cells seen in this entity (194, 200).
Parenthetically, mucinous areas in type 1 CPAM are reported to lack EGFR expression, whereas adjacent
epithelial cystic linings are strongly positive, suggesting that EGFR may play an important role in the
pathogenesis of CPAM but not in the associated malignant transformation (198). Bronchioloalveolar carcinoma
has also been reported in a child with hepatoblastoma (385). Radiographically, the mass is usually localized to
the midlung or peripheral lung fields. Grossly, the tumor is a moderately firm mucoid mass, which may display
central necrosis. Microscopically, the tumor presents a characteristic lepidic growth pattern with columnar cells
extending along alveolar walls, with little apparent destruction of the walls (Figure12-53A,B). Mucinous cells may
line alveoli, replacing normal alveolar lining cells and filling the lumen with mucin, which may extend into and fill
adjacent normal alveoli. Aerogenic spread (with resultant secondary lesions) may occur to other lobes of both
lungs. Treatment is by resection, and prognosis is favorable if resection is complete and no metastatic disease is
present at the time of diagnosis (386). Invasion of alveolar septa, pleura, or vascular spaces precludes a
diagnosis of bronchioloalveolar carcinoma and should be considered as an invasive adenocarcinoma even in the
presence of a predominant bronchioloalveolar component (384). Pulmonary adenocarcinoma has also been
reported in a child with prior tuberculosis (387).
FIGURE 12-53 ▪ Bronchioloalveolar carcinoma. A: A large, peripherally placed tumor nodule is composed of
dilated alveoli filled with mucin and clusters of tumor cells. (H&E, ×30.) B: Alveolar septa are lined by papillary
growths of columnar epithelial cells with irregular basal nuclei and apical mucin. Note the mucin lying free in the
alveoli. (H&E, ×300.)
Squamous cell carcinoma has been reported in children from 2 to 21 years of age with a nearly equal male-
female incidence (348, 388). Presenting symptoms include cough, hemoptysis, chest pain, or indication of
extrapulmonary metastatic disease (Figure 12-54A). Radiographically, the lesion may be central or peripheral,
occasionally involving an entire lobe or lung. Microscopically, sheets of cells range from small to large
undifferentiated cells to keratinizing squamous epithelial cells (Figure 12-54B). Areas of necrosis may be
extensive. Squamous cell carcinoma must be differentiated from disseminated laryngeal papillomatosis, which
may “seed” squamous cells throughout the lobes, which then grow into nodules. Malignant transformation of
papillomatosis has been reported in children (347). Squamous cell carcinomas of the lung may also arise in a
setting of bronchogenic cyst or teratoma. Treatment of squamous cell carcinoma is by resection, chemotherapy,
and radiation; prognosis is poor (348).
Pulmonary blastoma, as first described (and named embryoma of lung) by Barnard in 1952 and subsequently
redefined by Spencer in 1961, is a primary lung tumor consisting of a mixture of immature, embryonic-like
mesenchymal and epithelial components (389). These tumors are typically seen in adults, are usually solid, and
morphologically show either a well-differentiated fetal adenocarcinoma pattern or a biphasic
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epithelial and mesenchymal pattern. Although, the older literature describes similar tumors in children (usually
over 10 years of age), most tumors diagnosed as pulmonary blastomas in children are now classified as PPBs
(see below) in view of their variable anatomic location, primitive embryonic-like blastema and stroma, absence of
a carcinomatous component, and potential for sarcomatous differentiation (390).
FIGURE 12-54 ▪ Squamous cell carcinoma. A: The entire right lung of this 10-year-old boy is infiltrated by dense
tan-white tumor. B: Clusters of well-differentiated stratified squamous epithelial cells invade the parenchyma.
(H&E, ×30.)
Sarcomas
Sarcomas of the lung account for a small but significant number of primary malignant tumors, and include
malignant fibrous histiocytoma, monophasic and biphasic synovial sarcoma, malignant peripheral nerve sheath
tumor, leiomyosarcoma, angiosarcoma, pulmonary vascular intimal sarcoma, fibrosarcoma, and epithelial
hemangioendothelioma (349).
Malignant fibrous histiocytoma accounted for seven (27%) of 26 primary pulmonary sarcomas in children in
Keel's series (349). All but one of the cases were of the storiformpleomorphic subtype with spindle-shaped cells
arranged in fascicles that intersected in a cartwheel-like fashion. Mitoses ranged from 6 to 10 per 10 high-power
fields, with some atypical forms. Areas of necrosis were consistently seen, occasionally comprising up to 90% of
the mass. Treatment consisted of resection (and chemotherapy in one case) and, of six with follow-up, all were
alive at 8 to 94 months, although one continued to have demonstrable disease at 53 months.
Primary pulmonary synovial sarcomas may be either monophasic or biphasic, and may be confused with
fibrosarcoma and other lesions. Monophasic synovial sarcomas are composed of hypercellular fascicles of
spindle cells that intersect randomly or are arranged in a herringbone pattern separated by collagen bundles.
Diagnosis often requires confirmation by electron microscopy (cells with cytoplasmic projections and intercellular
spaces that contain amorphous extracellular matrix) or immunohistochemistry (positive for keratins, EMA, CD99,
BCL2, and CD34) (391). The presence of the t(X;18) translocation is confirmatory. Compared with soft-tissue
synovial sarcoma, primary pulmonary and mediastinal synovial sarcoma has less calcification, less obvious mast
cell influx, less radiologic vascularity but similar magnetic resonance imaging features, percentage of poorly
differentiated tumors, and number of t(X;18)-positive tumors (391). Survival with resection is relatively good,
although late metastases may occur.
Malignant peripheral nerve sheath tumors are uncommon in children and almost 50% of the cases occur in
patients with neurofibromatosis 1 (392). These tumors are composed of spindle cells with wavy hyperchromatic
nuclei arranged randomly, in fascicles or in a storiform pattern. Areas representing more conventional
neurofibroma may be seen at the periphery. Immunostains may not be helpful in diagnosis; electron microscopy
shows prominent basal lamina. Despite resection and chemotherapy, death usually occurs in 6 to 20 months
(349) (see Chapter 24).
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FIGURE 12-55 ▪ Leiomyosarcoma. Interlacing bundles of fusiform cells extend along alveolar septa. (H&E, ×75.)
Leiomyosarcoma occurs primarily in young children (5 to 10 years of age) but may also be seen in the newborn
period. Infants may present with respiratory distress, whereas older children display cough, anorexia, weight
loss, hemoptysis, pneumonia, or a combination of symptoms. The lesion may develop in a bronchus or in the
pulmonary parenchyma, frequently attaining a size of 7 cm or greater (393). Tumors arising in
immunosuppressed individuals (394), may be related to EBV infection (313, 351). Microscopically, the firm, gray-
yellow mass is composed of interlacing bundles of fusiform cells with oval vesicular nuclei, scanty cytoplasm, and
ill-defined cellular borders (Figure 12-55). Collagen is abundant, and actin-like filaments with focal condensations
(i.e., dense bodies) can be seen by electron microscopy (349). With total resection, prognosis is very good.
FIGURE 12-56 ▪ Fibrosarcoma. A: The entire lower right hemithorax is filled with a homogenous mass. B: The
tumor infiltrating the normal lung (at left) is composed of individual spindle cells in a somewhat woven pattern.
(H&E, ×30.)
Primary pulmonary fibrosarcoma is a very low-grade malignancy seen in newborns presenting with respiratory
distress or in older children presenting with cough, fever, and chest pain (395). The lesions may be
endobronchial or parenchymal and vary in size from 1.0 to 7.5 cm. The firm gray, yellow, or white lesions may
display areas of hemorrhage or cyst formation. Microscopically, the tumors are highly cellular with sheets and
interlacing bundles of densely packed spindle cells arranged in a herringbone-like pattern (Figure 12-56A,B).
Mitotic activity is lower in endobronchial lesions (0 to 3/10 hpf) than in parenchymal lesions (8 to 12/10 hpf). The
tumor cells display strong, diffuse cytoplasmic staining for vimentin but are nonreactive for musclespecific actin,
desmin, Ulex europaeus, NSE, S-100 protein, Leu-7, EMA, or factor VIII-related antigen. Prognosis with complete
resection is excellent (395). Those in infants may represent congenital peribronchial myofibroblastic tumor.
In a study of six HIV-positive children (aged 18 months to 10 years) with pulmonary Kaposi sarcoma, Theron et
al. (396) found predominantly perihilar and lower lobe involvement, and reported that pleural effusion, air space
involvement, and lymphadenopathy (mediastinal and axillary) were much more common in children than in adults
with pulmonary Kaposi sarcoma.
Primary intrathoracic rhabdomyosarcoma occurs rarely as mediastinal, pleural, pulmonary, or endobronchial
solid tumors with typical embryonal or alveolar patterns (397). More frequently, rhabdomyosarcoma-like foci are
noted in association with PPB (see below).
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Pleuropulmonary Blastoma
PPB is a rare distinct embryonic primary pulmonary tumor in children, with over 220 confirmed cases in the
International PPB registry (www.ppbregistry.org). There is an equal gender incidence, and the vast majority
(94%) present in the first 6 years of life (398), although cases have been reported in older children and even in
adulthood (399). Presenting symptoms include respiratory distress, nonproductive cough, fever, chest pain, or a
combination of symptoms of days to weeks duration (398). The lesion is considered to be part of a hereditary
tumor predisposition syndrome, and there is often a positive family history of childhood neoplasms, including
PPBs in siblings, cousins, and other close relatives. Other associations in PPB patients include familial cystic
nephroma and other renal tumors, medulloblastoma, ovarian tumors (germ cell tumor and sex cord stromal
tumor), seminoma, Hodgkin lymphoma, leukemia, thyroid neoplasia, and intestinal polyps (400, 401, 402, 403,
404, 405 and 406). Imaging studies may show a solid and/or cystic lesion that may be intrapulmonary, pleural
based, or mediastinal; a cystic component is more prominent in younger children consistent with the supposed
progression of lesions with increasing age. Tumors are classified as type I (cystic, 14% of cases, median age 9
months), type II (cystic and solid, 48% of cases, median age 36 months), and type III (solid, 38% of cases,
median age 42 months (398, 407). The multilobated masses measure 8 to 23 cm in diameter and weigh up to
1,100 g.
Type I PPB (Figure 57A to D) occurs as a delicate multilocular cyst with variable numbers of primitive
mesenchymal cells beneath a benign respiratory (bronchial or alveolar) epithelium, with the presence of
rhabdomyoblasts (49% of cases) and cartilage nodules (40% of cases) (407). Rhabdomyoblasts may be seen as
a subepithelial cambium layer (403). Tumors in patients less than 2 months of age are more uniform in
composition and cellularity compared with those in older groups, and have a subtle transition between normal
developing lung and tumor, showing bland interstitial mesenchymal cells uniformly expanding the alveolar septa.
Presumed regressive changes including cyst wall necrosis are common, and may explain the variable and
sometimes sparse tumor cellularity seen in some type I PPBs. Factors that control the balance between
progression and regression may be important in predicting tumor behavior and determining which patients will
benefit from adjuvant chemotherapy (407). The solid areas of type II (Figure 12-58A to C) and III PPB (Figure 12-
59A to D) consist of blastomatous islands of loose mesenchyme blending into fibrosarcoma-like foci; nodules of
benign-appearing to overtly malignant cartilage; rhabdomyosarcomatous component; and areas of large, bizarre,
pleomorphic, multinucleated mesenchymal cells (408). Immunohistochemical staining is variable from one tissue
type to another (390, 409). Cytogenetic analyses have revealed complex abnormalities with gain in chromosome
8q being the most frequent single abnormality (410). PPB families harbor heterozygous germ-line mutations in
DICER1, a gene encoding an endoribonuclease critical to the generation of small noncoding regulatory RNAs;
loss of DICER 1 in the epithelium of the developing lung may alter the regulation of diffusible factors that promote
mesenchymal proliferation and sarcomatous transformation (411).
Local recurrence may developed in fewer than 15% of type I PPBs but is seen in over 45% of type II and III
PPBs. Metastatic disease occurs in about 25% of patients (all with type II or III PPB), chiefly in the brain, spinal
cord, or bone. Cerebral metastasis is more frequent in PPB than in other childhood sarcomas (412). The 5-year
survival rate in a series of 50 cases reported by Priest et al. (398) was 83% for type I and 42% for types II and III.
Gender, side, tumor size, preexisting lung cysts, and extent of surgical resection at diagnosis do not impact
prognosis, whereas incomplete resection and extrapulmonary involvement at diagnosis result in a significantly
worse prognosis (413). As a final note, it is necessary to rule out type I PPB before a diagnosis of CPAMIV is
made.
Miscellaneous Tumors of Pleura and Thorax

Calcifying fibrous pseudotumor is a rare benign tumor of the pleura, characterized by a dense hyalinized
collagenous tissue interspersed with benign spindle cells, lymphoplasmacytic infiltrate, and, particularly,
psammomatous and/or dystrophic calcifications (414, 415). Mesenchymal hamartoma of the chest wall usually
arises from the posterior or lateral portions of the rib and usually involves many ribs. Chest wall mesenchymal
hamartoma associated with a massive fetal pleural effusion has been detected antenatally by ultrasound (416).
Multifocal bilateral mesenchymal hamartomas of the chest wall have been reported in a neonate (417) (see
Chapter 27). Pleuropulmonary desmoid tumor is rare, shows nuclear positivity for β-catenin, and may resemble
solitary fibrous tumors. However, unlike the latter, desmoids are negative for CD34 (418).
Malignant small cell tumor of thoracopulmonary region (Askin tumor) is a tumor with a neural phenotype and is
presently included in the Ewing sarcoma family (see Chapter 24). Malignant mesothelioma of the pleura is seen
in children as young as 5 years of age and accounts for about 10% of childhood mesotheliomas (419).
Miscellaneous Other Disorders of Lung

Sarcoidosis
Sarcoidosis is a chronic multisystem disorder characterized by the formation of noncaseating granulomata (420).
Although the condition is uncommon in children, when mass screening is performed, the incidence of the disease
approaches that of adults with similar demographics. The peak occurrence of the disease is between 20 and 40
years of age, but in children, most cases occur between 9 and 15 years of age, with a separate cluster seen in
children younger than 4 years of age in whom a distinct clinical triad of rash, uveitis, and arthritis is seen (421,
422 and 423). It is occasionally seen in children as young as 2 months of age. Childhood sarcoidosis is seen
with near-equal frequency in boys and
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girls and is significantly more common in blacks (55% to 80% of cases) and American Indians (5%) (424, 425).
FIGURE 12-57 ▪ PPB, type I, cystic. A: A large air-filled cyst resembles the gross appearance of CPAM type 1 or
4. B: When opened, the lesion consists of a large, very thin-walled cystic with a small area of hemorrhage. C:
The cyst walls are lined by interrupted lines of cuboidal/columnar epithelial cells that overlie a fibromuscular
stroma that displays/contains vessels of varying sizes. (H&E, ×40) D: The cells beneath the epithelium form a
dense cambium layer of a rhabdomyosarcoma, displaying staining characteristics of rhabdomyoblasts. (H&E,
×80.)
FIGURE 12-58 ▪ PPB, type II, cystic/solid. A: A large cystic lesion occupies the entire right hemithorax. Note the
opacity just above the right leaflet of the diaphragm. B: The opened resected specimen is composed of multiple
cysts and a small, irregular partially solid nodule. C: The nodule is covered by cuboidal epithelium and is
composed of rhabdomyosarcoma, chondrosarcoma and other areas of undifferentiated sarcoma. (H&E, ×20.)
Although the condition is asymptomatic in 5% to 16% of cases, older children frequently present with cough,
dyspnea, weight loss, lethargy, adenopathy, and headache. At presentation, 50% of children have characteristic
functional changes of restrictive lung disease. Nearly all patients, on radiographic observation, have bilateral
hilar lymphadenopathy, which is frequently associated with bilateral paratracheal adenopathy (Figure 12-60A).
Pulmonary parenchymal involvement is noted radiographically in about 50% of cases, but extrapulmonary lesions
(e.g., skeletal, CNS, skin, GI) are seen in 10% to 15% of cases, a number more frequent than in adults (426,
427). Sarcoidosis may be confused with tuberculosis in its early stages (428).
Clinically, lymphadenopathy is the most common sign, with firm, movable, nontender nodes palpable in over 60%
of cases. Skin and eye changes are also noted in 40% to 50% of patients, and hepatosplenomegaly is found in
about 35%. Findings noted in less than 20% of patients include fever, pulmonary signs (e.g., rales, rhonchi), joint
effusions, muscle masses, meningitis, and seizures.
Laboratory findings are nonspecific but include hypercalcemia, hypercalciuria, high serum immunoglobulins,
leukopenia, eosinophilia, and proteinuria. Angiotensin-converting enzyme elevation is noted in approximately
50% of childhood cases, and although not specific for sarcoidosis, its presence does correlate with disease
activity (420, 429). Bronchoalveolar lavage displays a threefold to fivefold increase in the number of
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lymphocytes, disproportionately represented by helper T-cells and causing the helper-suppressor ratio to be as
high as 10:1 (normally <1:1.8) (430). Bronchoalveolar lavage cytokine expression patterns may be helpful in
evaluating disease activity and planning treatment (431).
FIGURE 12-59 ▪ PPB, type III, solid. A: A large mass fills the anterior portion of the right hemithorax of a one-
year-old boy. B: A multilobulated mass of hemorrhagic and focally necrotic tissue is seen in situ attached to the
pleura. (Courtesy of Louis P. Dehner, M.D., St. Louis Children's Hospital, St. Louis, Missouri.) C: Immature
cartilage blends with blastemal and mesenchymal components. (H&E, ×75.) D: Clusters of anaplastic blastemal
cells displaying marked mitotic activity are separated by fibrovascular septa. (H&E, ×150.)
Although it is observed in less than 25% of children younger than 4 years of age, pulmonary involvement is a
consistent finding in older children. It begins as alveolitis consisting of inflammatory cells and immune effector
cells in the interstitium and alveolar areas of the lung. As the alveolitis progresses, epithelioid cells develop, and
the typical noncaseating granuloma is formed. Necrotizing granulomas may rarely be seen. The more typical
noncaseating granulomas are sharply circumscribed and composed of a focal collection of radially arranged
epithelioid cells and multinucleated giant cells surrounded by a rim of lymphocytes (Figure 12-60B,C). The large
epithelioid cells have pale, eosinophilic cytoplasm with round or oval nuclei. The giant cells, 150 to 300 g in
diameter, are of the Langhans cellular type formed from the coalescence of epithelioid cells. These cells may
contain large, nonspecific, concentrically laminated, basophilic inclusion bodies (Schaumann bodies) or small
star-shaped inclusion bodies with a central core of multiple radiating curved spines (asteroid bodies) (432). The
granulomata contain fibroblasts and varying amounts of amorphous hyaline or reticular material, which increases
with the age and maturation of the granulomata.
FIGURE 12-60 ▪ Sarcoidosis. A: Bilateral hilar lymphadenopathy is noted in the 14-year-old black female. B: In
these classic sarcoid granulomas, the central core of epithelioid cells and multinucleated giant cells is
surrounded by a rim of lymphocytes. (H&E, ×40.) C: The granulomas contain multinucleated giant cells (black
arrows), some of which may display asteroid bodies (blue arrow). (H&E, ×125.)
Although seen primarily in the interstitium of the lung, the granulomata may also be peribronchial or perivascular.
Granulomata may completely resolve to leave normal lung parenchyma or, with hyalinization and fibrosis, give
rise to nonspecific interstitial pulmonary fibrosis and, rarely in children, end-stage honeycomb lung. Involvement
of the upper airway is unusual. Granulomata also develop in lymph nodes, liver, spleen, eye, skin, parotid gland,
brain, heart, skeletal muscle, kidney, and bone (433).
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With early recognition and treatment with corticosteroids, complications such as blindness, pulmonary
insufficiency, and renal impairment can be diminished. Although longterm sequelae occur in 10% to 20% of
childhood cases, the mortality rate is only about 5% (434). Transplantation is a treatment option in severe cases,
but recurrence in the transplanted lung may occur (435).
Cystic Fibrosis
CF is a multisystem disorder of children and adults, the most common lethal genetic disease of the white
population, and the major cause of severe chronic lung disease of children (see Chapters 5 and 6). CF is
inherited as an autosomal recessive disorder, with 70% of cases caused by mutations in the CF transmembrane
conductance regulator gene located on chromosome 7 at ΔF508 (436, 437 and 438). The other 30% of the
mutations number over 1,000 (439, 440 and 441).
CF is characterized by high viscosity of the mucoid secretion products in the lungs, pancreas, liver, and
gastrointestinal tract, which causes plugging and secondary damage to these organs (see Chapters 14, 15 and
16). Nasal and sinus polyposis are commonly seen in patients with CF, and pulmonary
infection has long been recognized to be the most common cause of morbidity and mortality in these patients
(442). The respiratory flora of patients with CF include Staphylococcus aureus and Haemophilus influenzae in
the early stages of the disease, but repeated and chronic infections with Pseudomonas aeruginosa frequently
occur (443). Eradication of P. aeruginosa is extremely difficult in CF patients, and the organism may be the
dominant respiratory pathogen for years even after lung transplantation.
FIGURE 12-61 ▪ Cystic fibrosis. A: Massively ectatic bronchi are filled with viscid mucus in the explanted lung of
a 22-year-old woman. B: Bronchi are obstructed by a mixture of mucus and inflammatory debris that expands the
airway and occasionally extends into the adjacent parenchyma (H&E, ×15). C: Hyperplastic submucosal glands
exude a tenacious mucus that fills and adheres to the bronchial mucosa. (H&E, ×25.)
Fungal infections are present in over 20% of CF patients as a result of extensive lung damage, long-term
antibiotic therapy, and repeated exposure to pathogenic microorganisms. The most frequently encountered
organisms include members of the Aspergillus and Candida species, with allergic bronchopulmonary
aspergillosis reported in up to 11% of patients (444).
Bronchiectasis is the predominant lesion of CF and begins in infancy as mucous plugging of bronchi, followed by
infection, inflammation, mucosal necrosis and ulceration, and ectasia (Figure 12-61A-C). The chronic bronchitis
maybe associated with bronchiolitis obliterans and pneumonia. Bronchiectatic changes can alter the bronchial
volume (normally 4% of a lobe's volume in healthy lungs) by increasing it to 10% to 20% of the total lung volume
and occasionally as high as 50%.
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Changes in bronchi are most prominent in the upper lobes and may be accompanied by the development of large
subpleural cysts that communicate with bronchi (bronchiectatic cysts), are confined to the interstitium, or distend
the visceral pleura and merge with emphysematous pneumothoraces in CF patients. Transplantation of one or
both lungs is increasingly successful in the treatment of chronic pulmonary CF (445, 446).
FIGURE 12-62 ▪ Asthma. A: A markedly thickened undulating basement membrane separates the lumen of a
bronchus from the muscular wall that is heavily infiltrated with eosinophils, lymphocytes, and plasma cells (H&E,
×100). B: The wall of the bronchus displays markedly thickened muscle layers (H&E, ×20).
Asthma
Asthma is an acute, usually reversible airway disease that results in spasmodic, diffuse airway narrowing, with
persistent airway hyperreactivity (447, 448). It affects 3% to 8% of the population and accounts for 2,000 to
3,000 deaths each year in the United States. In autopsy specimens of patients dying during an acute attack, the
lungs show alternating areas of atelectasis and hyperexpansion. Mucus plugs composed of soft, gelatinous, or
rubbery grey material fill mediumto-small bronchi. The smooth muscle of bronchi is markedly thickened, often
2.5-fold or more than normal (Figure 12-62A,B) (449). There is also a prominent thickening of the basal lamina of
the mucosa, and the submucosa shows edema, vessel dilatation, and an inflammatory infiltrate of eosinophils,
plasma cells, lymphocytes, and neutrophils (304, 450). The bronchial mucosal lining and the submucosal glands
display an increased number of goblet cells. Microscopically, the mucus plugs in bronchioles and smaller bronchi
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may contain small linear whorled strands of material that are twisted in a common direction with a central highly
refractile densely coiled or braided coil, called a Curshmann spiral. Inflammatory cells are admixed with the
material in the lumen, and degranulated eosinophils may form crystals, called Charcot-Leyden crystals. Sloughed
segments of respiratory epithelium may also be present as Creola bodies. These structures—Curshmann spirals,
Charcot-Leyden crystals and Creola bodies—may also be found in sputum specimens of asthmatic patients
(304). Similar findings are seen in allergic bronchopulmonary aspergillosis.
FIGURE 12-63 ▪ Diaphragmatic hernia. A: A large defect in the left leaflet of the diaphragm allows herniation of
the liver and portions of gastrointestinal tract into the right hemithorax. B: A similar defect of the right leaflet
allowed the liver to herniate and shifted the mediastinum to the left side. When the liver is returned through the
diaphragm to the abdomen at the time of autopsy, the profound hypoplasia of the right lung can be seen.
Transplantation
Lung transplantation in children is becoming increasingly common for treatment of a variety of pulmonary
diseases, especially CF, but also PVOD, congenital surfactant deficiency, BPD, and other forms of interstitial
lung disease with fibrosis, pulmonary vein stenosis (often in association with congenital heart disease), and
pulmonary hypertension (446, 451, 452, 453 and 454) (see Chapter 8).
FIGURE 12-64 ▪ Diaphragmatic eventration. A: The thorax in this newborn is markedly reduced in size by the
elevation of the diaphragm and protrusion upward of the abdominal organs. B: At autopsy, the diaphragm
consisted only of a thin and largely translucent membrane. C: A section of the diaphragm displays only vessels
and a few strands of muscle between the thoracic and abdominal membranes. (H&E, ×50.)
DIAPHRAGM
Abnormalities of the diaphragm are both congenital and acquired. Developmental anomalies include accessory
diaphragm, agenesis of one or both leaflets, defective formation with herniation, and aplasia or hypoplasia of
muscle with eventration. Acquired diseases such as traumatic rupture, denervation, and muscular atrophy are
also seen.
The diaphragm develops from the septum transversum, pleuroperitoneal membranes, and dorsal mesentery
during the first 6 to 8 weeks of gestation. Under normal circumstances,
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the diaphragm separates the thoracic and abdominal contents completely by weeks 8 to 9 of gestation. Rarely,
the septum transversum fails to descend completely, and an accessory diaphragm divides the hemithorax into
upper and lower compartments. Always unilateral, and right-sided in more than 90% of cases, the accessory
diaphragm can produce respiratory distress in infants but may be asymptomatic and seen only incidentally (192).
Associated cardiovascular anomalies are seen in more than 40% of cases, and the entrapped portion of lung
may be hypoplastic.
Table 12-11 ▪ ANOMALIES ASSOCIATED WITH DIAPHRAGMATIC HERNIA
Pulmonary
Hypoplasia
Extralobar sequestration
Tracheoesophageal fistula
Congenital pulmonary airway malformation
Cardiovascular
Tetralogy of Fallot
Endocardial cushion defect
Atrial and ventricular septal defects
Ectopia cordis
Coarctation of the aorta
Pulmonic stenosis
Gastrointestinal
Imperforate anus
Omphalocele
Pyloric stenosis
Stomach duplication
Malrotation of bowel
Genitourinary
Hydronephrosis
Multicystic kidney
Duplicated collecting system
Chromosomal
Trisomy 18 and 21
Other
Arthrogryposis
Cleft lip and palate
Meningomyelocele
Hemivertebrae
Fetal alcohol syndrome
Cornelia de Lange syndrome
Syndactyly
Ullrich-Turner syndrome
Modified from Stocker JT. Congenital and developmental diseases. In: Dail DH, Hammer SP, eds.
Pulmonary pathology. Heidelberg: Springer-Verlag, 1994, with permission.
Complete absence of the diaphragm has been reported in a family, and agenesis of a hemidiaphragm is
occasionally seen (455).
Diaphragmatic hernia is one of the most frequently occurring anomalies of the lungs and thorax, seen once in
every 2,000 to 5,000 births. Herniation of abdominal contents into the thoracic cavity through a defect in the
diaphragm occurs early in gestation and results in varying degrees of pulmonary hypoplasia. The defect is
usually in the posterolateral (i.e., foramen of Bochdalek) aspect of the diaphragm. The size of the defect and
location on the right (20% to 35% of cases) or left (65% to 80% of cases) side influence the degree of pulmonary
hypoplasia and the clinical presentation.
Right-sided diaphragmatic hernias are often partially or completely occluded by the liver, and the degree of
pulmonary compromise is mild. Delayed presentation has been reported in infants with right-sided diaphragmatic
hernia whose symptoms are masked by a group B streptococcal sepsis (456).
Infants with the more typical large, left-sided hernia may present in the first minutes to hours of life with severe
respiratory distress. Herniation of abdominal contents including liver, spleen, and loops of intestine may result in
severe pulmonary hypoplasia (Figure 12-63A,B). Survival rates for infants with congenital diaphragmatic hernia
(CDH) have increased dramatically with the increased availability of surgical repair of the hernia (both in utero
and after birth) and the development of ECMO to support infants with mild-to-moderate pulmonary hypoplasia.
Current survival rates are up to 75% to 95% of liveborn infants with CDH (457). Those with the poorest
prognosis have the most severe pulmonary hypoplasia and are unable to maintain adequate oxygenation after
repair of the hernia. Pulmonary hypoplasia is life threatening when the lung weights are less than 30% to 40% of
expected weight. Infants with more than 45% to 50% of expected weight, including those whose entire right or
left lung has been removed because of a congenital malformation (e.g., CPAM), often survive with little
respiratory difficulty. Associated anomalies are noted in approximately 25% of cases of CDH and include a
variety of pulmonary, cardiovascular, gastrointestinal, and genitourinary malformations (Table 12-10). In addition,
tracheobronchomalacia may be seen in 5% to 10% of infants with CDH. CDH is also part of the phenotype of
Fryns syndrome.
Aplasia or hypoplasia of musculature within the leaflets of the diaphragm, either partial or complete, produces
eventration of the diaphragm (Figure 12-64A to C). Congenital eventration, usually seen in boys (62%), is
unilateral in 85% of cases, with the right side involved in 67% and the left in 33% (458). Phrenic nerve palsy from
birth injury or iatrogenic damage can lead to diaphragmatic elevation mimicking eventration (459)]. Associated
anomalies, present in over 30% of cases, are similar to those seen with diaphragmatic hernia (Table 12-11) but
also include cases of arthrogryposis. The involved segments of the diaphragm display normal parietal thoracic
and abdominal mesothelium separated by delicate fibrovascular connective tissue either devoid of muscle or with
only a few skeletal muscle fibers present.
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Chapter 13
The Cardiovascular System
Kathleen Patterson
CONGENITAL MALFORMATIONS OF THE CARDIOVASCULAR SYSTEM

Incidence
The heart, first recognizable at 15 days of gestation, develops from a single tube into a four-chambered structure via an
extraordinary series of loopings and septations (e1-e3). Given the complexity of cardiac development, it is not surprising that
congenital cardiac defects account for the vast majority of cases of cardiac disease in childhood. The reported incidence of
congenital heart disease (CHD) varies widely, ranging from 2.4/1,000 to 8.8/1,000 live births (1) (e4-e12). The incidence statistics
vary depending on the age of the patient population, the criteria used for diagnosis and inclusion in a study, and the length of study
group follow-up. Inconsistency in the methods of classifying hearts, especially when the lesions are multiple or complex, introduces
an additional level of variability to the reported relative frequency of individual defects (Table 13-1).
Etiology
The etiology of congenital heart malformations is multifactorial, with both genetic and environmental factors playing a role (2) (e3).
From 15% to 45% of patients with CHD have additional developmental anomalies, including chromosomal and nonchromosomal
syndromes, malformation associations or sequences, and teratogen-associated defects (e13-e15). Genetic factors have long been
recognized as a major player; approximately 10% of patients with CHD exhibit a trisomy, monosomy, duplication, or deletion on
routine cytogenetic study, with trisomy 21 being the most common (e16,e14). Over the past 15 years, microdeletions and mutations
of single genes have been identified in many of the developmental syndromes that include CHD as a major factor, with the 22q11
deletion, characteristic of the diGeorge/velocardiofacial syndrome, being the most prevalent (2) (e17,e18). The ever-increasing
number of identified chromosomal abnormalities linked with heart malformations unfortunately does not mean that genetic screening
can predict a specific form of
CHD. Instead, it has become clear that mutations at multiple genetic loci can cause the same cardiac malformation, and that
mutations at a single locus can cause multiple different malformations. Meanwhile, molecular and biochemical analysis of normal
gene products from many of these CHDassociated genes is leading to increased understanding of the developmental mechanisms
in the heart (e19).
Pathophysiology
Simplistically, two major factors, shunting and obstruction to flow, are central to an understanding of the pathophysiology of CHD. In
the normal heart, the pulmonary and systemic vascular circuits are completely separate, functioning as two parallel circuits. Loss of
this separation results in shunting of blood between the two circuits. The size and the predominant direction of the shunt (i.e., right
to left or left to right) are dynamic and determined by a variety of factors, including the
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relative resistance to flow in the two circuits and the presence or the absence of an associated obstructive lesion. Shunting lesions
are often not apparent at birth, when resistance in the two circuits is similar, but become clinically evident over time with the normal
decrease in pulmonary vascular resistance (PVR). In general, right-to-left shunts are associated with cyanosis, and left-to-right
shunts with increased pulmonary blood flow, congestive heart failure, and a risk for the development of pulmonary artery
hypertension.
Table 13-1 ▪ PREVALENCE OF CONGENITAL HEART DEFECTS
Type Range (%)a M:F
VSD 30-52 1:1
PDA 2.5-8.5 1:2
TOF 3.5-10.5 1:1

COTA 4.5-6.5 3:2
TGA 2.5-6.5 2:1
ASD 6.0-8.0 1:2
PS-IVS 2.5-9.0 1:1
AVSD 1.5-9.5 2:3
HLHS 2.0-5.0 3:2
AS 3.0-6.0 2:1
aPercentage of total for ten most common defects; see references (e4-e8,e10-e12,e14).
VSD, ventricular septal defect; PDA, patent ductus arteriosus; TOF, tetralogy of Fallot; COTA, coarctation of the aorta;
TGA, transposition of the great arteries; ASD, atrial septal defect; PS-IVS, pulmonary stenosis with intact ventricular
septum; AVSD, atrioventricular septal defect; HLHS, hypoplastic left-heart syndrome; AS, aortic stenosis; TAPVC, total
anomalous pulmonary venous connection; DORV, double-outlet right ventricle; PA-IVS, pulmonary atresia with intact
ventricular septum; SV, single ventricle; TA, tricuspid atresia; TRUN, truncus arteriosus.
Table 13-2 ▪ CONGENITAL HEART DEFECTS: MAJOR CLINICAL FINDINGS
SHUNT PBF
L→R R→L INC DEC Cyanosis Ductus Dependent Complications
ASD X — X — — — PVOD
ECD X — X — — — PVOD
VSD X — X — — — PVOD
TGV — — X — X X PVOD
TRUN X — X — — — PVOD
EBS — X — X X — Arrhythmia
TOF — X — X X — Polycythemia
HRHS — X — X X X —
HLHS — — — — — X Shock
PDA X — X — — — PVOD
PBF, pulmonary blood flow; L→R, left to right; R→L, right to left; INC, increased; DEC, decreased; ASD, atrial septal
defect; ECD, endocardial cushion defect; VSD, ventricular septal defect; TGV, transposition of the great vessels; TRUN,
truncus arteriosus; EBS, Ebstein malformation; TOF, tetralogy of Fallot; HRHS, hypoplastic right-heart syndrome; HLHS,
hypoplastic left-heart syndrome; PDA, patent ductus arteriosus; PVOD, pulmonary vascular obstructive disease.
Obstructive lesions can occur at almost any site in either of the circuits, with the cardiac chambers proximal to the site of obstruction
showing marked hypertrophy in response to the increased work load. In general, right-sided obstruction produces decreased
pulmonary blood flow and cyanosis; left-sided obstruction results in decreased systemic blood flow. In cases of severe obstruction,
the obstructed circuit is often dependent on blood flow across the ductus arteriosus (i.e., ductus-dependent lesions), and symptoms
characteristically appear at the time of ductus closure in the 1st day or two of life.
These generalizations presuppose normal connections between the respective cardiac chambers and the great vessels. When
these connections are abnormal, as in transposition of the great vessels, additional pathophysiologic consequences develop, which
are discussed in the context of the individual lesions. A summary of the clinicopathologic features in some of the more common
congenital cardiac defects is presented in Table 13-2.
Classification
An accurate classification of congenital heart malformations requires knowledge of the normal anatomy of the heart and a careful
systematic approach to the examination (e20,e21). Normal values for heart weight relative to age and body size are readily available
(see Appendix); normal values for the ventricular wall thickness and valve sizes have been reported for fetuses and newborns by
Oyer et al. (3) and for infants and children by Rowlett et al. (4) and Scholz et al. (5). Following a careful external examination of the
shape and the position of the heart, relationships of the great vessels, and venous drainage pattern, a sequential evaluation of the
three segments of the heart (atria, ventricles, and great vessels) is undertaken. The connections of the segments, the relationship
between the chambers within a segment, and the morphology of the segments are all assessed (6) (e20,e22-e24) (Table 13-3).
When this segmental approach is used for
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classification, normal connections, relationships, and morphology are not incorporated into the diagnosis. In the majority of cases of
CHD, a single defect is present [e.g., a ventricular septal defect (VSD)], and the heart is classified on the basis of that solitary
anomaly. With the increasing use of cardiac transplantation for complex congenital heart defects, both before and after repair,
accurate morphologic diagnosis of cardiac explants becomes a challenge. Although the atrial anatomy and vascular connections
can no longer be evaluated, careful examination of atrioventricular (AV) connections, the ventricular anatomy, and the
ventriculoarterial connections is still warranted (e25).
Table 13-3 ▪ SEQUENTIAL EXAMINATION OF HEART SEGMENTS
Atrial situs: determined by position of morphologic RA
Situs solitus: RA on right; LA on left
Situs inversus: RA on left; LA on right
Situs ambiguus: indeterminate atrial situs
Bilateral right-sided atria
Bilateral left-sided atria
Atrioventricular connections
Atrioventricular concordance: RA → RV; LA → LV
Atrioventricular discordance: RA → LV; LA → RV
Ambiguous (indeterminate) connection Double inlet ventricle: RA + LA → one ventricle

Absence of one atrioventricular connection
Ventricular organization
Normal or D-looped: morphologic RV to the right of the morphologic LV
Inverted or L-looped: morphologic RV to the left of the morphologic LV
Ventricular morphology
Three normal components: inlet = trabecular = and outlet
Trabecular component determines morphologic right and left ventricles
Rudimentary chamber: absent inlet portion
Ventriculoarterial connections
Ventriculoarterial concordance: RV → PA; LV → Ao
Ventriculoarterial discordance: RV → Ao; LV → PA
Double outlet ventricle: one ventricle → PA + Ao
Single outlet of heart: includes truncus = pulmonary atresia = aortic atresia
RA, right atrium; RV, right ventricle; LA, left atrium; LV, left ventricle; PA, pulmonary artery; Ao, aorta.
Septal Malformations
Malformations of the Atrial Septum
The atrial septum forms from three distinct embryonic structures: the septum primum, endocardial cushions, and septum secundum
(e1,e2,e26). In the fetus, blood flows freely between the right and the left atria via the foramen ovale, bordered by the superior right-
sided septum secundum (limbus of fossa ovalis) and the inferior left-sided septum primum (valve of fossa ovalis). This opening
normally fuses during the 1st year of life. However, in 25% to 30% of people, this fusion never occurs, leaving a “probe-patent” or
“valvularcompetent” foramen ovale (e27).
Atrial Septal Defect

An atrial septal defect (ASD), the most common atrial septal malformation, can occur in one or more of four sites (Figure 13-1 and
Table 13-4). A secundum ASD, the most common of the four types, manifests as multiple perforations of, a deficiency in, or absence
of the fossa ovalis flap valve (eFigure 13-1) (7) (e26). In patients with a probe-patent fossa ovalis, a secondary functional secundum
ASD may appear following atrial dilatation. An isolated secundum ASD usually remains asymptomatic through childhood with 80% to
90% closing spontaneously, especially when of a small size (≤ 4 mm diameter) (8) (e28,e29). Defects greater than 8 mm on the
other hand rarely close spontaneously, requiring instead surgical closure (8) (e28,e29). Complicating pulmonary vascular
hypertension, which develops in 10% of adult patients, occurs rarely in the pediatric population (e30-e32).
FIGURE 13-1 ▪ The positions of various atrial septal defects from the perspective of the right atrium, which has been opened
laterally. A: Secundum defect. B: Primum defect. C: Sinus venosus defect.
Table 13-4 ▪ TYPES OF ATRIAL SEPTAL MALFORMATION
Septal defects
Secundum atrial septal defect
Primum atrial septal defect
Sinus venosus atrial septal defect
Coronary sinus atrial septal defect
Single atrium (cor triloculare biventricularis)
Premature closure of foramen ovale
Primum defects are discussed later with AV septal defects. Sinus venosus defects result from a deficiency of the posterior superior
aspect of the atrial wall that normally separates the right pulmonary veins from the superior vena cava/right atrium junction. A defect
in this area therefore almost always occurs in conjunction with partial anomalous pulmonary venous return (9) (e33,e34). Coronary
sinus defects result from an unroofing or a fenestration of the coronary sinus on the posterior aspect of the left atrium and occur
most often in association with a persistent left superior vena cava (LSVC) (10) (e35,e36). Although most isolated ASDs occur
sporadically, they can occasionally be inherited as an autosomal dominant anomaly with or without associated conduction system
abnormalities (e37). In a small subgroup of these patients with associated radial limb defects (Holt-Oram syndrome), the underlying
genetic abnormality on chromosome 12q2 has been identified (e38,e39) (see Chapter 27).
Single Atrium
Complete absence of the atrial septum, a rare anomaly, results in a single atrial cavity, also termed common atrium or cor
triloculare biventricularis. The single atrium usually accompanies other severe cardiac anomalies and is often associated with AV
septal defects and situs ambiguous (7) (e40,e41).
Premature Closure of the Foramen Ovale

Premature closure of the foramen ovale manifests as a normally positioned but imperforate foramen ovale, an unidentifiable fossa
ovalis, or an aneurysmal pouch bulging into the left atrium. The restricted mixing in utero can be complicated by hydrops fetalis and
the hypoplastic left-heart syndrome (7) (e42-e44).
Malformations of the Ventricular Septum

The ventricular septum is a complex structure that can be divided into four components: inlet, trabecular, outlet, and membranous
(e45-e47). From the right ventricular aspect, the inlet septum lies superiorly and posteriorly behind the septal
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tricuspid valve, the trabecular septum occupies the apex, the outlet septum sits between the crista supraventricularis and the
pulmonary valve, and the membranous septum lies at the anteroseptal tricuspid commissure, where the tricuspid, mitral, and aortic
valves converge (Figure 13-2). From the left ventricular aspect, the inlet septum lies posteriorly adjacent to the mitral valve, the
trabecular septum occupies the apical region, the outlet septum sits beneath the right cusp of the aortic valve, and the membranous
septum lies below the right and posterior aortic valve commissure (Figure 13-2).
FIGURE 13-2 ▪ The positions of the ventricular septal components and the corresponding ventricular septal defects from the lateral
perspectives of the opened right and left ventricles. A: Membranous septum and perimembranous defect. B: Inlet septum and
defect. C: Trabecular septum and trabecular muscular defects. D: Outlet septum and defect.
VSDs, the most common type of congenital heart defect, can occur anywhere in the ventricular septum (11) (e47). VSDs are
subclassified according to the nature of the defect rim and its anatomic position in the septum (Table 13-5).
Perimembranous (membranous, infracristal) defects account for up to 80% of VSDs (11) (e45,e47). These defects are most easily
seen from the left side, where they lie in the left ventricular outflow tract just beneath the aortic valve (Figure 13-3). In the right
ventricle, they reside beneath the crista supraventricularis and behind the papillary muscle of the conus, partially obscured by the
septal leaflet of the tricuspid valve (12) (e46,e47) (Figure 13-4).
Table 13-5 ▪ VENTRICULAR SEPTAL DEFECTS: CLASSIFICATION
Defect Rim Defect Location Historical
Perimembranous Inlet Posterior

AV canal
Outlet Supracristal
Infundibular
Trabecular Membranous
Infracristal
Muscular Inlet AV canal
Outlet Infundibular
Subpulmonic
Supracristal
Central Trabecular Infracristal
Remote Trabecular Muscular
Doubly committed subarterial Outlet Infundibular
Supracristal
AV, atrioventricular.
Outlet (infundibular) defects account for 5% to 7% of VSDs in the Western world but nearly 30% of VSDs in Japan and the East
Asia (e45,e48,e49). These defects are often roofed by pulmonary and aortic valve tissue (i.e., doubly committed subarterial) (11,
12) (e47) and can be complicated by prolapse of the right coronary cusp of the aortic valve into the defect; 40% to 60% are
complicated by aortic regurgitation (e49,e48). Outlet and occasionally perimembranous trabecular defects may be associated with
malalignment between the outlet and the trabecular portions of the ventricular septum. Anterior malalignment results in aortic
override and posterior malalignment results in pulmonic override. Either can be complicated by subaortic stenosis, often with
associated arch anomalies (13) (e45,e50-e52).
Inlet defects, which account for 5% to 8% of VSDs, reside beneath the septal leaflet of the tricuspid valve, but posterior and inferior
to the position of perimembranous trabecular defects (e45). Although inlet defects are similar in location
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to the VSD component of AV septal defects, hearts with isolated inlet defects do not show the other characteristic features of AV
septal defects (11) (e45).
FIGURE 13-3 ▪ Ventricular septal defect. An opened left ventricle with the free wall reflected laterally contains a perimembranous
defect, visible in the outflow tract inferior to the aortic valve. The probe visible in the right ventricle in figure 13-4 traverses the
defect opening.
FIGURE 13-4 ▪ Ventricular septal defect. An opened right ventricle with the free wall reflected superiorly contains a perimembranous
defect hiding beneath the septal leaflet of the tricuspid valve. The probe traversing the defect is visible from the left ventricular
aspect in Figure 13-3.
Muscular trabecular defects, representing 5% to 20% of VSDs, often are multiple and may be difficult to see beneath the
trabeculations on the right ventricular aspect of the ventricular septum (e45,e47,e53). What appear to be multiple muscular defects
on the right ventricular side (“Swiss cheese” septum) may coalesce to form what appears to be a single defect in the left ventricular
aspect of the septum (e53).
Clinical manifestations of a VSD usually first appear at the age of 2 to 6 weeks, when the normal drop in PVR results in the onset of
a harsh holosystolic murmur at the left sternal border. The size of the defect and the state of the PVR rather than the anatomic
location of the defect determine the nature of the symptoms (e54,e55) (Table 13-6). In VSDs with large shunts, congestive heart
failure may be resistant to therapy, and the risk for pulmonary vascular obstructive disease is substantial (e54).
Spontaneous closure occurs in 25% to 40% of all VSDs and in up to 85% if the defect is small (e10,e54,e56,e57). Closure of
membranous VSDs by overgrowth of fibrous connective tissue or adherence of the tricuspid valve septal leaflet can result in the
formation of a ventricular septal “aneurysm” (e58,e59). Ventricular septal aneurysms, with or without complete defect closure, are
seen in more than 40% of patients with VSD, usually appearing after 2 years of age (e59).
Table 13-6 ▪ VENTRICULAR SEPTAL DEFECTS: CLINICAL GROUPS
Group Defect Size Shunt PVR Complications Prognosis/Therapy
1 Small L→R Nl SBE Spontaneous closure 75%-83%
2 Moderate L→R Nl SBE Surgical closure required 15%-20%
CHF
3 Large L→R Nl SBE Surgical closure <2 years
Inc CHF
4 Large R→L Inc CHF Inoperable
Cyanosis
PVR, pulmonary vascular resistance; L→R, left to right; R→L, right to left; Nl, normal; Inc, increased; SBE, subacute
bacterial endocarditis; CHF, congestive heart failure.
The penetrating and branching bundles of the conduction system traverse the membranous portion of the ventricular septum (12,
14). This relationship is of particular concern during the surgical repair of perimembranous and inlet defects. Anomalies in the
conduction system or an ill-placed suture can result in postoperative bundle branch block (12) (e60) or, rarely, sudden death (e61).
Malformations of the Atrioventricular Septum

The AV septum is a structure at the crux of the heart that separates the right atrium from the left ventricle (15) (e62). In the embryo,
the AV septum forms at the site of fusion of the four endocardial cushions (16) (e2,e40). The spectrum of lesions resulting from
defects of the AV septum has been traditionally called endocardial cushion defects (Figure 13-5). All forms of AV septal defect
display the following features (15) (e41,e63,e64):
1. Disproportion between the inlet and the outlet dimensions of the interventricular septum, which gives the inlet septum a “scooped
out” appearance
2. Elongation of the left ventricular outflow tract, which creates the “gooseneck” deformity
3. Abnormal formation of the AV valves, with a characteristic “cleft” in the left-sided anterior leaflet
AV septal defects are subdivided into partial and complete forms, depending on the morphology of the AV valve leaflets.
Partial Atrioventricular Septal Defect

Partial AV septal defect is defined by the presence of two discrete AV valve annuli. Partial AV septal defects account for
approximately one-third of cases (17) (e65-e69).
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Most hearts in this group contain an ostium primum ASD (Figure 13-6) in conjunction with a cleft in the anterior mitral valve leaflet
(15, 17) (e64) (eFigure 13-2). Although these hearts have traditionally been considered to have ASDs, echocardiographic studies
demonstrate that the atrial septum is, in fact, normal in size and that the defect is located in the ventricular septum, with inferior
displacement of the AV valves causing interatrial shunting (e70). The cleft mitral leaflet inserts, commissure-like, on the ventricular
septum. The septal leaflet of the tricuspid valve displays variable degrees of deficiency. When both the mitral and the tricuspid
valves are cleft, the valves insert, commissure-like, onto the rim of the ventricular septum; a connecting tongue of valve tissue
covers the ventricular septum and closes the ring (e63,e41). The size of the defect in the ventricular septum also varies in these
hearts, and in some cases the valve leaflets may be less firmly adherent to the ventricular septum so that some interventricular
shunting occurs (17) (e63). Hearts with associated interventricular shunting are said to have “intermediate” AV septal defects in
some reports (e71,e40).
FIGURE 13-5 ▪ Diagrammatic representation of the atrioventricular valves as viewed from the atria in a normal heart and various
atrioventricular septal defects. A, anterior leaflet; P, posterior leaflet; S, septal leaflet; AB, anterior bridging leaflet; PB, posterior
bridging leaflet; LA, left anterior leaflet; RA, right anterior leaflet; RL, right lateral leaflet; LL, left lateral leaflet.
FIGURE 13-6 ▪ Complete atrioventricular septal defect. A complete atrioventricular septal defect is readily visible centrally in this
opened left atrium and ventricle. At the upper rim of the defect a band of atrial septal tissue marked by the ^ separates the upper
secundum ASD from the lower ostium primum ASD. The lower rim of the defect marked by the * represents the upper rim of the
ventricular septum. The anterior and the posterior bridging leaflets of the common AV valve extend over the defect without chordal
insertion.
Complete Atrioventricular Septal Defect

In the complete AV septal defect (endocardial cushion defect, complete AV canal ) (Figure 13-6), the single AV orifice is guarded by
five valve leaflets: posterior (inferior) bridging, right lateral, left lateral, right anterior, and left anterior (superior, bridging) (17)
(e68,e72). The complete AV septal defect is further subclassified according to the extent of septal bridging of the left anterior leaflet
and the site of medial insertion (e40, e41,e68,e72).
Rastelli A: minimal bridging, attachment to right rim of septum or medial papillary muscle
Rastelli B: moderate bridging, attachment to an aberrant right apical papillary muscle
Rastelli C: marked bridging, attachment to the anterolateral papillary muscle of the right ventricle
Rastelli types A and C account for the vast majority of cases.
Additional cardiovascular anomalies occur in up to 50% of hearts with either partial or complete AV septal defects (e67,e73-e76).
The commonly associated anomalies vary in the different subtypes of AV septal defect, as outlined in Table 13-7. At least 50% of
patients have trisomy 21 with a variety of other syndromes in another 25% including the heterotaxy syndromes in particular
(e67,e73,e77). The ventricles are “unbalanced” in approximately 10% of AV septal defects, with dominant right and dominant left
ventricles occurring in nearly equal numbers (17) (e69,e73).
Clinically, the large left-to-right shunt precipitates severe congestive heart failure. Pulmonary hypertensive vascular changes can
appear in the 1st year of life, further complicating the clinical picture (18) (e77,e78). Without intervention, almost 50% of infants with
a complete AV septal defect die by 6 months of age, and only 15% survive to 2 years (e79). Early surgical repair, usually in the 1st
year of life, is recommended (e40,e77,e78,e80,e81).
Malformations of the Conus and Truncus

The conotruncal structures of the heart arise embryologically from the distal aspect of the heart tube and represent the outflow
region of the developing heart (e1,e2,e82). Embryologic research demonstrates the importance of cells derived from the neural
crest in normal conotruncal development (e75,e83-e85). This finding is reflected in humans by the close association between
conotruncal malformations and
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the diGeorge/velocardiofacial syndrome and its associated 22q 11.2 chromosomal deletion (e86,e87). Table 13-8 outlines the
spectrum of malformations that occur in the conotruncal region.
Table 13-7 ▪ ATRIOVENTRICULAR SEPTAL DEFECTS: ASSOCIATED CARDIAC ANOMALIES AND

SYNDROMES
Type AVSD Relative Incidence %Totalc Associated Syndromea Associated CV Anomaliesb
Partial 35% 33% with Trisomy 21 All AVSD:
Complete 65% 48% with Trisomy 21 Subaortic stenosis
Subtypes % Completed Coarctation of the aorta
Parachute mitral valve
Tetralogy of Fallot
Double outlet right ventricle
Common atrium
Rastelli A 55%-70% Trisomy 21 Subaortic stenosis
Rastelli B <10% — Valvular aortic stenosis
Coarctation of aorta
Hypoplastic left ventricle
Rastelli C 25%-40% Heterotaxy Tetralogy of Fallot
Common atrium
aSee references (17) (e67,e69).
bSee references (e40,e69,e73,e75,e76).
cSee references (17) (e64-e69).
dSee references (e66,e72,e75,e76).
AVSD, atrioventricular septal defect; CV, cardiovascular.

Transposition of the Great Vessels
In transposition of the great vessels, the aorta arises from the right ventricle and the pulmonary artery from the left ventricle (i.e.,
discordant ventriculoarterial connections). The transposed aorta thus originates in an anterior position, either to the right
(dextrotransposition, or D-transposition) or the left (levotransposition, or L-transposition) of the pulmonary artery. Hearts with
transposition are further classified according to their AV connection, as depicted in Figure 13-7.
Complete Transposition
Complete transposition, the most common form, accounts
for 2.5% to 6.5% of all congenital heart malformations (see Table 13-1), with a 2:1 male predominance (e88). The D-transposed
aorta ascends parallel and to the right of the pulmonary artery rather than following its normal, twisted course (Figure 13-8). Internal
examination reveals AV concordance with ventriculoarterial discordance (19). The aorta originates from the normally positioned
morphologic right ventricle, with a muscular band separating the aortic and the tricuspid valves; the pulmonary artery arises
posteriorly from the normally positioned morphologic left ventricle and is in fibrous continuity with the anterior mitral valve leaflet.
The coronary arteries originate from one or both of the “facing sinuses” of the aorta (i.e., the sinuses adjacent to the
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pulmonary artery) (19, 20) (e89,e90). The anatomic course traversed by the coronary arteries varies considerably, a feature of
significance when arterial switch surgery is planned (20, 21) (e88,e89,e91).
Table 13-8 ▪ TYPES OF CONOTRUNCAL MALFORMATIONS
Transposition of the great vessels
Complete transposition (D-transposition)
Corrected transposition (L-transposition)
Double outlet ventricles
Double outlet left ventricle
Persistent truncus arteriosus
Aortopulmonary window (aortopulmonary septal defect)
D, dextro; L, levo.
FIGURE 13-7 ▪ Diagrammatic representation of normal blood flow (top), blood flow through complete transposition (middle), and
blood flow through “corrected” transposition (bottom).
FIGURE 13-8 ▪ Complete transposition of the great vessels from the anterior aspect of the heart. The aorta, marked with *, is
situated to the right and slightly anterior to the pulmonary artery, marked with ^. The two vessels ascend in a parallel course.
A VSD accompanies the complete transposition in approximately 40% of cases, with 40% to 60% of the VSDs showing septal
malalignment (22) (e92). Anterior (rightward) malalignment, present in 20% to 25% of cases with VSD, results in subaortic (right
ventricular outflow tract) obstruction, often with associated coarctation (22) (e92-e94). Pulmonary (left ventricular outflow tract)
obstruction occurs in 25% to 30% of hearts with or without VSD, secondary to a malaligned VSD or subvalvular fibrous or
fibromuscular tissue bundles (21, 22) (e88,e95).
Patients with complete transposition of the great vessels can be divided into three clinical groups based on the status of the
ventricular septum and pulmonary outflow tract (Table 13-9). In groups 1 and 2, massive pulmonary blood flow is associated with a
high rate of early and accelerated pulmonary hypertensive vascular disease (e96-e98). A variety of surgical repairs have been
devised for transposition of the great vessels, depending in part on the group. Uncomplicated transposition was in the past most
commonly repaired with an atrial baffle (Mustard or Senning) procedure, which shunted systemic venous return to the left ventricle
and pulmonary venous return to the right ventricle. The atrial baffle repairs result in good long-term survival, but these hearts are
prone to late right ventricular dysfunction and arrhythmias
(e99-e103). The more anatomically correct arterial switch procedure is therefore the currently favored repair for hearts in groups 1
and 2 (e100,e104). In hearts with VSD and significant left ventricular outflow tract obstruction (group 3), repair focuses on directing
the left ventricular flow into the aorta and creating a shunt between the right ventricle and the pulmonary circulation (Rastelli
procedure) (21) (e95,e105).
Table 13-9 ▪ TRANSPOSITION OF THE GREAT VESSELS: CLINICAL GROUPS

Group 1 Group 2 Group 3
Ventricular septum Intact Large defect Defect
Small defect
Outflow tract Not obstructed Not obstructed LVOT Obstructed
RVOT obstructed
Presentation Cyanosis Heart failure Cyanosis
Surgical repair Arterial switch Arterial switch + Rastelli
VSD closure
LVOT, left ventricular outflow tract; RVOT, right ventricular outflow tract; VSD, ventricular septal defect.
Corrected Transposition
In the much less common corrected transposition, also known as L-transposition or ventricular inversion, an L-transposed aorta
ascends parallel to and to the left of the pulmonary artery. Internally, both AV and ventriculoarterial discordance are present. The
right atrium is in continuity with a rightsided morphologic left ventricle from which the pulmonary artery arises; the left atrium is in
continuity with a left-sided morphologic right ventricle from which the aorta originates; blood flow is thus anatomically “corrected”
(e106,e107) (Figure 13-7). The defect is frequently associated with other congenital anomalies, including tricuspid valve dysplasia,
pulmonary outflow tract obstruction, and VSDs (e106-e108). When corrected transposition is present as an isolated defect, patients
are initially asymptomatic but prone to late right ventricular dysfunction and arrhythmias, which reflect the limited ability of the right
ventricle to support the systemic circulation (e109). AV discordance can occur with other types of ventriculoarterial connections,
including double outlet right ventricle and ventriculoarterial concordance (e110).
Double Outlet Ventricle
Double Outlet Right Ventricle

Of the two forms of double outlet ventricle (Table 13-8), double outlet right ventricle (DORV) is the more common, accounting for 1%
to 1.5% of congenital heart defects (1) (e9,e10). The term DORV denotes a heart in which both great vessels originate from the
right ventricle. This seemingly straightforward term incorporates a currently controversial
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range of morphologic entities. The strictest criterion requires that both great arteries arise exclusively from the right ventricle, with
no fibrous continuity between the mitral and the outflow valves (23) (e111,e112). A somewhat less strict criterion requires only that
both great vessels arise exclusively from the right ventricle; 50% to 60% of such hearts display at least focal fibrous continuity
between the mitral and the outflow valves (24) (e113,e114). The broadest criterion requires only that more than 50% of one great
vessel override the ventricular septum; when this criterion is used, DORV overlaps with tetralogy of Fallot (25) (e115-e117). A VSD
almost always accompanies the double outlet right ventricle, serving as the only site for left ventricular outflow. The variable location
of the VSD relative to the pulmonary and aortic valves serves to define pathologic subcategories (23, 26) (e111,e118) (Figure 13-9).
The anatomic relationship between the great arteries also varies, as outlined in Table 13-10. In earlier reports, the side-by-side
relationship of the great arteries was described as the most frequent one, but more recent series describe the posterior normal
pattern as the most common (23, 24) (e114,e117). The wide variety of coronary artery anomalies that are associated with side-by-
side or malposed great arteries affect surgical repair options and procedures (e117,e119-e121).
The Taussig-Bing malformation, first described in 1949 (e122), is an uncommon variant of double outlet right ventricle in which the
VSD is subpulmonic; no pulmonary stenosis is present. When strict criteria are used, fewer than 10% of DORV are of the Taussig-
Bing variant (23). When broader criteria are used (“a spectrum of anomalies unified by a juxtapulmonary VSD with malalignment of
the infundibular septum”), some hearts can be classified both as the Taussig-Bing variant and as transposition of the great vessels
with a malaligned VSD (25) (e123).
FIGURE 13-9 ▪ The sites, D, of the ventricular septal defects in a double outlet right ventricle. A: Subaortic (60% to 65% of total
cases). B: Subpulmonic (25% to 30%). C: Doubly committed (5% to 15%). D: Remote (10% to 15%). Ao, aorta; PA, pulmonary
artery.
Table 13-10 ▪ DOUBLE-OUTLET RIGHT VENTRICLE: RELATIONSHIP OF GREAT ARTERIES
Root of Ao Relative to Root of PA Descriptive Terms Ascending Ao and PA
Posterior and right Normal or dextroposition Spiral
Parallel and right Side by side Parallel
Anterior and right D-malposition Parallel
Anterior and left L-malposition Parallel
Ao, aorta; PA, pulmonary artery; D, dextro; L, levo.
Various other cardiac malformations accompany many double outlet right ventricles. Pulmonary infundibular stenosis with or without
valvular stenosis occurs in 40% to 70% of hearts (23, 24) (e112,e113,e116). ASDs are not uncommon; complete AV septal defects
are less common (e112,e117,e124,e125). Left-sided inflow and outflow obstructive lesions may be accompanied by left ventricular
hypoplasia (24)(e112,e113,e116,e117).
The clinical presentation of DORV depends on the location of the VSD and the presence of associated malformations, particularly
pulmonary stenosis (e111,e126) (Table 13-11). Surgical correction varies depending on the anatomic configuration of the heart
(e118,e121,e126,-e129) (Table 13-11).
Double Outlet Left Ventricle

Double outlet left ventricle (DOLV) is a rare malformation in which both great vessels arise predominantly from the morphologic left
ventricle; a VSD accompanies the vast majority (27) (e130-e132). Similar to DORV, the DOLV is classified by the location of the
VSD relative to the great vessels (27). The DOLV with subaortic VSD is frequently complicated by pulmonary outflow tract
obstruction and DOLV with subpulmonic VSD by aortic outflow tract obstruction (27) (e132).
Persistent Truncus Arteriosus

Persistent truncus arteriosus is defined as a single arterial trunk that originates from a single semilunar valve and supplies the aorta,
one or both pulmonary arteries, and the coronary arteries (Figure 13-10). The truncal valve is tricuspid in 50% to 70% of cases,
quadricuspid in 25%, and bicuspid in most of the rest (e133-e137). The truncal vessel overlies and usually overrides an infundibular
VSD, although occasionally it is predominantly committed to one ventricular chamber (e133, e137,e138). The truncal valve is
always in fibrous continuity with the mitral valve; fibrous continuity may also be present between the truncal and tricuspid valves
(e134,e137). The truncal valve leaflets are frequently thickened and myxomatous, with valvular insufficiency
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present in approximately 15% to 30% (e134,e138-e140). The coronary arteries originate from the sinuses of the truncal valve in a
variable pattern, with a single coronary artery present in 15% to 20% (e133-e135,e137,e141).
Table 13-11 ▪ DOUBLE-OUTLET RIGHTVENTRICLE: CLINICOPATHOLOGIC CATEGORIES
Clinical Type VSD Location RVOTO LVOTO Surgical Repair
VSD Subaortic Absent Absent Intraventricular tunnel
Doubly committed
TOF Subaortic Present Absent TOF type
Doubly committed
TGA Subpulmonic (Taussig-Bing) Absent Often present Arterial switch + VSD closure
Intraventricular tunnel
Damus-Kaye-Stansel
Remote VSD Noncommitted Absent Often present Intraventricular tunnel
Single ventricle repair
VSD, ventricular septal defect; TOF, tetralogy of Fallot; TGV, transposition of the great vessels; RVOTO, right ventricular
outflow tract obstruction; LVOTO, left ventricular outflow tract obstruction.
Truncus arteriosus is subclassified according to the pattern of origin of the pulmonary arteries from the truncal root (Figure 13-11).
However, this classification by Collet and Edwards (28) (e142) has two problems:
1. It classifies hearts in which both pulmonary arteries arise directly from the descending aorta as type 4 truncus. The “type 4
truncus” instead represents a variant of pulmonary atresia (PA) with VSD (e134).
2. It does not address the aberrant origin of one pulmonary artery from the ascending or descending aorta.
In approximately 15% of cases, one pulmonary artery arises from the truncus and the other from the ductus or ascending aorta,
so that a pulmonary artery is “absent” (e143).
FIGURE 13-10 ▪ Truncus arteriosus. The left ventricle free wall has been lifted to uncover the smooth surfaced left ventricular
outflow tract with a VSD opening at the top. Above the VSD lies a somewhat nodular truncus arteriosus valve. The main pulmonary
artery almost immediately branches to the left from the common trunk; the aorta continues ascending posteriorly.
A classification devised by van Praagh creates a separate subtype for this latter finding and also acknowledges the rare case in
which there is no VSD (e144). The classification was subsequently revised by van Praagh to simplify the scheme in a surgically
meaningful fashion (29) (Table 13-12).
Associated anomalies most frequently involve the aortic arch and include absent ductus arteriosus (>50%), right-sided aortic arch
(20% to 35%), and interrupted aortic arch type B (10%) (e133,e134,e140). Extracardiac anomalies, especially those related to
diGeorge syndrome, occur in 20% to 30% (e145,e146). The diGeorge syndrome-associated chromosome 22q11 deletion can be
detected by fluorescence in situ hybridization (FISH) in 35% to 50% of infants with persistent truncus arteriosus (e87,e147,e148).
The early clinical manifestation of congestive heart failure results from intracardiac shunting and markedly excessive pulmonary
blood flow. The excessive pulmonary blood flow also produces rapidly progressive pulmonary hypertensive vascular disease; early
surgical repair is therefore recommended (e141,e149,e150).
Aortopulmonary Window
Aortopulmonary window, or aortopulmonary septal defect, is a rare malformation characterized by a defect in the vessel wall
between the ascending aorta and the main pulmonary artery. The defect may lie proximally (just above the aortic and the pulmonary
valves), distally (in the upper ascending aorta adjacent to the right pulmonary artery), or as a combined opening that involves the
majority of the ascending aorta (7) (e151,e152). Associated anomalies, present in more than 50% of cases, commonly include a
VSD, interrupted aortic arch type A, and anomalous origin of one pulmonary artery from the ascending aorta (30) (e153-e155).
Although aortopulmonary window occurs in the same general region as persistent truncus arteriosus, it is not seen in the
chromosome 22q11 deletion syndromes (30) (e156,e157).
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FIGURE 13-11 ▪ Truncus arteriosus. A: A window in the right ventricle shows a tricuspid truncal valve through an infundibular
ventricular septal defect, with an associated right aortic arch. There are three main truncus types. B: In type I, a single pulmonary
trunk arises from the truncus and divides into two pulmonary arteries. C: In type II, two pulmonary arteries originate from closely
apposed orifices in the common trunk. D: In type III, the two pulmonary arteries originate from widely separated orifices in the
common trunk. Ao, aorta; LPA, left pulmonary artery; RPA, right pulmonary artery.
Malformations of the Ventricular Inflow Tracts

Tricuspid Valve Malformations
Tricuspid Atresia
Tricuspid atresia, in which the only outlet to the right atrium is via a patent fossa ovalis or an ASD, accounts for 1% to 1.5% of
congenital heart malformations (e4-e6,e10,e15). The markedly hypoplastic right ventricle, positioned along the right anterosuperior
border of the heart, has no inlet segment. The markedly dilated right atrium contains no grossly identifiable valvular tissue in more
than 85% of cases (31, 32) (e158). A dimple in the muscular atrial floor, presumably marking the site of the missing valve, may have
a fibrous attachment to the right ventricle, but often is instead in continuity with the left ventricle by transillumination and pin prick
studies (32) (e158,e159). The remaining 5% to 15% of hearts display a tricuspid valve remnant in the form of an imperforate fibrous
membrane (e158,e160). A muscular VSD, termed the outlet foramen, allows communication between the dominant left ventricular
chamber and the rudimentary right ventricle; however, the VSD or the infundibular outflow tract may be restrictive (31) (e161,e162).
Tricuspid atresia is subclassified according to the size of the VSD, concordance or discordance of the great vessels,
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and the presence or absence of pulmonary stenosis/atresia (e163,e164) (Table 13-13). The clinical symptoms depend on these
anatomic variables; more than 50% of cases present with cyanosis and murmur in the newborn period (e162,e164,e165). In the vast
majority of hearts, the right ventricle is too small to function adequately as a pumping chamber (eFigure 13-3), and repair relies on
the Fontan operation or one of it modifications (e162,e165,e166).
Table 13-12 ▪ TRUNCUS ARTERIOSUS CLASSIFICATION
Collet and Edwards(e142) Van Praagh (e144) Modified van

Praagh (29)
Type 1 Type 1 Large aorta type
PAs arise as a single main artery and then divide PAs arise as a single TA with confluent
main artery and then or near confluent
divide PAs
Type 2 Type 2
PAs arise separate but next to each PAs arise separately
Type 3
PAs arise widely separated
Type 3 TA (large aorta type)

with absence of one
One PA branch PA
“absent”
Arises from ductus or

aorta
Type 4 Large pulmonary

artery type
Aortic arch hypoplastic TA with IAA or

or interrupted severe COTA
Type A = VSD present
Type B = VSD absent
PA, pulmonary artery; VSD, ventricular septal defect; TA, truncus

arteriosus; IAA, interrupted aortic arch; COTA, coarctation of the
aorta.
Table 13-13 ▪ TRICUSPID ATRESIA: CLINICAL CLASSIFICATION
I. Normally related great vessels (60%-70%)

A. Intact ventricular septum with pulmonary atresia
B. Small VSD with pulmonary stenosis
C. Large VSD without pulmonary stenosis
II. D-transposition of the great arteries (25%-30%)
A. VSD with pulmonary atresia
B. VSD with pulmonary stenosis
C. VSD without pulmonary stenosis
III. Malposition other than D-transposition of the great arteries (5%)
Ebstein Malformation
Ebstein malformation accounts for fewer than 1% of all cases of CHD, but is the most common cause of isolated tricuspid stenosis
or insufficiency (1, 33) (e11). It is characterized by adherence of variable portions of the septal and posterior tricuspid valve leaflets
to the right ventricular wall, with “atrialization” (i.e., downward displacement of the functional annulus) of a portion of the right
ventricle (Figure 13-12) (33, 34) (e167). The anterior valve annulus insertion is normally positioned, with a large, redundant, and
often muscularized leaflet. The margin of the leaflet may be attached to the posteroinferior right ventricular wall and produce
obstruction and in some cases complete occlusion of the AV orifice (Figure 13-13) (34) (e167,e168). Tricuspid regurgitation occurs
across the dilated AV junction (true annulus). Right-to-left shunting across a patent fossa ovalis or ASD and supraventricular
arrhythmias due to accessory conduction pathways frequently complicate Ebstein malformation (33) (e169,e170). A variety of other
associated cardiovascular defects, most commonly pulmonary valvular stenosis, PA, or a VSD, occur in 30% to 40% of cases (33)
(e169,e171e172). Abnormalities of the left ventricle include not only valvular dysplasia, but also noncompaction of the myocardium
(e170).
FIGURE 13-12 ▪ Mild form of Ebstein anomaly. The opened right atrium and right ventricle display a markedly thickened ventricular
wall. The septal (*) and posterior leaflets of the tricuspid valve are fixed to the underlying ventricular wall.
FIGURE 13-13 ▪ Severe form of Ebstein anomaly. The opened right atrium uncovers a markedly enlarged and dysplastic anterior
tricuspid leaflet attached to the apical myocardium by tiny chordae. A probe placed in the pulmonary artery traverses the remaining
right ventricular cavity and appears at the base of this dysplastic valve, illustrating the severe obstruction to pulmonary inflow and
outflow created by this defect.
Given the broad range of anatomic alterations encompassed by Ebstein malformation, it is not surprising to find a broad range of
clinical manifestations for the disorder. One-third to one-half of patients present in the newborn period with cyanosis and a murmur;
the mortality rate among such infants is high, particularly when the malformation is associated with additional cardiac anomalies
(e171-e174). In many patients, however, the diagnosis is delayed until the second decade of life or later, when arrhythmias often
represent the major clinical problem (33) (e169,e174). Surgical repair, required in approximately 40% of patients, includes either
tricuspid valvuloplasty or valve replacement with concomitant repair of associated lesions, most commonly ASD closure (e175-
e178).
Mitral Valve Malformations
Mitral Stenosis
The normal mitral valve apparatus is a complex structure with four primary components: annulus, anterior and posterior valve
leaflets, chordae tendineae, and anterolateral and posteromedial papillary muscles. A variety of malformations
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affecting any or all of the valve components result in congenital mitral stenosis and insufficiency (e179-e181) (Table 13-14). A
supramitral ring, a ridge of connective tissue at the atrial surface of the mitral leaflets, usually occurs with deformities of the mitral
valve apparatus (e180,e182-e184). Valve hypoplasia, in which the valve components are small but otherwise normally formed, most
commonly associated with left ventricle hypoplasia, VSDs, and coarctation of the aorta (COTA) (7) (e180). The “typical” mitral
stenosis manifests as lesions at both the valvar and the subvalvar areas including valve dysplasia with commissure fusion,
obliteration of the intrachordal spaces, and shortening of the chordae tendineae and papillary muscles. Associated malformations
include tetralogy of Fallot, COTA, and subaortic stenosis with a near-normal-sized left ventricle (7) (e180). The double orifice mitral
valve results when excessive valve tissue bridges between the anterior and posterior valve leaflets to create two, usually unequally
sized, orifices, both supported by chordal attachments that insert into often abnormally positioned papillary muscle (eFigure 13-4)
(e185,e186). The double orifice valve almost always occurs in company with other cardiac malformations, especially AV septal
defects (50% of cases) or left-sided obstructive lesions (40%) (e187,e186) Two forms of cleft mitral valve without associated
primum ASD or VSD have been described (e188):
1. Associated with normally related great vessels and a shortened inlet septum (i.e., forme fruste of an AV septal defect)
2. Associated with TGA or DORV and a normal inlet septum.
Table 13-14 ▪ MITRAL VALVE MALFORMATIONS
Supravalvar lesions
Supramitral ring
Valvar lesions
Valve hypoplasia
Valve dysplasia
Commissural fusion
Valve leaflet excess or agenesis
Double orifice valve
Cleft mitral valve
Subvalvar lesions
Parachute deformity (Single papillary muscle)
Funnel deformity (shortened fused chordae)
Arcade deformity (Papillary muscle fused with valve)
Mixed
Shone syndrome
Parachute deformity of the mitral valve, defined as insertion of all the chordae into a single papillary muscle group, also usually
occurs with other malformations of the heart, particularly VSDs and obstructive lesions of the aortic valve and arch (7, 35)
(e180,e182,e189). The eponym Shone syndrome denotes the association of a parachute mitral valve with a supramitral ring,
subaortic stenosis, and COTA (35) (e190). Repair strategies include balloon dilation and mitral valve reconstruction, mitral valve
replacement surgery, and the more recent pulmonary valve autograft (Ross II) (e191-e193).
Mitral Atresia
Mitral atresia, defined as the absence of a left AV connection, is marked on the left atrial aspect by muscular atrial floor with or
without a visible dimple or, less commonly, by an imperforate membrane (7, 36, 37) (e194). The microscopic examination of hearts
with no grossly obvious membrane between the left atrium and the left ventricle uniformly reveals a fibrous connection at the
presumed site of the absent valve (36). The outlet for pulmonary venous return is by way of a patent fossa ovalis or less commonly
an ASD (7). Rarely, the fossa ovalis is prematurely closed, and pulmonary venous return is shunted to the right side of the heart by
anomalous venous connections (7, 38) (e195). When the great vessels are normally related, mitral atresia is most commonly
associated with aortic atresia and, as such, is included in the hypoplastic left-heart syndrome. The left ventricle exists as a
diminutive chamber lined by translucent endocardium, which in some cases is evident only on microscopic examination of the
posterosuperior aspect of the hypertrophic right ventricle (7, 37). Less often a VSD is present and a patent aortic valve arises from
either the right (DORV) or left ventricular chamber (7, 37) (e194). Repair strategies are similar to those employed for other
hypoplastic left ventricles (see below).
Floppy Mitral Valve
Floppy mitral valve represents the central defect in the floppy mitral valve (FMV)/mitral valve prolapse (MVP)/mitral valve
regurgitation (MVR) triad. The primary defect in the “floppy” valve is deposition of acid mucopolysaccharides and dissolution of the
collagen in the pars spongiosa and fibrosa of the valve (39) (e196,e197). The accumulation of myxoid material leads to thickened
and enlarged valve leaflets often with increased chordal insertions on the ventricular surface, elongation of the chordae tendenae,
and dilatation of the valve annulus (39). With prolapse, the valve becomes “hooded,” defined as the presence of ballooning to a
height of at least 4 mm and involving at least one-half of the anterior or two-thirds of the posterior mitral leaflets (40). The
myxomatous degeneration in the valves leaflets is without inflammation and does not lead to fusion of the valve commissures,
distinguishing these valvular changes from those of rheumatic fever. Similar myxomatous changes occur elsewhere in the heart,
including the conduction system; a feature that likely explains the associated arrhythmias and the conduction defects (e198).
The reported incidence of FMV/MVP/MVR varies considerably, with less than 1% to 5% of children exhibiting clinical or
echocardiographic features of MVP (41) (e199,e200). In the pediatric population, the incidence increases with age; MVP is
extremely rare before 2 years of age (e201,e200). Most children are asymptomatic, presenting with the characteristic late systolic
“click” on ascultation; an occasional child presents with chest pain of unclear etiology (41) (e201,e200). Skeletal anomalies,
especially pectus
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excavatum, are common (42) (e196,e201). The 2:1 female predominance described in adults is also observed in some but not all
groups of children studied (41) (e200,e201). Progressive MVR, a major problem in adults with FMV, occurs rarely during childhood.
Other complications, including infectious endocarditis, thromboembolism, arrhythmias, and even sudden death, do occur
occasionally in the pediatric population (e196,e200,e202).
The disorder frequently occurs in families, following either an autosomal dominant or X-linked inheritance pattern (e196). The
linkage of MVP to loci on chromosomes 11, 13, and 16 has so far failed to yield an identifiable underlying genetic mutation (e203). A
small subgroup of patients with FMV/MVP/MVR do have an associated connective tissue disorder such as Marfan or Ehler-Danlos
syndrome (e196).
Univentricular Atrioventricular Connection

The term univentricular AV connection denotes the connection of the AV valves to a single ventricular cavity (37) (e204,e205).
Table 13-15 lists at least some of the terms previously used for this condition and outlines the range of anomalies encompassed.
The anatomy of such hearts can be highly complex and variable, so that sequential segmental analysis is an essential tool for
accurate classification (37) (e206,e207).
Double Inlet Ventricle

In double inlet left ventricle, the most common of the double inlet malformations, both the left and right AV valves open into a
dominant left ventricular cavity (e205,e208,e209). The rudimentary right ventricle occupies the right anterosuperior border of a
normally related, D-looped left ventricle or the left anterosuperior border of an inverted, L-looped left ventricle. The rudimentary right
ventricle communicates with the dominant left ventricle via a variably sized VSD. The great vessels are transposed in the vast
majority of cases but may be normally related, atretic, or in a double outlet configuration.
Table 13-15 ▪ UNIVENTRICULAR ATRIOVENTRICULAR CONNECTION
Common synonyms
Single ventricle
Common ventricle
Holmes heart
Univentricular heart
Cor triloculare biatriatum

Primitive ventricle
Anatomic subtypes
Double-inlet ventricle
Double-inlet left ventricle
Double-inlet right ventricle
Double-inlet ventricle of mixed morphology (absent ventricular septum)
Double-inlet ventricle of indeterminate morphology
Single-inlet ventricle
Mitral atresia
Tricuspid atresia
Common-inlet ventricle
Overriding atrioventricular valves
Common Inlet Ventricle

In common inlet ventricle, both atria communicate with a single ventricle via a common AV valve (e205,e210). Many of these hearts
represent the extreme form of unbalanced AV septal defect and thus include a dominant right or left ventricular cavity and a
rudimentary second ventricle. Less often, the common AV valve communicates with a single ventricular chamber of indeterminate
type without an identifiable rudimentary ventricle. These hearts frequently have abnormal ventriculoarterial connections as well.
Straddling and Overriding Atrioventricular Valves

An AV valve annulus may override, or its chordal insertion may straddle, the ventricular septum (43) (e205). In hearts with valve
annulus override, the AV connection is assigned to the ventricle to which more than 50% of the valve annulus is attached (e211).
Straddling of the chordae without valve annulus override does not change the AV connection designation.
Malformations of the Ventricular Outflow Tracts

Pulmonary Outflow Tract and Valve Malformations
Tetralogy of Fallot
Four components comprise the tetralogy of Fallot (TOF): infundibular pulmonic stenosis, VSD, aortic valve dextroposition, and right
ventricular hypertrophy (Figure 13-14). However, the morphologic detail surrounding these four components can vary considerably
(7, 44) (e212,e213). Infundibular pulmonic stenosis, the consequence of anterosuperior malalignment of the outlet septum, leads to
decreased pulmonary blood flow with an associated small pulmonary artery (Figure 13-15). Over time, the stenosis becomes
exacerbated by hypertrophy of the infundibular septum or cristal structures (e212,e213). The invariably large and nonrestrictive
VSD is perimembranous in 75% of cases, located in the muscular outlet in 20%, and subarterial only rarely (44) (e212,e214). The
degree of aortic override varies from 15% to 95%. In the extreme situation, the differentiation of TOF from double outlet right
ventricle depends on the presence of the characteristic infundibular stenosis and fibrous continuity between the aortic and mitral
valves; some investigators classify hearts with more than 50% aortic override as TOF with double outlet right ventricle (25).
The pulmonary valve is abnormal in 66% to 75% of cases. It is most often bicuspid but may be unicuspid or stenotic by virtue of
thickened dysplastic valve leaflets (7, 44) (e215). The 20% to 25% of cases with an imperforate pulmonary
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valve orifice are classified as PA with VSD, discussed in more detail later. The pulmonary arteries show a range of accompanying
abnormalities that includes localized stenosis at the origin of the pulmonary artery branches, central pulmonary artery discontinuity,
absent left pulmonary artery branch, and pulmonary hilar artery hypoplasia (45) (e216). When the pulmonary artery stenosis is
severe, pulmonary artery hypertension may develop after surgical repair of the TOF (46) (e217). In 3% to 6% of cases, the
pulmonary valve is absent (e216) and the pulmonary arteries are dilated; this dilatation may be massive.
FIGURE 13-14 ▪ An opened anterior right ventricle illustrates the four primary features of tetralogy of Fallot: marked narrowing of the
pulmonary infundibulum (between arrows); a large perimembranous ventricular septal defect (white asterisk); dextroposed
overriding aorta, visible through the ventricular septal defect; and hypertrophy of the right ventricular myocardium (black asterisk).
FIGURE 13-15 ▪ Heart and lungs removed at autopsy with an unrepaired tetralogy of Fallot. An incision through the anterior right
ventricle ends at the base of a small pulmonary artery. The markedly enlarged aorta arises behind and to the right of the pulmonary
artery.
A variety of other cardiovascular defects occur with TOF. Commonly associated anomalies include right-sided aortic arch (20% to
30%) and absent ductus arteriosus (20% to 25%) (e216,e215). Although a patent fossa ovalis occurs commonly in infants with TOF,
a true ASD is present in only 20% to 25% (e216,e215). A complete AV septal defect accompanies TOF in 1% to 2% of cases, most
often in children with trisomy 21 (e218,e219).
TOF accounts for 3.5% to 10.5% of all CHD and represents the most common cyanotic CHD. In approximately 33% of cases, TOF
occurs as part of a recognizable syndrome, most commonly DiGeorge syndrome or trisomy 21 (45).
Hypoxia and cyanosis are the principal symptoms of TOF; their severity varying with the degree of pulmonary obstruction (45). In
the presence of marked stenosis or atresia, cyanosis is evident in the neonatal period. More commonly, cyanosis appears in the first
6 months of life, associated with increasing infundibular stenosis. Required treatment consists of widening of the outflow tract by
surgical resection of outflow tract muscle and, in severe cases, insertion of a transannularpatch (45) (e165).
Pulmonary Atresia with Ventricular Septal Defect

Although the designation PA with VSD (PA/VSD) is frequently used as a synonym for TOF with PA, not all hearts with PA/VSD
display the hypoplastic right ventricular infundibulum characteristic of TOF (47) (e220). Most commonly, a dimple (more rarely
recognizable fused bicuspid valve cusps) marks the site of the pulmonary valve (e221). Abnormalities of the pulmonary arteries
include a connection of confluent, bilateral pulmonary arteries to the right ventricle by an atretic cord, absence of the left pulmonary
artery, and absence of all intrapericardial pulmonary arteries (48) (e222-e224). This latter group was classified as truncus arteriosus
type IV in the past. The ductus arteriosus supplies blood to one or both of the pulmonary arteries in 40% to 65% of cases. The
lungs also receive blood via collateral arteries that originate from the descending aorta and supply the pulmonary arteries via
intrapulmonary, hilar, or extrapulmonary anastomoses (48) (e225,e226). Recent studies demonstrate that the chromosome 22q11
deletion increases the likelihood of an absent ductus arteriosus and a major aortopulmonary collateral artery supply (e227,e228).
With a major aortopulmonary collateral artery supply, hypoplasia and arborization of the pulmonary arteries make surgical
management problematic (49) (e229,e222). Early unifocalization of the pulmonary blood supply seems to improve pulmonary
circulation and makes later corrective surgery possible in some patients (e230-e233).
Absent Pulmonary Valve

Absent pulmonary valve is a rare anomaly usually associated with TOF (e234-e236). At the site of the expected valve, a narrow
valve annulus is rimmed by rudimentary, nodular, gelatinous tissue with massive poststenotic dilation
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(e234). More complex pulmonary artery anomalies, including discontinuity of the main pulmonary arteries, anomalous origin of the
pulmonary arteries, and absence of the left pulmonary artery, occur less often (50) (e235,e237). The ductus arteriosus is frequently
absent (e238,e235). Dilated pulmonary arteries can compress the adjacent bronchi and cause respiratory compromise;
abnormalities of the intrapulmonary arteries and bronchi may exacerbate these pulmonary problems (50) (e239).
Pulmonary Stenosis with Intact Ventricular Septum

Pulmonary stenosis with intact ventricular septum (IVS) accounts for 2.5% to 8.0% of all cases of CHD (Table 13-1). The
obstruction is usually valvular, with secondary right ventricular hypertrophy and poststenotic dilation of the pulmonary trunk. The
valve is most often dome shaped with fused cusps and a single central orifice, but it may be unicuspid, bicuspid, or tricuspid with
partially fused commissures (51) (e240). Thickened dysplastic valve leaflets with nonfused cusps occur sporadically (e240,e241),
and as one form of pulmonary stenosis in Noonan syndrome (e242). The pulmonary artery trunk usually exhibits poststenotic
dilation; pulmonary artery hypoplasia is rare even with critical stenosis (e243,e244). Symptoms depend on the severity of the
stenosis. Critical stenosis, presenting as cyanosis in infancy, requires early balloon angioplasty, surgical valvotomy, or transannular
patch (e243-e246). More commonly, infants are asymptomatic; stenosis develops and worsens in early childhood in approximately
15%, and many remain asymptomatic into adulthood (52) (e247-e249). Secondary infundibular stenosis secondary to right
ventricular hypertrophy can further complicate the course over time (e248).
Subvalvular Stenosis
Pulmonary subvalvular or infundibular stenosis, which accounts for fewer than 10% of cases of pulmonary stenosis with IVS, occurs
when fibrous thickening at the junction of the trabecular and outlet segments divides the right ventricle into two chambers; it may
also be caused by tubular hypoplasia of the infundibulum (7) (e244). Double chamber right ventricle is a closely related anomaly in
which hypertrophied muscle bands cross the right ventricular cavity just proximal to the infundibulum and divide a high-pressure
proximal chamber from a low-pressure infundibular chamber (53) (e250,e251). The majority of hearts with double chamber right
ventricle exhibit other anomalies, most often (65% to 75%) a VSD (53) (e252,e253).
Table 13-16 ▪ SUPRAVALVAR AND PERIPHERAL PULMONARY ARTERY STENOSIS
Type I Single central stenosis of:
A Main pulmonary artery
B Right main pulmonary artery
C Left main pulmonary artery
Type II Bifurcation stenosis
A Short, localized stenosis
B Long, narrow segments of stenosis
Type III Multiple stenoses of peripheral segmental arteries Type IV Multiple stenoses of peripheral and central arteries
Supravalvular and Peripheral Pulmonary Artery Stenosis

Supravalvular or pulmonary artery stenosis occurs as a localized area of narrowing in the pulmonary trunk or branch or as multiple
areas of narrowing throughout the pulmonary artery tree (54) (e254). The stenosis is subclassified into four types, based on the site
of the obstruction, with type III accounting for approximately one-third of cases (e254) (Table 13-16). Associated cardiac
malformations, present in approximately 66% of cases, include VSD, ASD, valvular pulmonary stenosis, and TOF (55) (e244). A
variety of malformation syndromes include pulmonary artery stenosis (Table 13-17).
Pulmonary Atresia with Intact Ventricular Septum

PA with IVS accounts for 1% to 3% of all cases of CHD. The atresia is usually valvular, with a fibrous membrane containing
commissural lines present at the expected site of the valve (7) (e221,e255,e256). The pulmonary artery is funnel shaped and
usually only mildly to moderately hypoplastic (7) (e221,e256). The right ventricular myocardium is hypertrophied and the cavity of
variable size, with the size of the tricuspid valve directly related to the size of the right ventricle (56) (e255-e257). The right ventricle
and tricuspid valve are usually small, often with associated pulmonary infundibular stenosis or atresia (56) (e256,e258). Right
ventricle-coronary artery fistulas develop in more than 50% of hearts with small ventricular cavities; in a subgroup of these hearts,
the volume of flow through the fistula results in right ventricle-dependent coronary blood flow (57) (e259-e262). Accompanying
coronary artery luminal stenosis and vessel atrophy proximal to the fistula site can result in myocardial ischemia and sudden death
(57) (e10,e263). At the opposite extreme, the right ventricular cavity may be dilated, with the tricuspid valve showing dysplasia and
often Ebsteinization (56) (e256,e257,e264).
Table 13-17 ▪ PULMONARY ARTERY STENOSIS-ASSOCIATED MALFORMATION SYNDROMES
Syndrome Location of Stenosis Reference
Rubella Peripheral pulmonary arteries e342
Williams Peripheral pulmonary arteries e308,e309
Alagille Peripheral pulmonary arteries e711
Noonan Main pulmonary artery e242
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PA with IVS is a severe form of CHD in which pulmonary blood flow depends on a patent ductus arteriosus; initial palliative therapy
therefore includes infusions of prostaglandin E2. The definitive surgical management varies according to the degree of right
ventricular hypoplasia, the presence and severity of coronary artery fistula, and the status of the tricuspid valve with options
including a variety of outflow tract (“biventricular”) repairs, univentricular (Fontan) repair, or transplantation (58) (e259,e265-e267).
Aortic Outflow Tract and Valve Malformations
Aortic Valvular Stenosis

The left ventricular outflow tract may be obstructed at any level, but the most common form of obstruction is aortic valvular stenosis.
The spectrum of abnormal valvular morphology is similar to that in pulmonary valvular stenosis, but a bicuspid valve is the most
common form (7) (e268,e269). Bicuspid aortic valves are not congenitally stenotic and many remain asymptomatic throughout
childhood, with a smaller subset progressing quickly to significant stenosis requiring intervention (e268-e271). Unicommissural and
the less common tricuspid dysplastic and dome-shaped valves are stenotic from birth and therefore more likely to be symptomatic in
early childhood (e272-e275). With congenitally stenotic valves, the left ventricle may be dilated, normal in size, or hypoplastic. In
infants with severe stenosis, endocardial fibroelastosis and subendocardial ischemic damage often further complicate the picture
(e275-e277). COTA commonly accompanies a congenitally malformed aortic valve in two clinical settings: critical aortic stenosis
(59) (e278,e276) and bicuspid valves related to Turner syndrome (e279,e280).
Clinical features vary, depending largely on the severity of the stenosis. In “critical” stenosis, newborn infants present with heart
failure and shock (e281-e283). Treatment then depends on the morphology of the left ventricle. With a normally sized ventricular
cavity, surgical or balloon valvotomy relieves the obstruction (e282-e284). Infants with left ventricular hypoplasia or other
confounding malformations require more complex surgical procedures (e285-e288). Outside the newborn period, aortic stenosis
usually presents as an asymptomatic murmur (e289). Complications of the stenosis include bacterial endocarditis, myocardial
ischemic damage, arrhythmias, and sudden death (e268,e289,e290). Initial treatment involves surgical or balloon valvotomy
(e282,e291,e292), with up to 40% of patients requiring valve replacement later in life (60) (e290,e291). Given the growth
requirement inherent in pediatric valve replacement surgery, the potential for continued somatic growth makes the pulmonary
autograft (Ross) procedure particularly attractive (e293,e294).
Subvalvular Aortic Stenosis

Discrete subvalvular aortic stenosis most frequently takes the form of a fibroelastic diaphragm just beneath the base of the aortic
valve; less common forms include a thickened diaphragm with a muscular base or a tunnel-like narrowing of the outflow tract (7)
(e295-e297). Commonly associated heart defects, present in 50% to 75% of cases, include a malaligned VSD, aortic valvular
stenosis or regurgitation, aortic coarctation, and AV septal defects (59) (e273,e296-e298). These subaortic lesions rarely present in
infancy, and current theories consider them to be acquired progressively, perhaps on the framework of an underlying subtle
deformation of the outflow tract (61) (e295,e296,e299).
Supravalvular Aortic Stenosis

Supravalvular aortic stenosis occurs in three forms (62) (e300-e302):
1. An hourglass deformity caused by a constrictive annular ridge at the superior margin of the sinuses of Valsalva (66%)
2. A discrete fibromuscular membrane with a central opening in the lumen of the aorta (12%)
3. A diffuse hypoplasia of the ascending aorta with involvement of the arch and the branches (23%)
The coronary arteries may also be involved in the process, with ostial stenosis or luminal narrowing resulting in myocardial ischemia
or even sudden death (62) (e303-e306). Supravalvar aortic stenosis occurs sporadically but may also be familial (e307) or part of
the Williams syndrome (e308,e309). The latter two disorders have both been mapped to the elastin gene on chromosome 7
(e310,e311).
Aortic Atresia
Aortic atresia, the most common defect seen in the hypoplastic left-heart syndrome (e312-e314), shows a 2:1 to 3:1 male
predominance (7, 63) (e313,e315-e317). In isolated aortic atresia, the mitral valve and the left ventricular cavity are hypoplastic,
with secondary left ventricular endocardial fibroelastosis and myocardial hypertrophy (eFigure 13-5) (e312,e313,e316). In the 30%
to 50% of cases with associated mitral atresia, the left ventricle is diminutive (Figure 13-16) visible only on serial sections or
definitively identified only on microscopic examination (63) (e313,e316,e318). VSDs, present in fewer than 10% of cases, may on
the other hand be associated with a more normally sized ventricular cavity, with or without endocardial fibroelastosis (7, 63)
(e317,e319,e320). The site of the aortic valve may be invisible, or the valve may be represented by an imperforate membrane
(e312,e313). The ascending aorta is represented by a narrow vessel functioning as a conduit to the coronary arteries, which arise
normally. In 60% to 80% of cases, a discrete COTA is present (63) (e315,e316,e321). The descending aorta may then appear to
arise from the ductus arteriosus, which is widely patent in most cases (63). Coronary artery changes and ventricle-coronary arterial
connections, similar to those complicating PA, have been described in the left coronary artery and ventricle of hearts with aortic
atresia and mitral stenosis (e320,e322,e323).
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FIGURE 13-16 ▪ Posterior view of a heart removed at autopsy with mitral atresia and a hypoplastic left ventricle. At the left an
incision opens into the large dilated right ventricle. Serial transverse sections across the thick posterior wall of the right atrium
reveal a tiny opening just beneath an atretic mitral valve that represents the residuum of the left ventricle.
Hypoplastic Left-Heart Syndrome

Hypoplastic left-heart syndrome is a clinicopathologic condition in which underdevelopment of the left side of the heart and the
ascending aorta results in obstruction to the pulmonary venous outflow and dependence on a patent ductus arteriosus for adequate
systemic blood flow (e324,e325). Without surgical intervention, severe congestive heart failure and death invariably ensue, usually
in the first 2 months of life (1) (e281,e326). The uniform right atrial and ventricular enlargement combined with the small left
ventricle, ascending aorta, and aortic arch give these hearts a characteristic external appearance (e313) (Figure 13-17). Pulmonary
venous outflow depends on a left-to-right shunt across the atrial septum, usually via a patent fossa ovalis or secundum ASD (7)
(e327). However, hypoplastic left-heart syndrome is associated with and possibly caused by premature closure of the foramen ovale
in 5% to 10% of cases; in this situation, the pulmonary venous return must to be shunted to the right side of the heart via anomalous
venous connections or intramyocardial sinusoids (38, 63) (e43,e323,e328). The closed fossa ovalis worsens the already present
pulmonary venous obstruction and predisposes an infant to significant pulmonary hypertension early in life (e328,e329). Stenotic or
atretic mitral and aortic valves, with or without COTA, are the usual malformations resulting in hypoplastic left-heart syndrome
(e312-e315,e327). Occasional reports describe instead left ventricular hypoplasia with a “contracted” form of endocardial
fibroelastosis and hypoplastic but otherwise normally formed valves (e330,e331). In recent years, staged surgical correction of the
hypoplastic left-heart syndrome (Table 13-18) and neonatal cardiac transplantation have dramatically improved the outlook for
infants with this otherwise uniformly fatal disorder (64) (e318,e332-e336).
FIGURE 13-17 ▪ External view of a heart with hypoplastic left-heart syndrome, as seen from the anterior aspect. A dilated right atrial
appendage hugs the large pulmonary artery and atrioventricular groove. A large right ventricle occupies the entire anterior
ventricular surface.
Malformations of the Aortic Arch System

Ductus Arteriosus
The ductus arteriosus differs from the aorta and pulmonary arteries in that its media is formed predominantly of smooth muscle
layers (65) (e337). Control of ductus patency during fetal life relies on many factors, including relatively low oxygen tension and high
circulating prostaglandins (65). In normal full-term infants, the rapidly rising oxygen and the falling prostaglandin levels result in
functional ductus closure within 15 hours of birth. In premature infants, the immature state of the ductal response combined with
relative hypoxia and prostaglandin excess lead to persistent ductal patency in 40% of infants weighing less than 2,000g and up to
80% of infants weighing less than 1,200 g (e338). In premature infants with an inherently normal ductal structure, the administration
of prostaglandin inhibitors such as indomethacin usually induces closure (e338).
Patent Ductus Arteriosus

A persistently patent ductus arteriosus (eFigure 13-6) beyond the first 2 or 3 weeks of life accounts for 2.5% to 8.5% of cases of
CHD with a 2:1 female predominance (Table 13-1). In these older infants, the persistence of ductal patency is
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probably caused by an inherent structural abnormality, and prostaglandin inhibitors rarely induce closure (e337,e339). Grossly, the
patent ductus is usually thin-walled with a smooth intima, in contrast to the thick-walled irregular appearance of the normally closing
ductus (7). Occasionally, the communication between the pulmonary artery and the aorta is in the form of a window at the expected
site of the ductus (7). Microscopically, the internal elastic lamina is intact rather than fragmented, and a paucity of the intimal
cushions present in the normal ductus can be seen (e339,e340).
Table 13-18 ▪ HYPOPLASTIC LEFT HEART: MULTISTAGE “NORWOOD” REPAIR
Stage 1: Initial palliative surgery at

age 1-3 weeks
Purpose Establish unobstructed systemic blood supply
Limit pulmonary blood flow and pressure to normal levels
Procedures Transect proximal main pulmonary artery
Allograft reconstruction of ascending aorta (i.e., neoaorta)
Pulmonary root-neoaorta anastomosis
Atrial septectomy
Blalock-Taussig shunt
Stage 2: Intermediate palliation at

age 4-6 months
Purpose Decrease right ventricular load
Procedure Superior vena cava-right pulmonary artery anastomosis (i.e., bidirectional

Glenn procedure)
Stage 3: Definitive repair at age 18

months-2 years
Purpose Complete separation of pulmonary and systemic venous blood
Procedure Tunnel anastomosis between inferior vena cava and right pulmonary artery
(i.e., Fontan variant procedure)
In children outside the newborn period, a patent ductus arteriosus without other structural heart defects raises the possibility of an
underlying infectious or genetic disorder. Patent ductus arteriosus is a frequent manifestation of the congenital rubella syndrome
(e341,e342). Familial recurrence has been documented approximately in 3% of cases, and abnormal neural crest development may
play a role (66) (e343,e344).
The clinical manifestations of patent ductus arteriosus relate to the size of the left-to-right shunt. Children with a small shunt are
usually asymptomatic, coming to medical attention because of the characteristic continuous murmur. With increasing shunt size,
congestive heart failure develops. Like patients with other right-to-left shunting lesions, these patients are at risk for the
development of pulmonary obstructive vascular disease (7, 65) (e337). Closure options include surgical ligation and nonsurgical
insertion via a catheter of an occluding device or coils (65) (e345,e346).
Obstructive Anomalies of the Aortic Arch

The aortic arch can be divided into three segments: proximal transverse, distal transverse, and isthmus (67) (e347) (Table 13-19).
Obstructive arch anomalies occur in any of the segments with varying frequency, depending on the type of obstruction.
Coarctation of the Aorta

COTA, defined as a discrete, shelflike area of narrowing in the descending aorta, accounts for 5% to 6.5% of congenital heart
defects (Table 13-1). In the vast majority of instances, the coarctation is located in the upper thoracic aorta opposite the ductus
arteriosus insertion site, and the previously used “preductal” and “postductal” designations have been replaced in the newer
literature with the broader “juxtaductal” designation (e348). The site of narrowing is formed by a shelf of fibroelastic tissue and
smooth muscle that is in continuity with similar tissue in the ductus (7) (e349,e350).
Associated cardiovascular anomalies, present in 50% to 60% of cases, include bicuspid aortic valve (40% to 50%), VSD (40% to
50%), and a variety of complex obstructive lesions of the left side of the heart (24%) (e351). The associated VSDs show
malalignment and abnormalities of the left outflow tract that could reduce aortic arch blood flow in utero (e352). Decreased arch flow
is postulated to play a role in the pathogenesis of the obstructive aortic arch lesions (e352-e354).
Clinical symptoms of coarctation are related to the severity of the obstruction. In infancy coarctation presents with heart failure;
these infants often have associated tubular hypoplasia (7, 68). Children less than 1 year old at diagnosis are usually asymptomatic
and present with upper extremity hypertension and decreased pulse and blood pressure. Surgical repair can be accomplished by
resection of the coarctation ridge and end-to-end anastomosis of the aorta or by means of a subclavian flap or patch aortoplasty
(e355,e356). Balloon aortoplasty and implantation of stents have also been used with some success in infants and older children,
particularly those with discrete coarctation sites (e357-e359). Restenosis, aneurysm formation, or aortic dissection can occur as a
complication in any of these procedures with the highest risk for complications in the very young infants (e349,e358,e360-e362).
Table 13-19 ▪ AORTIC ARCH SEGMENTS
Arch Segment Anatomic Location Diametera
A. Isthmus Between L subclavian and ductus ≥40%
B. Distal transverse Between L carotid and L subclavian ≥50%
C. Proximal transverse Between R innominate and L carotid ≥60%
a% of diameter compared with ascending aorta. L, left; R, right
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Table 13-20 ▪ COARCTATION OF THE AORTA: SURGICAL CLASSIFICATIONa
Type I Primary coarctation
A. With ventricular septal defect
B. With other major cardiac defects
Type II Coarctation with isthmus hypoplasia
Type III Coarctation with tubular hypoplasia of isthmus and transverse arch

aAll types with or without patent ductus arteriosus.
Tubular Hypoplasia
The term tubular hypoplasia denotes an elongated (>5 mm) and hypoplastic segment of aortic arch, usually with an associated
discrete coarctation site (7, 68) (e363). In normal infants, the diameter of the aortic arch is smaller than that of the ascending aorta
(67) (e347). The normal values used to determine whether true arch hypoplasia is present are outlined in Table 13-20. In the vast
majority of instances, tubular hypoplasia is associated with other complex heart malformations, most often left ventricular hypoplasia
or DOLV (67). Amato et al. (69) have proposed a classification system for coarctation that incorporates many of these anatomic
variables (Table 13-20).
Interruption of the Aortic Arch

Complete obstruction of the aortic arch is subdivided into two lesions. In aortic arch atresia, an imperforate membrane or cord
occludes the arch isthmus (7) (e347). Interruption of the aortic arch refers to a complete loss of aortic arch continuity, which is
subclassified according to the interruption site (70) (e364-e367) (Table 13-21). Blood flow to the lower body depends on a patent
ductus arteriosus; most hearts also contain a VSD (70) (e364,e366,e368,e369). In a smaller number (one-third to two-third),
additional anomalies accompany the interrupted arch (70) (e369,e370). DiGeorge syndrome and its associated chromosome 22q11
deletion are identified in approximately 30% of cases with 90% of the cases of deletion occurring with the type B interruption (70)
(e369-e371).
Table 13-21 ▪ INTERRUPTED AORTIC ARCH SUBCLASSIFICATION
Type Interruption Site Relative Incidence
A Isthmus 15%-30%
B Distal transverse arch 70%-85%
C Proximal transverse arch 0%-8%
Aortic Arch Branching Anomalies

The normal aortic arch, ductus arteriosus, and main pulmonary arteries develop from a sequence of six paired vessels, which then
persist or disappear (71). The most significant of the myriad of anomalies that can result will be discussed briefly here.
Left Aortic Arch with Aberrant Right Subclavian Artery

The aberrant right subclavian artery originates distal to the left subclavian artery as a fourth branch of a left aortic arch, coursing
behind the esophagus to the right arm. It occurs as an asymptomatic and usually isolated anomaly in 0.5% of the general population
(72). It also accompanies other anomalies, appearing in 0.9% of children undergoing cardiac catheterization for other heart disease
(72). During its retroesophageal course, the aberrant artery compresses the esophagus; it rarely causes symptoms, but the anomaly
is visible on barium swallow. Rarely, a right-sided ductus arteriosus attaches to the anomalous right subclavian artery to form a
vascular ring (71, 72) (e372).
Right-sided Aortic Arch

A right-sided aortic arch, defined by a rightward sweep of the aorta as it arches into the posterior mediastinum, may display mirror-
image branching or may be accompanied by a variety of additional branching anomalies (72). A right aortic arch with mirror image
branching, the most common arch anomaly, is by itself of no clinical significance but almost always accompanies other cardiac
malformations, especially TOF, which is present in 50% of cases (72) (e373). Hearts with mirror-image branching usually retain a
left-sided ductus.
An aberrant left subclavian artery originating distal to the right subclavian artery as a fourth arch branch is the most frequent right-
sided arch branching anomaly. The aberrant subclavian artery follows a retroesophageal course to enter the left side of the chest,
where it usually attaches to a leftsided ductus arteriosus or ligamentum arteriosum to form a vascular ring. The origin of the aberrant
subclavian artery from the aortic arch frequently appears dilated—hence the designation of Kommerall diverticulum (71, 72).
Additional cardiac malformations accompany the right-sided arch with aberrant left subclavian artery in less than 20% of cases
(e372,e374,e375).
Vascular Rings
Vascular rings are malformations of the aortic arch structures that encircle and compress the trachea and the esophagus, causing
respiratory symptoms and dysphagia (e374-e377). The most common vascular rings are formed by a double aortic arch, a right
aortic arch with aberrant left subclavian artery and left ductus, or an anomalous left pulmonary artery (pulmonary sling) (71) (e374-
e376,e378). The most common of these, the double aortic arch, occurs with associated cardiac
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anomalies in less than 20% of cases (e379). The anomalous left pulmonary artery (pulmonary sling) originates from the right
pulmonary artery anterior to the right main bronchus and then passes between the trachea and the esophagus to enter the hilum of
the left lung (73) (e380,e381). A variety of tracheobronchial and cardiovascular anomalies occur in at least 50% of these infants with
tracheal cartilaginous rings or tracheal stenosis being the most common (73) (e382,e383).
Malformations of the Coronary Arteries

Anomalous Origin of the Left Coronary Artery
A variety of coronary artery anomalies have been described, but the only one of clinical significance is anomalous origin of the left
coronary artery from the pulmonary trunk, a rare malformation (74) (e384). Beyond the newborn period, blood from the low-pressure
pulmonary artery inadequately perfuses the high-pressure left ventricular myocardium. The resulting myocardial ischemia manifests
clinically as congestive heart failure and pathologically as extensive subendocardial fibrosis or fibroelastosis and anterolateral wall
infarction (74, 75). The clinical course depends largely on the adequacy of the collateral flow that develops during the first weeks of
life (75) (e385,e386). Most infants become symptomatic in the 1st months of life, and 65% to 85% die in early childhood if the
anomaly is not corrected by surgery (7, 74) (e386,e387). An anomalous origin of the right coronary artery is usually inconsequential
because the low-pressure right ventricle is adequately perfused by blood from the low-pressure pulmonary artery (7, 74).
Malformations of the Venous System

Systemic Venous Anomalies
Systemic venous blood returns to the heart via five sources: superior vena cava, coronary veins, hepatic veins, inferior vena cava,
and azygos veins. With normally lateralized situs (situs solitus or situs inversus), anomalies of the systemic venous system are not
uncommon but usually of little clinical significance. With situs ambiguus, complex systemic venous malformations are the rule.
Persistent Left Superior Vena Cava

A persistent LSVC is present in 0.3% to 0.5% of the general population and up to 10% of patients with other cardiovascular
anomalies (7, 76) (e388,e389). Absence of the innominate vein serves as a clue to the presence of a persistent LSVC in
approximately 40% of cases (7) (e390). The LSCV traverses the posterior surface of the left atrium to enter the coronary sinus in
the AV sulcus. Occasionally, the coronary sinus becomes dilated to the point of compressing the posterior wall of the left atrium,
mimicking cor triatriatum (e391,e392). The wall between the coronary sinus and the left atrium becomes unroofed in approximately
8% of cases, resulting in drainage of the LSCV into the left atrium (76). This latter morphology occurs most frequently in the setting
of the heterotaxy syndromes (10).
Coronary Sinus Ostium Atresia

With atresia of the coronary sinus ostium, a rare anomaly, cardiac venous drainage relies on a persistent LSVC with a patent
innominate vein or other left-to-right connection (e393). The obstructed coronary sinus ostium by itself creates few clinical problems,
but ligation of the persistent LSVC should be avoided during heart surgery (10) (e394).
Interruption of the Inferior Vena Cava with Azygos Continuation

Anomalies of the inferior vena cava are much less common. Infrahepatic interruption of the inferior vena cava with azygos
continuation results in an absence of the inferior vena cava between the renal and hepatic veins (7, 76). The inferior vena cava
below the renal veins drains via an enlarged azygos vein, which enters the thorax through the aortic hiatus and joins the superior
vena cava just superior to its junction with the right atrium. This anomaly is usually associated with other cardiovascular
malformations and is frequently present in the polysplenia syndrome (7, 77) (e395-e397).
Pulmonary Venous Anomalies
Pulmonary venous anomalies are listed in Table 13-22.
Partial Anomalous Pulmonary Venous Connection

Anomalous pulmonary venous connection refers to a group of conditions in which the pulmonary venous drainage is routed partially
or totally to the right atrium. In the more common anomaly, partial anomalous pulmonary venous connection, blood from one or
more, but not all, of the pulmonary veins drains into a systemic vein or right atrium. This anomalous drainage is right sided in more
than 80% of cases and most frequently enters the superior vena cava or the right atrium (7) (e398-e400). More than 80% of cases
occur in the setting of sinus venosus ASDs as described earlier (e33,e34,e398). The Scimitar syndrome represents a variant of
partial anomalous pulmonary venous connection characterized by anomalous pulmonary venous drainage into the inferior vena
cava with a variety of associated cardiopulmonary anomalies. The most frequent associations include right lung hypoplasia,
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dextrocardia, systemic arterial supply to the lung, and abnormal bronchial anatomy (78) (e401,e402).
Table 13-22 ▪ PULMONARY VENOUS MALFORMATIONS
Partial anomalous pulmonary venous connection
Sinus venosus atrial septal defect
Scimitar syndrome
Total anomalous pulmonary venous connection
Supradiaphragmatic
Supracardiac
Intracardiac
Infradiaphragmatic
Pulmonary vein atresia
Cor triatriatum
Table 13-23 ▪ TOTAL ANOMALOUS PULMONARY VENOUS CONNECTION: CLASSIFICATION
Site of Connection % Total % Obstructed
Supracardiac 45% 45%
Left innominate 25%-35%
vein
Right SVC 10%-15%
Cardiac 25% 0%-20%

Coronary sinus 15%-20%
Right atrium 5%-15%
Infracardiac 25% 80%-90%
Portal vein 15%-25%
Mixed 5%-10% 35%-60%
Total Anomalous Pulmonary Venous Connection

Total anomalous pulmonary venous connection, in which all the pulmonary veins drain to the systemic circuit, is subclassified
according to the route of the abnormal venous drainage and the presence or absence of obstruction to that drainage (79) (e403-
e405) (Table 13-23). The most common route of drainage is through a vertical vein that arises from a confluence of the pulmonary
veins posterior to the left atrium, traverses superiorly along the left side of the mediastinum, and drains into the innominate vein at
its junction with the left subclavian vein (7) (e403,e404). Less frequently, the common trunk drains into the superior vena cava, right
atrium, coronary sinus, or subdiaphragmatic portal venous system (Figure 13-18). In up to 10% of cases, the pulmonary veins drain
to multiple different sites (80) (e406,e407). Drainage is obstructed in approximately 60% of cases of total anomalous pulmonary
venous connection, with the cardiac sites having the lowest risk and the infracardiac the highest risk for obstruction (80) (e408)
(Table 13-23). The venous drainage can be obstructed by intrinsically small vessels, external compression, or interposition of a
capillary bed (7) (e403,e404). The pulmonary venous obstruction leads to early and often severe pulmonary hypertensive changes,
manifested as medial hypertrophy of the pulmonary arteries and veins combined with intimal proliferation and eventually
arterialization in the pulmonary veins (e409-e411). Total anomalous pulmonary venous connection is associated with other cardiac
anomalies in approximately one-third of cases, particularly with the heterotaxy syndromes (7) (e403,e412). Occasionally, it occurs in
families; the inherited form has been linked to chromosome 4p13 (e413,e414). The clinical manifestations of total anomalous
pulmonary venous connection vary with the degree of obstruction and the resultant PVR (79) (e407,e415,e405). With significant
obstruction and high levels of resistance, cyanosis, heart failure, and death occur in the 1st months of life (79). With low resistance,
infants may be asymptomatic at birth, and right-sided heart failure is the predominant manifestation (79). Surgical correction in the
modern era yields more than 90% short-term survival with only rare late deaths, usually caused by pulmonary venous stenosis (80)
(e405,e407,e415). In large series, risk factors for death include young age at surgery, cardiac or infracardiac connection sites, and
preoperative pulmonary venous obstruction (80) (e407).
FIGURE 13-18 ▪ Total anomalous pulmonary venous connection, infradiapragmatic type seen from the posterior view. A confluence
of the pulmonary veins (*) is isolated from the left atrium and drains into a vertical vein. This vein traverses the diaphragm to enter
the portal venous system of the liver.
Pulmonary Vein Atresia/Stenosis

In pulmonary vein atresia, the entire pulmonary venous system drains into a common chamber from which there is no site for egress
(7, 79). In pulmonary vein stenosis, which is less severe, luminal narrowing occurs at the venoatrial junction of one or more of the
pulmonary veins (e416).
CorTriatriatum
In cor triatriatum, the left atrium is partitioned by a fibromuscular shelf separating the pulmonary venous compartment from the atrial
appendage and the mitral valve orifice compartment (eFigure 13-7) (7, 79). The dividing membrane contains a variably sized
opening, which results in most instances in pulmonary venous obstruction (7) (e417,e418). The foramen ovale may open into either
compartment; when the opening is proximal to the obstruction, it can function as an escape valve for the pulmonary venous
obstruction (7) (e417,e418).
Malformations of Position and Situs

Dextrocardia
Dextrocardia, in which the heart is located in the right side of the chest with a right-sided apex, occurs with situs inversus,
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situs ambiguous, and as an isolated finding (7) (e419,e420). Except when occurring with situs inversus, the incidence of associated
intracardiac and extracardiac anomalies is high (1) (e420). Dextrocardia should be distinguished from dextroposition, in which the
heart is displaced to the right side of the chest with a left-sided apex (7).
Ectopia Cordis
Ectopia cordis, a rare anomaly in which the heart is partially or totally outside the chest (Figure 13-19), is subclassified according to
the location of the defect (7, 81) (e421). Thoracic ectopia cordis, the most common type, is the result of a sternal cleft. The heart is
usually located on the anterior surface of the chest without skin or a pericardial covering (81) (e421). Thoracoabdominal
(abdominal) ectopia cordis is associated with a defect in the lower sternum, diaphragm, and abdominal wall; the heart is usually
located with the abdominal viscera in a common omphalocele sac (81) (e421-e423). Intracardiac defects occur frequently but are
not inevitable (81).
Situs Ambiguous
Situs ambiguous (heterotaxia) occurs when the usual markers of situs are disorganized or missing as a result of disruption of the
left-right axis determination early in development (77) (e424-e426). The two best-described forms of situs ambiguous are asplenia
(bilateral right sidedness) and polysplenia (bilateral left sidedness). The “sidedness” of the heart is determined by the atrial
appendage morphology (82) (e427).
FIGURE 13-19 ▪ Infant with multiple congenital anomalies including cleft lip seen at the top of the photograph and an anterior defect
in the chest and abdomen through which the heart and liver protrude.
The heterotaxic syndromes are frequently associated with complex congenital heart and venous malformations and a variety of
extracardiac defects (77) (e412,e424,e428,e429). Heterotaxic syndromes occasionally complicate maternal diabetes (e430), and
the familial recurrence suggests a genetic factor (e431). Recent molecular studies have identified a variety of genes involved in left-
right patterning during development (83) (e432). The heterotaxy syndromes are likely multifactorial in origin.
Juxtaposition of Atrial Appendages
Juxtaposition of the atrial appendages, diagnosed when both atrial appendages reside partially or completely on the same side of
the great vessels, is a harbinger of underlying heart malformations (e433). Left-sided juxtaposition accounts for 86% of cases, with
tricuspid atresia and transposition of the great vessels the most common associated malformations (e433). On the flip side, 11% of
hearts with tricuspid atresia and 3% of hearts with D-transposition exhibit left-sided juxtaposition of the atrial appendages (32)
(e434).
HEREDITARY AND NONHEREDITARY FUNCTIONAL CARDIOVASCULAR DISEASES

Myocardial Disease
Cardiomyopathies
The designation cardiomyopathy (CMP) encompasses a heterogeneous group of diseases with dysfunction of the myocardium,
unaccompanied by structural malformations, as the defining pathophysiologic abnormality. Their classification, once largely
descriptive, continues to evolve with the recent explosion in understanding of the underlying molecular aspects of these diseases
and the ability to identify associated genetic mutations.
In 1980, the World Health Organization (WHO) presented a consensus definition of CMP as “heart muscle disease of unknown
etiology,” which was then divided into four subcategories (e435) (Table 13-24). Myocardial dysfunction of known etiology was
considered a “specific disease of heart muscle” rather than a CMP (e435). In 1995, with improved understanding of disease
pathogenesis, a revised WHO classification redefined CMP as “diseases of the myocardium associated with cardiac dysfunction”
(e436). These diseases were then classified into five categories based largely on the dominant pathophysiologic abnormality.
Myocardial diseases associated with specific cardiac or systemic disorders were reclassified as specific cardiomyopathies (Table
13-24). Over the ensuing 10 years, the explosion in molecular biology and genetic techniques resulted in dramatic advances in the
knowledge of disease pathogenesis and ability to make more specific diagnoses. With this advance in understanding, the prior
classification system has become increasingly incomplete and unwieldy. In response to this, the American Heart Association
recently proposed a new classification scheme as outlined in Table 13-24 (84).
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Table 13-24 ▪ CARDIOMYOPATHY: CLASSIFICATION
WHO 1980 (e435) WHO 1995 (e436) AHA 1006 (84)
Cardiomyopathy Cardiomyopathy Primary cardiomyopathy
Dilated Dilated Genetic
Hypertrophic Hypertrophic Hypertrophic
Restrictive Restrictive Arrhythmogenic RVC/D
Unclassified Arrhythmogenic RV LV noncompaction
Endocardial Unclassified Conduction system disease

fibroelastosis
Histiocytoid Fibroelastosis Ion channelopathies
Fiedler myocarditis Non compacted Mixed (genetic and nongenetic)

myocardium
Specific heart muscle disease Mildly DCMP Dilated

Infective Mitochondrial CMP Primary restrictive
nonhypertrophied
Metabolic Specific cardiomyopathy Acquired
General system disease Ischemic CMP Myocarditis (inflammatory

CMP)
Heredo-familial Valvular CMP Stress (Taku-Tsubo) CMP
Sensitivity and toxic Hypertensive CMP Others

reaction
Inflammatory CMP Secondary cardiomyopathy
Metabolic CMP Infiltrative Storage
Others Endomyocardial Toxicity
Endocrine Cardiofacial
Inflammatory
(granulomatous)
Neuromuscular/neurological
Nutritional deficiencies
Autoimmune/collagen
Electrolyte imbalance
Consequence of cancer
therapy
CMP occurs rarely in children, with 0.74 to 1.24 cases per 100,000 children in a year (e437-e439). In the pediatric population,
dilated CMP (DCMP) accounts for 50% to 60% of cases and hypertrophic CMP (HCMP) another 25% to 40% (e437-e441). The
clinical approach to a child presenting with CMP has been nicely summarized by Schwartz et al. (85).
Primary Cardiomyopathies
Hypertrophic Cardiomyopathy
HCMP is characterized by left ventricular hypertrophy, either symmetric or asymmetric, with a small ventricular cavity in a
structurally normal heart (Figure 13-20). Idiopathic hypertrophic subaortic stenosis, hypertrophic obstructive CMP, and muscular
subaortic stenosis are among the more than 50 synonyms used in the past (86).
At explant or autopsy, the heart is massively enlarged, weighing as much as two to three times the normal weight. The thickening of
the left ventricular free wall and the interventricular septum may be either symmetric (concentric) or asymmetric. With asymmetric
hypertrophy, which accounts for approximately two-thirds of cases, the thickness of the interventricular septum at its base measures
greater than 1.3 times the thickness at the posterior left ventricular free wall (S/P ratio) (87) This asymmetric hypertrophy is often
accompanied by an enlarged elongated mitral valve (86, 87). The resulting abnormal mitral valve movement contributes to left
ventricular outflow tract obstruction. This physiologic state may be marked by a fibrous imprint of the mitral valve septal leaflet on
the apposing septal endocardium (87).
At the microscopic level, HCMP manifests a triad of features: myocyte hypertrophy, interstitial fibrosis, and myofiber disarray defined
by whorled and intertwined clusters of myocytes surrounding a central fibrotic core (86, 87). At the ultrastructural level, the
myofilaments also display “disarray” (e442). Myofiber disarray is unfortunately not pathognomonic of HCMP, but can occur in
secondary hypertrophy, or even normal hearts, and the finding of “extensive” (i.e., >10% and usually ≥30% of septum) disarray is
therefore
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needed to make a diagnosis (87) (e443). The intramyocardial arteries in HCMP often display dysplastic changes similar to those
seen in fibromuscular dysplasia (86, 87). This small vessel disease may contribute to the myocardial ischemia, interstitial fibrosis,
and development of a dilated phase late in the course of the disease (e443).
FIGURE 13-20 ▪ Coronal section through an explanted heart with hypertrophic cardiomyopathy as viewed from behind. A catheter
marks the right atrium and right ventricle. Two cusps of the aortic valve are visible above the markedly thick walled left ventricle.
The hypertrophic interventricular septum narrows and distorts the left ventricular outflow tract.
It should be noted that the S/P ratio of greater than 1.3 is not an appropriate criteria in stillborn or newborn infants. In the developing
heart, the ventricular septum is disproportionately thick and an S/P ratio greater than 1.3 occurs in greater than 90% of embryos
and young fetuses, in 65% of older fetuses, and in 25% of normal-term newborns (e444).
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Table 13-25 ▪ HYPERTROPHIC CARDIOMYOPATHY
Autosomal dominant inheritance
Familial HCMP Gene
Defect in cardiac myosin-binding protein C MYBPC3 11p11.2
Defect in cardiac β-myosin heavy chain MYH7 14q12
Defect in cardiac troponin T TNNT2 1q32
Defect in cardiac troponin 1 TNNI3 19q13.4
Defect in myosin light chain 2 MYL2 12q23-

q24.3
Defect in myosin light chain 3 MYL3 3p
Defects in α-tropomyosin TPM1 15q22.1
Defect in cardiac α-actin ACTC1 15q14
Defect in titin TTN 2q31
HCMP with Wolff-Parkinson-White syndrome PRKAG 7q36
Noncompaction of the left ventriclea
Syndromic disorders
Noonan syndrome
Friedreich ataxia
Myotonic dystrophy
Cardiofaciocutaneous syndrome
Leopard syndrome/lentiginosis/multiple lentigines
Neurofibromatosis
Beckwith-Wiedemann syndrome
Telecanthus, multiple congenital anomalies
Deaf mutism
Rubinstein-Taybi syndrome
Autosomal recessive inheritance
Total lipodystrophy, insulin resistance, leprechaunism
Costello syndrome
Sporadic
Infant of diabetic mothera
Infiltrative (storage) disorders
Disorders of glycogen metabolism
Glycogen storage disease type II (Pompe disease: acid maltase deficiency)

Glycogen storage disease type IIb (Danon disease: lysosome-associated membrane
protein-2)
Glycogen storage disease type III (Cori disease: debranching enzyme)
Glycogen storage disease type IX (cardiac phosphorylase kinase deficiency)
Disorders of mucopolysaccharide degradation
Mucopolysaccharidosis type I (Hurler syndrome)a
Mucopolysaccharidosis type II (Hunter syndrome)
Mucopolysaccharidosis type III (Sanfilippo syndrome)
Mucopolysaccharidosis type IV (Morquio syndrome)
Mucopolysaccharidosis type VII (Sly syndrome)
Disorder of glycosphingolipid degradation (Fabry disease)
Disorder of glycosylceramide degradation (Gaucher disease)
Disorder of N-glycosylation
Disorder of phytanic acid oxidation (Refsum disease)a
Disorders of combined ganglioside/mucopolysaccharide and oligosaccharide degradation
GM1 gangliosidosisa
GM2 gangliosidosis (Sandhoff disease)a
Diminished energy production (mitochondrial disorders)
Disorders of pyruvate metabolism
Pyruvate dehydrogenase complex deficiency (Leigh disease)
Disorders of oxidative phosphorylation
Complex I deficiency
Complex III deficiency (histiocytoid CM)
Complex IV deficiency (muscle and Leigh disease forms)
Complex V deficiency
Mitochondrial transfer RNA mutation
MERRF syndromea
MELAS syndrome
Mitochondrial DNA deletions and duplications
Kearns-Sayre syndrome
Barth syndrome (3-methylglucurconic aciduria type II)a
Senger syndrome
Disorders of fatty acid metabolism
Primary carnitine deficiencya
Very-long-chain acyl-CoA dehydrogenase deficiency
Long-chain acyl-CoA dehydrogenase deficiency
Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiencya
Multiple acyl-CoA dehydrogenase deficiency (glutaric acidemia type II)
Toxic intermediary metabolite
Disorders of amino acid or organic acid metabolism
Tyrosinemia
aCauses both HCMP and DCMP.
DCMP, dilated cardiomyopathy; HCMP, hypertrophic cardiomyopathy; AR, autosomal recessive; CM, cardiomyopathy.
Modified from Schwartz ML, Cox GF, Lin AE, et al. Clinical approach to genetic cardiomyopathy in children. Circulation
1996;94:2021-2038.
See also Callis TE, Jensen BC, Weck KE, et al. Expert Rev Mol Diagn 2010;10:329-351.
Primary HCMP represents a common autosomal dominant disorder with an estimated incidence of 1:500 in the general population
(86). Causative mutations in at least ten different genes encoding sarcomere proteins have been identified in families with HCMP,
with mutations in the myosin-binding protein C or b-myosin heavy chains accounting for 80% of cases (86) (e445). Mutations can
also be identified in up to 60% of adults with sporadic HCMP (e445). In children under 10 years of age, underlying metabolic or
syndromatic causes, including Noonan syndrome in particular, account for 20% to 35% of cases of HCMP (88) (e446). The
spectrum of diseases that can present with HCMP is broad, as outlined in Table 13-25.
In primary HCMP, the symptoms of hypertrophy most commonly develop only after adolescent growth has been completed (86).
However, up to one-third of cases can present in infancy (e447). In infants, the hypertrophy tends to cause restriction of right
ventricular outflow in addition to obstruction of left ventricular outflow (86) (e446). When this occurs, HCMP may masquerade
clinically as pulmonary valvular stenosis, congenital mitral insufficiency, VSD, endocardial fibroelastosis, or myocarditis. Sudden
death may occur in 1% to 2% of affected children, whether they are symptomatic or not (86) (e447).
Arrhythmogenic Right Ventricular Dysplasia

Arrhythmogenic right ventricular dysplasia (ARVD) is characterized by partial or massive transmural fibrofatty replacement of the
right ventricular myocardium with associated ventricular arrhythmias (89) (e448).
Hearts removed at transplant or autopsy are large with the right ventricle appearing yellow or white. The fatty replacement occurs
initially in the anterior free wall of the right ventricle adjacent to the septum, with progressive involvement extending to the lateral
ventricular wall (89) (e448) (Figure 13-21). In the most severe cases, the entire right ventricle may be involved. In areas of thinning,
the ventricle wall may display focal aneurysm formation (89) (e448). At microscopic examination, fat mixed with variable amounts of
fibrous tissue and inflammatory cells replaces the normal myocardium (89) (e448). The extension of fat and fibrosis into the
conduction pathways correlates the histopathology with the clinical course (89). The left ventricle may be thickened in 15% of cases
and shows histologic features of patchy fibrosis with or without subepicardial fat infiltration in up to 50% of cases (89) (e448).
Distinguishing the normal fatty infiltration of the right ventricle, which occurs with increasing age and obesity, from ARVD can be
difficult. Suggested criteria include the association of fat with disorganized myocardium and presence of fibrosis with the fat (90)
(e449).
ARVD presents as a familial disease in 50% of cases with both autosomal dominant and autosomal recessive (Naxos
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disease) inheritance patterns (e450). Search for a gene has yielded a variety of mutations related to desmosomal junction proteins
and calcium receptors without a well-defined unifying disease mechanism (91) (e450). Apoptosis in the condition suggests that the
genes involved may play a role in programmed cell death and that apoptosis may precede the infiltration (e451).
FIGURE 13-21 ▪ A close-up view of the right ventricular wall cut surface showing near complete replacement of the normally deep
red myocardium by pale yellow fibrofatty tissue.
ARVD clinically presents with an arrhythmia or sudden death in young adults, especially males (M:F, 2.7:1) and has been reported
in children as young as 5 years (89, 90). The overall disease prevalence is estimated at 1:5,000 with certain regions (e.g., Greek
Island of Naxos) having an increased prevalence (90). ARVD accounts for up to 5% of sudden unexpected deaths in young adults.
Patients with known disease experience an annual mortality rate of approximately 2% due to arrhythmia or right-heart failure (90).
Therefore, treatment often requires aggressive measures such as radiofrequency ablation, implantable defibrillators, or
transplantation (90).
Noncompaction of the Ventricular Myocardium

Noncompaction of the ventricular myocardium (NCVM), also called persistence of spongy myocardium, refers to a luminal
meshwork of interlacing endomyocardial trabeculae intersected by irregular endocardial-lined sinusoids that communicate with the
ventricular lumen (92) (e452,e453). A similar pattern of spongy myocardium occurs in the very early stages of heart embryogenesis
(92) (e454,e455). During normal development, as the coronary arteries and veins develop, the sinusoids involute and the
surrounding myocardium becomes compacted, proceeding from the epicardium to endocardium and base to apex
(e452,e454,e455). The earliest description of noncompaction included hearts both with and without associated complex
malformations (e456). The noncompacted sinusoids in hearts with severe malformations, PA with IVS in particular, connect with the
subepicardial coronary arteries, whereas the sinusoids in hearts with isolated noncompaction communicate with the ventricular
lumen (92). Isolated noncompaction, recognized with increasing frequency in recent years, occurs at all ages (92) (e452,e453). In
recent studies, it accounts for up to 10% of heart lesions presenting to cardiology clinics (92).
The clinical diagnosis of NCVM hinges on echocardiographic findings with the diagnostic criteria varying between institutions (e457-
e460). The echocardiagraphic features of dilated, restrictive, or HCMP may accompany the noncompaction (e460,e461). These
echocardiographic findings correlate with the pathologic changes described in hearts examined at autopsy or following transplant
(92, 93). The left ventricular cavity contains poorly defined papillary muscles as an initial clue to the diagnosis. The excess
trabeculation may or may not be grossly visible on the luminal surface. Full thickness sections from the ventricular apex and/or free
wall will, however, yield the histologic picture of deep invaginations of endocardial-lined spaces with variable degrees of associated
fibroelastosois. The normal luminal trabeculae are of variable thickness and a definitive pathologic diagnosis of noncompaction
requires that the trabeculae and intervening sinusoids account for at least 50% of the myocardial wall thickness (Figure 13-22) (93).
Although left ventricular involvement represents the hallmark of this disease, the right ventricle is also involved in approximately
40% of cases (e460,e462).
FIGURE 13-22 ▪ Coronal section through an explanted heart with noncompaction as viewed from the front. Both the right and left
ventricles of this globular heart appear thick walled and dilated. The endocardium appears whitened due to endocardial
fibroelastosis, particularly in the left ventricle. At the apex of the left ventricle, only the external 25% of the wall has the appearance
of normal deep red compact myocardium. The fine trabeculations characteristic of noncompaction occupy the majority of the wall.
Clinically, NCVM manifests as arrhythmias and congestive heart failure. In adults, thrombi within the sinusoids often lead to systemic
emboli; this complication occurs less frequently in children (e460,e463,e464). Although in adults the disorder is reported more
commonly in males, in the pediatric population the M:F ratio is near equal (e452,e458, e460,e462,e463,e465). A variety of
extracardiac manifestations have been described, with neuromuscular disorders being the most frequent (92) (e466). Genetic
studies of family cohorts have yielded several associated gene mutations, the most frequent being the tafazzin gene on
chromosome Xp28, which is also associated with Barth syndrome (Table 13-26).
The underlying pathogenesis of NCVM remains unclear. Abnormal embryonic development currently represents the most popular
theory (e454). However, this theory does not explain the full clinical spectrum of disease, and the noncompaction phenotype likely
represents a final common pathway for a variety of etiologic factors.
Table 13-26 ▪ LEFT VENTRICULAR NONCOMPACTION GENETIC MUTATIONS

Gene Chromosome Reference
Tafazzin Xp28 (e712,e713)
a-dystrobrevin 18q12.1-q12.2 (e712)
CSX 5q del (e714)
CypherZasp 10q22.2-q23.3 (e715)
Lamin A/C 1q12.1-q23 and 10 (e716)
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Dilated Cardiomyopathy
DCMP represents a spectrum of disorders in which a dilated, poorly contracting, failing heart exhibits systolic and diastolic
dysfunction. DCMP represents the common endpoint for multiple underlying conditions (Table 13-27).
Table 13-27 ▪ DILATED CARDIOMYOPATHY
Nongenetic conditions
Infectious or postinfectious condition
Enteroviruses
Mumps
Corynebacterium diphtheroides
Endocrine/vitamin/mineral disorders
Thyrotoxicosis
Hypothyroidism
Vitamin E and selenium deficiency
Infants of diabetic mothersa
Cellular toxicity
Anthracycline toxicity
Hemochromatosis
Alcohol
Cyclophosphamide
Genetic/familial conditions
Infiltrative (storage) disorders
Glycogen storage disease type IV (Andersen disease: branching enzyme deficiency)
Mucopolysaccharidosis type I (Hurler syndrome)a
Mucopolysaccharidosis type VI (Maroteaux-Lamy syndrome)
Disorders of oxidative phosphorylation
Complex I deficiency
Mitochondrial transfer RNA mutations MERRF syndromea
Mitochondrial DNA deletions and duplications Barth syndrome (3-methylglucuronic aciduria type II)a
Disorders of fatty acid metabolism
Primary or systemic carnitine uptake deficiencya
Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiencya
Toxic intermediary metabolite disorders
Proprionic acidemia
Ketothiolase deficiency
Familial and neuromuscular conditions
Familial DCMP
Familial DCMP with conduction defects
Isolated ventricular noncompactiona
Muscular dystrophies
Duchenne and Becker muscular dystrophy
Emery-Dreifuss muscular dystrophy
Myotonic dystrophya
Limb-girdle muscular dystrophy
Congenital muscular dystrophy
Congenital myopathies
Centronuclear (myotubular) myopathy

Nemaline rod myopathya
Minicore-multicore myopathy
Friedreich ataxiaa
Refsum diseasea
aCause both DCMP and HCMP
DCMP, dilated cardiomyopathy; HCMP, hypertrophic cardiomyopathy; AR, autosomal recessive; AD, autosomal dominant.
Modified from Schwartz ML, Cox GF, Lin AE, et al. Clinical approach to genetic cardiomyopathy in children. Circulation
1996;94:2021-2038.
FIGURE 13-23 ▪ Coronal section through an explanted heart with dilated cardiomyopathy. Both ventricles appear dilated with
minimally thickened myocardium.
The gross appearance of a heart with DCMP is the same regardless of the cause (94). The key feature is biventricular dilation
(Figure 13-23), and often all four cardiac chambers are dilated. The enlarged heart may weigh 25% to 50% more than normal and
has a globular appearance. The dilated flabby, pale left ventricular wall is of normal thickness or appears thinned despite
hypertrophy of the myofibers. Stasis in the large enddiastolic atrial and ventricular cavities results in the formation of mural thrombi.
Interstitial myocardial fibrosis is the histologic feature common to all cases of DCMP, whatever the cause (94). Because these
histologic features are generally nonspecific, the diagnosis of DCMP based on biopsy material is difficult.
In childhood, an underlying etiology can be identified in 33% to 60% of cases of DCMP with lymphocytic myocarditis accounting for
15% to 45% (95) (e441,e467,e468). Biopsy early in the course of disease leads to a higher number of myocarditis diagnoses
(e467). Under the new classification scheme, these cases would be termed inflammatory CMP and are discussed further later.
Familial DCMP accounts for 10% to 45% of cases depending on the study methods used (95) (e467,e469,e470). Familial forms of
DCMP cover a broad spectrum of disease processes (Table 13-27). The most common familial diseases are neuromuscular, with
the majority having a known underlying muscular dystrophy. In a small subgroup of patients, however, the CMP represents the
presenting feature of the underlying neuromuscular disorder (e471,e472). The identification of clinical features such as weakness,
elevated creatine kinase, lactic acidosis, ptosis, granulocytopenia, and conduction abnormalities can help focus the search for the
underlying genetic defect (85) (e473).
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In a large combined prospective and retrospective review of DCMP in childhood, the majority of children (>70%) presented in
congestive heart failure with the median age at diagnosis of 1.5 years; 42% were under 1 year. There was an M:F ratio of 3:2 and a
2 to 3× increased incidence in the black versus white populations (95). Fifty percent of children in this study died or required heart
transplant.
Restrictive Cardiomyopathy
Restrictive CMP (RCMP) represents a heart in which the ventricular diastolic volume is decreased with near-normal systolic function
and wall thickness. This wall “stiffness” results from infiltrative or fibrotic disorders that may be primary in the heart or secondary to
a systemic disorder. RCMP occurs rarely in childhood accounting for less than 5% of all cardiomyopathies (e438-e440), with the
majority of cases being familial isolated CMP (e441).
Hearts from patients with the echocardiographic features of RCMP include three pathologic forms (96) (e474). The “pure” restrictive
form manifests a normal weight with small ventricular size and no hypertrophy; the hypertensive restrictive form manifests increased
weight, with free wall and septal hypertrophy; the dilated restrictive form manifests increased weight without hypertrophy and with
mild ventricular dilatation. Microscopic examination similarly displays overlapping features including fibrosis, hypertrophy, and even
myofiber disarray (96). With the restricted ventricular filling, atrial dilatation is often striking (97) (e475,e476). The increased left
ventricular filling pressure leads to pulmonary hypertension and associated right ventricular hypertrophy.
In children, RCMP most often occurs as a primary myocardial disease rather than secondary to infiltrative processes (97) (e477).
Although the majority of primary and familial RCMP are idiopathic, some have now been linked to some of the same genetic
mutations found in HCMP (e478). CMP associated with underlying genetic disorders, such as Noonan syndrome, can also present
as RCMP rather than HCMP.
Although children with RCMP can present at any age, in most series the mean age is under 5 years (97) (e475,e476). Symptoms at
presentation often reflect the increased PVR. The long-term prognosis in these children is poor, with up to 60% dying within 5 years
of diagnosis (97) (e476). Cardiac transplantation early in the course of disease offers the best opportunity for long-term survival (97)
(e475,e479).
Endocardial Fibroelastosis
Endocardial fibroelastosis (EFE) is a focal or a diffuse proliferation of fibroelastic tissue beneath the endocardium of any chamber of
the heart, but predominantly the left ventricle. EFE occurs in both structurally normal and structurally malformed hearts. In the past,
EFE in a structurally normal heart was considered a form of primary CMP. In recent years, with the overall improved understanding
of the cardiomyopathies, EFE is no longer considered a primary form of CMP and has instead been relegated to the status
“associated finding” in a wide variety of cardiomyopathic processes. In infants with primary EFE, mumps and/or adenovirus have
been identified in 90% of cases by PCR, suggesting in utero viral infection as an etiology for this disorder (e480).
FIGURE 13-24 ▪ Posterior view of an infant heart with windows opened into the left atrium and the left ventricle. The left ventricle
endocardium appears white due to the diffuse endocardial fibroelastosis.
EFE gives the normally thin translucent endocardium a white opaque appearance (Figure 13-24). Microscopic examination reveals
subendocardial layers of dense collagen and elastic fibers that extend into all the crevices of the chamber walls and even into the
myocardium to surround vessels and groups of myocytes. Focal dystrophic calcification and necrosis may also occur. The elastic
fibers in EFE often appear larger, more darkly staining, and more uniformly oriented than those found in the subendocardial fibrosis
that follows ischemic heart disease (e481).
Myocarditis (Inflammatory Cardiomyopathy)

Although the term myocarditis denotes inflammation of the myocardium, experienced physicians and pathologists recognize that the
clinical and morphologic diagnosis of myocarditis can be exceedingly difficult. The current widely accepted criteria for a morphologic
diagnosis of myocarditis requires the presence of an inflammatory infiltrate directly associated with myocyte damage that occurs in
the absence of ischemic changes associated with vascular disease (Figure 13-25) (98) (e482).
The macroscopic appearance of the heart, clinically or at autopsy, depends on the age of the patient, causative agent, time course
of the infection, and associated complications. In acute fulminant myocarditis, the myocardium is often flabby; it may have a gray
and glassy cast and be studded with scattered hemorrhagic foci. Although their overall shape may not be altered, the ventricular
walls are usually much softer than expected (99) (e483). With more longstanding disease,
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multifocal fibrosis of the ventricular wall and septum and endocardial thickening are common.
FIGURE 13-25 ▪ Acute myocarditis. Mononuclear cells infiltrate frayed and damaged cardiac myocytes. (Hematoxylin and eosin
stain, original magnification 400×.)
Microscopic features include inflammation and myocyte damage. Myocyte damage, best seen in longitudinal section, consists of
necrosis and myocyte debris; degenerative changes and altered staining characteristics (especially with Masson trichrome);
vacuolization, which causes a ragged, frayed appearance of the margins of the myocytes and cellular disruption with infiltration of
inflammatory cells. The nature of inflammatory infiltrates varies with the time course and underlying etiology; infiltrates may be
diffuse or focal and may include neutrophils, lymphocytes, macrophages, plasma cells, eosinophils, and/or giant cells (GCs) (98)
(e482). The histologic appearance of the inflammatory infiltrate, coupled with the type and extent of the myocyte damage, may offer
clues to the cause of myocarditis (99).
Endomyocardial biopsy currently serves as the major tool for diagnosing myocarditis based on the Dallas criteria (Table 13-28).
These criteria are however fraught with problems of sampling and interobserver variability (e484,e485). In an attempt to address the
biopsy interpretation difficulties, among other things, a new set of diagnostic criteria have recently been advanced (Table 13-28)
(e486,e487).
Myocarditis presents clinically in one of three patterns: sudden unexpected death, acute heart failure, and more insidious heart
disease that can mimic DCMP. In one large autopsy series, myocarditis accounted for 7% of sudden deaths (e488). Luckily,
myocarditis presents more commonly as acute heart failure, manifesting as a wide spectrum of clinical symptoms. Diagnosis relies
on EKG and echocardiographi c features, with identification of the underlying organisms usually requiring serologic studies (e489).
The incidence of acute myocarditis is best estimated from a large prospective study of myocarditis in Finnish military conscripts with
a mean age of 20 years that yielded an incidence of 0.17/1,000 person-years (e490). With aggressive clinical support, the death
rate in this group is less than 10% and the vast majority recover normal heart function (e489,e491). A young age of onset renders
the best long-term prognosis. The one exception to this overall good outlook is idiopathic GC myocarditis, discussed later.
Table 13-28 ▪ MYOCARDITIS
Dallas Criteria
Myocarditis—requires both inflammation and myocyte damage
Borderline myocarditis = Inflammation without myocyte damage

Inflammation = Lymphocytic ± neutrophils ± giant cells ± eosinophils
Myocyte damage = Frank fiber necrosis and/or intracellular lymphocytes and/or fiber vacuolization or disruption
First biopsy
Myocarditis with/without fibrosis
Borderline myocarditis
No myocarditis
Subsequent biopsy (requires myocarditis dx on first biopsy)
Ongoing (persistent) myocarditis with/without fibrosis
Resolving (healing) myocarditis with/without fibrosis Inflammation still present; no myofiber necrosis; reparative
changes present
Resolved (healed) myocarditis with/without fibrosis No inflammation in myocardium (may be in center of scar)
German Criteria
Myocarditis = ≥ 14 leukocytes/mm2 or clusters of ≥ 3 T-cells in myocardium; leukocytes quantitated using IHC
First biopsy
Acute (active) myocarditis—≥14 leukocytes/mm2 + myocyte damage ± fibrosis
Chronic myocarditis—≥ 14 leukocytes/mm2 without myocyte damage ± fibrosis
Subsequent biopsy
Ongoing (persistent) myocarditis—may be acute or chronic
Resolving (healing) myocarditis—acute or chronic but “sparser” than first biopsy
Resolved (healed) myocarditis—no inflammation in myocardium
Myocarditis has been linked to most human pathogens and also to a variety of noninfectious conditions (88, 100) (Table 13-29).
Bacterial myocarditis occurs rarely, usually as a complication of septicemia. Streptococci, staphylococci, and Neisseria result in
suppurative myocarditis (7); in rickettsial infections organisms directly invade the endothelium of myocardial vessels (e492).
Bacterial exotoxins have been implicated as the causative mechanism in diphtheria-( Corynebacterium diphtheriae) related
myocarditis (7, 100). The suggestion that a bacterial infection could elicit myocarditis through antigenic mimicry has received
support from studies of Chlamydia spp. infections and heart disease (e493).
Protozoal myocardial infections, rare in North America and Europe, lead to significant diseases in many parts of the world. Chagas
disease (Trypanosoma cruzi ), an endemic infection in South and Central America, represents the most common form of myocarditis
worldwide (88). Acute disseminated infection occurs predominantly in children following a focal lesion. Chronic Chagas disease is a
leading
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cause of cardiac failure and sudden death in endemic areas (7). Toxoplasmosis ( Toxoplasma gondii ) is widespread and may be
acquired or occur in utero (e494), but isolated myocardial disease is uncommon. Necrotizing inflammation with edema, lymphocytes,
histiocytes, and plasma cells is typical (e495). Occasionally, one finds pseudocysts or sporozoites in the site. Toxocara canis
causes severe granulomatous inflammation with an occasionally intense eosinophilic infiltrate (100) (e496). Trichinella spiralis
infection may lead to cardiac failure with a focal or a diffuse infiltration by lymphocytes and eosinophils; the parasites are however
rarely found in the sites of myocardial injury, having been either destroyed or passed directly into the circulation (7). Echinococcal
heart disease is rare in North America but is common in countries with large sheep-grazing programs.
Table 13-29 ▪ AGENTS AND CONDITIONS ASSOCIATED WITH MYOCARDITIS
I. Infections
A. Viruses
Enterovirus
Coxsackie A Coxsackie B Echovirus Poliovirus
Adenovirus
Herpes virus
Cytomegalovirus Herpes simplex Varicella Ebstein-Barr

virus
Influenza A or B
Paramyxovirus
Respiratory syncytial virus Measles Mumps
Parvovirus
Human immunodeficiency Hepatitis B Hepatitis C

virus
Rubella Dengue virus Yellow fever virus
B. Bacteria
Gram positive
Streptococcus Staphylococcus Corynebacterium

diphtheriae
Clostridium
Gram negative
Meningococcus Brucella Neisseria Salmonella
Hemophilus influenza Serratia marcescens

Acid-fast
Myocobacteria tuberculosis
Spirochetes
Leptospira Treponema pallidum Borrelia
Rickettsia
Rickettsia rickettsii Coxiella burnetii Rickettsia prowazekii
Other
Chlamydia Mycoplasma
pneumoniae
Actinomycetes Nocardia
C. Fungi
Candida Histoplasma Aspergillus Coccidioides
Cryptococcus Blastomyces Mucormycoses
D. Parasites
Trypanosoma cruzi Toxoplasma gondii Amoebiasis
Toxocara canis Schistosoma Visceral larva migrans
Trichinella Echinococcus Cystocercosis
II. Noninfectious
A. Connective tissue disease
Rheumatic heart diseases Systemic lupus

erythematosus
Rheumatoid heart disease Mixed connective tissue
Ulcerative colitis
Scleroderma
B. Drugs and toxins
Anthracyclines Acetazolamide Antibiotics
Cyclophosphamide Amphotericin B Indomethacin
Phenytoin Heavy metals Cocaine

C. Other
Giant cell myocarditis Kawasaki disease
Sarcoidosis Thyrotoxicosis
Viral infections account for most cases of infectious myocarditis, with a wide number of agents implicated (Table 13-29).
Coxsackievirus B is the most commonly recognized cause in infants and children (e497). Although polymorphonuclear leukocytes
may predominate initially, lymphocytes, plasma cells, and eosinophils soon replace them, followed by fibroblasts attempting repair
(7) (e483).
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The diagnosis of viral myocarditis can be established by (a) isolation and identification through cytopathic effects in culture, (b)
identification of pathognomonic tissue changes with light or electron microscopy, (c) tissue identification of specific viral antigens
with monoclonal antibodies, (d) recognition of a fourfold rise in specific antibodies in acute and convalescent serum samples, and
(e) specific identification with molecular methods (100). Most acute cases are identified by serologic study. In recent years,
molecular methods, in particular PCR, have become widely used to test for viral genome in inflamed myocardial tissue (e498,e499).
Using these techniques, viral genome can be detected in 23% to 46% of cases(e499,e500).
The role viral infection plays in biopsy-proven chronic myocarditis remains unclear. Using PCR, viral nuclei acid can be detected in
biopsy material from 10% to 60% of patients presenting with DCMP (e501). In infants with primary EFE, mumps virus and
adenovirus have been identified by PCR, suggesting in utero viral infection as an etiology for this disorder (e480). Early studies
suggested that treatment with steroids and other immunoregulatory drugs improved the clinical outcome; more recent studies call
this into question (e502).
Idiopathic GC myocarditis represents a distinct clinical entity with a rapidly progressive course leading to death or cardiac
transplant in 89% of patients (101) (e503). The pathologic features include three phases of disease, which may all be present
simultaneously within the same heart (102). The acute phase includes extensive zones of myocardial necrosis with an associated
mixed inflammatory infiltrate including CD8 T-lymphocytes, eosinophils, and macrophages including multinucleated GCs. Despite
the GCs, granulomas are not seen, distinguishing this disorder from infectious and sarcoid-related GC disease. In the healing
phase, granulation tissue containing inflammatory GCs mixed with myocardial GCs replaces the necrotic regions. Healed areas
contain fibrous scar tissue without GCs. Although predominantly an adult disease, GC myocarditis does occur in the pediatric age
range, predominantly the second decade. A variety of features have led to the speculation that GC myocarditis represents an
autoimmune disorder: (a) About 20% of patients have an underlying autoimmune disease, especially inflammatory bowel disease
(101) (e504). (b) GC myocarditis occurred in a child with common variable immunodeficiency, a disorder prone to the development
of autoimmune disorders (e505); and (c) recurrent disease occurs in approximately 25% of transplanted hearts (101).
Secondary Cardiomyopathies
Glycogen Storage Disorders

The glycogen storage diseases (GSD) are predominantly autosomal recessive conditions characterized by a deficiency in one of
the enzymes involved in the synthesis or degradation of glycogen. Significant cardiac involvement occurs in GSD types IIa (Pompe
disease) and IIb (Danon disease).
Pompe disease (type IIa GSD) results from a deficiency in α-1,4-glucosidase (acid maltase) causing accumulation of lysosomal-
bound glycogen in the heart and skeletal muscle. The disease manifests as infantile and late forms, depending on the severity of
the enzyme deficiency (e506). With complete loss of enzyme (infantile Pompe), glycogen accumulates in the heart at a rapid rate,
leading to onset of disease in the 1st month or two of life and death in the 1st year (e507). Involved infants invariably display
cardiomegaly on chest x-ray and a hypertrophic left ventricle by EKG and echocardiogram (e507). At autopsy, the heart weighs
three to ten times the expected weight for age and the walls of all the chambers appear thickened, giving the heart a globular
appearance. Mild degrees of EFE may be present. At microscopic examination, the cardiac myocytes appear markedly distended by
vacuolated and lacy cytoplasm due to the accumulation of PAS-positive digestible glycogen displacing the myofibrils (103).
Ultrastructural exam shows the glycogen to be at least partially membrane bound, a feature that distinguishes Pompe disease from
other forms of glycogen storage disease. The deficiency of a-1, 4-glucosidase (acid maltase) can be readily proved in muscle,
fibroblasts, lymphocytes, or urine (see Chapter 5).
Danon disease (type IIb GSD) represents an X-linked disorder caused by primary deficiency of lysosome-associated membrane
protein-2 (LAMP-2). Affected males present during childhood with muscle weakness, HCMP, and frequently (70%) mental
retardation with death from cardiac failure in the second or the third decade (104) (e508). Affected females also almost invariably
manifest disease, but at an older age. At the time of diagnosis, EKG displays features of both left ventricular hypertrophy and
rhythm disturbance including Wolff-Parkinson-White syndrome and bundle branch block. The pathologic changes are best
described in skeletal muscle biopsies (104). Muscle fibers contain PAS- and acid phosphatase-positive inclusions that exhibit
dystrophin and sarcoglycan staining of their membranes. Ultrastructural study shows the intracytoplasmic membrane-bound
vacuoles to contain glycogen mixed with cytoplasmic debris (104).
Mucopolysaccaridoses
Mucopolysaccharidoses (MPS) represent a group of lysosomal storage disorders caused by defects in the intralysosomal
degradation of acid mucopolysaccharides (glycosaminoglycans). Seven forms of MPS have been identified, all but one of which are
transmitted in an autosomal recessive fashion (e509,e510) (Table 13-30). Cardiovascular abnormalities occur in most forms of
MPS, with the degree of involvement varying between forms and over time for any one form (105) (e511). During life valvular
insufficiency due to thickening of the mitral, or less often aortic valve represents the most significant cardiovascular complication
(105). At autopsy, more extensive involvement can be identified. These cardiovascular changes are best described in MPS I (Hurler
syndrome). (106). The valves and the endocardium
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of all four cardiac chambers are thickened, the mitral valve especially so, with irregular nodules along its free margin (Figure 13-26).
The coronary arteries appear thickened with luminal narrowing; the aorta and systemic vessels exhibit substantial intimal plaque
formation. Subendocardial fibrosis may be severe, especially in the left ventricle with occasional patients presenting as newborns
with EFE (e512,e513). Histologically, the thickened connective tissues of the cardiovascular system and other sites are populated
by vacuolated “Hurler” cells containing large vesicles of soluble acid mucopolysaccharides and glycolipids. Ultrastructurally,
membrane-bound vacuoles contain concentric and parallel lamellae (106) (see Chapter 5).
Table 13-30 ▪ MUCOPOLYSACCHARIDOSE
Type Chromosome Stored Material Deficient Enzyme

Locus
MPS IH Hurler 4p16.3 Dermatan sulfate α-L-Iduronidase
Heparin sulfate
MPS IS Scheie 4p16.3 Dermatan sulfate α-L-Iduronidase
Heparin sulfate
MPS IH/S Hurler- 4p16.3 Dermatan sulfate α-L-Iduronidase

Scheie
Heparin sulfate
MPS II Hunter Xq28 Dermatan sulfate Iduronate sulfatase
Heparin sulfate
MPS IIIA Sanfilippo A 17q25.3 Heparan sulfate Heparan N-sulfatase
MPS IIIB Sanfilippo 17q21 Heparan sulfate α-N-Acetyl-glucosaminidase

B
MPS IIIC Sanfilippo NK Heparan sulfate Acetyl-CoA:α-glucosaminide

C acetyltransferase
MPS IIID Sanfilippo 12q14 Heparan sulfate N-Acetylglucosamine 6-sulfatase
D
MPS IVA Morquio A 16q24.3 Keratan sulfate chondroitin 6- Galactose 6-sulfatase

sulfate
MPS IVB Morquio B 3q21.33 Keratan sulfate α-Galactosidase
MPS VI Maroteaux- 5q13-114 Dermatan sulfate Arylsulfatase B

Lamy
MPS VII Sly 7q21.1 Dermatan sulfate, β-Glucuronidase
Heparin sulfate
Chondroitin 4-,6-sulfates
MPS IX 3p21.2-21.3 Hyaluronan Hyaluronidase
See references (e509,e510).
FIGURE 13-26 ▪ Mucopolysaccharidosis type IV. A thickened, nodular mitral valve is characteristic of most mucopolysaccharidoses.
Mucolipidosis
Mucolipidosis II (I-cell disease) (gene map locus 4q21-23), an autosomal recessive disorder caused by a deficiency of multiple
lysosomal hydrolases that degrade lipids and mucopolysaccharides, leads to a Hurler-like clinical presentation (e514). Fibroblasts
accumulate storage material leading to thickened and nodular valvular leaflets and abnormal chordae (107). The coronary artery
intima may contain foam cells (e515). Progressive left ventricular hypertrophy can contribute to the risk for sudden death in some
patients (e514).
Gangliosidoses
The gangliosidoses are autosomal recessive enzymatic defects of glycosphingolipid metabolism. Although manifesting
predominantly as disorders of neuronal tissues, accumulation of storage material in the myocardium mimicking that seen in the MPS
may cause significant disease in at least two of these disorders (see Chapters 5 and 10).
GM1 gangliosidosis resulting from a deficiency in acid β-galactosidase causes storage of GM1 ganglioside material in neuronal
tissue and glycosaminoglycans and glycopeptides in visceral organs (108). Cardiac involvement occurs in a subgroup of infants,
manifesting as CMP or valve insufficiency (e516,e517). Foamy histiocytes containing periodic acid-Schiff and alcian blue-positive
storage material accumulate in the heart valves, subendocardial regions, and vessel adventitia (108) (e518).
GM2 gangliosidosis type II (Sandhoff disease) results from deficiency of the hexosaminidase β-subunit (e519). Storage material,
described in the connective tissue cells throughout the heart, consists of membrane-bound concentric bodies
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(109) (e520). The resulting cardiac disease manifests clinically as HCMP and valvular insufficiency (109) (e519).
Fabry Disease
Fabry (Anderson-Fabry) disease, an X-linked recessive inborn error of glycosphingolipid metabolism (gene locus 3p21-23), results
from a deficiency of lysosomal a-galactosidase A (ceramide trihexosidase). The neutral glycosphingolipids deposit in lysosomes of
cells throughout the body, with the renal, cardiovascular, and peripheral nervous systems taking the largest “hit” (110). In cardiac
muscle cells, the deposits occupy the central, perinuclear areas and displace the contractile elements toward the periphery. In
frozen tissue, the storage material appears as PAS positive and birefringent. At electron microscopic study, the deposits form
intralysosomal aggregates of concentric or parallel lamellae (110). Cardiac disease manifests most commonly as left ventricular
hypertrophy, with less common clinically significant valve and conduction system alterations. The disease occurs with an incidence
of 1/40,000 to 117,000 male live births and accounts for 3% to 4% of unexplained LVH in young adult males (110). Although
commonly considered an adult disease, symptoms begin in childhood. The majority of patients manifest neurologic pain and/or skin
angiokeratomas and nearly 40% have cardiac manifestation in the second decade (e521-e523). Female heterozygous patients are
affected, though usually with a less severe and more delayed course compared with the male hemizygous patients. With the
possibility of affective enzyme replacement therapy, early diagnosis has become more important (110) (e521).
N-Glycosylation Disorders
N-glycosylation disorders refer to a group of multisystem diseases caused by at least 12 different defects in the attachment of N-
linked oligosaccharide chains to glycoproteins (e524). Hypertrophic or DCMP complicates at least a small subgroup of these
patients (111). Cardiac manifestations including pericardial effusions and HCMP may be the presenting symptoms in some patients
(111). Endomyocardial biopsy in one patient with DCMP revealed nonspecific findings of myocyte hypertrophy and interstitial
fibrosis without inflammation (111).
Fatty Acid Oxidation Defects

Fatty acid oxidation is a complex metabolic pathway that can go awry at a variety of points leading to cardiac dysfunction (Table 13-
31) (112) (e525). The majority of theses enzyme defects present in infancy, with abnormal free carnitine levels and acylcarnitine
profiles serving as diagnostic clues (112). Primary carnitine deficiency presents with hypoglycemia and CMP (113) (e526-e528).
The CMP may be either dilated or hypertrophic and there is often associated EFE (113) (e527). In both biopsy and autopsy
material, the cytoplasm of skeletal muscle and cardiac myocytes contain accumulations of neutral lipid vacuoles with associated
large aggregates of mitochondria (114) (e527). The CPT II and translocase deficiencies often present with an arrhythmia with or
without associated CMP (e525,e529) (see Chapter 5).
Table 13-31 ▪ FATTY ACID OXIDATION DISORDERS WITH CARDIOMYOPATHY
Enzyme Gene
Carnitine transporter OCTN2
Carnitine/acylcarnitine translocase CACT
Carnitine palmitoyltransferase II CPT-II

Very-long-chain acyl CoA dehydrogenase VLCAD
Electron transfer flavoprotein dehydrogenasea ETF-DH
Electron transfer flavoprotein-αa α-ETF
Electron transfer flavoprotein-βa β-ETF
Short-chain L-3-hydroxyacyl CoA dehydrogenase SCHAD
Mitochondrial trifunctional protein MTP
Long-chain 3-ketoacyl-CoA thiolase LKAT
aThese deficiencies also known as glutaric aciduria type II. CoA, coenzyme A.
Mitochondrial Electron Transport Chain Disorders

The mitochondria, home of energy production via oxidative phosphorylation (OXPHOS), represent a unique structure within cells.
The electron transport chain pathways involved in OXPHOS include five enzyme complexes, each including multiple proteins
produced by a mix of mitochondrial DNA (mtDNA) and nuclear DNA (nDNA). Mitochondrial enzyme deficiencies can be derived from
either mtDNA or nDNA mutations. This fact combined with the ability of mitochondria to divide independent of cell division
(heteroplasmy) results in an exuberant array of phenotypic variations for the electron transport chain deficiencies (e530,e531). Out
of this phenotypic array, a group of mitochondrial enzyme deficiency syndromes have been identified, some of which include cardiac
manifestations, especially hypertrophic or DCMP and conduction defects, as an important element (e530,e531). Up to 40% of
patients with mitochondrial cytopathy manifest cardiac disease, including both hypertrophic and DCMP with approximately 10% of
patients presenting as an isolated CMP (115) (e532). Patients whose mitochondrial disorder includes CMP follow a more severe
clinical course compared with patients without CMP (115, 116) (see Chapter 5).
The pathologic features of mitochondrial disease in cardiac muscle include replacement of the cardiac myofibers by pools of
mitochondria that ultrastructually may contain closely compacted stacks or circular arrays of cristae (116). In a subgroup of patients,
negative COX staining of frozen tissue serves as a clue to the diagnosis (116). Making a definitive diagnosis of a mitochondrial
cytopathy is, however, not straightforward requiring not only light and electron microscopic examination of the endomyocardial
biopsy material, but also mtDNA and nDNA analysis of blood or tissue and frequently electron transport chain analysis of
fresh/frozen skeletal muscle.
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Iron Overload
The most common form of severe iron overload in childhood is transfusional siderosis, or secondary hemochromatosis, caused by
the repeated transfusions (e533). The excess iron is primarily deposited in the mononuclear phagocyte system and by itself it does
not cause significant cellular dysfunction or injury. During long-term transfusion therapy, cardiac iron deposits become readily
demonstrable but usually cause no clinical problems.
Hereditary hemochromatosis occurs as a juvenile form due to mutations in hemojuvelin (HJV, 1q21) or hepcidin (HAMP, 19q13.1)
genes, rather than the HFE gene common in adult hemochromatosis (e534,e535). Clinical features are similar to those in the adult
disease but with earlier onset of cardiac symptoms and endocrine dysfunctions. Iron accumulation begins early in life and causes
clinical symptoms including arrhythmias and DCMP before the age of 30 years. The heart may be two or three times the normal
weight with a rusty brown discoloration (117). On microscopic exam, both biopsy and heart explant iron deposits, readily identified
by histochemical staining, are visible in both myocardial connective tissue cells and in cardiac myocytes (117) (e533).
Neuromuscular Disorders
Given the similar myofibrillar structure in skeletal and cardiac muscle fibers, it is not surprising that cardiac involvement occurs as a
part of many neuromuscular diseases (118). DCMP is the most common form. Conduction abnormalities and arrhythmias without
apparent cardiac histopathology are also common (see Chapter 26).
Muscular Dystrophies
Both the Duchenne and the Becker forms of muscular dystrophy involve mutations in the dystrophin gene (Xp21.2). At autopsy,
most patients with this X-linked recessive disorder have a DCMP with epicardial and extensive interstitial fibrosis (119) (e536).
Dystrophic changes may also develop in the left ventricular papillary muscles with MVP or involve the conduction system
(e537,e538). In a small subgroup of people with a dystrophin gene mutation, the DCMP may be the presenting feature of the
disease (e472).
Emery-Dreifuss muscular dystrophy represents a slowly progressive form of muscular dystrophy that presents with contractures at
the elbows and the ankles. This phenotype occurs in both X-linked (emerin gene Xq28) and autosomal dominant (lamin gene
1q21.2-q21.3) forms (118). Cardiac involvement manifests as conduction defects with DCMP occurring less frequently (e539).
Mutations in the lamin gene also cause a DCMP with conduction defects without the skeletal muscle disease (e540).
Myotonic dystrophy, an autosomal dominant disorder characterized by muscle delayed muscle relaxation (myotonia), results from an
abnormal expansion of a cytosine-thymineguanine (CTG) trinucleotide repeat in chromosome 19 (120).
The dystrophic changes in the heart manifest most frequently as conduction defects, though left ventricular hypertrophy, dilatation,
and valve prolapse also occur (120) (e541). Ventricular noncompaction has also been described in occasional families (e542). The
CTG repeat length is unstable with a trend toward increased length over time. This phenomenon is important as repeat length
correlates with disease severity in the heart as well as the muscle (e543).
Congenital Myopathies
Myofibrillar myopathy presents in the second decade of life with muscle weakness, cramps, or exercise intolerance. The finding of
abnormal accumulations of desmin material in the muscle fibers led to identifying mutations in the desmin gene in many of the
patients (121). A DCMP frequently accompanies and may predate the myopathy in affected families (e544). The disorder is
transmitted as an autosomal dominant trait with variable penetrance.
Central core disease represents a slowly progressive form of congenital myopathy diagnosed by the distinctive pathologic absence
of central mitochondria in skeletal muscle. Most cases can be linked to a mutation in the ryanodine receptor (RYR1) gene on
chromosome 19 and are without associated heart disease (e545). The central core phenotype has, however, also been identified in
skeletal muscle from patients with HCMP and a mutation in the β-myosin heavychain (MYH7) gene on chromosome 14 (e546).
Mutations in a different region of the MYH7 gene have been identified in the myosin storage myopathy in which myofibers contain
aggregates of myosin myofilaments beneath the cell membrane (e547). Patients with myosin myopathy present in childhood with
slowly progressive limb weakness; CMP is usually not a part of this disorder.
Friedreich Ataxia
Friedreich ataxia, the commonest form of inherited ataxia, results from an expansion of the GAA trinucleotide repeat in the frataxin
gene on chromosome 9q13 (122) (e548-e550). The frataxin gene is involved with mitochondrial iron metabolism and mitochondrial
dysfunction is believed to be the mechanism behind this disorder (e550). Cardiac disease, usually manifesting as HCMP,
complicates the clinical course in 65% to 75% of patients (e548-e550) and occasionally young patients present with CMP (e551).
The severity of the cardiac manifestations correlates with the number of GAA repeats (122). Examination of hearts at autopsy
reveals myocyte hypertrophy and fibrosis with myocyte degeneration and iron deposition (123) (e552).
Inflammatory/Autoimmune Disorders
Systemic Lupus Erythematosus
Most of the classic autoimmune diffuse connective tissue diseases, including systemic lupus erythematosis (SLE), rheumatoid
arthritis, scleroderma, polyarteritis, and dermatomyositis,
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occur in children and adolescents. These diseases manifest overlapping clinical features, with heart involvement occurring as a
component in many. SLE, which includes a wellstudied significant cardiac component, will be the focus of the discussion here.
In the pediatric population, SLE usually occurs after the age of 9 years with a striking (6:1) female predominance (124). The patients
present with a bewildering febrile illnesses that over time involve the joints, skin, serosal membranes, and kidneys. There is a large
clinical and serology overlap between the various autoimmune connective tissue disorders with a positive double-stranded DNA
antibody helping to discriminate SLE from the other forms. A transient similar condition may occur in infants born to mothers with
active SLE (see later). Cardiac disease, involving any and all portions of the heart, occurs commonly.
Pericardium: In collected autopsy series, pericarditis occurs in 65% of SLE cases (125). By echocardiography evidence, 35% to
40% of patients have evidence of pericardial effusions and/or pericardial thickening (e553). Clinical evidence of pericardial disease
occurs in even fewer patients, up to 30% (124, 125). The pericardial effusion is typically neutrophilic with a decreased glucose,
mimicking bacterial pericarditis (124). The histopathologic changes in the pericardium include mesothelial proliferation and necrosis
with a fibrinous exudate and underlying inflammation and granulation tissue formation. Fibrous obliteration of the pericardial space
occurs infrequently.
Myocardium: Autopsy series identify myocarditis in up to 40% of hearts though clinical evidence of myocarditis occurs in 2% to 25%
of patients (124, 125) (e554). Echocardiographic studies identify left ventricular hypertrophy and/or abnormal wall motion in 20%
(e553,e555). The pathologic features include small-vessel inflammation, interstitial inflammation with or without necrosis, and
interstitial fibrosis (124). The demonstration of immune complex deposition in intramyocardial vessels indicates that the myocarditis
can be attributed, at least in part, to the underlying autoimmune disorder (e555). The presence of hypertension and coronary
vascular narrowing in many patients suggests however that at least some of the myocardial disease occurs as a secondary
complication.
Endocardium and Valves: In his classic descriptions, Gross (126) described discrete vegetations of three types on the valves and
endocardium in SLE: the “pyramidal ridge type,” similar to that seen in rheumatic fever; the “massive thrombotic type” around
commissures, similar to that seen in nonbacterial endocarditis; and the “flat spreading type,” which he considered the most
characteristic form. These latter lesions represent the most notable gross cardiac feature in SLE, “nonbacterial verrucous” or
“Libman-Sacks” endocarditis. Libman-Sachs endocarditis manifest as smooth and friable vegetations, up to about 4 mm in greatest
diameter, which can be flat and granular, warty, or nodular, resembling mulberries. These vegetations, found most often on mitral
and aortic valves, are located on the valve surface impacted by blood away from the line of closure. Similar vegetations often
spread along the chordae tendinae and onto the endocardium. Microscopic changes begin on the surface of the valve leaflets and
include hematoxylin bodies, valvular necrosis without bacterial presence, widespread multinucleated eosinophilic coalescent bodies,
and a characteristic valvulitis with plasma cells and thick granulation bud capillaries (126). The valve under the vegetations is
minimally deformed and these small lesions may be difficult to identify by echocardiogram. A second type of valve abnormality in
SLE, diffuse thickening without discrete vegetations, has been described with increasing frequency in recent years, predominantly
in adults with longstanding disease (e556). Valvular disease is identified at autopsy in up to 65% of patients (124) and by
echocardiography in 20% to 35%, including a group of patients with mitral and/or aortic regurgitation without visible structural
defects (e553,e556,e557). The Libman-Sacks vegetations have been attributed, at least in some case to antiphospholipid antibody
deposition (125) (e555).
Neonatal SLE
Neonatal SLE manifests as characteristic skin lesions and cardiac involvement, especially heart block, with other systemic organ
involvement occurring only rarely. The skin rash may not be present at birth, appearing on the scalp and elsewhere by 2 months of
age and disappearing by 6 months of age. The heart disease frequently manifests in utero, with bradycardia frequently detectable
before 30 weeks of gestation (e558). Virtually all infants and their mothers have demonstrable 48-kD SSB/La, 52-kD SSA/Ro,
and/or 60-kD SSA/Ro autoantibodies. Despite the serologic abnormalities, approximately 40% of the mothers are without
autoimmune disease symptoms (e559). The antibodies apparently cross react with fetal cardiac tissue resulting in permanent
damage to the conduction system. Histologic studies of the conduction system in these infants show the AV node and parts of the
bundle branches to be replaced by fibrous scar tissue; a few lymphocytes may also be present as a residuum of prior inflammatory
damage (127, 128). When diagnosed in utero, complete heart block as a result of maternal autoantibodies leads to death in
approximately 40% of cases (129), with fetal hydrops a particularly poor prognosis marker. When diagnosed in the neonatal period,
morbidity is much lower (∽5%), but nearly all infants who survive require placement of a pacemaker (129) (e560,e558). Secondary
DCMP and EFE may further complicate the long-term cardiac function in these children (129) (e561).
Rheumatic Fever and Rheumatic Heart Disease

Rheumatic fever (RF), once the leading cause of death in young people age 5 to 20, has nearly disappeared from the developed
world, but it remains the leading cause of acquired heart disease in the developing world (e562,e563). RF represents a delayed
autoimmune reaction to group A β-hemolytic
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streptococcal pharyngitis. Whether strep throat leads to RF depends on several factors, including the M-protein type of the infecting
bacteria, the genetic background of the infected individual, and the socioeconomic environment surrounding the infection (130). The
appropriate infecting agent evokes a T- and B-cell response against the myosin-like M-protein, which then crossreacts with
antigens in myocardium, endothelium, and neurons (130) (e564). The resulting acute RF is characterized by (a) migratory
polyarthritis of large joints, (b) carditis, (c) erythema marginatum, a striking evanescent skin rash, (d) subcutaneous nodules, and (e)
Sydenham chorea, a neurologic disorder with features of involuntary, purposeless, rapid movements (130, 131) (e565). The
presenting symptoms in an individual patient may vary greatly, making a definite diagnosis of RF difficult. In 1944, T. Duckett Jones
published clinical criteria for making the diagnosis; the Jones criteria withstood the test of time and remain in use today with only
minor modifications (Table 13-32) (131) (e565,e566).
Table 13-32 ▪ DIAGNOSIS OF RHEUMATIC FEVER AND HEART DISEASE 2002-2004 WHO CRITERIA (BASED
ON REVISED JONES CRITERIA)
Major manifestations
Carditis
Polyarthritis
Chorea
Erythema marginatum
Subcutaneous nodules
Minor manifestations
Clinical: fever, polyarthralgia
Laboratory: elevated acute phase reactants (ESR, WBC)
Supporting evidence of preceding streptoccocal infection within last 45

days
Electrocardiogram: prolonged P-R interval
Elevated or rising antistreptolysin-O or other streptococcal antibody
OR
Positive throat culture
OR
Rapid antigen test for group A streptococci
OR
Recent scarlet fever
Diagnostic categories Criteria
Primary episode of RF 2 major or 1 major + 2 minor manifestations
Plus
Evidence of preceding group A streptococcal

infection
Recurrent attack of RF in patient without established RHD 2 major or 1 major + 2 minor manifestations
Plus

infectiona
Recurrent attack of RF in patient with established RHD 2 minor manifestations

Plus

infectiona
Rheumatic chorea Other major manifestations or evidence of group

A streptococcal infection not required
Insidious onset rheumatic carditis
Chronic valve lesions of RHD (presenting with pure mitral stenosis or Do not require any other criteria to be diagnosed
mixed mitral valve disease and/or aortic valve disease) as RHD
RF, Rheumatic fever; RHD, Rheumatic heart disease.
The pathologic response to this autoimmune disorder results in formation of Aschoff nodules, comprising central fibrinoid necrosis
surrounded by inflammatory cells. Included in the inflammation are lymphocytes, plasma cells, and a characteristic histiocytic cell
with ragged edges, and vesicular nucleus containing a dense central spiculated bar of chromatin named the Anitschkow cell (Figure
13-27) (e565). The carditis, which occurs in roughly 50% of RF patients, involves all layers of the heart. Rheumatic endocarditis
induces injury to the heart valves, representing the most clinically significant disease. The mitral valve is involved alone in 40% to
50% of cases, the aortic and the mitral valves together in 35% to 40%, the aortic valve alone in 15% to 20%, and the mitral, aortic,
and tricuspid valves together in 2% to 3% (e567). On gross examination, the valves display a nearly continuous row of translucent
verrucae near the closure margins of swollen, focally hemorrhagic valve leaflets. On the mitral and the tricuspid valves, the verrucae
lie on the atrial surfaces, a few millimeters from the free edges; the attached
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chordae may also be involved (Figure 13-28). Verrucae on the aortic and pulmonic valves involve the ventricular surface.
Microscopically, the entire leaflet is usually inflamed and edematous with focal formation of Aschoff nodules; new vessels extend
from the base of the valve into the leaflet. In about half of the patients, a series of thickened subendocardial ridges, the MacCallum
patch, develops in the left atrium immediately above and perpendicular to the posterior leaflet of the mitral valve (7, 131). The
ventricular endocardium is rarely involved. Rheumatic myocarditis results in left-heart dilatation with minimal associated
inflammation early in the course (131). With time, mononuclear cell infiltrates and characteristic Aschoff bodies are often found in
the edematous perivascular or subendocardial interstitium of the interventricular septum and left ventricle. Rheumatic pericarditis
develops only in people with underlying endocarditis and myocarditis (131). Characteristically, a fibrinous exudate thickens the
pericardium, binding the visceral and parietal layers together and obliterating the pericardial space. Microscopically, fibrin layers
containing scattered neutrophils cover the reactive mesothelial surfaces. Beneath this layer lie infiltrates of lymphocytes, plasma
cells, macrophages, and polymorphonuclear leukocytes. With resolution of the acute phase, the exudate may resolve completely or
leave variable patterns of adhesions.
FIGURE 13-27 ▪ Acute rheumatic carditis. A characteristic Aschoffbody is seen in this section of the left ventricle. There is a central
degenerating fiber surrounded by large mononuclear cells (200×).
FIGURE 13-28 ▪ Acute rheumatic carditis. Characteristic dark, nodular excrescences line the margins of closure on the mitral valve
leaflets. Rarely seen today, these sites often heal as fibrous nodules with associated valvular distortion. (Courtesy of Roma
Chandra, M.D., National Children's Hospital, Washington, DC.)
Infant of Diabetic Mother Cardiomyopathy

Maternal diabetes places an infant at significantly increased risk of heart disease at birth, including both a 5 to 10× greater relative
risk of having a malformed heart and a 15% to 40% risk of HCMP in an otherwise normally formed heart (132) (e568,e569). The
HCMP remains asymptomatic in the majority of infants but may also present with congestive heart failure or even stillbirth (132)
(e570). The HCMP is often asymmetric and in an occasional infant, this hypertrophied septum causes a transient left ventricular
outflow obstruction. The ventricular hypertrophy usually resolves completely in 2 to 12 months (e570). Given the transient and
rarely fatal nature of this disorder, histologic examination of involved myocardium is limited. In the few studied cases, the
myocardium appears hypertrophic with EFE in some cases (132) (e571,e572). Myofiber disarray similar to that seen in familial
HCMP, which occurs in some cases, is believed to be a normal feature of development.
Ischemic Myocardial Necrosis

Ischemic myocardial necrosis is relatively common in the normal and malformed hearts of infants and children, especially those in
which profound hypoperfusion develops for any reason. Ischemia primarily damages the papillary muscles and ventricular
subendocardium of either ventricle. Thus, sampling the infant heart requires examination of all the papillary muscles, the adjacent
free ventricular and atrial walls, and ventricular septal myocardium near the membranous septum. Given that the myocardial fibers
of the papillary muscles and much of the subendocardium are arrayed longitudinally, sections taken in the long axis of the heart give
the best sampling yield (133) (e573). Small deep red or yellow ischemic foci near the apex of a papillary muscle represent the
earliest and the mildest form of injury. The more severe the injury in papillary muscles, the greater the likelihood of subendocardial
necrosis in the ventricular free walls (134). Longitudinal sections through the septum may expose unsuspected large infarcts near
the AV ring that may involve the AV node or the bundle of His (134).
For the most part, the histopathology of ischemic myocardial injury in children resembles that in adult humans. However, in very
young infants, the chronology of injury repair does not follow that of adults and is in fact not well defined. Using birth as the time of
injury yields the following chronology of pathologic changes (134):
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0 to 9 hours—histopathologic evidence of ischemic injury in the myocardium may be entirely lacking. The
ninth component of complement (C9), part of the C5-9 membrane attack complex, has been used to identify
sites of very early perinatal myocardial injury (e574).
24 to 48 hours—Myocyte necrosis manifests as cytoplasmic eosinophilia and nuclear pyknosis; cross-
striations may persist in necrotic fibers; marginated cells in capillaries may be the only neutrophilic response.
72 to 96 hours—neutrophils infiltrate the margins of necrotic foci; the mononuclear, vascular, and fibroblastic
responses may be slowed by associated systemic problems.
Dystrophic calcification is common in the most frequent sites of perinatal myocardial injury, such as the papillary muscles and
ventricular subendocardial myocytes, and calcium may provide a sharp outline of the sarcolemmal membranes when no other signs
are present (133) (e575). Massive myocardial calcification may occur in perinates subjected to various causes of hypoxic-ischemic
injury (e576).
Endomyocardial Biopsy and Heart Transplant
Diagnostic Biopsy
The usefulness of myocardial biopsy in the evaluation of CMP remains controversial. The initial evaluation includes history, physical
examination, electrocardiography, echocardiography, and metabolic/genetic screens (85) (e577). When these studies fail to identify
an underlying etiology, especially in the setting of DCMP, cardiac catheterization with endomyocardial biopsy often becomes
indicated (e578). Given the broad differential diagnoses encompassed, appropriate handling of the biopsy material requires (a)
communication with the cardiologists regarding the patient's clinical picture; (b) information about specific disorders being
addressed by the biopsy; and (c) adequate tissue samples. In our laboratory, all endomyocardial biopsies are received fresh
accompanied by a standardized form (Table 13-33) outlining briefly the reason for the biopsy. The biopsy material is then divided
with one piece snap frozen in OCT for viral PCR or special stains (including PAS, oil-red-O, NADH reductase, cytochrome C
oxidase, succinate dehydrogenase, and immunohistochemical stains for dystrophin and sarcoglycans), one piece placed in
glutaraldehyde for electron microscopy, and the remaining three to four pieces fixed in formalin for routine light microscopy.
Heart Explants
At the time of heart transplantation, examination of the explanted heart yields an opportunity to confirm, further delineate, or change
the prior clinical diagnosis. In our laboratory, 40% of heart transplants are currently performed for a cardiomyopathic process and
60% for congenital malformations. Examination of the cardiomyopathic hearts has yielded previously unknown diagnoses of
noncompaction and arrhythmogenic right ventricular CMP.
Table 13-33 ▪ HEART BIOPSY
Clinical (to be completed by Cardiologist)
_______ Diagnostic
Clinical Suspicion: _____
_______ Transplant Biopsy
Date of transplant: _____
Indications for Transplant: _____
Current Rx: _____
Reason for Biopsy
_______ Rejection Surveillance (cellular and humoral)
_______ Clinical Rejection, cellular (describe indications: ________________________________________________)
_____ Clinical Rejection, humoral (describe indications: _________________________________________________)
Results to: ______________________ Pager _______________
Specimens submitted (to be completed by Cardiologist)
Right ventricular endomyocardial biopsies
# large _____ # medium _____ # small _____
_____ Routine processing (results reported the next business day)
_____ Stat processing. NOTE—Stat processing requires the following:

1. The pathologist on call must be consulted at the time the biopsy is scheduled
2. The specimen must be in the histology laboratory by 10:00 AM for same day results
Special requests:
Transplant Biopsy
Transplant biopsies play an integral role in the management of cardiac transplant patients. To be considered adequate, the biopsy
must include at least three fragments of myocardial tissue with the myocardium occupying greater than 50% of the tissue in the
biopsy fragment (135). Rejection may be either cellular or humoral. Cellular rejection manifests as interstitial lymphocytic
inflammation with or without myocyte necrosis. The extent of the inflammation and necrosis determines the grade of rejection. The
grading schemes for rejection have changed over the years with often poor interobserver concordance (e579,e580). In response to
this, the 2004 ISHLT working formulation (135) (e581) significantly modified the prior 1990 ISHLT/2001 Banff formulation (Table 13-
34). Humoral rejection refers to antibody and complement mediated graft dysfunction. Humoral rejection most commonly occurs in
the 1st month following transplant, but may persist for several months with an associated poor outcome (135, 136). Diagnostic
criteria for humoral rejection include evidence of endocapillary injury manifest as endothelial swelling, capillary neutrophil or
macrophage infiltrates, and interstitial edema and/or hemorrhage (135). These morphologic changes can however be difficult to
identify in biopsy material (e582). Positive immunocytochemical staining for C4d, when diffuse and intense, can serve as an aid in
the evaluation for humoral rejection (136) (e583).
Transplant biopsies can also display nonrejection alterations. In the early post-transplant period, differentiating harvest injury from
acute rejection may be problematic. Ischemic harvest injury results in contraction band or coagulative
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myocyte necrosis with the inflammatory response relatively mild in comparison to the degree of injury, whereas in acute rejection the
opposite relationship occurs (inflammation > necrosis) (135). The “Quilty” lesion, defined as nodular lymphocytic infiltrates in the
endocardium with or without extension into the adjacent myocardium, occurs frequently in biopsies (e584). Recognizing the invasive
form of a Quilty lesion where the myocardial lymphocytic inflammation is in direct continuity with the endocardial infiltrates often
requires examination of serial sections. This distinction is however important, as “invasive” Quilty is not considered rejection and
requires no treatment. The possibility of posttransplant lymphoproliferative disorder must also be kept in mind when examining the
biopsy transplant material.
Table 13-34 ▪ 1990 WORKING FORMULATION FOR THE STANDARDIZATION OF NOMENCLATURE INTHE
DIAGNOSIS OF HEART REJECTION
2004 ISHLT Banff (2001)/1990

ISHLT
No Rejection Grade 0 Grade 0
Interstitial and/or perivascular infiltrate without myocyte damage Grade 1 R, mild Grade 1, mild
A—Focal
B—Multifocal or diffuse
sparse
Single focus of dense infiltrate with myocyte injury Grade 1 R, mild Grade 2, moderate
(focal)
Multifocal (≥2 foci) of dense infiltrate with myocyte injury Grade 2 R, Grade 3, moderate
moderate
A—Focal
Diffuse infiltrate with multifocal myocyte damage Grade 3 R, Grade 3, moderate

severe
B—Diffuse
Diffuse infiltrate with multifocal myocyte damage, ± edema, ± Grade 3 R, Grade 4, severe
hemorrhage, ± vasculitis severe
Conduction System Abnormalities

The conduction system represents a part of the heart that is functionally important but anatomically difficult to identify. The
physiology of the conduction system and its broad range of abnormalities are beyond the scope of this chapter (e585). A basic
understanding of the conduction system anatomy is however important for pathologists, especially if they examine the hearts of
children at autopsy or abnormal native hearts removed during cardiac transplantation. This section will focus on the examination of
the conduction system and its abnormalities that can lead to sudden death.
The cardiac conduction system includes the sinus (SA) node, AV node, bundle of His, and bundle branches (137, 138) (e586). The
sinus node lies in the right atrium at its junction with the superior vena cava. Electrical impulses from the sinus node are transmitted
via the atrial myocardium to the AV node. The AV node lies just above the septal leaflet of the tricuspid valve in the apex of the
“triangle of Koch.” From the AV node, the electrical impulse is transmitted via the bundle of His through the central fibrous body and
membranous system to the superior margin of the muscular interventricular septum. Here the conduction tract divides, forming
bundle branches that extend along the right and the left sides of the septum. These bundle branches pass the impulse on to an
interweaving network of subendocardial Purkinje fibers, which transmit the signal simultaneously to the entire right and left
ventricular myocardium.
Histologic examination of the AV node and its connections to the ventricular myocardium represents the key to most pathologic
examinations of the conduction system. The Histology for Pathologists text and a recent forensic paper nicely describe in detail
methods for examination of conduction system (138, 139). Under the microscope, the nodal tissue includes three zones: (a) an inner
zone of pale staining specialized myocardial cells containing sparse myofibrils and inconspicuous striations embedded within a
fibrous stroma, (b) an outer zone of normal atrial myocardium, and (c) an intermediate zone containing transitional cells with a mixed
appearance. Both the SA and the AV nodes are innervated by sympathetic and parasympathetic nerve fibers. With formation of the
penetrating bundle and bundle branches, the specialized cells become surrounded by a fibrous tissue coat.
Interfering with this normal conduction at any point along the pathway can result in arrhythmias. Current classification divides the
arrhythmias into three basic types: supraventricular tachycardia, AV conduction disorders, and ventricular tachycardias (140) (Table
13-35).
The supraventricular tachycardias are predominantly medical disorders that rarely cause sudden death, with Wolff-Parkinson-White
(WPW) being the main exception (e587,e588). WPW results from persistence of cardiac muscle strands connecting atrial and
ventricular muscle. These accessory pathways cross the AV sulcus, bypassing the normal AV node (141) (e589). The pathologic
evaluation for accessory pathways requires systematic study of both AV rings (139). The faster conduction through these accessory
pathways leads to pre-excitation of the ventricular muscle and the diagnostic ECG findings of a short PR interval, delta wave, and
widened QRS complex (e585). Pre-excitation due to accessory pathways occurs with a prevalence of 0.1 to 3.1/1,000 in the general
population (e589).
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Table 13-35 ▪ ARRHYTHMIA CLASSIFICATION
Supraventricular tachycardia Neonate Childhood
Primary atrial tachycardia 10%-15% 10%-15%
Sinus tachycardia
Atrial flutter
Atrial fibrillation
Atrial reentry
AV nodal tachycardia <5% 5%-30%
AV nodal reentry
Junctional ectopia
aAccessory connection >80% >60%
mediated
Wolff-Parkinson-White
aAV Conduction Disorders
(AV block)
Congenital
Acquired
Ventricular tachycardia
Scar mediated
Cardiomyopathy related
aLong QT syndrome
Idiopathic
aDisorders discussed in text.
AV, atrioventricular.
Data from Calder L, Van Praagh R, Van Praagh S, et al. Truncus arteriosus communis: clinical, angiocardiographic, and
pathologic findings in 100 patients. Am Heart J 1976;92:23-38.
These abnormal conduction circuits can result in supraventricular tachycardia that manifests in young infants as congestive heart
failure or collapse and in older children as anxiety, chest discomfort, syncope, or cardiac arrest (e590). In symptomatic children, the
lifetime risk for sudden death is estimated as 3% to 4% (e591). However, not all individuals with pre-excitation ECG changes
develop symptomatic arrhythmias. Asymptomatic adults have a very low risk of cardiac arrest/sudden death (e592). In contrast, the
risk for cardiac arrest or sudden death in asymptomatic children is currently not known. In one multicenter study, 48% of WPW
deaths occurred in children without prior cardiac events (e593). In children with “high-risk” features (positive family history, multiple
accessory pathways), ablation of the accessory pathways resulted in improved survival (e587,e588). The appropriate use of
invasive treatment in asymptomatic children will depend on ascertaining appropriate risk stratification criteria.
Although usually sporadic, in 10% to 20% of instances WPW occurs in association with other congenital abnormalities (e589),
especially Ebstein malformation. Its association with an unusual form of glycogen storage type HCMP led to the identification of a
mutation in the PRKAG2 (e594,e595). Less commonly, ventricular pre-excitation result from accelerated conduction through a
hypoplastic AV node (e596,e597).
AV conduction disorders (AV block), due to interruption of impulse conduction from the atrium to the ventricle, are further
characterized as to the degree (first, second, and third or complete) of block. Congenital AV block (CAVB) is associated with a
variety of heart malformations, in particular AV septal defects and left atrial isomerism, and may also occur following surgical repair
of heart defects or ablation of arrhythmogenic foci (142) (e585,e598,e599). In the fetus CAVB can lead to hydrops, with an
associated high risk for fetal or neonatal death (129, 142) (e560,e599).
When not associated with underlying heart disease, CAVB occurs most commonly (85%) in the setting of maternal autoimmune
disease with anti-SSA/Ro and/or anti-SSB/La antibodies (e560,e600). Despite this high association between fetal CAVB and
positive maternal antibodies, the reverse is not true. Fetal CAVB complicates pregnancy in only 2% of women with positive anti-
SSA/Ro or SSB/La and the risk for recurrence following a pregnancy with CAVB is less than 20% (e558-e600). In fetuses and
infants with antibody-induced CAVB, histopathologic examination reveals fibrosis and calcification with or without inflammation in the
AV nodes as well as other sites along the conduction pathway (127, 128) (e601). Given the severity of these pathologic changes, it
is not surprising that antibodyinduced CAVB is permanent with 60% to 90% of children requiring pacemaker implantation (129)
(e558-e560).
Ventricular tachycardia can occur with a broad spectrum of precipitating causes of which the long QT syndrome (LQTS) is of
particular interest. The LQTS, manifest as QT prolongation and slowed repolarization on ECG, encompasses a group of disorders
affecting the cardiac muscle potassium, sodium, and calcium channels (channelopathies). Currently, LQTS can be subdivided into
eight major genotypes (LQTS1-8) (143), with some of the genotypes also expressing extracardiac abnormalities (Table 13-36).
Individuals with LQTS carry a significant risk of syncope and sudden death with the relative risk and event triggers (exercise,
emotional stress, sleep) correlating with the genotype (e602-e604). Recent molecular studies have identified LQTS mutations in up
to 9.5% of SIDS cases (144) (e605) and up to 20% of sudden cardiac deaths in older children (e606).
Catecholaminergic polymorphic ventricular tachycardia (CPVT), a form of ventricular tachycardia with a distinctive
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bidirectional polymorphic pattern on ECG, leads to recurren episodes of stress-related syncope in childhood (e607). Following
identification of mutations in the human cardiac ryanodine receptor gene (hRyR2) in CPVT, a molecular autopsy study identified the
same mutations in 14% of sudden unexpected childhood deaths (145).
Table 13-36 ▪ LONG QT SYNDROME
Gene Locus Phenotype
LQT1 KvLQT1, 11p15.5 RWS JLNS

KCNQ1
LQT2 HERG, 7q35-36 RWS

KCNH2
LQT3 SCN5A 3p21-24 RWS Brugada

syndrome
LQT4 ANKB, ANK2 4q25-27
LQT5 Mink, IsK, 21q22.1- RWS JLNS

KCNE1 2
LQT6 MiRP1, 21q22.1 RWS

KCNE2
LQT7 Kir2.1, 17q23 Anderson

KCNJ2 syndrome
LQT8 CACNA1C 12p13.3 Timothy

syndrome
RWS, Roman-Ward syndrome (143): ECG changes only.
Brugada syndrome (143): ECG changes only.
JLNS, Jervell and Lange-Nielsen syndrome: ECT changes +

sensorineural hearing loss.
Anderson syndrome (e717): Ventricular arrhythmia, periodic paralysis, dysmorphic facies, syndactyly, clinodactyly, cleft
palate, scoliosis.
Timothy syndrome (e718): Ventricular arrhythmia, heart malformation, syndactyly, immune deficiency, dysmorphic facies,
autism.
These recent molecular genetic findings in infant and childhood sudden deaths serve to emphasize the importance of a
comprehensive autopsy examination including molecular and genetic testing in cases of unexpected sudden death (e608,e609).
Extracardiac Vascular Disease
Pulmonary Hypertension
Pulmonary hypertension, defined as a mean pulmonary artery pressure at rest of greater than 25 mm Hg, represents a common
pathophysiologic state arrived at from a variety of etiologic pathways. Advances in understanding the underlying vascular biology,
physiology, and genetics, combined with new treatment modalities led in 2003 to a proposed revision in the classification of these
disorders (Table 13-37) (146 (e610,e611). In pediatrics, persistent pulmonary hypertension of the newborn (PPHN), and pulmonary
hypertension complicating L→R shunts represent the most common etiologies with primary (idiopathic or familial) PHT accounting
for many of the remaining cases. Before discussing the specifics of these conditions, it seems prudent to review (a) normal
pulmonary vascular development and (b) the pathologic changes associated with pulmonary artery hypertension.
Table 13-37 ▪ PULMONARY HYPERTENSION-WHO CLASSIFICATION
I. Pulmonary artery hypertension
A. Idiopathic
B. Familial
C. Associated with:
1. Collagen vascular disease
2. Congenital heart disease with L→R shunt
3. Portal hypertension
4. HIV disease
5. Drugs and toxins
6. Other
D. Associated with significant venous or capillary involvement

1. Pulmonary veno-occlusive disease
2. Pulmonary capillary hemangiomatosis
E. Persistent pulmonary hypertension of the newborn
II. Pulmonary hypertension with left-heart disease
A. Left-sided atrial or ventricular disease
B. Left-sided heart valve disease
III. Pulmonary hypertension associated with respiratory disorders or hypoxemia
IV. Pulmonary hypertension caused by chronic thrombotic/embolic disease
V. Miscellaneous (sarcoid, Langerhans cell histiocytosis, compression of pulmonary vessels, lymphangiomatosis)
Bold indicates disorders discussed in this section.
Normal Pulmonary Vascular Development

The vasculature in fully developed lung includes preacinar and intra-acinar arteries. The preacinar arteries travel in parallel with the
pulmonary airways and contain a well-developed muscle wall. The preacinar pulmonary arterial tree develops in synchrony with the
pulmonary airways becoming completely formed by 16 to 17 weeks of gestation (147) (e612). The intra-acinar arteries arise as a
network of supernumerary vessels that supply the terminal airspaces, and in the adult carry up to 40% of the pulmonary blood flow
(147). The development of the terminal airspaces and the intra-acinar arteries begins in utero but is incomplete at birth and
continues for many months thereafter. The formation of a muscle layer around the intra-acinar arteries lags behind the development
of the alveoli, and the medial muscle of these intraacinar arteries normally extends into the alveoli only after 8 to 10 years of age
(148) (e612).
The pathophysiology of the pulmonary vasculature clearly differs before and after birth. In the fetus, there is little pulmonary blood
flow with the high PVR due to increased thickness of the artery walls. At birth, this high PVR rapidly falls due to release of nitric
oxide and prostacyclin from the pulmonary artery endothelial cells, with resultant dilatation of the pulmonary arteries. Subsequent
thinning of the muscular media requires time and is complete only at 4 months of age (147).
Pulmonary Artery Hypertensive Changes

Pulmonary artery hypertension results in a constellation of changes outlined in Table 13-38 (149). The pulmonary artery changes
were traditionally graded by the Heath-Edwards scoring system (e613) (Table 13-38). However, with the advances in drug
treatment modalities gained from improved understanding of the underlying pathophysiology of pulmonary hypertension, this scoring
system has lost most of its value. The complex lesions are a marker of severe disease. The plexiform lesion is defined by
“glomeruloid” endothelial proliferation that extends through an area of vessel wall destruction into perivascular tissue (eFigure 13-
8). These lesions frequently occur adjacent to an area of concentric intimal fibrosis at an artery branch point. The plexiform lesions
are not only specific to primary/idiopathic pulmonary hypertension but also occur in the setting of cardiac shunting lesions. They are
however not a feature of PPHN (149). The dilation lesion refers to an area of artery wall thinning, often located distal to a plexiform
lesion. These dilated areas can serve as a focal point of hemorrhage.
Persistent Pulmonary Hypertension of the Newborn

PPHN occurs when the fetal circulation fails to adapt normally at birth due to underdevelopment, maldevelopment, or maladaptation
(148). The failed drop in PVR leads to right→left shunting at the foramen ovale and ductus
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arteriosus with resultant central cyanosis. PPHN most often occurs as a hypoxia-related maladaptation secondary to underlying
pneumonia, sepsis, or meconium aspiration. In infants with meconium aspiration, there is also abnormal extension of smooth muscle
into the media of the more peripheral, normally nonmuscular intra-acinar arteries suggesting in utero onset of the vascular
dysfunction (148). In approximately 20% of cases, peripheral muscle extension occurs without an obvious underlying etiology
resulting in “idiopathic” PPHN (146). Epidemiologic studies suggest that nonsteroidal anti-inflammatory drugs taken by the mother
may play a role in this vasculopathy. PPHN also occurs when the lungs fail to grow normally, resulting in a parallel
underdevelopment of the pulmonary vasculature. This manifests most dramatically in infants with congenital diaphragmatic hernias
who often develop severe respiratory insufficiency following repair (148). In recent years, the use of vasodilators, prostacyclin, and
ECMO has dramatically improved the survival of these infants.
Table 13-38 ▪ PULMONARY ARTERY HYPERTENSION PATHOLOGIC GRADING
Pulmonary Arteriopathy Heath and Edwards Grade
Pulmonary arteriopathy with isolated Grade 1
medial hypertrophy
Pulmonary arteriopathy with medial
hypertrophy + intimal thickening
(cellular or fibrotic)
Concentric laminar intimal fibrosis Grade 2 or 3

Eccentric or concentric nonlaminar
intimal fibrosis
Pulmonary arteriopathy with complex
lesion
Plexiform lesion Grade 5
Dilation lesion Grade 4
Arteritis Grade 6
Pulmonary arteriopathy with isolated Grade 6
arteritis
Compiled from references (149) (e613).
Congenital Heart Disease with Left→Right Shunt

A left→right shunt, particularly at the post-tricuspid valve level, results in increased volumes of blood flow at an increased pressure
in the lungs. This stimulates smooth muscle hyperplasia along with the intimal changes of pulmonary hypertension. An additional
feature seen in the left→right shunt scenario is the decrease in numbers of peripheral arteries (150) (e614). If the shunt can be
repaired early enough, these pulmonary hypertensive changes are reversible (150). However, if the repair is delayed, the
pulmonary vascular changes can become nonreversible and the pulmonary hypertension will continue to progress following surgery.
Predicting in an individual patient how quickly pulmonary hypertensive changes will progress to a nonreversible stage remains
problematic (e611). Age at surgery does play a role. In one study, in infants with a variety of shunting lesion operated upon before 9
months, the PVR uniformly returned to normal postoperatively, irrespective of pulmonary vascular histology. In older infants, medial
wall thickening greater than 2× normal resulted in an increased risk of persistent PHT following surgery (150). In the worst-case
scenario, the pulmonary hypertension progresses to a point where PVR exceeds systemic vascular resistance and the shunt
becomes right→left with resultant cyanosis (Eisenmenger syndrome) (e611).
Familial and Idiopathic Pulmonary Artery Hypertension

Primary pulmonary hypertension (PPH) occurs as a familial disease in 6% to 12% of cases with the remainder occurring
sporadically. The striking 1:2 female predominance observed in adults with PPH is less obvious in children where the M:F ratio is
1:1.3 to 1.5 (146, 151). The familial disease follows an autosomal dominant inheritance pattern with incomplete penetrance and
genetic anticipation (successive generations with worse disease) (151). In 50% of families, a mutation in the gene encoding bone
morphogenetic protein receptor-2 (BMRP-2) can be identified; similar mutations occur in approximately 25% of sporadic cases (146,
151) (e615). The involved pulmonary arteries display the range of pathologic changes described earlier. Improved understanding of
the pathophysiology of pulmonary hypertension in recent years has led to significant improvements in treatment for patients with
PPH. This disease, which once led to death within a year of diagnosis, can now be managed in most patients by initially using
vasodilator therapy with lung transplantation as a final option (e610).
Pulmonary Hypertension with Obstructive Left-Heart Disease

In the face of obstruction to pulmonary venous return, the vascular changes in the lungs include not only arterial but also venous
thickening. With extrapulmonary venous obstruction, the pulmonary arteries develop medial hypertrophy and intimal fibroplasia
without the more complex lesions. The pulmonary veins also develop medial hyperplasia with “arterialization”; that is, formation of a
discrete internal and external elastic lamina (e616). These venous changes, though striking, are reversible following repair of the
obstructive defect. The long-term outcome of the pulmonary hypertension depends instead on the severity of the associated arterial
disease.
Systemic Artery Disease

Arteriopathy
Idiopathic infantile arterial calcification (IIAC) represents a metabolic disorder of the arteries resulting in deposition of calcium
hydroxyapatite in and around the internal elastic lamina and intimal fibrous proliferation (e617). The calcification
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occurs in any artery, with the coronary arteries involved in greater than 75% of cases and the cerebral arteries involved only rarely
(e617). The calcification may elicit an inflammatory response including lymphocytes, eosinophils, and foreign body GCs.
Ultrastructural exam identified hydroxyapatite deposition in the elastic laminae, collagen fibers, smooth muscle, and fibroblasts
(e618). This rare condition most often presents as heart failure in early infancy, but may manifest prenatally as nonimmune hydrops.
The artery luminal narrowing caused by the calcification and intimal proliferation can lead to ischemic injury in the involved organ,
most commonly the heart, with 85% of infants dying in the first 6 months (e617). IIAC occurs in an autosomal recessive pattern in
some families. The presence of associated periarticular calcification in a few instances led to the finding of low nucleotide
pyrophosphatase (NPP) activity and inorganic pyrophosphate levels in some patients (e619). These observations in turn led to
identification of loss-of-function mutations in ENPP1, a gene encoding for NPP protein, in 8 of 11 kindreds with IIAC (e620).
Fibromuscular dysplasia represents a noninflammatory disorganization and fibrosis of large muscular arteries leading to segmental
luminal narrowing with the renal, internal carotid, coronary, celiac, hepatic, and mesenteric arteries most frequently involved. The
pathologic changes can involve all layers of the vessel wall. Several forms have been described, including medial fibroplasia,
perimedial fibroplasia, medial hyperplasia, and intimal fibroplasia (152) (e621). In medial fibroplasia, disorderly arrays of medial
smooth muscle cells form luminal ridges that narrow the artery lumen. Between the ridges, the artery contains abnormally thin layers
of otherwise normal smooth muscle cells. This alternating thick and thin luminal diameter gives an angiographic appearance of a
“string of beads.” Perimedial fibroplasia is characterized by layers of circumferential elastic-like tissue between the media and the
adventitia. With intimal fibroplasia, the intima may be thickened and the internal elastic lamina duplicated and fragmented.
Alternatively, the internal and the external elastic laminae may be disrupted and the intima and the media may merge. Fibromuscular
dysplasia accounts for up to 45% of renal hypertension in childhood with multifocal vascular involvement present in many (e622-
e624). A form of severe arterial dysplasia involving the aorta with its main branches has been described in both stillborn infants and
infants with sudden death. The vessels in these infants display medial thickening due to hyperplasia of the elastic fibers
(e625,e626).
Aneurysms
Aneurysmal dilatation of vessels, rare in childhood, occur both as primary defects in vessel wall structure and secondary to
underlying inflammatory or infectious disease (153). Inherited/genetic causes of aortic aneurysm are the focus here.
Dilatation and dissection of the ascending aorta occurs most commonly in Marfan syndrome but may also complicate a spectrum of
other disorders (Table 13-39). In all these disorders, microscopic examination reveals cystic medial degeneration with accumulation
of mucopolysaccharides in the tunica media in the involved aorta (153) (e627-e629). At the ultrastructural level, the elastic lamella
appears torn with loss of the connection between elastic lamella and smooth muscle cells (e630,e629).
Table 13-39 ▪ ANEURYSMS OF AORTA
Syndrome Gene Reference
Marfan's Fibrillin-1 (155)
Loeys-Dietz TGFBR-1 (156)
TGFBR-2
Ehlers-Danlos COL3A1 (158)
Turner Monosomy X (157)
Marfan syndrome represents an autosomal dominant disorder of connective tissue with high penetrance but variable phenotype due
to the broad spectrum of organ involvement. A set of diagnostic criteria, combining clinical and genetic features, has been devised
to aid in accurate diagnosis (Table 13-40) (154) (e627). Marfan syndrome occurs from mutations in the fibrillin-1 gene ( FBN1, gene
15q21.1), with approximately 25% of cases representing new mutations. Signs of the disease can appear at any age but most
patients come to diagnosis in the second or third decade. There is, however, a “neonatal” form of Marfan, associated with mutations
in exons 24 to 32 of the fibrillin-1 gene (155) (e628) that presents in infancy with aortic dilatation accompanied by mitral and
tricuspid regurgitation with death often occurring in the first 2 years (e63 1,e632).
Loeys-Dietz syndrome mimics many of the clinical features of Marfan syndrome, with craniofacial features of hypertelorism, low set
ears, and bifid uvular or cleft palate serving to distinguish the two (154, 156). Loeys-Dietz also represents an autosomal dominant
disorder, due to mutations in transforming growth factor beta receptors 1 or 2 ( TGFBR1/2). The aortic dilatation progresses to
dissection at a younger age in these patients, with a mean age of death at 26 years (156).
Ascending aortic dilatation also occurs with increased frequency in Turner syndrome and in isolated bicuspid aortic valve patients
(157) (e633,e634). The distribution of the aneurysms in the bicuspid aortic valve patients is however somewhat different from that of
Marfan. In Marfan, the dilatation occurs predominantly at the level of the aortic valve cusps, whereas in the bicuspid aortic valve
group the dilatation extends for longer distance up the aorta (e633).
Ehlers-Danlos syndrome is a heterogeneous group of at least ten generalized disorders of connective tissue synthesis, many
involving different forms of collagen and their genes (158). Ehlers-Danlos type IV (vascular type) manifests thin-walled vessels and
a diffuse decrease in elastic tissue in the media, deposition of acid mucopolysaccharide material between the medial elastic
lamellae, and a decrease
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in adventitial and medial collagen. Aortic dilatation with dissection and rupture, often intra-abdominal in location, can complicate
clinical course and even cause death (158). This form of Ehlers-Danlos syndrome is caused by a mutation in the COL3A1 gene
transmitted in an autosomal dominant fashion.
Table 13-40 ▪ MARFAN SYNDROME: GHENT DIAGNOSTIC CRITERIAa
System Major Criteria Minor Criteria
Family history Diagnosis in first degree relative None
Genetics Mutation FBN1 None
Cardiovascular Aortic root dilatation Mitral valve prolapse
Dissection of ascending aorta Mitral valve calcification (<40 years)
Pulmonary artery dilatation
Descending aorta dilatation/dissection
Ocular Ectopia lentis 2 of the 3
Flat cornea; elongate globe; myopia
Skeletal At least 4 major: 2-3 major or 1 major + 2 minor
Pectus excavatum with surgery
Pectus carinatum Minor:
Pes planus Moderate pectus excavatum
Positive wrist or thumb sign High arched palate

Scoliosis >20° or spondylolithiasis Typical facial features
Arm span/height ratio >1.05 Joint hypermobility
Protrusio-acetabulae
Elbow extension <170 degrees
Pulmonary None Spontaneous pneumothorax
Apical bulla
Skin None Stria
Recurrent or incisional hernia
Central nervous system Lumbosacral dural ectasia
aNeed major criteria in two organ systems + minor criteria in a third system.
See references (154) (e627).
Menkes steely hair syndrome (gene Xq12-13) is an X-linked, recessively transmitted deficiency state associated with the defective
intestinal absorption of copper. The disease manifestations result from reduced activities of the numerous copper-dependent
enzymes. One such enzyme, lysyl oxidase, plays a role in formation and repair of extracellular matrix material. With impaired enzyme
activity, vessel wall tensile strength is diminished leading to aneurysm formation in the high flow vessels (e635,e636). Arterial walls
exhibit abnormalities of the internal elastic lamina at both the light and electron microscopic level (159) (see Chapter 5).
Table 13-41 ▪ INHERITED HYPERCHOLESTEROLEMIAS
Disorder Inherit. Gene Product Gene CVD

Childhood
Homozygous familial AD Low-density lipoprotein receptor LDLR Yes

hypercholesterolemia
Heterozygous familial AD Low-density lipoprotein receptor LDLR No

Familial defective apolipoprotein B AD Apoplipoprotein B-100 APOB No
PCSK9 gain- of function AD Proprotein convertase PCSK9 No

subtisilin/kexin 9
Autosomal recessive AR ARH adaptor protein ARH

Variable
Phytosterolemia AR ATP-binding cassette g5, g8 ABCG5ABCG8 Yes

Inherit., Inheritance pattern; CVD, cardiovascular disease; AD, autosomal dominant; AR, autosomal recessive.
From Rahalkar AR, Hegele RA. Monogenic pediatric dyslipidemias: classification, genetics and clinical spectrum.
Mol Genet Metab 2008;93: 282-294.
Atherosclerosis
A spectrum of inherited disorders cause congenital hypercholesterolemia; the complicating atherosclerotic cardiovascular disease
occurs in childhood in a subgroup of these disorders (160) (Table 13-41). Atherosclerosis in childhood has been best described in
familial hypercholesterolemia caused by a mutation in the gene encoding the receptor for low-density lipoprotein located on
chromosome 19p13.2 (e637). Mutations in this gene occur frequently with heterozygotes identified at a 1:500 frequency (160)
(e638). About one person in a million is a homozygote resulting in plasma cholesterol levels in excess of 650 mg/dL from infancy
(e637). Study of an involved 20-week fetus revealed lipid deposits already present in the aorta intima (e639). Aortic atherosclerosis,
although generalized, tends to be worse in the ascending aorta near the coronary arteries, and in the thoracic segment
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and can result in supravalvular aortic stenosis as well as stenosis of the coronary artery ostia (161) (e640). Deposits of foam cells in
the aortic and mitral valves with fibrosis and cholesterol clefts also cause valvular stenosis or insufficiency (161) (e640). Disease is
widespread throughout the coronary arteries, and death from coronary artery disease can occur as early as 3 years of age (e641).
Treatment modalities include plasmapheresis, high-dose statins, and bypass surgery to treat the coronary artery disease as well as
the possibility of liver transplantation to reverse the metabolic defect (e8).
Vasculitis
Vasculitis by definition is an inflammatory, even destructive, process involving arteries and veins that can occur as one of many
manifestations in a broad spectrum of infectious and inflammatory disorders. Involvement of the heart and great vessels occurs
predominantly in two of these vasculitic disorders: Kawasaki disease and Takayasu arteritis.
Kawasaki Disease (Mucocutaneous Lymph Node Syndrome)

Kawasaki disease (KD), also called mucocutaneous lymph node syndrome, is an acute febrile exanthematous vasculitis that affects
infants and young children. First reported in Japanese children in 1967 (e642), KD is now recognized worldwide. Although the initial
febrile illness is self-limited, the vasculitic damage to the coronary arteries can lead to ischemic heart disease and occasionally
death. In the developed world, KD now represents the most common cause of acquired heart disease in childhood (e643).
KD occurs almost exclusively in young children with the peak incidence at 6 months to 1 year, and greater than 75% of cases
occurring before 5 years of life. There is a slight male predominance (M:F 1.5:1) and a prominent racial trend (Table 13-42) (162,
163) (e643,e644). This racial trend is reflected in the reported incidence of KD in Japan of 137.7 cases per 100,000 children
compared with that in the United States of 17.1 per 100,000 children (e645,e644).
Although the epidemiologic features of KD, including its acute febrile nature, seasonal occurrence, age of onset, and temporal and
geographic clustering point to an infectious etiology, a causal agent continues to elude detection. The possibility that it represents a
response to superantigens of group A Streptococcus or Staphylococcus aureus has attracted a lot of attention but has not been
proven (e646). An alternative theory suggests an antigen-mediated immune response with IgA plasma cells playing a central role
(e644). Epidemiologic data (Table 13-42) also suggest an underlying genetic predisposition to developing KD (e647).
Table 13-42 ▪ KAWASAKI DISEASE EPIDEMIOLOGY: GENETIC FACTORS
Seasonal incidence
Japan—biphasic winter and summer peak
United States—winter/spring peak
Race-specific incidence (highest to lowest)
Asia or Pacific Island

African American
Hispanic
Caucasian
Sibling risk 2.1 % = 10 × increase
Twin risk 13%
Data compiled from references (162, 163) (e643,e644,e645,e647).
The disease typically presents as a sudden febrile illness in young children between 6 months and 5 years of life. The fever, which
lasts 5 days or more, is accompanied by the development of bilateral conjunctivitis, erythematous changes of the lips and oral
cavity, a nonvesicular polymorphous rash of the trunk, erythematous desquamation of the palms and soles, and cervical
lymphadenopathy (162, 163). This constellation of features (Table 13-43) evolves over a 10-day period of time, often obscuring the
diagnosis particularly in the early stages. Affected children often manifest a marked increase in acute-phase reactants and the
erythrocyte sedimentation rate is generally elevated (162). This initial febrile illness is self-limited, but in 20% to 35% of patients the
underlying vasculitis leads to coronary artery aneurysm formation (164, 165) (e648). The risk of coronary artery disease is highest
in young infants, a group in which the symptoms are also most likely to be incomplete (e649). The natural history of the aneurysms
depends on their size and shape (164). Overall, 50% of the aneurysms resolve in the first 2 years; 20% become stenotic. Giant
aneurysms (≥8 mm), which account for 20% of all aneurysms, do not resolve and 45% become stenotic. Although the death rate
from Kawaski disease overall is much below 1%, up to 40% of children with stenotic vessels experience myocardial infarction, with
death in 18% (164). The advent of intravenous immunoglobulin therapy has dramatically reduced the incidence of coronary artery
aneurysms, particularly when given in the 10 days of illness (e650).
Table 13-43 ▪ CLINICAL CRITERIA FOR KAWASAKI DISEASE DIAGNOSIS
Fever persisting for ≥5 days
Presence of at least four of the following clinical features:
Bilateral nonpurulent conjunctivitis
Oral mucosal changes
Erythema, cracked lips, strawberry tongue or pharyngeal injection
Polymorphous exanthemous skin rash
Extremity changes
Acute = desquamative erythema of palms and soles; edema of hands and feet
Subacute = periungual peeling of digits
Cervical lymphadenopathy (usually unilateral)
Patients with fever and less than four clinical features
can be diagnosed with Kawasaki disease if coronary

artery changes are identified with echocardiography or
angiography
Compiled from references (162, 163).
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FIGURE 13-29 ▪ Kawasaki disease. The heart from this 4-year-old is enlarged, has excessive fat deposition, and shows thick
dilated coronary arteries.
The pathologic features of Kawaski disease are limited to autopsy studies and therefore represent the most severe pathologic
changes. Within the first 10 days, vessel walls appear edematous with acute inflammation in the perivascular soft tissue and vasa
vasorum without inflammation or necrosis in the media. From 12 to 25 days, the inflammation extends into the artery wall with a
mixed inflammatory infiltrate including lymphocytes, plasma cells, and eosinophils accompanied by necrosis, thrombosis, and
granulation tissue formation. Healing of the inflammation and organization of thrombi lead to aneurysm formation and luminal
stenosis (166) (e651) (Figure 13-29). In the acute phase, pericarditis, myocarditis, and endocarditis are often present with
involvement of the conduction system (166) (e652,e651). Overtime myocardial fibrosis and EFE appear. Endomyocardial biopsies
from Kawasaki disease patients also reveal evidence of myocardial fibrosis with or without inflammation although progression to
CMP is not a described feature (e653). In resected regressing aneurysms and coronary arteries from former Kawasaki patients
dying of unrelated disease, intimal thickening with or without organizing thrombus material raises the speculation that KD may lead
to early-onset coronary vascular disease (e654,e655).
Takayasu Arteritis
Takayasu arteritis (167) (e656) represents a form of large vessel vasculitis first described as “pulseless disease” because of
subclavian artery occlusion. The vasculitis manifests in the aorta and its main branches initially as chronic granulomatous
inflammation in the media and adventia of the vessel wall. Subsequent thrombosis and intimal and wall fibrosis may lead to vessel
occlusion; alternatively, the damaged vessel may aneurysmally dilated. Symptoms of fever, malaise, arthralgias, and myalgias
reflect the underlying inflammatory process. Occlusive symptoms, which vary depending on the anatomic location of the involved
vessels, include seizures or stroke, renal hypertension, extremity claudication, aortic regurgitation, and pulmonary hypertension.
Virtually all patients manifest multifocal bruits and/or absent pulses at presentation as a clue to the underlying disease. Takayasu
arteritis has a striking 80% to 90% prevalence in young woman, and occurs most commonly in southeast Asia and Mexico (167)
(e656). The underlying etiology for this rare disorder remains unknown.
Endocardial Diseases
The major pathologic process that affects the endocardium is endocarditis, defined by the presence of inflammatory cells within the
endocardium. With few exceptions, the surface of an inflamed endocardium is marked by friable or partly healed excrescences
termed vegetations. Although the heart valves are the most common sites, endocarditis also occurs on atrial walls, along the
chamber trabeculae, and on the papillary muscles or chordae tendineae. Endocarditis due to microbial infection has classically been
termed bacterial endocarditis and without infection nonbacterial endocarditis. With the increasing incidence of fungal endocarditis in
recent years, the more general terms infective and noninfective endocarditis are replacing the classic language.
Noninfective Endocarditis
Noninfective endocarditis does not occur commonly in childhood. In a review of large published series of nonbacterial thrombotic
endocarditis, only 3.2% of the reported cases occurred under 20 years of age (168). Noninfective endocarditis can be further
subdivided into three groups (Table 13-44) with the rheumatic and Libman-Sacks forms discussed previously.
Nonbacterial thrombotic endocarditis is believed to occur in the setting of endothelial/endocardial injury serving as a nidus for
platelet aggregation and thrombus formation. The associated vegetations, characterized by single or multiple, white-tan to pink,
friable, verrucous projections of variable size, lie along the contact margins of the valve leaflets (Figure 13-19). Vegetations may
occur as obvious warty, nodular or, sessile lesions occupying part or all of a valve leaflet, or they may be so small as to escape
detection until coming to light under the microscope. They consist of fibrin strands among which lie trapped platelets, scattered
erythrocytes, and occasional leukocytes. The underlying valves may appear normal or may be thickened and fibrotic (168) (e657).
The actual valvular inflammatory reaction is minimal, in contrast to the pronounced reaction seen in infective endocarditis (IE).
Visceral emboli are common, occurring in about 40% of cases, and resemble the parent lesions on the heart valve (168).
P.563
Table 13-44 ▪ FEATURES OF INFECTIVE AND NONINFECTIVE ENDOCARDITIS
Infective Endocarditis Noninfective Endocarditis
Bacterial Fungal Rheumatic Thrombotic Libman-Sacks
Underlying
disease
CHD Surgically repaired Rheumatic fever Indwelling arterial Systemic lupus

CHD catheter erythematosis
Rheumatic heart Prosthetic valve Malignancy

disease
Prematurity Immune deficit Hypercoagulable

states
IV drug abuse Severe burns

Valve before onset of endocarditis
Normal Normal Abnormal Normal Normal Normal

Prosthetic
Abnormal
Appearance of vegetations
Variable size Large Small (<4mm) Small, uniform size Variable size
Tan and friable Friable Row near cusp Friable Ventricular surface
margin
Patchy along cusp

margin
Valve ulceration and perforation
Ulceration ± Ulceration ± No No No
Perforation ± Rare perforation
Mural involvement
Rare Rare Rare Rare Common
Common sites
Septal defects Prosthetic valve Mitral valve Neonates—right-heart Tricuspid valve

valves
Suture lines Suture lines Aortic valve Others—left-heart Mitral valve

valves
Damaged valve
Peripheral embolization
Common Common Rare Occasional Rare
Often large
Neonates primarily have right-sided lesions associated with the use of intracardiac catheters, persistent fetal circulation, and
disseminated intravascular coagulation (e658,e659). In older children and adults, the vegetations occur more often on the aortic and
mitral valves and are associated with underlying malignancy, hypercoagulable states, septicemia, and extensive burns (168).
Infective Endocarditis
IE also occurs infrequently in children, making epidemiology data difficult to ascertain. The overall incidence of IE seems to be on
the rise (e660) and the few pediatric studies available suggest a similar trend in children (e661). In the past, rheumatic fever served
as the major underlying condition. Although this remains true in much of the developing world (169), in developed countries
underlying congenital heart defects serve as the nidus for infection in the majority of pediatric patients (Table 13-45) (170) (e662-
e664). The degree of risk for developing IE varies with the type of defect; the highest risk occurs in patients with complex cyanotic
heart defects, prior episodes of endocarditis, or repairs that include placement of shunt or prosthetic valve material (171) (e665).
Neonates with IE do not usually have underlying congenital heart defects; risk factors include prematurity and the presence of
central vascular catheters (e666,e667).
The clinical presentation for children with IE includes fever and malaise with a new or changing heart murmur, when detectable,
serving as a clue (e661,e665). Embolic phenomenon occurs in approximately 15% of patients (169, 170) (e662). With longstanding
disease, splenomegaly and immunologic stimulation leading to hypergammaglobulinemia, autoantibody formation, and immune
complex deposition may further confuse the clinical picture (e661).
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Neonates more often present with a septic picture including thrombocytopenia, disseminated intravascular coagulation, and septic
emboli (e661). Echocardiographic demonstration of vegetations aids in the diagnosis of IE, particularly in children without complex
heart defects (170) (e661,e665). Diagnosis depends on clinical or pathologic findings (Table 13-46) that include positive blood
cultures and echocardiographic identification of vegetations (e668).
Table 13-45 ▪ PEDIATRIC INFECTIVE ENDOCARDITIS CARDIAC ANATOMY
New York City, USA (170)
Complex cyanotic heart disease 35%
Normal anatomy 31%
Ventricular septal defect 15%
Other acyanotic heart disease 8%
Mitral valve prolapse 6%
Rheumatic heart disease 5%
Lahore, Pakistan (169)
Rheumatic heart disease 53%
Ventricular septal defect 18%
Tetralogy of Fallot 9%
Patent ductus arteriosus 9%
Aortic stenosis 4%
Complex congenital heart disease 4%
Myocarditis 2%
The formation of infected vegetations begins with endothelial injury or erosion, often at a site of turbulent blood flow. The injury site
serves as a nidus for fibrin clot formation. Gram-positive organisms, which account for 90% of identified organisms, have a
propensity to adhere to fibronectin and lamin within the clot material and activate further clot formation (e661). In the past,
Streptococcus viridans was the most common organisms causing IE; in more recent years, S. aureus has become nearly as
common (170) (e661,e662). Gram-negative, fastidious, and fungal organisms are identified infrequently, occurring most often in the
setting of prior heart surgery, underlying immune deficiency, or central line placement (Figure 13-30) (e661).
Table 13-46 ▪ DIAGNOSIS OF INFECTIVE ENDOCARDITIS
Modified Duke Criteria for Pathologic or Clinical Diagnosis
Pathologic Criteria
Organisms identified in vegetation, embolized vegetation, or cardiac abscess by either culture or histology
OR
Pathologic lesions present (vegetation or cardiac abscess) + histologic confirmation of endocarditis
Clinical Criteria
Two major criteria
OR
1 major and 3 minor criteria
OR
5 minor criteria
Major Criteria
1. Positive blood culture
—Typical IE microorganisms from ≥ 2 blood cultures
OR
—Microorganisms consistent with IE from
blood cultures drawn ≥12 hours apart or
all of 3 or majority of 4 or more blood cultures with
first and last ≥1 hour apart
2. Evidence of endocardial involvement by
—Positive echocardiogram of IE
Oscillating intracardiac mass or
abscess or
New partial dehiscence of prosthetic valve
OR
—New valvular regurgitation
Minor Criteria
1. Predisposing heart condition or intravenous drug use
2. Fever <38°F
3. Vascular phenomena
Major artery emboli, pulmonary infarct, intracranial
hemorrhage
4. Immunologic phenomena
Glomerulonephritis, Osler nodes, Roth spots, rheumatoid
factor
5. Microbiologic evidence
Positive blood culture not meeting major criteria or
serologic evidence of active infection with consistent
organism
6. Echocardiographic evidence
consistent with IE but not meeting major criteria
IE, infective endocarditis.
See references (e661,e665,e668).

FIGURE 13-30 ▪ Close-up view of the left ventricular outflow tract from a child with bicuspid aortic valve and Streptococcus
endocarditis. Two large irregular vegetations obscure and partially destroy the aortic valve leaflets.
The infected vegetations tend to occur on the atrial surface of the AV valves and the ventricular surface of the outflow valves (172).
In neonates without underlying heart malformations, the vegetations are often right sided (e666). With underlying heart
malformations, the vegetations may occur at the edge of VSDs or at the site of flow turbulence on the ventricular or malformed valve
surface (172). The infective organisms elicit an acute inflammatory response leading to destruction and perforation of the valve
tissue. The infection may spread into the adjacent vessel or heart tissue leading to abscess or fistula formation. Microscopically,
acute vegetations consist of granular, heaped-up layers of fibrin, platelets, necrotic materials, and polymorphonuclear leukocytes.
Organisms may or may not be identified. Similar neutrophilrich infiltrates with granulation tissue formation help distinguish infected
from noninfected prosthetic valve specimens (e669). With time (and antibiotic treatment), organisms are lost and the damaged valve
tissue undergoes calcification, chronic and at times granulomatous inflammation, and granulation tissue formation. Whether a native
valve was initially normal or not, the subsequently damaged valve becomes a potential site for recurrent endocarditis.
Pericardial Diseases
Pericarditis
The two-layered pericardium forms a sac around the heart that normally contains less than 30 mL of serous fluid. However, when
the pericardium becomes inflamed, the normally smooth mesothelium lining the sac becomes rough. A fibrinous exudate, rich in
fibrinogen and other plasma proteins,
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accumulates a dull-colored film over the pericardial surface. Friction between the two roughened surfaces results in the pericardial
friction rub that serves as a clinical marker of pericarditis. In many instances, pericarditis also leads to increased fluid volume in the
pericardial sac. The serous versus purulent versus hemorrhagic nature of this fluid varies with the underlying etiology.
Table 13-47 ▪ CAUSES OF PERICARDITIS
Infectious agents
Viral
Bacterial
Fungal
Parasitic
Immunologically mediated
Rheumatic fever
Systemic lupus erythematosus
Scleroderma
Postcardiotomy syndrome
Drug hypersensitivity
Other
Uremia
Postsurgical
Neoplasia
Trauma
Radiation
Pericarditis occurs in a wide variety of clinical settings (Table 13-47) (173). When the etiology is viral or noninfectious, a serous
effusion, rich in protein containing lymphocytes, accompanies a fibrinous exudate. Viral pericarditis typically follows a respiratory or
gastrointestinal illness with enteroviruses the common responsible viral agent (173). Noninfectious pericarditis may complicate a
variety of systemic illnesses, including rheumatic fever, systemic lupus erythematosis, juvenile rheumatoid arthritis, and KD (173,
174). Postoperative pericardial effusions occur in approximately 15% to 25% of children undergoing open heart surgery (175)
(e670). Approximately 25% of these postoperative effusions become symptomatic (postpericardiotomy syndrome) (175) (e670).
Purulent pericarditis is largely caused by pyogenic bacteria, with the most common cause in North American children being S.
aureus (173, 174) (e671). Purulent pericarditis usually results from a primary infection spreading to the pericardium either by direct
extension from an adjacent purulent pneumonia, mediastinitis, or empyema, or by hematogenous seeding from pyelonephritis or
osteomyelitis (174). Most patients are acutely ill with fever, tachypnea, and even chest pain. A shaggy, thick, yellow or gray exudate
covers the pericardial surfaces (Figure 13-31). Organisms are numerous, and large numbers of neutrophils infiltrate the strands and
local tissues.
Tuberculous pericarditis results as a direct extension of infection from tracheobronchial lymph nodes or from hematogenous spread.
The clinical onset may be insidious with fever and chest pain. Pericardiocentesis returns often bloody fluid containing numerous
lymphocytes and few neutrophils. Acid-fast bacilli can be identified in fluid smears from 15% to 40% of patients, and biopsy of the
thickened pericardium often reveals caseating granulomas (173).
FIGURE 13-31 ▪ The opened pericardium in an immune-compromised patient with disseminated Aspergillus infection. The
pericardium appears thickened and shaggy due to the intense inflammatory response to the infection.
Chronic or healed pericarditis manifests in two major patterns, adhesive (obliterative) and constrictive. Adhesive pericarditis is
characterized by the presence of small nodules of vascularized granulation tissue between fibrin aggregates and mesothelial cell
proliferations leading eventually to partial or complete obliteration of the pericardial cavity. In the more clinically significant
constrictive pericarditis, the heart becomes encased in a dense fibrous and even calcified shell, the rigidity of which may
mechanically interfere with cardiac diastolic function and venous return to the atria (173).
Developmental Abnormalities
Congenital aplasia of the pericardium occurs either as complete or partial absence of the parietal pericardium. When complete, the
defect is usually asymptomatic. Partial deficiency, which occurs mostly on the left side of the heart, may be complicated by
herniation and strangulation of myocardium (176). Small defects may be associated with other congenital mediastinal lesions, such
as bronchogenic cysts, pulmonary sequestration, and ectopia cordis. The defect is thought to result from a failure of the normal
pleuropericardial foramen to close in the pleuropericardial membrane.
Pericardial cysts are thin-walled, generally unilocular structures filled with clear fluid that tend to be benign and asymptomatic. They
are encountered in children only rarely at autopsy. The cysts are most often located at the costophrenic angles but may also appear
higher in the mediastinum. The cysts vary markedly in size with mesothelium lining the thin walls of fibrous tissue. Though believed
to be
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developmental, the embryonic origin of these cysts remains unclear (e672).
Table 13-48 ▪ TUMOR INCIDENCE BY AGE
Age Other
Ref. Range Rhabdomyoma Fibroma Teratoma Myxoma Histiocytoid benign Malignant
No. (No.) (%) (%) (%) (%) (%) (%) (%)
186 Fetal 64 11 22 0 0 6 0
(89)
Neonatal 47 16 15 4 11 5 2
(135)
194 <1 year 54 23 3 0 6 6 8

(35)
1-16 5 24 0 19 0 14 38
year (21)
e719 <1 year 58 13 19 0 0 6 4

(48)
1-15 39 14 12 14 12 9
year (89)
177 0-17 79 11 2 0 0 8 —
year (56)
(Benign
only)
Cardiac Tumors
Primary cardiac tumors occur rarely in both adults and children. In a review of 22 large autopsy series, Reynen (e673) identified a
frequency of 0.02%, or 200 primary tumors in 1 million autopsies in a general population. A large autopsy series of infants and
children yielded an estimated frequency of 0.08% (e674). Even among infants presenting for evaluation of cardiac disease, tumors
account for only 0.2% to 0.4% of lesions (1, 177). New imaging techniques have led to increased numbers of tumors identified
during life. Fetal echocardiographic studies report cardiac tumors in 0.11% to 0.14% of referred pregnancies (178) (e675) with
prenatal ultrasound identifying 21% of congenital cardiac tumors in one study (177).
The type of primary cardiac tumor present varies considerably with age (Table 13-48). In fetuses and newborn infants,
rhabdomyomas account for the vast majority followed by pericardial teratomas (178). During the first 2 years of life, rhabdomyomas
continue to be the most common tumor, with fibromas representing the second most common (179) (e676). Myxomas, the most
common tumor in adults, account for 6% of tumors in children, occurring almost exclusively in adolescents (179). In rare instances, a
cardiac tumor (especially rhabdomyoma) occurs in association with a heart malformation (180) (e677,e678). The space-occupying
aspect of these tumors suggests that they in fact may play a role in inducing the associated malformation.
Primary Tumors
Rhabdomyomas
The most common cardiac tumor in infants occurs as a solitary or, more often (77% to 90%) (181, 182) (e679-e681) multiple,
nodules any where in the heart (Figure 13-32). They are highly associated with tuberous sclerosis (TS), occurring in 40% to 60% of
patients examined echocardiographically (180, 181). Rhabdomyomas may be the first clue to the diagnosis of TS, with 70% to 80%
of fetuses and infants carrying rhabdomyomas subsequently having a diagnosis of TS confirmed (181) (e679,e680,e682).
Rhabdomyomas display a striking propensity to regress spontaneously. When identified in infancy, 50% to 70% will regress,
especially when associated with TS (180) (e675,e679,e683). Tumors identified in older children are less likely to regress (180).
Clinically, many rhabdomyomas remain asymptomatic. Larger rhabdomyomas may project from the ventricular wall or septum into
the cardiac cavity and obstruct cardiac flow or valvular motion (181) (e680,e681). Disruption of the conduction system with resultant
arrhythmias also frequently occurs (180, 181). The tumors come to light in fetuses due to nonimmune hydrops, arrhythmias, a mass
noted on routine prenatal ultrasound, or a family history of TS (178) (e679). Postnatally, tumors may present clinically with a
murmur, heart failure, arrhythmia, or sudden death. When symptomatic, partial resection to relieve symptoms may be required, but
more aggressive surgical intervention is contraindicated (e684).
Grossly, rhabdomyomas appear as well-circumscribed, yellow-to-gray nodules that vary in size from microscopic to 10 cm in
diameter (183). Usually multiple, they occur with about equal frequency in either ventricle. They also may occur in the atrial walls;
they have not been described in cardiac valves (182). Microscopically, the typical rhabdomyoma cells are much larger (up to 80
mm) than those of normal myocardium, due to accumulation of glycogen within
P.567
the cell cytoplasm, resulting in the formation of “spider cells”. By electron microscopy, the “spider cells” contain diffuse glycogen,
few myofibrils, scattered leptofibrils, and poorly developed sarcoplasmic reticulum. Well-formed intercalated disc-like intercellular
junctions surround the periphery of the cells, mimicking cardiac myoblasts (182) (e681,e685). The clinical pattern of multiple tumors
that tend to regress, combined with the ultrastructural appearance of the tumor cells, led to the conclusion that cardiac
rhabdomyomas represent a hamartomatous rather than a neoplastic process (182) (e681).
FIGURE 13-32 ▪ Multiple small rhabdomyomas. In this opened left ventricular cavity, the trabeculae appear thickened and
somewhat pale due to multiple small rhabdomyomas highlighted by asterisks. These rhabdomyomas were asymptomatic in this child
with tuberous sclerosis.
Cardiac Fibroma
The second most common tumor outside the fetal period presents most frequently (>1/3 of cases) in the 1st year of life with the
remaining spread out over the following two decades (184). These fibromas characteristically arise as a single ventricular mass in
an otherwise normal child, although a small subgroup occur with Gorlin syndrome (184) (e686,e687). They present with
cardiomegaly, arrhythmias, and heart failure; sudden death occurs in one-third of cases, probably as the result of arrhythmias or
outflow obstruction (182, 184). The tumors arise most frequently in the left ventricle or intraventricular septum, but may also occur in
the right ventricle and occasionally in the atria (183, 184). They reach large size, occasionally exceeding 10 cm (182). On cut
surface, these firm white trabeculated tumors grossly resemble a leiomyoma (Figure 13-33). Microscopically, the tumors display a
monomorphic population of bland spindled cells embedded within a variably collagenized stroma that often appears infiltrative at the
periphery (182). Tumors from young infants tend to appear more cellular and mitotically active, features that do not denote more
aggressive behavior (183, 184). In older children, calcification and focal cystic degeneration become more prevalent. Although
benign, cardiac fibromas do not regress and in fact tend to slowly increase in size (183). Surgical excision is the treatment of choice
(183) (e688); unresectable tumors may require transplantation (183) (e689).
FIGURE 13-33 ▪ Transverse section of an explanted heart as viewed from the back. A white firm trabeculated mass replaces the
interventricular septum and protrudes into the left and right ventricular cavities. A probe inserted into the aortic valve exits into the
left ventricular chamber under the mitral valve leaflet, highlighting the obstruction to the left ventricular outflow caused by this large
cardiac fibroma.
Teratoma
Teratoma represents the second most common cardiac tumor in fetuses, with 50% of this rare tumor being diagnosed before or
during the 1st month of life and two-thirds in the 1st year (185, 186). In its more common intrapericardial location, the tumor
originates from the external surface of the heart base and gives rise to an often marked pericardial effusion (185, 187).
Compression of the heart by the mass combined with the effusion leads to nonimmune hydrops in the fetus and cardiac tamponade
in infants (186). Intrauterine pericardiocentesis is reported to effectively relieve the fetal distress (e690-e692). Surgical excision is
curative (186) (e693). Rarely teratomas occur in the intraventricular septum where they clinically mimic cardiac rhabdomyomas and
fibromas (185) (e693,e694). The pathologic features of the tumor, when reported, are similar to benign teratomas occurring
elsewhere in the body (186) (e693,e695). Intrapericardial bronchogenic cysts overlap clinically with teratomas and may be included
as teratomas in the older literature (185, 187).
Myxomas
They present the most common (50% to 75%) cardiac tumor in adults (182) (e696,e694) but account for only 5% of tumors in
infancy and 15% to 20% of tumors in older children and adolescents (179, 182, 186). They arise from endocardium, usually
adjacent to the fossa ovalis, in the left (75%) or right (18%) atrium (182, 188). Presenting symptoms include one or more
components of a clinical triad (Table 13-49) (182, 188, 189). Although the vast majority of atrial myxomas occur sporadically,
approximately 5% occur in families as part of the familial atrial myxoma syndrome (Carney complex) (Table 13-49) (190, 191)
(e697,e698). The Carney
P.568
complex, an autosomal dominant disorder, results from a mutation in the PRKAR1A gene on chromosome 17q2 in 90% of cases
(190, 191). The syndromic form of atrial myxoma tends to occur at a younger age, in atypical locations, and with a higher frequency
of multiple recurrent tumors as compared with the sporadic form (192) (e699). Atrial myxomas occurring in children and adolescents
should therefore elicit a search for other manifestations of this complex in both patients and other family members.
Table 13-49 ▪ ATRIAL MYXOMAS—CLINICAL FEATURES

Clinical Triad (188, 189)
Valvular obstruction
Tumor emboli
Constitutional symptoms
Malaise, fever, weight loss, anemia, elevated ESR, hypergammaglobulinemia
Carney Complex (190, 191) (e698)
Skin lesions: Lentigines, blue nevi, Myxomas: cardiac, breast, skin, mucus membranes, bone
Endocrine abnormalities:
Adrenal pigmented cortical hyperplasia
Pituitary adenoma
Thyroid nodules
Breast adenomas
Testicular large cell calcifying Sertoli cell tumors
Identical pathologic features occur in syndromic and sporadic forms of atrial myxoma (182, 183, 188, 193). Grossly, the tumors
appear gelatinous with either a narrow or a broad base, and a frond-like or smooth surface. Cut surface appears variegated with
scattered gritty calcification. Microscopically stellate or elongate cells with scant eosinophilic cytoplasm disperse singly or as small
nests, trabeculae or perivascular rings in an acid mucopolysaccharide-rich myxoid matrix. With immunocytochemical stains, the cells
mark reliably with vimentin and variably with endothelial, actin, and cytokeratin markers (189, 193).
Histiocytoid Cardiomyopathy
This represents a rare myocardial disease of infancy and early childhood characterized by cardiomegaly, incessant ventricular
tachycardia, and sudden death. More than 70 cases have been reported under a variety of synonyms, including isolated cardiac
lipidosis, xanthomatous CMP, foamy myocardial transformation of infancy, oncocytic CMP, myocardial hamartoma, and Purkinje cell
tumor (194, 195) (e700-e702). The lesion presents almost exclusively in the first 2 years of life with a 75% predominance in girls
(194). Both cardiac and noncardiac malformations occur in a subgroup of these patients including atrial and VSDs, EFE, hypoplastic
left heart, corneal opacities, microphthalmia, cataracts, cleft palate, hydrocephalus, agenesis of the corpus callosum, and renal
cysts (194) (e702).
At surgery or autopsy, the heart is often enlarged with the left ventricular surface studded by multiple flat to round, smooth, yellow to
tan-white nodules that may or may not be visible to the naked eye. Similar nodules may also occur on the papillary muscles, right
ventricle, atria, and all four heart valves (194, 196) (e703). Histologically, the nodules contain cells that differ from the adjacent
myocardial cells in both their larger size (20 to 40 mm diameter) and the pale foamy nature of their cytoplasm, which gives them their
histiocytic appearance (Figure 13-34). Nodules of similar cells are also often present in the conduction system, the midmyocardium,
and beneath the epicardium. Immunocytochemical stains identify the cells as myocardial in origin based on positive muscle-specific
actin and myosin, and negative lysozyme and CD68 (197). These foamy cells contain only small amounts of glycogen, and lipid; the
mitochondria-rich nature of the cytoplasm becoming evident only at the ultrastructural level. By electron microscopy, the large
unusual cells have the configuration of swollen abnormal myocytes that contain abundant mitochondria with only rare peripherally
placed myofibrils, scattered leptofibrils, no T tubules, and decreased numbers of the usual desmosomes (196, 197) (e702,e704).
FIGURE 13-34 ▪ Histiocytoid cardiomyopathy. Enlarged, granular-appearing myocytes on the upper left contrast with the normal
compact myocytes on the lower right. (Hematoxylin and eosin stain, original magnification 100×.)
The pathogenesis for this unusual pathologic condition remains controversial. The ultrastructural features suggest a relationship
with primitive Purkinje cells (196) (e705) or primitive myocardial cells and support a hamartomatous process. Comparable cellular
changes may occur in other organs of infants with the cardiac lesions (e704,e706). The possibility of an underlying mitochondrial
disorder has been raised by the finding of respiratory chain enzyme deficiencies and mtDNA mutations in a few cases (195)
(e701,e707). The possibility of an X-linked chromosomal abnormalities has been suggested in a few other cases (198) (e708).
Unfortunately, the diagnosis of histiocytoid CMP is most often made at autopsy. When the presenting ventricular arrhythmias can be
initially controlled medically, subsequent electrophysiologic mapping and surgical ablation of the lesions can lead to long-term
survival (e590,e709). Cardiac transplantation in a single case has also been reported (e700).
Other Benign Tumors

A variety of other benign tumors and malformations have been described in the heart (Table 13-50). The vascular tumors may occur
in the setting of multiple cutaneous hemangiomas (186).
Table 13-50 ▪ OTHER BENING CARDIAC TUMORS AND MALFORMATIONS
Hemangioma and vascular malformation 4%
Mesothelioma of AV node 3%
Neurofibroma <1%
Bronchogenic cysts
Lipoblastoma
Inflammatory pseudotumor
Lipoma
Multicystic hamartoma
See references (179, 182, 183) (e710,e720).
P.569
Table 13-51 ▪ MALIGNANT CARDIAC TUMORS
Primary (e510,e721-e724)
Rhabdomyosarcoma
Fibrosarcoma
Undifferentiated sarcoma
Angiosarcoma
Malignant germ cell tumor
Leiomyosarcoma
Malignant nerve sheath tumor
Pleomorphic sarcoma
Synovial sarcoma
Myxosarcoma
Secondary (metastatic) (199) (e725,e726)
Non-Hodgkin lymphoma
Neuroblastoma
Wilms tumor
Hepatoblastoma
Hepatoma
Rhabdomyosarcoma
Undifferentiated sarcoma
Osteosarcoma
Adrenal carcinoma
Ewing sarcoma
Endodermal sinus tumor
Brain tumor
Hodgkin disease
Pleuropulmonary blastoma
Malignant Tumors
Malignant tumors account for less than 1% of the primary cardiac tumors in the fetus and newborn. In older infants and children,
malignancies become more prevalent, accounting for 10% to 20% of primary cardiac tumors (179, 182) (e710). A broad range of
diagnoses encompass the remaining reported cases (Table 13-51).
In children, as in adults, metastatic tumors outnumber the primary cardiac malignancies. In a review of records from Hospital for Sick
Children in Toronto over a 62-year period, Chan et al. (199) identified 16 primary cardiac tumors of which only one was malignant.
Over the same time period, 59 secondary malignant tumors were identified in the heart, of which 45 were distant metastases and 14
resulted from direct extension (199). Table 13-51 outlines the range of metastatic tumor types identified.
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Chapter 14
The Gastrointestinal Tract
John Hart
Rebecca Wilcox
Chrisopher R. Weber
EMBRYOLOGY
The gastrointestinal tract is derived largely from the endodermal germ layer. During the 3rd week of embryonic
development, cephalocaudal and lateral folds of the trilaminar germ disk develop and progressively incorporate parts of
the endoderm-lined yolk sac into the body cavity to form a tubelike gut. By the end of week 3 of gestation, an open
connection between the anterior portion of this tube, the foregut, and the amniotic cavity is established at the site of the
future mouth. During early embryonic life, the vitelline or omphalomesenteric duct provides an open connection between
the midgut and the yolk sac (Figure 14-1). This connection becomes progressively longer and narrower as gestation
proceeds and eventually forms part of the umbilical cord. By week 10, the communication between the lumen of the
midgut and the umbilicus becomes obliterated and soon disappears (e328).
The laryngotracheal diverticulum develops from the ventral foregut during week 4 of gestation (Chapter 12). Gradual
formation of an esophagotracheal septum along the length of the laryngotracheal diverticulum separates the ventral
respiratory and the dorsal digestive tubes (Figure 14-2).
FIGURE 14-1▪During week 4 of gestation, head and tail folds of the embryo surround portions of the yolk sac. An open
connection between the primitive midgut and the yolk sac exists. After this connection narrows, it is known as the
vitelline or omphalomesenteric duct.
During the 2nd month of embryonic life, rapid cellular proliferation within the digestive tube causes a transient partial
obliteration of the duodenal lumen, the so-called solid stage of development. Recanalization occurs by week 8 of
gestation. Rapid midgut growth within the relatively small body cavity results in a temporary herniation of the lengthening
midgut into the umbilical stalk during weeks 6 to 11 (Figure 14-3). During this physiologic herniation, the intestinal loops
rotate counterclockwise, a process that continues as the intestinal loops return to the abdominal cavity during weeks 10
and 11, so that the cecum comes to lie in the right side of the abdomen. If this orderly process fails to occur or is
anomalous, the locations of the small and large intestine, mesentery, and fixation points of the intestine to the body wall
will be abnormal. The hindgut, or posterior portion of the primitive digestive tube, initially ends posteriorly in the
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cloaca, separated from superficial ectoderm by the cloacal membrane (Figure 14-4). A transverse ridge, the urorectal
septum, grows posteriorly from the umbilical stalk and gradually divides the cloaca into a ventral portion, the urogenital
sinus, and a dorsal portion, the future rectum, and anus. This division is normally complete at the end of week 6 of
gestation. The membrane covering the anal canal disappears by week 9, so that communication between the digestive
tract and the amniotic cavity is established caudally.
FIGURE 14-2▪Development of the respiratory system from the foregut at week 4 of gestation. The esophagotracheal
septum develops from the two lateral folds that migrate toward the midline to separate the developing respiratory
diverticulum from the primitive gut.
FIGURE 14-3▪Physiologic gut herniation in an embryo at week 8 of gestation. Rapid elongation of the intestine in a
relatively small abdominal cavity causes the gut to herniate into the umbilical cord. This herniation resolves at the end of
the 3rd month of gestation. A failure in the normal events at this stage explains the omphalocele and malrotation.
FIGURE 14-4▪Primitive hindgut region in an embryo at 6 weeks of gestation. The urorectal septum grows posteriorly to
divide the cloaca into a urogenital portion separate from the intestinal portion. Note the intact cloacal membrane.
DISORDERS OF THE ESOPHAGUS

Congenital Abnormalities
Persistent Embryonic Epithelium
The embryonic and early fetal esophagus is lined by ciliated stratified columnar epithelium. The transformation to
stratified squamous epithelium is usually complete by week 25 of gestation, but occasionally a patch of superficial
columnar epithelium persists at birth, especially in premature infants. Persistent embryonic epithelium is usually found
incidentally at autopsy as microscopic foci in either the proximal or distal end of the esophagus and is of little clinical
consequence. This change is limited to surface epithelium only; glandular mucosa, as in gastric heterotopia of the
esophagus, is not present. Because it is not usually found after early infancy, persistent embryonic epithelium is
presumed to be replaced by squamous epithelium.
Heterotopic Gastric Mucosa (Inlet Patch)

Single or multiple small patches (5 to 30 mm) of gastric cardiac or fundic type mucosa can sometimes be found
incidentally in the cervical esophagus (eFigure 14-1). The incidence in patients undergoing
esophagogastroduodenoscopy has been reported to be 3.6% (120). These patches of heterotopic gastric epithelium
usually are not clinically important and are rarely biopsied since most endoscopists are familiar with them. They can be
colonized by Helicobacter pylori organisms (120). Confusion with Barrett esophagus can occur if the endoscopic
findings are not communicated to the surgical pathologist.
Esophageal Duplication
Duplication of the esophagus is rare. The duplicated segment may be a separate cylindrical tube alongside part of the
normal esophagus with a complete mucosa, submucosa, and two-layered muscularis externa (double esophagus).
Alternatively, a spherical, intramural esophageal cyst may form and share a portion of muscularis propria with the
adjacent esophageal wall. Esophageal duplication occurs most often in the thorax adjacent to the distal two thirds of the
esophagus, but it may also occur in the lateral cervical area. Esophageal duplication cysts may be asymptomatic and
discovered incidentally, or they may cause tracheal or esophageal compression. The epithelium is either stratified
squamous or columnar; the latter is derived from persistent embryonic esophageal ciliated columnar epithelium.
Distinction between esophageal and bronchogenic cysts may be difficult because they occur at similar locations in the
mediastinum and show similar ciliated columnar epithelium. The diagnosis of esophageal cyst is made if a two-layered
muscularis externa is present. A bronchial origin is favored if cartilage or respiratory glands are identified (e396). The
generic designation of “foregut cyst” is used in cases in which the lining epithelium is primitive columnar without the
distinguishing features cited.
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Enteric Cyst of the Mediastinum
Mediastinal enteric (gastroenteric) cyst is distinct from the esophagus; however, because of its location, it may be
confused with esophageal duplication cyst. Mediastinal enteric cyst is found in the right posterior mediastinum in a
retrocardiac position, often extending into the right thorax. Vertebral anomalies, especially cervical hemivertebra, are
associated in a high percentage of cases (e33,e36). A small number of enteric cysts extend through an intervertebral
space into the spinal canal, in which case the designation of neurenteric cyst is given. Enteric cysts are often lined
partially or completely by gastric mucosa, and some present with peptic ulceration, perforation, and hemorrhage. Small-
intestinal mucosa and primitive columnar epithelial lining have also been described.
Esophageal Atresia and Tracheoesophageal Fistula

Esophageal atresia and tracheoesophageal fistula occur together in most cases (e24,e438). The dual anomalies, which
occur approximately once in 3,000 births, result from faulty division of the foregut into tracheal and esophageal channels
during the 1st month of embryonic life. Additional congenital anomalies (usually midline) occur in 50% of these infants,
directly affecting the prognosis. Congenital heart disease, especially ventricular septal defect, patent ductus arteriosus,
and tetralogy of Fallot, are seen in 30% of cases of esophageal atresia, and imperforate anus occurs in approximately
10%. In babies with multiple malformations, the VATER (vertebrae, anal, tracheoesophageal, radial, and renal
anomalies) association or the VATERL (vertebrae, anal, tracheoesophageal, renal, and limb) association should be
considered (e374). Approximately one-third of the infants with esophageal atresia are born prematurely, so that morbidity
is further increased.
Variations in the anatomy of esophageal atresia and tracheoesophageal fistula are diagrammed in Figure 12-10 (see
Chapter 12). Esophageal atresia without tracheoesophageal fistula occurs rarely, and tracheoesophageal fistula without
esophageal atresia (H-type fistula) is even more unusual. The most common type is esophageal atresia with distal
tracheoesophageal fistula, which accounts for 85% of the cases (Figure 14-5). The esophagus ends in a blind pouch in
the upper chest, and the lower portion of the esophagus is connected to the trachea at or near the carina by a
tracheoesophageal fistula less than 0.5 cm in diameter (Figure 12-10A). During breathing, air enters the stomach
through the fistula, and as the stomach becomes distended, gastric secretions pass through the fistula into the lungs,
causing pneumonia. The infant cannot swallow oral secretions or food; attempts at feeding produce regurgitation and
aspiration. The diagnosis is suggested by the inability to pass a tube from the mouth to the stomach and is confirmed by
plain x-ray films of the chest and abdomen. Treatment is surgical and consists of extrapleural transection of the fistula
and anastomosis of the two ends of the esophagus.
Isolated esophageal atresia without an associated fistula, found in 7% to 8% of cases, is usually characterized by blind
proximal and distal esophageal pouches, often separated by a wide gap. Multiple surgical procedures are usually
required for repair.
FIGURE 14-5▪Tracheoesophageal fistula at autopsy (posterior view of thoracic organ block). The blind esophageal
pouch is at the upper left, and the tracheoesophageal fistula arises at the tracheal bifurcation.
In the relatively rare cases of isolated tracheoesophageal fistula without esophageal atresia (H-type fistula), the
diagnosis is often delayed beyond the newborn period. Patients with this anomaly present with coughing or choking
during feeding and with recurrent pneumonia. Histologic study of the tracheoesophageal fistula often reveals foci of
primitive ciliated columnar epithelium, respiratory glands, and even cartilage. These tracheobronchial elements and
abnormalities in smooth muscle may extend for some distance into the distal esophagus.
Esophageal Stenosis
In most cases, esophageal stenosis is an acquired lesion caused by gastroesophageal reflux with severe peptic
esophagitis. However, rare forms of congenital esophageal stenosis have been described resulting from membranous
mucosal rings and webs. Stenotic segments surrounded by respiratory epithelium, submucosal glands, and cartilage
rings derived from remnants of the embryonic tracheoesophageal bud may occur rarely (e220). Sloughing of esophageal
mucosa occurs in inherited epidermolysis bullosa, which is complicated by stenosis.
Acquired Diseases
Gastroesophageal Reflux and Reflux Esophagitis
Gastroesophageal reflux is common during the first few months of life, as evidenced by the frequent occurrence of
effortless regurgitation at this age (e53,e205,e348).
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It is considered a physiologic process secondary to immature esophageal peristaltic and lower esophageal sphincter
function and gradually improves during the 1st year of life. If reflux is excessive during infancy or persists beyond that
period, peptic esophagitis may ensue.
The symptoms of reflux esophagitis differ with the age of the patient. Infants show effortless regurgitation and sometimes
forceful vomiting, excessive irritability, and failure to thrive as a consequence of caloric losses. Children present with
vomiting and poorly characterized abdominal or chest pain. Patients of any age may exhibit gastrointestinal blood loss
(from esophageal ulceration), failure to thrive, and recurrent pulmonary problems (e.g., asthma, pneumonia, and night
cough). Most children with reflux esophagitis are otherwise normal, but certain groups of children are predisposed,
including those with mental retardation, cystic fibrosis, and bronchopulmonary dysplasia, and those who have
undergone repair of esophageal atresia and tracheoesophageal fistula in infancy (e53,e166,e218,e348). Esophageal pH
monitoring, esophageal manometry, and barium esophagography may be used in patient evaluation. If a patient has
signs of esophagitis, such as pain, gastrointestinal blood loss, or failure to thrive, esophagoscopy and esophageal
biopsy are indicated. However, histologic changes are found in only 40% to 50% of symptomatic infants and children
with clinical evidence of reflux esophagitis (e101,e501).
Histologic features in children with reflux esophagitis are similar to those widely described in adults with the same
condition (e42,e89,e101,e154,e382,e421) (Figure 14-6). Diagnostic histologic findings include intraepithelial
lymphocytes (IELs), neutrophils and eosinophils, basal cell hyperplasia (>15% to 20% of total epithelial thickness),
papillary elongation (>50% to 66% of epithelial thickness), and dilated intraepithelial spaces (2, 100). Basal cell
hyperplasia and papillary elongation are the most sensitive histologic features of reflux, but unfortunately these features
cannot be assessed accurately in poorly oriented specimens. They are also common in biopsies obtained from near the
squamocolumnar junction (Z-line) in patients without reflux. Dilated intracellular spaces between the squamous cells
(spongiosis) are also a sensitive marker of epithelial cell injury, usually best seen in the lower cell layers, and can be
evaluated even in poorly oriented biopsies (110). Intraepithelial eosinophils and neutrophils are very specific features of
esophagitis but are not sensitive indicators of reflux as they are usually present only at the more severe end of the
spectrum, when erosions are evident endoscopically. Of course, eosinophils are also present in fungal and pill
esophagitis and in eosinophilic esophagitis. There is considerable controversy regarding whether rare eosinophils are
present in the esophageal squamous mucosa of normal individuals. A recent well-performed study that included 20
healthy adult controls with normal pH monitoring did find one to two eosinophils in two of them (110). Lymphocytes and
Langerhans cells are normally present in the esophageal squamous mucosa, so the determination of an abnormal
increase in these cells is subjective. Also, the presence of increased IELs alone is not diagnostic of esophagitis, since
this finding has also been reported in patients with Crohn disease, celiac disease and other autoimmune disorders, and
H. pylori infection (126). In a more recent study esophageal squamous intraepithelial lymphocytosis was not found to be
associated with any particular pathologic condition, including reflux esophagitis (121).
FIGURE 14-6▪Reflux esophagitis. Note the presence of basal cell hyperplasia and lengthening of the papillae. There are
also scattered intraepithelial eosinophils (Hematoxylin and eosin, 200×).
In otherwise healthy pediatric outpatients, occasional cases of infectious esophagitis, particularly herpes simplex
esophagitis, present with signs and symptoms mimicking those of reflux esophagitis. Ingestion of caustic substances,
Crohn disease, and dermatologic conditions, such as bullous pemphigoid and Stevens-Johnson syndrome, are rare
possibilities, and other suggestive clinical findings are usually present. In children who are immunosuppressed or
severely debilitated from another illness, infectious esophagitis is an important diagnostic consideration (Table 14-1).
Reflux esophagitis is managed with thickened, small feedings; maintenance of an upright posture after meals; and
antacids, histamine H2-receptor antagonists (e.g., cimetidine and ranitidine), and proton pump inhibitors. In the few
cases resistant to medical therapy, surgical fundoplication procedures are performed to increase the efficacy of the
lower esophageal sphincter mechanism (e363). Sequelae of gastroesophageal reflux include ulcers (usually of the distal
one third of the esophagus and often associated with blood loss), stricture, and Barrett esophagus.
Table 14-1 ▪ CAUSES OF ESOPHAGITIS IN CHILDREN
Reflux esophagitis
Eosinophilic esophagitis
Ingestion of drugs or caustic substances
Chemotherapy induced injury
Trauma (e.g., nasogastric tube)

Crohn disease
Dermatologic conditions
Graft-versus-host disease
Eosinophilic gastroenteritis
Infections (Candida, herpes simplex virus, and CMV)
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Barrett Esophagus
Barrett esophagus, in which columnar epithelium replaces the normal squamous lining of the distal esophagus, is an
acquired metaplastic condition caused by chronic gastroesophageal reflux. It is now established that even in children,
Barrett esophagus is invariably found in association with severe reflux esophagitis; however, it is rare in children,
occurring in only a small percentage who undergo biopsy for symptomatic gastroesophageal reflux (e96,e102,e201).
Usually older children, not infants, are affected. Barrett esophagus cannot be predicted by the clinical presentation; the
symptoms are those of the associated reflux esophagitis.
The changes in Barrett esophagus affect the lower portion of the esophagus (Figure 14-7A) and involvement may be
either circumferential or patchy. The usual squamous lining is transformed to a columnar mucosa. Several types of
columnar-lined mucosa have been described in Barrett esophagus (e354). These include intestinal type mucosa with
absorptive cells and goblet cells (Figure 14-7B), gastric fundie type mucosa with parietal and chief cells, a junctional
type resembling gastric cardia; and a mixed type with tall columnar surface cells and a mixture of fundic and mucous
glands, but no goblet cells. In all types of Barrett mucosa, inflammation and glandular distortion and atrophy are often
noted. The squamous mucosa proximal to the affected esophagus often shows changes of reflux esophagitis.
FIGURE 14-7▪Barrett esophagus. A:Esophagectomy specimen exhibiting a 5-cm circumferential segment of Barrett
mucosa. B: Barrett mucosa, characterized by specialized columnar mucosa with goblet cells (Hematoxylin and eosin,
200×).
In the United States, a consensus panel of experts has required the presence of specialized columnar mucosa
containing goblet cells in biopsies confirmed by the endoscopist to have been obtained from the tubular esophagus to
make a diagnosis of Barrett esophagus (151). Thus, the location of the biopsy site in relation to the lower esophageal
sphincter must be known by the pathologist before Barrett esophagus can be diagnosed. In Great Britain, columnar type
mucosa without goblet cells is accepted as diagnostic of Barrett esophagus, provided the endoscopist is certain that the
biopsies were obtained from the tubular esophagus and not the proximal stomach (20). However, because the
endoscopic landmarks used to separate esophagus and stomach (primarily the upper extent of the gastric rugal folds)
are not precise, particularly in the presence of a hiatal hernia, confusion between distal esophagus and gastric cardia is
possible. The use of a CDX2 immunostain to confirm the presence of intestinal metaplasia even in the absence of goblet
cells has been proposed but has not been widely adopted to date (118). In one study of pediatric Barrett esophagus
CDX2 immunoreactivity was evident only when goblet cells were also present, and not in epithelium comprised entirely
of gastric cardiac or cardio-oxyntic type epithelium (32).
Well-formed barrel-shaped goblet cells are usually easily recognizable with ordinary hematoxylin and eosin staining, but
recognition can be enhanced and confirmed by staining with Alcian blue at pH 2.5, which imparts a blue color to
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intestinal-type acidic mucins (e173). This histologic type of Barrett mucosa has the potential to progress to dysplasia
and, after many years, to adenocarcinoma in about 1% to 2% of cases (e191,e201,e382). Adenocarcinoma in Barrett
esophagus in children is very rare but has been reported (e203,e209).
Eosinophilic Esophagitis
Recently, it was recognized that esophageal biopsies containing large numbers of intraepithelial eosinophils and
exhibiting basal cell hyperplasia can represent an allergic reaction to dietary or inhaled allergens, rather than
representing severe gastroesophageal reflux disease (88) (e488). This disorder, termed eosinophilic esophagitis, often
presents in childhood, although diagnosis in adults is also possible (81). Children are usually unable to distinguish
between heartburn due to reflux and dysphagia due to eosinophilic esophagitis. In young children presenting symptoms
include difficulty feeding, prolonged irritability and crying, failure to thrive, and growth delay. Characteristic endoscopic
findings include wrinkled or thickened esophageal squamous mucosa, sometimes with circumferential rings, linear
furrows, or tiny vesicles. Although this disorder has only recently been recognized, retrospective studies have shown
that in the past cases were interpreted as severe reflux esophagitis. Treatment with an elimination diet or topical or oral
steroids is usually effective, but esophageal stricture can develop in refractory cases (30).
FIGURE 14-8▪Eosinophilic esophagitis. A: Low power showing basal cell hyperplasia. Note the fibrosis of the
subepithelial stroma. B: There are more than 40 eosinophils per high power field. C: An eosinophilic microabscess in
the superficial epithelium (Hematoxylin and eosin, A: 100×, B: 200×, C: 400×).
Endoscopic biopsies of the esophagus typically reveal a heavy but patchy infiltrate of eosinophils, including clusters of
eosinophils (microabscesses), often near the luminal surface (Figure 14-8A to C). Basal cell hyperplasia is usually quite
prominent. Originally a cutoff of 24 eosinophils per high power field was suggested as useful in distinguishing between
eosinophilic esophagitis and reflux esophagitis, since in most cases of reflux esophagitis the density of intramucosal
eosinophils is less than seven per high power field. More recently an expert panel suggested that 15 eosinophils in any
single high power field (400×) should be regarded as consistent with eosinophilic esophagitis in the proper clinical
context (46). However, it is likely that in some patients even fewer eosinophils are present, or at least that limited
sampling will not identify areas of high eosinophil density. A trial of proton pump inhibitors as treatment for presumptive
reflux esophagitis before endoscopy is usual clinical practice, something the surgical pathologist should keep in mind
when evaluating esophageal biopsies. It must also be recognized that patients may have both gastroesophageal reflux
and eosinophilic esophagitis. In fact, reflux could conceivably predispose to the development of eosinophilic esophagitis,
and the presence of eosinophilic esophagitis may make the mucosa more susceptible to reflux injury (145).
The presence of inflammatory changes that are equally severe in biopsies from the midesophagus and distal
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esophagus is a useful finding in making the diagnosis of eosinophilic esophagitis (since reflux changes are typically
more severe distally than proximally). The presence of admixed neutrophils, on the other hand, favors the presence of
reflux esophagitis, since in general only eosinophils are present in eosinophilic esophagitis unless ulceration has
occurred. Biopsies of the gastric cardia can also be useful in distinguishing between reflux esophagitis and eosinophilic
esophagitis. In reflux esophagitis the cardia is uniformly inflamed (i.e., “carditis”), while in eosinophilic esophagitis the
cardia is typically not inflamed. The presence or absence of increased eosinophils in any gastric or duodenal biopsies
obtained during the endoscopy should also be mentioned in the surgical pathology report, to address the possibility of a
more generalized eosinophilic gastrointestinal disorder.
In some cases it is not possible to make a firm histologic distinction between eosinophilic esophagitis and severe reflux
esophagitis. Correlation with the clinical history and the endoscopic appearance is often sufficient to arrive at the proper
diagnosis, but 24-hour esophageal pH monitoring may be necessary in some patients.
FIGURE 14-9▪Herpes simplex virus esophagitis. A: Endoscopic appearance of a midesophageal ulcer. B: Viral
inclusions in squamous epithelium at the edge of the ulcer 200×. C: High power to demonstrate typical intranuclear
inclusions and multinucleated cells 400×.
Infectious Esophagitis
Infectious esophagitis is rare except in hospitalized, immunosuppressed, and debilitated children, who are at significant
risk for the development of esophagitis in association with infection by Candida species, herpes simplex virus, and
cytomegalovirus (CMV). Bacterial infection is a practical consideration only as a superinfection.
Herpes Simplex Esophagitis

Herpes esophagitis presents as odynophagia and is often accompanied by gingivostomatitis (e313,e333). Multiple small,
discrete ulcers separated by normal mucosa are distributed throughout the esophagus (Figure 14-9A). In severe cases,
confluent ulceration can occur. Microscopically, severe necrotizing esophagitis, abundant neutrophils, and ulceration are
found. Epithelial cells at ulcer margins often demonstrate discrete eosinophilic intranuclear inclusions (Cowdry type A) or
ground-glass intranuclear inclusions (Cowdry type B) (Figure 14-9B,C). Only the squamous cell can be infected by the
herpes simplex virus; so if the biopsy consists only of granulation tissue and necrotic debris, no comment can
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be made regarding the presence or absence of this infection. Although it occurs most often in immunosuppressed
persons, herpetic esophagitis may occasionally be found in otherwise normal children.
Candida Esophagitis
Immunosuppression, premature birth, cancer chemotherapy, and AIDS are the most significant risk factors for the
development of esophageal candidiasis in infants and children (e256,e409). Esophageal involvement is common in
children with mucocutaneous candidiasis. In debilitated patients, esophageal infection may lead to systemic candidiasis.
The gross appearance is usually a combination of white plaques and ulcerations. Histologically, the plaques consist of
masses of pseudohyphae and yeast forms admixed with inflammatory debris and fibrin (eFigure 14-2A to C).
Cytomegalovirus Esophagitis
CMV esophagitis is uncommon and limited to immunosuppressed persons. It rarely occurs alone; it is usually part of a
systemic CMV infection or an infection involving the whole gastrointestinal tract. In contrast to herpes simplex
esophagitis, CMV cannot infect squamous epithelial cells; so if the biopsy consists only of squamous epithelium, no
comment can be made regarding the presence or absence of this infection.
DISORDERS OF THE STOMACH

Congenital Anomalies
Hypertrophic Pyloric Stenosis
Although pyloric stenosis is diagnosed as early as 2 weeks of age, it is not a congenital anomaly in the usual sense
because it has rarely been demonstrated at birth (e437). Pyloric stenosis is a common condition, seen in 1 of 200 infant
boys. The male-to-female ratio is 5:1 or greater, and white, firstborn boys are at greatest risk. A definite familial
incidence has been noted, but there is no definite inheritance pattern. Neurons supplying the circular muscle layer of the
pylorus lack activity of the enzyme nitric oxide synthase (e476). The circular muscle layer undergoes hypertrophy and
elongation, and gastric outlet obstruction ensues. Progressive nonbilious vomiting, the primary manifestation,
commences at 2 to 6 weeks of age in an otherwise healthy infant. The diagnosis is suggested when the hypertrophic
pyloric muscle mass, approximately the size of an olive, is palpated in the right upper quadrant after a feeding.
Abdominal x-ray films show marked gaseous distension of the stomach, and barium studies demonstrate a narrow and
elongated pyloric channel (“string sign”). Treatment is surgical. At operation, the hypertrophic pyloric muscle appears as
an elongated sphere (“olive”), approximately 2.5 cm long and 1.5 cm in diameter. A longitudinal surgical incision of the
hypertrophic muscle down to the submucosa (pyloromyotomy) immediately and efficaciously relieves the obstruction.
Occasional postmortem observations indicate that the circular layer of muscularis propria is hypertrophic, hyperplastic,
and disorganized in appearance. The outer, longitudinal muscle layer is attenuated and of variable thickness. The cause
of infantile hypertrophic pyloric stenosis is unknown.
Antral Web
A very unusual cause of gastric outlet obstruction in young infants is an antral web (antral diaphragm) of fibrous tissue
and gastric mucosa obstructing the antrum a few centimeters proximal to the pylorus (e34). A small, central aperture,
usually no more than several millimeters in diameter, permits passage of some stomach contents; variability in the size of
the opening explains the variability in age at presentation. The diagnosis is made by barium studies, and endoscopy is
often difficult.
Duplication
Gastric duplication presents as a cystic mass on the greater curvature or at the pylorus and may present with bleeding,
rupture, or obstruction. The pathologic features are similar to those of the more commonly encountered small-intestinal
duplication. The mucosa of a gastric duplication resembles stomach mucosa most of the time, but primitive or simplified
gut epithelium or intestinal mucosa is also encountered.
Pancreatic Heterotopia
An island of ectopic pancreas may occur as an intramural nodule or mass on the greater curvature near the antrum
(e443). It is often detected incidentally on imaging studies or at autopsy. A central depression may be seen,
corresponding to the opening of the pancreatic duct draining the heterotopic tissue. Occasionally, ulceration develops in
the overlying mucosa and causes epigastric pain.
Acquired Diseases
Spontaneous Gastric Perforation in the Neonate
Spontaneous perforation of the body of the stomach occasionally develops in premature neonates, especially those
under intensive care (e221). The cause of the perforation is inapparent, although it often occurs in an area of
hemorrhagic or coagulative necrosis and may be ischemic or traumatic in origin. The usual presentation is sudden
abdominal distension and pneumoperitoneum.
Gastritis
A list of the types of gastritis in children is much shorter than a similar list in adults because of the absence of many of
the atrophic, metaplastic, and dysplastic conditions of the adult stomach. However, it is clear that gastritis occurs with
considerable frequency in children and adolescents. Numerous classification schemes exist (e119), but for practical
purposes, gastritis is categorized by etiology if apparent.
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Hemorrhagic and Erosive Gastritis
The etiology of acute hemorrhagic gastritis is multifactorial, with ischemia, stress, and drug therapy playing contributory
roles. Drugs known to damage the gastric mucosa include aspirin, corticosteroids, alcohol, and nonsteroidal anti-
inflammatory drugs (NSAIDs), such as indomethacin. The ingestion of corrosive substances also causes a similar
picture. At endoscopy, a diffusely injected and edematous mucosa, often with petechial hemorrhages and small
erosions, is seen. In severe cases, which usually occur in very ill children hospitalized for sepsis, hemorrhagic shock,
major surgery, burns, central nervous system disorders, or other severe illness, the changes are most severe in the
gastric body and fundus.
Biopsies are usually not obtained in these severely ill patients, and therefore this condition is usually seen at the time of
autopsy. The histologic changes essentially represent a chemical injury to the gastric mucosal caused by reduced host
defense against the injurious action of gastric acid and digestive enzymes. Hemorrhage and mucosal edema dominate
the histologic picture. Significant inflammation is not present except directly adjacent to areas of ulceration (eFigure 14-
3A,B).
Helicobacter pylori Gastritis

Since the early 1980s, it has been recognized that diffuse antral gastritis is caused by infection with H. pylori, a small
Gram-negative bacillus (e124,e202,e248,e300,e506). This organism, which is the pathogen responsible for the
associated symptoms and pathologic changes, is not an opportunist or a commensal. Children with H. pylori infection
usually present with nausea, vomiting, and epigastric pain. Endoscopy shows erythema, particularly in the antrum, and
in the more severe cases erosion, antral nodularity, and thickened gastric folds. However, there is not a good correlation
between the endoscopic and histologic findings of gastritis. That is, in many cases where endoscopic findings of gastric
mucosal erythema, granularity, or erosion are described, biopsies are entirely unremarkable. Conversely, in many cases
where the gastric mucosa is described as endoscopically normal, gastritis is actually evident histologically.
Currently, endoscopy and biopsy are the most widely used methods for the diagnosis of H. pylori infection. Culturing the
endoscopy specimen directly for H. pylori is difficult and not performed routinely. A commercial test is available in which
the presence of the organism in a fresh biopsy specimen causes a change in the color of a solution. This reaction is
based on the production of urease enzyme by the organism. A number of commercially available enzyme-linked
immunosorbent assay (ELISA) kits are also available for serologic testing, but they lack the sensitivity and specificity of
biopsy. On biopsy, the organisms are most reliably found in the antrum, although the fundus and cardia of the stomach
may also be affected. The bacilli can be seen faintly on ordinary preparations stained with hematoxylin and eosin, but
they are more easily seen with Giemsa, Genta (e160), Warthin-Starry, or immunoperoxidase staining; they appear as
small curved or slightly twisted rods, 4 to 5 μm in length, within the mucous coat overlying the surface or superficial
foveolar epithelium (Figure 14-10). Organisms are usually most easily found in areas of active inflammation.
FIGURE 14-10▪Helicobacter pylori organisms over antral mucosa (Warthin-Starry stain, 400×).
The antral mucosa exhibits a diffuse superficial infiltrate composed primarily of plasma cells and lymphocytes. Active foci
of neutrophilic infiltration may be seen in the lamina propria or in glandular or surface epithelium. Although lymphoid
aggregates are normal in the gastric mucosa, the presence of lymphoid follicles with germinal centers is highly
suggestive of past or current H. pylori infection (e159). In patients on a proton pump inhibitor for dyspepsia or symptoms
of gastroesophageal reflux, the H. pylori organism may migrate to cause active gastritis of the gastric body mucosa,
resulting in an inactive appearance of the antral gastritis.
Treatment of H. pylori with antibiotics results in prompt disappearance of the organisms and the neutrophilic component
of the mucosal inflammatory cell infiltrates. By contrast, it may take many months for the lymphocytic and plasma cell
infiltrates to disappear. Biopsies obtained during this period may be diagnosed as inactive gastritis. The diagnosis of
inactive gastritis can be difficult, as there are a small number of lamina propria lymphocytes and plasma cells in the
gastric mucosa normally. As a general rule of thumb, when the density of plasma cells is such that they are clustered
and touching each other, this can be regarded as indicative of inactive gastritis. In some patients with inactive antral
gastritis there may not be an antecedent diagnosis of H. pylori gastritis, as the infection may have been treated
incidentally during antibiotic treatment of infection elsewhere (e.g., otitis media).
Even though H. pylori causes duodenal ulcers, the organism is not found in duodenal mucosa except in instances of
gastric metaplasia of the duodenum, which is rare in children. The mechanism of duodenal ulcer formation in H. pylori
infection is thought to involve increased acid secretion as a response to the gastric infection, as well as direct damage
by the organism in the areas of duodenal gastric foveolar metaplasia.
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In addition to the immediate morbidity of gastritis and ulcer disease in children and adults, infection with H. pylori is
known to carry a risk for future adenocarcinoma of the stomach (e16,e351) and gastric lymphoma arising in mucosa-
associated lymphoid tissue (MALT) (e352). H. pylori infection can be difficult to eradicate. Bismuth preparations and
multiple antibiotic regimens are effective, but relapses are common.
The histologic differential diagnosis of H. pylori gastritis includes a small number of unusual conditions of the stomach
with distinctive clinical and histologic findings, including involvement by eosinophilic gastroenteritis, Crohn disease
(e505), or, less frequently, Langerhans cell histiocytosis (e182,e243), chronic granulomatous disease (e9,e117), and
Henoch-Schönlein purpura (e460). Lymphocytic gastritis, characterized by increased lymphocytes in the gastric foveolar
and glandular epithelium, can occur in patients with celiac disease and has also been reported as a consequence of H.
pylori infection. An increase in IELs above 1 per 25 gastric foveolar or glandular epithelial cells is generally regarded as
abnormal and diagnostic of lymphocytic gastritis. (eFigure 14-4A to C) (e6,e112,e503).
Helicobacter heilmannii (Gastrospirillum hominis) Gastritis

Helicobacter heilmannii infection of the stomach is much more rare and not as serious or chronic a disease as H. pylori
gastritis. The clinical presentation and histologic picture are similar except that H. heilmannii is a much larger organism
than H. pylori, much more obviously spiraled, and more readily seen on slides stained with hematoxylin and eosin
(Figure 14-11). It also resides on the gastric epithelial surface and does not invade tissue (e3,e111,e343).
Peptic Ulcer Disease

The widespread use of fiberoptic endoscopy has led to the realization that ulcer disease in children is not as rare as was
formerly thought (e127,e224). Peptic ulcers are of two types: acute (stress) and chronic. Nearly all peptic ulcers occur in
the stomach and duodenum, but they may occur in any location where acid- and pepsin-secreting gastric mucosa is
found, including Meckel diverticulum.
FIGURE 14-11▪ Helicobacter heilmannii organisms over antral mucosa (Giemsa stain, 1,000×).
Nearly all cases of childhood and adult chronic ulcers have been shown to be caused by infection with H. pylori in the
stomach (e15,e202,e224,e248,e300,e506). Chronic (or primary) peptic ulceration in children is the same acid-peptic
disease that is so common in adults. This condition can develop in children as young as 4 or 5 years old, although it is
more common in preadolescents and adolescents of either sex. It is most common in adolescent boys. Duodenal ulcer is
much more common than gastric ulcer. Chronic abdominal pain is the most frequent presenting symptom. More than
50% of the patients have hematemesis, melena, or occult bleeding at the time of presentation. At endoscopy, chronic
peptic ulcers are usually round to oval, less than 2 cm in diameter, well delineated from the surrounding mucosa by
sharp margins, and covered by exudate at the base.
Microscopically, granulation and scar tissue form the ulcer base, which often extends deep into the muscularis propria.
The stomach invariably shows active antral gastritis, and H. pylori is usually readily identified. If the ulcer is duodenal,
active duodenitis is usually present in surrounding, nonulcerated mucosa. Chronic peptic ulcers usually heal with a
medical regimen. Zollinger-Ellison syndrome, characterized by peptic ulceration resistant to therapy, giant gastric rugal
folds, and increased serum levels of gastrin, is very rare in children; fewer than 30 cases have been reported in this age
group. Ulceration due to mucosal injury caused by NSAIDs or other medications is also a diagnostic consideration in
older children.
Ménétrier Disease
Ménétrier disease is found primarily in adults, but cases in children have been described (e79,e80,e371). The disease
appears similar symptomatically and pathologically, but the clinical course and etiology are different. In adults, the cause
is unknown and the disease is usually severe and often requires gastrectomy. Childhood cases are often self-limited,
and most are caused by CMV infection. Classic Ménétrier disease presents with epigastric or abdominal pain, weight
loss, and peripheral edema. The edema is caused by protein loss in the stomach with resultant hypoalbuminemia.
Radiographs and endoscopic examination reveal prominent or “giant” gastric folds of the corpus; the antrum is usually
spared. Histologic features include mucous cell hyperplasia, pronounced elongation and tortuosity of the usually short
gastric pits (foveolae), glandular atrophy, and reversal of the usual pit-to-gland ratio. Cysts lined by superficial mucous
cells are found deep in the mucosa. Inflammation is more prominent in children than in adults, reflecting the infectious
etiology in most children. CMV inclusions are often evident in biopsy material in children. If they are not seen,
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polymerase chain reaction testing may be positive for CMV (e80,e371). Not all pediatric cases of Ménétrier disease are
caused by CMV. Formula protein intolerance has been suggested as an alternate cause in young infants (e145).
Ménétrier disease is difficult to diagnose in superficial mucosal biopsy specimens. The differential diagnosis includes
other causes of large gastric folds: H. pylori gastritis, peptic ulcers, Crohn disease, eosinophilic gastroenteritis, and
gastric lymphoma (e19). Foveolar hyperplasia of the antrum in neonates may be caused by prostaglandin therapy
administered to maintain patency of the ductus arteriosus in certain forms of congenital heart disease (e356). Usually,
the clinical setting, antral location, and presence of hypoalbuminemia make it possible to distinguish this group of
neonates from those with Ménétrier disease.
Eosinophilic Gastroenteritis
Eosinophilic gastroenteritis can occur at any age but is rare in infants and children. It is characterized by striking
eosinophilic infiltration of any part of the gastrointestinal wall (e44,e171,e278,e425,e453,e465,e495). A poorly
understood allergic reaction is thought to be responsible for the disease because most patients have an allergic history
and increased serum IgE levels. However, specific allergens have not been implicated in every case, and patients do not
respond to food elimination diets. Symptoms vary depending on which part of the gastrointestinal tract is affected and on
whether the disease is mucosal or transmural. The gastric antrum and proximal small intestine are affected in most
cases; isolated small intestinal, colonic, or esophageal involvement accounts for the remainder. In the transmural form of
the disease, submucosal edema and eosinophilic infiltration compromise the lumen and obstruct the intestine or gastric
outflow tract. Abdominal pain, vomiting, and weight loss are frequently the presenting complaints in this form (e453).
Disease limited to the mucosa may have a more insidious onset. Malabsorption and weight loss are found with small-
intestinal mucosal disease. Radiologic studies are often helpful in the diagnosis by demonstrating either antral narrowing
with a “mass” of inflammation and edema, or nodularity and thickening of the small-intestinal wall.
FIGURE 14-12▪ Eosinophilic gastritis. A: A pure infiltrate of abundant eosinophils 200×. B: Eosinophils infiltrate the
surface epithelium 400×.
Histologically, the mucosal form of the disease is characterized by prominent and diffuse inflammatory infiltration in the
lamina propria, with eosinophils accounting for the majority of the inflammatory cells. Eosinophils infiltrate and damage to
the surface and glandular epithelium, and eosinophilic glandular abscesses are occasionally found. Infiltration of the
muscularis mucosae is also a useful feature indicating true pathology (Figure 14-12A,B). The presence of increased
numbers of eosinophils in the lamina propria alone is insufficient to make the diagnosis of eosinophilic gastroenteritis
because the same phenomenon may occur in Crohn disease or infection, as a drug response, or as a normal finding in
some persons. Normal mucosal architecture is preserved; ulceration is unusual. In transmural forms of the disease,
eosinophil infiltration is often maximal in the submucosa, with lesser numbers seen in the muscularis externa and serosa.
Rarely, eosinophilic ascites and eosinophilic infiltration of regional lymph nodes are found. Most patients with
eosinophilic gastroenteritis have a chronic waxing and waning of symptoms. Steroids are often necessary to control the
symptoms. Because the disease is quite patchy and may not affect the mucosa, full-thickness biopsy is sometimes
necessary for diagnosis.
Crohn Disease of the Stomach

Involvement of the stomach by Crohn disease usually occurs in association with disease in the more usual locations—
the distal ileum and colon (e192,e280,e302,e338,e392,e400, e505). On occasion, the initial presentation of Crohn
disease
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is as a gastroduodenal process. In such cases, the antrum is usually involved, often in continuity with the proximal
duodenum. Obstruction of the gastric outlet is a feature shared with eosinophilic gastroenteritis and some cases of H.
pylori gastritis. The histology of gastric Crohn disease is similar to that in other sites. Particularly suggestive of
gastroduodenal Crohn disease is the combination of distinctly focal acute inflammation causing destruction of glandular
epithelium plus spotty chronic inflammation similar to the characteristic focal involvement of the distal gastrointestinal
tract in Crohn disease. This focally enhanced pattern of active gastritis in Crohn disease is usually distinct from the more
diffuse, superficial and plasma cell predominant pattern of gastritis due to H. pylori infection (e338,e505). The presence
of granulomas is very helpful in addition to these nonspecific inflammatory features.
Granulomatous Gastritis
Granulomatous gastritis not associated with Crohn disease has rarely been described in adults. Tuberculosis, fungal
infections, chronic granulomatous disease (e117), and sarcoidosis are other rare causes of gastric granulomas (e131).
In biopsy specimens, most granulomas associated with chronic inflammation in the stomach of a child or adolescent
prove to be Crohn disease.
Polyps and Tumors of the Stomach

Gastric polyps are rare in children. Juvenile polyps and Peutz-Jeghers polyps may occur in the stomach as part of a
generalized polyposis syndrome. Gastric hyperplastic polyps are rare in children but can occur in the setting of H. pylori
gastritis. Fundic gland polyps are a more common clinically insignificant consequence of chronic administration of proton
pump inhibitors used to treat gastroesophageal reflux disease and dyspepsia (72, 116). They are usually small and often
multiple and are restricted to the oxyntic mucosa of the proximal stomach. Histologically they can be difficult to
distinguish from normal gastric fundic mucosa as the histological features can be subtle, despite the endoscopic
appearance of a polypoid lesion. The diagnostic histologic features include dilatation of the fundic glands and parietal
cells with cytoplasmic protrusions extending into the glandular lumina. Cytoplasmic vacuolization of parietal cells is also
common. The complete absence of lamina propria inflammation and edema is a striking feature of these polyps (eFigure
14-5). The surrounding flat fundic mucosa often exhibits histologic features similar to but not as pronounced as those
evident in the polyps. Fundic gland polyps also develop commonly in patients with familial polyposis coli. Thus, if a
fundic gland polyp is identified in a young patient not taking a proton pump inhibitor, colonoscopy to exclude colonic
polyposis may be indicated. Dysplasia does occur in fundic gland polyps associated with familial polyposis coli but is
exceedingly rare in the sporadic setting (142). For this reason it is not necessary to remove multiple sporadic fundic
gland polyps.
Gastric teratomas are large, bulky multicystic masses that project into the gastric lumen or outward into the peritoneal
space (35). Heterotopic pancreatic tissue should be considered in the differential diagnosis of gastric tumors. This is
usually a sessile mass in the antrum and easily recognized histologically as acinar and endocrine pancreas (e443). The
term adenomyoma has been applied to a variant characterized by a predominance of pancreatic duct structures
interlaced with smooth muscle bundles but without pancreatic parenchyma (e157).
Malignant tumors of the stomach are quite rare in children. MALT lymphomas associated with H. pylori infection and
Burkitt lymphomas are the most common types of lymphoma reported (35) (e53). Adenocarcinoma of the stomach is
distinctly rare but has been reported in otherwise normal children (35) (e82,e294,e444). It is also known to occur in
ataxia-telangiectasia and other primary immunodeficiency disorders (e190). Rare examples of inflammatory
myofibroblastic tumor (e84) and rhabdomyosarcoma have also been reported (35) (e444).
Gastrointestinal Stromal Tumors

Gastrointestinal stromal tumors present occasionally in children, either as sporadic tumors or in the setting of a
syndrome. The vast majority of, but not all, gastrointestinal stromal tumors in children occur in the stomach. Iron
deficiency anemia is the most common presenting symptom in sporadic cases, while abdominal pain, a palpable mass,
or vomiting occurs rarely (77). Carney triad is used to describe patients with paragangliomas, pulmonary chondromas,
and gastric gastrointestinal stromal tumors (e69). About 85% of patients with Carney triad are female, and the
gastrointestinal stromal tumors are often multifocal, which is unusual for sporadic tumors. Despite extensive molecular
analysis, a specific underlying genetic defect has not been identified in patients with Carney triad (144). Carney-
Stratakis syndrome designates a separate group of patients with gastric gastrointestinal stromal tumors and
paragangliomas but no pulmonary chondromas. In these patients, autosomal dominant transmission has been
demonstrated and germline mutations in any of three mitochondrial complex II succinate dehydrogenase (SDH) enzyme
subunits (SDHB, SDHC or SDHD) have been documented (144). Gastrointestinal stromal tumors can also develop in
individuals with neurofibromatosis type 1, although usually not in childhood. Lastly, individuals with germline mutations in
the KIT or PDGFRA genes are at risk for the development of gastrointestinal stromal tumors, although again the tumors
usually occur outside of the pediatric age range (114).
Gastrointestinal stromal tumors are presumed to develop from the interstitial cells of Cajal, which are thought to
represent the pacemaker cells throughout the gastrointestinal tract. These cells are normally located within the
myenteric plexus and the muscularis propria and have an important role in the regulation of peristalsis. A gain of function
mutation in
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either the c-kit or platelet-derived growth factor receptor A gene can be detected in about 85% of gastrointestinal stromal
tumors in adults (92). By contrast, mutations in these two genes are rare in pediatric tumors, with reported rates from 0%
to 10%. Thus, the molecular pathogenesis of pediatric gastrointestinal stromal tumor is distinct from the adult
counterparts, and the underlying mechanisms are currently undefined (1, 114).
Pediatric gastrointestinal stromal tumors can be of spindle cell or epithelioid morphology, and mixed forms are also
common. Among sporadic tumors, epithelioid tumors are more common overall, but spindle cell morphology is more
common in boys (154). The epithelioid tumors are composed of round to polygonal cells, which may have little or
abundant cytoplasm. Cytoplasmic vacuolization is common in these tumors and sometimes can be so prominent as to
produce a signet-ring cell-like appearance (Figure 14-13A,B). The vacuoles do not stain for mucosubstances, glycogen,
or fat and appear to represent an artifact of formalin fixation. The spindle cell variant of the tumor resembles smooth
muscle tumors, but the cells are usually not as long and slender. Areas of hyaline fibrosis are common in both spindle
cell and epithelioid variants of the tumor.
The diagnosis of gastrointestinal stromal tumor is confirmed by immunohistologic detection of cytoplasmic reactivity in
tumor cells with the c-kit antibody. Even in pediatric tumors where 10% or less of the tumors have mutations in either the
c-kit or PDGFRA genes, most of the tumors still express c-kit by immunohistochemistry. In adults immunohistologic
detection utilizing a recently developed antibody designated DOG1 has been reported as highly sensitive and specific
for the diagnosis of gastrointestinal stromal tumors, including those that are nonreactive with the c-kit antibody (104,
154). The antigen detected by the DOG1 antibody is uniformly present in Cajal cells throughout the gastrointestinal
tract, but not in mast cells, unlike the c-kit protein (104). In one study 9 of 11 pediatric gastrointestinal stromal tumors
were reactive with the DOG1 antibody (91).
FIGURE 14-13▪ Gastric gastrointestinal stromal tumor. A: This example demonstrates epithelioid histology, which is
more common in the pediatric age group 100×. B: Cytoplasmic vacuoles are sometimes prominent, as seen here 200 ×.
Various schemes have been utilized to stratify the risk of prognosis of gastrointestinal stromal tumors (76). Most
systems, including a consensus scheme developed under the auspices of the National Institutes of Health, rely primarily
on tumor size and mitotic rate (mitotic figures per 50 high power fields) (44). However, it has been known for some time
that the site of tumor origin also has an important influence of the risk of poor outcome. Therefore, tumor site has been
incorporated into several subsequent iterations of risk assessment schemes proposed by various groups (41, 49, 76). In
addition, tumor rupture appears to be an important risk factor for tumor spread (68). These schemes have not been
applied specifically to pediatric gastrointestinal tumors. Retrospective data suggests that the prognosis of these tumors
in children is more difficult to predict (1, 114).
DISORDERS OF THE SMALL AND LARGE INTESTINE

Omphalocele
Omphalocele (exomphalos) is a developmental defect of the anterior abdominal wall in which the abdominal
musculature, fascia, and skin are absent in the midline at the point of insertion of the umbilical cord (e301,e510).
Abdominal organs extrude anteriorly through the defect and are covered by a saclike membrane consisting of amnion
externally and parietal peritoneum internally (Figure 14-14). Omphalocele results from failure of the intestine to return to
the body cavity after its normal herniation into the umbilical stalk during
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embryonic life (e301). Omphaloceles vary in size; the defect may be a few centimeters in diameter, or most of the
anterior abdominal wall may be lacking. Depending on the size of the defect, small intestine, liver, spleen, and pancreas
may be in the sac. The umbilicus usually inserts at the dome of the sac, and umbilical vessels ramify across the
membrane. Intrauterine rupture of the sac may occur; exposure of the gut to amniotic fluid results in edema, bowel wall
thickening, and matting of intestinal loops. Such cases must be distinguished from gastroschisis. The intestine is nearly
always malrotated and shorter than normal.
FIGURE 14-14▪ Omphalocele. A translucent membrane covers the abdominal organs, which are protruding through an
abdominal wall defect in this newborn. Note the insertion of the umbilicus into the center of the omphalocele sac.
(Courtesy of Robert J Izant Jr, M.D., Case Western Reserve University, Cleveland, Ohio.)
Other congenital anomalies are found in at least one third of these infants, including gastrointestinal malformations,
congenital heart disease, genitourinary anomalies, imperforate anus, and central nervous system defects. The incidence
of omphalocele is increased in infants with trisomy 18, trisomy 13, and trisomy 21. Omphalocele is a key feature of
Beckwith-Wiedemann syndrome (gigantism, macroglossia, hemihypertrophy, visceromegaly, and hypoglycemia) (33)
(e164).
The prognosis in omphalocele is usually determined by the other anomalies and the size of the defect. The omphalocele
sac is excised surgically just after birth, with closure of the defect primarily or with temporary prosthetic material.
Gastroschisis
Gastroschisis occurs much less frequently than omphalocele (e115,e301,e461). In gastroschisis, a relatively small
paraumbilical abdominal wall defect (right side-to-left side ratio of 9:1) is distinctly separate from the normally placed
umbilicus. Loops of bowel, not covered by a membrane, extrude through the opening (Figure 14-15). Because the
extruded intestine has been bathed in amniotic fluid in utero, it appears abnormally thickened and edematous and may
be coated with fibrin. The intestine is usually not rotated and is much shorter than normal. Jejunoileal atresia is another
recognized association. In contrast to omphalocele, gastroschisis is rarely associated with concurrent major congenital
anomalies (e115,e461).
FIGURE 14-15▪ Gastroschisis. Loops of intestine extrude through an abdominal wall defect located to the right of the
normally placed umbilicus. The intestines are not covered by a sac. (Courtesy of Robert J Izant Jr, M.D., Case Western
Reserve University, Cleveland, Ohio.)
Gastroschisis is believed to result from failure of the umbilical cord to form properly, so that the elongating midgut
ruptures into the amniotic cavity during the first trimester (e301). Treatment consists of surgical closure of the defect at
birth or staged procedures with the use of prosthetic material.
Malrotation
The term malrotation includes a group of congenital positional and associated abnormalities of the intestine and
mesentery resulting from nonrotation or abnormal rotation and fixation of the developing embryonic gut (e150,e462).
During the most rapid period of growth, the embryonic intestine extends outside the abdominal cavity (Figure 14-3).
During weeks 10 and 11 of gestation, the intestine returns to the abdomen in sequential stages, the first of which is a
270-degree counterclockwise rotation of the midgut around the superior mesenteric artery until the duodenum comes to
rest in its usual position posterior to the superior mesenteric artery. After that, the cecum and right colon rotate, first
entering the abdomen on the left side, then crossing to the right and descending into the right lower quadrant anterior to
the superior mesenteric artery. At week 11, fixation of the gut to the abdominal wall occurs. A broad-based mesentery
extending from the ligament of Treitz to the ileocecal area attaches the intestine to the posterior abdominal wall and
stabilizes it. The right and left portions of the colon become fixed retroperitoneally.
Failure of this sequence to take place at all (nonrotation) or failure at any step produces a spectrum of malrotation
abnormalities. Any arrest in the process of rotation also tends to interfere with the normal mesenteric fixation of the
bowel and results in a narrow mesenteric base and a mobile intestine that
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is predisposed to volvulus. A person with an incompletely rotated bowel is likely to have abnormal mesenteric fixations
and associated extrinsic intestinal obstruction and volvulus. Malrotation often occurs together with other congenital
anomalies, including duodenal atresia, omphalocele, gastroschisis, jejunoileal atresia, and Meckel diverticulum.
Variations of malrotation are diagrammed in Figure 14-16. In the case of nonrotation (Figure 14-16A), the duodenum is
directed inferiorly and lacks the usual sweep to the left. The distal portion of the duodenum and the ascending colon lie
together in the midabdomen and are attached to the abdominal wall posteriorly by a very short mesenteric root
containing the superior mesenteric artery. The descending colon is not fixed. The narrow mesenteric root and nonfixed
descending colon result in midgut volvulus and duodenal obstruction (Figure 14-16C). The rapid progression of volvulus
causes the most dreaded and lethal complication of malrotation, which is cessation of mesenteric artery blood flow at the
base of the twisted mesentery and infarction of the entire midgut. Midgut volvulus usually presents in the 1st month of
life with intestinal obstruction. Normal rotation of the duodenal loop with nonrotation of the colon is associated with the
same potential for midgut volvulus (Figure 14-16B).
In the variation of normal colonic rotation with nonrotation of the duodenum, abnormal mesenteric bands may
intermittently obstruct the duodenum. In another variation, both the duodenum and the colon rotate normally, but the
ascending colon does not become fixed. Abnormal peritoneal (Ladd) bands between the hepatic flexure and lateral
abdominal wall overlie the duodenum and may obstruct it (Figure 14-16D).
FIGURE 14-16▪Normal rotation and variations in position of stomach and intestines due to malrotation. A: Nonrotation of
duodenum and colon. B: Nonrotation of colon. C: Midgut volvulus resulting from a narrow mesenteric root and nonfixed
descending colon in malrotation. Occlusion of mesenteric blood flow leads to midgut infarction. D: Ladd (peritoneal)
fibrous bands (upper left) may extend from the lateral abdominal wall to the right colon, compressing and obstructing
the duodenum.
Intestinal Atresia and Stenosis

Intestinal atresia is the complete absence of a segment of the intestine or complete occlusion of the intestinal lumen.
Either situation is a common cause of neonatal intestinal obstruction, with a prevalence of 2 in 10,000 live births (1). The
rates of atresia in the duodenum and in the more distal jejunum and ileum are approximately equal; colonic atresia is
much less frequent. Multiple jejunoileal atresias are found in approximately 10% of cases.
Clinical, pathologic, histologic, and experimental observations indicate that most jejunoileal atresias and stenoses are
secondary malformations. The disruptions are caused by intrauterine vascular accidents, with infarction and subsequent
resorption or scarring of the affected segment (e114). The fact that bile and squamous epithelial cells are often found
distal to the obstruction indicates that the lumen was patent early in gestation. The presence of serosal fibrosis and
meconium indicates previous (intrauterine) intestinal perforation and peritonitis. Experimental occlusion of portions of the
mesenteric circulation in fetal animals results in identical atretic lesions. Atresias are associated with known vascular
insults, such as intrauterine malrotation with volvulus, intussusception, internal hernia, and constricting gastroschisis.
Familial patterns in some cases of multiple jejunoileal atresias suggest that not all cases result from vascular accidents.
Some arise from abnormal development of the mesenteric vasculature, probably genetically-based (134, 138). A
particularly distinctive form of multiple intestinal atresias occurs in French Canadians, although it is not limited to this
ethnic group (e273,e415).
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FIGURE 14-17▪ A: Classification of intestinal atresia. I: mucosal (membranous) atresia with intact bowel wall and
mesentery; II: blind intestinal ends attached by a fibrous cord; III A: blind intestinal ends separated by a V-shaped
mesenteric defect without an intervening cord; IIIb: “apple-peel” atresia; and IV: multiple atresia. (From Grosfeld JL.
Jejunoileal atresia and stenosis. In: Welch KJ, Randolph SG, Ravich MM et al., eds. Pediatric surgery. 4th ed. Chicago:
Year Book, 1986:843, with permission.) B: Photomicrograph of a “fibrous cord” of intestinal atresia reveals an intact
muscularis propria, but there is fibrous obliteration of the lumen and submucosal calcification, consistent with intrauterine
ischemia and healing (H&E, original magnification 20×).
The embryologic basis of duodenal atresia probably differs from that of jejunoileal and colonic atresia. Because most
cases of duodenal atresia are of the membranous type, they probably result from a lack of central vacuolization during
the solid cord stage of duodenal development. The rate of associated anomalies in infants with duodenal atresia is high.
One-fourth of infants with duodenal atresia have Down syndrome, an association not noted with atresia at other sites.
Additional congenital anomalies associated with duodenal atresia include cardiac and renal malformations, esophageal
atresia, imperforate anus, and vertebral anomalies. Annular pancreas and malrotation are each found in approximately
one-fourth of infants with duodenal atresia (e103). Jejunoileal atresia is less likely to be associated with other anomalies,
although an association between cystic fibrosis and jejunoileal atresia has been noted (e40).
The symptoms of intestinal atresia depend on the level of gastrointestinal tract affected. Duodenal and proximal jejunal
atresia cause maternal polyhydramnios (secondary to reduced absorption of swallowed amniotic fluid), vomiting, and
abdominal distension in the first 24 hours of life; these symptoms are delayed with more distal obstruction. Abdominal
radiographs show gaseous distension of the stomach with duodenal atresia and, in lower intestinal atresias, air-fluid
levels. Many cases of jejunoileal atresia are now detected by prenatal ultrasonography.
Intestinal atresias have been classified according to their gross appearance (e184) (Figure 14-17A). Type I has an intact
intestinal wall and mesentery but a septal or membranous luminal obstruction. Because the proximal segment is
obstructed, its diameter greatly exceeds that of the distal segment. In type II, two intestinal segments with blind ends are
separated by a fibrous cord. In type III, the most common, two blind ends are present without an intervening cord; a
wedge-shaped mesenteric defect is also present. In the “apple-peel” or “Christmas tree” variety of extensive jejunal
atresia, the intestine is very short and the distal ileal segment is coiled around its arterial blood supply (the ileocecal
artery). Type III may also be associated with a congenitally short small intestine.
Histologic examination of a type II atresia usually shows a recognizable intestinal wall with muscularis layers but fibrous
obliteration of the mucosa and submucosa. The frequent presence of luminal granulation tissue, fibrosis, and
calcification suggests previous ischemia and healing (Figure 14-17B).
Intestinal segments adjacent to regions of atresia or stenosis should be examined for changes suggestive of
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cystic fibrosis: dilated glands with eosinophilic inspissated secretions, unusually viscid secretions in the lumen, and
hyperplasia of goblet cells (e121). In one study, 8% of neonates with jejunoileal atresia had cystic fibrosis (4).
Congenital intestinal stenosis is less common than intestinal atresia; it may be solitary or multiple and may affect a short
or long segment. The bowel diameter is greatly reduced, although the lumen is patent throughout. Histologic
examination of the intestinal wall often shows evidence of previous ischemia and healing, including mucosal atrophy,
submucosal fibrosis, and scarring of smooth muscle. As in intestinal atresia, most cases are presumed to result from an
intrauterine ischemic insult, although a history of an untoward event during pregnancy is often lacking (e114).
Duplications of the Gastrointestinal Tract

Gastrointestinal (enteric) duplications are tubular or cystic structures that lie alongside the intestinal tube. The
duplication and the intestinal tube often share a muscular wall (intramural); less often, the duplication is separated from
the intestine proper but in close proximity to it (extramural) (e51,e183). Duplications may occur anywhere near the
gastrointestinal tract from the neck to rectum; the single most common site is the ileum (Figure 14-18A).
No single theory of embryogenesis satisfactorily explains the origin of all duplications. They are thought to result from
aberrant diverticula in embryonic life (e469) or as a consequence of incomplete intestinal infarction in the fetus with
isolation of a viable portion of intestine adjacent to the regenerated intestinal tube (e138).
Symptoms vary widely, depending on the location of the duplication. Thoracic enteric duplication cysts are usually
extramural and found in the posterior mediastinum; they present with respiratory symptoms in infancy and may
communicate across the diaphragm with the intraabdominal gastrointestinal tract. Abdominal duplications may present
with pain, a palpable mass, intestinal obstruction, and, if peptic ulceration occurs in ectopic gastric mucosa, intestinal
bleeding.
FIGURE 14-18▪A: Locations and incidence of gastrointestinal duplication cysts. B: Ileal duplication cyst located near the
ileocecal valve. The appendix and cecum are on the left, and the dilated obstructed ileum is on the right. (Courtesy of
Robert J. Izant Jr, M.D., case-Western reserve University, Cleveland, Ohio.)
Multiple duplications are found in 5% of patients. The usual intestinal duplication is a cystic mass located on the
mesenteric border. It ranges in size from 2 to 7 cm in diameter, although much larger ones may also be found (Figure 14-
18B). The cyst lumen usually does not communicate with the intestinal lumen. Occasionally, tubular duplications
paralleling a long segment of intestine are found; these form a blind pouch proximally but communicate with the intestinal
lumen distally. Noncommunicating cysts are filled with mucoid material and histologically mimic normal gastrointestinal
tract with enteric mucosa, submucosa, muscularis propria, and a myenteric plexus. Intramural duplications usually do not
have a complete muscularis layer but rather share a muscularis layer with the adjacent intestine. The mucosa may
resemble adjacent normal gastrointestinal mucosa, but it is often very simplified and difficult to categorize except that
columnar epithelium bears a generic resemblance to intestinal surface epithelial cells. Cilia may be present, as in
embryonic intestinal epithelium. Gastric mucosa is found in approximately 20% of duplications and may cause peptic
ulceration in unlikely sites, such as the ileum and posterior mediastinum. Intestinal duplications in the abdomen must be
distinguished from a Meckel diverticulum and other vitelline duct remnants, mesenteric cyst (which lacks intestinal wall
morphology), and cystic lymphangioma.
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Meckel Diverticulum and Other Vitelline Duct Anomalies
The vitelline (omphalomesenteric) duct usually becomes obliterated by week 10 of embryonic life and subsequently
disappears completely (e226,e477). In approximately 2% of the population, however, it remains in various forms (Figure
14-19A-E). These include Meckel diverticulum or, less commonly, a fibrous cord extending from ileum to umbilicus, a
cyst, or an umbilical sinus. Many of these remnants are asymptomatic, but others cause symptoms that develop most
frequently in the first few years of life.
Meckel diverticulum is the most common vitelline duct remnant and also the most common congenital anomaly of the
gastrointestinal tract. It results from incomplete obliteration of the vitelline duct at the ileum and appears as a 1- to 5-cm
fingerlike protrusion of the intestine on the antimesenteric surface of the middle ileum (Figure 14-19D). When found
incidentally at autopsy or surgery, most Meckel diverticula are lined by small-intestinal epithelium. Those causing
symptoms are likely to contain heterotopic gastric mucosa (Figure 14-20A,B), which secretes acid and leads to peptic
ulceration of adjacent intestinal mucosa with subsequent abdominal pain, rectal bleeding, and occasionally intestinal
perforation. Approximately 25% of all Meckel diverticula contain foci of gastric mucosa. Occasionally, a Meckel
diverticulum may invert into the intestinal lumen and serve as the lead point of an ileal intussusception.
Other vitelline duct remnants are much less common than Meckel diverticulum. A vitelline cyst (Figure 14-19B) results
from partial obliteration of the vitelline duct and presents as a mass subjacent to the umbilicus. Microscopically, the cyst
wall resembles that of the intestine and is lined by mucus-secreting intestinal epithelium. A vitelline band is a fibrous cord
that persists after obliteration of the vitelline duct (Figure 14-19E). These bands extend from umbilicus to ileum, a
Meckel diverticulum, or a vitelline cyst and they may serve as a fulcrum for volvulus. Persistence of part of the vitelline
duct at the umbilicus causes an umbilical sinus, which presents with mucous discharge from the umbilicus (Figure 14-
19C). This must be distinguished from the very rare persistence of the entire vitelline duct (Figure 14-19A). Vitelline
cysts and sinuses at the umbilicus are distinguished histologically from urachal remnants at the same site by the
presence of intestinal or columnar epithelium. Urachal remnants have an urothelial lining.
FIGURE 14-19▪ Vitelline (omphalomesenteric) duct anomalies. A: Persistence of the entire vitelline duct from the ileum
to umbilicus. B: Vitelline duct cyst. C: Vitelline duct and umbilical sinus. D: Meckel diverticulum. E: Vitelline band.
Meconium and Meconium Abnormalities

Meconium is the dark green-to-black mucoid material that fills the neonatal colon and distal small intestine. It consists
predominantly of water (75%) admixed with mucous glycoproteins, swallowed vernix caseosa, gastrointestinal
secretions, bile, pancreatic enzymes, plasma proteins, minerals, and lipids. More than 90% of healthy term newborns
pass a meconium stool averaging 200 mL within the first 24 hours of life, and nearly all have done so by 48 hours.
Abnormalities of meconium (e.g., in cystic fibrosis) or of intestinal motility (e.g., in Hirschsprung disease) result in a
delayed meconium passage (e18).
Meconium Ileus
Meconium ileus is neonatal obstruction of the ileal lumen by abnormally viscid and inspissated meconium containing an
abnormally high level of albumin (e122,e211,e345,e513). Most but not all cases occur as the initial manifestation of
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cystic fibrosis; 10% to 15% of patients with cystic fibrosis are born with meconium ileus. Rarely, infants with congenital
pancreatic or pancreatic duct abnormalities have meconium ileus without cystic fibrosis, but these account for fewer than
5% of cases. The diagnosis of cystic fibrosis should be pursued in every infant with meconium ileus. In the classic case,
the distal one third of the ileum has a nearly normal diameter, but the lumen is filled with dense gray beadlike or solid
meconium having the consistency and appearance of putty. The middle one third of the ileum, proximal to the
obstructing meconium, is dilated and filled with dark gelatinous or tarlike meconium. Because the colon in meconium
ileus is empty throughout the fetal life, its diameter is smaller than normal.
FIGURE 14-20▪ AB: Meckel diverticulum. Ectopic gastric fundic and pancreatic tissue in the mucosa lines the
diverticulum. A: 40×. B: 100×.
Microscopically, the distal ileal lumen is filled with hypereosinophilic, focally calcified meconium. Intestinal glands are
dilated, often V-shaped, and plugged with hypereosinophilic secretions that are continuous with the luminal meconium. If
intrauterine intestinal perforation has occurred, the infant will also have meconium peritonitis. Approximately 50% of the
patients with meconium ileus have meconium peritonitis or other complications of meconium ileus, which include
intestinal atresia (e40) and volvulus.
The overall survival rate of infants with meconium ileus exceeds 80%, although they often have a prolonged hospital
course.
Meconium Peritonitis
Intestinal perforation in utero causes meconium to be released into the peritoneal space. The result is a distinctive
chemical peritonitis, with sterile inflammation, fibrosis, and characteristic calcifications (e151,e287). Between 33% and
50% of patients with meconium peritonitis have meconium ileus and cystic fibrosis. In half of the remaining patients,
perforation is the result of intrauterine intestinal obstruction resulting from atresia, malrotation with volvulus, mesenteric
hernias, or congenital bands. In the others, the bowel perforation and its cause are no longer apparent at birth, but it is
believed that intrauterine vascular insufficiency has caused the intestinal perforation (e459). Meconium peritonitis is
usually seen just after birth and is temporally remote from the intrauterine intestinal perforation that caused it. At gross
examination, the peritonitis is usually organized, with fibrosis, calcifications, and often dense intestinal adhesions.
Occasionally encountered is a meconium pseudocyst, a collection of soft meconium walled off by peritoneal fibrosis.
Microscopically, collections of squames and bile pigment in the peritoneal space, florid fibrosis, and calcifications
indicate the presence of meconium. Inflammation is usually chronic, and a well-developed foreign body response to
squames and calcifications may be noted. Because the fetal gut is sterile, the degree of inflammation is much less than
in the usual case of postnatal peritonitis. If meconium is released into the peritoneal space during intrauterine life, when
the inguinal canal to the scrotum is patent, the migration of meconium into the paratesticular area results in a condition
called meconium periorchitis (e113). Inguinal and even labial meconium masses may also occur, although more rarely, in
girls (e251).
Meconium Plug
Meconium plug is a syndrome of neonatal colonic obstruction caused by a plug of desiccated meconium, usually in the
ascending colon or, in infants with a very low birth weight, the ileum or proximal colon (e345,e484). It is a much less
serious condition than meconium ileus, but it may present with a similar clinical picture. The plug is usually passed after
a Gastrografin enema, and the infant has no further problems. Meconium plug syndrome may rarely occur in patients
with cystic fibrosis. It is essential that Hirschsprung disease be excluded. However, most infants with a meconium plug
have neither of these conditions.
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Gastrointestinal Involvement in Cystic Fibrosis
Cystic fibrosis, the most common lethal genetic disease in white children, is an autosomal recessive condition with an
incidence of 1 in 3,000 live births in the United States and Canada (e260,e422) (Chapters 5, 12, 15). The past decade
has seen great advances in the understanding of its pathogenesis. It is now known that mutations in the gene that
encodes the cystic fibrosis transmembrane conductance regulator result in faulty electrolyte transport across epithelial
surfaces and subsequent dehydration of luminal contents, which in turn leads to obstruction of glands and ducts by
thick, viscid secretions. The pancreas, intestines, and lungs are the chief organ systems affected. Gastrointestinal
symptoms may be present at birth and almost invariably appear during the first few months of life. Malabsorption
resulting from exocrine pancreatic insufficiency is a prominent manifestation in nearly all children and adults with cystic
fibrosis. Many gastrointestinal tract abnormalities are also characteristic signs of cystic fibrosis (e260,e346,e350,e422)
(Table 14-2).
Meconium ileus is the first sign of cystic fibrosis in approximately 10% to 15% of patients (e122,e211,e513). It usually
presents as intestinal obstruction in the 1st hours or days of life but has also been diagnosed antenatally by obstetric
ultrasonography. It should be considered a manifestation of cystic fibrosis until proven otherwise. Up to one-half of the
infants with meconium ileus have concurrent gastrointestinal manifestations of cystic fibrosis, including meconium
peritonitis, small-intestinal atresias and stenoses, duplication, volvulus, microcolon, and mesenteric bands or adhesions.
Distal intestinal obstruction syndrome, formerly called meconium ileus equivalent, is partial or total distal intestinal
obstruction by inspissated fecal material occurring in older children and adults with cystic fibrosis. It has nothing to do
with meconium. Viscid intestinal contents, a change in dietary habits, dehydration, and temporary disturbances in motility
are all thought to be etiologic (e81,e198). Up to 33% of all patients with cystic fibrosis are affected at one time or
another. The incidence of meconium plug syndrome is increased in neonates with cystic fibrosis, although most cases
occur in infants without cystic fibrosis (e345,e388). Fibrosing colonopathy is a rare distinctive stricturing process of the
colon first described in patients with cystic fibrosis in the 1990s, when it was linked to the ingestion of new preparations
of high-dose pancreatic enzyme replacement capsules. The condition usually presents with partial or complete intestinal
obstruction, and symptoms may mimic those of distal ileal obstruction syndrome (meconium ileus equivalent) or chronic
inflammatory bowel disease (e254,e355,e408,e433). Fibrosing colonopathy usually affects a long segment of the
ascending colon but may involve the entire colon. The lumen is compromised by circumferential submucosal fibrosis
along the length of the strictured segment. Fibrosis of the lamina propria, mucosal ulceration, acute and chronic mucosal
inflammation, and granulation tissue can also be seen. One series noted increased numbers of eosinophils in the
mucosa (e355). Because of the strong association between fibrosing colonopathy and high-strength pancreatic enzyme
supplements, it was recommended that the daily dose be reduced (e146).
Table 14-2 ▪ GASTROINTESTINAL MANIFESTATIONS OF CYSTIC FIBROSIS
Gastroesophageal reflux and Barrett esophagus

Malabsorption and pancreatic insufficiency
Meconium ileus, peritonitis, and meconium plug syndrome
Microcolon
Small-intestinal obstruction and atresia
Intussusception
Volvulus
Rectal prolapse
Brunner gland hyperplasia
Fibrosing colonopathy (due to pancreatic enzyme replacement)
Pneumatosis intestinalis
Mucocele
Gallbadder disease
HIRSCHSPRUNG DISEASE
Hirschsprung disease is characterized by an absence of intramural parasympathetic ganglion cells in the distal
gastrointestinal tract in association with a loss of tonic neural inhibition, persistent contraction of the affected segment,
and subsequent colonic obstruction. The condition usually results from defective craniocaudal migration of vagal neural
crest cells (the progenitors of ganglion cells) between gestational weeks 5 and 12. Interruption of craniocaudal migration
of neural crest cells explains the distal aganglionosis in Hirschsprung disease, but development of the ganglia is also
influenced by local factors in the intestinal wall (e234). The physiology of the disease is more complex than can be
explained by colonic ganglion cell absence alone; abnormalities of both adrenergic and cholinergic fibers are
demonstrable in the aganglionic bowel, and function of the internal anal sphincter is also abnormal.
Hirschsprung disease is a congenital disorder with an incidence of one per 5,000 live births and is much more common
in male infants (85%) than in female infants (e48). Most cases are sporadic, although a familial component has been
noted in approximately 10% of cases (13). Long-segment Hirschsprung disease and total colonic aganglionosis are the
types most likely to be familial conditions. It has a complex genetic basis, involving defects in at least ten genes, with
mutation in the RET gene playing a central role (4). In most cases, Hirschsprung disease is an isolated congenital
anomaly, but associations have been noted with Down syndrome (10% of patients with Hirschsprung disease have
Down syndrome), congenital heart disease, genitourinary anomalies, Waardenburg syndrome, congenital deafness,
intestinal atresia, and Ondine curse.
More than 90% of patients with Hirschsprung disease are born at full term with a normal birth weight. Presenting
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symptoms in neonates include delayed passage of meconium, vomiting, abdominal distention, and enterocolitis.
Hirschsprung enterocolitis, a grave complication that may develop rapidly, results from vascular compromise caused by
the distal obstruction and superimposed bacterial infection. More than 90% of infants with Hirschsprung disease fail to
pass meconium within 24 hours after birth (39).
The aganglionic segment in Hirschsprung disease begins at the anal sphincter and extends proximally (Figure 14-21A to
E). In 80% of cases, aganglionosis is limited to the rectum and distal sigmoid colon; this situation is sometimes referred
to as short-segment Hirschsprung disease. In the remaining patients, the aganglionic segment is longer (long-segment
Hirschsprung disease) and extends as far proximally as the splenic flexure or transverse colon in 10% and the cecum in
5% (total colonic aganglionosis or Zuelzer-Wilson disease). In rare cases, aganglionosis extends into the small intestine
and may reach as far as the proximal duodenum (e46). In the usual case, barium enema shows a narrow rectum and
rectosigmoid colon with a proximal funneling transition to a very dilated sigmoid colon. Neonates and infants with long
aganglionic segments do not have this diagnostic radiologic picture. Anal manometry is another valuable diagnostic tool
used in certain clinical settings. Despite the relative rarity of Hirschsprung disease, it enters into the differential
diagnosis of many other conditions because of its varied modes of presentation and the common occurrence of
functional constipation in children.
Although radiographic and manometric studies are routine diagnostic screening procedures, microscopic evaluation of
rectal biopsies is regarded as the gold standard for the diagnosis of this disorder. Suction biopsies are preferable over
routine forceps biopsies in order that an adequate sample of submucosal tissue is obtained (e512). The biopsy should
be performed at least 2 cm above the dentate line in order to avoid the normal zone of hypoganglionic distal rectum
(e4,e490). Thus, the first task of the surgical pathologist is to assess the adequacy of the biopsy material. A biopsy that
contains squamous or anal transitional epithelium should be reported as inadequate and the absence of ganglion cells in
such a specimen is disregarded. The biopsy must also contain an adequate thickness of submucosa in order to evaluate
for loss of ganglion cells. An accepted rule of thumb is that in a well-oriented biopsy the portion of submucosa sampled
should be at least as thick as the overlying mucosa.
FIGURE 14-21▪Distribution of affected colon in Hirschsprung disease (stippled area). A: Rectosigmoid aganglionosis.
B: Ultrashort Hirschsprung disease affected the distal rectum near the anal sphincter. C: Long-segment Hirschsprung
disease with involvement of the hepatic flexure. D: Total colonic aganglionosis. E: aganglionosis involving the entire
colon and the distal small bowel (in exceptional cases the distal duodenum may be involved).
Ganglion cells in neonates can have an immature morphology that can be difficult to recognize in H&E sections,
particularly in the submucosa (Meissner plexus) (e483). Confusion between endothelial cells and neuronal cells may
lead to a false negative diagnosis (Figure 14-22A,B). Careful examination of a large number of serial sections of each
biopsy is necessary before a diagnosis of Hirschsprung disease is made.
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The absence of ganglion cells in an adequate biopsy is diagnostic of Hirschsprung disease, but the presence of multiple
submucosal hypertrophic extrinsic nerve fibers is regarded as a helpful additional “positive” feature in making the
diagnosis. These abnormally thick fibers should be at least 40 μm in diameter (Figure 14-23). This feature is present in
more than 90% of the rectal suction biopsies of patients with Hirschsprung disease (e326). Thick submucosal nerve
fibers are reportedly less common in short segment Hirschsprung disease and in total colonic aganglionosis (112).
Immunohistologic stains to highlight these nerve fibers utilizing nerve growth factor receptor, glial fibrillary acidic protein,
or glutose transporter-1 antibodies can be performed on paraffin embedded biopsies, but the sensitivity and specificity
are not as good as that for acetylcholinesterase stains (79, 82) (e235,e507).
FIGURE 14-22▪ Normal submucosal ganglion cells A: In this 7-monthold child ganglion cells are easy to identify 200 ×.
B: In this 11-day-old infant the ganglion cells are immature appearing and therefore more difficult to identify 200 ×.
(Courtesy of Dr. Aliya N. Husain, University of Chicago Medical Center.)
FIGURE 14-23▪ Hypertrophic submucosal nerve fibers in a patient with Hirschsprung disease 200×. (Courtesy of Dr.
Aliya N. Husain, University of Chicago Medical Center.)
Histochemical demonstration of acetylcholinesterase positive cholinergic nerve fibers within the lamina propria provides
supportive evidence for the diagnosis of Hirschsprung disease, since they are not present in normal individuals (Figure
14-24). The presence of thick ropy fibers in the lamina propria between the crypts is regarded as highly specific for the
diagnosis (99) (e75). However, this change may not be clearly evident in patients under the age of 6 months, particularly
in short-segment Hirschsprung disease (112). In these patients the abnormal fibers may be present only in the
submucosa and muscularis mucosae, and since some positively stained thinner fibers are also present in these
locations in healthy infants, the diagnosis in this circumstance is problematic (99, 112). In addition, this technique can
only be performed on frozen sections, requiring the clinician to obtain extra biopsies, and the staining procedure must be
followed meticulously using freshly prepared reagents (98) (e272). The frozen sections should be cut at 15 μm thickness
so that the nerve fibers can be properly highlighted. Recently, a prepackaged kit for acetylcholinesterase staining has
become available, and this may increase usage of this methodology (99).
FIGURE 14-24▪Acetylcholinesterase stain of a rectal biopsy in a patient with Hirschsprung disease. Note the presence
of abnormal ropey nerve fibers in the lamina propria 400×.
There are also a variety of histochemical and immunohistologic stains that can be used to visualize submucosal ganglion
cells in suction rectal biopsies. The histochemical stains, which must be performed on frozen sections, highlight enzymes
present in ganglion cells, including lactic dehydrogenase, alpha-naphthyl esterase, and NADPHdiaphorase. These
stains are not commonly performed in the United States, but the availability of a prepackaged kit may increase their
usage in the future (99). Immunohistologic stains have the advantage of being performed on sections from paraffin
embedded biopsies, obviating the need to obtain extra biopsies. However, they all suffer from the distinct disadvantage
of having to be performed on multiple levels to ensure that no ganglion cells are present. Antibodies that have been
utilized to highlight the presence of ganglion cells include NSE, bcl-2, bone morphogenic protein 1 A, and RET (19, 85)
(e492). In addition, S-100 immunostains can highlight the presence of hypertrophic submucosal nerve fibers and
highlight the presence of ganglion cell bodies by their lack of staining (e386,e451).
Recently, the observation was made that patients with Hirschsprung disease lacked calretinin immunoreactive
submucosal nerve fibers in the aganglionic segment of colon (13). This observation has led to a proposal to use
calretinin immunostains on rectal suction biopsies as a diagnostic test. In the single report to date, calretinin reactive
nerve fibers were absent from all rectal biopsies from Hirschsprung patients and present in all patients where ganglion
cells were present in the biopsy (Figure 14-25A,B). The authors stressed that for unclear reasons the density of
calretinin reactive submucosal fibers varied greatly among the patients without Hirschsprung disease and that at the
anorectal junction no reactive fibers are normally present (84). If the utility of this staining pattern can be confirmed by
other authors, this technique may represent a valuable diagnostic adjunct in Hirschsprung disease.
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FIGURE 14-25▪Calretinin immunostains performed on sections from suction rectal biopsies. A: In a normal infant,
calretinin reactive submucosal nerve fibers are prominent 400×. B: In an infant with Hirschsprung disease, there is no
reactivity in submucosal nerve fibers in the aganglionic segment 400 ×. (Courtesy of Dr. Aliya N. Husain, University of
Chicago Medical Center.)
A histologic diagnosis of Hirschsprung disease made from examination of a suction rectal biopsy should be confirmed by
frozen section at the time corrective surgery is performed. A seromuscular biopsy containing the full thickness of the
muscularis propria from the abnormally narrowed aganglionic segment allows for examination to confirm the absence of
ganglion cells within the myenteric (Auerbach) plexus. The myenteric (Auerbach) plexus and ganglion cells within it are
much larger and easier to interpret than those in the submucosal (Meissner) plexus. Normal myenteric plexus, found
between the inner circular and outer longitudinal layers of the muscularis externa, consists of unmyelinated nerve fibers
and clusters of rounded Schwann cells and large ganglion cells around the perimeter of the nerve fibers. Myenteric
ganglion cells can be recognized by their polygonal shape, abundant cytoplasm, round and eccentric nucleus, and
prominent nucleolus. These features are apparent even on frozen section. In Hirschsprung disease, by contrast, nerves
are present but ganglion cells are completely absent.
If a full-thickness biopsy is performed, the submucosa can be examined for the presence of ganglion cells in Auerbach
plexus. The ganglion cells are contained within “neural units”, first described by Yunis is a seminal 1976 publication,
consisting of vaguely organoid structures containing two to ten nuclei in a horseshoe-shaped array surrounding a central
core of pale, bubbly neural tissue. The ganglion cells are located at the periphery of these neural units (e512).
There is often a hypoganglionic colonic segment of variable length just proximal to the aganglionic segment (e162).
Thus, frozen sections are also performed on muscularis propria from above the grossly narrowed segment to confirm
that normal numbers of ganglion cells are present. This allows the surgeon to identify the proper level at which to
transect the colon. However, it must be admitted that the confident recognition of immature ganglion cells in frozen
sections is problematic, and both false positive and negative errors are possible (97).
In some patients the distal functional obstruction leads to the development of diversion-like colitis changes in the
aganglionic segment and obstructive-like colitis in the segment just proximal to it (e372). Colitis is a major cause of
morbidity and mortality in patients with untreated Hirschsprung disease (e456).
Chronic Intestinal Pseudo-Obstruction

The term intestinal pseudo-obstruction denotes greatly impaired or absent peristalsis without mechanical obstruction to
luminal flow (e390,e480). Pseudo-obstruction is a clinical syndrome, not a pathologic diagnosis. A recent consensus
workshop of pediatric gastroenterologists defined the clinical features of chronic intestinal pseudo-obstruction as
intermittent or continuous bowel obstruction, including radiologic documentation of dilated bowel with air-fluid levels but
without a lumen-occluding lesion. A wide clinical spectrum comprises more than 70 known entities, all of them rare. The
diagnosis is often elusive and difficult for both gastroenterologists and pathologists (e167). Both congenital and acquired
forms exist, and the pseudo-obstruction may occur in any region of the gastrointestinal tract. By the above definition,
cases of Hirschsprung disease would be included, but by convention, they are excluded. However, Hirschsprung
disease is considered in the differential diagnosis of colonic pseudo-obstruction presenting early in life.
The two major subclasses of pseudo-obstruction are myopathic and neurogenic, with primary and secondary
subcategories in each (Table 14-3). In primary chronic intestinal pseudo-obstruction, manifestations of the disease are
limited chiefly to the gastrointestinal tract; in the secondary form, the
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gastrointestinal manifestations are part of a systemic disease. A panel of immunohistologic stains can be used to
systematically evaluate both the muscular and neural elements of the bowel wall in specimens submitted for diagnostic
purposes (5).
Table 14-3▪ CHRONIC INTESTINAL PSEUDO-OBSTRUCTION IN CHILDREN
Primary visceral myopathies
Congenital megacystis-microcolon-hypoperistalsis
Other familial visceral myopathies
Secondary visceral myopathies
Muscular dystrophies
Connective tissue disorders
Ehlers-Danlos syndrome
Primary visceral neuropathies
Familial visceral neuropathies

Neuropathies associated with malrotation and gastro
schisis
Myenteric plexus neuropathies
Intestinal neuronal dysplasia
Secondary visceral neuropathies
Familial dysautonomia
Myotonic dystrophy
Postviral pseudo-obstruction
Visceral myopathies can be appreciated by conventional microscopy, although full-thickness specimens of intestinal wall
are necessary because the abnormalities are in the muscularis propria. Degeneration, atrophy, and sometimes fibrosis
of intestinal smooth muscle are revealed by hematoxylin and eosin stain and enhanced by Masson trichrome stain. The
outer, longitudinal layer of the muscularis propria is almost always more affected than the inner, circular layer. The most
widely recognized of the visceral myopathies is congenital megacystis-microcolon-intestinal hypoperistalsis syndrome,
also called hollow visceral myopathy (e13,e37,e405,e502). It is transmitted as an autosomal dominant trait and is
diagnosed at birth or by prenatal ultrasonography. Many other familial visceral myopathies without megacystis also
occur. The inheritance of these varies with type and from family to family (e267,e336,e405,e432). Smooth muscle
degenerative changes also occur secondarily in the muscular dystrophies, particularly Duchenne muscular dystrophy
(e281), and in connective tissue disorders, such as polymyositis, lupus erythematosus (e65), and scleroderma. Genetic
defects of the mitochondrial oxidative phosphorylation pathway have been determined to be responsible for a small
subset of the myopathic form of pseudo-obstruction (6). Structural malformation of the intestinal muscularis propria has
also been reported as a rare cause of pseudo-obstruction (83).
Study of the visceral neuropathies is less easily accomplished because conventional light microscopy of paraffin-
embedded sections of intestine stained with hematoxylin and eosin often does not demonstrate any changes. Special
techniques such as electron microscopy and acetylcholinesterase staining of frozen sections may demonstrate
abnormalities. Even more technically difficult and not often performed in routine practice is the sectioning and staining
technique devised by Smith to demonstrate subtle changes in the myenteric plexus. With this method, a large surgical
biopsy specimen of muscularis propria is embedded flat so that the myenteric plexus is sectioned longitudinally or
obliquely rather than in the more conventional perpendicular plane. Much thicker sections are used than in conventional
microscopy, and these are prepared with a special silver stain (e406,e431). By means of this technique, Schuffler and
others have described a number of rare abnormalities of the myenteric plexus (e266,e334,e406). The submucosal
plexus is usually normal. The onset of symptoms in the pediatric visceral neuropathies is usually between birth and the
first few months of life. Depending on the location and length of the affected intestinal segment(s), symptoms include
abdominal distension, vomiting, and constipation. Many of the visceral neuropathies in children show no familial pattern
(e266), although familial forms are also known (e29,e68,e135,e267,e390). Familial forms of visceral myopathy involving
the gastrointestinal tract have been reported (125).
Intestinal Neuronal Dysplasia

Isolated intestinal neuronal dysplasia has been described in the distal colon and rectum, and its clinical presentation,
with constipation and intestinal obstruction, mimics that of Hirschsprung disease (e136,e399). Intestinal neuronal
dysplasia type A is extremely rare, if it exists at all. The term has been used variously to describe either
hypoganglionosis or complete aganglionosis, involving either the myenteric plexus alone or affecting both the myenteric
and submucosal plexi (101) (e210,e255). Accepted diagnostic criteria have not been agreed upon. Neuronal intestinal
dysplasia type B was first described and strictly defined by Meier-Ruge and Scharli (e399) in newborns with colonic
obstruction, hyperplasia of the submucosal and myenteric plexuses, giant ganglia, and increased nerve fibers in
acetylcholinesterase-stained frozen sections of bowel wall. The original definition required visualization of the myenteric
plexus in a full-thickness biopsy specimen plus the demonstration of increased numbers of nerve fibers in
acetylcholinesterase-stained frozen sections. Since the entity was first described, others proposed adjustments to the
criteria so that the diagnosis could be made by rectal suction biopsy alone. This resulted in variable diagnostic criteria,
continued controversy, and wide variations in the reported incidence of intestinal neuronal dysplasia (99) (e92). Some
investigators believe that histologic features of intestinal neuronal dysplasia type B can be seen in suction biopsy
specimens in a variety of clinical settings and that it is more a descriptive entity than a specific disease requiring surgical
intervention (e90,e257,e322,e402,e403). Many patients in whom this condition was diagnosed by suction biopsy have
improved over time with conservative measures exclusive of surgery (e90,e402,e427).
Diagnostic criteria developed by a consensus panel of European pathologists that were published in 1991 included
obligatory features (hyperplasia of submucosal nerves,
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increased acetylcholinesterase activity in submucosal nerve fibers, and giant submucosal ganglia) as well as facilitative
features (increased acetylcholinesterase activity in lamina propria nerve fibers and heterotopic ganglia in the lamina
propria) (e50). At this time the disorder was recognized to affect only the submucosal nerve plexus, and thus diagnosis
by sampling of the myenteric plexus is not possible. The criteria, which have been adopted by many clinical studies,
have subsequently been revised and updated (140) (e317,e318). For instance, the number of ganglion cells defining a
giant ganglion has varied from seven to nine (Figure 14-26). The subsequent recognition of age-dependent changes in
the histologic features of the disease has led to further refinement of the diagnostic criteria. Specifically, the number of
acetylcholinesterase-positive submucosal nerve fibers has been shown to decrease with age in affected patients (140).
Currently, some experts in the field have stated that the histologic diagnosis of intestinal neuronal dysplasia type B rests
primarily upon the identification of giant submucosal ganglia, defined as a ganglion containing eight or more identifiable
ganglion cells (102). If more than 20% of the submucosal ganglia in a rectal biopsy are giant ganglia, a diagnosis of
intestinal neuronal hyperplasia is appropriate. However, since it is now recognized that giant ganglia are more numerous
in premature infants and neonates, this diagnostic criterion is felt to be diagnostic only in patients more than 1 year of
age. In addition, the criteria above were developed based on the examination of well-oriented frozen sections cut at 15
μm thickness and utilizing enzymatic histochemical stains for dehydrogenases to highlight ganglion cells. The
applicability of these criteria to the evaluation of H&E stained 4 μm-thick paraffin sections is uncertain (102). The
reproducibility of the histologic diagnosis of intestinal neuronal dysplasia type B remains poor (e258). It appears that
even as currently defined the symptoms of patients with intestinal neuronal dysplasia type B improve with age and
therefore conservative therapy is more appropriate that surgical treatment (131) (e257).
FIGURE 14-26▪ Giant ganglion in a patient with intestinal neuronal dysplasia 400 ×.
Histological features overlapping with those of intestinal neuronal dysplasia type B have been reported in some patients
with neurofibromatosis type I (von Recklinghausen disease) (e99,e140,e155,e414) and multiple endocrine neoplasia
type IIB (e70,e99,e414). Intestinal neuronal dysplasia has also been described in association with Hirschsprung disease,
at sites proximal to the classic aganglionic segment of Hirschsprung disease (e58,e369).
Acquired Diseases
Intussusception
Intussusception, or the invagination of a portion of the intestine into itself, is a relatively common pediatric surgical
problem (e132). Infants, particularly those between 5 and 9 months of age, are most commonly affected. More than 90%
of cases of childhood intussusception begin at the ileocecal valve, and the intussusceptum may reach as far as the
descending colon or rectum. Progressive compression of the mesentery and blood supply of the invaginated bowel
causes edema, hemorrhage, and ischemic necrosis. In the classic case, severe, intermittent, colicky pain begins
suddenly in an infant, followed after a few hours by vomiting and the passage of blood and mucus from the rectum.
Barium enema is both diagnostic and therapeutic. The obstructing mass of invaginated bowel can be recognized, and if
the congestion and edema are not too advanced, the application of hydrostatic pressure by the radiologist reduces the
intussusception. Operative reduction is required if barium enema reduction fails, as happens in 20% to 30% of cases
(e511).
Gangrene of a portion of the intussusceptum necessitates segmental intestinal resection in approximately 10% of cases.
These specimens exhibit edema, congestion, and coagulative and hemorrhagic necrosis indicative of combined
ischemia and venous outflow obstruction.
In most cases, the cause is unknown. Large Peyer patches have been proposed as the possible lead point in many
cases. Lymphoid hyperplasia due to adenovirus infection has been implicated in a subset of patients (Figure 14-27A to
C) (16) (e327). Use of the first commercially available oral rotavirus vaccine was associated with an increased risk of
intussusception in children, leading to withdrawal of the vaccine from the market (117). Newer generation vaccines do
not appear to have this risk (34).
Approximately 10% of cases of childhood intussusception do not conform to the typical picture. In children past infancy
and in atypically located intussusceptions (i.e., those not in
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the ileocecal valve region), a discrete lead point is usually identified. Meckel diverticula, Peutz-Jeghers polyps, juvenile
polyps, small-intestinal duplications, and Burkitt lymphoma have been implicated (70).
FIGURE 14-27▪ Small-bowel intussusception in an infant due to adenovirus infection. A: Histologic section from the lead
point of the intussusception demonstrating lymphoid hyperplasia 20×. B: High power demonstrates smudgy intranuclear
inclusion within enterocytes 200×. C: Immunostain utilizing an adenovirus antibody confirms the diagnosis 200×.
GASTROINTESTINAL INFECTIONS
Infections of the gastrointestinal tract are the leading cause of morbidity in infants and children in all parts of the world
(e188) (Table 14-4). Mortality resulting from this group of illnesses is common in infants in underdeveloped countries
where malnutrition contributes to the poor outcome. In developed countries, gastroenteritis and diarrhea are frequent
causes of illness, but they seldom cause death because nutrition is better and medical care and intravenous fluids are
available (e165).
In the 1970s, viruses, particularly rotavirus, were identified as the causal agents in more than half the cases of
gastroenteritis and diarrhea worldwide. In developed countries, bacterial infections account for approximately 15% of
hospitalized cases of diarrhea. Many of the causative strains have been identified only relatively recently; these include
Clostridium difficile, Campylobacter jejuni, H. pylori, Yersinia enterocolitica, and the enteroinvasive, toxigenic, and
hemorrhagic strains of Escherichia coli. The AIDS epidemic and advances in immunosuppressive chemotherapy have
led to recognition of “new” organisms. At the same time, rapid advances in microbiologic, serologic, and molecular
diagnosis have made it possible to identify them in patients.
Viral Diarrhea
Most cases of acute infection, gastroenteritis, and diarrhea in infants and children are caused by viruses (e43,e66).
Typically, the viruses localize in the small intestine and cause a noninflammatory, watery diarrhea; neutrophils and red
blood cells are not found in the stool. Patients usually have nausea, vomiting, and low-grade fever. Infants are often
quite ill and may become severely dehydrated.
Rotaviruses
Rotaviruses have been extensively studied (e43, e148,e467). Group A rotaviruses are responsible for
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approximately 50% of hospitalized cases of diarrhea in all parts of the world (e105,e148). Children younger than 2 years
are most susceptible; they are usually ill for 4 to 7 days and are most likely to be admitted to a hospital for treatment of
dehydration resulting from watery diarrhea and vomiting. Rotavirus was first identified by electron microscopy in small-
intestinal epithelial cells and later in the stools of infants with diarrhea. Rotavirus infection was formerly diagnosed by the
ultrastructural identification of virus particles in stools, but this method has been replaced by ELISA of stool samples.
Biopsies are almost never performed during the acute illness, but several morphologic studies have shown proximal
smallintestinal mucosal injury with surface enterocyte necrosis, partial villous atrophy, and chronic inflammation in the
lamina propria (e27,e105,e501). The loss of enterocytes greatly reduces the capacity of the intestine to absorb fluid and
electrolytes, and the effect is compounded when damage to brush border enzymes results in malabsorption. The
mucosa takes 3 to 8 weeks to recover; during this time, malabsorption may persist (e105). Immunodeficient patients may
take months to clear the virus and suffer a more chronic illness. Rotavirus vaccines are now available.
Table 14-4 ▪ PEDIATRIC GASTROINTESTINAL INFECTIONS
Means of
Organism Location Symptom/Syndrome Histology Diagnosis
Bacteria
Helicobacter pylori Stomach, Epigastric abdominal Active chronic Histologic

especially antrum pain in surface nonspecific gastritis; identification
organisms visible of bacilli on
muous coat biopsy,
culture of
endoscopic
biopsy
Salmonella (S. Distal SI, especially Gastroenteritis, Acute enteritis with Stool culture,
enteritidis, S. ileum, colon inflammatory bloody, exudation, blood culture
typhi, S. mucoid stools; enteric hemorrhage, focal (S. typhi)
cholerasuis) (typhoid) fever ulceration; acute
infective colitis;
hypertrophy,
necrosis and
macrophage
infiltration of Peyer
patches, mesenteric
LN
Shigella Colon, distal Bloody, mucoid stools, Acute infective Stool culture
SI diarrhea, cramps, colitis.
fever, convulsions
Vibrio cholerae SI Massive watery Minimal change Stool culture

diarrhea and
dehydration
Escherichia coli SI Diarrhea Enteritis; may show Stool culture

Enteropathogenic villous atrophy and
serotyping
Enterotoxigenic SI Watery diarrhea, Minimal change Stool culture

traveler's diarrhea and
serotyping
Enteroinvasive Distal SI, colon Bloody, mucoid Acute infective Stool culture
diarrhea colitis and
serotyping
Enterohemorrhagic Colon, distal Bloody diarrhea Acute infective Stool culture

SI hemolytic uremic colitis on selective
syndrome medium,
serotyping,
toxin assay
Campylobacter SI, colon Abdominal pain, Acute enteritis, Stool culture

jejuni diarrhea, bloody stools acute infective
colitis, acute
appendicitis,
mesenteric adenitis
Yersinia Entire GI tract Diarrhea, abdominal Enteritis with Stool culture

enterocolitica especially ileum, pain, fever ulcerations and
appendix, colon microabscesses;
terminal ileitis
mimicking Crohn
disease; necrotizing
appendicitis; acute
infective colitis with
apthoid ulceration;
mesenteric adenitis
Clostridium Colon Pseudomembranous Pseudomembranous Toxin assay

difficile colitis, colitis; acute colitis on stools
antibioticassociated
diarrhea
Listeria SI, colon; systemic Fever, gastroenteritis ND for GI tract Stool culture,
monocytogenes spread in rectal swab
immunosuppressed on selective
media
Aeromonas Colon, SI Acute watery diarrhea; Acute colitis Stool culture

dysenteric-like illness,
colitis
Mycobacterium SI, colon Diarrhea, abdominal Acid-fast bacilli in Identification

avium complex pain, malabsorption in macrophages of acid-fast
AIDS throughout lamina bacilli in
propria macrophages
in lamina
propria of
intestinal
biopsy
Fungi All GI tract Depends on location Pseudohyphae and Fungal stain

Candida yeast forms with on biopsy
acute inflammation
Protozoa Proximal SI Diarrhea, Proximal SI changes Stool

Giardia lamblia malabsorption; failure range from minimal examination
to thrive change to chronic for cysts;
inflammation and mucus
villous atrophy; smears of
organisms on H&E intestinal
biopsy;
identification
visible of
trophozoites
on proximal
SI biopsy
Crytosporidium Small intestine in Watery diarrhea Minimal change or Stool

normal hosts; mild, nonspecific examination
stomach, SI and enteritis; organisms for cysts;
colon in AIDS visible on H and E identification
patients by histologic
examination
of proximal SI
biopsy
Entamoeba Colon Hematochezia; Diffuse acute colitis, Stool

histolytica diarrhea; bloody, microulcerations; examination
mucoid diarrhea deep ulcers for
undermining trophozoites
mucosa, organism and cysts;
visible on H and E identification
of
trophozoites
on histologic
examination
of colonic
biopsy;
serology;
identification
of organism
on biopsy
Viruses SI Watery diarrhea, Enterocyte necrosis, Stool ELISA

Rotavirus vomiting partial villous
atrophy,
mononuclear
inflammation
Norwalk virus SI Watery diarrhea, See text Stool ELISA,

vomiting PCR on stool
Adenovirus Colon, SI Diarrhea Inclusions in surface Identification

epithelium of inclusion
on biopsy
Astrovirus ND Diarrhea ND ELISA on

stool
Calicivirus ND Diarrhea ND
Cytomegalovirus All parts of GI tract GI bleeding; Focal necrotizing Identification

hemorrhagic colitis; colitis, esophagitis, of inclusions
esophagitis gastritis; inclusions on
visible in endoscopic
endothelium and biopsy,
mesenchymal cells culture of
biopsy or
stool
Herpes simplex Esophagus, colon Esophagitis, colitis Small focal Identification

(apthous) of inclusions
ulcerations, on biopsy,
inclusion in culture of
epithelium biopsy
GI, gastrointestinal; H and E, hematoxylin and eosin; LN, lymph node; SI, small intestine; ND, not described;
ELISA, enzyme-linked immunosorbent assay; RT-PCR, reverse transcriptase polymerase chain reaction.
Norwalk Virus
Norwalk virus is the second most commonly encountered cause of viral gastroenteritis. Cases tend to occur in clusters,
and epidemics are more frequent in children of school age than in infants. This is a briefer, less severe illness
characterized by vomiting and watery diarrhea. No method to diagnose this infection is readily available, although
immune electron microscopy and recently developed ELISAs to detect viral antigen and antibody responses are used in
reference laboratories. Only samples taken from patients during diarrhea epidemics are likely to be studied by these
means. Histologic studies are sparse, but villous blunting, infiltration of mononuclear cells and neutrophils into the lamina
propria, and vacuolization of enterocytes have been described (e2,e404).
Electron microscopic studies of stool specimens during outbreaks of gastroenteritis and diarrhea have led to the
identification of other viruses, although none of these is encountered as often as rotavirus and Norwalk virus.
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FIGURE 14-28▪ CMV colitis in a child status post stem cell transplantation. A: Mild colitis is evident in this biopsy 400×.
B: Several classic intranuclear viral inclusions are present 400×.
Enteric Adenovirus
Enteric adenoviruses are serologically distinct from respiratory adenoviruses, and for a long time they eluded detection
except by electron microscopy and difficult immunologic methods not widely available (e293). Several outbreaks of
enteric adenovirus gastroenteritis have been described in normal infants, and this infection is probably a common cause
of pediatric viral gastroenteritis. Immunocompromised patients, particularly those with AIDS, are highly susceptible to
adenovirus infection (e264,e292,e508). Patients with solid organ and bone marrow transplants are also at risk
(e147,e418,e442).
Nonspecific watery diarrhea with vomiting, dehydration, and abdominal pain characterizes the illness (e467). Lactose
intolerance and other malabsorptive states may follow adenovirus enteritis and last for months. Adenovirus nuclear
inclusions can be identified by light microscopy within infected surface epithelial cells, but the presence of adenovirus
should be confirmed by immunohistochemical stains or electron microscopy (e508). Enteric adenovirus infection can
result in small intestinal mucosal lymphoid hyperplasia, which can form the lead point of an intussusception (Figure 14-
27A-C).
Cytomegalovirus
Gastrointestinal infection with CMV has become a significant clinical problem in persons with bone marrow or solid organ
transplants and in patients with AIDS or other conditions associated with immune compromise (e368,e389). Occasional
cases have been reported in patients with ulcerative colitis and Crohn disease. Any site in the gastrointestinal tract can
be affected, from esophagus to colon. In the most severe cases, the patient often also has evidence of a systemic
infection, with CMV pneumonia, hepatitis, and retinitis. Symptoms vary according to the affected site of the
gastrointestinal tract. Particularly characteristic is a fulminant hemorrhagic colitis with multiple focal ulcerations resulting
from CMV vasculitis and thrombosis; this sometimes leads to toxic megacolon, necrotizing colitis, and intestinal
perforation (e14,e152,e319). Esophageal involvement is usually distal, with ulcerations and erythema. Gastric and small-
intestinal involvement is also generally manifested as erosions and ulcerations. Because it tends to affect blood vessels,
CMV infection often presents with gastrointestinal bleeding. The diagnosis is made by endoscopic biopsy, with typical
inclusion bodies usually seen in vascular endothelium, mesenchymal cells of the lamina propria, and more rarely in
glandular epithelial cells (Figure 14-28A,B). Inclusions are not seen in squamous cells (e389). Variable degrees of acute
and chronic inflammation, vasculitis, thrombosis, and ulceration are present, depending on the extent of the infection and
the degree of ulceration.
Herpes Simplex Virus

Gastrointestinal infection with this group of viruses is usually limited to immunosuppressed patients, although herpes
esophagitis occasionally develops in normal children. In the gastrointestinal tract, herpesvirus most commonly causes
esophagitis (see previous section) or proctocolitis. At both sites aphthous and more extensive ulceration is
characteristic. Viral cytopathic changes and inclusions are most commonly seen in squamous epithelium, but glandular
epithelium and mesenchymal cells may also be involved. The accompanying inflammation commonly contains
neutrophils and histiocytes (e305,e389).
Other Viruses
Other recently described viruses associated with gastroenteritis include coronaviruses, astroviruses, (e206,e323) and
caliciviruses (e28,e323).
Bacterial Diarrhea
Bacteria cause diarrhea through multiple pathophysiologic mechanisms that are categorized as inflammatory or
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noninflammatory (e176,e379). Salmonella species, Shigella species, and Campylobacter jejuni are the most common
causes of inflammatory infectious diarrhea in children (e85), with C. difficile, Y. enterocolitica, enteroinvasive E. coli,
and the protozoan Entamoeba histolytica causing a similar picture. Most of these organisms invade the mucosa, usually
in the colon and distal small intestine, and cause epithelial necrosis and a neutrophilic response. The same inflammatory
response may be elicited by the cytotoxins of some noninvasive pathogens, such as C. difficile, and some toxin-
producing E. coli, including enterohemorrhagic E. coli 0157:H7 (e85). Dysentery is said to be present if inflammatory
diarrhea is accompanied by systemic manifestations such as fever, abdominal pain, and prostration. The stool contains
neutrophils, mucus, and blood. A mucosal biopsy is not usually obtained if the organism is identified by stool culture, but
a biopsy may be performed in a patient with infectious colitis before the organism is cultured or if rectal bleeding
persists. The pathologist may be asked to distinguish infection from ulcerative colitis or Crohn disease (e337,e448).
In contrast, organisms causing noninflammatory diarrhea exert their effect through a toxin or other mechanism without
penetration of the intestinal mucosa. Watery diarrhea is characteristic, and neutrophils and blood are not found in the
stool. The tissue response is less pronounced and usually localized to the small intestine. Examples of organisms
causing noninflammatory infectious diarrhea include Vibrio cholerae, C. jejuni, enteropathogenic E. coli, enterotoxigenic
E. coli, and some Salmonella organisms. Giardia lamblia, Cryptosporidium, and viruses, especially rotaviruses, are
nonbacterial causes of noninflammatory diarrhea.
Salmonella
Salmonella-induced diarrhea is a worldwide food-borne and waterborne illness. In the United States and Canada,
infants and children are most often affected. In a large series of infants with diarrhea at the Hospital for Sick Children,
Toronto, Salmonella organisms were the most common bacterial pathogens isolated. Infants may present with the acute
onset of watery diarrhea, abdominal pain, and fever, but a dysenteric presentation with mucus, pus, and blood in the
stools is also encountered (e108,e311). Salmonella organisms penetrate the intestinal mucosa and invade the
submucosa, stimulating a neutrophilic response, epithelial necrosis with focal ulceration, hyperemia, and sometimes
hemorrhage. In typhoid fever (Salmonella typhi infection), the organisms are carried by macrophages to intestinal
lymphoid tissues, particularly Peyer patches, which become hyperplastic and necrotic. From there, bacilli enter the
bloodstream. Morbidity and mortality are high (e457). The more common childhood enteric infections with other strains
of Salmonella (S. enteritidis and S. typhimurium) are usually acquired through the ingestion of contaminated eggs,
poultry, and other animal products, and their course is more self-limited. The histology of the usually self-limited acute
enteritis caused by Salmonella species is rarely observed in clinical practice. However, a colonic mucosal biopsy
specimen is occasionally encountered and shows nonspecific edema, neutrophilic exudate in the lamina propria and
epithelium, and crypt abscesses (e180). Rarely does a colonic biopsy in salmonellosis show the florid crypt abscess
formation, goblet cell depletion, and chronic crypt alterations characteristic of ulcerative colitis. Patients with AIDS are
especially susceptible to severe Salmonella infections, sometimes with enterocolitis and bacteremia that are resistant to
therapy (e284).
Shigella
Shigella dysenteriae is the prototype organism producing dysentery (the frequent passage of bloody mucoid stools with
fever and abdominal cramps) (e282). Shigella dysenteriae and Shigella flexneri are the species responsible for most
infections in developing countries (e11), and Shigella sonnei is usually isolated in industrialized countries. Direct
invasion of the colonic epithelium and lamina propria by the organism causes cell death, ulceration, and hemorrhage.
Shigella also produces potent toxins, known as Shiga toxins, which compound the intestinal damage. Shigella colitis is
characterized by superficial ulceration, purulent mucosal exudate, and, in severe cases, confluent hemorrhagic necrosis
of large areas of mucosa with pseudomembrane formation. The microscopic picture is that of an acute colitis with
ulceration, crypt abscess formation, and goblet cell depletion (eFigure 14-6). In fulminant cases distinction from
ulcerative colitis can be difficult in the absence of culture results (e436).
The potent Shiga toxins produce watery diarrhea in some cases and also have far-reaching effects throughout the body
(e242,e282). Hemolytic-uremic syndrome is known to occur after shigellosis in a small percentage of children
(e263,e288). Thrombotic thrombocytopenic purpura, a similar illness with more central nervous system manifestations, is
the adult counterpart of hemolytic-uremic syndrome. Both these conditions are caused by Shiga toxin and a group of
similar toxins, Shiga-like toxins, produced by enterohemorrhagic strains of E. coli, particularly 0157:H7. Shiga toxin not
only affects the gastrointestinal tract but also exerts a cytotoxic effect on endothelial cells throughout the body and is a
neurotoxin. The toxin causes extensive platelet fibrin thrombi to form in small blood vessels, impairing perfusion to vital
organs. Hemolytic-uremic syndrome, a microangiopathic hemolytic anemia, results from the effect of the toxin on the
kidneys. In the most severe cases, acute renal failure may follow the thrombotic microangiopathic renal process (see
Chapter 17).
Vibrio Cholerae
Cholera is rarely encountered in developed countries, but it is an important cause of morbidity and mortality in children
worldwide. Cholera is a classic example of enterotoxigenic diarrhea, in which massive fecal fluid losses rapidly lead to
dehydration in the absence of any tissue invasion by
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the organism, whose enterotoxin stimulates the secretion of water and electrolytes and inhibits absorption by epithelial
cells (e375). Morphologic changes are minimal. Surface epithelium of the small intestine remains intact, and at most, a
mild increase in cellularity of the lamina propria and vascular congestion are observed (e104).
Escherichia coli
Various strains of E. coli were first recognized as stool pathogens after the development of specific serotyping, which
made it possible to differentiate them from normal gut flora. More recent molecular, genetic, and biochemical differences
have led to the identification of many classes of E. coli that cause diarrhea, with a different pathogenesis and clinical
picture for each (e176).
Enteropathogenic E. coli was the first of these to be identified in association with epidemics of diarrhea in infant hospital
wards, nurseries, and day care centers. It is one of the major causes of bacterial diarrhea in infants worldwide and is
also one cause of traveler's diarrhea in adults who visit underdeveloped countries. It produces a toxin that acts on the
small-intestinal epithelium to produce profuse watery diarrhea. Numerous serotypes have been identified, but serologic
testing is usually performed only in epidemic clusters and not in sporadic cases. Electron microscopy of infected small
intestine shows adherence of the bacteria to the brush border of enterocytes, with dissolution of microvilli. Adherent E.
coli can sometimes be identified on the intestinal epithelial surface, associated with villous atrophy and inflammation of
the lamina propria. Enterotoxigenic strains of E. coli produce secretory enterotoxins and commonly cause watery
diarrhea in children and adults in developing countries, in addition to traveler's diarrhea. Occasional outbreaks from
contaminated food or water are reported in developed countries. A choleralike illness characterized by noninflammatory
watery diarrhea results from the effect of bacterial toxin on the small intestine. The illness is usually self-limited and
resolves in a few days unless the child is malnourished or very young. Enteroinvasive E. coli affects the colon rather
than the small intestine. It invades the mucosa, much like Shigella, and in severe cases produces a similar dysenteric
disease, with bloody and mucoid diarrhea, neutrophils in the stool, and systemic symptoms, including fever, headache,
myalgia, and abdominal pain.
Enterohemorrhagic E. coli was first identified in the early 1980s in association with outbreaks of hemorrhagic colitis in
children and adults in North America (e52,e85,e180,e393). It is a commensal in a small percentage of beef cattle and is
usually spread to humans through the ingestion of undercooked ground beef or contamination of fruits or vegetables. It
produces a Shiga-like toxin (verotoxin) that shares many features with the Shiga toxin of Shigella (e237,e242,e393).
The most notorious enterohemorrhagic serotype is E. coli 0157:117, which has been responsible for numerous
epidemics and sporadic cases of hemorrhagic colitis in the past decade (e52,e56,e85,e237,e335,e380). In contrast to
most E. coli strains, E. coli 0157:117 does not ferment sorbitol and can thus be recognized by a characteristic pattern on
a selective growth medium, sorbitol-MacConkey agar. Since its recognition and the development of a relatively easy
method of identification, E. coli 0157:117 has been detected more frequently than Shigella, averaging approximately
21,000 infections and 240 deaths per year in the United States alone (e52). The Shiga-like toxin causes endothelial
damage in the kidney, which results in hemolytic-uremic syndrome in 6% of infected persons, usually young children. It
is the most common cause of hemolytic-uremic syndrome in North America (e52,e56,e237,e380). In older patients, the
toxin may cause thrombotic thrombocytopenic purpura. In epidemics in the United States, approximately one-fourth of
infected persons become ill enough to be hospitalized. The toxin produces watery stools at first, which progress to
bloody diarrhea over several days. Abdominal pain, diarrhea, and rectal bleeding may mimic ulcerative colitis or
appendicitis. Stool is negative for leukocytes in mild infections but positive in more severe cases. Endoscopy shows
colonic edema, hyperemia, superficial ulcers, and, in the most severe cases, pseudomembranous colitis.
The early histopathology consists of focal hemorrhagic colitis with ischemic changes, edema, and acute inflammation in
the superficial mucosa, which progress to confluent ulceration and pseudomembrane formation in the most severe cases
(Figure 14-29). Small blood vessels in the lamina propria and submucosa may contain platelet-fibrin thrombi, and
occasional vasculitis is responsible for the superficial ischemic changes and hemorrhage (108) (e180,e245,e380). Most
of the fatalities are associated with the complications of hemolytic-uremic syndrome. Although E. coli 0157:117 has
achieved notoriety, at least 100 other serotypes of enterohemorrhagic E. coli have been reported to cause bloody
diarrhea via Shiga toxin production, and several of these
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also cause hemolytic-uremic syndrome (e381). Many of the non-0157:H7 enterohemorrhagic E. coli strains are not
detected on sorbitol-MacConkey agar but demonstrate production of Shiga-like toxin. Assay for Shiga toxin is indicated
in the clinical situation of bloody diarrhea, especially if culture on sorbitol-MacConkey agar does not yield growth
characteristic of E. coli 0157:H7.
FIGURE 14-29▪ Colitis due to E. coli 0157:H7. The combination of histologic features of infectious colitis seen at each
end of the biopsy and ischemic changes in the middle of the biopsy is typical of this infection 200×.
Unfortunately, none of the pathogenic strains of E. coli can be identified on routine stool culture. Some microbiology
laboratories routinely screen for E. coli 0157:H7 with selective media, but some do not. A sporadic case of
enteropathogenic or enterohemorrhagic E. coli can easily elude diagnosis. E. coli is rarely serotyped in sporadic cases
of noninflammatory diarrhea; only reference laboratories perform this procedure with any degree of regularity, and most
detailed investigations are saved for outbreaks or clusters.
Campylobacter jejuni
Campylobacter jejuni is now the major cause of acute bacterial diarrhea in older infants and children in developed
countries (e45,e236,e385,e430). Virtually unknown as recently as the early 1970s because of the unique conditions
required for it to grow in culture, C. jejuni is now isolated as often as Salmonella, Shigella, and enteropathogenic strains
of E. coli from children with diarrhea. Ordinary laboratory stool culture techniques are unsatisfactory to isolate this
fastidious organism; a microaerophilic environment and selective media must be used. Campylobacter infection is
acquired from animals in which the organism is a commensal, including pets and poultry, and from contaminated water
or milk. The infection presents with abdominal pain, low-grade fever, and diarrhea that becomes bloody after a few days.
Direct examination of the stool usually shows neutrophils. The illness is usually self-limited and lasts approximately one
week, but it may linger for 5 or 6 weeks or relapse after initial improvement. Jejunum, ileum, colon, rectum, and appendix
may all be affected. Campylobacter colitis may be sufficiently severe to mimic Crohn disease, with cobblestone mucosa
and aphthous ulcers. Toxic megacolon has also been described. Rectal and colonic biopsy specimens show an infective
proctocolitis with edema, neutrophils in the lamina propria, and crypt abscesses (eFigure 14-7) (e86,e367,e499).
Yersinia enterocolitica
Yersinia enterocolitica is a Gram-negative coccobacillus known to cause diarrhea, gastroenteritis, and mesenteric
adenitis in older children; more rarely, it causes enterocolitis, appendicitis, arthritis, erythema nodosum, and sepsis
(e54,e57). In children younger than 5 years of age, a mild, self-limiting, febrile gastroenteritis lasting 1 to 2 weeks is
characteristic (e297). In more severe cases in older children, fever, leukocytosis, and abdominal symptoms may be
mistaken for appendicitis and a laparotomy performed unnecessarily; in this situation, inflammation of the terminal ileum,
cecum, appendix, and mesenteric lymph nodes is found, often with an inflammatory mass in the ileocecal region,
mimicking Crohn disease (e54,e60,e91,e168,e478). In severe cases, extensive ulceration and inflammation of the small
intestine and colon, sepsis, and extraintestinal abscesses may develop. Microabscesses and neutrophilic infiltrate
characterize the tissue response in Yersinia infection; the sarcoidlike granulomas of Crohn disease do not occur
(eFigure 14-8A,B). Gram-negative coccobacilli may be found in microabscesses and areas of mucosal necrosis and
within the generally enlarged and sometimes necrotic lymphoid tissue (e54,e60).
Y. enterocolitica is usually identified by stool culture. The organism grows on standard selective stool culture media,
such as MacConkey agar, but overgrowth of normal flora makes identification difficult unless specific subculturing and
other identification techniques are used. The anatomic pathologist may encounter relatively severe or prolonged cases
of Yersinia infection in a variety of circumstances: acute inflammation and mucosal ulceration of the colon, necrotizing
appendicitis and periappendicitis, terminal ileitis thought to be Crohn disease, or even severe mesenteric lymphadenitis
mistaken for intestinal lymphoma.
Clostridium difficile
Clostridium difficile is best known as a cause of pseudomembranous colitis, but it is also responsible for many cases of
antibiotic-associated diarrhea without pseudomembranous colitis, in addition to occasional cases of diarrhea unrelated
to antibiotic exposure (e30,e244). It is a common nosocomial pathogen that is notoriously difficult to eradicate once
established (e244,e310). Most of the work implicating this organism as a human enteric pathogen dates from the late
1970s. Before this, C. difficile was extremely difficult to isolate and culture from fecal flora, hence its designation
“difficile.” Since then, it has been shown that this Gram-negative anaerobe can be recovered from the intestine of only
2% to 3% of normal adults. In adults and children over the age of 2 years, antibiotic treatment alters normal gut flora and
allows intestinal overgrowth of C. difficile from endogenous and exogenous sources, which leads to diarrhea. In this age
group and setting, C. difficile is a pathogen. More rarely, C. difficile causes diarrhea, with or without
pseudomembranes, in an older child without antecedent antibiotic therapy. Neutropenic patients and those with
inflammatory bowel disease are susceptible to such infections, although healthy children are also occasionally affected
(31, 115). The C. difficile strains that cause disease produce potent toxins that induce fluid secretion and colonic
mucosal necrosis and inflammation. The diagnosis of C. difficile infection is usually established by demonstrating C.
difficile toxins in stool with a commercially available enzyme immunoassay (e244). Stool culture for the organism is also
performed in many centers.
The situation in infants is more complex. C. difficile is harbored in 30% to 70% of healthy neonates, in whom the
organism may produce toxin without causing disease
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(23, 147) (e123). The carriage rate decreases abruptly in the first 5 months of life but does not decline to adult levels
until approximately 2 years of age (e440,e491). However, C. difficile is known to cause severe disease in certain groups
of young infants, particularly those with Hirschsprung disease and malignancies, in whom organisms can be
demonstrated invading colonic mucosa (e373). Recently, a hypervirulent strain of C. difficile has emerged as an
important cause of nosocomial colitis in hospitalized patients, including children (23, 147).
C. difficile colitis ranges in severity from mild watery diarrhea noticed shortly after the initiation of antibiotic therapy,
which can be controlled merely by discontinuing antibiotic therapy, to severe colitis with or without pseudomembranous
colitis (see “Pseudomembranous Colitis”). A characteristic form of necrotizing enteropathy may also be seen, in which
focal superficial or deep areas of coagulative necrosis and ulceration are surrounded by neutrophils, with sparing of
intervening areas. Marked submucosal edema is also characteristic. The Gram-positive organisms can be seen invading
tissue; the large size of the bacilli and spore formation are unique in this setting. Metronidazole and vancomycin are
used to treat the more severe cases. Relapse of infection following therapy and the development of vancomycin-
resistant strains are emerging problems. Surgical intervention may be required in extreme cases.
Aeromonas
In the 1990s, Aeromonas species were increasingly implicated in a variety of gastrointestinal illnesses, although they are
still relatively uncommon isolates in the microbiology laboratory. In young children, acute watery diarrhea (e76) or
gastroenteritis (e397) is the usual presentation. More rarely, especially in adults, an acute dysenteric illness is seen,
sometimes with a severe colitis mimicking chronic inflammatory bowel disease (e137).
Protozoal Infections
Giardia lamblia (Intestinalis)
Giardia lamblia is a flagellated protozoan capable of causing diarrhea and malabsorption in human hosts (e128) (Table
14-4). In some parts of the world, cyst forms of the organism can be frequently identified in the stools of asymptomatic
carriers, but in developed countries, ingestion of the organism usually leads to clinically apparent illness. Toddlers and
children and persons with selective IgA deficiency and other primary immunodeficiencies are more susceptible to
infection. Case clusters of giardiasis have been reported in day care centers and residential institutions. Travelers
drinking untreated water in Rocky Mountain areas and in the Soviet Union are also at risk.
Affected children manifest diarrhea, nausea, weight loss, malabsorption, and failure to thrive. The host ingests the cyst
form of the organism. In the proximal small intestine, the cyst wall dissolves and trophozoites are released; these adhere
to the brush border of epithelial cells, damaging the microvilli. In small-intestinal biopsy specimens, trophozoites are 10
to 18 μm long, have an arched or curved appearance at high levels of magnification, and are visible at the surface of
enterocytes or in the mucous coat (eFigure 14-9). They do not invade tissue. The trophozoites can be highlighted with a
trichrome stain or CD117 immunostain.
In immunocompetent hosts, the degree of intestinal reaction varies from insignificant to severe. The villous architecture
is usually normal, but increased numbers of chronic inflammatory cells and eosinophils in the lamina propria and
increased numbers of IELs may be seen. Patients with severe diarrhea and malabsorption show variable degrees of
villous atrophy and more severe chronic inflammation (e339). Patients with selective IgA deficiency or other
immunodeficiency syndromes are highly susceptible to giardiasis, and villous atrophy and inflammation are usually more
severe in these cases (e200). Identification of Giardia in the intestine should prompt consideration of an
immunodeficiency, although normal infants and children can also become infected.
In approximately one-third of patients, giardiasis becomes chronic and causes secondary effects of malabsorption,
including macrocytic anemia, lactose malabsorption with bloating, and growth impairment. The diagnosis is best
accomplished by microscopic examination of stool specimens for Giardia cysts. Commercial ELISAs are available to
detect Giardia antigens in stool. All too often, however, microscopic examination of a duodenal biopsy specimen will
provide the first clue that a patient has giardiasis.
Cryptosporidium
Cryptosporidium was first identified as a human pathogen in 1976, as a rare cause of self-limited, watery diarrhea in
immunocompetent persons (e95,e223,e279). Clusters of diarrhea in day care centers and families have been described.
In the early 1980s, Cryptosporidium was identified with increasing frequency as a cause of severe chronic diarrhea in
patients with AIDS, and cryptosporidiosis as one of the opportunistic infections that often heralds the onset of AIDS
(e187). Once immunodeficient patients are infected, they are plagued with chronic diarrhea, which is often severe and
difficult to eradicate. Cryptosporidium species are present in a large number of domestic and wild animals. Both zoonotic
and personto-person spread occurs. In 1993, a widely publicized outbreak in Milwaukee, Wisconsin, was traced to
contamination of the municipal water supply (e290). Oocysts are ingested orally and progress through stages of the life
cycle in the proximal small intestine where they are recognized on the surface of epithelial cells in mucosal biopsy
specimens as a line of spherical basophilic structures 3 to 4 μm in diameter. They are recognizable with hematoxylin and
eosin stains, but they also may be stained with Giemsa. They usually cause minimal morphologic changes in the
intestine except for chronic inflammatory infiltration of the lamina propria
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(Figure 14-30). Electron microscopy reveals destruction of enterocyte microvilli. Diagnosis is accomplished by
microscopic examination of stool specimens for oocysts with an acid-fast stain. ELISA kits are commercially available to
aid in the detection of oocysts. The infection is self-limited in normal hosts. No satisfactory treatment is available for
chronic infection in immunosuppressed persons.
FIGURE 14-30▪ Colonic cryptosporidium infection. Numerous small dotlike organisms line the crypt luminal surfaces
400×.
Entamoeba histolytica
Entamoeba histolytica infection (amebiasis) is a major cause of diarrhea in third world countries (e320,e361,e364). In
the United States, it is diagnosed most often in southwestern states, especially in Hispanic patients and those who have
recently traveled to Latin America. Infection may be asymptomatic (carrier state), or it may cause isolated hematochezia
or a dysentery-like syndrome with diarrhea and blood and mucus in the stools. Children often acquire the organism by
fecal-oral transmission from an asymptomatic adult and are more likely than adults to become ill when infected. The
diagnosis is made by finding cysts or trophozoites in fresh stool smears or trophozoites in biopsy material. The diagnosis
may also be made serologically by an elevated indirect hemaglutination titer.
In acute amebic infection, the organism invades the colon and causes a diffuse acute inflammation that may be difficult
to distinguish from ulcerative colitis or Crohn disease, both endoscopically and pathologically. Rectal biopsy specimens
often show no more than edema, scattered intraepithelial and lamina propria neutrophils, and a mild increase in lamina
propria cellularity. Superficial microscopic ulcerations usually indicate invasion of the trophozoites into the lamina
propria. Organisms resemble large, pale histiocytes, 15 to 30 μm in diameter, with a pale nucleus and ingested red blood
cells in the cytoplasm. They may be found in the surface mucous coat or in the superficial lamina propria beneath a
microscopic ulceration (Figure 14-31A to D). Organisms are not found in up to 50% of rectal biopsy specimens from
patients with acute-onset amebiasis (e361). Examination of stool smears is a more sensitive method of diagnosis. In
advanced cases, amebiasis causes multiple ulcerations, particularly in the cecum and ascending colon. Microscopically,
these have a characteristic flask shape at low levels of magnification, a consequence of epithelial undermining. At this
stage, abundant trophozoites are found in the intestinal wall. Sequelae include intestinal perforation, peritonitis,
lymphatic and hematogenous dissemination, and systemic amebiasis.
Fungal Disease of the Gastrointestinal Tract

Candida is regarded as part of the normal flora in healthy persons. It becomes a pathogen only when immunodeficiency,
immunosuppression, debilitation, or prolonged antibiotic therapy supervene. Since the onset of the AIDS epidemic,
esophageal candidiasis has been recognized as an AIDSdefining condition in many patients. Candidiasis develops in
most patients with AIDS sometime during their illness (e389). Zygomycosis and Aspergillus infection are also usually
limited to debilitated and immunosuppressed patients (e321). The gastrointestinal tract is often the portal of entry for
fungal septicemia in these people. Any portion of the gastrointestinal tract may be infected by these three groups of
fungi. Multiple fungal microabscesses a few millimeters in diameter or focal ulcerations are grossly identifiable on
mucosal surfaces. Histoplasma capsulatum may cause gastrointestinal disease in immunocompetent persons in endemic
areas (e435).
MALABSORPTION
Malabsorption in children has many causes, only some of which have anatomic correlates of concern to the pathologist
viewing an abnormal biopsy specimen. Conditions associated with a failure to absorb nutrients but without diagnosable
histologic abnormalities include pancreatic and liver diseases, enterocyte enzyme deficiencies, alterations of normal
bacterial flora, some infections, some immunodeficiency states, and decreases in intestinal surface area. These
extraintestinal, enzymatic, metabolic, and other nonstructural causes of malabsorption in children are covered in
standard textbooks and review articles (e384).
Children with malabsorption of any cause usually present with growth failure, bulky or diarrheal stools, and anemia.
Edema and hypoalbuminemia occur if inadequate protein is absorbed or if enough serum protein is lost through the
intestine. Steatorrhea results in a failure to absorb fat-soluble vitamins, associated with a prolonged prothrombin time
and manifestations of bleeding (vitamin K deficiency), rickets and hypocalcemia (vitamin D deficiency), and night
blindness (vitamin A deficiency). Zinc malabsorption produces a characteristic dermatitis. Anemia results from
malabsorption of iron, folate, or vitamin B12. The laboratory diagnosis of malabsorption is complex but usually includes
documentation of fat, carbohydrate, and protein loss in the stools.
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FIGURE 14-31▪Entamoeba histolytica colitis. A: Colonoscopy revealed scattered ulcers. B: Trophozoites can be seen
within mucus and debris at the luminal surface 200×. C: The trophozoites are slightly bigger than histiocytes and contain
a nucleus 400×. D: The organisms are highlighted in a PAS stain 400×.
A 72-hour quantitative stool fat excretion is used to quantify steatorrhea. Carbohydrate absorption is evaluated by
means of the H2 breath test, fecal pH, and D-xylose absorption. Serum proteins, immunoglobulins, calcium, carotene,
folic acid, and vitamin B12 are all subject to intestinal loss and can be measured directly. Stool is cultured and examined
for ova and parasites, especially G. lamblia. Because cystic fibrosis is a common cause of malabsorption in children in
North America and northern Europe, a sweat test is often performed. Antigliadin and antiendomysial antibodies are
sought in serum to rule out celiac disease.
A small-intestinal biopsy specimen is evaluated for inflammation, plasma cells, and the ratio of villus height to crypt
length (normal, 3:1 in infants; 4:1 in older children) (Figure 14-32). Duodenal biopsy specimens should be examined for
G. lamblia and cryptosporidium within the surface mucous coat.
Causes of malabsorption in children with morphologic abnormalities of the small intestine are listed in Table 14-5.
Several excellent review articles are available on the examination of small-intestinal mucosal biopsy specimens
(e357,e481). Of the causes of intestinal malabsorption, the most commonly encountered in developed countries are
celiac disease, temporary postgastroenteritis syndrome (postenteritis enteropathy), cow's milk protein intolerance, short-
gut syndrome (postoperative), Crohn disease, and immunodeficiency states.
Celiac Disease
Celiac disease, or gluten-sensitivity enteropathy, is the most common small-intestinal mucosal disease causing
malabsorption in white children. It can be diagnosed at any age after institution of gluten into the diet. One recent
screening
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study of unselected healthy infants reported that 9 of 484 had a positive anti-tissue transglutaminase test at age 2 1/2
years. In seven of these children duodenal biopsies confirmed the diagnosis of celiac disease (27). Presentation with the
classic symptoms of malabsorption (diarrhea, steatorrhea, abdominal bloating and pain, weight loss, poor weight gain,
failure to thrive, fatigue, and metabolic bone disease) is becoming less and less frequent. Instead, a wide (and ever
expanding) range of “atypical” presenting symptoms is being reported, including low serum folate, calcium, magnesium
or phosphorus levels, intracranial calcifications causing seizures, and growth retardation (94). Unexplained iron
deficiency anemia is now one of the leading presenting signs of celiac disease, particularly among adolescent patients
(e55,e247). One study proposes that gastrointestinal blood loss is partially responsible for the anemia, in addition to the
obvious decrease in iron absorption (e143). There has also been a significant increase in diagnosis through screening
of patients with Down syndrome, juvenile-onset diabetes mellitus, and other autoimmune disorders. Virtually every
patient with dermatitis herpetiformis has or will develop celiac disease. About 90% of patients with celiac disease carry a
HLADQ2 of HLA-DQ8 allele. There is a 70% concordance rate in identical twins (94).
FIGURE 14-32▪Normal duodenal mucosa. Although scattered IELs are evident in the lower portions of the villi, they
become progressively less numerous toward the tips of the villi. This normal “decrescendo” pattern is helpful to rule out
a borderline increase as seen in various pathologic conditions 200×.
Celiac disease occurs because of the ingestion of alphagliadin within gluten-containing foods by sensitive individuals.
Gluten is present at high levels in wheat, rye, and barley but is absent in corn and rice. Oats (in moderate amounts) has
been shown to be tolerated by some celiac patients without adverse effects (e225). Gliadin injures the enterocytes in
celiac disease patients, causing them to aberrantly express HLA antigens and secrete IL-15. This in turn may lead to the
intraepithelial infiltration of CD8+ T-cells that is so characteristic of celiac disease. The gliadin is deaminated by tissue
transglutaminase in the interstitium, resulting in a peptide that is recognized by an expanded population of CD4+ T-cells
(DQ2 and DQ8 restricted) in the lamina propria (56).
Table 14-5▪CAUSES OF MALABSORPTION IN CHILDREN
Celiac disease
Postviral enteropathy
Cow's milk and other dietary protein intolerance
Eosinophilic gastroenteritis
Immunodeficiency states (e.g., common variable immunodeficiency)
Bacterial overgrowth/stasis
Bacterial infection (e.g. mycobacterium avium intracellular)
Parasitic infections (Giardia and Cryptosporidium)
Crohn disease
Autoimmune enteritis
Microvillous inclusion disease
Tufting enteropathy
Intestinal lymphangiectasia
Abetalipoproteinemia
Cystic fibrosis
Langerhans cell histiocytosis
Chronic granulomatous disease
In the past elevation of serum antigliadin and antiendomysial antibody titers were required to establish a diagnosis of
celiac disease. In patients with a high clinical suspicion of celiac disease (i.e., those with classic symptomatology) the
sensitivity and specificity of these assays were in the range of 90%. However, as screening tests in asymptomatic adult
blood donors, for example, the positive predictive value for a positive antigliadin antibody test was only 20% (e181).
Moreover, the value of these serologic tests varied by geographic area and ethnicity, even among high-risk patient
populations (e349,e470). It is important to remember, though, that the antiendomysial antibody test only detects IgA
antibodies, while both IgA and IgG antigliadin antibody tests are available. This is important because the frequency of
selective IgA deficiency is more than ten times higher in patients with celiac disease than in the general population (e94).
Recently, it was demonstrated that the primary antigen detected by the antiendomysial indirect immunofluorescent test is
a peptide portion of tissue transglutaminase (tTG) (e118). Automated ELISA assays have become the primary test for
celiac disease and have supplanted the more time-consuming and subjective antiendomysial antibody test (e445) (130).
However, no single test is 100% sensitive and specific in all testing situations, and currently a panel
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including anti-tTG, antiendomysial, and antigliadin antibody tests is usually performed (133).
The classic histologic features of celiac disease in patients ingesting gluten include villous blunting, crypt hyperplasia,
an increased number of mitotic figures in the crypts, dense mixed inflammatory cell infiltration of the lamina propria, and
an increased number of IELs. The lamina propria inflammatory cell infiltrates are composed mostly of plasma cells and
lymphocytes, with scattered admixed eosinophils. Although not characteristic, a few scattered neutrophils may be
present as well. Disorganization, flattening, and/or vacuolization of the surface epithelium are also evident in some cases
of celiac disease (eFigure 14-10A,B). After withdrawal of gluten from the diet, there is slow resolution toward normal
villous architecture. The mucosa of the most distal portion of the small bowel recovers most quickly, while the duodenum
is the last to normalize. It may take several months or longer of a strict gluten-free diet before the biopsy appearance
returns to normal. Ileal biopsies obtained during a colonoscopy may exhibit the same histologic features seen in
duodenal biopsies, although significant villous blunting is very unusual.
The degree of villous blunting in celiac disease varies in individual patients and even among duodenal biopsies from a
single patient. Multiple biopsies are recommended because the pathologic changes can be patchy, and because some
of the biopsies may be poorly oriented or artifactually distorted, interfering with proper interpretation. The number of
duodenal biopsies that are obtained have been shown to influence the likelihood of identifying flat mucosa (e359,e410).
Since a flat mucosa with increased IELs is much more specific for celiac disease than an increase in IELs alone, the
procurement of multiple biopsies is clearly desirable (e463). If the duodenal mucosa is completely flat the diagnosis of
celiac disease is almost ensured, although rarely autoimmune enteropathy, viral enteritis, and tropical sprue can result in
a flat mucosa. Villous architecture is difficult to accurately assess in the proximal duodenum because Brunner glands
can cause mild villous architecture distortion and shortening. In addition, the inflammatory changes of peptic duodenitis
are most severe in the proximal duodenum and when present, interfere with the recognition of the histologic features of
celiac disease. For these reasons surgical pathologists should encourage gastroenterologists to obtain small bowel
biopsies for the evaluation of malabsorption from as far distally in the duodenum as is practical.
As more patients with “atypical” symptoms underwent duodenal biopsy to rule out celiac disease, it became clear that a
range of pathologic abnormalities could be expected. Marsh proposed a classification for the morphologic appearance of
duodenal biopsies in celiac disease patients (e298,e299), which can be briefly summarized as follows:
Type 0—normal crypt and villous architecture with no increase in IELs

Type 1—normal crypt and villous architecture with greater than 40 IELs/100 enterocytes
Type 2—crypt hyperplasia but normal villous length; greater than 40 IELs/100 enterocytes
Type 3—crypt hyperplasia and villous blunting (mild to flat) with greater than 40 IEL/100
enterocytes
Type 1 morphology, also known as the “infiltrative lesion,” which represents the earliest recognizable light microscopic
change, was first documented in biopsies from first-degree relatives of celiac disease patients and in patients with
dermatitis herpetiformis (Figure 14-33A to D). These patients had no gastrointestinal complaints and were considered to
suffer from a form fruste of celiac disease (e298). It has been shown that the infiltrative lesion can be induced in
fullfledged celiac disease patients who have been on a gluten-free diet (with a documented entirely normal duodenal
mucosa) by administering a low dose of dietary gluten. Increasing the load of dietary gluten can produce evolution to a
flat mucosa (e141,e153). It has recently been estimated that only 30% of “gluten-sensitive” patients exhibit a flat mucosa
(e298). It appears that many of the “asymptomatic” first-degree relatives of celiac disease patients often do have subtle
symptoms (e.g., iron deficiency anemia) related to abnormal small bowel morphology. It is important to realize that 10%
of patients with dermatitis herpetiformis actually exhibit a flat mucosa on duodenal biopsy and still do not suffer diarrhea
or significant malabsorption (e298). Obviously, the clinical presentation is dependent to some extent on the length of the
small bowel mucosa that is severely affected.
It is currently unknown what percentage of patients with the “infiltrative lesion” will go on to develop flat mucosa and the
full blown clinical syndrome of celiac disease. It is important to recognize this morphologic expression of gluten sensitivity
because the atypical symptoms of these patients will respond to dietary gluten withdrawal. For that reason, and because
of the greater long-term risk of lymphoma in untreated patients, a gluten-free diet is recommended by most
gastroenterologists for all celiac patients, regardless of the presence or absence of villous blunting. On the other hand, it
is clear that celiac disease is not the only cause of intraepithelial lymphocytosis. Other disease states in which an
increased number of IELs may occur include tropical sprue, autoimmune enteropathy, cryptosporidiosis, giardiasis,
microsporidiosis, bacterial overgrowth, other food allergies, viral enteritis, Crohn disease, Zollinger-Ellison syndrome,
and systemic autoimmune states. It is also possible that NSAIDs can cause intraepithelial lymphocytosis (22, 50, 78).
Severe H. pylori gastritis may also cause a mild increase in IELs in biopsies of the duodenal bulb, but usually not more
distally (103).
Most cases of flat duodenal mucosa and increased intraepithelial lymphocytosis are due to celiac disease, but common
variable immunodeficiency, autoimmune enteritis, and severe viral enteritis can also produce this pattern of injury. With
lesser degrees of villous blunting, the differential diagnosis broadens considerably. Given the large number of disorders
that can cause intraepithelial lymphocytosis and villous
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blunting, it is clear that a diagnosis of celiac disease is not possible solely by histologic examination of duodenal
biopsies. Instead, correlation of the serologic tests results and the biopsy findings is necessary to establish a firm
diagnosis of celiac disease. Resolution of symptoms or abnormal laboratory tests after a gluten-free diet is an important
confirmatory sign. Re-biopsy after a gluten-free diet is instituted or after gluten rechallenge is no longer standard clinical
practice.
FIGURE 14-33▪ Celiac disease. A: Scalloped duodenal folds seen by endoscopy. B: Normal villous architecture is
maintained 100×. C: Prominent intraepithelial lymphocytosis 200×. D: Mild lymphocytosis 200×. See Figure 14-32 for
normal morphology.
The number of IELs that separate normal individuals from those with small bowel disease have, surprisingly, not been
studied extensively. Forty IELs per 100 enterocytes were adopted as the cutoff for the diagnosis of celiac
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disease by Furguson and Murray because the highest level in their control group of normal individuals was 40 and the
mean value plus two standard deviations (SD) was 36.1 (e156). However, a recent larger study of a Swedish population
concluded that a value of 20 IELs per 100 enterocytes was more appropriate (their mean plus 3 SD was 18.5). The
authors calculated that using that cutoff no more that 1 in 1000 healthy persons would be falsely diagnosed with
intraepithelial lymphocytosis (149). They also studied the use of CD3 immunostains to highlight IELs and calculated a
cutoff of 30 per 100 enterocytes, with 25 to 29 IELs reported as “borderline” (149). The authors stress the importance of
not performing counts of IELs in the epithelium anywhere in the vicinity of even small lamina propria lymphoid
aggregates, since increased numbers of IELs are normal there. This is particularly critical in the evaluation of biopsies of
the terminal ileum, since lymphoid aggregates and follicles often occupy large portions of such samples.
Immunohistologic studies to highlight IELs utilizing a CD3 antibody may aid in the recognition of intraepithelial
lymphocytosis in biopsies without villous blunting (105), but the use of this strategy is generally not required in daily
practice.
Recently, it has been proposed that an abnormal distribution of IELs along the length of the villi, even if the overall
number is not significantly increased, is suggestive of celiac disease (52). This proposal is based on the observation
that in healthy individuals there is a progressive decrease in the density of IELs from the base of a villus to its tip. In
contrast, in a subset of patients with celiac disease this normal “decrescendo pattern” of IEL distribution is lost, and
instead, the number of IELs is similar along the entire length of the villus or is actually higher at the tip than at the base.
However, the author emphasizes that this pattern of IEL distribution merely suggests the need for serologic testing to
rule out celiac disease, as there are other causes of this histologic finding, and some patients may have no disease
state at all (51).
Gastric antral biopsies from patients with celiac disease, particularly children exposed to gluten at a young age, may
also exhibit an intraepithelial lymphocytosis, and the term “lymphocytic gastritis” has been applied in such cases. More
than 25 IELs per 100 foveolar epithelial cells is considered abnormal. There is conflicting data on whether patients with
lymphocytic gastritis experience upper gastrointestinal symptoms, such as nausea and vomiting or dyspepsia, more
often than patients without this finding (e6,e112,e503).
Some celiac patients who are asymptomatic on a glutenfree diet sudden redevelop symptoms of malabsorption. Most of
these patients are ultimately discovered to have discontinued the gluten-free diet, inadvertently or not. Some patients,
however, relapse despite strict adherence to the proper diet and are said to suffer from refractory sprue. The most
feared complication of celiac disease is the development of small bowel lymphoma, which is sometimes heralded by the
redevelopment of malabsorption. These lymphomas are unusual in that they are almost always of T-cell phenotype
(e329), while almost all sporadic gastrointestinal lymphomas are of B-cell origin. In many patients gene rearrangement
studies are necessary to confirm the diagnosis of a clonal T-cell proliferation, since significant cytological atypia may not
be present. The relative risk of small bowel lymphoma in celiac patients has been variously estimated at 40- to 100-fold
greater than that for the general population (e134), but there is some evidence that strict adherence to a gluten-free diet
may prevent the development of lymphoma (e88). The mucosa in celiac patients with lymphoma often appears atrophic,
with both crypt hypoplasia and total villous blunting (Marsh type 4 morphology). The evolution to lymphoma was initially
overlooked in some celiac patients who developed diffuse ulceration of the small bowel mucosa (the so-called ulcerative
jejunoileitis), making it difficult to discern the underlying clonal lymphoid infiltrate, especially in biopsies (28) (e73). There
has been some confusion in the literature, however, in that the term ulcerative jejunoileitis is also used to describe large
areas of small bowel mucosal ulceration in nonceliac patients. In this population there is no association with lymphoma.
A small number of patients also develop lymphocytic or collagenous colitis, which may manifest simultaneously, before
or after the diagnosis of sprue (e504). In one study of 21 patients with “refractory sprue,” collagenous colitis was
responsible for the development of diarrhea in three patients on a strict gluten-free diet (e144).
Gastroenteritis and Postenteritis Enteropathy

Most cases of acute gastroenteritis in children are caused by viruses. Biopsies are usually not performed during the
acute infection. Of more concern is the infant or child who apparently recovers from an episode of acute viral
gastroenteritis but then lapses into a malabsorptive state lasting weeks to months. These children may undergo
endoscopic examination and their biopsy findings must be distinguished from those of celiac disease. Cases of
postenteritis enteropathy show villous atrophy and chronic inflammation, but the changes characteristically vary in
severity from one piece of tissue to another (e458). Cow's milk protein intolerance is sometimes unmasked by acute
gastroenteritis and should be considered.
Enteropathy Induced by Cow's Milk Proteins

Enteropathy associated with malabsorption is one of several gastrointestinal symptom complexes in infants caused by
cow's milk proteins. The others are occult gastrointestinal blood loss and iron-deficiency anemia, protein-losing
enteropathy, and allergic proctocolitis. Enteropathy, with or without protein loss, is well-known
(e149,e269,e270,e295,e487). Other dietary proteins, including soy protein and even casein hydrolysate in formulas,
may also cause the syndrome (e195,e475). Symptoms usually develop in the first 6 months of life in a bottle-fed baby.
The onset may be sudden, with vomiting and diarrhea, or more gradual, with failure to thrive and chronic loose stools. In
some cases, hypoproteinemia
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and edema (protein-losing enteropathy) dominate the picture. Steatorrhea and carbohydrate malabsorption can be
demonstrated, and some patients have a mild peripheral eosinophilia. Small-intestinal biopsy specimens (Figure 14-34)
show villous blunting and inflammation of the lamina propria, which is often quite patchy, which necessitates
procurement of multiple biopsies for diagnosis. Lamina propria cells are usually a mixture of mononuclear cells and
eosinophils; polymorphonuclear leukocytes are rare. IELs are not increased in number. Sigmoidoscopic biopsies are
easier to obtain in infants than duodenal biopsies and are therefore the most common specimens seen by surgical
pathologists. Normal crypt architecture is well preserved in all cases. The inflammatory infiltrates are usually eosinophil
predominant, always distinctly patchy, and sometimes also include neutrophilic infiltrates. In fact, some of the biopsies
from a given case may be entirely normal. More than 60 eosinophils/10 hpf has been used as a diagnostic cutoff for
colonic biopsies, while others have suggested 20 eosinophils/hpf. However, most biopsies (from any site) also exhibit
focal eosinophilic cryptitis and/or infiltration of the muscularis mucosae, and these features are useful to make a firm
diagnosis. It must be emphasized that increased eosinophils are not present in every case, and the presence of a
neutrophilic predominant colitis is also consistent with the diagnosis. However, in that situation infectious colitis and
Hirschsprung disease-associated colitis must also be ruled out clinically.
FIGURE 14-34▪Cow's milk protein intolerance. This duodenal biopsy demonstrates villous blunting (without crypt
hyperplasia) and an intense infiltrate of eosinophils 200×.
Infants with cow's milk protein-induced enteropathy usually respond to a diet free of cow's milk with resolution of
symptoms and morphologic abnormalities. Identical symptoms of malabsorption and enteropathy often develop in infants
who cannot tolerate cow's milk protein when they are switched to a soy protein formula or even a casein hydrolysate
formula (e424,e475). Such patients respond to an elemental formula containing amino acids. In any case, milk-sensitive
enteropathy is a temporary state. By the age of 1 year, most patients can ingest products containing cow's milk without
difficulty.
Intestinal Lymphangiectasia
Intestinal lymphangiectasia is a disease category rather than a single entity. It is characterized by greatly dilated
lymphatic vessels in the lamina propria of the small intestine with leakage of lymph into the intestine and consequent
proteinlosing enteropathy (e1,e479). Primary (congenital) forms often are associated with extraintestinal lymphatic
abnormalities. Secondary forms are caused by lymphatic obstruction resulting from cardiac failure, pericarditis,
abdominal tumors, inflammatory bowel disease, and other conditions. Patients with both primary and secondary forms
present with diarrhea and protein-losing enteropathy (i.e., intestinal protein loss, hypoalbuminemia, and edema).
Lymphocyte and immunoglobulin losses into the intestine through the lymphatics also produce lymphopenia and
hypogammaglobulinemia. The dilated lymphatics can often be seen through the endoscope as multiple, white, pinhead-
sized spots on the smallintestinal mucosa. On biopsy specimens, the abnormally dilated lymphatic vessels are often
grouped at the tips of villi (Figure 14-35A,B) but may appear elsewhere in the lamina propria. A distinct endothelial lining
helps differentiate lymphatic vessels from artificial tears caused by biopsy trauma. Because intestinal lymphangiectasia
is a focal abnormality, multiple biopsies and serial sections are indicated if this diagnosis is suspected. Unaffected villi
are normal or show a mildly increased cellularity in the lamina propria.
Immunodeficiency
Immunodeficiency diseases may present in infancy and childhood as malabsorption, diarrhea, and failure to thrive
(e8,e10). The most common disorders are selective IgA deficiency, which may present at any age; common variable
immunodeficiency, which usually presents in the older child or adult; and AIDS. In all three conditions, villous atrophy
and inflammation mimicking celiac disease may occur. Plasma cells are conspicuously absent from the lamina propria,
and the enteropathy is patchy in common variable immunodeficiency and severe combined immunodeficiency. Plasma
cells are also absent in X-linked agammaglobulinemia. Exacerbation of malabsorption in many immunodeficient patients
often indicates superimposed giardiasis.
IPEX (immune dysregulation, polyendocrinopathy, enteropathy, and X-linkage) syndrome is due to a mutation in the
FOXP3 (scurfin) gene, which codes for a transcription factor involved in the development and proliferation of CD4+ T-
cells. Affected infants may develop severe enteropathy, diabetes mellitus, eczematous ichthyosis, hemolytic anemia, and
thyroid and/or adrenal dysfunction. The duodenal morphology resembles autoimmune enteropathy. Although
immunosuppressive therapy may ameliorate symptoms for a time, death will occur without stem cell transplantation (14).
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FIGURE 14-35▪Small-bowel lymphangiectasia. A: This duodenal biopsy demonstrates dilated lymphatic channels within
the lamina propria 200×. B: In this resection specimen the dilated lymphatic vessels are within the superficial
submucosa 100×.
Short-bowel Syndrome and Bacterial Overgrowth

Extensive surgical resection of the intestine is the usual cause of short-bowel syndrome, defined as malabsorption in the
presence of reduced intestinal length, usually 50 cm or less of small intestine in a neonate. The normal small-intestinal
length in the term neonate is 239 ± 67 cm (e426). Extensive intestinal resection is performed in patients with such
conditions as neonatal necrotizing enterocolitis, malrotation with volvulus, multiple intestinal atresias, and, in older
children, Crohn disease. Very rarely, the intestine is congenitally very short (e197,e454). In any case, diarrhea results
from a shortened transit time, and malabsorption of nutrients results from an inadequate absorptive surface area. In
some patients, a reduction in peristalsis leads to intestinal dilation. Bacterial overgrowth in the intestinal lumen
contributes substantially to malabsorption in many cases of short-bowel syndrome (e241,e474). Small-intestinal biopsy
specimens often show nonspecific partial villous atrophy, crypt hypertrophy, and inflammation with lymphocytes, plasma
cells, and eosinophils. In severe cases of bacterial overgrowth, an acute enteritis with polymorphonuclear leukocytes in
the lamina propria or crypts may be present. The colon is often also involved (e455). Bacterial overgrowth can be
diagnosed by culture of proximal intestinal fluid; organisms are rarely seen on biopsy specimens. Intravenous
alimentation with amino acid and lipid preparations has made long-term survival possible in these patients. Antibiotics
often decrease the bacterial overgrowth. Newer bowel-lengthening procedures have been used to correct selected
cases, and in the most severe cases, small-bowel transplantation is possible.
Bacterial overgrowth is most often seen in children with short-bowel syndrome (see above). However, the same bacterial
overgrowth and subsequent enteropathy may also occur in children with a normal intestinal length if they have severe
malnutrition, an alteration of peristalsis and stasis, as in one of the pseudo-obstruction syndromes, or a surgically
created blind loop. The clinical picture is that of malabsorption (e241,e417).
Malnutrition
Kwashiorkor and marasmus may both produce villous atrophy and inflammation, although this is rare in developed
countries. Patients with protein-calorie malnutrition and gastrointestinal symptoms often have a superimposed infection
(e59).
Abetalipoproteinemia
Patients with this rare autosomal recessive metabolic disease are unable to synthesize and transport low-density
lipoproteins (β-lipoproteins) and manifest numerous extraintestinal abnormalities, including red cell acanthocytosis,
retinitis pigmentosa, and neuromuscular degeneration. Malabsorption and diarrhea are conspicuous within the 1st year
of life and are often the earliest manifestations of the disease (152) (e494). On small-intestinal biopsy specimens,
surface epithelial cells are markedly vacuolated by lipid that has been absorbed but cannot be normally transported out
of the cells (Figure 14-36). Villous architecture is otherwise normal (e376).
Microvillus Inclusion Disease

Microvillus inclusion disease (congenital microvillous atrophy and familial enteropathy) is a rare, lethal, familial disease
that presents at birth with relentless secretory diarrhea (e97,e98,e106,e358). Small-bowel transplantation is required in
many cases (127). Small-intestinal biopsy specimens show villous atrophy, hypoplasia of crypts, an inappropriate lack of
compensatory mitosis, and vacuolization of enterocyte apical
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cytoplasm (Figure 14-37A,B). The lack of inflammation is striking in comparison with other enteropathies. Similar
changes are also evident in colonic and gastric mucosa (e401).
FIGURE 14-36▪Abetalipoproteinemia. There is prominent accumulation of small droplets of lipid within the enterocytes
from this duodenal biopsy 200×.
FIGURE 14-37▪Microvillous inclusion disease. A: This duodenal biopsy exhibits both villous blunt and crypt hypoplasia,
resulting in atrophic appearing mucosa 100×. B: A well-developed brush boarder is not visible 400×. C: Electron
microscopy demonstrates a microvillous inclusion body in the cytoplasm of an enterocyte.
Electron microscopy of surface enterocytes shows distinctive intracytoplasmic inclusions and absent or shortened
microvilli (Figure 14-37C) (e35,e97,e98,e358). Although electron microscopic demonstration of the unique cytoplasmic
inclusions confirms the diagnosis, paraffin-embedded sections can also be evaluated; affected enterocytes contain
periodic acid-Schiff (PAS)-positive and diastase-resistant material (e98) and show distinctive inclusions utilizing a CD10
antibody by immunohistochemistry (58).
Autoimmune Enteropathy
To date, approximately 100 infants have been described in the world literature with a severe protein-losing enteropathy
refractory to treatment except for potent immunosuppressive agents such as cyclosporine or FK506 (tacrolimus) or
smallbowel transplantation (137) (e72). The onset of diarrhea and protein-losing enteropathy may be at any time
between several weeks after birth to approximately 2 years of age. Many of the infants have had an associated
autoimmune disease of some type, including diabetes, thyroid disease, atopic dermatitis, glomerulonephritis,
autoimmune hemolytic anemia,
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polyarthritis, and autoimmune hepatitis. Some of the cases are familial (e87,e93,e98,e175,e391). Some of the patients
have serum antibodies against either enterocyte or goblet cell antigens, but these assays are available only in
specialized centers. Small-intestinal biopsy changes are variable, ranging from partial to total villous atrophy with
lymphoplasmacytic infiltration in the lamina propria and crypt elongation. IELs in surface enterocytes, similar to the
characteristic finding in celiac disease, are also present in some cases. Absence of goblet cells is an easily overlooked
feature in some cases (Figure 14-38A,B). Colonic and gastric mucosa may be involved (e98,e208).
FIGURE 14-38▪ Autoimmune enterocolitis. A: This duodenal biopsy exhibits total villous blunting and mild intraepithelial
lymphocytosis, but serologic tests for celiac disease were negative. A serum antienterocyte antibody titer was elevated
100×. B: This colonic biopsy from a different child exhibits mild colitis with prominent epithelial cell apoptosis and a
complete absence of goblet cells. The serum antigoblet cell antibody titer was elevated 200×.
Tufting Enteropathy
This rare genetic disorder is responsible for some cases of intractable congenital diarrhea (e98,e174,e378).
Smallintestinal biopsy specimens show moderate to severe villous atrophy and crypt hyperplasia without significant
inflammatory cell infiltrates (Figure 14-39A to C). The diagnostic histologic feature is the disorganization of the surface
epithelium, with crowding, tufting, and shedding of enterocytes (54). Decreased epithelial cell adhesion molecule
expression due to an underlying gene mutation has been found in some patients (139).
GASTROINTESTINAL MANIFESTATIONS OF IMMUNODEFICIENCY

Gastrointestinal symptoms, infections, and morphologic abnormalities figure prominently in many primary and secondary
immunodeficiency syndromes (see Chapter 22). Excellent review articles on this subject are available
(e9,e10,e120,e216,e389). Biopsy specimens from immunodeficient patients may come to the pathologist masquerading
as malabsorption, inflammatory bowel disease, giardiasis, or lymphoid hyperplasia. The pathologist evaluating an
intestinal mucosal biopsy specimen or resected tissue for any of these clinical indications may be the first to suspect
AIDS, hypogammaglobulinemia, agammaglobulinemia, or, if plasma cells are absent in the lamina propria, severe
combined immunodeficiency or X-linked agammaglobulinemia (e489).
At times, a primary gastrointestinal abnormality may result in a secondary immunodeficiency. Leakage from intestinal
lymphatics, as in intestinal lymphangiectasia and Crohn disease, may lead to lymphopenia and functional T-cell
deficiency (e120). Protein-losing enteropathy in cow's milk protein intolerance can produce hypogammaglobulinemia and
lymphopenia. Structural defects, such as malrotation and cavernous hemangioma of the jejunum, have been associated
with defects of both humoral and cellular immunity, postulated to result from intestinal losses of protein and lymphocytes
(e139).
Immunosuppression caused by steroids and cytotoxic agents, especially in children with malignancies, increases the risk
for fungal or viral infection of the gastrointestinal tract. Necrotizing inflammation of the cecum or typhlitis (neutropenic
colitis) is likely to develop in children being treated for leukemia (e413,e482).
Primary Immunodeficiencies
Selective IgA deficiency is the most common primary immunodeficiency in the general population, with an incidence of 1
to 2 in 1,000. Diarrhea and steatorrhea may occur at any age and are often the initial manifestations of
immunodeficiency. The incidence of celiac disease is increased in IgA-deficient patients, and the diagnosis may be more
difficult than usual
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because the serum levels of antigliadin and antiendomysial IgA antibodies are not elevated. Intestinal giardiasis may
also cause malabsorption, but malabsorption persists in some IgA-deficient patients even after elimination of gluten from
the diet and treatment of Giardia infestation. Various chronic inflammatory bowel diseases, morphologically identical to
Crohn disease and ulcerative colitis, have also been reported in IgA-deficient patients. Nodular lymphoid hyperplasia of
the small intestine occurs in both selective IgA deficiency and common variable hypogammaglobulinemia, but is rare in
children.
FIGURE 14-39▪Congenital tufting enteropathy. A: Duodenal biopsy reveals villous and crypt hypoplasia 200×. B:
Tufting of the surface epithelium 400×. C: Note the lack of intraepithelial lymphocytosis 400×.
Common variable immunodeficiency (common variable hypogammaglobulinemia) may also first come to clinical attention
with gastrointestinal symptoms in older children (148). The diagnosis is often delayed because a pattern of recurrent
infections involving multiple organs is not recognized (122). They are susceptible to a host of gastrointestinal
complications, which often become the dominant clinical problem. Infections are common, with giardiasis, bacterial
infections, and chronic viral infections reported. The diagnosis rests upon the findings of abnormally low serum
immunoglobulin (IgA, IgM, and IgG) levels without another explanation. A poor or absent response to immunization is
helpful to confirm the diagnosis (80). Malabsorption states, nonspecific colitis, gastritis, and chronic inflammatory bowel
diseases resembling Crohn disease and ulcerative colitis are also found.
Gastrointestinal plasma cells are absent or markedly decreased in most but not all cases. Duodenal biopsies in patients
with malabsorption may exhibit villous blunting, crypt hyperplasia, and intraepithelial lymphocytosis, closely resembling
the histologic features of celiac disease (Figure 14-40A,B). The proper diagnosis rests on the recognition of the lack of a
dense lamina propria infiltrate of lymphocytes and plasma cells, as expected in celiac disease. Since patients with
common variable immunodeficiency are at particularly increased risk of Giardia infection, this possibility should be
excluded by special stain (trichrome or CD117 immunostain). In some duodenal biopsies epithelial apoptosis is
prominent, resulting in an appearance similar to severe graft-versus-host disease or autoimmune enteritis. Esophageal
biopsies may reveal Candida esophagitis. Severe diffuse nonspecific gastritis may be seen in antral or gastric body
biopsies. Colonic
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biopsies may reveal features consistent with lymphocytic or collagenous colitis or exhibit crypt architectural distortion
and active inflammation resembling inflammatory bowel disease. Granulomas may also be present (37). Again, the
absence of plasma cells is a clue to the proper diagnosis in most cases.
FIGURE 14-40▪ Common variable immunodeficiency. A: This duodenal biopsy exhibits complete villous blunting and
crypt hyperplasia 200×. B: There is also a mild intraepithelial lymphocytosis, similar to that seen in celiac disease.
However, the complete absence of lamina propria plasma cells suggests the correct diagnosis 400×.
X-linked agammaglobulinemia presents in the first 6 months of life with severe respiratory infections and meningitis.
Diarrhea, malabsorption, giardiasis, and colitis are frequent manifestations and may dominate in any given case (e489).
Examination of the lamina propria reveals an absence of plasma cells.
Severe combined immunodeficiency is fatal in the first few months of life unless a bone marrow transplant is successful.
Malabsorption, villous atrophy, diarrhea, and severe failure to thrive regularly develop in untreated patients.
Gastrointestinal plasma cells are lacking.
Immunodeficient patients are predisposed to gastrointestinal infections by usual and unusual pathogens (e480). G.
lamblia infection of the small intestine has been found in up to 50% of symptomatic patients with primary
immunodeficiency syndromes and is responsible for many of the cases of chronic diarrhea and malabsorption in patients
with common variable hypogammaglobulinemia, selective IgA deficiency, and X-linked agammaglobulinemia. Eradication
of the parasite usually relieves the symptoms.
Gastrointestinal Involvement in Pediatric AIDS

Gastrointestinal disorders, especially infections, are the chief cause of morbidity and mortality worldwide in patients
infected with HIV. Some of the conditions may occur anywhere in the digestive tract, including Candida, CMV, and
Mycobacterium avium complex infections, smooth muscle tumors, and atypical lymphoid proliferations. Most of the
infections listed are discussed in the section on gastrointestinal infections. Several excellent reviews on this topic are
available (e232,e389).
Much of the chronic failure to thrive, diarrhea, and malabsorption seen in infants and children with AIDS result from a
condition known as AIDS enteropathy, with or without infection(s). AIDS enteropathy is a poorly understood syndrome of
chronic diarrhea and weight loss associated with small-intestinal changes of villous atrophy, mononuclear cell infiltration
of the lamina propria, and crypt hyperplasia, all in the absence of a known pathogen. The etiology is currently unknown;
theories include intestinal infection by the HIV virus or an agent not yet identified, an immune dysregulation, or a reaction
to luminal antigens. A deficiency of brush border enzymes in AIDS enteropathy results in lactose and fat malabsorption
(e32,e177,e232,e468). Infections develop in many children with AIDS that also occur in immunocompetent children, but
they are more severe or sustained than in the normal host; agents include rotavirus, Salmonella, Shigella,
Campylobacter, Cryptosporidium, and Giardia. In addition, AIDS patients are unusually susceptible to infections with
certain opportunistic agents, including Candida albicans, CMV, Mycobacterium avium complex, and Microsporium.
Candida infection of the mouth and esophagus is the most common opportunistic infection in children with AIDS. More
than half of all children with AIDS carry CMV; in some, it causes a fulminant hemorrhagic, ulcerative, and necrotizing
gastrointestinal illness to erupt suddenly that is often fatal.
A number of atypical proliferations may develop in gastrointestinal lymphoid tissue in AIDS patients, including the
following: (a) endoscopically visible lymphoid aggregates of duodenal mucosa, (b) a polyclonal lymphoproliferative
process resembling posttransplant lymphoproliferative
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syndrome (e230,e232,e394), and (c) AIDS-associated non-Hodgkin lymphoma (e222,e394). Children with AIDS have a
disproportionate number of smooth muscle tumors in the gastrointestinal tract and extraintestinal sites. Most are
leiomyomas, sometimes multiple; more rarely, leiomyosarcoma has been diagnosed. These tumors have been
demonstrated to be Epstein-Barr virus (EBV)-related (e74,e307,e314).
FIGURE 14-41▪Colonic acute graft-versus-host disease. A: Lamina propria cellularity is decreased from normal due to
the effect of induction chemotherapy prior to the stem cell transplantation 200×. B: Note the characteristic epithelial cell
apoptosis 400×.
Graft-versus-Host Disease (GVHD)

The intestinal tract is one of the three major target organs in graft-versus-host disease. The skin and the liver are the
other two organs affected when donor lymphoid cells are transfused into an immunosuppressed host. Donor T-
lymphocytes target epithelial cells in these organs and initiate an immune response that destroys them. GVHD is usually
diagnosed following allogeneic bone marrow or stem cell transplant to treat leukemia and other malignant and
nonmalignant diseases, but it may also rarely occur following the transfusion of nonirradiated blood into patients with
primary or secondary immunodeficiency disorders (e411). GVHD develops in two phases: acute, which begins 1 week to
4 months after transplantation, and chronic, which begins approximately 4 months or more after the transplant. The
clinical and pathologic features of the two phases are distinctly different.
The gastrointestinal tract is affected in at least half of the patients with acute GVHD. The skin and liver may be involved
at the same time, at different times, or not at all. Intestinal GVHD is usually heralded by profuse watery diarrhea, which
indicates involvement of the small intestine and colon. Occasionally, the upper gastrointestinal tract will be involved first
or exclusively; the symptoms are nausea, vomiting, and anorexia (e434). Acute intestinal graft-versus-host disease is
diagnosed by colonoscopic or upper endoscopic biopsies. The earliest histologic change is the development of
apoptosis of individual epithelial cells in the regenerative (stem cell) compartment, characterized by vacuolization of the
cytoplasm and nuclear karyorrhexis (Figure 14-41A,B). The stem cell population in the small bowel and colon resides in
the lower portions of the crypts. In the esophageal squamous mucosa apoptosis is seen in the basal cell layer, similar to
that seen in skin involvement. In the stomach the stem cell population is located in the neck zone of the mucosa.
Inflammatory cell infiltrates are typically quite sparse, and lymphocytic infiltration of the epithelium in areas exhibiting
apoptosis is usually not apparent. Scattered eosinophils are usually present. Diagnostic histologic features of graft-
versus-host disease are often quite patchy, and many biopsies are necessary to exclude the diagnosis (21). Grading
schemes for acute graft-versus-host disease have been proposed, usually based on features best seen in the colonic
mucosa, but there is not a close relationship between clinical findings and grade. If the process is not arrested by
appropriate medical therapy, it progresses to glandular attenuation, destruction, and dropout. Areas of complete
glandular loss and extensive mucosal denudation occur in severe graft-versus-host disease (e308,e309,e395,e434).
Apoptosis can also occur as a consequence of the induction chemoradiation therapy regime used prior to stem cell
transplantation. Thus, histologic distinction between therapy-related mucosal damage and graft-versus-host disease is
usually not possible in the first 20 to 30 days after induction therapy is instituted. CMV infection can cause epithelial cell
apoptosis and therefore the diagnosis of concurrent GVHD is problematic. In addition, mycophenolate mofetil can cause
diarrhea and produce a graft-versus-host-like appearance in the gastrointestinal mucosa, and this agent is used in some
stem cell transplant patients. After appropriate therapy the mucosa has a regenerative appearance with mucosal
architectural distortion but absence of ongoing apoptosis (153).
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Chronic graft-versus-host disease is a more insidious process that primarily affects skin and liver. The intestinal tract is
largely spared except for the esophagus, in which a scleroderma-like fibrosis and dysmotility develop (e308,e309,e446).
In the evaluation of all the phases of intestinal graftversus-host disease, the differential diagnosis must include infection,
particularly with opportunistic organisms.
Henoch-Schönlein Purpura and Other Systemic Vasculitides

Henoch-Schö nlein purpura is a systemic vasculitis affecting mainly the skin, gastrointestinal tract, joints, and kidneys
(e239,e387,e460). It is believed to be triggered by a humoral response to a variety of antigens, including viruses,
bacteria, and some drugs. Gastrointestinal involvement is usually in the stomach or small intestine and presents with
abdominal pain and bleeding. These may be the heralding symptoms or may follow the characteristic purpuric rash. The
underlying gastrointestinal pathology is an acute leukocytoclastic vasculitis of small blood vessels in the submucosa or
deep lamina propria (Figure 14-42). Only rarely is this sampled by endoscopic biopsy; instead, nonspecific focal mucosal
hemorrhage, edema, erosions, and aphthous ulceration are seen, and endoscopic biopsy shows nonspecific
inflammation. These nonspecific histologic features often lead to an erroneous diagnosis of inflammatory bowel disease
or nonspecific colitis, resulting in a delay in proper diagnosis (21).
Other forms of vasculitis affecting the intestinal tract include systemic lupus erythematosus, Churg-Strauss syndrome,
Wegener granulomatosis, and microscopic polyangiitis (e62). Some cases in the past classified as polyarteritis nodosa
affecting the gastrointestinal tract actually represent microscopic polyangiitis using current diagnostic criteria. Some
infectious agents, notably enterohemorrhagic strains of E. coli, including serotype 0157:H7, and CMV, may target blood
vessels and cause small-vessel vasculitis, platelet-fibrin thrombi, and patchy hemorrhage and necrosis.
FIGURE 14-42▪Henoch-Schönlein purpura. Leukocytoclastic vasculitis involving small submucosal arterioles of the
colon 200×.
Table 14-6 ▪ CAUSES OF COLITIS IN PEDIATRIC PATIENTS
Idiopathic disorders
Ulcerative colitis
Crohn disease
Lymphocytic/collagenous colitis (rare)
Infections (see Table 14-4)
Viral (e.g., CMV, adenovirus, and enteric viruses)

Bacterial (e.g., Shigella, Salmonella, E. coli, Clostridium difficile, etc.)
Fungal (e.g., Zygomycoses)
Parasitic (e.g., Strongyloides)
Autoimmune and immunodeficiency
Autoimmune enterocolitis
Common variable immunodeficiency
Chronic granulomatous disease
Typhlitis (neutropenic enterocolitis)
Miscellaneous
Diversion colitis
Hirschsprung enterocolitis
Allergic colitis
Vasculitides
COLITIS
The numerous causes of colitis in infants and children are listed in Table 14-6. A comprehensive review of this topic is
available (e17).
Inflammatory Bowel Disease (IBD)

The term inflammatory bowel disease, as it is commonly used, encompasses only the chronic idiopathic conditions of
ulcerative colitis and Crohn disease. Specifically excluded are numerous other gastrointestinal inflammatory processes,
including infections, antibiotic-associated colitis, ischemia, and allergic diseases (e78,e246,e250). Ulcerative colitis and
Crohn disease have many similar features—enigmatic etiology, familial predisposition, clinical presentation, chronic
course, extraintestinal manifestations, and response to treatment. However, important differences make it possible to
differentiate between the two entities in most patients, and it is desirable for long-term prognostic and therapeutic
purposes to make this distinction if possible. Differentiating Crohn disease from ulcerative colitis may be difficult if the
disease is limited to the colon. The clinical findings, pattern of involvement in the gastrointestinal tract, radiologic
studies, and histopathology must all be integrated for a diagnosis to be made. In some cases, the distinction is
impossible, even after surgical resection and careful pathologic examination; in these cases, the designation
inflammatory bowel disease, indeterminate type is used (see below.)
Crohn disease and ulcerative colitis are far from rare in children. Among all cases of inflammatory bowel disease,
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20% to 30% are diagnosed before the age of 20 years. The incidence of Crohn disease and ulcerative colitis was
approximately equal until the 1970s, but since then, the incidence of Crohn disease in children has risen steadily while
the incidence of ulcerative colitis has remained relatively stable (e31,e185). Caucasians are at greatest risk, but other
races are also affected. Although most cases in children are diagnosed in the second decade, toddlers and young
children may also be affected (e100,e185,e186,e246). The symptoms of inflammatory bowel disease in children are
similar to those in adults, but in addition, growth retardation and delayed puberty are commonly encountered, particularly
in children with Crohn disease. Cessation of growth resulting from steroid therapy is also an important consideration in
the treatment of Crohn disease and ulcerative colitis in children.
Before idiopathic inflammatory bowel disease is diagnosed, intestinal infections with organisms such as Salmonella,
Shigella, Campylobacter, Yersinia, pathogenic strains of E. coli, and E. histolytica must be ruled out by appropriate
cultures and stool examination. The pathology of inflammatory bowel disease in children is identical to that in adults in
most respects (e17,e96,e228).
Ulcerative Colitis
Ulcerative colitis is an idiopathic chronic inflammatory disease that begins in the rectum and extends proximally and
contiguously for a variable distance. In a given patient disease may be limited to the rectum, involve only the left colon,
or involve the right colon as well. A fluctuating clinical course with exacerbations and remissions is typical. A fulminant
presentation with toxic megacolon is also seen. Ulcerative colitis is limited to the colon, although in patients with active
pancolitis mild inflammation may also involve the mucosa of the distal few centimeters of the terminal ileum (the so-called
backwash ileitis).
Diarrhea and rectal bleeding are the presenting symptoms in nearly all cases, although abdominal pain, cramping,
anorexia, and weight loss are also frequently seen. A small percentage of patients have a fulminant presentation, with
acute abdominal signs and toxic megacolon. As many as 20% of children have extraintestinal manifestations, with
arthritis of the large joints being the most common; uveitis, growth failure, skin involvement, and liver disease are more
unusual. Infections (e.g., with Shigella, Salmonella, C. difficile, Yersinia, and E. histolytica) must be ruled out, and
radiologic investigation, including barium enema and radiography of the upper gastrointestinal tract with smallbowel
follow-through, is undertaken to determine the extent and type of disease. Endoscopic features of ulcerative colitis
include mucosal hyperemia, friability, and ulceration beginning at the rectum and extending proximally. Biopsy
specimens taken at multiple levels during colonoscopy are important in diagnosing the disease, monitoring its progress,
and evaluating the response to therapy.
Ulcerative colitis can usually be well controlled medically, although powerful immunosuppressive drugs are sometimes
necessary. Surgery, usually a total proctocolectomy, cures the disease. Surgery is performed in ulcerative colitis for both
acute and chronic indications, including massive bleeding, acute fulminant colitis with megacolon, a chronic course with
severe disability or complications of medical therapy, and retardation of growth and sexual maturation. Sphincter-sparing
ileal reservoir (ileal “pouch”) operations spare the patient a permanent ileostomy (e17,e129). Patients with ulcerative
colitis of more than 10 years' duration are advised to undergo periodic surveillance colonoscopy with biopsies to monitor
for the development of dysplasia. Cancer is usually preceded by histologic evidence of dysplasia in biopsy specimens
(e383).
Pathologic findings in the first endoscopic biopsy specimens from a given patient may not be diagnostic by themselves,
but they are extremely helpful in arriving at a diagnosis when integrated with clinical and radiologic findings. Inmost
cases of untreated ulcerative colitis, the mucosal biopsy specimen shows diffusely increased numbers of chronic
inflammatory cells (plasma cells and lymphocytes) and acute inflammatory cells (polymorphonuclear leukocytes and
eosinophils) in the lamina propria. Plasma cells dominate the inflammatory response, often densely packing the lamina
propria and extending beneath crypts (basal plasmacytosis). Crypt abscesses and intraepithelial neutrophils may be
present at the initial diagnosis and during exacerbations (Figure 14-43). Superficial ulcerations may be seen, but even in
their absence, damage to the surface epithelium is nearly always indicated by the presence of regenerating epithelial
cells without goblet cells.
In normal colonic mucosa the crypts are arranged in straight and evenly spaced rows. Even at the time of the initial
presentation of ulcerative colitis, with symptoms of short duration, biopsies of involved segments will usually exhibit
distortion of this normal crypt architecture. This is typically
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manifested by scattered branched and irregularly shaped crypts, as well as crypts that no longer extend all the way
down to the muscularis mucosae. Assessment of crypt architecture is much easier in well-oriented biopsies. In poorly
oriented biopsies the crypts are usually seen in cross section as doughnut-shaped profiles, which makes it difficult to
evaluate branching and foreshortening. However, irregular spacing and variation in crypt diameter may still be observed
in tangential sections. A feature often associated with crypt architectural distortion is the presence of Paneth cell
metaplasia. Paneth cells are normally present in the mucosa throughout the small intestine but in the colon are limited to
crypts of the cecum and ascending colon. In IBD Paneth cells may be present more distally, and their presence is a good
marker of chronic colitis. In patients with inactive disease of very long duration crypt architectural distortion may become
very subtle, to the point where the histologic (and endoscopic) appearance may be indistinguishable from normal. In this
situation review of biopsies obtained during previous colonoscopic procedures may be necessary to confirm a diagnosis
of IBD.
FIGURE 14-43▪ Colonic biopsy demonstrating active ulcerative colitis. Note the presence of crypt architectural distortion
and a basal infiltrate of lymphocytes and plasma cells between the bases of the crypts and the muscularis mucosae
100×.
In the relatively recent era of routine colonoscopy and effective medical therapy for IBD it has become clear that healing
ulcerative colitis can appear quite patchy endoscopically, simulating the appearance of Crohn colitis (e38,e39,e249).
Fortunately, microscopic examination of these apparent “skip areas” of endoscopically normal mucosa in patients with
treated ulcerative colitis usually reveals evidence of quiescent disease, as indicated by the presence of (sometimes
subtle) crypt architectural distortion. However, patchy areas of completely normal mucosa have been documented in
long-standing ulcerative colitis. Often this is a result of intensive long-term medical therapy, but it can also be seen
before therapy is instituted. There are also cases in which skip areas of normal mucosa are definitely present from the
onset (typically a segment in the transverse or descending colon), and yet all other clinical and histologic features are
consistent with the diagnosis of ulcerative colitis. The clinical course in such a patient is almost always that of typical
ulcerative colitis (e249,e252). Topical steroid therapy delivered via enema has been convincingly demonstrated to result
in complete resolution of active inflammation and regression of crypt architectural distortion in rectal biopsies from
ulcerative colitis patients (e283,e340).
Histologically, rectal sparing at the onset of symptoms has been documented to occur in a subset of pediatric patients
with ulcerative colitis (48, 123, 152) (e296). These patients may also have histologically patchy disease at presentation
(59, 155). No clinical feature appears to separate children who present with rectal sparing from those who do not,
although atypical histology may be more common in the youngest children (124).
The distinctive features of ulcerative colitis are better visualized in colonic resection specimens. Ulcerative colitis is most
often characterized by uninterrupted mucosal involvement beginning at the rectum and extending proximally in a
circumferential and contiguous manner.
FIGURE 14-44▪Total abdominal colectomy specimen from a patient with ulcerative colitis involving the left colon.
The mucosa is usually diffusely hyperemic and granular, with areas of superficial or deep ulceration in patients under
poor medical control at the time of colectomy (Figure 14-44). Inflammatory polyps may be present, and in some cases
are numerous (Figure 14-45). The ileal mucosa is generally grossly unremarkable. The rectum and descending colon
may show more chronic changes, such as loss of the haustral folds and a smooth or granular mucosal surface.
Conspicuously absent are skip (uninvolved) areas, strictures, fistulas, and fibrotic thickening of the colonic wall, all of
which are commonly seen in Crohn disease.
Histologic examination reveals inflammation that most severely affects the mucosa and submucosa, with lesser severity
or sparing of the muscularis layers and serosa. Extensively ulcerated areas show mucosal and submucosal destruction,
with replacement by granulation tissue. Inflammatory polyps are composed of islands of surviving mucosa with
pronounced glandular distortion, inflammation, and capillary dilation. After the acute inflammation has subsided and
healing has occurred, evidence of ulcerative colitis remains as a loss of crypt parallelism, crypt atrophy and shortening,
hypertrophy of the muscularis mucosae, and metaplasia of Paneth cells. The appendix is commonly involved in resected
specimens (e170,e233) even when the cecum is spared, an
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exception to the diffuse contiguous involvement characteristic of ulcerative colitis (e107,e268).
FIGURE 14-45▪ Ulcerative colitis with inflammatory polyps.
Crohn Disease
In contrast to ulcerative colitis, Crohn disease may arise anywhere in the gastrointestinal tract, from mouth to anus. In
approximately 50% of children with Crohn disease, the classic distal ileal and proximal colonic involvement is seen.
Approximately 15% of children have only diffuse smallbowel disease, another 15% have only distal ileal involvement,
and 10% have isolated colonic disease. The remaining 10% have disease in another site, as in gastroduodenal Crohn
disease (e280,e302,e392,e505), or combination of sites.
Symptoms depend on the site of involvement, but in general the presentation of Crohn disease is more insidious than
that of ulcerative colitis, so that the diagnosis is often delayed. Vague abdominal pain, diarrhea, growth failure, and
anorexia are common. Small-bowel involvement may present as diarrhea and malabsorption. Colonic involvement may
present as bloody diarrhea and mimic ulcerative colitis. Endoscopic and radiologic studies of the upper and lower
gastrointestinal tract are important in determining the extent of involvement.
Unlike ulcerative colitis, Crohn disease is characterized by a segmental or skip pattern, in which involved areas of
intestine are often separated by normal intestine. Another important distinguishing feature is that the inflammation in
Crohn disease is transmural rather than mucosal, so that fissures, fistulas, intramural abscesses, strictures, and fibrous
adhesions develop (Figure 14-46). Thickening of the bowel wall as a result of edema and fibrosis occurs at the expense
of the lumen and causes intestinal obstruction. Inflammation, edema, and fibrosis of the bowel and regional lymph nodes
may cause adjacent structures to mat together and form an ileocecal mass. Perianal fissures, skin tags, and rectal-
perineal fistulas and abscesses are common in children with Crohn disease.
Endoscopic examination in Crohn disease often reveals patchy involvement and skip areas of normal mucosa.
Ulcerations are often linear, with intervening preserved mucosal islands, resulting in a cobblestone appearance. Small
(<5 mm), round, superficial “aphthoid” ulcerations are common in otherwise normal mucosa at the periphery of more
severely involved segments. In Crohn colitis the right side of the colon is often more severely affected than the left, and
the rectum may be completely spared.
FIGURE 14-46▪ Crohn enteritis with cobblestoned mucosa.
The histologic hallmark of Crohn disease is the presence of noncaseating epithelioid granulomas (eFigure 14-11).
Unfortunately, granulomas can be identified in biopsy specimens in less than 50% of Crohn disease patients, limiting the
utility of this feature. The routine examination of serial sections increases the likelihood of the identification of
granulomas (e271). Poorly formed granulomas can occur in association with ruptured crypt abscesses in ulcerative
colitis, presumably in response to extravasated mucin. Examination of serial sections may be necessary to demonstrate
the relationship between the damaged crypt and the granuloma (eFigure 14-12). Also, in a tangential section the
pericryptal fibroblast sheath can resemble a small granuloma. Distinction between Crohn disease and intestinal
tuberculosis can also be problematic (86).
Mucosal biopsy specimens in Crohn disease show increased numbers of chronic and acute inflammatory cells in the
lamina propria, crypt abscesses, and superficial ulcerations, all of which are nondiagnostic in the absence of
granulomas. In many cases the degree of crypt architectural distortion is less severe in Crohn disease than is typical of
ulcerative colitis, but confident distinction between the two diseases cannot rest on the assessment of this feature.
Relative preservation of the mucin content of goblet cells, even in cases of severe inflammation, is also more
characteristic of Crohn colitis than ulcerative colitis (9).
The histologic features of Crohn ileitis are essentially identical to those evident in colonic biopsies. There is usually
clear-cut distortion of normal villous architecture at least focally. Mucous (pyloric) gland metaplasia is a reliable marker
of long-standing inflammation and is common in ileal biopsies from patients with Crohn disease (Figure 14-47). However,
mucous gland metaplasia has also
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been documented in biopsies of ileal ulcers from patients taking NSAIDs (90). Although ulcerative colitis is classically
limited to the colon, some patients with pancolitis may exhibit the so-called “backwash ileitis.” “Backwash ileitis”
generally consists only of scattered neutrophils in the lamina propria and surface epithelium, with relative preservation of
the mucosal architecture. However, the spectrum of ileal mucosal damage in backwash ileitis has not been well-defined
in the current era of routine colonoscopic ileal biopsies (36).
FIGURE 14-47▪ Crohn ileitis. The inflammatory cell infiltrates are distinctly focal and destructive of the crypt epithelium
(100×).
Transmural chronic inflammation is the most helpful histologic feature in a resected intestinal specimen from a patient
with Crohn disease. Deep knifelike fissures, fistulas lined by granulation tissue, and fibrous strictures are also
characteristic. Submucosal fibrosis and the presence of many lymphoid aggregates or follicles also suggest Crohn
disease rather than ulcerative colitis (e17,e228,e366).
One of the best ways to distinguish between ulcerative colitis and Crohn disease is by examination of biopsies from the
upper gastrointestinal tract. The presence of significant patchy inflammatory changes of the esophageal, gastric, or
duodenal mucosa, while usually not diagnostic in isolation, can be very helpful in confirming a diagnosis of Crohn
disease (89) (e505).
The medical treatment of Crohn disease is similar to that of ulcerative colitis, although anti-tumor necrosis factor
monoclonal antibody therapy plays a more central role in Crohn disease. Surgery is not curative in Crohn disease and is
generally undertaken only when intestinal obstruction, fistulas, massive hemorrhage, or abscesses supervene. Growth
failure while the patient is on medical therapy and failure of medical therapy may also be reasons for a limited surgical
resection.
Indeterminate Colitis
The term indeterminate colitis is descriptive rather than diagnostic and is applied to cases of chronic inflammatory bowel
disease in which ulcerative colitis cannot be distinguished from Crohn disease. The term was first used by Price (e365)
in a description of fulminant pancolitis with overlapping pathologic findings, but it has gradually come to encompass
other cases with a gradual onset. In up to one-fourth of patients with a colitic presentation of chronic inflammatory bowel
disease, the distinction between Crohn disease and ulcerative colitis cannot be made, even when the endoscopic,
imaging, and biopsy findings are known. The term indeterminate colitis is often used as a temporary designation until
evolution of the disease provides further clues, such as the development of granulomas, fistulas, or gastroduodenal or
perineal involvement in Crohn disease. In a small percentage of patients, the distinction between Crohn disease and
ulcerative colitis is extremely difficult or impossible, even after a chronic course and colonic resection.
It is important to make the distinction between ulcerative colitis and Crohn disease if possible because the surgical
treatment of ulcerative colitis is significantly different from that of Crohn colitis. Patients with severe ulcerative colitis who
fail medical therapy undergo a total proctocolectomy with creation of an internal ileal reservoir (J-pouch) and ileal pouch-
anal anastomosis, which allow defecation through the anus. Patients with Crohn colitis often do poorly after the creation
of an ileal reservoir, and the procedure is contraindicated in them (e17).
Lymphocytic Colitis
Lymphocytic colitis was originally called microscopic colitis when it was first described in adults with chronic diarrhea
and normal colonoscopy findings, but demonstrable mucosal inflammation on colonic biopsy specimens. In the past
decade, the definition has been refined and the name changed to lymphocytic colitis with the recognition that patients
often have other autoimmune diseases, such as diabetes and arthritis, and that the colonic inflammation is characterized
by an increase in T-lymphocytes. On biopsy specimens, characteristic findings are increased numbers of IELs, surface
epithelial damage, and dense mononuclear cell inflammation of the lamina propria in the absence of crypt architectural
distortion and acute cryptitis (e49,e227,e276,e509). Similar findings are encountered in some patients with celiac
disease (e504). Lymphocytic colitis is seldom diagnosed in children, although occasional cases have been described
(e303).
Collagenous Colitis
Collagenous colitis has many of the same clinical and histologic characteristics as lymphocytic colitis (see above), with
the additional histologic finding of a distinct subepithelial collagen band that is obvious with hematoxylin and eosin stain
and highlighted by Masson trichrome stain. The collagen band represents a thickened basement membrane that is
unevenly distributed in specimens from different areas of the colon and is probably thickest in the proximal colon. A
basement membrane thickness of at least 10 μm is suggested for the diagnosis of collagenous colitis, measured in well-
oriented sections in which crypts are longitudinally sectioned. In adults, the thickness of the basement membrane in
collagenous colitis is variable up to 50 μm (e49,e227,e276,e509). The many similarities of lymphocytic and collagenous
colitis suggest a similar pathogenesis. This condition is almost never diagnosed in children, but an occasional report is
the exception (e179). Crypt architectural distortion and acute cryptitis are found in Crohn disease and ulcerative colitis,
neither of which is seen in microscopic or collagenous colitis.
Acute Self-limited Colitis (Infectious Colitis)

Acute self-limited colitis is defined clinically as a condition in which diarrhea, often bloody and with a sudden onset,
resolves spontaneously after several weeks. It is presumed to be bacterial in nature but since stool cultures are not
routinely obtained in every case of diarrhea a specific
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causal organism is not identified in a given patient. The histologic features include neutrophils in the lamina propria,
cryptitis, and, in the most severe cases, erosions and microscopic ulcerations, but normal crypt architecture is well
maintained (e17,e178,e447,e448). In some cases the superficial portion of the mucosa is focally necrotic and
hemorrhagic (eFigure 14-13). The lamina propria lacks the basal lymphoplasmacytosis seen in chronic inflammatory
bowel disease (e126,e277,e337,e407,e447,e448). Despite these differences, in the absence of a stool culture positive
for organisms, it is still sometimes difficult to assign an initial biopsy to the self-limited infectious category rather than to
chronic inflammatory bowel disease. Further clinical studies and a follow-up period of observation usually clarify the
situation.
Pseudomembranous Colitis
The term pseudomembranous colitis refers to a gross or endoscopic appearance of the colonic mucosa in which
numerous discrete, irregular, yellow plaques, 0.2 to 2.0 cm in diameter, appear anywhere on the colonic mucosal
surface. In the most severe cases, the membranes coalesce and become nearly confluent, and the process spreads to
involve most of the colon. The membranes are tightly adherent to the mucosal surface; wiping does not remove them.
Formerly thought to represent C. difficile infection in nearly all cases, pseudomembranous colitis is now known to occur
in infection with E. coli 0157:H7 (e380), other toxin-producing strains of E. coli, and Shigella, and in ischemia, ulcerative
colitis and Crohn colitis, uremia, fungal infections, neonatal necrotizing enterocolitis, and Hirschsprung disease-
associated enterocolitis (e373). However, antibiotic-associated C. difficile infection is still the most common cause.
When C. difficile infection is responsible, pseudomembranous colitis typically develops during a course of antibiotic
therapy or up to 6 weeks afterward (e30,e64,e244,e316,e485). C. difficile overgrows in the colon after antibiotic
alteration of normal flora. Clindamycin, ampicillin, penicillin, cephalosporins, and many other antibiotics have been
implicated. The onset of watery diarrhea is usually abrupt and accompanied by systemic signs, including fever,
abdominal pain, and leukocytosis.
Histologically, pseudomembranes are composed of inflammatory cell exudate, necrotic debris, and desquamated and
apoptotic epithelial cells, admixed with red blood cells and mucus. The pseudomembrane overlies acutely inflamed
colonic mucosa. In cases of pseudomembranous colitis due to due to C. difficile infection there is a characteristic lesion
that has been likened to a mushroom or volcano erupting from the crypts (Figure 14-48). The surface epithelium is often
destroyed, and in severe cases, much of the mucosa is necrotic. The intervening areas of mucosa are normal or show
nonspecific colitis while the submucosa is often edematous the deeper bowel layers are usually normal
(e17,e64,e244,e316).
FIGURE 14-48▪ Clostridium difficile infection. The classic “erupting volcano” appearance with a pseudomembrane
composed of desquamated epithelial cells, inflammatory cells, and red blood cells admixed with mucus and fibrin 100×.
Colitis Associated with Antibiotics

By altering the normal gut flora, antibiotics can cause a wide variety of gastrointestinal symptoms; these range from
innocuous diarrhea that ceases with the antibiotic therapy to fatal pseudomembranous colitis (see preceding section)
(e244,e316,e485). Tissue alterations, when they occur at all, can be manifested as an enteritis or colitis. When the
colon is affected, findings range from acute self-limited colitis to hemorrhagic colitis (e238) and pseudomembranous
colitis. C. difficile infection is sought by either toxin assay or selective culture in the most severe cases because it is
responsible much of the time.
Diversion Colitis
Diversion colitis is a chronic inflammatory process in an intestinal segment that has been bypassed by ileostomy or
colostomy and left in place, as in Hirschsprung disease, Crohn disease, ulcerative colitis, or other conditions that are
treated surgically. The cause is unknown but is thought to be an interplay between altered bacterial flora and a
deficiency of short chain fatty acids in the bypassed segment (e17,e199). In milder cases, the findings are identified
incidentally during pathologic examination of a bypassed segment removed during a “pull-through” procedure for
Hirschsprung disease. Other patients may become symptomatic and demonstrate endoscopic mucosal abnormalities,
including erythema, friability, and aphthous ulcerations. The most characteristic histologic finding is mucosal and
submucosal follicular lymphoid hyperplasia. Chronic mucosal inflammation, acute cryptitis, crypt abscesses, and
epithelial injury are seen in the most severe cases. The clinical setting usually suggests the diagnosis, but in patients
with chronic inflammatory bowel disease, these findings in the rectosigmoid colon may pose a diagnostic dilemma when
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ulcerative colitis must be distinguished from Crohn disease (e17,e169,e194,e199,e259,e289).
Typhlitis (Neutropenic Enterocolitis)

The term typhilitis is derived from the Greek word meaning “blind sac” (referring to the cecum). It is a necrotizing
enterocolitis typically centered around or limited to the cecum. It was first described in children with leukemia (e486), but
other susceptible persons are patients undergoing cancer chemotherapy or being treated with immunosuppressive
drugs following bone marrow or solid organ transplantation, children with AIDS, and those with neutropenia secondary to
hematologic diseases. Symptoms include fever, diarrhea, and right lower quadrant pain. Bacterial sepsis with recovery
of enteric bacteria is common, and polymicrobial sepsis is not unusual. The involved bowel is edematous, hemorrhagic,
and ulcerated. Transmural necrosis not uncommonly leads to bowel perforation. Antibiotics are sometimes effective in
arresting the infection, but intestinal resection is necessary in many cases (e240,e413,e482,e486).
Neonatal Necrotizing Enterocolitis

Neonatal necrotizing enterocolitis is a distinctive common disease of premature infants in the neonatal intensive care
unit characterized by coagulative and hemorrhagic necrosis and inflammation of portions of the small and large intestine
(e214,e253). Despite decades of research, the precise pathogenesis of this disease remains enigmatic (93, 107, 132).
Important contributing factors include altered bowel motility and digestion, immature intestinal circulatory regulation,
abnormal bacterial colonization, immature intestinal mucosal barrier, and enteral formula feedings. Intestinal ischemia
results from reduced splanchnic perfusion, systemic hypoperfusion, systemic hypoxia, or local factors such as intestinal
gaseous distension. Bacterial colonization is nearly always present, although neonatal necrotizing enterocolitis is not
primarily an infectious process in the usual sense and no specific organisms or group of organisms have been
implicated. Immature innate intestinal immune function likely contributes to the process of bacterial colonization.
Inflammatory mediators, especially platelet-activating factor and tumor necrosis factor, are endogenously induced by the
presence of bacterial toxins and play an important role in the pathophysiology of intestinal necrosis in neonatal
necrotizing enterocolitis. The role of oral feeding strategies in the pathogenesis of neonatal necrotizing enterocolitis has
been debated for years. There does appear to consensus that human breast milk feeds have a beneficial role, perhaps
by aiding in establishing a healthy intestinal bacterial flora and by augmenting cellular and humoral immunity. The
feeding of hyperosmolar formulas, in contrast, may result in the proliferation of abnormal gut flora and may adversely
affect intestinal perfusion (93, 107, 132).
Neonatal necrotizing enterocolitis occurs primarily in premature infants with birth weights ranging from 1,000 to 1,500 g
who are more than 2 weeks of age and who are severely ill with respiratory distress syndrome (Chapter 12). However,
up to 10% of infants with neonatal necrotizing enterocolitis are born at term, and the disease may develop as early as
the 1st day of life (143). Manifestations include abdominal distension, bloody stools, diarrhea, gastric retention of
feedings, shock, and apnea. As many as one-third of the affected infants have a fulminant course with intestinal
perforation, and a similar number have bacterial sepsis. The overall mortality is approximately 15% to 30%. The
diagnosis of neonatal necrotizing enterocolitis requires a suggestive clinical picture and radiographic demonstration of
pneumatosis intestinalis (i.e., gas within the bowel wall) or gas in the portal or hepatic veins. However, positive
radiologic signs may be lacking in one-third of the patients in whom the diagnosis is confirmed at surgery or autopsy (93,
107, 132).
In most cases, the most severely affected portions of the gastrointestinal tract are the terminal ileum and cecum (80%)
and the ascending colon, although either the small intestine or colon alone may be affected, or the entire small intestine
and colon. The gross appearance of neonatal necrotizing enterocolitis in 50% of the cases is that of a patchy segmental
necrosis with intervening spared areas; half of the cases show a continuous segment of intestine with circumferential
necrosis, dilation, and friability. If perforation has occurred, peritonitis is present. The mucosa shows a combination of
coagulative necrosis, inflammation, and hemorrhage. Focal necrotic pseudomembrane formation is seen in
approximately 10% of cases. Overall microscopic features are similar in most cases of neonatal necrotizing enterocolitis,
but the findings vary considerably from one microscopic field to another. Coagulative and hemorrhagic necrosis is
always present; it is limited to the mucosa in the early stages but at least focally transmural in the surgical or autopsy
cases (Figure 14-49). Acute and chronic inflammation is commonly
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found, limited to the mucosa in some foci but transmural in others. Inflammation and coagulative necrosis often occur
together in a given segment, but in some instances, one or the other may predominate (e26).
FIGURE 14-49▪Necrotizing enterocolitis. This section from a resected portion of small bowel reveals extensive mucosal
necrosis and submucosal hemorrhage. Note the large air spaces in the submucosa consistent with pneumatosis
cystoides 40×.
Mixed intestinal bacteria are often visible in the lumen or within the necrotic superficial mucosa. Fungal growth is
unusual, occurring in 3.5% of cases in one large series (e26). Pneumatosis intestinalis is found in approximately one-
half of surgical specimens with neonatal necrotizing enterocolitis, usually limited to the submucosa. These gas bubbles
have been shown to contain hydrogen, a product of bacterial fermentation. More than 50% of the cases of neonatal
necrotizing enterocolitis undergoing laparotomy show focal reparative epithelial changes and other evidence of healing,
such as the formation of granulation tissue and crypt distortion. Villous atrophy may be observed (e26,e231). Such
changes suggest that neonatal necrotizing enterocolitis evolves gradually before a catastrophic event, such as
perforation, brings it to clinical attention.
Intestine compromised by neonatal necrotizing enterocolitis, but not resected during the acute phase of the disease, may
develop progressive circumferential submucosal fibrosis during healing, causing intestinal stricture (e35,e262,e265).
Strictures are found in 10% to 20% of infants between 3 and 10 or more weeks after neonatal necrotizing enterocolitis
has been diagnosed. Before oral feedings are resumed, strictures are routinely sought by barium enema.
The treatment of neonatal necrotizing enterocolitis includes cessation of oral feedings, administration of antibiotics, and
surgery or percutaneous peritoneal drainage for perforation or other evidence of severe bowel compromise. Lengthy
intestinal resection may produce short-bowel syndrome. Other complications include peritonitis, sepsis and its
complications, and compromised nutrition. Long-term parenteral nutrition is required in many cases (65).
Spontaneous Perforation of the Gastrointestinal Tract

Isolated spontaneous perforation of the gastrointestinal tract in premature and term neonates occurs occasionally as a
clinical event distinct from neonatal necrotizing enterocolitis. It is usually an unexpected event in an infant not known to
have any prior gastrointestinal compromise (e23). The perforation develops in a single location in almost any part of the
stomach (e221), small intestine, or colon, and at laparotomy, the damage to surrounding tissue is inapparent or minimal.
Some cases have been explained by prior exposure of the infant to indomethacin to close a patent ductus arteriosus or
arrest maternal preterm labor (e5,e419). In other cases, localized segmental absence or thinning of the muscularis
externa has been observed (e217,e286) and is thought to represent a congenital abnormality. This view is not
universally accepted; however, an opposing view is that the thinning of muscle layers occurs secondary to excessive
distension, tearing, and retraction of muscle fibers. A final common pathway in these cases is probably localized
ischemia (e217), caused by a drug, a transient local decrease in splanchnic circulation, or a combination of these
factors. One case report noted the simultaneous occurrence of intestinal atresia, known to be ischemic in origin in most
cases, and a segmental absence of muscle coats (e7). Defects in muscle have also been described in strictures
developing after neonatal necrotizing enterocolitis and after ischemic bowel disease in older patients.
Allergic Colitis (Allergic Proctocolitis)

Allergic proctocolitis is a common cause of rectal bleeding and diarrhea in infants younger than 6 months to 1 year of
age. Most of those affected have been fed artificial formulas, usually based on cow's milk protein, or have recently been
switched from breast- to bottle-feedings. However, any dietary protein can be responsible, and cases have been
described in infants fed with soy milks, casein hydrolysate formulas, and even breast milk; in the latter cases, the
offending protein is thought to originate in the mother's diet and be transmitted in breast milk
(e171,e207,e229,e291,e341,e362, e475,e500).
The presenting symptom is usually blood streaks on the surface of stools or bloody diarrhea in an otherwise healthy
infant. Constipation is a presenting symptom in some patients. Fever, leukocytosis, and other signs of infection are
lacking. A peripheral blood eosinophilia is characteristic, but is not present in every patient (109) (e219,e291,e500).
Colonoscopy may be normal, but most often there is mucosal erythema, erosions, and loss of the normal vascular
pattern (e291).
Colonic biopsies usually reveal preservation of the normal crypt architecture. The most characteristic histologic feature
is a patchy or diffuse increase in eosinophils. Various studies have used different cutoff values to separate normal
controls from patients with allergic colitis (109) (e171,e500). A frequently cited rule of thumb regarding the number of
eosinophils is that 60 eosinophils should be seen in 10 high power fields in the lamina propria plus eosinophils within the
epithelium (e109). The presence of focal eosinophilic cryptitis with damage to crypt epithelium, eosinophilic crypt
abscesses, or infiltration of fibers of the muscularis mucosa, are features helpful in confirming the diagnosis when sheets
of lamina propria eosinophils are not present. (Figure 14-50A,B).
The differential diagnosis includes infectious colitis, which may present with similar symptoms but is characterized by
polymorphonuclear leukocytes rather than eosinophils. Eosinophilic gastroenteritis should also be considered in the
differential diagnosis if tissue eosinophils are very prominent, although this is much less likely to occur in infants.
The treatment of allergic proctitis consists of a dietary change to eliminate the offending protein. A large number of
special formulas are commercially available. The diagnosis of allergic proctitis is not considered confirmed unless the
symptoms and rectal eosinophilia resolve on the elimination diet and recur with challenge feedings of the offending
protein.
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FIGURE 14-50▪ Eosinophilic colitis due to food allergy in an infant. A: Normal crypt architecture is maintained 100×. B:
Eosinophilic infiltrates can be quite patchy 400×.
INTESTINAL NEOPLASMS
Intestinal tumors are uncommon in children, and most of them are not malignant. Many childhood intestinal masses
prove not to be tumors at all but rather inflammatory processes, such as ileocecal Crohn disease, or developmental
anomalies, such as duplication cyst or pancreatic heterotopia. Except for juvenile and Peutz-Jeghers polyps, epithelial
lesions are unusual, in contrast to their frequent occurrence in the adult intestine. The most common category of
intestinal malignancy in children is non-Hodgkin lymphoma, particularly Burkitt lymphoma. Hereditary syndromes should
be kept in mind when certain types of gastrointestinal polyps and tumors appear in children. An excellent comprehensive
review of pediatric gastrointestinal tract polyps and neoplasms is available (e82).
Polyps
Juvenile polyps of the rectosigmoid colon are the most commonly encountered gastrointestinal neoplasms in children.
Other polyps of the gastrointestinal tract are rare in children, yet they merit precise identification because of potentially
important long-term implications to both the children and their families. Most polyposis syndromes are hereditary and
associated with an increased risk for gastrointestinal and other malignancies (e82,e193).
Juvenile Polyps and Juvenile Polyposis Syndrome

Juvenile polyposis is an autosomal dominant syndrome characterized by the development of multiple hamartomatous
gastrointestinal hamartomatous polyps. The prevalence is approximately 1 in 100,000 (47, 69, 73). Germline mutations
in either of two genes of the TGF-beta signaling pathway, the SMAD4 gene located on chromosome 18q21, or the
BMPR1a gene on chromosome 10q23 are identified in about 45% of affected patients. Between 25% and 50% of cases
there is no family history of the disorder (25, 29).
Polyps usually develop during childhood and number between 5 and 100. Presenting symptoms and the gastrointestinal
distribution of polyps have led to the clinical subclassification of juvenile polyposis. There is a rare infantile form in which
severe polyposis of the entire gastrointestinal tract leads to clinically significant protein-losing enteropathy, rectal
bleeding, intussusception or prolapse of polyps, and involvement of other organ systems. In some affected probands an
autosomal recessive pattern of inheritance has been suggested. Another group of patients present later in childhood
with a milder form of generalized gastrointestinal involvement. There is also a subset of patients who develop only
colonic polyps. Finally, there is a small group of patients with both hereditary hemorrhagic telangiectasia and juvenile
polyposis syndrome who develop vascular ectasias throughout the body, including the gastrointestinal tract, as well as
gastrointestinal juvenile polyps (47, 69).
Because isolated colonic juvenile type polyps occur in up to 2% of children who do not have juvenile polyposis
syndrome, criteria for the diagnosis of the syndrome have been developed. The diagnosis requires either (a)
documentation of five juvenile polyps, (b) the presence of juvenile polyps in the stomach or small bowel, or (c) the
presence of any juvenile polyp and a positive family history of juvenile polyposis syndrome. Because there is some
overlap of the histologic features of juvenile polyps and other types of gastrointestinal hamartomatous polyps [especially
the phosphatase and tensin homolog (PTEN) hamartoma syndromes, discussed below], the diagnosis also requires the
absence of extraintestinal manifestations of any other polyposis syndrome (47, 69).
Juvenile polyps develop as a disordered overgrowth of mucosal elements. Colonic juvenile polyps consist of
hyperplastic and cystically dilatated crypts set in an abundant
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edematous and markedly inflamed stroma. Isolated juvenile polyps are usually sessile and extensively eroded, resulting
in the development of abundant superficial granulation tissue (Figure 14-51A,B) (73). This produces a highly
characteristic strawberry-like endoscopic appearance. The polyps in patients with the juvenile polyposis syndrome, in
contrast, often lack this extensive surface erosion and may have a more pedunculated configuration. In addition, there is
usually a greater amount of the epithelial component and less of the stromal elements in the syndromic polyps (Figure
14-52). In contrast to colonic Peutz-Jeghers polyps an arborizing core of smooth muscle is usually not present, although
a few smooth muscle fibers may be evident if the polyp has been prolapsing. In small polyps the crypt hyperplasia and
stromal edema and inflammation may be minimal, making accurate recognition difficult. Dysplasia does occur rarely in
the polyps of patients with colonic juvenile polyposis polyps, but great care must be taken to avoid overcalling reactive
changes related to the inflammatory background (Figure 14-53A,B).
FIGURE 14-51▪ AB: Sporadic juvenile polyps usually exhibit cystically dilated crypts, abundant edematous and inflamed
stroma with numerous eosinophils, and surface erosion. A: 40×, B: 100×.
FIGURE 14-52▪Juvenile polyposis syndrome in which the polyps typically exhibit greater epithelial proliferation, less
stroma, and an intact surface epithelium 20×.
Gastric juvenile polyps are histologically similar to their colonic counterparts. There is disorganized hyperplasia and
cystic dilatation of the gastric foveolar epithelium set in a background of inflamed and edematous stroma (Figure 14-
54A,B). Unfortunately, these same features also characterize sporadic gastric hyperplastic polyps, and histologic
distinction is generally not possible. Sporadic gastric hyperplastic polyps can be multiple and do not always occur in a
background of diffuse gastritis, which makes separation from gastric involvement by juvenile polyposis even more
problematic. Furthermore, gastric Peutz-Jeghers polyps often have a very poorly developed core of arborizing smooth
muscle fibers and therefore can also closely resemble gastric juvenile polyps. These confounding factors suggest that
histologic classification of hamartomatous polyps is best performed by analysis of small-bowel or colonic polyps. If
gastric polyps are discovered first, the prudent course for the surgical pathologist is to suggest the possibility of a
polyposis syndrome and to recommend examination for small-bowel or colonic polyps. Gastric juvenile polyps may also
develop dysplastic changes, but once again care must be taken not to mistake reactive epithelial changes due to
inflammation for dysplasia (73).
Involvement of the small bowel by juvenile polyposis is less common than colonic and gastric involvement, and the
polyps are less often sampled endoscopically. Small intestinal juvenile polyps lack the well-developed core of smooth
muscle of Peutz-Jeghers polyps and are generally much more inflamed, so accurate distinction is usually not
problematic.
There is a significant lifetime risk of malignancy in patients with juvenile polyposis syndrome, including cancers of the
pancreas, stomach, small bowel, and colon. One study of the risk of colorectal cancer yielded an absolute risk of 38.7
per
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100 affected persons and a relative risk of 34 times compared to the general population (18).
FIGURE 14-53▪Juvenile polyposis syndrome. A: Colonic polyp with a focus of high-grade dysplasia 40×. B: Focus of
invasive signet ring adenocarcinoma in a colonic polyp from an adult patient 200 ×.
PTEN Hamartoma Tumor Syndrome

A number of clinical syndromes including hamartomatous gastrointestinal polyps have been linked to mutations in the
PTEN tumor suppressor gene on chromosome 10q23.3 (17, 67). The best characterized of these disorders is Cowden
syndrome, an autosomal dominant disorder with hamartomatous lesions involving multiple organ systems, as well as a
substantially increased risk of thyroid, breast, and endometrial cancer. Mucocutaneous lesions, including multiple facial
trichilemmomas, acral keratosis, and papillomas (particularly of the oral cavity) are pathognomic features of the
syndrome. However, because these lesions can also occur sporadically in the general population, diagnosis requires
finding multiple such lesions or additional features of the syndrome. Macrocephaly and a large variety of benign lesions
of the breast, thyroid, and brain are also recognized as major manifestations of the syndrome. These lesions may begin
to develop in childhood and are usually diagnosed by the third decade of life. A consensus panel of diagnostic criteria
have been developed and more than 80% of individuals fulfilling these criteria harbor a mutation in the PTEN gene.
Screening and surveillance strategies for early detection of the various types of tumors have been advocated (17, 67,
119).
FIGURE 14-54▪ Juvenile polyposis syndrome. A: The gastric polyps in this syndrome closely resemble sporadic gastric
hyperplastic polyps 40x. B: This duodenal polyp lacks the central core of smooth muscle typical of smallbowel Peutz-
Jeghers polyps 40x.
Bannayan-Riley-Ruvalcaba syndrome is also caused by mutation in the PTEN gene. Cardinal clinical features include
macrocephaly, pigmented penile macules, lipomas,
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hemangiomas, and gastrointestinal hamartomas. Additional described features include developmental delay, thyroiditis,
proximal muscle myopathy, and joint hyperextensibility. Consensus criteria for clinical diagnosis have not yet been
formulated. About 70% of patients with this syndrome have mutations or large deletions in the PTEN gene. While an
increased risk of malignancy has not been firmly documented in Bannayan-Riley-Ruvacaba syndrome, affected
probands with overlap between this disorder and Cowden disease have been reported to have an increased risk of
breast cancer, and therefore the same cancer screening and surveillance recommendations have been advocated for all
affected individuals (17, 67).
Because the gastrointestinal hamartomas are usually asymptomatic and documentation of their presence is not
necessary to establish a diagnosis of either Cowden syndrome or Bannayan-Rubalcava-Riley syndrome, the incidence
of polyps in these disorders is not known precisely. In one review of reports of patients with Cowden syndrome in the
literature, gastrointestinal polyps were identified in 85% of patients who underwent endoscopic screening (67). The
hamartomatous polyps in both syndromes resemble those present in juvenile polyposis syndrome, and therefore
distinction between these disorders rests upon the presence of other diagnostic clinical features and genetic testing.
The polyps in Cowden syndrome have been reported to exhibit more stromal myofibroblastic proliferation and less
edema than juvenile polyps, and scattered lamina propria ganglion cells have also been described (73). Inflammatory
type polyps and lipomas have also been reported. Dysplasia and malignant degeneration of the hamartomatous polyps
do not appear to occur in these syndromes. Colonic adenomas have been reported in affected individuals, but currently
it is thought that they are sporadic and do not occur at increased incidence compared to the general population (17, 67)
(see Chapter 24 for other PTEN findings).
Peutz-Jeghers Polyposis Syndrome

Peutz-Jeghers syndrome is an autosomal dominant disorder with an incidence of between 1:8,300 and 1:280,000 in the
general population. It is characterized by the development of mucocutaneous hyperpigmentation, hamartomatous polyps
throughout the gastrointestinal tract, and an increased risk of malignancy at many sites. The median age of onset of
symptoms caused by the gastrointestinal polyps is 13 years of age. Presenting symptoms include bowel obstruction,
intussusception, and gastrointestinal bleeding or anemia. Recognition of the characteristic hyperpigmented macules can
also lead to proper diagnosis. They occur most often on the lips, buccal mucosa, or periorbital skin, but can also develop
on the skin of the fingers, palms and soles, genitalia, and perianally. The macules may fade with age (47, 67). Diagnosis
and follow-up with new technologies such as video capsule endoscopy and double balloon enteroscopy likely will be
beneficial to the clinical management of affected individuals (150).
More than 90% of patients have a detectable mutation in the SKT11 gene on chromosome 19p13.3. About 10% to 20%
of patients present with de novo mutations. The protein product is a serine/threonine kinase that is expressed
ubiquitously in human tissues. It regulated a number of downstream kinases and has important roles in the cellular
response to energy stress and in the establishment of cell polarity (66, 75, 135).
The hamartomatous polyps occur primarily in the small bowel (92%) but can also develop in the colon (30%) and
stomach (25%). Hamartomatous polyps may also occur in the nasal cavity, bladder, and lungs. The burden of
gastrointestinal polyps is usually lower than in juvenile polyposis syndrome; often less than ten polyps are present. A
clinical diagnosis of Peutz-Jeghers syndrome is made when two of the following three criteria are met: (a) two or more
small bowel Peutz-Jeghers type polyps; (b) characteristic hyperpigmented macules of the nose, lips, nose eyes,
genitalia, or fingers; and (c) a family history of Peutz-Jeghers syndrome (47, 69, 73). Peutz-Jeghers polyps are quite rare
outside the setting of the syndrome, with less than 50 cases reported in the literature (146).
The characteristic histologic features are best developed in Peutz-Jeghers polyps of the small intestine. The arborizing
central core of haphazardly arranged smooth muscle bundles is the most distinctive feature. The epithelial elements are
hyperplastic and disorganized. The inflammatory component is sparse when compared to juvenile polyps, and stromal
edema is not prominent (Figure 14-55). Displacement of the hyperplastic epithelial component into the deeper bowel
layers is not uncommon, particularly in larger polyps that have caused bowel obstruction or intussusception (Figure 14-
56). At frozen section the herniation of the epithelium into submucosa or muscularis propria can be confused with
invasive adenocarcinoma by the unwary surgical pathologist (e416,e493).
Peutz-Jeghers polyps of the stomach and colon often do not exhibit a prominent central core of arborizing smooth
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muscle and therefore can be confused with the more common juvenile polyps at these sites. They are less inflamed and
edematous than juvenile polyps, but accurate diagnosis is problematic unless small-bowel polyps are also present.
While there is a significant increased risk of gastrointestinal malignancy in patients with Peutz-Jeghers syndrome,
dysplasia and cancer development within the polyps themselves is extraordinarily rare (38, 43).
FIGURE 14-55▪Peutz-Jeghers polyp. Jejunal polyp with hyperplastic and disorganized mucosal elements and the
characteristic central arborizing core of smooth muscle 40×.
FIGURE 14-56▪ Jejunal Peutz-Jeghers polyp. Displacement of epithelial elements into the muscularis can be confused
with invasive adenocarcinoma, particularly in frozen sections, but the epithelium is clearly benign 40×.
Individuals with Peutz-Jeghers syndrome have a significantly increased risk of malignancy compared to the general
population (e163). Gastrointestinal, pulmonary, breast, gynecological, and pancreatic malignancies all occur with
increased incidence. The cumulative incidence of malignancy is reported to reach 85% by the age of 70 years (63). A
number of screening and surveillance programs have been advocated to monitor patients with Peutz-Jeghers syndrome
(29, 47, 69).
Adenomatous Polyps and Adenocarcinoma

Adenomatous polyps are true neoplasms and are rare in children. When identified in a child, even a single adenomatous
polyp should prompt consideration of familial adenomatous polyposis (96) (e82) and related polyposis syndromes. An
adenomatous polyp may be grossly sessile or pedunculated and microscopically exhibit a tubular or villous growth
pattern. Microscopically, it exhibits both architectural and cytologic features of dysplasia. Architectural features include
crowded crypts or cribriforming. Cytologic features of dysplasia include elongation and stratification of nuclei, nuclear
contour irregularity, and nuclear hyperchromasia. Adenomas lack the cystic dilatation of crypts and abundant
inflammatory stroma of juvenile polyps and the arborizing core of smooth muscle of Peutz-Jeghers polyps.
Familial adenomatous polyposis (adenomatous polyposis coli), the most common of the polyposis syndromes, is an
autosomal dominant disorder with an incidence of 1 in 8,000 persons. Approximately one-third of the cases are sporadic
(96). In 1991, the defective gene in familial adenomatous polyposis was localized to chromosome 5, and shortly after,
Gardner syndrome and Turcot syndrome were mapped to the same locus. In patients with familial adenomatous
polyposis, hundreds of adenomatous polyps usually carpet the colonic mucosa (Figure 14-57). The disease may
become symptomatic in adolescents, usually causing diarrhea and abdominal pain. The incidence of colonic
adenocarcinoma is very high in patients with familial polyposis, approaching 100% by age 50. Malignancy may occur as
early as the second decade. For this reason, colectomy is recommended whenever symptoms develop or in early
adulthood (12, 96). Multiple colonic adenomas also occur in the MUTYH polyposis syndrome, another autosomal
recessive disorder with a significantly increased risk of colonic adenocarcinoma. In this condition, however, colonic
adenomas almost never develop during childhood (128).
FIGURE 14-57▪Prophylactic colectomy specimen from a 27-year-old female with familial adenomatosis polyposis. No
invasive adenocarcinoma was identified.
After colectomy continued surveillance is necessary since small intestinal adenomas will almost always develop,
frequently in the area of the ampulla of Vater. Patients also commonly develop gastric fundic gland polyps. Dysplasia
has been reported to develop in these polyps, but progression to invasive gastric adenocarcinoma is exceedingly rare
(15, 142).
Patients with familial polyposis coli may exhibit a variety of extraintestinal malignancies, including thyroid and pancreatic
carcinomas, hepatoblastoma, and fibromatosis (desmoid tumor). There is also an increased incidence of a variety of
benign lesions, including dermatofibroma, lipoma, and bone lesions (e.g., osteoma, exostosis, cortical thickening of long
bones, and dental cysts). Congenital hypertrophy of the retinal pigment is the most common extracolonic manifestation
of familial adenomatous polyposis and may be detected before the gastrointestinal polyps.
Gastrointestinal and extraintestinal components of the syndrome may appear in different members of a family.
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FIGURE 14-58▪Invasive colonic adenocarcinoma arising in an adult patient with familial adenomatosis polyposis.
(Courtesy of Richard R. Anderson, M.D., Laboratory & Pathology Diagnostics, LLC.)
The designation of Turcot syndrome has been applied to patients with familial adenomatous polyposis who also develop
malignant central nervous system tumor. Glioblastoma and medulloblastoma usually cause death, although
ependymoma has also been reported (57) (e466).
Adenocarcinoma of the colon and rectum remains a rare diagnosis in children, with an incidence of only 1 in several
million (e82). Recognized antecedent conditions, such as familial adenomatous polyposis, familial juvenile polyposis,
and ulcerative colitis, account for a minority of the cases (Figure 14-58). Hereditary nonpolyposis colon cancer (Lynch
syndrome) (95) and other syndromes account for a few more, but most childhood cases appear sporadically. Presenting
symptoms of pain, vomiting, weight loss, and constipation are similar to those in adults. The diagnosis tends to be
delayed in children and therefore many have advanced disease and a rapidly fatal course shortly after presentation.
Involvement of the right colon where early disease is clinically silent is more frequent in children than in adults.
Histologically, the tumor in children tends to show poor differentiation with abundant mucin and often “signet ring”
features (e274,e377).
In addition to true polyps, other conditions may present as polypoid masses in the gastrointestinal tract. These include
inflammatory pseudopolyp in inflammatory bowel disease, pancreatic or gastric heterotopia, and tumors such as
leiomyoma, adenocarcinoma, lipoma, neurofibroma, and ganglioneuroma.
Nonepithelial Gastrointestinal Tumors

In children with congenital immunodeficiency or AIDS, smooth muscle tumors may develop in association with EBV
infection in either gastrointestinal or extraintestinal sites (e74,e307,e314). The tumors are commonly multifocal and are
uniformly reactive by in situ hybridization with probes to the EBV small noncoding RNAs (EBER) (40, 42).
Spindle cell tumors of smooth muscle origin must be distinguished from others with similar histology, including
inflammatory myofibroblastic tumor and fibromatosis (e82,e83).
Inflammatory fibroid polyp can occur at any age in the gastric or intestinal wall or adjacent mesentery and can become a
large mass (e84). The histologic picture is variable but usually includes loose fascicles of bland spindle cells admixed
with a mixed inflammatory cell infiltrate with a prominent component of eosinophils (111). The spindle cells often exhibit a
perivascular whorling orientation that is characteristic. Because of its large size, a malignancy may be considered
clinically, but the low cellularity of the lesion and lack of significant cytologic atypia usually lead to the correct diagnosis.
Recently, gain of function mutations of the PDGFRA gene has been documented in a subset of small-bowel and gastric
inflammatory fibroid polyps (87). Ganglioneuroma is yet another intestinal spindle cell tumor that is usually easily
distinguished by a frequent polypoid configuration and a positive reaction with neural immunocytochemical markers
(e99,e414).
Mesenteric or omental cysts, although not strictly gastrointestinal tumors, should be considered in the differential
diagnosis of abdominal masses in children (e71,e204,e325). Ultrasonography reveals their typical unilocular or
multilocular cystic nature. Most are located in the mesentery immediately adjacent to the small intestine. Mesenteric cyst
is lined by a single layer of cells or consists only of fibrous septa. It may be confused with a similar cystic lesion, cystic
lymphangioma, which has an endothelial lining in addition to lymphoid tissue and smooth muscle in its walls
(e172,e261,e452). Cystic lymphangioma often spans both the bowel wall and adjacent mesentery, so that a segmental
bowel resection is required; in contrast, mesenteric cyst is usually easily separated from the bowel wall.
Lymphoma
The intestine is the most common site of primary extranodal lymphoma (e41), and non-Hodgkin lymphoma is the most
common malignant intestinal tumor in children. Boys from 5 to 10 years of age account for most of the affected children,
and the usual clinical presentation is abdominal pain and a palpable right lower quadrant mass. Burkitt lymphoma is by
far the most common gastrointestinal lymphoma of childhood. It usually arises in the submucosal lymphoid tissue of the
ileocecal region and extends transmurally to involve local mesenteric lymph nodes and form a bulky tumor mass. Less
advanced cases may present with intussusception or intestinal obstruction. Histologically, the mucosa and submucosa
are replaced by sheets of uniform lymphoblastic cells with very regular, round, noncleaved nuclei, usually arranged in a
“starry sky” pattern. Appropriate hematopathologic evaluation of Burkitt lymphoma reveals a B-cell lineage; a
translocation, t(8;14), is characteristic.
The prognosis is related to the extent of abdominal or systemic tumor spread. If the intestinal and nodal masses
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are amenable to resection and appropriate chemotherapy is administered, approximately 80% of these children are
cured. Other non-Hodgkin lymphomas, in addition to Burkitt lymphoma, have been reported in the gastrointestinal tract
but are less frequent in otherwise healthy children (e82).
In immunodeficient patients, malignant lymphoma occurs anywhere in the intestinal tract and does not demonstrate a
preference for the ileocecal area. Unusual large-cell lymphoproliferative disorders have been reported in the
gastrointestinal tract in primary immunodeficiency diseases (e133). A number of lymphoproliferative processes in
addition to AIDS-associated non-Hodgkin lymphoma develop in patients with AIDS (e222,e230,e232,e394). A spectrum
of posttransplant lymphoproliferative disorders associated with EBV infection, which involves the gastrointestinal tract in
many cases, may develop in recipients of solid organ and bone marrow transplants (e142,e332,e450). These include
polyclonal and monomorphic B-lineage lymphomas at the most advanced end of the spectrum.
Langerhans cell histiocytosis (formerly called histiocytosis X) may affect any portion of the gastrointestinal tract to
produce malabsorption, diarrhea, ulceration, or bleeding. Gastrointestinal involvement occurs as a component of
widespread systemic infiltration. The infiltrate is usually mucosal and consists of the characteristic histiocyte-like cells
with grooved nuclei admixed with a mixed inflammatory cell infiltrate including a prominent component of eosinophils
(Figure 14-59A,B). Immunohistochemical reactivity for S-100 protein and CD1a confirms the diagnosis. Because of the
presence of multinucleated giant cells in some cases, this condition may be mistaken for a granulomatous infectious
process or Crohn disease (62) (e47,e158,e182,e243).
Systemic mastocytosis develops due to a specific activating mutation (codon 816) in the c-kit gene. Gastrointestinal
involvement occurs in about 70% to 80% of patients with systemic mastocytosis. The stomach and duodenum are the
most commonly involved. Common symptoms include abdominal pain, diarrhea, and bleeding. Peptic ulcer disease can
develop due to hypergastrinemia stimulated by the release of histamine from the mast cells. Serum tryptase levels
greater than 20 ng/mL are considered abnormal (74).
FIGURE 14-59▪ Colonic involvement by Langerhans cell histiocytosis. A: Histiocytic infiltrate in the lamina propria 200×.
B: Higher power reveals mixture of histiocytic cells with grooved nuclei, multinucleated giant cells, and a few admixed
eosinophils 400 ×.
Mast cells are cytologically bland and are inconspicuous in H&E sections of normal mucosa. With the use of special
stains, scattered mast cells can be seen in the lamina propria. In patients with systemic mastocytosis, endoscopy may be
normal or reveal thickened mucosal folds and erosions. In most cases the infiltrate of mast cells is dense (eFigure 14-
14A,B). Eosinophils are often also increased in number. Chloracetate esterase, toluidine blue, or Giemsa stains can be
used to highlight the mast cells. Immunohistologic stains utilizing mast cell tryptase or CD117 antibodies are probably
more sensitive and easier to perform (eFigure 14-14C) (74).
APPENDIX
Normal Anatomy and Histology
The appendix is present at the tip of the cecum at birth, but as the cecum grows the appendix moves to a position on the
posteromedial wall below the ileocecal valve (e498). However, aberrant takeoff from the cecum is not unusual, and both
anterior and retrocecal positions are particularly commonly encountered (e189). The average length of the adult
appendix is 9 cm, with a reported range of 2 to 25 cm (e12,e498).
The overall gross anatomy of the appendix is most similar to the colon. There is a serosa, muscularis propria with an
outer longitudinal and inner circular layer, submucosa,
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muscularis mucosae, and mucosa (e498). The mucosa closely resembles its colonic counterpart, although branched
crypts are regarded as a normal finding in the appendix. Endocrine cells and Paneth cells are scattered throughout the
mucosa. The lymphoid tissue component is also exaggerated in the appendix, more akin to that seen in the terminal
ileum. In children lymphoid follicles may be confluent over large areas of the mucosa. Within the lamina propria there are
numerous ganglion cells, Schwann cells, and nerve fibers, as well as endocrine cells, which may be the origin for
carcinoid tumors (e116).
Congenital and Neuromuscular Disorders

Diverticula of the appendix may be congenital or acquired, with acquired lesions being ten times more common
(e285,e464). Acquired diverticula may easily rupture during bouts of acute appendicitis. Inflammation of the diverticula
(akin to sigmoid diverticulitis) may present as a mild form of appendicitis (e285). Appendiceal diverticula are particularly
common in patients with cystic fibrosis (e161). Appendiceal intussusception is usually the result of a pathologic process
involving the appendix itself, such as a tumor, endometriosis, cystic fibrosis, or virally induced lymphoid hyperplasia
(e15,e312). Adenovirus infection has specifically been implicated in some cases.
Fibrous obliteration of the appendiceal tip, a common finding in adults, is uncommon in childhood and is of no clinical
significance. In some cases a disorganized hyperplasia of neural elements is evident within the fibrous tissue, including
nonmyelinated nerve fibers and Schwann cells, and in some cases endocrine cells (156) (e25,e344,e439). Some
authors have applied the term “appendiceal neuroma” to this finding (e439). It is unclear whether the presence of these
elements is indicative of prior acute appendicitis. A lesser degree of neural proliferation is sometimes present within the
mucosa in appendices without luminal obliteration (e344).
Acute Appendicitis
Acute appendicitis is the most common indication for emergent surgery in the United States, with about 250,000
appendectomies performed each year (136). The epidemiology, pathogenesis, and clinical features of acute appendicitis
have been extensively investigated for many decades, but certain aspects of this very common disorder remain
controversial. The peak age of incidence is from 10 to 30 years of age, although cases in infants and the elderly do
occur (26). The lifetime risk of the development of acute appendicitis is about 7% in the United States (136). About twice
as many women as men undergo appendectomy, although the disorder has about an equal incidence in females and
males. The overlap in symptomatology and laboratory findings between acute appendicitis and a variety reproductive
tract diseases accounts for the high frequency of unnecessary appendectomy in women (7). The rate of appendectomy
in which acute appendicitis is not confirmed pathologically is currently approximately 15% (45). The rate has not
decreased significantly in the past 70 years, despite the use of even more sophisticated and expensive laboratory and
imaging techniques (26).
The classic early symptoms of acute appendicitis include abdominal pain, anorexia, nausea, and vomiting. McBurney
described progression from vague periumbilical pain to localized pain in the right lower quadrant more than 100 years
ago (e306). Physical examination typically reveals mild tachycardia, low-grade fever, decreased bowel sounds, and
tenderness to palpation in the right lower quadrant. Laboratory evaluation usually reveals a mildly elevated white blood
cell count with a left shift (136). The use of abdominal ultrasound and helical computed tomography has been proposed
to increase the sensitivity and specificity of the diagnosis of acute appendicitis (136). Appendectomy remains the
mainstay of treatment for acute appendicitis. However, when perforation has already occurred at the time of diagnosis,
some surgeons advocate delaying surgery until after a course of antibiotics.
The pathogenesis of acute appendicitis is still a matter of debate (e498). Many investigators are convinced that luminal
obstruction (usually by a fecalith or lymphoid hyperplasia) leads to distension and ischemia followed by bacterial
invasion (136) (e21,e22,e275). Ligation of the appendix in animals has consistently resulted in the development of acute
appendicitis (e360). However, there are some data that suggest that this mechanism is not operative in the majority of
human cases. Alternative hypotheses include primary viral infection or local ischemia producing microscopic ulcers, thus
allowing for bacterial invasion (26).
Although ultimately bacterial invasion plays a central role in the pathogenesis of acute appendicitis, microbiologic studies
have shown that any of a variety of enteric organisms could be responsible in an individual case. In a recent study
involving peritoneal swabs performed at the time of appendectomy for perforated appendicitis, E. coli was cultured in
about 75%, with P. aeruginosa and Streptococcus responsible for the majority of the remaining cases (141).
The gross appearance in acute appendicitis varies depending on the severity of the acute inflammatory process. In
classic cases with transmural involvement the serosa appears dull, discolored, and shaggy. The wall is edematous and
swollen and retracts when incised. Acute inflammatory cell exudate may be evident at the site of a perforation. If the
appendix is removed before transmural inflammation has developed, the appendix may grossly appear normal or exhibit
only mild serosal hyperemia.
The histologic features of acute appendicitis reflect the gross appearance. In severe acute appendicitis there may be
transmural necrosis with perforation and acute peritonitis.
There is debate, however, regarding the minimal degree of neutrophilic inflammation that is required to render a
diagnosis of acute appendicitis. Most authors will accept neutrophilic mucosal infiltrates if associated with at least focal
ulceration as diagnostic (e77,e130,e498). However, others
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point to data suggesting that such changes can be present in incidental appendectomies performed in asymptomatic
patients. These authors insist that for this reason a diagnosis of acute appendicitis is not warranted until neutrophilic
inflammation extends into the muscularis propria (26). The point has been made that if multiple additional sections are
obtained from an appendix that on initial examination exhibits only superficial inflammation, there is a high likelihood of
finding involvement of the muscularis propria (e63,e429). There is uniform agreement that luminal neutrophils or focal
infiltration of the surface epithelium alone is insufficient ground for a diagnosis of acute appendicitis (assuming that the
entire appendix has been examined). The possibility of an enteric infection (such as Campylobacter ileocolitis) should be
considered in such patients (26) (e498).
Interval Appendectomy
It has become accepted practice in many centers to delay appendectomy in patients in whom perforation and abscess
formation have occurred. Instead, the patient is treated conservatively with supportive care and antibiotics, and
appendectomy is thus delayed for 4 to 8 weeks, at which time the patient is clinically stable and the complication rate is
therefore lower. In fact, some surgeons question the need for appendectomy at all if conservative management is
successful (8).
Histologic examination of interval appendectomy specimens most often reveals mural thickening and fibrosis, transmural
lymphoid aggregates, and mucosal architectural distortion (e304) (Figure 14-60A,B). Not infrequently granulomas are
also evident, resulting in a histologic appearance that closely mimics Crohn disease. Occasionally, xanthogranulomatous
inflammation is evident (61) (e304). In some cases the appendix is histologically normal or exhibits only mild serosal
fibrosis.
FIGURE 14-60▪Interval appendicitis. A: There is fibrosis of the serosa with lymphoid follicles, consistent with resolution
of a prior episode of acute appendicitis with perforation 20×. B: The acute inflammation has completely resolved 40×.
Unusual Infections of the Appendix

A variety of viral pathogens can produce inflammation of the appendix and many produce clinical features similar to
acute appendicitis. Measles virus infection involving the appendix is very rare but is well described. In most patients
appendectomy is performed during the prodromal stage of the illness, before the diagnosis of measles is established.
The histologic hallmark is the presence of Warthin-Finkeldey cells similar to those seen in the tonsillar tissue of infected
patients. Lymphoid hyperplasia is also prominent, but there is little evidence of acute inflammation (113). However, if
appendectomy is delayed superimposed bacterial appendicitis of the traditional type may develop (113). Adenovirus and
rotavirus infection has already been mentioned as a cause of appendiceal intussusception. Smudgy appearing viral
inclusions can be identified in epithelial cells, but rotavirus does not produce inclusions visible by light microscopy (60).
Yersinia, Campylobacter, or Salmonella infection can cause ilocecitis that results in symptoms, laboratory test
abnormalities, and radiographic findings difficult to distinguish from acute appendicitis (e370,e472). Histologic
examination in cases where an appendectomy is performed usually reveals only mild mucosal neutrophilic infiltrates,
without involvement of the submucosa (e331,e420,e473). However, there are also cases of true acute appendicitis
caused by Yersinia infection (e420). Histologic examination in these cases has revealed severe neutrophilic infiltrates
extending into the muscularis, as well as prominent granulomatous inflammation. In these cases isolated Crohn
appendicitis must also be considered, and distinction between these two disorders is usually not possible on histologic
grounds alone.
Enterobius vermicularis (pinworm) is infrequently seen in the lumen of the appendix, but this parasite is not thought to
cause acute appendicitis (e61,e428,e497). On occasion ova that become embedded in the mucosa may produce mild
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mucosal inflammation (e497) (Figure 14-61A,B). In some parts of the world, appendiceal infection by E. histolytica,
schistosomiasis, tuberculosis, and Toxoplasma are not uncommon (53) (e342).
FIGURE 14-61▪ A and B: Appendix with Enterobius vermicularis. The adult worms can be seen in cross section within
the appendiceal lumen. A: 40×. B: 200×.
Miscellaneous Conditions
Mucosal melanosis has been documented in 46% of appendices removed from pediatric patients. It has no clinical
significance and does not appear to be related to the use of laxatives (55). In pregnancy submesothelial deposits of
decidualized cells may develop in the absence of endometriosis (e449). Confusion with metastatic tumor is avoided by
use of immunohistologic stains, since the decidualized cells are uniformly negative with keratin antibodies. The
relationship between deciduosis and symptoms of acute appendicitis is unclear, as no neutrophilic infiltrates are present
(e449). Endometriosis of the appendix is not uncommon. The appearance is histologically similar to that seen with
involvement in other pelvic sites. Although appendiceal endometriosis usually does not cause acute inflammation, it can
produce cyclic abdominal pain (e67).
FIGURE 14-62▪ Appendix in a patient with cystic fibrosis. A: Thick mucinous secretions fill the appendiceal lumen 40×.
B: The crypts are distended by thick mucinous secretions 200×.
Cystic fibrosis has already been mentioned as a cause of appendiceal intussusception and diverticular disease. Gross
examination usually reveals a dilated appendix that is filled with thick, tenacious mucus. Histologic examination reveals
densely eosinophilic mucin filling the lumen and sometimes causing dilatation of the crypts (e330,e423) (Figure 14-
62A,B).
Both Crohn disease and ulcerative colitis often involve the appendix (e233). In some cases of ulcerative colitis, the
appendix is involved even when the proximal colon is not, an exception to the rule that ulcerative colitis does not “skip”
segments (e268). Appendiceal inflammation clinically
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mimicking acute appendicitis may be the heralding event that leads to the diagnosis of Crohn disease. In the absence of
known chronic inflammatory bowel disease, it is rarely possible to diagnose either Crohn disease or ulcerative colitis
when an abnormal appendix is encountered. However, because appendicitis is ordinarily an acute suppurative process,
the presence of unusual or chronic features, such as predominantly chronic inflammation, fibrosis, fissuring ulcers, or
granulomas, should lead to a consideration of another diagnosis. Noncaseating granulomas, in addition to being
characteristic of Crohn disease, are also found in idiopathic granulomatous appendicitis, an entity that is felt to be
different from Crohn disease (e20,e125,e215). Granulomatous appendicitis presents with appendiceal symptoms, and
the histology shows chronic appendicitis with granulomas. It is possible that most of these cases represent a chronic
resolving phase of acute appendicitis, as the histologic features are identical to those seen at the time of interval
appendectomy for resolved acute appendicitis with perforation.
Appendiceal Carcinoid Tumors

About 70% of carcinoids are located at the tip of the appendix, allowing for appendectomy with a negative surgical
margin. Generally, a well-circumscribed firm yellowish nodule is evident. The tumor is centered in the submucosa but
may extend into the overlying mucosa and into the underlying muscularis propria. A precise size should be recorded in
the gross description (106). Histologic examination usually reveals an insular or trabecular arrangement of tumor cells.
Depth of invasion and the presence or absence of angiolymphatic invasion are important prognostic factors.
Immunohistologic studies are usually not required for diagnosis. Classic carcinoid tumors are usually reactive with
neuroendocrine markers such as synaptophysin, chromogranin A, neuron specific enolase, and CD56. About 15% of the
tumors are reactive with the CK20 antibody, but they are consistently CK 7 nonreactive. About 85% of appendiceal
carcinoids are reactive with the CDX2 antibody, even in metastatic sites. As expected, appendiceal carcinoids are
nonreactive with the TTF-1 antibody, which is useful in the distinction in a metastatic site from pulmonary primaries,
which are usually positive with this marker (3, 11, 24, 71, 129).
FIGURE 14-63▪ Classification of anorectal malformations. Anomalies are classified as low or high depending on their
relationship to the pubococcygeal line on x-ray film (broken line). Fistulas tracts between the rectum and other structures
are indicated by stippling. A: low anomalies are usually associated with an external fistula to the vestibule (in girls) or
the skin. B: In high anomalies, fistulas are internal, usually to the posterior vagina in girls or the proximal urethra in boys.
Carcinoids less than 1 cm in dimension are regarded as benign since lymph node metastasis and distant metastasis are
vanishingly rare. Appendectomy with a negative margin is regarded as curative for these tumors. In contrast, lymph node
or distant metastasis occurs in about 5% of carcinoids greater than 2 cm in size and therefore right hemicolectomy is
recommended for these tumors. The proper management of carcinoids between 1 and 2 cm is unclear since prospective
data regarding the natural history of these tumors is limited. Many authors recommend right hemicolectomy if tumor
invades the mesoappendiceal fat or if angiolymphatic invasion is detected. However, it should be noted that some
advocate simple appendectomy even for carcinoids larger than 2 cm, given the indolent natural history of even
metastatic carcinoid tumor, and the lack of a proven survival advantage for right hemicolectomy (10) (e324). Goblet cell
or adenocarcinoid is a more treacherous neoplasm.
DISORDERS OF THE ANUS

Anorectal anomalies comprise a spectrum of malformations. These range from a thin membrane obstructing an
otherwise normal rectum and anus (true imperforate anus) to atresia of the entire distal rectum, in which the proximal
rectum ends as a blind pouch in the pelvis (e496). The term imperforate anus is a misnomer because the anomaly
usually consists of considerably more than an imperforate anal membrane. The anal canal and rectum are usually both
affected. The incidence of these malformations is approximately 1 in 5,000 births.
The classification of anorectal malformations has long been confusing and nonstandardized. The malformation is usually
classified as high, intermediate, or low, depending on the location of the distal rectum in relation to the levator ani
muscle, which can be demonstrated radiologically in relation to bony landmarks (e.g., the pubococcygeal line on a lateral
radiograph) (e398). Fistulas between the rectal pouch and perineal skin or various locations in the urinary and genital
systems are often found. The most common variations are
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shown in Figure 14-63. Persons with a low imperforate anus have an intact anal sphincter and usually a fistula from the
rectal pouch to the perineal skin anterior to a covered anal dimple, although in up to 25% of the cases, a fistula is not
present. High malformations, formerly known as anorectal agenesis, are associated with absence of the anal sphincter
and complex abnormal interval anomalies. A rectourethral fistula is present in most boys with this malformation; girls
usually have a fistula between the rectal pouch and the vagina, bladder, or urethra. The term persistent cloaca
describes the condition in which a girl has a single perineal opening draining an internal pouch comprised of the terminal
portions of rectum, vagina, and urinary tract structures.
Other congenital anomalies are found in as many as 50% of infants with high anorectal malformations. The most
frequent associations are genitourinary and skeletal abnormalities (especially of vertebrae and pelvis), congenital heart
disease, and esophageal atresia with tracheoesophageal fistula. A diagnosis of VATER syndrome, VATERL syndrome,
or caudal regression syndrome (e353,e441) should be considered in every infant with an anorectal anomaly.
Acquired Diseases
Condylomata Acuminata
Condylomata acuminata, or venereal warts, are being increasingly reported in prepubertal children (e110,e412,e471). In
boys, they occur in the perianal region, and in girls, they are found in the perianal or genital regions. The etiologic agent
is human papillomavirus of the same DNA sequence types that are responsible for condylomata acuminata in adults
(e471). In many but not all cases, a history of sexual abuse of the affected child is obtained (e412) (see Chapter 7).
Alternatively, the mother of an affected child may have transmitted the virus. Histologically, the lesions are identical to
those seen in adults.
Perianal Abscess and Anal Fistula

Perianal abscesses, usually found in infants, result from breaks in the skin or anal mucosa or an infection in the anal
glands. Treatment consists of surgical incision and drainage. Anal fistulas between the anal canal and skin may develop
secondarily, requiring surgical excision of the fibrous and granulation tissue tract in perianal soft tissues and muscle.
Older children with leukemia, Crohn disease, and immunodeficiency states are especially susceptible to perianal
abscesses and fistulas (e411).
All References are listed on the Stocker Website.
Chapter 15
The Liver, Gallbladder, and Biliary Tract
John Hicks
Haresh Mani
J. Thomas Stocker
DEVELOPMENT
Hepatobiliary morphogenesis occurs during the first 10 weeks of gestation (e473,e685,e686,e697). The liver
primordium appears in week 3 as a tubular evagination of the future duo-denal segment of the foregut endoderm.
The hepatic diverticulum differentiates cranially into the proliferating hepatic cords and caudally into the
extrahepatic bile ducts and the gallbladder. The hepatic diverticulum branches dichoto-mously, and thick
anastomosing sheets of epithelial cells grow into the mesenchyme of the septum transversum, and the
mesenchymal cells form the connective tissue elements of the hepatic stroma and capsule. As the hepatic sheets
extend outward in the septum transversum, they are penetrated by the capillary plexus derived from the vitelline
veins, which arise from the primitive hepatic sinusoids (e695).
In the 10-mm embryo, bile canaliculi appear as intercellular spaces between sheets of presumptive hepatocytes.
The epithelial lining of the extrahepatic bile ducts is continuous with the primitive hepatic sheets that give rise to
the epithelium of the intrahepatic bile ducts. The epithelium of the intrahepatic bile ducts is probably generated
by interaction of the primitive hepatic epithelium and the mesenchyme surrounding the developing and branching
portal vein. The epithelial layer, which is in direct contact with the mesenchyme around the portal vein,
transforms into bile duct-like cells, after which a second layer transforms into bile duct epithelial cells (e694). At
around 8 weeks' gestation, the ductal plate develops, appearing as a cleft in the shape of a cylinder around the
mesenchyme of the progressively developing and branching portal vein (e741). The ductal plate (Figure 15-1)
undergoes gradual remodeling to form the interlobular bile ducts in the portal tract, undergoing a balanced
process of cell proliferation and apoptosis (e696). Intrahepatic bile ducts are recognized in the 20- to 30-mm
embryo. The hepatocytes and bile duct epithelial cells are structurally and functionally distinct. The canals of
Hering, which connect the canaliculi to the bile ducts, consist of both typical hepatocytes and bile duct epithelial
cells (e742).
The development of the liver is associated with changes in the primordial vitelline veins, which give rise to the
portal, hepatic, and umbilical veins. The definitive pattern of veins within the liver is established in the 10-mm
embryo. The proximal end of the right vitelline vein forms the terminal part of the inferior vena cava. The portal
vein arises from persistence of segments of both right and left vitelline veins and three anastomotic channels
between the two. The right umbilical vein disappears, and all blood from the placenta enters the liver from the left
umbilical vein. The coalescence of some of the hepatic sinusoids produces an oblique channel, the ductus
venosus, which connects the left umbilical vein to the right vitelline vein, diverting some of the oxygenated blood
directly to the heart.
The right side of the liver receives blood predominantly from the portal vein, and the left lobe is supplied mainly
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by oxygenated blood from the left umbilical vein. This may account for the difference in the appearance of the
two lobes. At birth, the left lobe is larger relative to its size in later life. Moreover, the right lobe shows more
hematopoiesis, and the hepatocytes contain more glycogen, lipid, and iron pigment than those in the left lobe.
Fetal blood flow through the hepatic artery is insignificant compared with that delivered by the umbilical and
portal veins.
FIGURE 15-1 ▪ Ductal plate in the fetal liver is formed by a collar of epithelial cells at the periphery of the portal
tract and abuts against zone 1 hepatocytes. Note the presence of extramedullary hematopoiesis (H&E stain,
200×).
The caudal part of the hollow diverticulum elongates and presumably becomes the common bile duct, hepatic
duct, cystic duct, and the gallbladder between weeks 5 and 7.5. The liver is the site of hematopoiesis between
weeks 6 and 7, and erythropoiesis dominates from week 12 until the beginning of the third trimester (e641).
During the third trimester, the bone marrow is the dominant site of hematopoiesis, and hepatic erythropoiesis
decreases, although it continues in the newborn period and may persist into the first few weeks of life.
HISTOLOGY
The conventional histologic unit of the liver is the hepatic lobule, which consists of a central efferent vein with
cords of hepatocytes radiating to several peripheral portal tracts. The portal tract contains the interlobular bile
ducts, branches of the portal vein, and hepatic artery and lymphatics. The functional unit of the liver is the
hepatic acinus (Figure 15-2). The hepatic acinus is a three-dimensional structure with the portal tract as the
central point (zone 1) where blood flows from terminal branches of the portal vein and hepatic arteries into the
sinusoids and empties into the terminal hepatic venules at the periphery of the acinus (centrilobular/zone 3). Bile
is secreted into the canaliculi and flows toward the portal areas into the interlobular bile ducts that are connected
to the canaliculi by canals of Hering. The acinus thus includes parts of several adjacent lobules.
FIGURE 15-2▪ Schematic view of hepatic lobule or acinus. The conventional view of the liver consisted of a
hepatic lobule with a central vein CV) surrounded by hepatocyte cords radiating to peripheral portal areas. The
functional unit of the liver, the acinus, however, consists of a threedimensional structure with a central portal tract
surrounded by concentric zones of hepatocytes (I, II, and III), with the most peripheral zone (III) lying near the
central vein.
The hepatocytes in children older than 5 or 6 years of age are organized into single-cell plates. In younger
children, the liver cells are arranged in two-cells-thick plates. In the preterm infant, the lobular structure of the
liver is poorly defined and hepatic plates are more than one cell thick. Canaliculi lie between adjacent
hepatocytes, and, ultrastructurally, tight junctions are present between the hepatocytes surrounding the
canaliculus. Microvilli from the hepatocytes project into the canalicular lumen. The hepatocytes in childhood
often have nuclear glycogen, and lipofuscin in the cytoplasm is usually scanty. The hepatic sinusoidal lining cells
include endothelial and Kupffer cells. The endothelial cells are supported by reticulin fibers, and between the
endothelial cells and hepatocytes is the space of Disse. Perisinusoidal cells (cells of Ito) are interstitial fat-storing
cells and appear to play a significant role in hepatic fibrogenesis.
CONGENITAL ANOMALIES
Agenesis of the liver is incompatible with life and is usually associated with other severe congenital anomalies in
stillborn fetuses. Agenesis of one lobe of the liver, usually the right, is seen infrequently and is rarely associated
with clinical symptoms (e327,e412). In situs inversus totalis, the liver, its peritoneal and vascular connections,
and the gallbladder and extrahepatic ducts have a mirror-image configuration to normal situs. In the asplenia-
polysplenia syndromes, the liver may be midline and bilaterally symmetrical (e468).
The liver may herniate through defects in the diaphragm (Figure 15-3). Diaphragmatic defects are more common
on the left side, and the liver often herniates into the left pleural
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cavity (61) (e159,e161,e453,e568). The herniated portion of the liver may be dusky, and a groove often marks
the site of compression by the margin of the diaphragm. The right lobe of the liver may bulge into the right pleural
cavity in association with eventration of the right hemidiaphragm (e453). In cases of omphalocele, the liver is
often herniated into the omphalocele sac. In large omphaloceles, there is often distortion of the liver and its
vascular and biliary connections. The liver may have signs of marked congestion and even hemorrhagic
necrosis. Intrapericardial herniation of the liver occurs rarely and may result in massive pericardial effusion in
neonates (e159). Nearly all cases of the thoracopagus type of conjoined twins show connections between the
two livers, ranging from a bridge to a common liver between the two twins (e652).
FIGURE 15-3 ▪ Herniation of liver through diaphragmatic defect. A large defect in the left leaflet of the diaphragm
has led to herniation of the left lobe of the liver and intestines into the left hemithorax, resulting in mediastinal
shift to the right and severe pulmonary hypoplasia.
FIGURE 15-4 ▪ A: Ectopic liver tissue within the diaphragm, B: lung, and C: umbilical cord (H&E, 40×). D:
Ectopic liver tissue in the umbilical cord with bile ducts (H&E stain, 200×).
Hepatic ectopia or heterotopia is extremely unusual, with only rare reports of distinct lobules of hepatic tissue
within the gallbladder wall, the substance of the diaphragm, lung, and umbilical cord (Figure 15-4) (101)
(e83,e520,e611). Often times, this liver tissue is seen in conjunction with congenital diaphragmatic hernias and
congenital heart disease. Ectopic pancreatic tissue within the liver or in the porta hepatis may also be seen,
occasionally obstructing the common hepatic duct (e611). Adrenal heterotopias are usually the result of adrenal-
hepatic adhesion or fusion depending on the presence (adhesion) or absence (fusion) of a capsule between the
organs. Liver tissue at these variable sites is at the same risk for viral hepatitis and subsequent hepatocellular
carcinoma (HCC) as an orthotopic liver tissue infected with hepatitis viruses.
TISSUE TRIAGING
The most important aspect of providing an accurate diagnosis is appropriate triaging of tissue to allow for optimal
evaluation (Figure 15-5). It is imperative that adequate tissue is obtained to perform all necessary tests for an
appropriate diagnosis to guide future therapy and to avoid repeat biopsy.
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FIGURE 15-5 ▪ Liver biopsy triaging consists of tissue submitted for for-malin-fixation and paraffin embedding,
freezing tissue at -70°C, viral or microbiologic culture submission, and glutaraldehyde fixation for electron
microscopy.
Fresh tissue can be obtained for microbiologic and viral cultures and polymerase chain reaction (PCR) testing.
Tissue should be obtained for routine histology (formalin fixation), histochemical stains (frozen in optimal
cryomatrix material [OCT] at -20°C and alcohol fixation), electron microscopy (glutaraldehyde), and
genetic/molecular evaluation (frozen at -70°C). It is especially important with glycogen storage diseases (GSDs)
to maintain optimal preservation of glycogen. With formalin fixation, up to 70% of glycogen is lost due to the
soluble nature of the predominant form of glycogen in the cytoplasm. Glycogen can be preserved with freezing
and/or alcohol fixation, allowing for quantitative evaluation by analytical techniques (frozen tissue) and qualitative
assessment by histochemical staining (PAS, PAS-diastase). Quantitative analysis of enzyme(s) responsible for
suspected metabolic and mitochondrial diseases must be done on frozen tissue. Assessment of gene mutation
and sequencing of the gene responsible for the enzyme defect or mitochondrial disease also require frozen
tissue. Preservation of the enzyme, enzyme activity, DNA, and RNA requires cryopreservation at -70°C and
maintaining this temperature until the tissue reaches the appropriate reference laboratory. Depending on the
testing required for a definitive diagnosis, tissue requirements may dictate an open biopsy of the liver or skeletal
muscle. Obtaining fibroblast cultures from a skin biopsy may also be necessary for genetic and enzyme studies.
Current trend in surgical and interventional radiology practice has been toward needle core biopsies for
diagnosis. The pathologist should be aware of necessary tissue requirements (tissue weights and preservation
methods) for appropriate testing to be completed. A single tissue core of 20 mm length from a 16-gauge needle
with a 1.5 mm diameter yields about 15 mg of tissue. Several metabolic disease tests require a minimum of 20
mg of tissue. With GSDs, 100 mg or more of tissue will be needed. This may necessitate numerous tissue cores,
or an open biopsy, to obtain adequate tissue for all tests. This emphasizes the importance of active
communication between the healthcare team and the pathologist. Because tissue will be preserved in a steady
state with cryopreservation (-70°C), comprehensive workup (histopathology, histochemistry, electron
microscopy) by the pathologist to determine which additional testing is most appropriate can be completed prior
to performing specialized testing on the frozen tissue.
PHYSIOLOGIC JAUNDICE
Hyperbilirubinemia in the neonatal period is one of the earliest postnatal events that requires clinical assessment
to determine its clinical significance (83, 147) (e158,e534). In the majority of cases, it is assessed to be
physiologic jaundice with an elevated unconjugated bilirubin, which resolves within the first 2 weeks of life.
However, in the presence of conjugated hyperbilirubinemia and other concurrent hepatic enzyme abnormalities,
a clinically serious underlying disorder must be given consideration. With infants and older children, development
of jaundice is a sign of hepatic or biliary tract disease of diverse etiologies, requires thorough clinical, imaging
and laboratory evaluation, and may need liver biopsy to determine the exact nature of the underlying disease.
Physiologic jaundice is characterized by an increase in serum unconjugated bilirubin of 5 to 6 mg/dL by 2 to 4
days of age. This is a result of increased bilirubin production following breakdown of fetal red blood cells,
combined with transient limitation in the conjugation of bilirubin by the liver. Levels of up to 12 mg/dL may be
seen in Chinese, Japanese, Korean, or Native American infants. Other risk factors include maternal diabetes,
prematurity, altitude, polycythemia, male sex, trisomy 21, cutaneous bleeding, cephalohematoma, oxytocin
induction, and vitamin K use (e499). Other causes of unconjugated hyperbilirubinemia are listed in Table 15-1.
Cholestasis is rarely present in the liver in the absence of other diseases.
HEREDITARY HYPERBILIRUBINEMIAS
Crigler-Najjar syndrome (CNS), an autosomal dominant disorder, results from a mutation in one of the five exons
of the UGT1A1 gene coding for the enzyme bilirubin-UDPglucuronosyltransferase (24)
(e143,e323,e506,e590,e760). UGTIA1 mutation leads to elevated unconjugated bilirubin levels. In type 1 CNS,
enzymatic activity is completely absent and the neonate presents with jaundice and frequently kernicterus with
death by 1 year of age. Liver transplantation has been successfully used in management. The liver may show
prominent canalicular bile or may appear normal. With type 2 CNS, there is only partial deficiency of glucuronyl
transferase, and this has milder clinical course with most affected individuals being asymptomatic. Gilbert
syndrome is a benign condition with minimal clinical manifestations, owing to greater preservation of enzyme
activity. Although the condition is occasionally seen in children, the diagnosis is usually made incidentally in
young adults or in later life (e242).
Dubin-Johnson syndrome, an autosomal recessive trait, may present in the neonatal period with conjugated and
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unconjugated hyperbilirubinemia and severe cholestasis (145, 181) (e249,e630,e713). This syndrome has
mutation in the ABCC2 gene that is responsible for synthesis of MRP2/cMOAT, an organic ion transporter. Zone
3 hepatocytes contain deposits of a granular golden-brown pigment, with staining characteristics of melanin.
Ultrastructurally, however, these granules do not have the features of melanosomes, but are lysosomes with a
distinctive appearance.
Table 15-1 ▪JAUNDICE IN INFANTS DUETO UNCONJUGATED HYPERBILIRUBINEMIA
Physiologic Features Associated Conditions
Overproduction of bilirubin Sepsis

Rh/ABO incompatibility
Erythrocyte defects
Hemoglobinopathies
Hematoma, birth trauma
Polycythemia, maternal fetal or fetal maternal transfusion
Drugs
Impaired transport of bilirubin Hypoxia

Acidosis
Hypoalbuminemia
Intralipid
Impaired hepatic uptake Decreased sinusoidal perfusion

Gilbert syndrome
Impaired conjugation Gilbert syndrome

Impaired conjugation
Breast milk jaundice
Hypoglycemia
Hypothyroidism
High intestinal obstruction
Glucoronyl transferase deficiency, types I and II
Drugs
Impaired enterohepatic circulation Low intestinal obstruction

Meconium ileus
Rotor syndrome is characterized by persistent elevation of conjugated and unconjugated serum bilirubin and
presents infrequently in children (181) (e132,e251,e329,e722). It differs from Dubin-Johnson syndrome clinically
and morphologically and can be distinguished from Dubin-Johnson syndrome by elevated urinary coproporphyrin
levels (2.5 to 5 times normal) (e132,e251,e722). The liver is normal histologically, but ultrastructurally immature
bile canaliculi and osmiophilic lysosomal granules have been described.
CONGENITAL AND ACQUIRED CHOLESTATIC DISORDERS IN THE NEWBORN

AND INFANT
Idiopathic Neonatal Hepatitis
Idiopathic neonatal hepatitis (INH) is largely a diagnosis of exclusion, because there are many infectious,
metabolic, toxic, and anatomic etiologies to explain neonatal cholestasis (Table 15-2) (15, 69, 153). Once other
disorders have been excluded, INH accounts for approximately 25% to 40% of
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cases, with an incidence of 1 in 4,500 to 9,000 live births. Although two subsets are seen, sporadic INH (85% to
90%) and familial INH (10% to 15%), it is likely that INH will become a better defined diagnostic category with the
elucidation of addition etiologies for cases considered to be INH. For example, alpha1-antitrypsin deficiency was
included in the idiopathic category prior to discovery of the clinical and genetic features of this disease. It is now
a separate and distinct entity, accounting for 25% to 30% of neonatal hepatitis cases (69). It should be noted that
there are some familial INH cases without a defined genetic pattern.
Table 15-2 ▪CHOLESTATIC DISEASE IN INFANCY
Extrahepatic obstruction
Biliary atresia
Bile duct stenosis
Sclerosing cholangitis
Stone
Neoplasm
Mucus/bile plug
Intrahepatic disorders
Giant cell hepatitis

Paucity of intrahepatic bile ducts
Syndromic (Alagille)
Nonsyndromic
Byler syndrome (progressive hepatocellular disease with persistent cholestasis)
Defects in bile acid metabolism (trihydroxycoprostanic acidemia)
Congenital abnormalities
CHF
Caroli disease
Toxic
TPN
Sepsis
Endotoxemia
Chromosomal
Down syndrome
Trisomy 17,18
Inborn errors of metabolism
Amino acid
Tyrosinemia
Carbohydrate
Galactosemia
Fructosemia
Glycogen storage disease, type IV
Lipid
Gaucher disease
Niemann-Pick disease
Wolman disease
Glycolipid
A1AT deficiency
Miscellaneous
CF
Neonatal iron storage
Copper overload Indian childhood cirrhosis
Cerebrohepatorenal syndrome of Zellweger
Hypopituitarism
Hypothyroidism
Infections
Viral
Cytomegalovirus
Hepatitis B
Herpes simplex
Rubella
Reovirus
ECHO
Coxsackie
Varicella
Bacterial
Mycobacterium
Listeria
Syphilis
Toxoplasmosis
Infiltrative disorders
Langerhans cell (histrocytosis X)

Familial erythrophagocytic lymphohistiocytosis
Other
Shock
Cardiac failure
Grossly, the liver in neonatal hepatitis may be enlarged, is usually smooth, and has a deep green bilious
appearance. Microscopically, cholestasis is usually seen in zone 3 hepatocytes and canaliculi and rarely in the
interlobular bile ducts. Giant cell transformation is usually prominent, but is a nonspecific finding, because it may
be seen in many disorders involving the neonatal liver (Figure 15-6). Hepatocytes may show ballooning,
acidophilic necrosis, and pseudoglandular or acinar formation. Lobular or portal mononuclear cells are generally
sparse, but a prominent inflammatory component and extramedullary erythropoiesis should suggest an infectious
etiology. The portal areas in INH are usually not expanded, and the bile ducts are normal or may be
inconspicuous. Rarely, there may be mild proliferation of the interlobular bile ducts (69). Histologic features
comparing INH with those of extrahepatic biliary atresia (EHBA) are listed in Table 15-3 (177).
FIGURE 15-6 ▪ A-C: Idiopathic neonatal hepatitis. Giant cell transformation with expansion of the portal region
by chronic inflammatory cells, prominent bile ducts, and readily identified cholestasis (A at 100×, B at 200×, C at
200×, H&E).
The prognosis of sporadic cases of INH is generally favorable (74% complete recovery, 7% chronic liver
disease, 19% death). Infants with the familial form (family history of neonatal cholestasis) have a considerably
poorer prognosis (22% recovery, 16% chronic liver disease, and 63% death) (69).
Extrahepatic Biliary Atresia

EHBA is a disorder of infants that occurs worldwide with an incidence of 1 in 8,000 to 12,000 live births (14, 66,
80) (e45,e46,e163,e270). EHBA presents in two forms: an embryonic or fetal type (10% to 35%) and a perinatal
form (65% to 90%). The embryonic or fetal form is characterized by early onset of neonatal cholestasis without a
jaundice-free
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period, unlike neonatal physiologic jaundice. This form is also associated with other anomalies, such as
polysplenia and asplenia, cardiovascular defects, abdominal situs inversus, intestinal malrotation, and portal vein
and hepatic artery anomalies. The perinatal form presents as late-onset neonatal cholestasis (4 to 8 weeks of
age) following a jaundice-free period with passage of normally pigmented stools at birth. There is no associated
anomaly with the perinatal form (14). Similar to neonatal hepatitis, EHBA is also considered to be a condition with
more than one etiology. In fact, INH and EHBA have been seen as sequential processes in the same infant over
a period of several weeks or months.
Table 15-3 ▪COMPARISON OF BILIARY ATRESIA AND NEONATAL HEPATITIS SYNDROME
Features Biliary Atresia Neonatal Hepatitis
Giant cell transformation Usually focal Diffuse; occasionally focal
Hepatocellular necrosis Variable Variable
Lobular disarray Usually mild May be marked
Cholestasis Hepatocytes, canaliculi, and ducts Hepatocytes and canaliculi; ducts

(rare)
Portal fibrosis In all portal areas Absent early in the course
Bile ducts Proliferation typically seen in all Normal; rarely focal proliferation
portal areas
Cellular infiltrate Variable; mononuclear Variable; mononuclear
Extramedullary Usually present Usually present

hematopoiesis
Fat Typically absent Typically absent

FIGURE 15-7 ▪ A: Extrahepatic biliary atresia liver biopsy. Histopathologic features include diffuse bile duct
proliferation in expanded portal region with canalicular cholestasis. B: Hepatocytes organized into pseudoacinar
pattern, C: giant cell transformation adjacent to fibrotic portal region. D: Cirrhosis may occur rapidly without
surgical intervention (H&E stains, A, B, C at 200×, D 40×).
The liver biopsy remains an integral component in the diagnosis of a neonate or young infant with persistent
conjugated hyperbilirubinemia and is a highly reliable means of establishing the diagnosis of EHBA in 85% to
97% of cases (14). Ductular proliferation is the most common finding and is considered a diagnostic feature of
EHBA, although modest bile duct proliferation may be seen in neonatal hepatitis (Figures 15-7, 15-8 and 15-9)
(e381). The interlobular bile ducts are
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tortuous and have distorted contours, readily demonstrated with pancytokeratin. The lining epithelium shows
degenerative changes, and periductal reactive fibrosis may occur with plump fibroblasts surrounded by a loose
edematous stroma. Lymphocytes and even neutrophils are found within the portal areas, with occasional
infiltration of the bile duct epithelium. Portal lymphocytes, which are usually few in number, should not be
confused with extramedullary hematopoiesis in younger infants. As the disease progresses in the first few weeks
of life, nearly all portal areas are expanded by fibrosis, with type IV collagen deposition. Bridging fibrosis occurs,
and early nodular transformation is evident as a prelude to the development of secondary biliary cirrhosis. The
progression to cirrhosis varies considerably from one case to another, but there is some direct relationship with
age.
FIGURE 15-8 ▪ Resection of residual common bile duct during Kasai procedure. A: Common bile duct remnant
with orientation by surgeon. CHD, hepatic duct, GB; gallbladder, CBD; common bile duct; plate, liver plate. B:
Near total obliteration of common bile duct lumen with no residual epithelial lining (H&E, 20×). C: Microscopic
residual common bile duct lumen with epithelial lining (H&E, 20×). D: Nests of bile duct epithelium in common
bile duct wall (H&E, 200×). The latter side chain structures should not be mistaken as evidence of a patent bile
duct.
Hepatocellular alterations include cholestasis (canalicular, hepatocellular, ductular), feathery

(pseudoxanthomatous) degeneration, pseudoacinar transformation, and focal giant cell transformation. These
features overlap with those of neonatal hepatitis. The cholestasis in EHBA is usually severe. The most prominent
cholestasis is in zone 3, but is also present in the ductules and bile ducts at the zone 1 interface. Hepatocytes
may form glandlike structures around bile plugs, imparting a “pseudoacinar” configuration, the so-called
cholestatic rosettes. Bile “lakes” consisting of amorphous collections of bile surrounded by inflammatory cells
and connective tissue are seen rarely in liver biopsies, unlike in adults with obstruction of the biliary tract.
Hepatocytes may display mild enlargement and rarefaction of the cytoplasm (feathery degeneration), but fatty
change is
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rarely seen. Giant cell transformation, if present, is generally restricted to zone 1 at the interface with the
expanded portal tracts (Table 15-3).
The most frequently observed changes within the liver in EHBA are the cholestasis, portal fibrosis, and ductular
proliferation. Other causes of obstruction (bile duct stenosis, choledochal cyst, mucous or bile plug) produce
similar changes, as will disorders such as alpha-1-antitrypsin deficiency and total parenteral nutrition (TPN)-
associated cholestasis. It is important to realize that other disorders can simulate patterns of liver injury similar to
those for EHBA.
FIGURE 15-9 ▪ Explanted liver with prior Kasai procedure. A: Explanted liver with micronodular cirrhosis. B:
Patent small bowel anastomosis site at liver hilum. C: Liver in cross section with close apposition of small bowel
anastomosis to liver hilum and micronodular liver parenchyma with diffuse bile staining. D: Small bowel
anastomosis separated by muscular wall of small bowel and fibrous tissue from the underlying liver parenchyma
(H&E, 10×).
The extrahepatic ducts may display a wide variety of histopathologic changes, ranging from a mild degree of
inflammation to complete obliteration (Figure 15-8). The epithelium
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of large, medium, and small ducts shows nuclear irregularity and pyknosis with cellular degeneration and
necrosis. Cellular debris and bile-stained macrophages may be present in the lumen. The duct lining is often
infiltrated by neutrophils and is ulcerated, with intraluminal and extraluminal fibrosis distorting the lumen. As the
epithelial inflammation and degeneration progresses, fibrosis increases and eventually obliterates the duct. With
active ductular destruction, the stroma around and between ducts becomes infiltrated by neutrophils,
lymphocytes, and macrophages, along with a prominent fibroblastic proliferation. As the ductular inflammation
diminishes and the ducts are destroyed, the stromal activity is replaced by dense fibrosis, containing a few
residual inflammatory cells and remnants of bile ducts. Choi et al. (e128) have used ultrasonography to define a
“triangular cord” of fibrous connective tissue in the portal hepatis of infants with EHBA. Rarely, islands of hyaline
cartilage may be found in the porta hepatis, suggesting a congenital malformation as the cause of the atresia in
these selected cases (e447). The gallbladder may be diminutive and exhibit varying degrees of fibrosis, epithelial
degeneration and destruction, and luminal compromise.
FIGURE 15-9 ▪ (continued) E: Large bile-filled lakes within the liver parenchyma and micronodular cirrhosis with
diffuse bile staining, and F: bile plugs distending portal legion with adjacent micronodular liver parenchyma
(H&E, 40×).
Biliary remnants have been classified by Gautier and Eliot (58) into three types:
1. 1. Absence of any lumen lined by biliary epithelium, with little or no inflammatory cells in the connective tissue
(Figure 15-8).
2. Presence of lumina lined by cuboidal epithelium. The remnants may be numerous, have a lumen less than 50
ìm, and be surrounded by a neutrophilic infiltrate. Cellular debris and bile may be present in the lumen, and
epithelial necrosis may be seen in ducts with a diameter exceeding 300 ìm.
3. The presence of a central altered bile duct incompletely lined by columnar epithelium, in addition to smaller
epithelial structures resembling those in the second type.
The size of the ducts tends to be larger in infants younger than 12 weeks of age, and beyond this age, total
obliteration of ducts is the common finding. Tan et al. (e687) noted that few or absent ductal remnants at the
porta hepatis and absence of portal inflammation were predictors of poor prognosis. However, this finding has
not been confirmed in other clinical studies. Age at operation (improved outcome at <60 days of age), the
surgical team's experience, and the degree of liver disease are factors associated with prognosis.
Persistent Intrahepatic Cholestasis

Once the presence of a normal biliary tract has been established through a variety of studies and procedures,
the differential diagnosis of persistent conjugated hyperbilirubinemia shifts in the direction of inherited and
infectious etiologies. The inherited disorders include those conditions of a primary nature affecting the structure
of intrahepatic bile ducts or bile secretion with secondary effects on the intrahepatic ducts. The first category is
represented primarily by the Alagille and Byler (progressive familial intrahepatic cholestasis [PFIC]) syndromes,
and the second by a diverse group of infectious, metabolic, and inherited disorders.
Alagille Syndrome (Syndromic Paucity of Interlobular Bile Ducts, Arteriohepatic Dysplasia)

Alagille syndrome is an autosomal dominant disorder associated with abnormalities of the liver, heart, eye,
skeleton, and a characteristic facial appearance (Table 15-4) (82) (e60,e320,e344,e360,e532,e584,e730). The
genetic defect for this syndrome is the JAG1 gene locus on chromosome 20p12. JAG1 encodes a ligand for the
Notch signaling
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pathway that is important in early cellular development, particularly in the liver, kidney, and heart. Alagille
syndrome is the most frequent condition associated with paucity of intrahepatic bile ducts and has been referred
to as syndromic paucity of interlobular bile ducts. The onset of cholestasis occurs in the first 3 months of life with
unconjugated hyperbilirubinemia and an obstructive pattern on laboratory evaluation and hepatobiliary
scintigraphy. Cutaneous manifestations occur later in the course and include pruritus (hyperbilirubinemia) and
xanthomas (hypercholesterolemia). The typical facies includes a prominent forehead, hypertelorism, flattened
malar eminence, and a pointed chin, although the specificity of the abnormal facies has been questioned.
Characteristic eye findings include a posterior embryotoxon. The cardiovascular anomaly most often reported is
pulmonic stenosis with a heart murmur (95%). Vertebral abnormalities (butterfly vertebrae, 60% to 70%) and
foreshortened fingers are skeletal anomalies associated with the syndrome. Renal abnormalities leading to renal
failure include interstitial nephritis and membranoproliferative glomerulonephritis with mesangial lipid deposits.
Unilateral renal cystic dysplasia, renal hypoplasia, ureteropelvic obstruction, and renal artery stenosis may also
be seen (e427). Other features include mental retardation, stunted growth, cerebrovascular accidents (15%),
pancreatic insufficiency, moyamoya, and middle aortic syndrome. Incomplete forms of the syndrome have been
described in which only some of the major features are present. The mortality rate is 17% to 28%, which is
largely determined by the presence of cardiovascular disease or progressive liver disease (e195).
Table 15-4 ▪FAMILIAL CHOLESTATIC SYNDROMES
Age at Associated
Syndrome Onset Anomalies Inheritance Outcome Pathologic Features
Alagille <3 Facies, Autosomal Mild Paucity of ducts,

months heart, dominant disease,cirrhosis cholestasis, giant
eye,bone, in 12%-14% cells; pigment in Golgi,
kidneys ER, and lysosomes
Byler 3-12 None Autosomal Fatal in Cholestasis, giant

months recessive childhood cells, fibrosis
Norwegian <3 Lymphedema Autosomal Mild disease Cholestasis, portal

months recessive fibrosis
Benign, 1-15 None Unknown No disease Cholestasis

recurrent years
North <3 None Autosomal Fatal cirrhosis Cholestasis, giant

American months recessive cells, actin filament
Indian hyperplasia
ER, endoplasmic reticulum.
Liver disease is noted in almost 95% of cases within the 1st year of life, with progression to cirrhosis. HCC is an
infrequent complication (e553). Transplantation has been performed in approximately 50% of patients in some
series, with approximately a 75% survival rate (e103,e552).
The characteristic histopathologic feature of Alagille syndrome is absence or paucity of interlobular bile ducts
(Figure 15-10). Because normal numbers of bile ducts may be present in early biopsies and even ductal
proliferation, it is assumed that the syndrome is characterized by progressive damage and subsequent loss of
intrahepatic ducts, as noted in liver biopsies from older children (e714). Loss of ducts through atrophy secondary
to decreased bile flow is an alternative explanation for the paucity of bile ducts. An optimal diagnostic liver biopsy
should contain 20 portal areas, which may require a wedge biopsy, but a needle biopsy containing at least six
portal areas may be adequate. Portal triads may be diminished in size and number and show no or mild fibrosis.
Cholestasis is usually present in zone 3, but may be seen in zone 1. Hepatocellular ballooning, pseudoacinar
transformation, focal giant cell formation, and lobular disarray are other nonspecific features. A quantitative
increase in hepatic copper may occur and is demonstrable by rhodamine or other copper stains in zone 1
hepatocytes, a finding also common in other obstructive or cholestatic hepatopathies. Ultrastructural changes
are distinctive with bile pigment retention in the cytoplasm, especially in lysosomes and in vesicles in the outer
convex region of the Golgi apparatus. Rarely, bile pigment is present in the bile canaliculi or immediate
pericanalicular region, suggesting a block in the bile secretory apparatus (e730).
Progressive Familial Intrahepatic Cholestasis (Byler Disease and Byler Syndrome)

PFIC is a group of severe genetic cholestatic hepatopathies of early life, including the archetypical PFIC1 (Byler
disease) first described in Amish children. This autosomal recessive disorder is heralded by infantile cholestasis,
which leads to hepatic fibrosis and death (90) (e18,e160,e243,e275,e352). Children who have a clinically similar
disorder, but are not members of the Amish kindred in which Byler disease was described, are said to have Byler
syndrome. The gene for Byler disease is at 18q21 locus of the ATP8P1 gene, which synthesizes an
aminophospholipid translocating ATPase on the bile duct epithelium. This same gene mutation is
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implicated in benign recurrent intrahepatic cholestasis (BRIC), which is associated with recurrent cholestasis with
pruritus. PFIC types 1 (ATP8B1 gene mutation at 18q21) and 2 (ABCB11 gene mutation at 2q24) (e311,e665)
are characterized by cholestasis and low serum gamma-glutamyltransferase (GGT) activity. With PFIC type 3,
serum GGT is elevated and is associated with mutation of the ABCB4 gene (7q21). This gene encodes the
canalicular protein
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MDR3 responsible for translocation phospholipids from hepatocytes to canalicular lumens. Intrahepatic
cholestasis of pregnancy occurs in heterozygotes with an ABCB4 gene mutation and is associated with elevated
aminotransferases, cholestasis with pruritus, and recurrent fetal losses.
FIGURE 15-10 ▪ A-D: Alagille syndrome (paucity of interlobular bile ducts). Absence of bile duct within the portal
tracts and presence of proliferating cholangioles at the periphery of the liver lobules as identified with cytokeratin
7 immunostaining (H&E staining, A,C: 20×; Cytokeratin 7 immunostaining, B,D: 20×). E: Micronodular cirrhosis
with bile plugs and diffuse bile staining.
Histologically, PFIC type 1 exhibits giant cell transformation and paucity of bile ducts, which progresses through
a spidery fibrosis beginning in zone 3 and extending to zone 1, eventually leading to cirrhosis (Figure 15-11).
The bile has a coarse granular appearance on electron microscopic examination. In contrast, non-Amish children
have neonatal hepatitis, amorphous to finely filamentous bile, and a more benign course, but with recurrent
cholestasis (33). PFIC type 2 is characterized by persistent neonatal cholestasis with features of neonatal
hepatitis and later biliary cirrhosis. PFIC type 3 displays periportal inflammation, extensive bile duct proliferation,
feathery hepatocyte degeneration, and fibrosis, which progresses to biliary cirrhosis (e664). Partial external
biliary diversion and transplantation have been helpful in 80% of patients (e307). Liver biopsies in Amish and
Mennonite children with familial hypercholesterolemia have bland intracanalicular cholestasis and low GGT and
improve with ursodeoxycholic acid treatment. The genetic defects in these children are associated with aberrant
tight junction proteins (claudin, TJP2 gene) and a defective bile acid conjugation enzyme (gene BAAT).
FIGURE 15-11 ▪ A-B: Progressive familial intrahepatic cholestasis (PFIC). Hepatic lobular disarray with giant cell
transformation and focal canalicular cholestasis (H&E, A: 100×, B: 400×). C: Diffuse cytoplasmic cholestasis of
hepatocytes with granular bile (H&E, C 400×). D: Central lobular fibrosis with fine feathery extension into the
peripheral zone toward the portal region (Trichrome, D: 100×).
FIGURE 15-11 ▪ (continued) E: Micronodular cirrhosis with portal to portal bridging fibrosis and loss of central
veins (H&E, E, 100× G; Gross appearance). F: Electron microscopic appearance of coarse granular bile
markedly distending a canalicular space between hepatocytes (Electron microscopy, F: 20,000×)
Other conditions may also present initially with cholestasis and end in cirrhosis. A disease that presents with
neonatal cholestasis and may mimic EHBA is North American Indian cirrhosis. This disease has progressive
fibrosis and usually culminates in cirrhosis early in life. The genetic defect has
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been localized to a mitochondrial protein gene, CIRHIN. A syndrome that is comprised of arthrogryposis, renal
tubular dysfunction, and cholestasis (ARC) may present initially as cholestasis with a low GGT, and is typically
fatal in the first few years of life.
Nonsyndromic Paucity of Intrahepatic Ducts

Paucity of intrahepatic bile ducts have been reported in several sporadic cases of neonatal cholestasis with
progressive liver disease, but rarely does the condition evolve into cirrhosis. Alpha-1-antitrypsin deficiency has
been associated with paucity of intrahepatic bile ducts in a subgroup of patients. Other conditions include
congenital syphilis, Turner syndrome, Down syndrome, cytomegaloviral infection, hepatitis B antigenemia,
hypopituitarism, medications, infections, toxins, immune-mediated injury, and graft-versus-host disease (79, 107,
111) (e664,e672). Ultrastructural evidence of bile duct destruction in nonsyndromic paucity of bile ducts has
been regarded as representing a primary ductal injury (79).
Recurrent Intrahepatic Cholestasis

Benign Recurrent Intrahepatic Cholestasis
BRIC and PFIC type 1 share the same locus on 18q21 (108) (e91,e92,e364). The gene PFIC1 has been
identified in both groups of patients, but the relationship between these two entities is unclear (91). BRIC is
characterized by recurrent episodes of cholestasis without permanent liver damage.
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Hereditary Cholestasis with Lymphedema (Aagenaes Syndrome)
Hereditary intrahepatic cholestasis with lymphedema (Aagenaes syndrome) is an autosomal recessive, inherited
syndrome with more than 75% of the cases occurring in Norwegians, and is associated with a genetic defect on
chromosome 15q (2, 184) (e225). Cholestasis with high serum GGT is present before or shortly after birth. With
modern treatment, the cholestasis usually improves considerably during the first 2 years of life, but periods of
recurrent cholestasis occur later. In some cases, lymphedema is present at birth, but this usually comes to light
during childhood. The prognosis for the liver disease is good, but cirrhosis develops in about 15% of Norwegian
cases (e2).
Anatomic Anomalies and Disorders of Biliary and Hepatic Ducts

Agenesis of the Common Bile or Hepatic Duct
Agenesis of the common bile duct or hepatic duct is extremely rare. With common duct atresia, the hepatic duct
enters directly into the gallbladder, and a long cystic duct drains into the duodenum (e426).
Congenital Bronchobiliary Fistula

Congenital bronchobiliary fistula (CBBF) is a rare anomaly with varied presentations, including aspiration
pneumonia and atelectasis, and may be associated with common bile duct abnormalities, including biliary atresia
and diaphragmatic hernia (e110,e112,e179,e192,e261,e296). CBBF usually arises from the proximal part of the
right main bronchus, a short distance below the carina, and joins the biliary system at the level of the left hepatic
duct. The intrahepatic portion is usually lined by squamous or columnar epithelium, whereas the proximal section
resembles a bronchus with respiratory epithelial lining and cartilage plates in the wall (e192).
Ciliated Foregut Cyst

Ciliated hepatic foregut cyst is usually seen in adults, but may rarely present in childhood with abdominal pain or
portal hypertension (38, 165) (e626,e668,e740,e787). The cyst is thought to arise from the migration of a
bronchiolar bud of the foregut through the pleuroperitoneal canal. The cyst is subcapsular, measuring from 1 to 4
cm in diameter, and is composed of a lining of ciliated pseudostratified columnar epithelium overlying connective
tissue, a layer of smooth muscle bundles, and a fibrous capsule.
FIGURE 15-12 ▪ Classification of congenital bile duct cysts.
Congenital Dilatation of the Bile Ducts

Choledochal cyst is a presumed congenital anomaly of the intrahepatic and extrahepatic ducts characterized by
segmental ductal dilatation, bile stasis, and hyperbilirubinemia (167, 168 and 169) (e338,e460). An association
with malunion of the pancreatic and distal common bile ducts is a common finding. The prevalence of
choledochal cysts is 1:15,000 live births and is higher in Asian populations. There is a female predilection.
Secondary causes of bile duct dilatation include cholangitis, biliary perforation, biliary tract carcinoma, acute
pancreatitis, and biliary cirrhosis (e704). The cysts are classified (Figure 15-12) as:
Type Ia—large cystic or saccular dilatation of the choledochus

Type Ib—segmental dilatation with no pancreaticobiliary malunion
Type Ic—diffuse cylindrical or fusiform dilatation
Type II—diverticulum of the common bile duct or gallbladder
Type III—choledochocele of the distal common bile duct that usually extends into the wall
of the duodenum
Type IVA—multiple choledochal cysts with intrahepatic and extrahepatic involvement
(Caroli disease)
Type IVB—multiple extrahepatic cysts
Type V—single or multiple intrahepatic dilatations (may belong to Caroli disease—see
later)
Choledochal cysts present most often with nonspecific symptoms. In 40% of patients, most of whom are children,
a classic clinical triad of pain, jaundice, and right upper quadrant mass is seen. Irritability, nausea, vomiting, and
a palpable abdominal mass may also be present. Affected infants often have large choledochal cysts, presenting
as abdominal
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masses (e759). Associated atresia or stenosis of the biliary tree is often present, and has a greater risk for
cirrhosis in infants (e673). Diagnostic imaging studies, including isotope scan, ultrasonography, CT scan, and
endoscopic or percutaneous cholangiography are useful in establishing a preoperative diagnosis (e345,e377).
With some, prenatal ultrasound examination may identify dilatation of the bile ducts, suggesting choledochal cyst
or extrahepatic biliary obstruction.
Intrahepatic histopathology is similar to that seen with EHBA. There is bile ductular proliferation and periportal
fibrosis, which may progress to biliary cirrhosis. Regression of biliary cirrhosis has been documented after
drainage of the choledochal cyst. Total excision of the cyst is recommended to avoid ascending cholangitis,
choledocholithiasis, chronic liver disease, pancreatitis, and carcinoma of the bile ducts, liver, or pancreatic ducts
that may be associated with internal drainage alone (e673). The excised cyst wall is usually 1 to 2 mm thick and
bile stained (Figure 15-13). It consists of dense fibrous tissue containing a few to no inflammatory cells. Only a
few smooth muscle fibers may be identifiable within the wall. An epithelium is generally lacking, but occasional
foci of residual columnar epithelium may be identified and some cysts may even have a complex epithelial
pattern.
FIGURE 15-13 ▪ Choledochal cyst. A: Choledochal cyst with portion of common bile duct. B: Choledochal cyst
with smooth glistening lining of cystic cavity. C, D: Lumen of choledochal cyst lacking an epithelial lining with the
wall formed by dense connective tissue with scattered chronic inflammatory cells and no residual smooth muscle
(H&E, C: 20×, D: 40
Congenital polycystic dilatation of the larger intrahepatic bile ducts is known as Carolidisease and has a marked
predisposition to cholangitis, liver abscess, and portal hypertension (109) (e28,e535,e627,e718,e784).
Carolidisease occurs most frequently in adults, but may be seen in children and
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infants if there are severe clinical symptoms (e335,e404). The diagnosis is based on cholangiographic findings
of polycystic segmental dilatation of the intrahepatic biliary tree, including multiple small saccular dilatations of
the peripheral segments of the intrahepatic biliary ductal system (e718). Histopathologically, a pattern of
dysplastic portal ducts and fibrosis resemble congenital hepatic fibrosis (CHF) in 90% of cases, even though
only 10% have clinical evidence of portal hypertension. The combination of intrahepatic bile duct cystic changes
and CHF has been termed Caroli syndrome. Medullary sponge kidney or renal tubular ectasia may be present in
approximately 50% of patients with Caroli disease (e718). Isolated hepatic polycystic liver disease may also
occur that histopathologically appears similar to autosomal dominant polycystic kidney disease (ADPKD). The
genetic defect lies at the 19p13.2 locus and is associated with mutation in the PRKCSH gene. This gene is
responsible for synthesis of hepatocystin, which modulates fibroblast growth factor receptor functions.
Predominantly, young adult females are affected.
Fibropolycystic Disease of the Liver Associated with Cystic Renal Disease

Ductal plate abnormalities of the liver and portal fibrosis may occur in the presence or absence of renal cysts in
several inherited syndromes (Table 15-5) (47, 49, 63) (e58,e276,e514,e721). These syndromes include
autosomal recessive polycystic kidney disease (ARPKD), ADPKD, hereditary renal dysplasia, and hereditary
tubulointerstitial nephritis. Morphometric studies show that the hepatic lesions in the infantile, juvenile, and adult
types of polycystic disease are identical initially as ductal plate malformations. Both liver and kidneys are
involved, and patients present with hepatomegaly and enlarged kidneys at birth or in early infancy (see Chapter
17).
Table 15-5 ▪CYSTIC LESIONS OF THE LIVER AND ASSOCIATED CONDITIONS
Infantile polycystic disease
Hereditary renal dysplasia
Meckel syndrome and congeners
Chondrodysplasia syndromes
Majewski and Saldino-Noonan short rib polydactyly syndromes
Jeune asphyxiating thoracic dystrophy syndrome
Ellis-van Creveld chondroectodermal dysplasia
Elejalde acrocephalopolydactylous dysplasia

Trisomy 9 syndrome
Trisomy 13 syndrome
Zellweger cerebrohepatorenal syndrome
Ivemark renal-hepatic-pancreatic dysplasia
Glutaric aciduria, type II
Hereditary tubulointerstitial nephritis
Juvenile nephronophthisis
Bardet-Biedl syndrome
Adult polycystic disease
Isolated nonsyndromatic disease
During the neonatal period, the liver has increased fibrous tissue in the portal areas and a striking increase in the
number of bile ducts (Figure 15-14). The bile ducts are dilated and have cleftlike or irregular contours. The
dilated bile ducts are most prominent at the periphery of the portal areas and may extend into the periportal
lobule. The ductal epithelium is bland and lacks epithelial degenerative changes or mitoses, in contrast to the
increased numbers of bile ducts associated with extrahepatic obstruction. Inflammation is usually not a feature.
Hepatocytes appear normal. Occasionally, cholestasis may be seen.
Congenital Hepatic Fibrosis

CHF presents in a seemingly healthy child or young adult with hematemesis from esophageal varices secondary
to portal
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hypertension. Cholangitis may also be seen on occasion. CHF has been noted in association with a variety of
renal lesions (Figure 15-14) (47, 49, 63) (e58,e276,e514,e721) including ARPKD (mutation of PKHD1-fibrocystin
at 6p21 locus), ADPKD (mutation of PKD1-polycystin-1 or mutated PKD2-polycystin-2), and rarely in Meckel-
Gruber syndrome (mutation of MKS1 at 17q, MKS2 at 1q, or MKS3 at 8q) and Jeune syndrome. Because Caroli
disease and choledochal cyst are associated with CHF in a small proportion of cases, a common pathogenesis is
worth consideration. CHF has also been seen in association with a variety of other syndromes including Joubert
syndrome (agenesis or hypoplasia of the cerebellar vermis, retinal dystrophy, chorioretinal colobomata,
oculomotor abnormalities, episodic hyperpnea, ataxia, mental retardation), Ivemark syndrome (renal, pancreatic,
hepatic dysplasia), Down syndrome, Laurence-Moon-Biedl syndrome (mental retardation, retinitis pigmentosa,
obesity), and COACH syndrome (hypoplasia of the cerebellar vermis, oligophrenia, congenital ataxia, coloboma,
hepatic fibrosis) (47, 49, 63) (e58,e276,e365,e514,e721).
FIGURE 15-14 ▪ Congenital hepatic fibrosis associated with autosomal recessive polycystic kidney disease. A,
B: Ductal plate abnormality with dilated and concentric arrangement of bile ducts in expanded and fibrotic portal
regions (H&E, A: 20×, B: 40×).
FIGURE 15-14 ▪ (continued) C: Autosomal recessive polycystic kidney disease with markedly enlarged kidney
due to numerous thin cystics extending from the cortical to medullary regions.
Desmet (49) suggested that CHF is caused by faulty development of the interlobular bile ducts with a
disturbance in epithelial-mesenchymal inductive interactions. As a result, the ducts are subject to progressive
destructive cholangiopathy of variable progression and duration that leads to biliary fibrosis. In addition, HCC
has been reported to arise in a case of CHF (e48).
There is nearly a 1:1 correlation between the frequency of liver and kidney disease in ARPKD, although the
degree of kidney involvement may vary considerably. The majority of ARPKD patients present in utero or shortly
after birth with abdominal masses, anuria, and oligohydramnios and frequently die within days. With the milder
(juvenile) ARPKD form in older children, the clinical picture may be dominated by cholestasis in the newborn
period or symptoms related to CHF (portal hypertension, bleeding esophageal varices). A number of diagnostic
imaging studies are available for the diagnosis of CHF.
The liver in ARPKD displays a gross pattern of interweaving white “streaks” beneath the capsule. The cut
surface may also show small cysts of a few millimeters in diameter. Microscopically, the portal areas contain
increased numbers of bile duct structures usually arranged in concentric rings around the portal area (Figure 15-
14). The anastomosing and branching ducts are associated with an increase in connective tissue, which is
minimal at first but expands to form broad fibrous bands over time. Unlike cirrhosis, the fibrosis does not have a
bridging appearance, and there are no regenerative nodules. However, there is the potential for the
misinterpretation of CHF for cirrhosis. The portal bile ducts in infants are lined by cuboidal to columnar
epithelium, which may form small polypoid projections. Pink or orange secretions are often present in bile duct
lumina.
Unlike in ARPKD, CHF is rare in ADPKD (e58). Hepatic involvement varies widely from one kindred to another,
with CHF reportedly causing death shortly after birth in one ADPKD family. Other ADPKD families have shown
little tendency for progression of the hepatic manifestations over long periods of clinical follow-up.
Several syndromes of inherited renal dysplasia are characteristically associated with hepatic changes that are
identical to CHF and carry the designation of biliary dysgenesis (e58,e59). These include Meckel-Gruber
syndrome, chondrodysplasia (short-rib polydactyly), Jeune asphyxiating thoracic dysplasia, trisomy 21, Bardet-
Biedl syndrome, Ivemark syndrome (renal-hepatic-pancreatic dysplasia), Zellweger cerebrohepatorenal
syndrome, and type II glutaric aciduria (e181,e509,e666). Central nervous system, ocular, and pancreatic
abnormalities are additional components of these syndromes. Compared with CHF, the differences in the hepatic
lesions in these syndromes are a matter of degree rather than type, with less severe fibrosis and bile duct
abnormalities being a general observation. The essential saclike structure of the biliary passages is similar, and
ductal dilatation resembling Caroli disease has been seen. Large intrahepatic cysts may be present. A similar
hepatic lesion has been described in some cases of vaginal atresia syndrome and tuberous sclerosis (e371).
Another condition that is associated with CHF is nephronophthisis (mutation in NPHP1 [nephrocystin], NPHP2
[inversin], NPHP3, or NPHP4). Ductal plate abnormalities in the liver and marked tubulointerstitial kidney disease
are the features of this familial condition. Progressive renal failure occurs during the first two decades of life.
METABOLIC DISORDERS
A wide variety of metabolic disorders involve the liver, in addition to other organs, and a number of these
disorders may present in the neonatal period as cholestasis and with
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neonatal hepatitis-like changes (Table 15-6). The overall incidence of metabolic disease is approximately 4 per
10,000 live births (e32). The following disorders are considered in some detail because of their association with
significant liver disease (see Chapter 5).
Table 15-6 ▪ DISTINCTIVE FEATURES IN METABOLIC DISORDERS WITH NEONATAL

HEPATITIS-LIKE CHANGES
Disorders Histologic Features
Galactosemia Moderate-to-severe fatty change, fibrosis, no other distinctive features
Fructosemia Moderate-to-severe fatty change, fibrosis, EM shows characteristic “fructose holes”
Tyrosinemia Moderate-to-severe fatty change and fibrosis; regenerative nodules and

hepatocellular dysplasia are suggestive
A1AT deficiency Stored A1AT in hepatocytes PAS-positive diastase-resistant globules;

immunohistochemistry and EM are diagnostic aids, especially in the neonate
Glycogenosis Early cirrhosis; stored structurally abnormal glycogen (diastase-resistant PAS-

(IV) positive material with characteristic fibrillar nonmembrane-bound dense material by
EM) in hepatocytes
CF “Focal biliary cirrhosis” manifests as proliferated bile ducts with intraluminal
(mucoviscidosis) inspissated material in expanded and fibrotic portal areas; rarely seen in the
newborn
Niemann-Pick Stored sphingolipids in Kupffer cells demonstrable histochemically; EM shows

disease characteristic pleomorphic lamellar inclusions in lysosomes
Idiopathic iron Hepatocellular necrosis with collapse; fibrosis; massive amounts of iron in
storage hepatocytes and duct epithelial cells with negligible amounts in Kupffer cells
EM, electron microscopy.
Carbohydrate Metabolism Disorders

Galactosemia
Hereditary galactosemia, an autosomal recessive disorder, is most commonly due to deficiency of galactose-1-
phosphate uridyl transferase, an enzyme encoded on the GALT gene on chromosome 9q13 (57)
(e76,e77,e391,e488,e701). Genetic defects in the enzymes galactokinase and uridyl diphosphate galactose-4-
epimerase are less common causes of galactosemia. These three enzymatic defects impair conversion of
galactose to glucose. The incidence varies from approximately 1.5 to 4 per 100,000 whites to 1 per 400,000 in
Chinese (e32,e39,e723,e751). The disorder exhibits considerable allelic heterogeneity, and more than 150
different mutations have been identified in 24 different populations and ethnic groups in 15 countries. The
mutations most frequently cited are Q188R, K285N, S135L, and N314D. Q188R is the most common mutation in
European populations or in those predominantly of European descent (e723). The clinical features in neonates
includes hepatomegaly, jaundice, hypoglycemia, generalized aminoaciduria, presence of reducing substances in
the urine, diarrhea, and vomiting. The differential diagnosis includes inborn errors of metabolism that manifest as
neonatal hepatitis (Table 15-6). Histopathologically, canalicular and intracellular cholestasis, pseudoacinar
transformation, bile ductular proliferation, focal giant cell transformation of hepatocytes, and presence of lipid are
the notable features (Figure 15-15). These features in the
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neonatal period are similar to those seen in tyrosinemia and fructosemia. Fibrosis occurs early and progresses to
cirrhosis if left untreated within the first 3 to 6 months of life (e765a). The ultrastructural features are diagnostic,
although the individual features are not specific. The diagnosis is made by demonstration of enzyme deficiency
in erythrocytes. Neonatal screening tests are available. Liver effects may be reversible with dietary galactose
restriction.
FIGURE 15-15 ▪ Galactosemia. A, B: Hepatocytes display medium to large droplet fatty metamorphosis, along
with pseudoglandular transformation (H&E, A: 100×, B: 200×).
Table 15-7 ▪HEPATIC FINDINGS IN GLYCOGEN STORAGE DISEASE
Disease Light Microscopic Findings Electron Microscopic Findings
Type I von Excess glycogen in enlarged hepatocytes and Accumulation of cytoplasmic

Gierke in nuclei; uniform mosaic pattern; lipid glycogen and lipid; nuclear glycogen
disease droplets
Type II Nonuniform, mild distension, and vacuolation Lysosomal monoparticulate glycogen

Pompe of hepatocytes
disease
Type III Cori Uniform mosaic pattern, resembling type I; Similar to type I; lipid and nuclear
glycogenosis portal fibrosis glycogen less pronounced
Type IV Inclusions in periportal hepatocytes; cirrhosis Nonmembrane-bound inclusions of

Andersen fibrillary material, glycogen, and
disease tubules
Type VI Her Nonuniform enlargement of Glycogen and finely granular

disease hepatocytes;periportal fibrosis material in cytoplasm
Type VIII Glycogen deposition Glycogenosis
Type IX Glycogen deposition Glycogen deposition with “starry-

sky” pattern
Type X Glycogenosis Glycogenosis

Fructosemia
Hereditary fructose intolerance is caused by catalytic deficiency of aldolase B (fructose-1,6-bisphosphate
aldolase) in the liver, intestines, and kidneys, and is a recessively inherited condition (193) (e16). Aldolase B
deficiency inhibits gluconeogenesis and glycogenolysis. Two mutations on chromosome 9q (A149P and A174D)
account for more than 70% of cases (193) (e372). The disease becomes manifest when fructose is introduced
into the diet and presents with vomiting, diarrhea, failure to thrive, jaundice, renal failure, and hepatomegaly.
Liver failure may be severe, with hepatic necrosis during the acute phase (e145). More commonly, there is
variable fibrosis involving the portal and lobular regions, as well as microsteatosis and macrosteatosis. Chronic
disease may be associated with severe fibrosis, potentially leading to cirrhosis. A neonatal hepatitis-like pattern
may be seen with diffuse hepatocellular steatosis, a frequent feature. Electron microscopy of hepatocytes shows
endoplasmic reticulum degranulation with membrane profiles. The response to dietary exclusion of fructose is
rapid, and when so treated, the disease is compatible with a normal life span (e145).
Glycogen Storage Diseases

The GSDs are a group of metabolic disorders with specific enzyme defects resulting in accumulation of abnormal
amounts of structurally normal or abnormal glycogen in the liver and other tissues (136, 176)
(e328,e632,e732,e770). The GSDs most frequently associated with hepatic manifestations include types I, II, III,
IV, VI, and IX. The mode of inheritance is autosomal recessive in all the types described, with the exception of
type IXb, which is inherited as a sex-linked recessive trait. Some forms of the disease present in infancy and
others in early childhood, with failure to thrive, developmental delays, acidosis, hypoglycemia, and
hepatomegaly. The morphologic changes have been reviewed by McAdams et al. (119), and the ultrastructural
features have been illustrated by Phillips et al. (141). Features of those glycogenoses that significantly involve
the liver are summarized in Table 15-7.
Type I Glycogenosis (von Gierke Disease)

GSD type I is the most common form of glycogenosis and potentially the most severe (77, 136, 176)
(e129,e328,e459, e632,e732,e770). There is no gender predilection. Children with this disease present in
infancy with hypoglycemia and hepatomegaly. There is lactic acidosis, seizures, and failure to thrive. The
subsequent course is characterized by the development of hyperlipidemia, xanthomata, hyperuricemia, cyclic
neutropenia with recurrent infections, nephropathy, and chronic bowel inflammation with type 1b GSD. Type 1a
GSD is due to a deficiency in the enzyme glucose-6-phosphatase (17q21). Type 1b GSD has a deficiency in a
transmembrane protein required for glucose-6-phosphate transport (11q23) into microsomes. Type 1c has a
deficiency in a phosphatase transporter (11q23-24.2). These deficiencies result in the accumulation of large
amounts of normal glycogen in the liver, kidney, and intestine.
Histopathologically, the liver shows marked distension of the hepatocytes with glycogen, resulting in a diffuse
mosaic pattern with compression of the sinusoids (Figure 15-16). Intranuclear glycogen is a common feature (77,
119). The glycogen is best demonstrated by periodic acid-Schiff (PAS) stains of unfixed frozen sections or
alcohol-fixed tissue. Lipid is also present, while fibrosis is typically absent. Rarely, Mallory bodies may be seen
(e309). Ultrastructurally, there are pools of monoparticulate glycogen in the cytoplasm and nuclei of hepatocytes.
Lipid vacuoles are found in the cytoplasm. The organelles are displaced, and the size of the mitochondria may
be increased. Hepatic adenomas have
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been reported with some frequency, and cases of HCC and hepatoblastoma have been described in type 1
glycogenosis (e67,e309,e318,e471). Hepatic transplantation has been used in the treatment of types I, III and IV
GSD (e432).
FIGURE 15-16 ▪ Glycogen storage disease, Type I. A: Hepatocytes distended by glycogen with obliteration of
the sinusoids, forming a mosaic pattern, and glycogenated nuclei. (H&E, 400×). B: Hepatocyte with
glycogenated nuclei, cytoplasmic monoparticulate glycogen, large lipid droplet, and abnormally shaped
mitochondria (Electron microscopy, 7,500×).
Type II Glycogenosis (Pompe Disease, Generalized Glycogenosis, or Acid Maltase Deficiency)

GSD type II is classified as a lysosomal storage disorder in contrast to the cytoplasmic storage disorder that
occurs in the other GSDs. It is the result of a deficiency in acid maltase caused by mutations in the alpha-1,4-
glucosidase (GAA, 17q25.2-25.4) gene (77, 136, 176) (e177,e328,e355, e459,e632,e732,e770). The major
manifestations are muscular and cardiac, and the liver shows changes as a component of the generalized
involvement. Three clinical types have been described (e177,e438,e459,e732). Infantile or classic Pompe
disease manifests in infancy with hypotonia and cardiomyopathy, leading to death in infancy. The second type
presents in childhood with predominant involvement of the skeletal musculature, and a third type is described
with an onset in the second to fourth decades. Cardiac involvement in these latter variants is minimal. Affected
hepatocytes are mildly enlarged and have finely vacuolated cytoplasm (Figure 15-17). Ultrastructural features
are characterized by the presence of monoparticulate glycogen within membrane-bound lysosomal vacuoles.
Acid phosphatase activity is associated with the lysosomal vacuoles.
FIGURE 15-17 ▪ Glycogen storage disease, type II. A: Hepatocytes demonstrating mosaic pattern with
obliteration of sinusoids (H&E, 40×). B: Hepatocytes in close proximity to one another, with thickened cell
membranes, fine cytoplasmic vacuolization, and indistinct sinusoids (H&E, 400×).
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FIGURE 15-17 ▪ (continued) C: Monoparticulate glycogen within membrane bound lysosomes (Electron
microscopy, 20,000×).
Type III Glycogenosis (Cori Disease, Forbes Disease, Limit Dextrinosis, Debranching Enzyme Disease)
GSD type III is the result of a deficiency in the amylo-1, 6-glucosidase (debrancher enzyme, 1p21) activity (77,
136, 176) (e328,e459,e632,e732,e770). This deficiency leads to abnormal glycogen formation with increased
branching points that accumulate in the liver and muscle. Hypoglycemia develops during stress or fasting due to
lack of conversion of the abnormal glycogen to glucose. Hepatic morphologic features are very similar to those
seen in type I GSD with panlobular cytoplasmic distension by glycogen and a uniform mosaic pattern.
Accumulated glycogen is demonstrable by the presence of diastase-digestible PAS-positive material in the
cytoplasm. Nuclear glycogen is not as prominent as in type I GSD, but is a distinguishing feature from other
types of GSD, especially types VI and IX (77). Hepatomegaly with hepatic fibrosis may be prominent and may
progress to cirrhosis by the third or fourth decade of life.
Type IV Glycogenosis (Andersen disease, Amylopectinosis; Glycogen Branching Enzyme Disease)

GSD type IV manifests at birth or in early infancy with failure to thrive and hepatosplenomegaly (77, 136, 176)
(e323,e632,e732,e770). In the absence of transplantation, there is progression to cirrhosis and death in early
childhood (e422). The brancher enzyme 1,4-1,6-glucon:1-4-glucan, 6-glycosyl transferase (3p12) is absent,
resulting in abnormal glycogen with decreased branch points that resembles amylopectin or starch. Deposits of
the abnormal glycogen are generalized with significant involvement of the liver, skeletal and cardiac muscles,
and intestine. Microscopically, the hepatocytes in the periportal region (zone 1) contain pale eosinophilic hyaline
inclusions surrounded by a clear halo, resembling Lafora bodies. These inclusions resist diastase digestion, but
are digestible with pectinase treatment (Figure 15-18). Colloidal iron staining is also seen in the cytoplasmic
inclusions. Ultrastructurally, the inclusions consist of central fibrillar glycogen surrounded by polyparticulate
glycogen rosettes (e109). Prenatal diagnosis is possible (e628).
Type VI Glycogenosis (Hers disease)

GSD type VI, a deficiency in hepatic phosphorylase E activity (14q21-22), presents with hepatomegaly in the
absence of the serious complications seen in other glycogenoses (77, 136, 176)
(e115,e328,e459,e632,e732,e770). Histopathologically, there is a mosaic pattern of hepatocellular distension
with glycogen in zone 1 hepatocytes. Mild portal fibrosis may be seen. Ultrastructurally, pools of monoparticulate
glycogen with interspersed glycogen rosettes displace the cytoplasmic organelles. A finely granular material of
low electron density that is devoid of organelles may be scattered in the glycogen aggregates, imparting a starry-
sky appearance (77).
Type VIII Glycogenosis

GSD type VIII, a deficiency in phosphorylase kinase (16q12-13), is accompanied by progressive neurologic
deterioration leading to death in early childhood secondary to glycogen accumulation within the central nervous
system (136, 176) (e328,e459,e632,e732,e770). Hepatic changes are those of glycogen accumulation with
nonspecific features, although a rare case of cirrhosis and adenomatous hyperplasia has been reported (e633).
Type IX, X, and XI Glycogenoses

Other glycogenoses with hepatic manifestations are GSD types IX (phosphorylase b kinase deficiency, Xp22.2-
22.1) and X (cyclic 3,5 AMP-dependent kinase deficiency, 17q23-24) (136, 176) (e328,e459,e632,e732,e770).
Hepatic glycogenosis with stunted growth (type XI, Fanconi-Bickel syndrome) is associated with renal glycogen
deposition. Type XI glycogenosis is caused by mutations in the glucose transporter 2 (GLUT2, 3q26.1-26.3)
gene (136, 176) (e328,e459,e632,e732,e770). Generalized glycogen deposition is accompanied by cirrhosis, but
with normal glycogen metabolism.
Other Glycogenoses
Hepatic involvement is not a feature of GSD types V (McArdle disease, muscle glycogen phosphorylase
[myophosphorylase], 11q13) and VII (Tarui disease, phosphofructokinase enzyme deficiency, 12q13), in which
skeletal muscle is primarily affected (136, 176) (e29,e328,e470,e632,e710, e732,e770). GSD type 0
(aglycogenosis) is an autosomal recessive disease with a deficiency in glycogen synthase (chromosome
12p12.2) (136, 176) (e328,e459,e632,e732, e770). Deficiency in glycogen synthase leads to a marked
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reduction in liver glycogen stores. The symptoms of GSD type 0 are those associated with hypoglycemia and
include lethargy, pallor, nausea, vomiting and, rarely, seizures in the early morning before breakfast. Liver biopsy
will demonstrate moderate steatosis and small amounts of glycogen (0.5% versus 1.6% for normal wet liver
weight) on quantitative analysis. There have also been reports of liver fibrosis in some GSD type 0 cases.
FIGURE 15-18 ▪ A: Glycogen storage disease, type I V. Hepatocytes with pale hyaline inclusions surrounded by
indistinct halos, resembling LaFora bodies (H&E, 200×). B, C: The inclusions stain intensely with PAS (400×).
and colloidal iron (400×). D: Hepatocyte with hyaline inclusion comprised of fibrillary glycogen (electron
Amino Acid Metabolism Disorders

Tyrosinemia
Tyrosinemia results from a deficiency of fumaryl acetoacetate hydrolase (FAH, 15q23-25) and presents as acute
fulminant disease in infancy or as a chronic liver disease later in childhood (157, 161) (e198,e257,e536).
Diagnosis is based upon serologic or urinary determination of succinylacetone level and FAH assays. Liver
biopsy in the acute form reveals cholestasis, pseudoacinar transformation of hepatocytes, fatty change, marked
intralobular fibrosis, and variable giant cell transformation (Figure 15-19) (78). These features are indicative of a
metabolic hepatopathy, but are not specific for tyrosinemia. Regenerative nodules may already be present in
early liver biopsies. The chronic form is characterized by cirrhosis with variable-sized nodules separated by thick
bands of fibrous connective tissue with little inflammation or bile duct proliferation. Hepatocytes may demonstrate
nuclear hyperchromasia, dysplasia, or adenomatous hyperplasia. The incidence of HCC is quite high with
tyrosinemia (e544). Liver transplantation is advisable soon after diagnosis and before 2 years of age, because of
the high risk of HCC. Treatment with NTBC [2(-nitro-4-trifluoromethylbenzoyl)-1-3-cyclohexanedione] prevents
accumulation of the toxic metabolites of FAH and the
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subsequent liver and neurologic effects, but does not entirely eliminate the risk of HCC.
FIGURE 15-19 ▪ Tyrosinemia. A, B: Hepatocytes with macrovesicular steatosis and indistinct sinusoid spaces
(H&E, A: 200×, B: 400×). C: Micronodular cirrhosis in chronic form of tyrosinemia (H&E, 40×).
Lysosomal Storage Diseases

Lipidoses
Wolman Disease
Wolman disease and cholesterol ester storage disease (CESD) are rare autosomal recessive lipoprotein-
processing disorders caused by mutations in the gene encoding human lysosomal acid lipase (10q24-25; Table
15-8) (e185,e281,e399,e724, e727,e771). Wolman disease is fatal in early life, presents with failure to thrive and
diarrhea, and is characterized by generalized accumulation of foam cells and adrenal calcifications. Because
there is partial enzyme activity, CESD is a milder clinical form of the disorder, generally limited to the
gastrointestinal tract and the liver. Liver pathology includes steatosis and numerous foamy macrophages that
contain cholesterol and lipid (Figure 15-20) and are similar in both diseases, although cirrhosis may occur in
CESD. Cholesterol accumulation is demonstrated with frozen sections using polarized light microscopy.
Ultrastructurally, hepatocytes, Kupffer cells, and portal macrophages are engorged with membrane-bound lipid
vacuoles with dense membranes. Cholesterol clefts are seen in the cytoplasm.
Mucolipidoses
The mucolipidoses are a group of disorders caused by defects of various lysosomal hydrolases and include
sialidosis (ML I, neuraminidase gene at 6p21.3), I-cell disease (ML II, GNPTAB gene at 12q23.3), pseudo-Hurler
disease (ML III, GNPTAB gene at 12q23.3), and sialolipidosis (ML IV, mucolipin-1 gene at 19p13.3) (60, 116)
(e37,e178,e548,e550,e667). Many of the clinical stigmata of mucopolysaccharidoses may be seen, but
mucopolysaccharides are not excreted in the urine. I-cell disease and pseudo-Hurler polydystrophy are
autosomal recessive disorders. Coarse facies, skeletal changes, hepatosplenomegaly, and delayed growth and
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development are some of the clinical features. The primary histopathologic and ultrastructural changes are
cytoplasmic vacuolization of hepatocytes, Kupffer cells, and less frequently, biliary epithelial cells. Inclusions
within clear vacuoles can be demonstrated within fibroblasts and peripheral nerves in skin and conjunctival
biopsies (141). Glomeruli and renal tubular epithelium contain similar inclusions, and the inclusions are also
present in the urine.
Table 15-8 ▪LYSOSOMAL DISORDERS
Light Electron
Enzyme Microscopic Microscopic
Disease Deficiency Findings Findings
Gaucher Glucocerebrosidase Gaucher cells Membrane-bound

with striated inclusions with
cytoplasm; twisted tubules in
fibrosis Kupffer cells
Niemann-Pick Sphingomyelinase Finely vacuolated Myelin figures in

cytoplasm of Kupffer cells and
Kupffer cells hepatocytes
Wolman and cholesterol ester Acid lipase Steatosis of Lipid droplets and
storage hepatocytes and lipolysosomes and
Kupffer cells; cholesterol clefts in
cholesterol clefts, hepatocytes and
fibrosis histiocytes
Mucopolysaccharidoses, Hurler, Iduronidases Swollen clear Membrane-bound,

Hunter, Scheie sulfatases cytoplasm of sharply delimited,
hepatocytes and electron-lucent
Kupffer cells; inclusions with some
fibrosis; cirrhosis granular material
Mucolipidoses, I-cell disease Acid hydrolases Vacuolated Membrane-bound

hepatocytes, vacuoles with
Kupffer cells flocculent material
Oligosaccharidoses Sialidase, Vacuolated Membrane-bound

mannosidase, hepatocytes and vacuoles with finely
fucosidase Kupffer cells granular material
Metachromatic leukodystrophy Aryl sulfatase A Metachromatic Lamellar prismatic

granules in portal inclusions within
macrophages macrophages,
hepatocytes, and
Kupffer cells
Farber Acid ceramidase β-galactosidase Vacuolated Membrane-bound

Lipogranulomatous infiltrates hepatocytes and vacuoles with
Curvilinear lysosomal material Kupffer cells granular material
Gangliosidosis GM1
Oligosaccharidoses (Glycoproteinoses)
Disorders of glycoprotein degradation resulting from defects in specific lysosomal enzymes lead to the
accumulation of oligosaccharides in tissues and urinary excretion of these substances. These are rare
autosomal recessive conditions with a phenotypic similarity to the mucopolysaccharidoses (e359,e522,e541).
These disorders include sialidosis (neuraminidase gene at 6p21.3), mannosidosis (mannosidase 2B1 gene at
19cen-q12), fucosidosis (FUCA1 gene at 1p34), and aspartylglycosaminuria (aspartylglucosaminidase gene at
4q32-33). The liver is involved in all forms and has enlarged vacuolated hepatocytes. Ultrastructurally, the foamy
appearance is due to cytoplasmic membrane-bound clear vacuoles (141). The vacuoles are of variable sizes,
may be molded by adjacent vacuoles, and fuse to form larger vacuoles. They are composed of finely granular to
flocculent material intermingled with membrane material. Kupffer cells, biliary epithelial cells, and endothelial cells
show similar vacuoles.
FIGURE 15-20 ▪ A, B: Cholesterol ester storage disease. Hepatocytes with diffuse microsteatosis (H&E, A
200×, B 400×).
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FIGURE 15-21 ▪ Metachromatic leukodystrophy. A: Gallbladder with markedly thickened mucosa with fine
cobblestone to papillary surface. B, C: Papillary fronds lined by columnar epithelial cells with amphophilic
cytoplasm (H&E, B: 100×, C: 200×). D: Lysosomal inclusions with closely packed herringbone appearance
(Electron microscopy, 25,000×).
Metachromatic Leukodystrophy
Metachromatic leukodystrophy is an autosomal recessive condition caused by a deficiency in lysosomal aryl
sulfatase activity (arylsulfatase A gene at 22q13.31-qter) (59) (e54,e68,e238,e239,e314,e428). This results in
accumulation of galactosyl sulfatide in the tissues and excessive urinary excretion of the metachromatic material
(e314). Demyelination occurs with excess storage of the substrate in the central and peripheral nervous system
(59) (e428). The storage material is metachromatic and shows brown granules with a characteristic birefringence
in cresyl-violet-stained, unfixed frozen sections. By light microscopy, foam cells are seen in the nervous system,
liver, kidneys, pancreas, adrenal cortex, and gallbladder. The gallbladder may show papillary fronds lined by
epithelial cells and with foam cells in the subepithelial stroma (Figure 15-21) (5). Ultrastructurally, the lysosomal
inclusions consist of prismatic structures with closely packed periodic leaflets that display a herringbone pattern.
In the liver, the inclusions are found in portal macrophages, fibroblasts, and Kupffer cells.
Farber Disease (Farber Lipogranulomatosis)
Farber disease is an autosomal recessive condition in which ceramide, a sphingolipid, accumulates in the tissues
due to a deficiency of the lysosomal enzyme acid ceramidase (N-acylsphingosine amidohydrolase gene at 8p22-
21.3) (e227,e392,e516,e735). Disseminated lipogranulomata are the morphologic findings. The liver is mildly
affected, with clear
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vacuoles in the hepatocytes similar to the membrane-bound vacuoles in mucopolysaccharidoses. The Kupffer
cells and portal macrophages have lysosomal comma-shaped, bananashaped, and curvilinear inclusions in
common with other tissues. Death occurs in adolescence or early adulthood.
FIGURE 15-22 ▪ Fabry disease. Membrane-bound lysosomal inclusions with lamellar and concentric pattern
(Electron microscopy, 12,000×).
Fabry Disease
Fabry disease is an X-linked recessive disorder caused by mutations in the alpha-galactosidase A gene (GLA
gene, Xp22) and results in globotriaosylceramide accumulation in the liver and other organs (70)
(e134,e194,e292,e786). Endothelial cells are the most commonly affected cell type. Ultrastructural findings are
characterized by pleomorphic, membrane-bound, osmiophilic lamellar and concentric inclusions (Figure 15-22).
Gangliosidoses
The gangliosidoses are a group of autosomal recessive disorders with impairment of ganglioside metabolism (28,
59, 110) (e207,e288). GM1 and GM2 gangliosidoses have several clinical variants in each group. Five types of
GM1 gangliosidosis have been described. The infantile type presents in infancy with coarse facies, skeletal
abnormalities, retinal cherry-red spot, hepatosplenomegaly, and progressive deterioration (beta-galactosidase-1
at 3p21.33). Lysosomal beta-galactosidase is deficient, and the substrate accumulates in the brain and the
viscera. Hepatocytes and Kupffer cells are foamy and vacuolated. Ultrastructurally, the cells are distended with
large lysosomes that appear as electron lucent vacuoles filled with reticular granular (141). Lamellar, concentric,
membrane-bound bodies may also be seen. GM2 gangliosidosis is a group of heterogeneous disorders that
includes Tay-Sachs disease (hexosaminidase A gene at 15q23-24) with a hexosaminidase A deficiency, and
Sandhoff disease with hexosaminidase A and B deficiencies (beta subunit hexosaminidase at 5q13). In Tay-
Sachs disease, the central nervous system is primarily affected. The liver appears normal by light microscopy,
but concentric membrane-bound inclusions (“zebra bodies”) may be seen on electron microscopic examination
(Figure 15-23).
FIGURE 15-23 ▪ Tay-Sachs disease. “Zebra bodies” comprised of concentric membrane-bound lysosomal
inclusions (Electron microscopy, 20,000×).
The mucopolysaccharidoses are a group of distinct genetic disorders with accumulation of acid
mucopolysaccharides (glycosaminoglycans), dermatan sulfate, heparan sulfate, chondroitin sulfate, and keratin
sulfate in the tissues with excretion of these substances in the urine (59, 129) (e135,e224,e562). Multiple clinical
types have been described, each associated with a specific enzyme defect. With the exception of Hunter disease
(type II), which is an X-linked recessive condition (Xq28), mucopolysaccharidoses are inherited in an autosomal
recessive pattern. The major clinical manifestations are caused by involvement of the brain, skeletal system,
liver, cornea, and other organ systems. Because the histopathologic and ultrastructural features are identical, the
various syndromes cannot be differentiated on morphologic grounds alone.
The liver is involved in all types with marked cytoplasmic vacuolization of the hepatocytes, Kupffer cells, and Ito
cells. Stored acid mucopolysaccharide can be demonstrated with colloidal iron staining, but requires frozen
sections or nonaqueous fixatives. Numerous electron lucent membranebound vacuoles are seen with electron
microscopic examination, corresponding to acid mucopolysaccharides that are extracted with routine tissue
processing. Finely granular to flocculent material may be seen in some of the vacuoles arranged in concentric
whorls. Hepatic fibrosis may occur.
Sphingolipidoses
Niemann-Pick Disease
Niemann-Pick disease is an autosomal recessive lysosomal disorder associated with a deficiency of
sphingomyelinase
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(type IA and 1B [type A and B], sphingomyelin phospho diesterase-1 gene at 11p15.4-15.1) or a defect in
cholesterol esterification (type II or type C, NPC gene at 18q11-12) (59, 77, 195)
(e178,e301,e503,e507,e508,e616, e671,e738,e774). This disease is characterized by sphingomyelin storage in
various organs (59, 77). Sphingomyelin accumulation varies in extent, but it is most pronounced in type A (type
1A), the acute neuropathic form, and in type B (type 1B), the chronic nonneuropathic form. Sea blue histiocytes
are seen in the bone marrow. The liver is enlarged and pale on gross examination. The lobular structure of the
liver is not disorganized, and fibrosis is generally not a feature. However, cirrhosis may rarely occur.
Type C (type II) disease usually presents with neurologic symptoms between 2 and 4 years of age (59, 77, 195)
(e178,e301,e503,e507,e508,e616,e671,e738,e774). However, it may present in the neonatal period with
jaundice, hepatosplenomegaly, and failure to thrive and progress to death in months. Foamy macrophages and
Kupffer cells may be infrequent initially, but there is progression to the more classic swollen, foamy vacuolated
appearance of the cytoplasm (Figure 15-24). Hepatocytes show similar alterations. Ceroid pigment, cholesterol,
and phospholipids accumulate in the cells. The stored material is best demonstrated by the Baker hematin
reaction for phospholipids. Histochemical staining for acid phosphatase activity reveals a reticular pattern.
Ultrastructurally, the appearance is distinctive (141). Large, pleomorphic, membrane-bound inclusions composed
of concentric or parallel osmiophilic lamellae are seen in the Kupffer cells and to a lesser extent in the
hepatocytes. Bone marrow transplantation has been reported to reverse the amount of storage material in the
liver, spleen, lung, and bone marrow, but it does not prevent progression of the neurologic changes.
FIGURE 15-24 ▪ Niemann-Pick disease, Type C. A: Hepatocytes and Kupffer cells with swollen, granular to
foamly vacuolated cytoplasm (H&E, 400×). B: Large pleomorphic membrane-bound lysosomal inclusions with
concentric to parallel lamellae (Electron microscopy, 15,000×).
Gaucher Disease
Gaucher disease is caused by glucocerebrosidase deficiency (acid beta-glucosidase gene at1q21) and leads to
glucosyl ceramide accumulation in various organs (39) (e63,e64,e97, e274,e319,e484,e635,e788). The disorder
is inherited in an autosomal recessive manner, and three clinical types have been described. Type I, the most
common, is the adult or chronic nonneuropathic form; type II is the acute neuropathic or infantile form; type III is
the juvenile or subacute neuropathic form. The liver has a similar appearance in all three clinical types. There is
massive hepatosplenomegaly with portal hypertension. Gaucher cells are the hallmark. These cells are
distended and have a characteristic striated, “wrinkled tissue paper” appearance of the cytoplasm (Figure 15-
25). The striations are accentuated with the PAS stain, and acid phosphatase activity can be demonstrated
histochemically (39) (e6). Hemosiderin and lipofuscin are frequently present. These macrophages are also seen
within the spleen and bone marrow. Clusters of Gaucher cells in the lobule and in portal areas may be
associated with fibrosis and cirrhosis in some cases (e519). The ultrastructural features are distinctive with
closely apposed, irregular lysosomal inclusions, which correspond to the wrinkled tissue paper light microscopic
appearance of Gaucher cells. The inclusions are composed of innumerable tubules with circular profiles on cross
section. “Pseudo-Gaucher” cells have been described in association with benign and malignant hematologic
diseases and HIV and mycobacterial infections (e657).
Bile Acid Metabolism Disorders

Bile acid synthesis defects are inherited in an autosomal recessive manner, have low to normal GGT, present in
infancy, and are progressive (e675). These diseases typically
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present with neonatal hepatitis and mimic other etiologies for this nonspecific disease process. In older children,
there is a more chronic hepatitis-like picture. Clinical signs and symptoms include pruritus with
hyperbilirubinemia, difficulty with lipid absorption, and failure to thrive. With some conditions, bile acid
substitution will reverse the clinical and histopathologic effects of the bile synthesis deficiencies.
FIGURE 15-25 ▪ Gaucher disease. A, B: Markedly enlarged Kupffer cells with cytoplasm with a striated, wrinkled
tissue paper appearance (H&E, A: 100×, B: 800×). C,D: Kupffer cells with cytoplasmic tubular inclusions with a
circular profile on cross section (Electron microscopy, C: 6,000×, D: 24,000×).
Zellweger Syndrome (Cerebrohepatorenal Syndrome)

Zellweger syndrome (cerebrohepatorenal syndrome) is an autosomal recessive disorder characterized clinically
by multiple congenital abnormalities, including craniofacial abnormalities, hypotonia, and psychomotor
retardation. Renal cortical cysts, cerebral dysgenesis, and hepatic abnormalities are present (188)
(e49,e176,e233,e417,e655,e678). Several different genes involved in peroxisome biogenesis occur in different
forms of Zellweger syndrome, including peroxin-1 (PEX1 at 7q21-q22), peroxin-2 (PEX2, 8q21.1), peroxin-3
(PEX3 6q23-q24), peroxin-5 (PEX5 12p13.3), peroxin-6 (PEX6 6p21.1), peroxin-12 (PEX12 on chromosome 17),
peroxin-14 (PEX14 1p36.2), and peroxin-26 (PEX26 22q11.21). Absence of peroxisomes in hepatocytes and
renal tubular cells is a distinctive feature (77, 156) (e325,e571). Death occurs in early infancy. The liver shows
hepatocellular disarray, biliary dysgenesis, portal inflammation, and striking iron deposition in Kupffer cells and
hepatocytes. Giant cell transformation, steatosis, and hepatic fibrosis or cirrhosis may be seen.
Other Bile Acid Synthesis Defects

Many genetic defects of bile acid synthesis are known (25) (e763). The most common defect among these is 3-
betahydroxy dehydrogenase deficiency caused by mutation in the gene encoding 3-beta-hydroxy-delta-5-C27-
steroid oxidoreductase (16p12-p11.2). This entity is referred to as
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Congenital Bile Acid Synthesis Defect Type 1. This leads to neonatal hepatitis and will progress to chronic liver
disease without appropriate diagnosis and bile acid substitution. Another form of a congenital defect in bile acid
synthesis is due to delta(4)-3-oxosteroid 5-beta-reductase deficiency (Congenital Bile Acid Synthesis Defect
Type 2). This is caused by mutation in the AKR1D1 gene (7q32-33). Congenital Bile Acid Synthesis Defect Type
4 is caused by mutation in the alpha-methylacyl-CoA racemase gene located at 5p13.2-q11.1. Neonatal hepatitis
with bile duct proliferation is associated with a bile synthesis defect in oxysterol 7-alphahydroxylase (gene at
6p21.1-p11.2). Cholesterol is converted into one of several oxysterols prior to being 7-alpha-hydroxylated by an
oxysterol 7-alpha-hydroxylase (gene at 6p21.1- p11.2). Lack of this enzyme leads to neonatal hepatitis and the
potential for progressive liver disease. A deficiency in 25-hyroxylase (gene at 10q23) results in a bile acid
synthesis defect. This is due to the role of this enzyme in expression of genes involved in cholesterol and lipid
metabolism. Liver fibrosis is somewhat variable, with a more prolonged course of fibrosis in affected neonates
and children. Familial hypercholanemia is characterized by elevated serum bile acid concentrations, itching, and
fat malabsorption (BAAT gene at 9q22.3). The defect in this condition is associated with the enzyme bile acid
CoA amino acid N-acyltransferase (BAAT). This enzyme produces N-acyl conjugates of cholanoates (C24 bile
acids) with glycine or taurine. The resulting bile acid-amino acid conjugates serve as detergents in the
gastrointestinal tract. Those affected with bile acid conjugation defects may present as neonatal hepatitis with
fibrosis or as mild chronic liver disease. There are several other conditions that may also have bile acid
synthesis defects, such as peroxisome diseases (Zellweger syndrome, Refsum disease, hyperpipecolic anemia,
adrenolipodystrophies) and cerebrotendinous xanthomatosis (CYP27A1 gene encoding sterol 27-hydroxylase at
2q33-qter).
Alpha-1-Antitrypsin Deficiency
Liver disease associated with alpha-1-antitrypsin deficiency (A1AT) was initially described by Sharp et al. and
has been extensively reviewed (140, 166, 176) (e201,e222,e350,e357, e529,e528,e624,e662). This is an
autosomal recessive disease caused by mutations in the protease inhibitor gene (Pi) on chromosome 14. Both
liver and lung diseases (emphysema) occur due to lack of neutralization of neutrophil elastase secondary to
absent or decreased protease inhibitor activity. Liver disease without pulmonary emphysema occurs when a
mutant but functional form of protease inhibitor is present that inhibits neutrophil elastase activity. This mutant
form of AIAT has a defect that does not allow for proper folding, resulting in failure of the material to be
translocated from the endoplasmic reticulum to the Golgi for further processing before release from the
hepatocyte. The AIAT continues to accumulate in the rough endoplasmic reticulum, leading to hepatocyte injury
and liver disease. Clinical presentations vary from neonatal hepatitis with cholestatic jaundice, to young adults
with recurrent hepatitis that may lead to chronic hepatitis and cirrhosis, and to older adults with a silent clinical
course and cirrhosis development.
A close association of A1AT deficiency has been noted with neonatal cholestasis, accounting for over 10% of
cases of neonatal cholestasis, making it the most common genetic cause of neonatal liver disease (78) (e527).
Bleeding diathesis, including intracranial hemorrhage, may be the presenting manifestation in the newborn,
probably related to an associated vitamin K deficiency (e308).
A1AT is a glycoprotein that is synthesized in the liver and secreted into the serum. Its biosynthesis is controlled
by a pair of genes at the protease inhibitor (Pi) locus (140, 166, 176) (e 202,e222,e350,e357,e528,e529,e662).
More than 25 alleles have been described and are responsible for A1AT variant molecules. The normal genotype
is PiMM. PiZZ is the most clinically significant genotype with respect to liver disease, and is due to a point
mutation with substitution of Lys for Glu. PiMZ genotype patients have 50% normal A1AT and 50% mutant A1AT.
Other mutant gene alleles include PiS with reduced A1AT level and no clinical disease, and PiNull with no
detectable A1AT. With electrophoresis, PiZ is the slowest of the A1AT variants. In the homozygous (PiZZ) state,
there is a marked reduction in the serum A1AT levels. Homozygous PiZZ A1AT has an incidence of 1 in 1,600 to
2,000 live births, making it nearly as frequent as cystic fibrosis (CF) (e527). A few cases of liver disease have
been reported in association with PiSZ (e679). Neonatal liver injury has occurred with the PiZ null phenotype
(e551). The risk of HCC is increased, especially in homozygous patients, with most cases being reported in
adults.
Liver morphology varies in the early phase of the disease. Hepatocellular injury is manifested principally as
cholestasis, pseudoacinar transformation, and giant cell transformation, similar to other metabolic hepatopathies
(Figure 15-26). Extramedullary hematopoiesis is usually seen. Cholestasis is hepatocellular and present in the
form of plugs within the canaliculi. Three morphologic patterns with prognostic significance have been described
for the early cholestatic phase. In group 1, portal areas show mild portal fibrosis and no bile duct proliferation,
which has a neonatal hepatitis-like appearance (Figure 15-26). In group 2, the portal triads are fibrotic and
expanded and contain proliferating bile ducts in which bile may be present. This pattern may be mistaken for the
obstructive changes seen in EHBA, and is associated with persistent hepatic disease leading to cirrhosis with a
higher frequency. With group 3, there is a paucity of extrahepatic ducts. The prognosis of this group is uncertain.
Extensive hepatocellular necrosis may also occur and lead to fulminant hepatic failure.
The morphologic hallmark of the disease is the presence of A1AT in the hepatocytes, predominantly in zone 1
and occasionally in bile duct epithelium. The stored material appears in the form of eosinophilic hyaline globules
that are PAS-positive and resist diastase digestion. The globules
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progressively increase in number and may not be visible by hematoxylin and eosin sections in biopsy specimens
obtained in the first 3 months of life (e527). The stored material may be demonstrable by immunohistochemistry,
even in the absence of appreciable globules. Ultrastructurally, the stored material appears as flocculent,
moderately electrondense material within dilated cisternae of rough endoplasmic reticulum.
FIGURE 15-26 ▪ A: Alpha-1-antitrypsin deficiency. Zone 1 hepatocytes with reactive changes and portal areas
with chronic inflammation and mild bile duct proliferation (A: H&E, 200×). B: Cirrhosis in late stage of disease
detection (B: H&E, 40×). C, D: Zone 1 hepatocytes with PAS-positive (C: 400×) cytoplasmic globules that are
diastase resistant (D: 400×). E: Immunostaining for alpha-1-antitrypsin reacts with the cytoplasmic globules (E:
400×). F: Granular, flocculent material distends cisternae of the rough endoplasmic reticulum (F: Electron
The frequency of progression to cirrhosis after neonatal presentation with prolonged cholestasis is variable. Only
about 15% of the PiZZ population develop liver disease in the first 20 years of life. If A1AT deficiency is
manifested in the neonatal period, as many as 50% of cases progress to cirrhosis, typically micronodular (Figure
15-26) (78). The presence of PAS-positive diastase-resistant globules is the pathologic hallmark, differentiating
the micronodular cirrhosis associated with A1AT deficiency from micronodular cirrhosis associated with other
disorders. The extrahepatic bile ducts are usually normal. A few cases of hypoplasia of the extrahepatic bile
ducts with A1AT deficiency have been described, and the hypoplasia has been ascribed to a low-flow state.
FIGURE 15-27 ▪ Cystic fibrosis. A: Appendix with dilated lumen and dense eosinophilic mucin in lumen (H&E,
20×). B: Appendiceal glands with insipissated densely eosinophilic mucin (H&E, 200×). C: Hepatocytes with
diffuse microsteatosis and focal macrosteatosis (H&E, 200×).
Cystic Fibrosis
CF is caused by mutations in the CFTR gene (CF transmembrane conductance regulator, 7q31.2) that regulates
a cyclic AMP-dependent chloride channel (55) (e401,e492,e589). CFTR gene mutation results in decreased
sodium and water content of bile with an increase in bile viscosity and reduction in bile low, leading to
intrahepatic bile duct obstruction and injury. The incidence of hepatic involvement in CF has increased over the
past several decades with increased life expectancy of CF patients. Although pulmonary complications are the
predominant clinical manifestations, up to 5% of CF patients may have substantial hepatic dysfunction and an
even larger proportion have the typical histologic lesions of CF in the liver without abnormal liver function tests
(41, 103, 128) (e138,e152,e180,e639). The liver may have multiple capsular depressed scars, with a
resemblance to hepar lobatum. Histopathologically, there are focal irregular areas of fibrosis with bile duct
proliferation and intraluminal inspissated eosinophilic or pale orange secretions (Figure 15-27). This
pathognomonic hepatic lesion is the so-called focal biliary cirrhosis. Mononuclear cell infiltration may be seen.
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Steatosis is confined to zone 3 or shows a panacinar distribution, especially in infants with newly diagnosed CF
whose pancreatic enzyme replacement has not yet been initiated.
FIGURE 15-27 ▪ (continued) D: Bile ducts in fibrotic portal region with lumenal insipissated densely eosinophilic
secretions (H&E, 400×). E-G: Explanted liver with macronodular and micronodular cirrhosis (H&E, G 40×).
The disease may present in the neonate with cholestatic changes with giant cell transformation and steatosis as
the feature of a metabolic hepatopathy. A liver biopsy in an infant with CF may not show the distinctive bile duct
lesion (focal biliary cirrhosis). The progression from neonatal cholestasis to focal biliary cirrhosis is not clear.
A coarsely nodular cirrhosis is present in 4% to 10% of cases, with the prevalence increasing through childhood
(41, 103, 128) (e138,e152,e180,e639). Interestingly, there is a diminished prevalence of cirrhosis in those
surviving to young adulthood, suggesting that liver disease may influence premature respiratory death in
teenagers. At the cirrhotic stage, the liver shows multiple, large nodules, with areas between the nodules
appearing depressed and presenting a finely nodular appearance. Portal hypertension and its complications may
occur and, rarely, death may ensue acute bleeding from esophageal varices. Combined liver and lung
transplantation are necessary in a minority of cases.
The gallbladder is frequently abnormal. The prevalence of gallbladder abnormalities increases with age (103,
128) (e152,e180). The gallbladder may be small, with the epithelium frequently having mucinous metaplasia.
Diagnostic imaging may show a diminutive or nonfunctioning gallbladder. Cholesterol gallstones are seen in 6%
to 12% of patients over 12 years of age, with the risk of developing calculi increasing with age.
Iron Storage Disease

Primary and secondary disorders of iron metabolism are characterized by excessive iron accumulation in the liver
(Figure 15-28) as a component of increased total body iron stores (16, 59, 77, 192)
(e15,e86,e216,e315,e481,e613,e750). Secondary iron overload may be the result of multiple transfusions for
hemoglobin disorders such as thalassemia and
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sickle cell disease, or may be due to excessive iron intake. Inherited iron storage disease or hemochromatosis is
most often an autosomal recessive condition characterized by a defect in the regulation of iron absorption in the
intestine and may present in childhood. There are several inherited forms of hemochromatosis caused by
different gene mutations. The clinical features of hemochromatosis include cirrhosis of the liver, diabetes,
hypermelanotic pigmentation of the skin, and heart failure. Pancreatic deposition of iron leads to diabetes, and
congestive cardiomyopathy is the result of iron deposition in the myocardium. Primary HCC, complicating
cirrhosis, is responsible for about one-third of deaths in affected homozygotes. Because hemochromatosis is a
relatively easily treated disorder if diagnosed early, this is a form of preventable cancer. At least five iron-
overload disorders labeled hemochromatosis have been identified on the basis of clinical, biochemical, and
genetic characteristics (6). Classic hemochromatosis (HFE), an autosomal recessive disorder, is most often
caused by mutation in a gene designated HFE on chromosome 6p21.3. It has also been found to be caused by
mutation in the gene encoding hemojuvelin (HJV), which maps to 1q21. Juvenile hemochromatosis or
hemochromatosis type 2 (HFE2) is also autosomal recessive. One form, designated HFE2A, is caused by
mutation in the HJV gene (1q21). A second form, designated HFE2B, is caused by mutation in the gene
encoding hepcidin antimicrobial peptide, which maps to 19q13. Hemochromatosis type 3, also an autosomal
recessive disorder, is caused by mutation in the gene encoding transferrin receptor-2 (TFR2), which maps to
7q22. Hemochromatosis type 4, an autosomal dominant disorder, is caused by mutation in the SLC40A1 gene,
which encodes ferroprotein and maps to 2q32. Most affected children and adolescents are asymptomatic with
periportal iron accumulations extending toward the central lobule during adolescents. With juvenile
hemochromatosis, organ failure with severe iron overload presents before age 30. Both the inherited
(hemochromatosis) and secondary (transfusion) forms of iron storage differ from hemosiderosis in that the iron,
in addition to being deposited in mononuclear phagocytic cells, is also deposited in the parenchymal cells. Iron
deposition in biliary epithelial cells is seen more often in inherited iron storage disease.
FIGURE 15-28 ▪ Secondary hemosiderosis due to chronic transfusion therapy. A: Occasional Kupffer cells with
iron pigment in their cytoplasm (H&E, 400×). B: Abundant iron storage in Kupffer cells revealed with Prussian
blue histochemical stain for iron (400×).
Neonatal Iron Storage Disease

Neonatal iron storage disease (NISD), a fatal neonatal disorder, is characterized by massive iron overload (192)
(e146, e353,e463,e743,e764,e765). The liver is the predominant organ affected, but iron is also deposited in the
pancreas, thyroid, adrenals, pituitary, heart, intestinal mucosa, salivary glands, and sweat glands. The basic
defect has not been defined, but most reports suggest that it is a metabolic error, unrelated to classic iron
storage disease or hemochromatosis. The condition should be differentiated from other disorders such as
tyrosinemia, galactosemia, and Zellweger syndrome, in which excess iron is usually present. More recently,
NISD has been considered a gestational disease in which fetal liver injury leads to the phenotype in the neonate.
NISD recurrence rate in siblings after the index case is 60% to 80%, implicating maternal alloimmune damage to
fetal liver. Pregnant mice injected with human IgG from women with NISD offspring had pups with extensive
hepatic injury and liver necrosis. Clinical investigations have evaluated treatment of pregnant women with a prior
NISD neonate with intravenous immunoglobulin (IV Ig). In these clinical studies, prior gestational histories
indicated a high risk for NISD occurrence, with 92% of at-risk pregnancies resulting in intrauterine fetal demise,
neonatal death, or liver failure necessitating transplant. With IV Ig gestational therapy during pregnancy, there
were only three failures, while 52 infants did not experience NISD (192).
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FIGURE 15-29 ▪ Neonatal iron storage disease. A,B: Hepatocytes and bile duct epithelium with readily identified
iron pigment accumulation in cytoplasm (H&E, 400×). C: Explanted liver with micronodular cirrhosis and green
and brown pigmentation from bile and iron accumulation, respectively.
In NISD, the hepatic architecture is markedly disorganized with lobular collapse and early fibrosis (Figure 15-29).
Scattered nests of hepatocytes with heavy iron deposits, pseudoacinar profiles, and multinucleated hepatocytes
are other microscopic features. Iron may also be demonstrable in biliary epithelial cells. With other organ
systems, the iron deposits tend to be within the reticuloendothelial system, with sparing of the parenchymal cells.
Minor salivary glands in the oral mucosa show iron deposition in NISD and may be biopsied for diagnosis in
suspected cases while awaiting genetic testing results for hemochromatosis.
Wilson Disease
Wilson disease is an inborn error of copper metabolism with an autosomal recessive pattern of inheritance. A
genetic defect in ATP7B on chromosome 13q14-21 has been described. This gene encodes a transmembrane
copper-transporting adenosine triphosphatase (ATPase) that is located on the canalicular membrane of
hepatocytes, and is also homologous with Menkes disease gene. The genetic defect results in reduced copper
excretion in the bile and decreased copper incorporation into ceruloplasmin. There are many different mutations
in ATP7B, which account for the variable clinical phenotypes.
In normal metabolism, copper is taken up by the stomach and duodenum, weakly bound to albumin, and
transferred to hepatocytes (113) (e12,e206,e213,e298,e437,e603). Within the hepatocytes, copper is
incorporated into the alpha-2- globulin of ceruloplasmin and released into the bloodstream. Senescent
ceruloplasmin is reabsorbed by the hepatocytes and undergoes lysosomal degradation and excreted into the
bile. In Wilson disease, copper accumulation occurs in the liver, brain, eyes, and other organs. Elevated levels of
serum and hepatic copper, increased urinary copper excretion, and diminished levels of serum ceruloplasmin are
the common laboratory abnormalities. In some cases, serum ceruloplasmin values may be within normal limits. A
normal serum level of copper excludes Wilson disease, but an elevated level is not always diagnostic, because
elevations in copper
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may be seen in other forms of liver disease, especially of cholestatic type, and in chronic hepatitis. Genetic
analysis is available for the diagnosis of Wilson disease in patients and their families.
The clinical presentation varies according to the age of the patient and the stage of the disease (113)
(e12,e206,e213,e298,e437,e603). The most frequent symptoms are related to hepatic involvement. Liver
disease may be chronic, and cirrhosis or chronic hepatitis may be evident at clinical presentation. Acute hepatitis
and fulminant hepatic failure may be the presenting features in a minority of cases, especially in the first two
decades of life. Hemolytic anemia is frequent. Central nervous system signs, neuropsychiatric symptoms
associated with basal ganglia involvement, and Kayser-Fleischer rings develop in the course of the disease. The
latter are characterized by green-brown deposits of copper in Descemet membranes of the corneal limbus.
Penicillamine therapy has been reported to alter the natural course of the disease, and, when instituted early,
can prevent progression of liver disease (113) (e12,e206,e213, e298,e437,e603). Controversy, however, exists
as to the timing of the use of penicillamine in treatment. Treatment using zinc and trientine has also been
studied. Transplantation may be necessary in some cases.
FIGURE 15-30 ▪ Wilson disease. A: Hepatocytes with variable cytoplasmic swelling and decreased cytoplasmic
eosinophilia (H&E, 200×). B: Infrequent hepatocytes with glycogenated nuclei, apoptotic (acidophil) bodies, and
fine granular cytoplasm with a certain degree of cytoplasmic swelling (H&E, 400×). C: Cytoplasmic copper
detection in periportal hepatocytes (Rhodamine stain, 400×).
FIGURE 15-30 ▪ (continued) D: Wilson disease with cirrhosis of liver (H&E, 100×). E: Variably sized and
relatively pleomorphic mitochondria and dense lysosomal deposits in Wilson disease (Electron microscopy,
3,000×).
Histopathologic features in the liver vary from mild to moderate fatty changes, focal cytoplasmic swelling,
glycogenated nuclei, and occasional acidophilic bodies in the early stages (Figure 15-30). Generally, portal tract
inflammation, lobular chronic inflammation, and fibrosis are not seen at this stage. Copper is diffusely dispersed
in the cytoplasm and usually cannot be demonstrated histochemically. In the symptomatic stage, the liver may
have features of chronic hepatitis (interface hepatitis, portal inflammation, fibrosis, and spotty acidophilic
necrosis of hepatocytes). Mallory bodies may be seen, especially in the zone 1 hepatocytes. Glycogenated
nuclei are a frequent, but nonspecific, feature. A mixed micronodularmacronodular cirrhosis is the consequence
of the chronic hepatitis. Rarely, massive liver necrosis is seen.
Copper can be demonstrated histochemically and is most pronounced in the periportal hepatocytes. The
rhodamine
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stain gives a brick red reaction product, while rubeanic acid stains the copper gray-black. The Shikata orcein
stain demonstrates associated copper-binding protein. Copper may be irregularly distributed in the hepatocyte
nodules and may be absent in the regenerative nodules by histochemical methods. Biochemical quantitation of
hepatic copper typically demonstrates marked elevations (>250 ug/g dry weight). This can be performed on fresh
tissue or a paraffin block. Ultrastructurally, the mitochondria show characteristic alterations appearing enlarged
and pleomorphic. Separation of the inner membranes, widening of the intracristal space with microcystic
formations at the tips of the cristae, crystalloid inclusions, disoriented cristae, and increased granules in the
matrix of the mitochondria are regarded as diagnostic of the disorder (77). Copper deposits are seen in the
lysosomes of zone 1 hepatocytes and appear extremely electron-dense. Peroxisomal deposition of copper has
also been described.
Porphyrias
Porphyrias are a group of disorders of porphyrin and heme biosynthesis (16, 59) (e30,e73,e370). Porphyria may
be inherited or acquired and is characterized by increased excretion of porphyrins and storage of abnormal types
of porphyrin pigments within tissues. Hepatic abnormalities may be seen in acute intermittent porphyria
(hydroxymethylbilane synthase 11q23.3), porphyria cutanea tarda (hemochromatosis gene at 6p21.3,
uroporphyrinogen decarboxylase gene at 1p34), and congenital erythropoietic protoporphyria (uroporphyrinogen
III synthase gene at 10q25.2-q26.3). The changes in acute intermittent porphyria and porphyria cutanea tarda
are similar, although the severity of hepatic injury is greater in porphyria cutanea tarda. Fatty changes and iron
overload are evident. Cirrhosis and hepatic failure may occur in porphyria patients, and HCC has been described
as a complication in later life. The uroporphyrin crystals are water-soluble, needle-shaped, and have a red
fluorescence on examination under ultraviolet light. The needle-shaped inclusions are seen in the hepatic cells
by electron microscopy. Additional ultrastructural features include abnormal mitochondria, autophagic vacuoles,
and myelin figures. In congenital erythropoietic protoporphyria, the hepatic findings consist of focal accumulation
of dark brown pigment in the canaliculi, bile duct epithelium, Kupffer cells, and connective tissue. The pigment is
birefringent with bright granules and central Maltese crosses. An intense red fluorescence is seen in frozen
sections examined under ultraviolet light. Ultrastructurally, the crystals are electron-dense, straight or curved,
and arranged singly or in a radiating star-burst pattern.
Urea Cycle Disorders

Hyperammonemia is characteristic of this group of disorders and should be differentiated from other conditions
with elevated ammonia levels (59) (e95,e150,e199,e200,e247, e386,e406,e441,e600,e644). In the newborn,
hyperammonemia may be found in premature infants or infants with birth asphyxia. In utero hepatic necrosis of
undetermined cause has been found to be associated with hyperammonemia (59). Defects of the urea cycle
include deficiency of ornithine transcarbamylase (Xp21.1), deficiency of carbamyl synthetase (2q35), citrullinemia
associated with deficiency of argininosuccinic acid synthetase (9q34.1), argininosuccinic aciduria due to
deficiency of argininosuccinase lyase (7cen-q11.2), argininemia associated with arginase deficiency (6q23), and
deficiency of N-acetyl-glutamate synthetase (17q21.31) (e95,
e150,e199,e200,e247,e386,e406,e441,e600,e644). With the exception of ornithine transcarbamylase deficiency,
which is inherited as an X-linked dominant condition, the other conditions have an autosomal recessive pattern of
inheritance.
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FIGURE 15-31 ▪ Urea cycle disorder—ornithine transcarbamylase deficiency. A, B: Explanted liver with no gross
abnormalities in ornithine transcarbamylase deficiency. C: Portal triad and zone 1 and 2 hepatocytes with no
histopathologic abnormalities (H&E, 200×).
Prenatal diagnosis is possible. Liver biopsy in urea cycle defects may be normal or may have only mild
nonspecific changes including steatosis, cholestasis, individual cell necrosis, and early fibrosis (Figure 15-31).
Liver transplantation may be necessary depending upon the specific urea cycle defect disorder.
Hepatic Steatosis and Steatohepatitis

Fatty change of the liver is a frequent, nonspecific finding associated with a variety of metabolic and nutritional
disorders (29, 104) (e17,e89,e118,e390,e416,e521,e565,e566, e674,e779). Diagnosis of the specific metabolic
disorder associated with this change requires the demonstration of pathognomonic biochemical and morphologic
features of that disease. Disorders of lipid and lipoprotein metabolism include abetalipoproteinemia,
hypercholesterolemia, congenital lipodystrophy, and fatty acid oxidation defects. The ultrastructural features of
these diseases have been described elsewhere (141). Various chemical agents, drugs (valproate, asparaginase,
steroids, amiodarone), and toxins (alcohol) are known to induce hepatosteatosis. Other causes of
hepatosteatosis in childhood include protein malnutrition, kwashiorkor, obesity, chronic illnesses, type I diabetes,
hepatitis C, TPN, mitochondrial disease, inborn errors of metabolism, and severe infection. In the case of
obesity, fatty change may be accompanied by inflammation in the lobules and portal tracts as features of
steatohepatitis.
This is a reversible form of cellular injury. The lipid may accumulate in the form of small droplets of
microvesicular fat, or in the form of large (macrovesicular) droplets that occupy most of the cytoplasm and
displace the nucleus to the periphery (Figure 15-32). Microvesicular fat leads to a foamy or clear appearance of
the hepatocyte cytoplasm, without displacement of the nucleus, and may not be obvious as fat in the usual
preparation. Fat stains on frozen sections and electron microscopy conclusively demonstrate the fat.
The marked increase in childhood obesity and type II diabetes has significantly increased the prevalence of
nonalcoholic fatty liver disease (NAFLD) in the pediatric population (29, 104)
(e17,e89,e118,e390,e416,e521,e565,e566,e674, e779). In fact, NAFLD has emerged as the leading cause of
chronic liver disease in children and adolescents in the United States. Elevated insulin, ALT levels, and
hyperlipidemia (increased cholesterol and triglyceride) are commonly present in these children. Further,
cardiovascular risk and
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morbidity in children and adolescents are associated with fatty liver.
FIGURE 15-32 ▪ Nonalcoholic fatty liver disease. A: Hepatocytes with macrovesicular and microvesicular
steatosis in an azonal pattern (H&E, 100×). B: Macrosteatosis and occasional hepatocytes with glycogenated
nuclei (H&E, 400×). C: Portal expansion by chronic inflammatory cells with extension into Zone 1 (H&E, 200×).
The characteristic histological features of NAFLD range from steatosis alone to steatohepatitis (NASH) with or
without fibrosis to cirrhosis (Figure 15-32). Liver biopsy remains the gold standard for the diagnosis of NASH.
NAFLD grading systems are based upon the proportion of hepatocytes demonstrating macrovesicular steatosis,
hepatocyte injury (ballooning degeneration), lobular inflammation, and stage of fibrosis (29) (e89,e779). In adults,
the histological features of NAFLD have been well-described and include macrovesicular steatosis,
perisinusoidal or pericellular fibrosis, foci of lobular inflammation, lipid granulomas, Mallory hyaline, and
megamitochondria (29). The combination of macrovesicular steatosis with ballooning change of hepatocytes
and/or perisinusoidal fibrosis constitutes a pattern of histology considered diagnostic of NASH in an appropriate
clinical context. However, pediatric fatty liver disease often displays a histologic pattern distinct from that found in
adults (104, 160). In a large study of 100 children with biopsy-proven NAFLD, Schwimmer et al. demonstrated
two different forms of steatohepatitis. While both types showed steatosis, “type 1” was characterized by
ballooning degeneration, and perisinusoidal fibrosis (as in adults) affecting 17% of subjects while “type 2” was
more common (affecting 51% of subjects) and was characterized by portal inflammation and portal fibrosis. Boys
were significantly more likely to have type 2 NASH than girls. Further, type 1 NASH was more common in white
children, whereas type 2 NASH was more common in children of Asian, Native American, and Hispanic ethnicity.
In cases of advanced fibrosis, the pattern was generally that of type 2 NASH (160).
Reye Syndrome
Reye syndrome (acute encephalopathy with hepatic fatty degeneration) is an acute disease of childhood that
presents as an encephalopathy, which may progress rapidly to irreversible coma and death (35)
(e240,e326,e549,e610). The disease has decreased dramatically since its association with salicylate use was
described and warnings issued about the use of salicylates in febrile children. The disease has a biphasic
clinical course with an initial febrile illness, usually associated with an upper respiratory viral infection, followed
by apparent recovery and the abrupt onset of protracted vomiting, delirium, and stupor. The basic damage
appears to be
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a widespread mitochondrial injury, especially in the liver, brain, and muscle, leading to abnormal metabolism of
lipids. Children with symptoms mimicking Reye syndrome may have metabolic disorders, such as organic acid
and betaoxidation defects, and urea cycle disorders. This emphasizes the need to evaluate these children
thoroughly, setting aside tissue appropriate for metabolic disorder investigations and molecular genetic studies.
Liver dysfunction is manifested by elevations in transaminases, hypoprothrombinemia, and hyperammonemia
(35) (e240,e326,e549,e610). Hypoglycemia may be present. Serum amino acid and free fatty acid levels may be
elevated. Grossly, the liver is enlarged and is yellow to pale due to increased parenchymal lipid. Microscopically,
the hepatocytes appear either normal or contain finely vacuolated microvesicular steatosis, which does not
displace the nucleus. Oil-red-O stains on frozen sections reveal the panlobular distribution of lipid, and virtually
all hepatocytes contain small droplets of lipid (34). Characteristically, there is no hepatocellular necrosis or
inflammation. Severe decrease or absence of succinate dehydrogenase enzyme activity is demonstrable
histochemically.
The ultrastructural features of microvesicular lipid droplets and typical mitochondrial abnormalities are
considered virtually diagnostic of the syndrome (141). The changes are reversible, and in children who recover,
the liver may show normal morphology, except for the presence of lipid in some hepatocytes and Kupffer cells,
and occasional large mitochondria.
Lipid accumulation is also seen in other organs, notably renal tubular epithelium, myocardial and skeletal
muscles, lungs, and pancreatic islets. The brain is edematous and mitochondrial changes similar to those in the
liver have been described.
Defects in Fatty Acid Oxidation
Defects in fatty acid oxidation, such as carnitine deficiency (SLC22A5 gene at 5q31.1) and acyl-CoA-
dehydrogenase deficiency (gene locus at 12q22-qter), may be associated with clinical features resembling Reye
syndrome (e356-e361). Episodes of a recurrent Reye-like illness or siblings similarly affected should raise the
distinct possibility of fatty acid oxidative disorder.
FIGURE 15-33 ▪ Fatty acid oxidation defect—carnitine deficiency. A-C: Variable lipid deposition from fine
cytoplasmic vacuolization (A) to microsteatosis (B) to macrosteatosis (C)
Carnitine has a role in the beta-oxidation of fatty acids by aiding in their transport across the inner mitochondrial
membranes (e75,e87,e402,e403,e449,e577,e599). Three clinical types of carnitine deficiency (SLC22A5 gene at
5q31.1) have been described: myopathic, systemic, and mixed. In the systemic form, carnitine levels are reduced
in the serum, liver, and muscle. During the acute episode, often initiated by a relative minor clinical event such as
gastroenteritis, the liver shows microvesicular steatosis with panacinar distribution (Figure 15-33) (e102).
Ultrastructurally, there is nonmembrane-bound lipid and proliferation of smooth endoplasmic reticulum, increased
numbers of lysosomes and accumulation of lipofuscin. Mitochondria may be abnormal in a nonspecific manner.
Between clinical episodes, the liver may appear normal. It is important to keep this group of metabolic disorders
in mind when a child dies rather abruptly during a seemingly innocuous febrile illness. Tissue and fluids should
be obtained at the time of autopsy and be appropriately preserved for possible biochemical and genetic analysis.
Glutaric aciduria type II (type IIA, ETFA gene at15q23-25; type II B, ETFB gene at 4q32-qter; type IIC ETFDH
gene at 19q13.3) is associated with deficiency of several mitochondrial acyl-CoA-dehydrogenases and is
characterized by acidosis, nonketotic hypoglycemia, organic aciduria, hyperammonemia, and accumulation of
lipid in the liver, myocardium, and renal tubular epithelium (59) (e248,e449). One of the unique aspects of this
inherited metabolic disorder is the presence of several congenital malformations, including renal cortical and
medullary cysts, cerebral pachygyria, pulmonary hypoplasia, and facial dysmorphism. A familial
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syndrome of hepatosteatosis, jaundice, and kernicterus has been described (e560,e595,e676). Death occurs in
the first 3 months of life. Histologically, the liver shows panlobular steatosis with variable cholestasis and portal
fibrosis. Lipid is also demonstrable in the renal tubular epithelium and myocardium. The basic mechanism of this
disease has not been defined, and there is a possibility that the disease may not be a distinct entity.
FIGURE 15-33 (continued) ▪ within hepatocytes (H&E, A: 400×, B: 200×, C: 400×). D: Nonmembrane-bound
lipid droplets within the hepatocyte cytoplasm (Electron microscopy, 4,000×).
VIRAL HEPATITIS
Viral hepatitis is the result of primary infection of the liver by specific hepatotropic viruses. These include
hepatitis A, B, C, D, E and possibly G (GB virus C) viruses. Many studies and reviews of clinical findings, the
nature of the viruses, morphologic findings, and immunopathology are available that discuss the disease as it
affects adult and pediatric age groups (e168,e476,e688,e768). Some characteristics of these viruses and the
associated hepatic diseases are shown in Table 15-9.
Hepatitis A
Infectious hepatitis, or hepatitis A, accounts for one-third of reported pediatric cases. This virus is a single-
stranded RNA virus (picornavirus). Transmission is by the fecaloral route because the virus is resistant to low
gastric pH (e609). Sexual transmission is prominent among homosexual men. Epidemics of the disease occur,
and there are endemic areas, especially in the tropics, with a high rate of infection. Institutionalized children are
at risk owing to poor hygienic conditions. In countries with poor sanitary conditions, most children are infected at
an early age. Seroepidemiologic studies have routinely shown that up to 100% of preschool children have
detectable anti-hepatitis A virus (HAV) antibodies, presumably reflecting previous subclinical infection (e265).
The average age of infection is rapidly increasing to 5 years and older, when symptomatic infection is more likely
(e304). In industrialized countries, there is a low prevalence of HAV infection among children and young adults.
Thus, in the United States, the prevalence of anti-HAV antibodies is approximately 10% in children and 37% in
adults (e1).
HAV causes acute inflammation of the liver, which resolves without chronic carrier status, chronic hepatitis, or
HCC in infected patients. The incubation period is 2 to 4 weeks, rarely up to 6 weeks. Histologically, acute
hepatitis manifests as lobular disarray with ballooned hepatocytes, apoptotic (Councilman) bodies,
lymphomononuclear inflammation, and zone 3 cholestasis. However, the diagnosis is established on serology
and biopsy is not required. Mortality rate is low in previously healthy individuals. The real impact of the disease is
in the morbidity it causes, usually a significant problem only in adults and older children. Approximately 11% to
22% of patients with acute HAV require hospitalization (e271). Young children (below 2 years of age) are usually
asymptomatic with only 20% developing jaundice, whereas most 5-year-old children (80%) develop symptoms.
Management includes only general supportive measures. A highly effective vaccine is available; however, it is
not recommended for children younger than 2 years of age.
Hepatitis B
The hepatitis B virus (HBV), a partially circular doublestranded DNA virus (hepadnavirus), is usually transmitted
perinatally, sexually, and parenterally by means of blood or
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other body fluids including semen, saliva, and breast milk (183) (e642). HBV has an incubation period of 6 to 8
weeks. Among children, those at increased risk include hemophiliacs and others who require frequent
transfusions, adolescent intravenous drug abusers, institutionalized children, and infants of mothers with chronic
HBV infection.
Table 15-9 ▪HEPATITIS VIRUSES AND LIVER DISEASE
Tissue Markers
Virus Characteristics Antigen Disease Nucleus Cytoplasm
HAV 27-nm RNA virus Hepatitis Acute viral None +

found in stool, blood, A hepatitis
and liver (HAAg)
HBV 42-nm DNA virus Hepatitis Acute viral None None

with envelope, 27-nm B hepatitis
core in nucleus and surface
22-nm coat found in (HBsAG)
blood and
hepatocyte
cytoplasm
Hepatitis Acute hepatitis 1+ HBcAg 1+ HBsAg

B core with bridging
(HBcAg)
Hepatitis Carrier state, no None 4+ HBsAg

E liver disease
(HBeAg)
CAH 1+ HBcAg 1+ HBsAg
Chronic hepatitis, 4+ HBcAg 1+ HBcAg

immune
suppressed
Carrier state, mild 4+ HBcAg 1+ HBsAg

hepatitis
Hepatitis Single RNA strand 6 Hepatitis Acute viral None None
C (non- genotypes, 80 C hepatitis CAH
A, non- subtypes
B)
Hepatitis RNA defective virus; Delta In the presence of 4+ delta 1+ delta

D virus 35-37-nm incomplete coinfection with
(HDV) virion, requires HBV, implicated in
delta HBsAg to be massive hepatic
agent infective necrosis; and
higher frequency
of chronic disease
Hepatitis 27-30-nm Hepatitis Acute self-limiting Immunofluores

E virus nonenveloped RNA E hepatitis cence positive
(HEV) virus in frozen
tissue
The prevalence of HBV infection varies in different geographic areas (e424). In most high-prevalence areas such
as Hong Kong and China, perinatal transmission is the major mode of spread, accounting for 40% to 50% of
chronic HBV infection (e400). However, horizontal spread during the first 2 years of life is the major mode of
transmission in other endemic areas including Africa and the Middle East (e340,e711). In intermediate-
prevalence areas, transmission occurs in all age groups, but early childhood infection accounts for most cases of
chronic infection (defined as persistent serum HBsAg for 6 or more months after initial diagnosis). In low-
prevalence areas, such as the United States, Western Europe, and Australia, most infections are acquired in
early adult life through unprotected sexual intercourse or intravenous drug abuse. Age at infection has a
significant impact on the clinical outcome, because chronic infection occurs in approximately 90% of infants
infected at birth, in 25% to 50% of children infected between the ages of 1 and 5 years, and in less than 5% of
those infected during adult life (183) (e51,e114,e144,e642,e656,e690).
Acute HBV infection has been estimated to account for 10% to 25% of all cases of childhood acute hepatitis
(183) (e113,e642). Acute hepatitis B can cause fulminant hepatitis. Acute and chronic hepatitis B morphologically
may resemble hepatitis of other etiologies and requires serology for definitive diagnosis. Chronically infected
patients may have acute hepatitis B flares; superinfection with hepatitis D virus (HDV) should be considered in
this setting. Chronic hepatitis B is an important risk factor for cirrhosis, dysplasia, and HCC.
Anti-HB confers long-term immunity. An effective and safe vaccine has been available since the early 1980s and
is now included in the routine pediatric immunization schedule. Following infection, treatment should be instituted
as early as possible, before there is irreversible liver damage. Extrahepatic manifestations including arthralgia,
arthritis, skin rash, and Gianotti-Crosti syndrome (papular acrodermatitis) are common (in 25% of patients). Many
new antiviral and immunomodulatory therapies have become available in recent years; however, these therapies
are efficacious in less than 50% of patients. Liver biopsy is also useful in confirming virologic clearance or
persistence; a part of the specimen should be routinely preserved for viral DNA quantitation.
Hepatitis C
The hepatitis C virus (HCV) infects over 100 million people worldwide, mostly adults, and is perhaps the most
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common cause of chronic hepatitis (126). Hepatitis C is a single-stranded RNA virus (flavivirus-like). The
development of serologic tests has led to donor screening and its decrease in transfusion recipients. A
population at risk is injection drug abusers. The most common transmittal route is parenteral, with an incubation
period of 6 to 12 weeks. Although transfusions were the most common mode of spread, nucleic acid-based
screening of blood and blood products has almost eliminated this route of spread. Intravenous drug abuse is
presently the most common route of transmission. Perinatal transmission is known to occur, but the predominant
route (transplacental or perinatal) and incidence of transmission are not known (183) (e214,e303,e496,e642).
Perinatal transmission has been documented only from anti-HCV women who are HCV-RNA positive.
Transmission is more efficient if mothers have acute HCV infection during pregnancy, high circulating HCV-RNA
levels, and/or HIV coinfection. HCV is not transmitted by breast-feeding. Since maternal HCV antibodies are
passively transferred to the neonate, diagnosis requires RNA-based tests.
Eighty percent of patients develop chronic hepatitis, with 20% developing cirrhosis and 20% developing HCC.
Histopathologically, chronic hepatitis C is characterized by predominant portal lymphomononuclear inflammation
with or without lymphoid aggregates and bile duct (Poulsen) lesions, lobular inflammation, and varying degrees
of fibrosis. The standard grading and staging systems in use for the histopathologic assessment of most chronic
hepatitis were originally developed for evaluating chronic hepatitis C and as such are best standardized in this
setting. Transplantation is not curative, and recurrent infection is universal. No vaccine is available, and antiviral
therapy is effective in only 25% to 40% of patients. Anti-HCV antibody does not confer immunity. Serum
transaminases fluctuate markedly and cannot be used as surrogate markers of infection or the degree of hepatic
injury (126, 183).
Hepatitis D
The HDV (or delta agent) is a unique defective passenger RNA virus requiring helper functions provided by the
HBV, including provision of the hepatitis B surface antigen coat for virion assembly and penetration into
hepatocytes (151) (e262). Transmission is via a parenteral route. In about 80% of those affected, chronic
hepatitis D progresses to cirrhosis. These individuals are also at risk for HCC. Survival after transplantation is
better than for other types of viral hepatitis, and reinfection is rare.
Hepatitis E
The hepatitis E virus (HEV; enterically transmitted non-A, non-B hepatitis) was identified in 1983 and cloned in
1990 (3) (e9,e111,e157,e342,e464). HEV is a single-stranded RNA virus that is water-borne and has an
incubation period of 6 weeks. It is responsible for large epidemics of acute hepatitis in parts of Asia, Middle East,
Africa, and Mexico. Transmission is fecal-oral, through contaminated water secondary to virion shedding in
stools. Young adults are most commonly infected. The illness is usually self-limiting, except in pregnant women
who tend to have severe disease and a high mortality rate (up to 25%). Chronic infection is unknown. Diagnosis
is based on serologic detection of anti-HEV antibodies. Liver biopsy is not usually performed for diagnosis.
Biopsy morphology is of acute hepatitis; fatal cases may show submassive to massive necrosis. No specific
treatment or vaccine is available.
Hepatitis G
The Hepatitis G virus (HGV or GB virus-C) is a flavivirus with global distribution that is transmitted primarily by
parental routes, but can also be transmitted sexually and perinatally (149) (e40,e61,e411). There is no
convincing evidence that HGV is a primary hepatotropic virus, and it has not been known to cause acute or
chronic hepatitis. There is controversy as to whether HGV should be included among the well-established
hepatitis viruses A through E; however, there is coinfection with HCV in up to 20% of patients with hepatitis E
and coinfection in patients with HIV infection.
Pathology of the Viral Hepatitides
Microscopic features of acute viral hepatitis, regardless of specific viral etiology, are characterized by lobular
disarray and inflammation (Figure 15-34). Liver injury is manifest by ballooning degeneration, individual cell
necrosis with dropout of hepatocytes, and acidophilic (Councilman) bodies. Concomitant regenerative activity is
evidenced by mitoses and binucleate or multinucleate cells. The cellular infiltrate is predominantly mononuclear
and has a lobular and portal distribution. Portal areas are uniformly infiltrated with lymphocytes. Plasma cells,
neutrophils, and eosinophils may be present. The infiltrate may extend into the periportal lobule, but in contrast
to chronic hepatitis with marked activity, periportal necrosis is not usual, and all portal areas are uniformly
involved. There is hyperplasia of the sinusoidal lining cells, and Kupffer cells may contain lipofuscin pigment.
Cholestasis is variable and usually mild, seen most often in zone 3. In the cholestatic form of hepatitis, prominent
cholestasis simulating extrahepatic obstruction may be seen. In subsiding hepatitis, the changes become less
prominent and may resemble chronic hepatitis with mild to moderate activity. Clusters of macrophages (PAS-
positive, diastaseresistant) may suggest a recent acute hepatitis in these cases. In more severe forms of acute
hepatitis, bridging necrosis with loss of hepatocytes may be accompanied by reticulin collapse and the formation
of passive septa between central veins and between central veins and portal areas. The presence of bridging
necrosis is an adverse prognostic factor that may be associated with a fatal outcome or progression to cirrhosis
(118) (e82). In a few cases, the course may be fulminant with a high mortality rate; at autopsy, submassive
necrosis with few surviving hepatocytes is seen in the
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periportal zones. The major portion of the lobule shows diffuse loss of hepatocytes accompanied by collapse and
approximation of the portal areas. Some degree of regenerative activity of the surviving periportal hepatocytes
may be evident in the form of pseudoductular or neocholangiolar proliferation (e531). Lymphocytes, plasma cells,
neutrophils, and eosinophils are seen in the sinusoids and space of Disse, and an endophlebitis may be seen,
particularly if more than 10 days have elapsed since the onset of the process. Inflammation is seen in the portal
areas. Kupffer cells contain cell debris and lipofuscin pigment. An etiologic distinction between the acute
hepatitis caused by hepatitis A, B, C, D, and E viruses is not possible on morphologic grounds alone, although
reports of acute hepatitis caused by hepatitis C describe the presence of lipid in the hepatocytes and a
prominent sinusoidal mononuclear cell infiltrate with marked hypertrophy of the sinusoidal lining cells (Figure 15-
35). The differences between hepatitis A and B infection are not readily appreciated. Perivenular cholestasis,
interface hepatitis with a dense portal infiltrate with frequent plasma cells, and extensive microvesicular steatosis
are considered to be more characteristic for HAV-associated acute viral hepatitis. With HBV acute viral infection,
hepatocytes with ground-glass cytoplasm is associated with abundant HBsAg production and may be somewhat
helpful in differentiating HBV from HAV.
FIGURE 15-34 ▪ Viral hepatitis—Hepatitis B. A: Expansion of portal region by chronic inflammatory cells with
extension into zone 1 by piecemeal necrosis (H&E, 100×). B, C: Zone 1 hepatocytes with deeply eosinophilic,
glassine cytoplasm, and chronic inflammatory cells extending into Zone 1 (H&E, B: 200×, C: 400×). D: Necrotic
hepatocytes (apoptotic/acidophil bodies) with pyknotic nuclei and densely eosinophilic cytoplasm (H&E, 400×).
E: Diffuse fibrosis extending from the portal region into the hepatic lobule (Trichrome, 40×).
FIGURE 15-34 ▪ (continued) F, G: Hepatocytes immunoreact with Hepatitis B core antigen in nuclear pattern (F:
400×) and with Hepatitis B surface antigen in cytoplasmic pattern (G: 400×). H: Hepatocyte with intranuclear
hepatitis B virus inclusions (Electron microscopy, 75,000×).
FIGURE 15-35 ▪ Viral hepatitis—Hepatitis C. A: Portal chronic inflammation with lymphoid aggregate with
germinal center (H&E, 200×). B: Hepatocytes with microsteatosis and occasional chronic inflammatory cells in
sinusoids (H&E, 400×)
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HBV and HCV infection may lead to chronic liver disease. Histopathologic features that predict progression to
chronicity include bridging necrosis, prominent portal infiltrate with periportal extension, and distortion of the
lamina limitans, lymphoid follicles, and early fibrosis. The concomitant demonstration of the surface and core
antigen of HBV by immunohistochemistry has been associated with progression to chronic liver disease. The
histopathology of hepatitis B infection has been recently comprehensively reviewed (118).
Chronic hepatitis is not a single disease, but rather a clinicopathologic syndrome that may have a variety of
causes (48, 126) (e166,e466,e677,e756). Traditionally, chronicity has been defined clinically as continuing
disease for at least 6 months. This definition still has some practical utility, but asymptomatic disease must also
be taken into account; for example, both HCV and autoimmune hepatitis (AIH) may remain asymptomatic for long
periods. The terms chronic active hepatitis (CAH), chronic persistent hepatitis (CPH), and chronic lobular
hepatitis (CLH) have become obsolete and should not be used.
The chronic hepatitides consist of chronic necroinflammatory diseases in which hepatocytes rather than biliary
structures appear to be the main target of attack. Chronic cholestatic diseases, such as primary biliary cirrhosis
(PBC) and primary sclerosing cholangitis (PSC), and metabolic disorders, such as Wilson disease and A1AT
deficiency, are not always included under the headings of chronic hepatitis (48). However, they may show similar
morphologic features, and there is thus practical merit in considering them in the broader spectrum of chronic
hepatitis (2).
Various etiologic types of chronic hepatitis share a number of histopathologic characteristics that may vary over
time in an affected individual. Most of these common morphologic features allow the pathologist to assess the
grade (severity of inflammatory activity) and stage (degree of fibrosis) of the disease process, but do not always
allow a definitive distinction between the various etiologies. In general, lobular inflammation predominates in
acute forms of hepatitis, and portal and periportal inflammation predominates in chronic hepatitis. Chronic
hepatitis with flares of disease activity commonly shows lobular hepatitis, together with portal and periportal
inflammation and fibrosis (48, 118) (e94).
Table 15-10 ▪ GRADING OF DISEASE ACTIVITY IN CHRONIC HEPATITIS
Grading Terminology Criteria
Lymphocytic Lobular Inflammation and

Semiquantitative Descriptive Piecemeal Necrosis Necrosis
0 Portal inflammation None None

only; no activity
1 Minimal Minimal, patchy Minimal; occasional spotty

necrosis
2 Mild Mild; involving some Mild; little hepatocellular

or all portal tracts damage
3 Moderate Moderate; involving Moderate; with noticeable

all portal tracts hepatocellular change
4 Severe Severe; may have Severe; with prominent diffuse

bridging fibrosis hepatocellular damage
aWhen a discrepancy exists between criteria, the more severe lesion should determine the grade.
Portal inflammation (Figures 15-34 and 15-35) is common to all forms of chronic hepatitis and is composed
mainly of a mixture of lymphocytes, plasma cells, and macrophages. Lymphoid aggregates with or without
germinal centers are more often seen in HCV-associated chronic viral hepatitis (Figure 15-35). Periportal
inflammation commonly accompanies local hepatocyte damage. This necroinflammatory process is referred to as
lymphocytic piecemeal necrosis or interface hepatitis. The composition of these inflammatory infiltrates is
identical to those in the portal tracts. As a consequence of the necroinflammatory process, collagen and elastin is
deposited. In contrast to portal and periportal inflammation, lobular inflammation usually consists of single small
clusters of mononuclear cells rather than confluent sheets. Lobular inflammation is usually accompanied by
hepatocellular damage. Hepatocellular damage is generally manifested by scattered necrotic hepatocytes
(acidophilic, apoptotic, or Councilman bodies), hepatocellular nuclear disarray (anisonucleosis), mitotic activity,
and hepatocellular swelling (ballooning degeneration). Apoptotic hepatocytes are characterized by pyknotic
nuclear remnants and dense retracted cytoplasm. Degenerative and regenerative hepatocellular changes are
frequently more impressive than the number of inflammatory cells.
Over time, chronic hepatitis leads to progressive fibrosis, which begins in portal areas, extends to periportal
zones, and eventually links portal tracts to other portal tracts and to terminal hepatic venules. After fibrous septa
have formed, regenerative nodules, indicative of cirrhosis, may appear. With the exception of HCV, in which
approximately 70% of cases show fatty change (12), steatosis is uncommon in chronic hepatitis. Steatosis in a
liver biopsy may be unrelated to viral hepatitis and may purely reflect of background fatty change. Chronic viral
hepatitis is rarely cholestatic.
Pathologic reporting of liver biopsies should include the etiology, grade, and stage of the chronic hepatitis in the
final diagnosis (Tables 15-10 and 15-11). Development of cirrhosis may be related to the duration of CAH.
In the asymptomatic patient with chronic hepatitis B, the liver may show no abnormality except for the
groundglass hepatocytes, which represent cells containing HBsAg (Figure 15-34). The ground-glass hepatocyte
is larger than
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the normal hepatocyte and has a smooth, uniform, pale, eosinophilic cytoplasm, often with a clear halo. The
nucleus may be displaced to the periphery. These cells show a positive staining reaction with orcein and
aldehyde fuchsin. Immunohistochemical staining is more sensitive and specific. The hepatitis B core antigen
(HBcAg) is identified predominantly in the nuclei (Figure 15-34), and may correspond with the so-called sanded
nuclei seen on hematoxylin and eosin stains. The distribution pattern of the tissue markers varies with the type of
hepatic disease and is related to the host's immune response. Immunocytochemical staining for HBsAg and
hepatitis B early antigen is also available. Electron microscopic examination may reveal HBsAg in the cytoplasm
of hepatocytes, as 22 nm spheres and rods (Figure 15-34).
Table 15-11 ▪ STAGING OF CHRONIC HEPATITIS
Staging Terminology
Semiquantitative Descriptive Criteria
0 No fibrosis Normal connective tissue
1 Portal fibrosis Fibrous portal expansion
2 Periportal Periportal or rare portal-portal septa

fibrosis
3 Septal fibrosis Fibrous septa with architectural distortion;no obvious

cirrhosis
4 Cirrhosis Cirrhosis
Nonhepatotropic viruses that may involve the liver as part of a systemic infection include herpes simplex, human
herpesvirus-6, varicella, adenovirus, ECHO virus, Epstein-Barr virus (EBV), parvovirus, and cytomegalovirus
(CMV). The liver may also be affected in acquired immune deficiency syndrome (AIDS), and a chronic hepatitis-
like disorder has been described in children with AIDS. Hepatic involvement can occur in rickettsial diseases.
The hepatic lesion in childhood cases of Rocky Mountain spotted fever has been described elsewhere.
FIGURE 15-36 ▪ Viral agents in fulminant hepatic failure. A, B: Adenovirus hepatitis with deeply eosinophilic
cherry-red homogenous smudgy inclusions with hepatocytes (H&E, A: 400×), and adenovirus particles in nuclei
by electron microscopy (B: 75,000×).
FULMINANT HEPATIC FAILURE

Fulminant hepatic failure is characterized clinically by altered mental status and coagulopathy of rapid onset (<8
weeks after jaundice) (88, 99) (e136,e203,e379,e380,e410). Its etiology is variable around the world and
includes viruses (most commonly HBV, EBV, herpes simplex viruses, CMV) (Figure 15-36), drugs (most
commonly acetaminophen, other drugs with idiosyncratic reactions), pregnancy-induced, metabolic (inborn errors
of metabolism), malignancy, and other rare causes. Ten to twenty percent of cases remain cryptogenic.
Prognosis depends on age, etiology, and rapidity of onset of disease. A liver biopsy reveals zones of
hepatocellular loss, a variable inflammatory reaction, and residual foci of hepatocytes (Figure 15-37). Residual
hepatocytes have abnormalities ranging from steatosis to ballooning degeneration. Small regenerative nodules
of hepatocytes may be seen at a somewhat later stage in the evolution of the process. In most cases, there are
few clues about the etiology in the biopsy findings. Liver transplantation is often successful. New experimental
treatments include use of extracorporeal liver assist devices and hepatocyte transplantation.
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FIGURE 15-36 ▪ (continued) C, D: CMV hepatitis with characteristic intranuclear “owl-eye” inclusions with bile
duct epithelium (H&E, C: 400×) and CMV/Herpes viral particles in nuclei by electron microscopy (D: 75,000×).
E: Paramyxoviral particles in the hepatocyte cytoplasm (electron microscopy, 55,000×).
FIGURE 15-37 ▪ Fulminant hepatic failure. A: Liver explant with tense, distended liver capsule. B: Liver explant
cross section demonstrating diffuse liver necrosis with red-brown fine punctate areas representing viable liver
parenchyma.
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FIGURE 15-37 ▪ (continued) C: Central area of hepatocyte necrosis with dense eosinophilia, hemorrhage in the
background and necrotic hepatocytes (H&E, 200×). D: Residual bile ducts, hepatocytes organized into
pseudoacini and necrotic hepatocytes (H&E, 400×). E: Residual bile ducts with rare hepatocytes and
background of chronic inflammatory cells (H&E, 400×). F: Trichrome stain highlights residual bile ducts, loss of
hepatocytes, and replacement by fibrotic tissue (200×).
PRIMARY SCLEROSING CHOLANGITIS AND AUTOIMMUNE HEPATITIS

PSC is a progressive hepatobiliary disease characterized by a cholestatic syndrome (98)
(e21,e35,e116,e246,e351,e375, e394,e407,e408,e491,e636,e637,e703). Diagnostic imaging abnormalities
consist of segmental narrowing and dilatation of intrahepatic and extrahepatic ducts. The disease is seen
primarily in young men in the third to fifth decades of life, but it also seen in the pediatric population including
neonates.
The pathogenesis is unknown, but a strong association with ulcerative colitis (UC) has been documented in most
cases. Inflammatory bowel disease (UC) has been associated with sclerosing cholangitis in 70% of cases (98)
(e21,e351,e394,e408). However, PSC is seen in only about 5% of patients with UC. Langerhans cell
histiocytosis occurs in 15% of cases, and immunodeficiency is associated with another 10% of children. No
apparent underlying disease is present in 24%, including cases of neonatal onset. The clinical presentation of
childhood PSC is highly variable and frequently without features of cholestasis. Typically, elevated alkaline
phosphatase (ALP) and bilirubin are noted. In addition, hypergammaglobulinemia, DRw52a (HLA subtype),
perinuclear-antineutrophil cytoplasmic antibody (p-ANCA), and IgM elevation may be identified by serologic tests.
Clinical similarity to AIH is common (152). Contrast digital imaging of the intrahepatic biliary tree by retrograde
endoscopy shows a characteristic beaded appearance. This is due to strictures and secondary dilation of the
affected bile ducts.
The histopathologic changes in the liver are not diagnostic in most cases (Figure 15-38). Portal fibrosis,
pericholangitis, fibrous obliterative cholangitis, and cirrhosis are the range of microscopic features. Fibrous
obliterative cholangitis consisting of concentric whorls of dense collagen, with an onion
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skin appearance, surrounding the bile ducts is regarded as a characteristic lesion. This is seen only early in the
evolution of the disease. Fibroinflammatory stricture of bile ducts may be seen at various sites from the ampulla
of Vater to the interlobular bile ducts. Histopathologic staging of PSC is based upon degree of involvement
(Stage I—portal; Stage II—periportal; Stage III—septal; Stage IV—cirrhosis). Stage I (portal) has concentric
periductal fibrosis with a lymphocytic infiltrate around bile ducts. Stage II (periportal) has fibrosis extending in the
periportal tissues with interface hepatitis and reactive bile ducts. Stage III (septal) has obliterated bile ducts and
bridging fibrosis. Stage IV (cirrhosis) has biliary type cirrhosis.
FIGURE 15-38 ▪ Primary sclerosing cholangitis. A: Severe portal chronic inflammation with bile duct proliferation,
fibrosis, and interface hepatitis (H&E, 100×). B, C: Concentric fibrosis around bile ducts (onion-skinning) and
portal fibrosis (H&E, 200×). D: Liver explant for primary sclerosing cholangitis with diffuse bile pigmentation and
biliary cirrhotic pattern.
Cholangiography is essential for diagnosis to evaluate medium to large intrahepatic ducts, since 40% of children
lack extrahepatic duct involvement (152). The most serious complication is adenocarcinoma of the bile duct and
colon in patients with concurrent PSC and UC (e376). The prognosis appears to be more favorable in children
than in adults. Liver transplantation is required for children who progress to biliary cirrhosis and hepatic
decompensation. Recurrence of PSC may occur in the transplanted liver.
AIH may be present in children with signs and symptoms of acute hepatitis (50% to 60%), fulminant liver failure
(10%), or a more chronic, insidious onset (30% to 40%) (111, 123)
(e26,e74,e153,e256,e443,e444,e663,e699,e739). This disease is more typically seen in young and middle age
women. Two types of AIH are recognized: type 1 with antinuclear antibodies (ANA), antismooth muscle
antibodies (SMA), antiactin antibodies, soluble liver antigen, and acute asialoglycoprotein receptor; and type 2
with anti-LKM1. Younger children present with anti-LKM1 with or without ANA or SMA antibodies. There is no
difference in clinical outcome between the types of AIH. Family history of autoimmune disorder is noted in 40% of
cases. Affected children may have other autoimmune disorders including lymphocytic (Hashimoto) thyroiditis,
rheumatoid arthritis, Sjögren syndrome, and UC. Hyperglobulinemia is a common feature. It is important to
ensure that viral serologic markers are negative. Liver biopsy demonstrates plasma
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cells within the chronic inflammatory infiltrate in the portal tracts and an aggressive interface hepatitis, which may
lead to collapse (Figure 15-39). Marked lobular chronic inflammatory infiltrates with plasma cells may be seen.
Hepatocellular injury with acinar (rosette) formation and syncytial giant hepatocytes can be features as well.
Plasma cells with or without hepatocyte rosette formation are considered to be highly suggestive of AIH.
However, one should remember that plasma cells can be seen in other chronic hepatitides as well. Because
lymphoid aggregates may be seen in HCV, it is important to eliminate this from consideration. Cirrhosis develops
in 90% of cases. In a certain proportion of children, serologic and histologic evidence is supportive of AIH at
initial diagnostic evaluation. However, diagnostic imaging and liver biopsy have features that support PSC. When
this occurs, the term autoimmune sclerosing cholangitis overlap syndrome is employed. These patients appear to
respond to immune suppression therapy.
FIGURE 15-39 ▪ Autoimmune hepatitis. A, B: Plasma cells within portal regions and within the hepatic lobules
(H&E, 400×). C: Hepatocytes arranged in pseudoacinar pattern with occasional plasma cells and increased
fibrous tissue (H&E, 400×). D: Explanted liver for autoimmune hepatitis with macronodular pattern of cirrhosis
and bile staining.
ABSCESSES
Pyogenic abscesses are uncommon in the liver in the pediatric patient and, when they occur, may be single or
multiple (76, 91) (e615,e623,e638). The infection may be hematogenous or ascend via the biliary tract. In the
neonate, umbilical vein catheterization complicated by septic omphalitis poses an additional hazard. Ascending
cholangitis may be associated with intrahepatic abscesses, especially after a portoenterostomy procedure for
EHBA. Hepatic abscess may occur in the setting of a systemic disorder. In patients with congenital or acquired
neutropenia or aplastic anemia, hepatic abscesses may show a paucity of neutrophils, and coagulative necrosis
without liquefaction may be seen. Hepatic abscesses may be the initial presentation of chronic granulomatous
disease (CGD) (91); as many as one-third of hepatic abscesses in children are a complication of CGD (72).
These abscesses show a central area of suppuration, often with a surrounding
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palisade of macrophages. Pigmented lipid-laden histiocytes in the portal tracts and sinusoidal lining cells are
characteristic of this process. Blunt trauma associated with hepatic necrosis may be complicated by abscess
formation. Occasional reports have documented hepatic abscess without a predisposing condition. Amebic liver
abscess may be seen in areas endemic for amebiasis.
Grossly, the abscesses may be multiple and range from small yellow foci scattered throughout the liver to large
cavitary lesions with purulent debris. Microabscesses consist of focal collections of neutrophils with no zonal
distribution. Larger abscesses show a central area of liquefaction necrosis in which degenerating neutrophils are
seen. At the periphery, there is characteristically a mixed cellular infiltrate consisting of neutrophils and
mononuclear cells and a variable fibroblastic proliferation.
Polymicrobial infection is present in about 80% of cases (2.4 isolates per specimen), with anaerobes and
microaerophilic streptococci being more common (76) (e42,e88,e638). The predominant anaerobes implicated
are Peptostreptococcus, Bacteroides sp., Fusobacterium sp., and Clostridium sp., whereas common aerobes
implicated are Escherichia coli , Streptococcus group D, Klebsiella pneumoniae, and Staphylococcus aureus.
Diminutive abscesses consisting of no more that a few neutrophils are seen in CMV hepatitis in
immunosuppressed children and adults. The mortality rate for pyogenic liver abscesses has decreased to less
than 10% with improved diagnostic imaging, percutaneous draining techniques, and antibiotics (e42).
PARASITIC DISEASES
A variety of parasitic diseases can involve the liver (144) (e250,e348,e395,e422,e588). Among the protozoal
infections are toxoplasmosis, malaria, leishmaniasis, and amebiasis. Toxoplasma infections have a worldwide
distribution. Infection may be transmitted through contact with house pets, such as cats. Congenital infections
are an important cause of illness with prominent hepatic manifestations. Giant cell transformation of hepatocytes
may be seen. Occasionally, the parasite may be demonstrable.
Acute Plasmodium falciparum malaria may be fatal. At autopsy, the liver is enlarged and tense with a dark red or
slate gray color. There is marked engorgement of the sinusoids and central veins, and erythrocytes may contain
parasites. There is Kupffer cell hyperplasia and phagocytosis of ruptured erythrocytes. Within Kupffer cells, the
dark brown malarial pigment is a characteristic cytoplasmic feature. This hemazoin pigment is formed by the
trophozoite from the breakdown of hemoglobin and does not give a positive Prussian blue reaction.
In leishmaniasis (kala-azar), hyperplastic Kupffer cells contain the parasites (Leishman-Donovan bodies).
Infiltration with lymphocytes, plasma cells, and histiocytes may be seen in portal areas and lobules, and
granulomas may form.
Amebic infection of the liver is the most frequent extraintestinal complication of the disease and it manifests
usually as a single abscess, most often involving the right lobe. The abscess cavity contains red-brown, thick
(“anchovy sauce-like”) material. The abscess wall consists of a layer of necrotic parenchyma, external to which a
mixed inflammatory cell infiltrate is seen. A fibrous capsule may be present, and the adjacent liver is compressed.
Amebae may be demonstrable in the necrotic zone or in the compressed parenchyma as PAS-positive round or
oval bodies about the size of macrophages.
Liver involvement may also occur in infestation by a variety of helminths. In schistosomiasis, liver injury results
through migration of ova in the portal venous system. The ova elicit a granulomatous response, and in severe
infection with Schistosoma japonicum, diffuse fibrosis and portal hypertension may result. Liver flukes
(Clonorchis sinensis, Fasciola hepatica) inhabit major intrahepatic ducts and cause inflammation and epithelial
injury. Biliary hyperplasia, cholangitis, and periductal fibrosis are common findings with liver flukes. Hydatid cyst
is caused by infestation with the larval stage of the cestodes Echinococcus granulosus and E. multilocularis.
The right lobe is more frequently involved. The cyst has a thick, white wall and a cavity in which the fluid
contains fine granular sediment (“hydatid sand”). The cyst may be unilocular or multilocular. The cyst has a
characteristic laminated outer layer and an inner layer containing multiple nuclei. Brood capsules are formed
from numerous scolices and arise from the inner germinal layer. Invaginations of the cyst give rise to daughter
cysts. Secondary cholangitis may result from intrahepatic bile duct obstruction. Toxocariasis (visceral larva
migrans) results from migration of the larvae of Toxocara canis or T. cati (144) (e395). Granulomas containing
larval fragments may be seen in the liver. Ascariasis infestation is associated with numerous foul-smelling
cavities in the liver upon gross examination. The liver tissue demonstrates necrotic debris with a granulomatous
and eosinophil inflammatory response to degenerated parasites.
GRANULOMATOUS HEPATITIS
Granulomas in the liver are associated with the same etiologic agents as granulomas at other sites (97)
(e65,e189, e330,e358,e748). The frequency of granulomas in the liver varies with geographic location, due to
variation in causative agents in different populations. The etiologic associations are shown in Table 15-12.
Nevertheless, tuberculosis and sarcoidosis account for the majority of cases.
Histopathologic evaluation includes a search for an etiologic agent with appropriate special stains, especially for
acid-fast bacilli and fungi. The auramine O stain for fluorescent microscopy is more sensitive in demonstrating
acid-fast bacilli than standard stains. PCR for mycobacteria is also possible from formalin-fixed and paraffin-
embedded tissue.
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Table 15-12 ▪ HEPATIC GRANULOMAS
Bacterial
Tuberculosis
Atypical mycobacteria
Listeriosis
Tularemia
Brucellosis
Rochalimaea henselae (cat scratch disease)
Mycotic
Candida
Histoplasmosis
Cryptococcosis
Blastomycosis
Coccidioidomycosis
Rickettsial and spirochetal

Q fever
Syphilis
Viral
Infectious mononucleosis
Cytomegalovirus
Parasitic and protozoal
Schistosomiasis
Visceral larva migrans
Ascariasis
Toxoplasmosis
Leishmaniasis (kala-azar)
Drug-related
Sulfonamides
Diphenylhydantoin
Sulfonyl urea compounds
Allopurinol
Miscellaneous
CGD of childhood
Sarcoidosis
Hodgkin lymphoma
Crohn disease
Foreign body
Undetermined etiology
VASCULAR DISORDERS
Cavernous Transformation of the Portal Vein
The most important entity in this group of disorders is portal vein obstruction due to thrombosis and cavernous
transformation resulting from recanalization of the thrombus (e25,e290,e299,e333,e783). This is the most
frequent cause of noncirrhotic portal hypertension in children. Extrahepatic causes of portal hypertension,
including portal vein obstruction, are reported in approximately 50% of cases (e25). Umbilical vein catheterization
and omphalitis have been incriminated most frequently, with other mechanisms including local infections,
portoenterostomy, sepsis, and chemotherapy (e299,e333). Hypercoagulopathy secondary to protein C, protein
S, and antithrombin III deficiencies are frequently found in children with portal vein obstruction (e186). The liver is
histologically normal in most cases of portal vein thrombosis or cavernous transformation of the portal vein.
Budd-Chiari Syndrome
Obstruction of the hepatic veins may occur in the main branches or ostia leading to Budd-Chiari syndrome (198)
(e38,e188,e264,e294,e537,e731). The lesion occurs most frequently in women in the third and fourth decades of
life and is uncommon in childhood. In young women, there is an association with contraceptive medications and
pregnancy. Paroxysmal nocturnal hemoglobinuria, sickle cell disease, nephrotic syndrome, TPN, blunt trauma,
myeloproliferative disorders, and coagulation abnormalities may also be associated with Budd-Chiari syndrome.
Venous occlusion by tumor occurs less frequently in childhood than in adults. Congenital webs and obliteration
of the suprahepatic inferior vena cava are seen in children. Budd-Chiari syndrome may occur after giant
omphalocele repair. Thrombosis of hepatic veins and retrohepatic inferior vena cava may result from direct
pressure on the hepatic venous outlet after visceral reduction and final abdominal wall closure. Gaucher disease
has also been implicated in rare instances.
Clinical features associated with Budd-Chiari syndrome include ascites and hepatomegaly (198) (e38,e188,
e264,e294,e537,e731). The liver, in early stages, shows severe centrilobular congestion, hepatocyte
degeneration and loss, and erythrocytes in the space previously occupied by the liver cells in zone 3 (Figure 15-
40). Central veins are not affected, but sublobular veins may contain thrombi. Pericentral fibrosis with extension
into adjacent parenchyma causes distortion of the architecture and may progress to cirrhosis.
Venoocclusive Disease
Venoocclusive disease (VOD) (18) (e50,e104,e142,e173, e287,e393) was initially described in Jamaican
children and ascribed to pyrrolizidine alkaloids in Senecio tea. Other etiologic associations are cytotoxic agents
used
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for malignant disease therapy and in preparation for bone marrow transplantation, hereditary tyrosinemia, familial
immune deficiency disorders, and irradiation (e500,e509). The condition has also been described in newborn
infants (e80,e167,e313).
FIGURE 15-40 ▪ Budd Chiari syndrome. Centrilobular ischemia and hepatocyte degeneration with less affected
hepatocytes away from Zone 3 (H&E, 200×).
FIGURE 15-41 ▪ Venoocclusive disease. A, B: Partial to nearly complete obliteration of central veins with
pericentral vein fibrosis (H&E, 400×).
Early in the course of the disease, there is massive centrilobular hemorrhage with hepatocyte degeneration or
loss. The abnormal central veins have narrowed lumina and widened subendothelial spaces, containing collagen
fibers, fragmented cells, cell debris, and hemosiderin-laden macrophages. At this stage, central vein
abnormalities are subtle and require special stains to demonstrate collagen deposition. Later in the course of the
disease, there is intimal thickening due to reticulin and collagen deposition, and presence of foam cells, causing
partial or complete obliteration of vessel lumens (Figure 15-41). Central hepatocytes (zone 3) are atrophic, and
cholestasis may be seen. Pericentral fibrosis with extension into the adjacent parenchyma distorts the
architecture, but true cirrhosis is infrequent. Allograft rejection may resemble VOD, and this needs to be taken
into consideration prior to making a diagnosis of VOD.
Peliosis Hepatis
Peliosis hepatis was initially described in adults with chronic debilitating diseases, steroid medications, HIV,
mycobacterial infection, and wasting conditions (186) (e173,e174,e334,e592,e729). This condition was
described in a child with CF who died at the age of 11 years, after which additional reports documented peliosis
hepatis in the pediatric age group, including the neonatal period. Two previously healthy young children in whom
peliosis hepatis presented as acute hepatic failure associated with E. coli pyelonephritis have also been
reported. Both patients had active intraperitoneal hemorrhage from the peliotic liver lesions (e312). Focal peliosis
hepatis has been found incidentally in five children succumbing to an asphyxiating death (e614). Androgenic
anabolic steroids, oral contraceptives, thiopurines, and danazol play a role in development of this lesion.
Resolution of the lesion tends to occur after discontinuing such medications. Liver infection by Bartonella
henselae in HIV-infected patients is known to lead to peliosis hepatis (186). Also, peliosis hepatis occurs with
increased frequency in renal transplant recipients (e107). The liver contains grossly identifiable multiple blood-
filled spaces, which, on microscopic examination, consist of pools of erythrocytes in the hepatic lobule with no
zonal predilection (Figure 15-42). A definitive endothelial lining is not identified. The early lesion consists of
localized areas of sinusoidal dilatation, likely due to disruption and injury to the sinusoidal endothelial cells.
Disruption of sinusoidal reticulin fibers may be demonstrated using typical reticulin stains. In HIV-infected patients
with Bartonella-associated lesions, there may be myxoid perisinusoidal stroma with granular clumped material.
Within the granular material, organisms may be detected with Warthin-Starry staining and PCR. Rupture and
hemoperitoneum are potential complications.
FIGURE 15-42 ▪ Peliosis hepatis. Early lesion of peliosis with widely dilated sinusoids, which tends to be
localized due to sinusoidal endothelial injury (H&E, 400×).
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Hepatic Hemorrhage
Hepatic hemorrhage may occur as a result of blunt or sharp trauma (e215,e223,e625). Spontaneous
subcapsular hemorrhage in the newborn occurs most frequently in premature infants and may be a cause of
morbidity. A review of infant autopsies showed a 15% incidence of subcapsular hemorrhage. At-risk infants tend
to be premature male infants with chronic problems during gestation and complications during labor and delivery,
as well as sepsis. Hemoperitoneum due to liver rupture may lead to hypovolemic shock.
TOTAL PARENTERAL NUTRITION RELATED INJURY

Hepatic abnormalities secondary to TPN were first described in a premature infant. The infant died after 71 days,
and the liver at postmortem examination showed cirrhosis, bile duct proliferation, and cholestasis. Subsequent
reports have confirmed the association of hepatobiliary dysfunction with TPN (62)
(e90,e259,e337,e451,e556,e706). The associated cholestasis is seen most frequently in the premature infant,
with low birth weight and low gestational age being the greatest risk factors. The incidence and severity of the
disease are greater in infants with gastrointestinal disease or intestinal resection.
FIGURE 15-43 ▪ Total parenteral nutrition. A: Portal tract expansion by fibrous tissue with bile duct proliferation
and cholestasis (H&E, 100×). B, C: Pseudoacinar arrangement of hepatocytes with obvious cytoplasmic
cholestasis, apoptotic hepatocytes (C), and increased sinusoidal fibrous tissue (H&E, 400×). D: Trichrome
staining highlights pseudoacinar pattern and lobular fibrosis (H&E, 200×).
Cholestasis increases with prolonged TPN infusion (62). Most infants with TPN-associated cholestasis and
subsequent cirrhosis have severe gastrointestinal disease, such as necrotizing enterocolitis, gastroschisis, and
volvulus, or have undergone intestinal resection. These infants are also subject to infection, cardiopulmonary
dysfunction, shock, and hypoxia. Toxicity of the infusate, especially amino acid composition and lipid content,
has been considered a factor in liver dysfunction associated with TPN.
The onset of jaundice is insidious, and the infant may manifest no other evidence of hepatic disease. The
earliest biochemical abnormality is the elevation of serum bile acid concentration, as early as 5 days after
beginning TPN, and routine study of serum bile acids may help in diagnosis. Hyperbilirubinemia is usually seen 3
to 4 weeks after TPN initiation.
Histopathologic changes noted in TPN-associated disease are nonspecific and quite variable (Figure 15-43).
Because
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there is no specific clinical, biochemical, or histopathologic marker, the diagnosis remains one of exclusion.
Canalicular and hepatocellular cholestasis, most pronounced in zone 3, is the initial finding and a constant
feature of TPN. There is lobular disarray with ballooned hepatocytes. Kupffer cell hyperplasia is present, with the
Kupffer cells containing lipofuscin pigment. Iron pigment is demonstrable within hepatocytes. Giant cell
transformation, pseudoacinar formation, and scattered foci of hepatocyte necrosis may be present.
Extramedullary hematopoiesis may be prominent. Focal inflammation is usually seen and may vary from mild to
severe. The cellular infiltrate is predominantly lymphocytic, but neutrophils and eosinophils may also be present.
A pericholangitis may be seen, along with focal fibrosis of variable degree. The vast majority of patients recover
with clearing of the jaundice after cessation of TPN, and commencement of enteral feedings. In repeat liver
biopsies, cholestasis usually clears. Hepatocyte ballooning, lobular disarray, and occasional cholestasis and
portal fibrosis may persist. However, cirrhosis and hepatic failure have been noted in infants receiving TPN.
CIRRHOSIS
Cirrhosis has been defined by the Working Group of the World Health Organization as a diffuse process
characterized by fibrosis and conversion of normal liver architecture into structurally abnormal nodules (9).
Cirrhosis is the end result of hepatic cell necrosis caused by a variety of injurious agents (9, 100) (e602).
Necrosis is associated with collapse, fibrosis, and regeneration, resulting in the formation of nodules.
The classification of cirrhosis may be etiologic or morphologic. Cirrhosis has many etiologies. Many metabolic
disorders are associated with cirrhosis and have been reviewed elsewhere (65). Alper disease, a putative
mitochondrial disorder, is characterized by progressive neuronal degeneration and cirrhosis in childhood
(e8,e191,e273). Alcoholic cirrhosis, a common cause of liver injury in adults, may rarely be seen in adolescents.
Hepatic changes resembling adult alcohol-associated injury has been described in the fetal alcohol syndrome
(e382). Cardiac cirrhosis in the pediatric population occurs most commonly in association with congenital heart
disease (e383). Hematologic conditions, such as hemophilia, can be associated with progressive liver disease
(e282). The role of trace metals in childhood cirrhosis has been detailed elsewhere (e241). Gallbladder
duplication has been described in association with childhood obstructive biliary disease and biliary cirrhosis
(e253). Etiologic associations with cirrhosis in childhood are presented in Table 15-13. Establishing the etiology
requires demonstration of the specific histopathologic characteristics of a disease, such as AIAT stored in
hepatocytes, ground-glass hepatocytes in HBV, or biochemical evaluation in metabolic disorders. As in the adult,
cirrhosis in childhood may be cryptogenic, with failure to identify an etiologic agent in the explanted liver.
Table 15-13 ▪CIRRHOSIS IN INFANCY AND CHILDHOOD
Causes of Cirrhosis Related Disorder
Infections Neonatal viral infection
Neonatal hepatitis
Viral hepatitis
CAH
Syphilis
Biliary obstruction EHBA
Choledochal cyst
Familial cholestatic syndromes
Paucity of intrahepatic bile ducts
Cholangitis
Vascular disease Hepatic vein occlusion
VOD
Constrictive pericarditis
Chronic congestive cardiac failure
Rendu-Osler-Weber disease
Hereditary syndromes Cerebrohepatorenal (Zellweger syndrome)
CF
Indian childhood cirrhosis
CHF
Metabolic abnormalities Galactosemia
Fructosemia
Tyrosinemia
Glycogenoses, types III and IV
A1AT deficiency
Gaucher disease
Niemann-Pack disease
Wolman disease
Cholesterol ester storage disease
Wilson disease
Hemochromatoses
Arginosuccinic aciduria
Cystinosis
Porphyria
Miscellaneous TPN
Malnutrition
Obesity
Alcohol
Sclerosing cholangitis
Histiocytosis X
Drugs
Morphologic classification is based on nodule size. In micronodular cirrhosis, nodules measure less than 3 mm in
diameter and are relatively uniform throughout the liver. Fibrous septa are delicate and extend from portal to
central areas or encircle the lobule. Macronodular cirrhosis is characterized by nodules larger than 3 mm, usually
with broad bands of fibrous tissue (Figure 15-44). Large nodules contain several lobules in which portal areas
and central veins are identifiable. In mixed type cirrhosis, the liver contains an approximately equal proportion of
small and large nodules. Transformation of one type to another can occur with continuing necrosis, collapse, and
fibrosis. In some conditions,
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cirrhosis is predominantly macronodular, such as after submassive bridging hepatic necrosis due to hepatitis or
toxic agents. A predominantly micronodular cirrhosis is associated with biliary atresia and cholestatic syndromes.
However, considerable overlaps exist owing to the transformation that may occur between the various
morphologic types of cirrhosis, and the etiology of cirrhosis cannot be ascertained from the morphologic type of
cirrhosis in all cases. This morphologic classification has therefore fallen out of favor.
FIGURE 15-44 ▪ Cirrhosis. A, B: Liver explant with a cirrhotic surface and cross section demonstrating numerous
macronodules and micronodules. C, D: Fibrous tissue separates nodules of hepatocytes lacking central veins
from each other. Note the variable size to the nodules (H&E, 200×).
Activity of cirrhosis is evaluated by identifying continuing hepatocellular necrosis and the degree of septal
inflammation. Portal hypertension with all its sequelae is a frequent complication (e405), although portal
hypertension may also be noncirrhotic in origin. Noncirrhotic portal hypertension may be suspected when the
patient presents with portal hypertension without parenchymal dysfunction (as reflected by maintained albumin
level and prothrombin time indicating preserved synthetic function) (e594).
Putative preneoplastic hepatic lesions may be found in cirrhotic livers. Liver cell dysplasia (large cell dysplasia) is
characterized by nuclear and cytoplasmic enlargement, nuclear hyperchromasia, prominent nucleoli, and
occasionally, multinucleation (8). Adenomatous hyperplasia (macroregenerative nodule) is a nodular lesion that
occurs in cirrhosis and is thought to progress to HCC through an intermediate lesion termed atypical
adenomatous hyperplasia (small cell dysplasia) (130). Atypical adenomatous hyperplasia occurs as an ill-defined
nodule within a cirrhotic nodule (the so-called nodule-in-nodule formation), identified by compression of
surrounding reticulin fibers and a different orientation of the liver plates. The evidence suggests that this lesion,
rather than liver cell dysplasia, is more likely the precursor of HCC in a cirrhotic liver. Although it typically takes
many years for HCC to develop, HCC may be associated with cirrhosis even in a neonate (e434).
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Table 15-14 ▪ HEPATICTUMORS IN PEDIATRIC PATIENTS, BIRTHTO 20 YEARS (AFIP 1970-
1999)
Type of Tumor N %
Hepatoblastoma 198 27.6
Hepatocellular carcinoma 135 18.9
Infantile hemangioendothelioma 119 16.5
Focal nodular hyperplasia 72 10.1
Mesenchymal hamartoma 57 8.0
Undifferentiated “embryonal” sarcoma 52 7.2
Nodular regenerative hyperplasia 32 4.5
Hepatocellular adenoma 27 3.8
Angiosarcoma 17 2.4
Embryonal rhabdomyosarcoma 7 1.0
TOTAL 716 100.0
HEPATIC TUMORS
Primary hepatic neoplasms account for 0.5% to 2.0% of all pediatric neoplasms and comprise a variety of benign
and malignant epithelial and mesodermal tumors. Incidences of these tumors change significantly from birth to 20
years of age (Tables 15-14, 15-15 and 15-16). Of 716 cases of the 10 most commonly occurring hepatic
neoplasms seen at the Armed Forces Institute of Pathology between 1970 and 1999, hepatoblastoma, HCC, and
hemangioendothelioma accounted for almost 65% (see Table 15-14).
FOCAL NODULAR HYPERPLASIA

Focal nodular hyperplasia (FNH) is a benign tumorlike lesion of the liver. Rather than a true neoplasm, it is
considered to be the result of a hyperplastic response to hemodynamic disturbance related to vascular
abnormalities. Although it most commonly occurs in women of childbearing and middle age, nearly 8% of cases
present in the first 15 years of life, with a slightly increased frequency in those 6 to 10 years of age (39%) and a
distinct female predominance of more than 3:1 (171) (Figure 15-45A).
Table 15-15 ▪ HEPATIC TUMORS IN PEDIATRIC PATIENTS, BIRTHTO 2 YEARS (AFIP 1970-
1999)
Type of Tumor N %
Angiosarcoma 4 1.4
Hepatocellular adenoma 0 0
Embryonal/rhabdomyosarcoma 0 0
TOTAL 285 100.0
Table 15-16 ▪ HEPATIC TUMORS IN PEDIATRIC PATIENTS, 5-12 YEARS (AFIP 1970-1999)
Type of Tumor N %
Hepatocellular adenoma 22 8.4
Angiosarcoma 6 2.3

Embryonal rhabdomyosarcoma 2 8
TOTAL 262 100.0
aPortions of this section were adapted from Stocker, JT Hepatic tumors in children. In: Suchy FJ, Liver
disease in children 2nd ed Philadelphia: Lippincott Williams and Wilkins. In press.
Pathogenesis
In 1985, Wanless and collaborators proposed that FNH is a hyperplastic response of the hepatic parenchyma to
a preexisting local arterial spiderlike malformation, likely with a developmentally abnormal origin (e752). FNH is
also related to well-known vascular diseases, such as hereditary hemorrhagic telangiectasia or congenital portal
vein absence (e24,e96,e165). Hepatocellular hyperplasia in FNH is thought to be secondary to increased arterial
flow and hyperperfusion of localized parenchyma (e228,e229,e752). An association between the use of oral
contraceptives in older children and adults and the development of FNH is still under debate. However, some
studies suggest that the use of contraceptive pills may increase the size of the nodules (e433,e601) or may
predispose to bleeding (e634).
A variety of associations have been anecdotally noted in children with FNH (Table 15-17) (e497,e586). FNH is
associated with vascular abnormalities, including hepatic hemangiomas, which supports the concept of a
vascular component in the pathogenesis of this lesion. FNH has also been reported in patients with a variety of
nonhepatic tumors and tumorlike conditions (21).
Clinical Features
The vast majority of lesions (90%) are asymptomatic, presenting as a mass on routine physical examination or as
an incidental finding at surgery or autopsy. Symptomatic cases may present with abdominal pain, weight loss,
vomiting, or diarrhea. Laboratory parameters in patients with FNH are rarely abnormal, and alpha-fetoprotein
(AFP) is not elevated.
Imaging studies can be extremely helpful in differentiating FNH from other benign or malignant hepatic lesions
(e20,e295), especially hypervascular lesions such as hepatocellular adenoma (HCA), HCC, and hypervascular
metastases. Color power Doppler allows, in most cases, its distinction from other focal liver lesions (e648). In
contrast
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to adenomas, imaging techniques are sufficient for diagnosis in 70% of cases. Magnetic resonance imaging
(MRI) has higher sensitivity and specificity for FNH than does ultrasonography or computed tomography.
Typically, FNH is isointense or hypointense on T1-weighted images, is slightly hyperintense or isointense on T2-
weighted images, and has a hyperintense central scar on T2-weighted images. FNH demonstrates intense
homogeneous enhancement during the arterial phase of gadolinium-enhanced imaging and enhancement of the
central scar during later phases (71). Arteriography often displays the prominent single or multiple feeder arteries
associated with FNH. Centrifugal filling from the feeder artery to the periphery of the lesion may be seen.
Ultrasonography may demonstrate a feeding artery with a radial vascular architecture, which, however, may not
be present in a lesion smaller than 3 cm in size. Cheon et al.
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(e125), however, noted that children often display a wide spectrum of imaging findings on various radiologic
examinations and that the typical centrally placed scar is not always seen. Superparamagnetic iron oxide (SPIO)-
enhanced MRI has been shown to be useful in differentiating benign lesions such as FNH and hepatic adenoma
from malignant hepatocellular lesions (e53).
FIGURE 15-45 ▪ Focal nodular hyperplasia. A: Age distribution in 79 cases. B: A well-circumscribed lesion is
subdivided into smaller nodules by bands of connective tissue. C: Arborizing septa of fibrous connective tissue
surround and subdivide nodules of hepatocytes (Reticulin stain, original magnification 15×). D: The edges of the
fibrous septa contain scattered small ducts along with small to large vessels, some displaying eccentric
subintimal thickening (H&E stain, original magnification 60×).
Table 15-17 ▪ ASSOCIATED ANOMALIES IN CHILDREN WITH FOCAL NODULAR

HYPERPLASIA
Glycogen storage disease

Gastroschisis, absent gallbladder
Cardiac hypertrophy, nodular hyperplasia of thyroid and adrenal cortex
Ovarian dysgerminoma
Persistent hypoglycemia
Multiple telangiectasia on arms and legs
Hypospadias, bilateral syndactyly of toes, bilateral hydrocele
Left-sided hemihypertrophy, syndactyly, absent distal phalanges of second and third fingers on left
hand, multiple telangiectasia over face and lips, umbilical hernia
Sickle cell disease
Biliary atresia with portoenterostomy
Fibrolamellar hepatocellular carcinoma
Adrenocortical tumor
Modified from Stocker J T, Ishak KG: Focal nodular hyperplasia of the liver: a study of 21 pediatric
cases. Cancer 1918;48:336-345, with permission.
Treatment
Symptomatic children are treated with resection of the lesion. However, since morbidity and death have been
associated with attempts at resection, Pain et al. (e510) suggested that asymptomatic lesions be observed with
regular ultrasonography and treated only if they enlarge or become symptomatic. Young girls with FNH should
be cautioned on the use of oral contraceptives, because bleeding may occur within the lesion (e204). FNH does
not undergo malignant transformation. Although Saul et al. (e596) described the association of FNH with
fibrolamellar HCC (FL-HCC), they also suggested that the FNH, usually found either in or adjacent to the FL-
HCC, is a phenomenon secondary to the highly vascular nature of FL-HCC. There is currently no proof of FL-
HCC arising in a preexisting FNH. However, the radiologist should be cautious about the similar radiographic
appearance of FL-HCC and FNH, both of which may contain central scars (e436).
Gross Appearance
FNH occurs most frequently (90%) as a single mass within the right or left lobe (Figure 15-45B). Bilateral
involvement by a large lesion may be present in 10% of cases. Multiple lesions within both lobes are seen in
10% of cases and often have a histologic appearance different from that in cases with a single lesion (see later).
The single lesions are firm, irregular in outline, and range from 1 to 17 cm in greatest diameter with weights as
high as 1,500 g (171) (e659). The lesions often bulge from the surface of the liver and may be pedunculated. On
cut section, the lesion is sharply demarcated from the surrounding liver and displays a nodular tanbrown
parenchyma subdivided by gray-white septa radiating from a central area of fibrosis. Prominent vessels may be
seen near the edge of the lesion arising within the normal liver parenchyma and ramifying within the lesion.
Areas of hemorrhage or necrosis may rarely be seen.
Histopathology
The typical histopathological features of classical FNH include a firm, well-delimited but not encapsulated lesion
composed of hepatocellular nodules with normal hepatocytes, a central scar, and radiating fibrous septa. The
central scars of the single lesions display broad bands of fibrous connective tissue typically containing large
dystrophic arteries (ectatic vessels with eccentric intimal thickening and medial hyperplasia) (Figure 15-45C, D).
Frequently, there is a lymphocytic infiltrate. The fibrous septa subdivide, partially or completely enclosing lobules
of parenchymal cells arranged in cords, almost imparting an appearance of a focal biliary cirrhosis. Numerous
small bile ducts, arterioles, and venules are present within the septa, along with varying numbers of lymphocytes
and neutrophils. Bile ductules are usually found at the interface between hepatocytes and fibrous regions.
VanEyken et al. (e733) demonstrated that hepatocytes within the liver express cytokeratins of bile duct type,
suggesting that the ductular proliferation of FNH is derived from ductular metaplasia of hepatocytes. Interlobular
bile ducts are usually absent. The cords within the nodules contain hepatocytes in 1- to 2-cell-thick plates that
may be slightly larger than those of the normal liver and may contain intracellular fat and variable amounts of
glycogen (22, 114). The cells within the FNH show no evidence of dysplasia (e596). The lesion often
compresses the adjacent parenchyma but is separated from it only by a discontinuous fibrous capsule. A large
feeder artery is frequently present within this capsule. Wanless et al. (190) demonstrated a connection between
this feeder artery and a spiderlike structure of smaller vessels supplying 1-mm nodules within the lesion.
The diagnosis of FNH is usually evident in a liver biopsy specimen. However, some cases of FNH may show
atypical clinical and/or histopathologic features and the diagnosis is difficult in these even in the resected
specimen, let alone on a biopsy (114) (e387,e483). In atypical FNH, the above key diagnostic features are either
lacking or inconspicuous. Differential diagnosis with adenomas may be difficult in these cases, especially when
the nodules are small (<10 mm) or associated with significant steatosis. Fabre et al. (52) have proposed a
scoring system for the reliable diagnosis of FNH with atypical features. In their study, most radiologically atypical
tumors also showed nonclassic histopathology.
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Some lesions have histological features of both adenoma and FNH. These variant lesions have often been
classified as the telangiectatic type of FNH (1) (e754). These tumors are often multiple and the cut surface
displays a spongy telangiectatic appearance with numerous small, blood-filled cavities. In these multifocal
telangiectatic lesions, connection(s) between the vessels within the connective tissue and dilated sinusoids
within the parenchymal nodules can be readily demonstrated. As a result of this connection, the telangiectatic
lesion displays markedly dilated sinusoids filled with red blood cells clearly separating the hepatic cords (190).
Foci of more firm tissue resembling the single FNH lesions may be present in the multiple telangiectatic lesions. It
is not clear if all nodules originally called telangiectatic FNH (e388,e483) and progressive FNH (e585) are
histologically the same lesions as those subsequently included in the studies that demonstrated monoclonality
(e70,e512) and/or were associated with syndromes such as meningioma, astrocytoma, telangiectasia of the
brain, and berry aneurysm (e754). Clinical and molecular evidence indicates that telangiectatic FNH should be
reclassified as adenomas (e70,e791).
Molecular Pathology
The molecular pathogenesis of FNH was recently reviewed by Rebouissou et al. (146). Of 33 FNH lesions
evaluated by the HUMARA assay in the literature, 9 (27%) showed a uniform pattern of X chromosome
inactivation consistent with clonality. Other studies analyzing chromosome gains and losses by comparative
genomic hybridization (CGH), allelotyping, or karyotype have identified chromosome alterations indicating a
clonal origin in 14% to 50% of cases (e70,e123,e336,e554) Although somatic gene mutations in b-catenin gene (
CTNNB1), TP53, APC or HNF1a (e70,e72,e124) have not been identified in FNH, mRNA expression levels of
the angiopoietin genes (ANGPT1 and ANGPT2) involved in vessel maturation are altered, with increased
ANGPT1/ANGPT2 ratio (e70).
Immunohistochemical assays of extracellular matrix proteins also support the hypothesis that FNH is merely a
hyperplastic response of liver parenchyma to local vascular abnormalities and have shown that the lesions of
perisinusoidal fibrosis associated with FNH are accompanied by the induction of integrin receptors on
hepatocytes and sinusoidal endothelial cells (e612).
NODULAR REGENERATIVE HYPERPLASIA

Nodular regenerative hyperplasia (NRH) of the liver is an uncommon condition characterized by the presence of
widely distributed to diffuse parenchymal nodules with little or no fibrosis. The condition is more common in
adults than in children and increases with age. In a study of 2,500 consecutive autopsies, Wanless (190) found a
prevalence of 2.6%, rising to 5.3% above 80 years of age at death. Over 30 cases of NRH have been reported in
children, including two cases in fetal livers (127) (e230,e715). In the pediatric age group, NRH has been
demonstrated in 4.5% of a large series of 716 pediatric liver tumors, but only in 2.1% of liver tumors from birth to
2 years of age. However, since liver tumors are in general rare, NRH remains the fourth most common “liver
tumor” from 5 to 20 years of age (Figure 15-46A), after HCC, FNH, and undifferentiated embryonal sarcoma
(UES) (174).
Pathogenesis
Originally described as “miliary hepatocellular adenomatatosis” in a patient with Felty syndrome (e558), NRH is
seen in association with other rheumatologic and autoimmune diseases, hematological disorders, drug therapy,
PBC, congestive heart failure, other hepatic circulatory disorders, metastases, tuberculosis, and CGD (112, 148)
(e413,e542) (Table 15-18). Three familial cases of NRH have been reported in literature (e187). In a series of 16
children with NRH, clinical associations included a history of anticonvulsant drug therapy (four patients),
Donohue syndrome, disseminated intravascular coagulation, renal angiomyolipoma, other intraabdominal tumors,
thrombocytopenia, and pancytopenia (127). Other pediatric reports have been associated with congenital heart
disease (e715,e719), Krabbe disease (e450), Still disease (e465), chronic inflammation (e530), sacrococcygeal
teratoma (e155), autoimmune disorders (112) (e22), and multiple organ malformation in fetuses (e230).
The etiopathogenesis of NRH is not fully understood. NRH may be a hyperproliferative response to an
obstructive portal venopathy resulting in an uneven perfusion of the hepatic parenchyma (148)
(e137,e472,e753). It is hypothesized that the portal venopathy leads to centrilobular (acinar zone 3) ischemic
atrophy with compensatory proliferation of zone 1 hepatocytes. The resultant “regenerative nodules” compress
the atrophic hepatocytes, yielding the characteristic pattern highlighted by reticulin stains. This hypothesis is
supported by the association of NRH with diseases that are known to cause vascular injury and the frequent
histologic finding of portal venous abnormalities in NRH. Vascular abnormalities such as atrial septal defects,
ventricular septal defects, abnormal junction of pulmonary veins, congenital absence of portal vein, and other
congenital anomalies are reported in children diagnosed with NRH, strengthening the argument that NRH may
result from microcirculatory derangements (e254,e756). However, other investigators have not confirmed these
findings (e670) and suggest that NRH is a primary generalized proliferative disorder of the liver (112). In cases
associated with drug therapy, it has been suggested that polymorphisms in genes encoding thiopurine
methyltransferase may be linked to development of NRH probably through altered drug metabolism (e85). Some
NRH cases have been suggested to result from chronic, cytotoxic CD8+ T-lymphocyte targeting of sinusoidal
endothelial cells (e790), and NRH has also been postulated to be an organ-specific form of antiphospholipid
syndrome (e349).
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FIGURE 15-46 ▪ Nodular regenerative hyperplasia. A: Age distribution in 25 cases. B: A large mass of different-
sized nodules occupies most of the liver. C: The nodules are composed of hyperplasic “regenerative”
hepatocytes, which are light staining and compress remnants of atrophic lobules into thin bands. (H&E stain,
original magnification 30×).
Clinical Features, Diagnosis, and Management

Most patients with NRH may remain asymptomatic for years before coming to clinical attention. The diagnosis of
NRH requires a high index of suspicion and awareness of its associations detailed above; NRH should be
considered in the differential diagnosis of patients who present with unexplained portal hypertension. NRH may
also clinically simulate metastates and should be considered in patients with history of malignancy treated with
chemotherapy and/or radiotherapy who develop single or multiple hepatic masses (e133).
Table 15-18 ▪ CONDITIONS ASSOCIATED WITH NODULAR REGENERATIVE HYPERPLASIA IN

CHILDREN
Donohue syndrome
Mental retardation
Anticonvulsant therapy
Vater syndrome
Renal angiomyolipoma
Disseminated intravascular coagulopathy
Krabbe disease
Portal hypertension
Wilms tumor
Down syndrome
Still disease
In symptomatic patients, portal hypertension and its complications dominate. However, ascites is relatively
uncommon since patients typically have normal hepatic synthetic function with normal albumin levels. Although
based on the vascular compromise hypothesis, portal hypertension should be presinusoidal in nature (190),
portal pressure measurements in a small number of patients have been more consistent with a sinusoidal portal
hypertension, possibly due to sinusoidal compression by the regenerating nodules in later stages of the disease
(148).
The radiological findings of NRH reflect clinical observations (e154,e670,e715). Liver size can be normal,
reduced, or increased; immense hepatomegaly leading to abdominal deformity is very rare. Nodules range in size
from 0.1 to 10 cm in diameter and are often hyperechoic on ultrasound, although they may be even undetectable
by this modality. CT scans generally show hypodense nodules with respect to the adjacent liver parenchyma,
without significant contrast
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enhancement (e27). On MRI, lesions are described as isointense to normal liver on T2-weighted images and
contain foci of high signal intensity on T1-weighted scans (e106). Kobayashi et al. report typical imaging findings
to include hyperintensity on T1-weighted MRI, hyperdensity on CT during arterial portography (CTAP), and
isointensity to hypointensity onSPIO-enhanced T2-weighted MRI (e356).
Laboratory parameters of liver function are also usually normal in NRH, although approximately 25% of cases
reported in the literature note an elevated ALP (148). Liver biopsy is essential for diagnosis. It has been
emphasized that the histologic findings of NRH may not be detected by a needle biopsy of the liver and a wedge
biopsy may be required (112) (e716). In the case of needle biopsy, the gauge of the needle is an important
consideration. Regenerative nodules may be missed if the needle is too narrow, as is often the case with
transjugular liver biopsy, thus making the diagnosis of NRH difficult.
The mainstay of treatment is to manage the underlying disease, remove offending drugs, if any, and control
portal hypertension. Given the uncommon nature of NRH, there is scant literature on the natural history of this
disease and treatment strategies are based on experience with other more common causes of portal
hypertension (148). It is not known whether NRH is a reversible process once the presumed cause is removed,
such as might occur with stopping a drug. Since the synthetic function of the liver is generally intact in NRH,
despite the potential for the development of significant portal hypertension, liver transplantation is not a
conventional therapy. The outcome of NRH depends on the presence of portal hypertension, associated
systemic disease, and the risk of rupture of a large hyperplastic nodule. Some investigators claim that NRH is a
premalignant condition, which may progress to hepatocyte dysplasia and HCC. Nzeako et al. (e489)
demonstrated that 23 of 342 patients without cirrhosis who had HCC also had NRH and also found that 73.9% of
their patients with NRH and HCC had liver cell dysplasia. Liver cell dysplasia is a common finding in NRH and
has been noted in 20% to 42% of cases (e489,e670).
The largest pediatric series of NRH (127) comprised 16 patients (10 girls and 6 boys) with a median age of 6
years (range 7 months to 13 years). Nine presented with hepatomegaly or splenomegaly, with and without signs
of portal hypertension. Follow-up was available for eight patients; six patients died of causes unrelated to the
nodular hyperplasia. Two patients were asymptomatic when last seen 5 and 18 years after the initial diagnosis of
nodular hyperplasia.
Pathology
Based on autopsy studies, the liver with NRH shows a diffuse transformation into nodules of 1 to 3 mm in size
(Figure 15-46). Unlike cirrhosis, there is no fibrosis separating nodules; each nodule presses directly against its
neighbor. Although nodules greater than 15 mm have been described grossly, these are frequently revealed to
be composed of smaller nodules when examined microscopically (190).
Histopathology is the only means of definitive diagnosis and is also required to rule out cirrhosis and HCC. By
definition, the nodules are less than 3 mm in thickness and perisinosoidal fibrosis is absent to minimal (1, 148).
At a minimum, to make the diagnosis of NRH, one should see the characteristic nodular zones of widened
hepatocyte plates bounded by narrowed and compressed plates. Parenchymal nodularity can be appreciated on
scanning magnification with a characteristic pattern of light and dark areas (127). The light areas are comprised
of swollen liver cells with empty to clear cytoplasm, whereas the dark areas correspond to compressed liver cell
plates between the nodules. The hepatocytes within the nodule may be arranged in plates that are more than
one cell thick. The individual hepatocytes may be enlarged and have hypertrophic nuclei. Between individual
nodules, the hepatocytes are small and atrophic and are pressed together into thin, parallel plates. This
compression is best visualized using a reticulin stain and may be associated with slitlike central veins and
sinusoidal dilation (in areas of hepatocellular atrophy). Immunohistochemical granular staining for alpha-1-
antitrypsin is reportedly increased in the regenerating (periportal) compartment and this may help in the
histological evaluation of difficult cases (e474). Whereas the larger portal veins may be widely patent, portal
venous structures in smaller radicals may be absent or occluded. Central veins may show venoocclusive
changes or may be compressed into narrowed slits. However, no vascular abnormalities were noted in Moran's
series (127). Fibrosis typical of chronic liver disease is usually not present, although there may be some degree
of periportal fibrosis or perisinusoidal fibrosis, the latter frequently associated with the atrophic areas. There is
usually little or no inflammation or cholestasis, and normal bile ducts and arteries can be easily identified. In
needle biopsies of the liver, the changes of regeneration and atrophy may be very subtle on routine hematoxylin-
eosin stains. Therefore, any “normal” liver biopsy specimens, particularly those from patients with portal
hypertension, should be investigated further using reticulin stains (148).
The differential diagnosis of NRH includes hepatic adenoma, FNH, partial nodular transformation, large
regenerative nodule, CHF, incomplete cirrhosis, cirrhosis, and HCC. The International Working Party has
published guidelines and definitions for these nodular hepatic lesions (1). It is important to remember that more
than one type of nodular lesion can coexist in the same liver since clinical portal hypertension may result from
NRH, whereas disabling pain or hemorrhage may be due to other pathology such as hepatic adenoma, with
different treatment options for each situation. Histologically, patients with portal hypertension not associated with
cirrhosis may present with NRH, hepatoportal sclerosis (portal venopathy), central venous obliteration, sinusoidal
dilatation, or some combination of these lesions (131). Histologic findings in these settings may be subtle and
awareness of these will prevent underdiagnosis.
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HEPATOCELLULAR ADENOMA
HCA is a rare benign tumor of the liver. In the pediatric age group, it is seen most frequently in teenage girls
(Figure 15-47A) but has also been described in younger children with GSD and galactosemia, in infants, and
even in utero (150) (e33,e477,e561,e762). Most patients, however, are older than 10 years of age and, like
adults, have a history of oral contraceptive use (e659).
Pathogenesis
In addition to oral contraceptive use, HCA has been described in a variety of conditions in children, including
GSD types I, III, and IV; galactosemia; Hurler syndrome; severe combined immunodeficiency; diabetes mellitus;
and androgen therapy for Fanconi anemia (150) (e23). Osteoporosis has been noted in some children with HCA
(e762).
Clinical, Laboratory, and Imaging Features

The lesion may be asymptomatic, produce mild episodic abdominal pain, or present as acute abdominal pain due
to hemorrhage into the tumor or peritoneal cavity. Laboratory studies are usually not helpful with normal or only
mildly elevated serum aminotransferases, ALP, and bilirubin values (150).
FIGURE 15-47 ▪ Hepatocellular adenoma. A: Age distribution in 18 cases. B: A poorly circumscribed light
yellow-tan mass occupies a large portion of the liver. Note the smaller nodules of similar colored tissue in the
adjacent normal liver parenchyma. C: The lesion is composed of trabeculae of uniform hepatocytes, some
surrounding canaliculli. Note the absence of portal areas and bile ducts. (H&E stain, original magnification 100×).
Although imaging studies are helpful in demonstrating the large single mass usually seen in this disorder, at
present, HCA cannot be conclusively identified by any currently available imaging technique. Arteriography
displays hypervascular masses that in some areas are hypovascular, presumably because of intratumor bleeding
or necrosis (e339). Ultrasound has detected the lesions in utero (150) (e33). Imaging findings of HCA and
adenomatosis are similar and vary according to the particular characteristics of the lesional tissue: there are fatty
patterns, peliotic patterns, and heterogeneous patterns with necrotic and hemorrhagic foci (e458). Currently,
imaging techniques are unable to detect early malignant transformation in HCA.
Treatment and Outcomes

HCAs require excision, in view of their propensity to bleed or rupture, association with osteoporosis, and the
inability to predict malignant transformation (e762). HCAs in girls using
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contraceptive steroids may regress after discontinuation of their use (e23).
There is, at present, inadequate data regarding the growth and involution of HCAs. Also, the risk of hemorrhage
in an HCA is not restricted to larger lesions and is unpredictable. There are no longitudinal studies evaluating
transformation of HCA to HCC, although a recent study found malignancy only in adenomas larger than 4 cm and
more often in men than in women (20).
Gross Pathology
HCA is usually a solitary, well-demarcated, globular to ovoid lesion measuring 0.1 to 15 cm in diameter, often
with large vessels coursing over its surface (150). The lesion is soft to firm in consistency and has a variegated
appearance ranging from light brown to tan, with or without areas of yellow necrosis or reddish-brown
hemorrhage. Multiple lesions may be present (Figure 15-47B). By definition, “adenomatosis” requires the
presence of at least ten adenomas in the liver (e126,e217). This definition theoretically excludes patients with
glycogenosis, or those taking contraceptives (e217), although some authors feel that this may be an unduly
restrictive definition (22).
Histopathology
HCAs can be solitary or multiple. They represent a heterogeneous group of tumors in which histopathological
features may vary according to the etiological background (e31). Microscopically, the tumor is composed of
sheets of neoplastic cells in trabeculae that are one to three cells thick, separated by compressed sinusoidal
spaces lined by endothelial cells and some Kupffer cells (Figure 15-47C). The tumor cells are the same size as
or slightly larger than the normal hepatocytes and may be either normal, clear (glycogenrich), or fatty. Some
lesions may be almost entirely steatotic, prompting a differential diagnosis including angiomyolipoma. The tumor
parenchyma is supplied by thin-walled arteries without other portal tract elements such as significant amounts of
connective tissue, bile ducts, or ductular reaction. Bile may be present in intracellular canaliculi. Foci of dilated
sinusoids may impart a “pelioid” appearance. Large vessels are often present near the periphery of the lesion,
displaying arterial intimal thickening and elastic lamina reduplication. Smooth muscle proliferation may narrow or
obliterate the lumen of veins, particularly in cases associated with contraceptive steroid use. Infarcts and
hemorrhage are frequent, especially in larger lesions. Hemorrhage may be internal to the lesion, usually admixed
with necrotic changes (this type is mostly observed in adenomas larger than 4 cm) or may result in spontaneous
rupture with resultant subcapsular hematoma and/or hemoperitoneum (e425). Internal hemorrhage may heal with
fibrosis, and this may simulate a central scar of FNH, making it difficult to differentiate the two, particularly in core
biopsy material. Hemosiderin-laden macrophages may also be seen. Foci of extramedullary hematopoiesis as
seen in cases of hepatoblastoma may be present, sometimes posing difficulty in differentiating the two lesions
(150). Foci of dysplastic hepatocytes may be present within the lesion, especially in patients with Fanconi
anemia, but malignant transformation is rare (150) (e208). Nuclear atypia, mitoses, and acinar
(“pseudoglandular”) growth pattern are rarely seen; these cases may also be extremely difficult to distinguish
from HCC. The term “atypical adenoma” is often used in these settings to indicate that the distinction between
HCA and HCC remains problematic and resection and/or close clinical follow-up may be needed. Cytogenetic
techniques such as FISH and CGH may help distinguish HCA from HCC, since the former usually does not show
chromosomal aberrations (e766). Resnick et al. suggest that immunostains for proliferating cell nuclear antigen
(PCNA) may be used to help differentiate HCA from hepatoblastoma and HCC; the PCNA labeling index was
significantly lower in hepatic adenomas (0.3% to 5.1%) than in HCCs (9.6% to 23.8%) and hepatoblastomas
(21.8% to 44.3%), in their study (150). The range of PCNA labeling is even lower in patients with adenoma who
do not have Fanconi anemia (0.3% to 1.7% for adenoma alone versus 3.2% to 5.1% for those with Fanconi
anemia) (e70). Care must also be taken to distinguish the usual solitary HCA from the multiple nodules of NRH of
the liver, which is associated with many other disorders.
As outlined above, the heterogeneous histopathology of HCA raises many differential diagnoses, the greatest
overlap being with FNH. Until recently, the presence of bile ductules (characterized immunohistochemically as
CK7 positive and usually CK19 negative) in a lesion precluded the diagnosis of HCA. However, molecular
studies have shown that HCA may contain bile ductules, especially when associated with sinusoidal dilatation;
these lesions traditionally referred to as telangiectactic FNH are being reclassified as adenomas (e70), although
there is a lack of consensus at this time. The problem of differentiating HCA and FNH is further compounded by
the fact that the two lesions are associated and may occur concurrently. Laurent et al. have shown that the
presence of FNH is significantly higher than expected in at least two circumstances: adenomatosis and multiple
inflammatory HCA (e373). Immunohistochemical stains with antibodies to CD34 (e252), cytokeratin 7, or hepatic
transporters (e737) have been suggested as adjunct techniques to help differentiate between FNH and HCA.
Molecular Pathology
The past decade has seen numerous advances in understanding the molecular basis of HCA. Based on two
molecular criteria (hepatocyte nuclear factor 1a [HNF1a] mutations and b-catenin mutations), and an additional
histological criterion (the presence/absence of inflammation), a molecular/histologic classification correlating the
genotype and phenotype of HCAs has been proposed (23) (e69,e791).
Typical HCA
These have classic histology with regular liver cell plates up to three cells thick and little cytologic atypia; thin-
walled arteries without other portal tract elements, bile ducts, or ductular
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reaction; may be monoclonal; and overlap with FNH. They are negative for HNF 1a and b-catenin mutations.
Variant 1
HNF1a biallelic somatic mutations are observed in 35% of the HCA cases. These patients are almost always
women. There is marked steatosis/clear cells and a lack of expression of liver fatty acid binding protein (LFABP)
on immunohistochemistry. An HNF1a germline (constitutional) mutation is observed in less than 5% of HCA
cases and is associated with MODY 3 diabetes, familial adenomatosis, and a younger age at presentation.
Variant 2
An activating b-catenin mutation is found in 10% of HCA. These b-catenin activated HCAs are observed in both
men and women and are associated with specific risk factors such as male hormone administration or
glycogenosis. There is cytological atypia and an acinar pattern (the so-called “atypical HCA” or “HCA/HCC
borderline lesion”) and steatosis is not prominent. Immunohistochemical studies show that these HCAs
overexpress b-catenin (nuclear and cytoplasmic) and glutamine synthetase. This group of tumors has a higher
risk of malignant transformation. The association in the same coalescent nodule of HCC and adenoma (b-catenin
and glutamine synthetase positive) can be explained by either malignant transformation of adenoma, or an HCC
with both very well differentiated and less differentiated areas. At present, this issue remains unresolved.
Variant 3
Inflammatory HCAs are observed in 40% of the cases; they are most frequent in women but are also found in
men. In this group, GGT is frequently elevated, with a biological inflammatory syndrome present. Also, there are
more overweight patients in this group. These lesions may be multiple and associated with other vascular or
neurological disorders (e754). The histology is of the so-called “telangiectatic FNH” and is characterized by
inflammatory infiltrates, dystrophic arteries, sinusoidal dilatation, and ductular reaction (CK7 positive ductules).
Although definitionally there is no mutation, 10% of inflammatory HCAs also express b-catenin, and behave as
variant 2, with higher risk of malignant transformation.
Variant 4
This group includes the (<10%) HCAs that are currently unclassified by the above schema. They lack any
specific trait in that there are no known mutations or specific association.
There is a higher risk of bleeding in the variant forms, although the degree of this risk in each of the different
categories is unknown. If molecular techniques are not available to test for b-catenin mutation on frozen or
formalin-fixed tissue, immunostains for b-catenin (e682,e708) on paraffin sections may help identify variant two
tumors, since these may have a higher risk of malignant transformation, although more studies are needed to
confirm this.
MESENCHYMAL HAMARTOMA
Hepatic mesenchymal hamartoma (HMH) is an uncommon benign tumor of childhood. Historically, mesenchymal
hamartoma has been described in the literature by various names including pseudocystic mesenchymal tumor,
giant cell lymphangioma, cystic hamartoma, bile cell fibroadenoma, hamartoma, and cavernous
lymphangiomatoid tumor; the unifying term mesenchymal hamartoma was coined by Edmondson in 1956 (e190).
The lesion makes up approximately 8% of all pediatric tumors and, after hemangioma, is the second most
common benign hepatic tumor in childhood.
Pathogenesis
The pathogenesis of HMH is still debated. A handful of series have shown an association with placental
abnormalities including mesenchymal stem villous hyperplasia of the placenta, thrombosis, or transient
honeycombed multicystic placental enlargement (56) (e105,e221,e347,e366,e712), raising the possibility of
synchronous abnormal mesodermal development rather than a true developmental abnormality. Alternatively,
placental dysplasia may be secondary to compression of the umbilical vein by the HMH. Given the similarities
between the bile duct abnormalities in MHL and those in von Meyenburg complexes, bile duct hamartomas,
Caroli disease, and CHF, a primary bile duct plate malformative etiology has also been proposed for MHL. In
fact, serial dissection studies have demonstrated a single portal tract as being the source of the lesion
(e384,e498).
Clinical Features, Laboratory Studies, and Imaging

Mesenchymal hamartoma is a lesion of infants; 55% of cases present in the 1st year of life and nearly 85% by 2
years of age (Figure 15-48A). Rare cases have been reported in children older than 5 years of age, with
anecdotal reports in adults. Intrauterine HMHs have been well documented in several reports. Cornette et al.
(43) reviewed 17 reported cases in the literature, with the earliest case having been incidentally detected on
ultrasonography at 15 weeks' gestation (e398). However, only 4 of 17 had been correctly diagnosed antenatally,
while in other cases, preoperative diagnoses entertained included ovarian masses, lymphangiomas,
pseudocysts, enteric duplication cysts, and choledochal cysts. Mesenchymal hamartomas have also been noted
as an incidental finding at autopsy.
Infants usually present with a history of a nontender enlarging abdomen over a period of days to months. Other
symptoms that rarely present are vomiting, decreased appetite, and
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respiratory distress. There is a slight male predominance but no apparent racial predilection. Physical findings
are those associated with the abdominal mass, including a protuberant abdomen and dilated superficial veins.
Large masses may eventually produce a mass effect such as vena cava compression, feeding difficulties, and
respiratory distress secondary to upward pressure on the diaphragm and may be complicated by ascites,
jaundice, and even congestive heart failure (e305). Occasionally, the mass will expand rapidly, most likely
because of rapid accumulation of fluid within cystic spaces (e316). Stocker and Ishak (172) reported other
anomalies or diseases in 5 of 30 patients, including adrenal cytomegaly,
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neonatal hyperbilirubinemia, endocardial fibroelastosis of the left ventricle, idiopathic thrombocytopenic purpura,
and diffuse endocrinopathy.
FIGURE 15-48 ▪ Mesenchymal hamartoma. A: Age distribution in 71 cases. B: Multiple cysts of varying sizes
(previously filled with clear yellow fluid) are surrounded by variegated, solid components. C: Near the edge of
the cystic portion (on the left side of the image), the lesion displays a diffuse infiltration and widening of the portal
areas (light tan areas), compressing the intervening hepatocytes lobules into thin brown strips. D: The cysts,
with no discernible cellular lining, are surrounded by loose to compressed connective tissue, which contains
scattered residual bile ducts. Note the compressed liver at top (Masson trichrome stain, original magnification
10×). E: Residual bile ducts within the lesion are surrounded by loose mesenchymal tissue containing scattered
neutrophils, lymphocytes, and small foci of extramedullary hematopoiesis (H&E stain, original magnification 75×).
Laboratory findings are noncontributory; tumor markers including AFP, b-human chorionic gonadotropin (hCG),
and vanillylmandelic acid are usually negative (e781), although rare cases may show elevated AFP levels
(e99,e726). Ultrasonography, except in the youngest infants, displays an echogenic mass that may be
pedunculated and which displays internal septation and cysts (e462,e781). MRI and CT also highlight the
multicystic nature of the lesion and can suggest the fluid nature of the cyst contents (e295). The typical CT scan
features are that of a well-circumscribed, multilocular, multicystic mass that contains low-density cysts separated
by solid septae and stroma. The stroma and septae may be vascular and occasionally show contrast
enhancement on CT scan similar to that seen in infantile hemangioma. When the cysts are small, the lesion may
appear solid on imaging. Selective arteriography most frequently displays an avascular mass (172).

Surgical resection of the lesion or partial hepatectomy is the treatment of choice. Partial resection with drainage
of the cysts and marsupialization has been successful in managing large lesions believed to be impossible to
resect completely, but may be associated with recurrence (e462). Less invasive techniques, such as
laparoscopic fenestration, have also been used successfully (e293). Spontaneous regression of the lesion has
been described, prompting some authors to suggest a policy of watchful waiting (e44,e324,e398). Others, on the
other hand, have used liver transplantation for lesions in those children who are highly symptomatic or are
considered to have an unresectable lesion (e693).
Prognosis is favorable in patients who undergo complete resection. Of 104 patients who underwent follow-up
examination, six had died from intraoperative and postoperative complications; the remaining 98 were alive and
well up to 15 years after surgery, except a 4-year-old boy who died of leukemia 2 years after surgery (e659). In
neonates with large antenatal tumors, vascular compression of great vessels may lead to ischemia complicated
by congestive cardiac failure, intraventricular hemorrhage, cystic encephalomalacia, and renal failure (e717).
Fluid loss to the cysts and reduced fetal albumin production by the liver can further increase the risk of hydrops
(e62,e324). Polyhydramnios is associated with upper intestinal tract obstruction, and elevation of the diaphragm
poses the fetus at risk for pulmonary hypoplasia. In their literature review, Cornette et al. observed intrauterine
demise or early neonatal mortality in 5 of 17 (29%) antenatally detected cases (43). In antenatally detected
cases, fetal intervention in the form of ultrasound-guided percutaneous cyst aspiration has been reported to
dramatically improve outcome (e717). Isaacs reports on 45 patients with mesenchymal hamartoma in the fetal
and neonatal period, of which 29 (64%) had surgical resections and 23 (79%) survived (75).
Gross Appearance
The lesions vary in size from a few centimeters, an incidental finding at autopsy, to as large as 30 cm in older
patients. The average weight is 1,300 to 1,900 g, but weights of 5,400 g have been reported (172). Cooper et al.
(e141) noted a 3,500 g lesion in a 1-month-old boy whose birth weight had been 8,300 g. The right lobe is
involved in 75% of cases, the left lobe in 22%, and both lobes in 3%. The tumor may bulge from the surface or
even be pedunculated in about 20% of cases, attached to the liver by a thin to broad pedicle (e659).
On cut surface, multiple cysts are present, ranging in size from a few millimeters to 15 cm in over 85% of cases
(Figure 15-48B, C). Clear amber to yellow fluid or gelatinous material fills the cyst and is similar to serum, except
for lower concentrations of total protein, albumin, immunoglobulin, cholesterol, and glucose (e182). The cysts
have gray-tan to yellow linings that may be smooth, long, or ragged. The surrounding tissue is yellow-tan to
brown and loose to moderately dense. Only in the youngest patients are the lesions without cysts.
Histopathology
Microscopically, the lesion consists of an admixture of mesenchyme, bile ducts, hepatocyte cords, and variable-
sized cysts (Figure 15-48D). The cysts may be no more than a loose, fluid-filled area of mesenchyme or dilated
lymphatics or bile ducts. More often, the cysts that are discernible grossly consist of an “unlined” wall of loose to
dense mesenchyme. In older patients, however (e.g., those older than 1 to 2 years of age), the cysts may be
lined by cuboidal epithelium. The mesenchyme consists of scattered stellate cells in a rich matrix. Collagen in the
form of fibrils or small bundles is often associated with vessels and bile ducts within the mesenchyme.
Extramedullary hematopoiesis is a consistent finding (more than 85% of cases) (Figure 15-48E), and scattered
plasma cells and lymphocytes, although rarely prominent, are seen throughout the lesion. In older patients, more
mature collagen bundles may be present. Nodules of mesenchyme may be separated by dense, highly vascular
connective tissue. Hepatocytes appear to be a passive component of the lesion, often seen near the periphery of
the lesion or as thin compressed strips between collections of mesenchymal tissues within the lesion. Bile ducts,
however, appear to be an active or proliferative component, with single ducts or intricately branching ducts
primarily near the periphery of the lesion. Bile is rarely present within the ducts. Atypical mitoses and invasion of
adjacent liver are absent. Cytologic sampling may result in misdiagnosis due to the heterogeneous nature of the
lesion. Although clusters of normal bile duct epithelium and hepatocytes admixed with bland mesenchymal cells
in a myxoid background are highly suggestive of HMH on fine needle aspiration, rare cases with elevated AFP
levels have been misdiagnosed as hepatoblastoma due to limited sampling of the hepatocellular component
(e99,e726). In a series of 17 cases of HMH, Chang et al. found 7 (41%) to be
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solid. The solid “variant” was associated with higher serum AFP levels, smaller bile ducts, and more frequent
vascular proliferation. Serum AFP level correlated with the proportion of hepatocytes. Two of seven solid cases
harbored a larger amount of evenly distributed hepatocytes and proliferation of small ducts with focal hepatocyte-
bile duct transition, suggesting that hepatocytes within HMH may be a truly neoplastic rather than an entrapped
component (37).
Immunohistochemistry may be used to rule out other entities. In HMH, bile ducts and hepatocytes are cytokeratin
positive, whereas the mesenchyme and pseudocysts are vimentin positive. Myxomatous infantile
hemangioendotheliomas can resemble HMH on fine-needle aspiration (FNA) biopsy, but the plump endothelium
of the former is positive for factor VIII-related antigen, CD31, and CD34 immunohistochemical stains; however, a
localized vascular proliferation within an HMH will stain similarly. Immunostains may not be helpful in
differentiating HMH from biphasic hepatoblastomas.
Molecular Pathology
Various authors have noted a balanced translocation involving chromosome 11 (band q11, q13 or q15) and
chromosome 19 (band 19q13.4) (e78,e430,e493,e555,e651). Talmon et al. (e684) reported a case with a
deletion involving chromosome 19q13.4. Sharif et al. (e622) reported tumor recurrence associated with
chromosome 19q translocation and suggested that these cases may require more radical surgical resection. In a
case of an undifferentiated (embryonal) sarcoma putatively arising from an HMH, Lauwers et al. demonstrated
that the transformed component had the 19q13.4 breakpoint in addition to several other numerical and structural
chromosomal abnormalities. Taken together, these findings suggest that a subset of HMH may be truly
neoplastic rather than hamartomatous (e374).
Relationship to Undifferentiated Embryonal Sarcoma

Ramanujam (e557) reported the “malignant transformation” of a mesenchymal hamartoma of the liver into a
“malignant mesenchymoma,” and de Chadarevian et al. (e164) noted an “undifferentiated (embryonal) sarcoma
arising in conjunction with mesenchymal hamartoma.” Lauwers et al. (e374) also described an undifferentiated
(embryonal) sarcoma (UES) “arising in” a mesenchymal hamartoma. In their case, the mesenchymal hamartoma
component was diploid by flow cytometry, while the UES showed a prominent aneuploid peak. Karyotypic
analysis of the UES showed structural alterations of chromosome 19, which have been implicated as a potential
genetic marker of mesenchymal hamartoma. We have also seen a case of UES surrounded with large areas of
more bland mesenchymal tissue but without the characteristic bile ducts and cysts of a mesenchymal
hamartoma. The question yet to be resolved in all these cases is whether the UES truly arises in a preexisting
“benign” mesenchymal hamartoma or if the entire lesion is primarily an UES with areas of bland-appearing
stroma. Begueret et al. reported a case of a large, cystic hepatic mass in a 17-year-old girl that had areas
characteristic of both embryonal sarcoma and HMH, with the intervening “transition zone” showing the
architectural features of a HMH but with the atypical mesenchymal cells of an embryonal sarcoma (19). Flow
cytometric analysis of DNA aneuploidy showed that all three areas had similar DNA indices, suggesting a
common lineage. O'Sullivan et al. have also described a case of UES arising within a mesenchymal hamartoma.
The mesenchymal hamartoma in their case had unusual features including large mesothelial-lined cysts and
adrenal cortical heterotopy (e493). While the association of UES and mesenchymal hamartoma is still tenuous, it
would be prudent, in practice, to extensively sample all mesenchymal hamartomas for histologic evaluation, so as
not to miss a focus of UES.
CAVERNOUS HEMANGIOMA
Although 1% of the population may harbor cavernous hemangiomas of the liver, the lesion is rare in children and
is usually asymptomatic when it does occur (e659). The lesions are often small (<2 cm), single, red-purple, and
spongy. Microscopically, the lesions are well-circumscribed collections of large channels with a thin layer of
endothelial cells. Fibrosis, thrombosis, and calcification may be present. Infantile hemangioendotheliomas
frequently contain foci of large vascular channels, resembling cavernous hemangioma.
INFANTILE HEMANGIOENDOTHELIOMA (IHE)

Infantile hemangioendothelioma is the most common benign hepatic neoplasm in the pediatric age group,
accounting for 17.7% of all liver tumors/pseudotumors, and 40% of all benign tumors/pseudotumors in children
(e305). It is a vascular tumor that is almost always seen in the 1st year of life.
Pathogenesis
The pathogenesis of infantile hemangioendothelioma is unclear.

Infantile hemangioendothelioma is seen most frequently in the first 6 months of life (86%) (Figure 15-49A) with
33% presenting in the neonatal period (e305). Patients are rarely older than 3 years of age (see Table 15-14);
only one such patient (a 15-year-old female) was noted among 91 patients seen at the Armed Forces Institute of
Pathology (163). There is a slight female predominance (1.7:1) but no racial predilection.
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FIGURE 15-49 ▪ Infantile hemangioendothelioma. A: Age distribution in 102 cases. B: Following injection of a
radio-opaque dye, the liver displays numerous nodules throughout both lobes. C: On cut section of the liver,
multiple blood-filled nodules are visible. D: The lesions of C are composed of trabeculae of loose fibrous
connective tissue covered by a single layer of uniform endothelial cells (Masson trichrome stain, original
magnification 75×). E: The epithelial cells lining the sinusoid of the lesion stain positively with the GLUT1 stain, a
feature seen most prominently in patients with asymptomatic hepatomegaly and multiple small hepatic lesions
(GLUT1 Immunoperoxidase stain, original magnification 100×).
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FIGURE 15-49 ▪ (continued) F: Areas of cavernous vascular change are often present at the margin of the
hepatic lesion. (H&E stain, original magnification 10×). G: Entrapped bile ducts (center) can often be found
within the fibrous septa. Note the thin covering of endothelial cells at top (H&E stain, original magnification 75×).
H: Some lesions display dense clusters or “tufts” of endothelial cells felt by many to represent involutional
changes within the lesion (H&E stain, original magnification 40×).
The classic presentation is a triad of hepatomegaly, congestive heart failure, and anemia (163). Most patients
present with an abdominal mass or distension. Congestive heart failure may be present in 10% to 15% of cases,
with increased cardiac output, elevated right and left end-diastolic pressure, small systolic pressure gradient
across the pulmonary outflow tract, and mild elevation of pulmonary artery pressure (e156). Other presenting
symptoms include failure to thrive, fever, jaundice (up to 20%), and (rarely) liver failure or tumor rupture with
death (163) (e156,e278). Skopec and Lakatua (e640) reported on a premature infant who presented with
nonimmune fetal hydrops, thrombocytopenia, and hypofibrinogenemia in association with an infantile
hemangioendothelioma. This association, the Kasabach- Merritt syndrome, is attributed to trapping and
increased destruction of platelets within the vascular tumor, often resulting in progression to disseminated
intravascular coagulation with activation of both clotting and fibrinolytic pathways (e773). Fetal hydrops is
attributable to large arteriovenous shunts created by the neoplastic vascular channels. In these cases, prenatal
US can depict a liver mass together with polyhydramnios, cardiomegaly, anasarca, and ascites. In general, the
prognosis is determined mainly by the amount of the shunt volume. Among 117 diagnosed antenatally (n = 33)
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and at birth (n = 84), the most common initial finding in the fetus was a hepatic mass detected by antenatal
sonography followed in rank by anemia, hydrops, hydramnios, congestive heart failure, thrombocytopenia, and
disseminated intravascular coagulation, which contributed to its demise. In the neonate, hepatomegaly was the
leading finding, followed by congestive heart failure, cutaneous hemangiomas, a murmur (bruit), respiratory
distress, cardiomegaly, and thrombocytopenia (75).
Hemangiomas of the skin have been reported in up to 70% of cases, but in an experience with 91 cases, they
were noted in only 11% (163). Isaacs noted cutaneous hemangiomas in 4 (5%) of 76 patients with focal liver
tumors, compared to 20 (49%) of 41 patients with multifocal liver lesions (75). Associated extrahepatic
hemangiomas may also be present in the brain, placenta, lungs, eyes, lymph nodes, pancreas, retroperitoneum,
adrenal, or bone as single or multiple lesions (75, 163) (e429). Pereyra et al. (e523) described the death of a
child from airway obstruction by a laryngotracheal hemangioma 4 months after resolution of a hepatic
hemangioma treated with steroids and radiotherapy.
Significant laboratory findings in infantile hemangioendothelioma include anemia in about 50% of cases,
hyperbilirubinemia in 20% of cases, and elevated aspartate transaminase (over 100 U/dL) in 32% of cases (163)
(e52). Although in Isaacs' review (75) AFP levels were elevated in 5 fetuses and in 11 neonates (16/117 or 14%)
with hemangiomas, the importance of this finding is unclear since even otherwise normal neonates may have
elevated AFP levels (e331). Even in the absence of a liver lesion, “adult” levels of AFP (<25 ng/mL) are not
reached until 6 months of age, and infants under 1 month of age may normally have levels as high as 2,500
ng/mL (e487). When adjusted for the age of the infant, AFP levels are not elevated with infantile
hemangioendothelioma. Hemangioendotheliomas have been reported to express type 3 iodothyronine
deiodinase and cause severe hypothyroidism (64). The hypothyroidism may be resistant to medical treatment,
but resolves following OLT (e378).
Diagnostic imaging is helpful in the evaluation of infantile hemangioendotheliomas. Hepatomegaly with a soft
tissue mass is usually visible on plain film of the abdomen, and speckled calcification of the lesion is present in
15% to 37% of cases (86). Chest radiography may demonstrate cardiomegaly with or without prominent
pulmonary vascular markings. Ultrasound examination may show single or multiple hyperechoic, complex, or
hypoechoic lesions. If significant arteriovenous shunting is present, a prominent Doppler signal flow is seen. On
CT imaging, hepatic hemangioendtheliomas manifest as a well-defined, hypoattenuating mass. Contrast
enhancement demonstrates peripheral pooling and central enhancement with variable delay. MRI is the most
useful single modality because it shows not only the extent of the hemangioendothelioma but also the flow
characteristics and the surrounding vascular structures (e572). Technetium-99m scans display a characteristic
early “blush”. Imaging studies demonstrate the hepatic origin of the lesion, multifocality, and extrahepatic lesions
(e55,e286,e295,e515,e545). Selective arteriography displays a diffuse angiomatous lesions with rapid filing of
the hepatic vein (e52) and can be used to determine the extent of the lesion and possible surgical approaches to
the large feeder vessels.

Infantile hepatic hemangioendotheliomas are vascular lesions that show a clinical course intermediate between a
hemangioma and an angiosarcoma. Treatment is determined by the severity of the presenting symptom and
whether the lesion is single or multifocal. Patients with congestive heart failure and a multifocal lesion are treated
with digitalis and diuretics. Although spontaneous regression may occur, steroid therapy is thought by some to
hasten the regression of the lesion and improve the platelet count but is considered by others not to be helpful
(e546,e591). Alpha-interferon therapy has also been used as a component of medical management. Radiation
therapy has been used in the past but is infrequently employed now, because of the potential long-term side
effects. Interventional therapies include hepatic artery ligation or embolization, resectional surgery, or OLT (e5).
Surgical excision of single lesions, even in the face of congestive heart failure, is frequently successful. Becker
and Heitler noted the survival of 46 of 50 infants (92%) who had localized lesions treated with hepatic lobectomy
or localized resection (52). For large single lesions or multifocal tumors, success has been achieved through
OLT, hepatic artery ligation, or transarterial embolization, often in association with the use of digitalis, diuretics,
and steroids (e5,e100,e435,e574).
Both success and complete failure have been reported variously with many agents including epsilon-
aminocaproic acid (e755), tranexamic acid (e455), low-molecular-weight heparin (e692), vincristine (e524),
cyclophosphamide (e418), and interferon-alpha (e692,e772). A treatment algorithm has been published by the
vascular tumors study group at Boston Children's Hospital (40).
Patients with infantile hepatic hemangioendothelioma usually have an excellent prognosis, especially with
spontaneous regression after the 1st year of life. Survival in 26 cases reviewed by Becker and Heitler was 65%
(e52). Of 71 patients studied at the AFIP who had been followed for at least 6 months, 50 (70%) were alive and
well approximately 24 years after diagnosis (mean 7.7 years). Of the 21 deaths, 19 occurred in the 1st month
after diagnosis, and two deaths occurred at 3 and 7 months after diagnosis. Presence of congestive heart failure,
jaundice, multiple tumor nodules, and absence of cavernous differentiation were significant predictors of death at
6 months (163). Because of the uncertainty about the behavior of “type 2”, resection if possible, is advisable.
Gross Appearance
The tumors are single in about 55% of cases and multiple in 45%. Single tumors measure from smaller than 0.5
cm to as large as 13 cm and are located equally in the right and
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left lobes, with an occasional single lesion involving both lobes (163). When more than one lesion is present,
they may be limited to one lobe but frequently involve large portions of the liver (Figure 15-49B). Lesions near
the hepatic surface often show central umbilication. In his review of fetal and neonatal cases, Isaacs found 76
solitary and 41 multifocal lesions, the latter also including diffuse or disseminated hemangiomatosis. Most focal
hemangiomas, 33 (43%) of 76, were found in the right lobe of the liver and 18 (24%) of 76 in the left. Among the
41 multifocal lesions, 11 were limited to the liver, 20 also had cutaneous hemangiomas, and 10 cases showed
noncutaneous extrahepatic involvement (75). On cut section, they are well demarcated, reddish brown to light
tan, and soft and spongy (Figure 15-49C). In large lesions, central areas of infarction, hemorrhage, fibrosis, and
yellowish gritty specks of calcification may be present. In cases preoperatively treated with hepatic artery ligation
or embolization, the entire lesion(s) may be infarcted.
Histopathology
Histologically, hemangioendotheliomas have traditionally been classified as type 1 or type 2 lesions (e169).
Hemangioendotheliomas are composed of vascular channels lined by a single continuous layer of plump
endothelial cells in a supporting fibrous stroma (Figure 15-49D to H), reflecting the “type 1” lesion defined by
Dehner and Ishak (e169). Also, in about 20% of cases, larger pleomorphic and hyperchromatic cells are present
along poorly formed vascular spaces, often displaying tufting or branching, the “type 2” lesion (Figure 15-49H).
However, the so-called “type 2” lesions are now grouped together with angiosarcomas. Well-preserved bile
ducts may be present in the supporting stroma, most frequently near the periphery of the lesion. Foci of
extramedullary hematopoiesis are noted within the vascular spaces in over 60% of patients. Mitoses are
infrequent but rarely may number 5 to 10 per high-power field. Larger vascular channels resembling cavernous
hemangioma may be found in 50% to 65% of patients (Figure 15-49F). Areas of hemorrhage, infarction, fibrosis,
and calcification may occupy small to large areas of the lesion, occasionally obliterating all but the margin of the
lesion. When hemorrhage or fibrovascular reaction dominates the biopsy, it is difficult to differentiate the stroma
of an infantile hemangioendothelioma from that of a mesenchymal hamartoma, and a discussion with the
pediatric radiologist may help sort this differential diagnosis (46). One should also remember that hemorrhagic
necrosis can be seen in IHE but is uncommon in mesenchymal hamartoma. The presence of hemorrhagic
necrosis in a biopsy should also raise the possibility of a hepatoblastoma. Another vascular lesion with a myxoid
stroma is hepatic epithelioid hemangioendothelioma, albeit rare in children. This is a multifocal tumor, comprised
of strands, clusters, and nests of CD31-positive epithelioid cells with intracellular lumina and sinusoidal infiltraton
(117).
Immunohistochemically, the endothelial cells of the lesion are positive for CD31, CD34, factor VIII-related antigen,
von Willebrand factor, vimentin, and Ulex europaeus I lecten (e108,e780). Cerar et al. (e108) noted that the cells
beneath the endothelial cells contained cytoplasm that was positive for alpha-smooth muscle actin and
antimuscle actin, negative for desmin, and were enveloped with basement membrane (BM) that they believed
were characteristic of pericytes. Electron microscopy displays vascular channels lined by endothelial cells with
irregular fine cytoplasmic processes along the luminal surface (e205). Fibroblasts and collagen fibers are present
in the stroma. The “type 2 lesion” with multilayered, hyperchromatic endothelial cells in a tufted or branching
pattern is now thought to represent a form of angiosarcoma (see later), but could represent degenerative
change.
Mo et al. (125) use the presence or absence of GLUT1 immunoreactivity of the endothelial lining in separating
the “true infantile hemangioma” (hemangioendothelioma) from a “hepatic vascular malformation with capillary
proliferation.” They note that with the GLUT1-positive infantile hemangioma patients have asymptomatic
hepatomegaly and an incidental finding of the lesion in the first few weeks or months of age. The lesion is
present in the liver as multiple small nodules of closely packed capillary vessels with involutional features. These
tumors undergo spontaneous involution over months or years and are usually unresponsive to corticosteroids or
interferon treatment. The GLUT1-negative hepatic vascular malformation with capillary proliferation is usually
symptomatic at birth or in the first few weeks of life with severe edema and congestive heart failure. The lesion is
usually a single mass with malformed irregular vessels commonly associated with infarction, hemorrhage,
calcification, and peripheral reactive capillary proliferation. Surgical resection is often required.
Molecular Pathology
Flow cytometry performed by Selby et al. on 21 cases showed 16 to be diploid, 3 to be aneuploid, and 2 with a
wide coef-ficient of variation (163). Ito et al. described an interstitial deletion of chromosome 6q in a 6-month old
boy with hepatic infantile hemangioendothelioma, microcephaly, hypertelorism, low-set ears, prominent nasal
bridge, cubitus valgus, overlapping fingers, cryptorchidism, and micropenis (e310). Other anomalies reported in
association with infantile hemangioendothelioma include trisomy 21, extranumerary digit, hydrocele, and
congenital heart disease (163). Shah et al. noted a newborn girl with a large left-sided diaphragmatic hernia who
had a heterotopic liver with an infantile hemangioendothelioma in the left hemithorax attached by a pedicle
through the diaphragm to the left lobe of the liver (e619).
TERATOMA
Teratoma is a rarely occurring benign neoplasm composed of a mixture of tissue of mesodermal, ectodermal, and
endodermal origin. Most pediatric cases occur in the 1st year of life, presenting as an abdominal mass, which on
plain film frequently
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displays areas of calcification (e559,e705,e757,e769). AFP may be elevated. Associated conditions include
anencephaly and trisomy 13. Resection may be curative. Care should be taken not to confuse teratoma with a
mixed hepatoblastoma with teratoid features (e139,e170). Intrahepatic fetus-in-fetu has been reported (e409).
FIGURE 15-50 ▪ Teratoma. A: A large irregular mass contains both solid and cystic components of varying color.
B: Random sampling of the lesion displays tissues of various somatic lines including, in this image, cartilage,
epithelial-lined ducts, and “immature” tissue (lower left). (H&E stain, original magnification 15×).
The lesions are large, with a variegated cut surface reflecting the various tissues of the tumor (Figure 15-50).
Microscopically, benign tissues of all three germ cell layers may be found, including well-differentiated squamous
epithelium, bone, cartilage, gastrointestinal mucosa and muscularis propria, renal glomeruli and tubules,
respiratory epithelium, and neural tissue. The presence of embryonal or fetal hepatoblastoma cells precludes the
diagnosis of teratoma and instead favors mixed hepatoblastoma with teratoid features or teratoid
hepatoblastoma.
HEPATOBLASTOMA
The incidence rate of primary hepatic malignancies in children 0 to 14 years of age is approximately 0.2 per
100,000 children in the United States, with hepatoblastomas accounting for 47% of the malignancies and nearly
27% of all pediatric hepatic tumors (42). By age group, hepatoblastoma accounts for 1% of all pediatric
malignancies in children under 15 years age, 1.5% of all malignancies in children younger than 5 years of age,
and 3.3% of all malignancies in white and black children under 1 year of age (e420,e575). The reported
incidence is 11.2 cases per million during the 1st year of life; nearly 90% of hepatoblastomas are seen in the first
5 years of life, with 68% discovered in the first 2 years and 4% present at the time of birth (Figure 15-51). Of 271
primary hepatic malignancies reported in the United States to Surveillance, Epidemiology and End Results
(SEER) data between 1973 and 1997 in patients below 20 years of age, 67% and 31% were HB and HCC,
respectively. In the group less than 5 years of age, HB accounted for 91%, whereas among those 15 to 19 years
of age, HCC represented 87% of the cases (45). The relative frequency of hepatoblastomas in younger children
is most apparent when noting that hepatoblastomas account for over 40% of all hepatic tumors (benign and
malignant) in children younger than 2 years of age, but only 7.5% of liver tumors in children 5 to 20 years old.
Although
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there is no racial predilection for hepatoblastoma, there is a distinct male predominance from 1.2:1 to 3.6:1 (e93).
FIGURE 15-51 ▪ Hepatoblastoma. Age distribution in 105 cases.
Pathogenesis
There appears to be a genetic predisposition to hepatoblastomas, with an increased incidence in a setting of
Beckwith-Wiedemann syndrome (macrosomia, macroglossia, visceromegaly, abdominal wall defects,
hemihypertrophy), hemihypertrophy, and familial adenomatous polyposis (FAP). The relative risk for the
development of hepatoblastoma in Beckwith-Wiedemann syndrome is 22.80 (e166,e654), while that for FAP is
12.20 (e236), suggesting a role for genetic aberrations of chromosomes 11 and 5, respectively, in the
pathogenesis of hepatoblastoma. Inactivation of the APC tumor-suppressor gene (found on chromosome 5) is
found in 67% to 89% of sporadic hepatoblastoma (e34,e291,e317,e761). This gene is known to regulate
betacatenin and modulate the wnt signaling pathway, suggesting a role for this signaling pathway in the
development of hepatoblastoma (e725). Additional biologic markers may include trisomies 2, 8, and 20 and
translocation of the NOTCH2 gene on chromosome 1. There is also an association of prematurity/low birth
weight and hepatoblastoma, with a relative risk of up to 15.64 in patients weighing less than 1,000 g, compared
with patients weighing 2,500 g (e505). In Japan, Ikeda et al. (e300) have noted an increasing incidence of
hepatoblastoma in very low birth weight infants from 0.7% of patients with birth weights less than 1,500 g with
tumors in 1985 to 1989 to 8.6% of patients with similar low birth weights in 1990 to 1993. In the United States,
Ross and Gurney (e576) have observed a similar increasing trend of 5.2% in hepatoblastoma incidence in
children 4 years and younger during the most recent two decades, a period corresponding with improved survival
for low birth weight children. Hepatoblastoma has been described in association with trisomy 18, including some
cases with abdominal wall defects. Hepatoblastoma has been noted in a number of sibling pairs including
identical male twins and two siblings with GSD type 1a (e309,e475,e563,e677). There are no known
environmental risk factors (e605). Zimmermann has recently reviewed putative pathways from ontogenesis to
oncogenesis as a possible basis for a molecular classification of hepatoblastomas (199).

Most patients present with an enlarged abdomen noted by a parent or discovered on routine physical
examination. Other symptoms such as anorexia, weight loss (less frequently), nausea, vomiting, and abdominal
pain may indicate advanced disease (e525). Jaundice is noted in about 5% of cases. Physical examination
reveals a firm, often irregular mass in the right upper abdomen that may cross the midline and extend down to
the pelvic rim. A variety of malformations and clinical presentations have been described in patients with
hepatoblastoma (Table 15-19). A striking presentation of hepatoblastoma is seen in children (particularly young
boys) whose tumors produce hCG, leading to precocious puberty with genital enlargement, the appearance of
pubic hair, and a deepening voice. The increased levels of hCG correlate with immunohistochemical staining and
are accompanied by increases in serum luteinizing hormone and plasma testosterone
(e284,e285,e457,e469,e478,e758).
Table 15-19 ▪CLINICAL SYNDROMES, CONGENITAL MALFORMATIONS AND OTHER

CONDITIONS ASSOCIATED WITH HEPATOBLASTOMA
Absence of left adrenal gland
Aicardi syndrome
Alcohol embryopathy
Beckwith-Wiedemann syndrome with opsoclonus, myoclonus
Bilateral talipes
Budd-Chiari syndrome
Cleft palate, macroglossia, dysplasia of ear lobes
Cystathioninuria
Down syndrome, malrotation of colon, Meckel diverticulum, pectum excavatum, intrathoracic kidney,
single coronary artery
Duplicated ureters
Fetal hydrops
Gardner syndrome
Goldenhar syndrome oculoauriculovertebral dysplasia, absence of portal vein

Hemihypertrophy
Heterotopic lung tissue
Heterozygous A1AT deficiency
HIV or HBV infection
Horseshoe kidney
Hypoglycemia
Inguinal hernia
Isosexual precocity
Maternal clomiphene citrate and Pergonal
Meckel diverticulum
Oral contraceptive, mother
Oral contraceptive, patient
Osteoporosis
Persistent ductus arteriosus
Polyposis coli families
Prader-Willi syndrome
Renal dysplasia
Right-sided diaphragmatic hernia
Schinzel-Geidion syndrome
Synchronous Wilms tumor
Trisomy 18
Type 1a glycogen storage disease

Umbilical hernia
Very low birth weight
From Ishak KG, Goodman Z, Stocker J T. Tumors of the liver and intrahepatic bile ducts. In: Rosai J,
Sobin L, eds. Atlas of tumor pathology, 3rd series, Washington, DC: Fascicle; Armed Forces Institute of
Pathology, 2000.
Anemia is common (70%) in patients with hepatoblastoma as is thrombocytosis (50% of cases). Platelet counts
of greater than 500 × 106/L were noted in 35% of 99 cases by Shafford and Pritchard (e618), with 29% having
counts over
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800 ×106. Along with AFP, thrombocytosis has been used as a measure of disease activity (e220,e777).
Approximately 90% of patients demonstrate elevated serum AFP levels, and there is a correlation between AFP
levels and extent of disease (e736), with a return to normal levels after complete resection of the tumor and a re-
elevation with recurrence (e658). However, the least well-differentiated hepatoblastomas, that is, the small cell
undifferentiated type, may in some cases show little or no elevation in AFP (e720). Van Tornout et al. (e736)
have noted that for unresectable or metastatic hepatoblastoma, AFP levels can reliably predict outcome and
identify poor responders to treatment. In studying patients who had undergone an initial surgery and
chemotherapy, those patients whose AFP failed to decrease by at least two logs had a much poorer prognosis.
In contrast, a large early decrease in AFP levels was a strong independent predictor of favorable outcome. It is
important to remember, however, that AFP is present at levels of 25,000 to 50,000 ng/mL at birth and does not
fall to “adult” levels of less than 25 ng/mL until 5 to 6 months of age (e487). AFP levels in infants with tumors
resected in the first 6 months of life may therefore be “appropriately” elevated even though the tumor has been
completely resected.
Isaacs reviewed 32 cases of hepatoblastomas reported in fetuses and neonates (75). Nine cases were
diagnosed antenatally and 23 at birth, with a female predominance (female to male ratio 1.6). Although the most
common presenting finding was an elevated AFP, this finding was present only in 50% of the patients,
suggesting that AFP levels may not be a reliable indicator of the tumor in the fetus and neonate as compared
with older children. Abdominal distension was the second common presenting finding followed in rank by a
palpable abdominal mass, hepatic or abdominal mass detected on antenatal sonography, and hepatomegaly.
Anemia, fetal hydrops, and respiratory distress were other initial findings. The most common site of origin was
the right lobe of the liver (47%) compared with the left (16%), or both lobes (6%). Four patients had more than
one hepatic tumor at the time of diagnosis. Most patients were classified as stage 1 (12 of 32, 37.5%), none as
stage 2, 4 (12.5%) as stage 3, and 6 (18.8%) as stage 4. In 10 patients (31.2%) the stage of disease was not
mentioned in the report. Survival rates for stages 1, 3, and 4 were 50%, 50%, and 0%, respectively. Sixty-three
percent of the patients were treated by the following modalities: surgical resection alone, surgical resection plus
chemotherapy, and surgical resection with hepatic artery embolization and chemotherapy; survival rates were 3
of 9 (33%), 3 of 5 (60%), and 1 of 1 (100%), respectively. Only one of four infants who received chemotherapy
alone after a biopsy survived. Fetal survival was slightly less than the neonatal diagnosed cases, 22% and 26%,
respectively. All 12 untreated patients died. Of the 20 treated infants, 8 (40%) lived. The overall survival for
hepatoblastoma group was poor, 8 of 32 (25%) survived. The main cause of death from hepatoblastoma was
mass effect by the tumor, producing abdominal distension, compression of portal vein and inferior vena cava,
fetal hydrops leading to stillbirth, and severe respiratory distress. Metastases to the placenta with occlusion of
umbilical vessels and to the lungs were other terminal events. Anemia resulting from bleeding into the tumor and
rupture of the tumor during delivery occurred in seven and four patients, respectively. There were a few
perioperative deaths related to immaturity and clinical condition of the patients. Female/male ratio was 1.6:1. Of
32 cases, 9 (28%) were diagnosed antenatally and 23 (72%) in the neonatal period. Tumors were more common
in the right (15/32, 47%) than in the left (5/32, 16%) lobe, with two patients (6%) having tumors in both lobes.
Tumors ranged in size from 3 to 16 cm (mean 8 cm) and weighed from 21 to 429 cm (mean 160 cm). The relation
of histology and survival was as follows: fetal 3/10 (30%); embryonal 1/6 (17%); fetal and embryonal 1/2 (50%);
and fetal, embryonal, and mesenchymal 3/8 (37.5%).
Imaging studies are helpful in diagnosing hepatoblastoma and differentiating it from other liver disorders seen in
young children (e286). CT demonstrates a solitary or occasionally multifocal mass(es) with attenuation values
between those of water and normal liver parenchyma. Speckled or amorphous calcification may be seen on CT
in more than 50% of cases (e446). Ultrasonography displays a mass with increased, inhomogeneous
echogenicity, punctate or amorphous calcification, and occasional cystic areas (e162). On antenatal
ultrasonography, hepatoblastomas are described as well-defined, solid, echogenic lesions, with a “spoked-
wheel” appearance (e631). Calcifications may be present, and a pseudocapsule gives the lesion(s) a
characteristic well-demarcated appearance (75). Using pulsed Doppler ultrasonography, Bates et al. (e47) found
peak systolic Doppler frequency shifts equal to or greater than 4 kHz and were also able to demonstrate
antegrade diastolic flow. Differentiation of hepatoblastoma from other childhood hepatic solid, cystic, or vascular
lesions such as mesenchymal hamartoma, infantile hemangioendothelioma and HCC can be aided by MRI with
standard spin-echo T1- and T2-weighted imaging enhanced by the application of advanced sequences such as
gradient-echo, fast spin-echo, and fat suppression techniques (e545). The histologic features of
hepatoblastomas can be differentiated by MRI as well, with the homogenous character of an “epithelial” lesion
contrasting with the heterogeneous character of a “mixed” hepatoblastoma with its fibrotic bands. Decreased
signal intensity compared with normal liver is noted on T1-weighted images, whereas increased signal intensity
is seen on T2-weighted images. Hypointense bands on MRI identify fibrotic bands, and the presence of vascular
invasion may be detected by gradientecho MRI (e545).
Staging
Most patients in the United States are staged postoperatively according to the Children's Cancer Study Group
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(CCSG) classification (see Table 15-20) (e268,e501). Other classifications include the TNM or variations of the
CCSG staging classification (173) (e279,e645). Based on these classifications, approximately 38% of
hepatoblastomas are stage I at the time of initial diagnosis and before any chemotherapy is administered. At this
same point, about 9% are stage II, 24% stage III, and 29% stage IV (42) (e211,e268). However, this traditional
staging system has been criticized for being rather subjective, depending to a large extent on the surgeon rather
than the tumor (e36,e439). In 1990, the International Society of Pediatric Oncology Liver Study Group (SIOPEL-
1) adopted a new preoperative staging system, Pretreatment Extent of Disease (PRETEXT), based exclusively
on images obtained prior to surgery, based on the branching pattern of the portal vein, which divides the liver
into eight segments. The system divides the liver into four sectors: (a) lateral sector (Couinaud segments 2 and
3); (b) medial sector (segment 4); (c) anterior sector (segments 5 and 8) and; (d) posterior sector (segment 6 and
7). Tumors are classified as one of four categories (PRETEXT-I to PRETEXT IV) by determining the number of
affected liver sector(s) on imaging. Extrahepatic growth of the tumor is indicated by adding a letter (V
involvement of hepatic vein, P involvement of portal vein, E for extrahepatic extension, M for the presence of
distant metastasis). The PRETEXT system has prognostic value for overall and disease-free survival and is
useful in defining treatment (27) (e606). Although the PRETEXT system was developed mainly to assess the
efficacy of neoadjuvant chemotherapy and to predict surgical resectability, it also had highly prognostic value for
both overall survival and event-free survival (27). Conceptually, however, both preoperative and postoperative
staging systems use the same parameters for staging, namely, size, vascular invasion, extension and complexity
of the primary tumor, and the absence or presence of metastases. Metastatic spread of hepatoblastoma is seen
most frequently to the lung but may also spread to bone, brain, eye, and ovaries (e79,e197,e255,e448,e567).
Local extension into hepatic vessels and the inferior vena cava may also occur (e681).
Table 15-20 ▪ STAGING OF HEPATOBLASTOMAS
Stage Complete resection

I
Stage Microscopic residual tumor Intrahepatic Extrahepatic

II
Stage Gross residual tumor Primary completely resected, nodes positive and/or tumor spill Primary
III not completely resected, and/or nodes positive and/or tumor spill
Stage Metastatic disease Primary completely resected Primary not completely resected
IV
From King D, Ortega J, Campbell J. The surgical management or children with incompletely resected
hepatic cancer is facilitated by intensive chemotherapy. J Pediatr Surg 1991;26:1074-1081, with
permission.

Although complete resection of hepatoblastoma is the mainstay of treatment and is the only chance of cure,
improvements in survival that have occurred over the last three decades have been a function of standardized
chemotherapy regimens that reduce tumor size and enable complete tumor excision, even permitting cure in the
presence of initially unresectable or metastatic disease. Treatment strategies currently combine surgery,
transplantation, and chemotherapy (adjuvant and neoadjuvant), as defined by the PRETEXT stage of the tumor.
Surgery remains the mainstay in the treatment of hepatoblastoma, with prognosis directly related to tumor stage.
Small, solitary lesions localized to a single lobe can be adequately treated by lobectomy. Larger lesions,
including those requiring preoperative chemotherapy to allow resection, may require more extensive surgery or
transplantation (e5,e193,e212,e234). Surgical complications, particularly hemorrhage, are noted in 14% of
primary resections and 29% of second resections (e744). If resection is possible and safe, an attempt to obtain
clear resection margins is essential; positive margins on pathology warrant a re-resection if possible. Although
elevated AFP immediately after resection is common, persistently elevated or rising AFP levels indicate the need
to evaluate further for disease recurrence or search for distant metastasis. Contraindications to immediate
resection include extensive bilateral liver involvement, presence of vascular invasion of major hepatic veins or
inferior vena cava, diffuse multifocal disease, and distant metastasis (e289).
At the time of diagnosis, 40% to 60% of hepatoblastomas are considered to be unresectable and 10% to 20% of
patients are found to have pulmonary metastases. Preoperative chemotherapy converts nearly 85% of these
“unresectable” lesions to ones that can be entirely grossly removed, that is, to stage I or II lesions (see Table 15-
20) with subsequent long-term survival (e193,e533,e658). Even if unresectable at diagnosis, most
hepatoblastomas are unifocal and chemosensitive, with cisplatin and adriamycin being the most commonly used
agents. Chemotherapy has been proven effective in both an adjuvant and neoadjuvant treatment and can shrink
tumors. It makes them less prone to bleed and delineates the tumor from the surrounding normal parenchyma
and vascular structures facilitating resection. In rare cases, patients may survive with chemotherapy alone
(e782). On the other hand, some tumors may become resistant to prolonged courses of chemotherapy (e747),
and the highest survival rates have historically been observed in patients with initially resected tumors—although
these tumors also tend to be the smaller more favorable tumors. Further, prolonged (>4 cycles) courses of
neoadjuvant chemotherapy are discouraged, since this may lead to cumulative chemotherapy toxicity and cause
tumor cells to become resistant to chemotherapy (122).
The 5-year survival rate for hepatoblastomas has improved to over 75% compared with a 5-year survival rate
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of 35% almost 30 years ago (122) (e201,e547). Preoperative chemotherapy has increased resectability of
hepatoblastoma from 40% to 60%, to 90%, with the more extensive tumors requiring transplantation to remove
the involved portions of liver (e196,e669). In fact, primary liver transplantation with neoadjuvant chemotherapy
may result in an 80% 5- to10-year disease-free survival rate, whereas the 10-year survival falls to 40% in those
undergoing transplantation as “rescue therapy” (135). The SIOPEL-1 study showed that, in patients undergoing
transplantation, only macroscopic venous invasion had a significant prognostic effect on survival (e504). A
worldwide electronic registry for liver transplant in childhood liver tumors (hepatoblastoma, HCC, and
hemangioendothelioma) has been established; this “pediatric liver unresectable tumor observatory (PLUTO)”
registry can be reached at http://transplant.test.cineca.it/.
Traditionally, tumor stage at the time of initial resection has been the key prognostic factor in determining the
survival of children and adults with hepatoblastoma (42) (e193,e268,e669,e745). Data from the recent COG
study, 9,645, show 3-year event-free survival of 90% for stage I to II, 50% for stage III, and only 20% for stage IV
(122). However, as noted earlier, preoperative and postoperative chemotherapy and aggressive treatment of
pulmonary and central nervous system metastases have significantly changed the survival rate in patients with
stage IV tumors (e209,e518,e567). Survival is independent of histologic subtype when adjusted for age, sex, and
stage (42) (e745). Only small cell undifferentiated hepatoblastoma may have a worse prognosis than others, but
the small number of cases of this unusual type makes analysis uncertain. More recently, two broad categories of
risk stratification have been advocated, namely standard risk and high risk. Standard risk tumors are PRETEXT
I, II, or III. SIOPEL high-risk tumors are defined as tumors involving all four hepatic sectors (PREVEXT IV), or any
tumor with metastasis (m), ingrowth of the vena cava (v), ingrowth of the portal vein (p) or contiguous
extrahepatic disease (e), and tumors that fail to express AFP with AFP less than 100 at diagnosis (155).
Gross Appearance
Hepatoblastomas are single masses in approximately 80% of cases. They occur in the right lobe in 58% of
cases, in the left in 15%, and in both lobes in the remaining 27%, either as a large single lesion extending across
the midline or as multiple lesions (e659). Distant metastasis are present in 20% of patients at the time of
diagnosis, with the lung as the most common site of metastasis; other common sites are the brain and bone and
metastasis occurs more commonly with disease relapse (e209).
Tumors may measure 15 cm or more in diameter and weigh in excess of 1,000 g. Grossly, they are coarsely
lobulated and frequently bulge from the surface of the liver (Figure 15-52A). On cut section, the lesions are tan to
light brown to green and display frequent areas of hemorrhage and necrosis. Various types of mesenchymal
tissues (e.g., osteoid, cartilaginous, fibrous) in the mixed type of hepatoblastoma may alter the color and
consistency of the gross appearance.
Table 15-21 ▪ HISTOLOGIC CLASSIFICATION OF HEPATOBLASTOMA

I.Epithelial type
A. Fetal pattern
B. Embryonal and fetal pattern
C.Macrotrabecular pattern
D.Small-cell undifferentiated pattern
II. Mixed epithelial and mesenchymal type
A.Without teratoid features
B.With teratoid features
Histopathology
Histologically, the tumor is traditionally classified into six patterns (Table 15-21) (Figures 15-52, 15-53 and 15-
54). The epithelial types account for approximately 56% of cases, including pure fetal (31%), embryonal (19%),
macrotrabecular (3%), and small cell undifferentiated (3%). The mixed pattern of epithelial and mesenchymal
components accounts for 44% of the cases, including 34% without teratoid features and 10% with such
components as squamous epithelium and striated muscle (see later).
The fetal pattern refers to cases in which 100% of the tumor is composed of small, round, uniform cells with
abundant cytoplasm and distinct cytoplasmic membranes (Figure 15-52B). The cells are arranged into thin
trabeculae, usually two to three cells thick, with alternating light and dark areas.
The embryonal pattern refers to cases of epithelial hepatoblastoma in which, in addition to fetal cells, part of the
tumor has cells arranged into sheets of irregular, angulated cells with a high nucleocytoplasmic ratio, increased
nuclear chromatin, and indistinct cytoplasmic membranes (Figure 15-52C). Pseudorosette and acinar formation
are common features. Foci of extramedullary hematopoiesis (EMH) are seen in both the fetal and embryonal
areas.
The macrotrabecular pattern refers to cases in which trabeculae more than 10 cells in thickness are present as a
repetitive pattern within the tumor (Figure 15-52D). The large trabeculae contain either fetal- or embryonal-type
cells; a third, larger cell type with cytoplasm that is more abundant than in normal hepatocytes or fetal-type cells;
or a combination of all three cell types. Thus, the term “macrotrabecular” refers more to a growth pattern rather
than a distinct subtype and these lesions form a heterogeneous group. Cases that have embryonal or
mesenchymal cells with an isolated macrotrabecular focus are classified based on the embryonal or
mesenchymal cell present and not as macrotrabecular. Tumors where the third (hepatocyte-like) cell type
predominates may be very difficult to distinguish from HCC. The presence of EMH is useful in a diagnosis of
hepatoblastoma.
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FIGURE 15-52 ▪ Hepatoblastoma, epithelial type. A: A large, wellcircumscribed mass (left) is composed of
irregular nodules of tissue resembling normal liver. B: A light (left) and dark (right) pattern is produced by
trabeculae of uniform small hepatocytes with clear (light) or granular (dark) cytoplasm. Note the foci of
extramedullary hematopoiesis in this well-differentiated fetal epithelial lesion (H&E stain, original magnification
100×). C: The embryonal component of the epithelial lesion is composed of single or small clusters of oval or
tapered cells with mild anisonucleosis and nuclear hyperchromasia. (H&E stain, original magnification 150×). D:
A “macrotrabecular” pattern is formed by solid sheets of hepatocytes, some with central areas of necrosis (H&E
stain, original magnification 40×). E: With “anaplastic” hepatoblastoma, sheet of small, round blue cells resemble
neuroblastoma cells (H&E stain, original magnification 200×).
The small cell undifferentiated or anaplastic pattern is composed of cells reminescent of neuroblastoma or other
small round blue cell tumors, with scanty cytoplasm and hyperchromatic nuclei (Figure 15-52E). Round or ovoid
cells predominate, with the occasional presence of spindle or stellate cells within a mucoid matrix. These cells
grow in sheets but lack cohesiveness. Mitoses are occasionally present, but the cells do not produce glycogen,
fat droplets, or bile pigment. Abortive or incompletely formed bile ductules may be present, but electron
microscopy or immunohistochemical studies, or both, may be needed to confirm the diagnosis of
hepatoblastoma. Particularly helpful in establishing the diagnosis is the presence of cytoplasmic staining with
polyclonal anticytokeratin antibodies. Gonzalez-Crussi believes that the small cell form represents the subtype
with the least differentiation within the highly variable morphologic spectrum of hepatoblastomas (e245). Medium-
and large-sized cells have been reported in undifferentiated hepatoblastomas; some undifferentiated tumors
have shown intermediate or large cells, leading to a proposal to subclassify undifferentiated hepatoblastomas as
small cell, intermediate cell, and large cell subtypes (94, 199). Immunostains are required to
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differentiate these from other large cell tumors such as lymphoma, large cell medulloblastoma (e235), large cell
neuroblastoma (e709), and Ewing sarcoma family tumors, although some hepatoblastomas may be positive for
CD99 (199). The histogenesis of these undifferentiated tumors is not known. Although hepatic stem cells have
been invoked in their pathogenesis (e583), this has been refuted by other authors (13). An alternative pathway
might involve regression to a primitive cell lineage of the hepatogenic foregut endoderm (199). These tumors
may show loss of INI1.
FIGURE 15-53 ▪ Mixed epithelial and mesenchymal hepatoblastoma. A: The tumor within the liver displays a
highly variegated appearance reflecting the presence of mesenchymal tissue and epithelial cells.B Osteoid-like
material (left) contains cells similar to the fetal and embryonal epithelial cells (right) The cells associated with
the “osteoid” are cytokeratin positive (H&E stain, original magnification 125×)
The mixed epithelial and mesenchymal type of tumor contains cells admixed with primitive mesenchyme and
various mesenchymally derived tissues (Figure 15-53). The highly cellular primitive mesenchyme consists of
elongated, spindle-shaped cells with a scanty cytoplasm, and elongated pump nuclei with rounded ends,
resembling fibroblastoid/myofibroblastoid tissue. Some areas may display parallel orientation of cells with definite
collagen fibers and young fibroblasts; other areas may have more loosely arranged cells leading to a
myxomatous appearance. Mature fibrous septa are also seen, along with areas of osteoid and cartilaginous
tissue. Cells within the osteoid foci have an irregular, angular outline and short processes that make them
indistinguishable from osteoblasts. Immunohistochemical studies, however, have identified this osteoid-like
material as being produced though a process of epithelial differentiation (e3). Osteoid stromal component is
reportedly more prominent following chemotherapy (e283,e598). The prognostic significance of these stromal
elements is unclear with studies reporting both improved survival or no effect on survival (e268). Approximately
20% of the mixed types of hepatoblastomas contain a variety of tissues, including stratified squamous epithelium,
melanin pigment, mucinous epithelium, cartilage, bone, and striated muscle in addition to the epithelial cells,
fibrous tissue, and osteoid-like material (Figure 15-54). These tumors have been termed teratoid
hepatoblastomas by Manivel et al. (e419).
In view of the histologic heterogeneity of hepatoblastomas, rare tumors with unique morphologies may be seen
that are difficult to classify into one of the above categories. This has led to anecdotal descriptions of “new”
variants of hepatoblastomas including mucoid anaplastic hepatoblastoma (e322), hepatoblastomas with
endocrine/neuroendocrine differentiation (e578,e579), cholangiocytic/cholangioblastic hepatoblastoma (e789),
and hamartoma-like hepatoblastoma/hepatoblastoma with organoid configuration or to the allocation of
problematic cases into a neutral category, such as hepatoblastoma, not otherwise specified (199). The biologic
significance, if any, of these morphologic variants is not known. Tumors that show mainly mesenchymal/stromal
tissue with apparent lack of an epithelial component have been termed “pediatric hepatic stromal tumors” and
have been proposed to resemble similar lesions described in childhood kidney cancer, that is, metanephric
stromal tumor/MST (199). It is likely that these may be related to the so-called nested epithelial and stromal
tumors (see later).
Notwithstanding the morphological differences in mixed HBs between epithelial components of any kind and the
stromal components, there is evidence that both have a common lineage. This is suggested by the observation
that b-catenin mutations visualized by nuclear reactivity occur in epithelial and mesenchymal components. The
pathogenic pathways causing the development of both epithelial and mesenchymal/stromal lineages within the
same tumor are not yet known. However, epithelial-mesenchymal transition or mesenchymal-to-epithelial
transition has been hypothesized to play a role in pathogenesis.
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FIGURE 15-54 ▪ Mixed hepatoblastoma with teratoid features. A, B: Together with the typical epithelial
component (right), the tumor is composed of mesenchymal tissue showing differentiation into fibrous tissue (left)
and stratified squamous epithelium (right). C, D: Striated muscle cells, and osteoid-like material containing
melanin pigment. (H&E stains, original magnification 30× [A], 100× [B], 300× [C], and 200× [D]).
Although open biopsy and needle biopsy often are adequate in establishing the diagnosis, the use of FNA may
prove dif-ficult, particularly in cases of small cell undifferentiated or embryonal epithelial lesions. FNA has been
reported to be accurate in diagnosing hepatoblastoma in approximately 65% (19 of 29) of cases, primarily fetal
epithelial and mixed hepatoblastomas (e66,e172,e263,e647,e728,e749). The diagnosis was most frequently
confused with metastatic tumor including Wilms tumor, neuroblastoma, and rhabdomyosarcoma (e647,e749).
Weir et al. have reported the cytologic features of hepatoblastoma in serous cavity fluids. All six specimens
examined showed hypercellular smears in a relatively clean background. Mixed embryonal and fetal subtypes of
HBL disclosed three-dimensional clusters of neoplastic cells that formed straight or branched cords and acinus-
like structures. The cells were moderately pleomorphic, had high nuclear-to-cytoplasmic ratios, rare intranuclear
inclusions, and numerous mitoses. The small cell subtype showed tight clusters of small, round, primitive cells
with hyperchromatic nuclei, high N/C ratios, and prominent nuclear molding. In addition, there were numerous
single cells with naked nuclei, often in an Indian-file configuration. Bile pigment, osteoid, and other mesenchymal
components were absent in all their specimens (191).
The prognostic impact of histology has been analyzed in a few studies (27, 42)
(e226,e267,e268,e415,e440,e746). Of the five histologic subtypes (pure fetal, embryonal, mixed epithelial-
mesenchymal, macrotrabecular, and small cell undifferentiated), the fetal subtype carries the most favorable
prognosis, and small cell undifferentiated the worst. In general, pure fetal histology is associated with an
improved prognosis, while undifferentiated histology is associated with a poor prognosis, with macrotrabecular
histology probably having an intermediate prognosis. In a study of 168 patients
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the estimated 24-month survival probability was 50% with the macrotrabecular type in comparison with 92%,
63%, and 0% for the purely foetal, embryonal, and SCUD histologies, respectively (e268). Small cell
undifferentiated histology predicts an increased risk of relapse (184) (e502). Even a focal (partial or
predominant) expression of small cell histology in completely resected HBL may have an unfavourable effect on
outcome (e267), drawing a corollary with focal versus diffuse anaplasia in nephroblastoma. Small cell
undifferentiated tumors appear to be biologically different from tumors with non-small cell histology and have
been reported to be similar to rhabdoid tumors at the immunohistochemical (INI1 negative), cytogenetic and
molecular level and in terms of their adverse adverse outcomes (184). Among the mixed epithelial/mesenchymal
type tumors, the presence of mesenchymal elements may be associated with improved prognosis (e268).
Immunohistochemistry
The various patterns of hepatoblastoma display differing immunoreactivity, probably based on their degree of
differentiation, with the fetal cell areas of an epithelial hepatoblastoma staining positively for a broad range of
epithelial markers and small cell undifferentiated hepatoblastomas showing positivity for only a few markers
(Table 15-22) (e3,e101,e490,e538,e580-e583,e643,e660,e734). Ruck et al. (e582) noticed a correlation
between the cytokeratin staining of normal biliary epithelium and liver parenchymal cells and the types of
epithelial cells in hepatoblastoma, with CK19 more prominent in small cell and embryonal epithelial cell areas
(and in biliary epithelium) and CK18 more prominent in fetal epithelial areas (and in normal hepatocytes). Osteoid
areas were positive for both CK18 and CK19, whereas spindle cells areas were not immunoreactive for any of
the cytokeratins. These characteristics have suggested to some authors that the primitive small cells give rise to
embryonal hepatoblastoma cells and, after further maturation, fetal hepatoblastomas (e3,e582). In a study of 12-
needle core biopsies in proven hepatoblastomas, Ramsay et al. reported variable antigen expression with
positivity for cytokeratins (10/12 cases), alpha-1-antitrypsin (5/12 cases) and AFP (7/12 cases), MIC-2 (CD99)
(8/12 cases), NCAM (CD56) (4/12 cases), neuroblastoma marker NB84 (3/12), desmin (2/12 cases), BCL2 (2/12
cases), and one case each for vimentin, NSE, and PGP 9.5. However, all tumors were negative for CD45, WT1,
and S-100. The authors concluded that hepatoblastoma shows no distinct immunohistochemical profile, and the
diagnosis requires a combination of the clinical, imaging, and pathologic findings, since they can express
antigens normally seen in other childhood malignancies (143). Insulinlike growth factor 2 and insulin-like growth
factorbinding protein expression have been noted in 11 hepatoblastomas, with their expression inversely
correlated with the degree of tumor cell differentiation. Akmal et al. (e11) suggest that these markers may be
used as an assessment of the degree of differentiation of the tumor. Interestingly, hypoglycemia has been noted
as a rare presenting symptom of hepatoblastoma with the hypoglycemia disappearing after removal of the tumor
(e266). Glypican 3, a heparin sulfate proteoglycan bound to the cell surface, is overexpressed both at the
genomic (by microarray studies) and at the protein (by IHC) levels in hepatoblastoma. In their series, Zynger et
al. found that 65 of 65 hepatoblastomas had cytoplasmic immunoreactivity for GPC3 with greater than 90% of
cases showing strong, diffuse positivity. There was no reactivity in benign
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liver tissue. Fetal, embryonal, and small cell undifferentiated patterns were diffusely positive in almost all cases,
whereas mesenchymal and teratoid patterns were nearly all negative (200). Immunohistochemical studies have
identified putative stem cells in hepatoblastomas. Cells in atypical ducts were found to express simultaneously
stem cell markers and hepatocytic or biliary lineage markers, suggesting a direct role for stem cells in the
histogenesis of hepatoblastoma (e210). The presence of stem cells in these tumors is also supported by the
occurrence of teratoid variant of hepatoblastoma.
Table 15-22 ▪IMMUNOHISTOCHEMICAL FINDINGS IN HEPATOBLASTOMA
Fetal Cell Embryonal Small-cell Mesenchymal “Osteoid”

Areas Cell Areas Areas Areas Areas
Keratin ++ ++ ++ ± +
α fetoprotein ++ ++ − +
α1-antitrypsin ++ ++ ± + ++
α1-antichymotrypsin ++ ++ + + +
Ferritin ++ + + +
Carcinoembryonic ++ ++ − − ±
antigen
Epithelial + − + ++
membrane antigen
Transferrin + ++
Human chorionic + +
gonadotropin
Vimenten − − − ++ ++
Serotonin ± ± − − −
Somatostatin ± ± − − −
NSE − ± − + ++
S-100 ± + ± + ++
Desmin − − − −
Chromogranin A ± ± − − +
Symbols: ++, Majority of cases strongly positive; +, moderately or weakly positive in some cases; ±,
positive in some reports and negative in others; -, negative in all reports.
From Ishak K, Goodman Z, Stocker J. Tumors of the liver and intrahepatic bile ducts. In: Rosai J, Sobin
L, eds. Atlas of tumor pathology, 3rd series ed. Washington, DC: Armed Forces Institute of Pathology,
2001.
Molecular Pathology
Deregulation of the APC/beta-catenin pathway occurs in a consistent fraction of hepatoblastomas, with mutations
in the APC and beta-catenin genes implicated in FAP-associated and sporadic hepatoblastomas, respectively.
Mutations of the beta-catenin gene are present in over 90% of hepatoblastomas, leading to activating
transcription of a number of target genes. b-Catenin is central to the convergence of the Wnt, b-catenin, and
cadherin signaling pathways, where it forms a signaling complex with axins, APC tumor suppressor protein,
glycogen synthase kinase 3b, and other proteins (e479). The Wnt signalling pathway prevents proteosomal
degradation of b-catenin and allows b-catenin to translocate to the nucleus and initiate gene transcription. In fact,
b-catenin can be immunohistochemically detected in the nucleus, following its translocation. Nuclear staining for
b-catenin in hepatoblastomas has been reported to correlate with poor histologic phenotype, higher stage
disease, and poor survival (e517,e683). Other components of the Wnt signaling pathway including Axin gene
mutation (e442) and loss of APC function (e700) have also been have been implicated in hepatoblastoma
tumorigenesis. Giardiello et al. (e237) identified an APC gene mutation in all eight hepatoblastoma patients of
seven FAP kindreds. Oda et al. (e495) have also noted genetic alterations in the APC (loss of heterozygosity
[LOH] or somatic mutations) in 9 of 13 cases of hepatoblastoma in non-FAP patients. Interestingly, a distinct
male predominance (nearly 75%) is seen in APC gene-related hepatoblastomas.
A host of other genetic alterations have been described in hepatoblastomas involving cell cycle-related genes
(e14,e776), apoptosis pathways (e621), p53 mutations (e151), mismatch repair defects (e151), FOXG1
overexpression (e7), and signal transduction pathways (e466), to name a few. It is possible that many of these
molecular aberrations may be late events in the clonal evolution of these tumors that indicate progressive
genomic instability rather than primary events (199). López-Terrada et al. have hypothesized that histologic
microheterogeneity in hepatoblastoma may correlate with molecular heterogeneity, reflecting different stages of
developmental arrest. They found Wnt activation to be most prevalent in embryonal and mixed types, whereas
Notch activation, needed for cholangiocytic differentiation at a more differentiated state, was predominant in pure
fetal hepatoblastomas (105). p53 protein expression is seen less frequently in hepatoblastoma than in other
childhood tumors. In 10 cases of hepatoblastoma, Chen et al. noted only one case of overexpression of p53
protein in a macrotrabecular type at stage IV (e122). Ruck et al. (e580) noted p53 protein immunoreactivity in two
small cell hepatoblastomas and in the embryonal areas of two fetal and embryonal epithelial hepatoblastomas,
but not in the fetal areas of eight fetal or fetal and embryonal epithelial tumors or the mesenchymal areas of four
mixed tumors. Somatic mutations, however, were detected in 9 of 10 cases of hepatoblastoma in the five to eight
exons of the p53 gene by Oda et al. (e494), who suggest that environmental mutagens may be involved in some
cases of hepatoblastoma.
Many aberrations have also been reported at the chromosomal level in hepatoblastomas. Genome-wide
allelotyping of hepatoblastomas have shown frequent allelic losses at many microsatellite loci implicating
chromosome instability as an important factor in development and progression of hepatoblastoma (178). Trisomy
2, trisomy 20, and 4q structural rearrangement are the most common chromosomal abnormalities in
hepatoblastoma (e13,e41,e43,e98, e218,e269,e272,e361,e397,e431,e511,e569,e608,e649,
e658,e680,e689,e707) (see Table 15-23). A derivative chromosome 4 from an unbalanced translocation
between the long arms of chromosomes 1 and 4 has been noted as a recurring abnormality in hepatoblastoma,
while it is rarely seen in other types of neoplasms (e608). In 32 cases, Kraus et al. have shown LOH on
chromosome 1p in seven cases, LOH on 1q in seven cases, and LOH on both 1p and 1q in three more,
suggesting that tumor suppressor genes at the telomeric region of chromosome arm 1p and different regions of
chromosome arm 1q may be involved in the pathogenesis of hepatoblastoma (e361). Albrecht et al. (e13) noted
LOH in 6 of 18 hepatoblastomas in 11p restricted to the telomeric region 11p15.5 and determined that the
parental origin was exclusively maternal. DNA analysis by flow cytometry has been reported in more than 70
cases, with a diploid pattern noted in the well-differentiated (fetal) portions of the tumors and an aneuploid
pattern present in embryonal portions or in small cell (anaplastic) tumors (e140,e280,e363,e604). Krober et al.
(e363) noted an aneuploid peak in tumors with embryonal and fetal components when the areas were analyzed
together and encouraged analysis of all differing areas of a tumor if ploidy is to be used in drawing conclusions
about the prognosis in individual cases. Hata et al. (e280) noted an increased incidence of vascular invasion and
a poorer prognosis in patients with an aneuploid tumor. Terracciano et al. studied 35 hepatoblastoma specimens
by CGH and found significant gains of genetic material. The most frequent alterations were gains of Xp (15
cases, 43%) and Xq (21 cases, 60%), while other common alterations were 1p-, 2q+, 2q-, 4q-, and 4q+. There
was no difference between different histologic subtypes, suggesting a common clonal origin for the different
components (179).
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Table 15-23 ▪CYTOGENETIC FINDINGS IN HEPATOBLASTOMAS
Case
No. Karyotype Histologic Type
1. 46,XY,-2,der(19)t(4, 19),+mar Epithelial; embryonal
2. 47,XY,+20,dmin Mixed mesenchymal-

epithelial; fetal
3. 47,XY,+20,dmin Epithelial; primary embryonal,

foci of fetal areas
4. 93,XXXX,+I(8q)/93,XXXX,del(1)(p22),+I(8q) Mixed mesenchymal-
epithelial; fetal
5. 50,XY,+2,+8,+20,+dic(1)(p12) Mixed mesenchymal-

epithelial; fetal and embryonal
6. 47,XX,+20,del(1)(q32.2)dup(2)(q21q35),dmin/50, Mixed mesenchymal-

XX,+5,+7,+20,+22,del(1),dup(2),I(8q),dmin epithelial; fetal and embryonal
7 47,XX,+20,dup(2)(q23q35)/47,XX,+20,dup(2),dup(6) Mixed mesenchymal-

(p11p24) epithelial; fetal
8. 54,Y,der(X)t(X;1)(p22;q21),+2,+6,+8,+8,+12,+15,+17, Mixed mesenchymal-

+20,inv(9)(p11q21)a epithelial; fetal and embryonal
9. 46,XYder(4)t(2;4)(q21;q35),t(9;?)(p24;?)/47,XY,+20, Mixed mesenchymal-

der(4),t(9?) epithelial; fetal
10. 51,XY,+2,+12,+20,+der(5)t(1;5)(q25;q35),+del(6)(q15) Epithelial; fetal
11. 48,XX,+2,+r/48,XX,+20,+der(2)t(1;2)(q23;p21),inv(5) Epithelial; fetal

(q22q35)/49,XX,+20,+der(2),inv(5),+r47,XX,+2
12. 47,XX,+2 Epithelial; primary fetal, foci of

embryonal areas
13. 47,XX,2q+,t(3;5)(p25;q31),dup(4)(q12q26),+20 Mixed mesenchymal-epithelial
14. 46,XY,t(10;22)(q26;q11) Small cell undifferentiated
15. 47,XY+2 Fetal and embryonal, possible

macrotrabecular
16. 47,XX,+20 Mixed mesenchymal-epithelial

fetal and embryonal
17 46,XX,del(17)(p12)/46,XX Mixed with teratoid features
ainv(9)(p11q21) was constitutional
Modified from Stocker J, Conran R, Selby D. Tumor and pseudotumors of the liver. In: Stocker J, Askin
F, eds. Pathology of solid tumors in children
London: Chapman & Hall, 1998:83-110, with permission.
HEPATOCELLULAR CARCINOMA
HCC is the third most frequently seen pediatric liver tumor and represents up to 20% of all pediatric liver
neoplasms (44, 95). It occurs primarily in the older pediatric patient, with over 65% of cases seen in children
older than 10 years of age (Figure 15-55A) (see Tables 15-14 and 15-16). Rare cases, however, have been
reported even in infants (e268,e332,e368). However, the fibrolamellar variant has not been reported in infants
(84) (e148). There is a slight male predominance, but no specific racial predilection, although there is an
increased incidence in populations with a high number of HBV carriers.
Pathogenesis
Underlying liver dysfunctions, especially viral hepatitis (HBV and HCV) and cirrhosis, are known predisposing
conditions, although children are less likely to have associated chronic liver disease than adults (44). In areas
hyperendemic for HBV, almost all children with HCC are HBV seropositive (e121,e775). In a study of 20
Taiwanese patients aged 8 months to 16 years (all but one older than 8 years of age) with HCC, Wu et al. (e775)
noted HBsAg positivity in all the patients, 70% of their mothers, and 52.9% of their siblings. In these children,
HBV is commonly acquired from their mothers, with malignancy developing in 7 to 8 years (e119). However,
exposure time may be less in immunocompromised hosts; an exposure time of only 3 years has been described
in a 10-year-old boy with HCC who contracted HBV in the course of chemotherapy for acute lymphoblastic
leukemia at age 7 (e120). The incidence of HBsAg seropositivity is higher in children with the usual histologic
type of HCC than in patients with the fibrolamellar variant (see later) in whom the incidence of HBsAg positivity is
only 5%. Unlike in adults, integration of HBV-DNA into the host genome may be a late event in children with
chronic HBV infection. Huang et al. found that HBV-DNA integration increased in parallel with the progress of
liver histology toward the neoplastic transformation, with 0% in the liver of chronic hepatitis, 22.2% in nontumor
livers of HCC patients, and 66.7% in tumor liver tissues of HCC patients. Fortunately, the introduction of the
hepatitis B vaccine has markedly reduced the incidence of HCC, especially in males. HCV, while becoming more
frequently associated with adult HCC, is only occasionally associated with that tumor in children (e277).
HCC is also associated with inborn metabolic errors such as alpha-1-antitrypsin deficiency, hereditary
tyrosinemia, Gaucher disease, urea cycle defects, CESD, glycogen storage disease, Alagille syndrome, and
congenital biliary atresia (e175,e343,e368,e564,e629). Recently, a familial cholestasis syndrome caused by a
bile salt export pump deficiency has been described as a previously unrecognized risk for HCC in children
(e354).

Most patients (nearly 80%) present with abdominal pain, an abdominal mass, or both. Other symptoms include
anorexia, malaise, fever, nausea, vomiting, and jaundice. Symptomatic patients (with abdominal discomfort or
mass) often have
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advanced stage disease at presentation (189). Tumor rupture with hemoperitoneum may be seen rarely. A wide
variety of associated conditions are seen in 20% to 25% of cases (e659) (Table 15-24), including a recent
association between Gardner syndrome and the fibrolamellar form of HCC (e258). One of the closest
associations is with hereditary tyrosinemia, with a 37% incidence of HCC in tyrosinemia patients surviving
beyond 2 years of age, and liver transplantation before 2 years of age has been suggested for these patients
(e171,e421).
FIGURE 15-55▪ Hepatocellular carcinoma. A: Age distribution on 98 cases. B: A single, large mass displays foci
of hemorrhage and necrosis. C: The tumor is composed of broad trabeculae of poorly to moderately
welldifferentiated hepatocytes. Note the pseudogland appearance of some trabeculae with central necrosis of
cells (H&E stain, original magnification 40×). D: The individual hepatocellular carcinoma cells may be moderately
differentiated and cluster around a canaliculus (center), but note the nuclear anisocytosis and multiple nucleoli
(H&E stain, original magnification 300×).
Laboratory findings in patients with HCC include mild anemia or erythrocytosis, and thrombocytosis. Serum
transaminases (ALT and AST), lactate dehydrogenase (LDH), ALP, and lipid levels may be elevated (e659).
Serum bilirubin levels may be increased in 15% to 20% of cases. Unlike in adults with HCC, where biochemical
liver function tests are often abnormal, abnormal results of ALT, bilirubin, and albumin are infrequent in pediatric
patients as well as in patients with advanced stage (189) (e121). Further, unlike in adults, elevated ALP in the
presence of a liver mass did not correlate with metastatic disease (189). Serum AFP is elevated in 50% to 100%
of children with HCC (189) (e482,e607), although AFP may be normal or only mildly elevated in patients with the
fibrolamellar variant (e56,e436,e659). Elevated AFP levels are especially common in Taiwanese children and
this has been attributed to the almost universal association with HBV in this cohort. HBV is both carcinogenic and
also independently reactivates the gene encoding AFP within hepatocytes (e454,e653). Alternatively, extremely
high levels of AFP may also be due to the advanced tumor stage in these HCC patients (e219).
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Table 15-24▪CONDITIONS ASSOCIATED WITH HEPATOCELLULAR CARCINOMA IN

CHILDREN
Acute lymphoblastic leukemia
A1AT deficiency
Ataxia-telangiectasia
Anomalies of abdominal venous drainage
Arteriohepatic dysplasia
Atypical retinitis pigmentosa
Biliary atresia
CHF
Cystinosis
Familial cholestatic cirrhosis (Byler disease)
Familial hepatocellular carcinoma
Familial polyposis
Fanconi anemia
Focal nodular hyperplasia of liver
Galactosemia
Gardner syndrome
Hepatic adenoma
Hepatitis B infection
Hepatitis C infection
Hereditary tyrosinemia
Hyperalimentation
Methotrexate therapy
Neurofibromatosis
Oral contraceptives
Osteogenesis imperfecta
Polycythemia
Soto syndrome
Types I and III glycogenoses
Wilms tumor
Wilson disease
Modified from Stocker JT: Hepatic tumors. In: Balistreri W F, Stocker J T, ed. Pediatric hepatology. New
York, NY: Hemisphere Publishing Company, 1990:399-488, with permission.
Imaging studies can delineate the mass and often help in determining whether resection is possible (Figure 15-
55B) (e545). Soyer et al. (e650) used CT scans to study patients with the fibrolamellar variant and noted a
hypodense single, bilobed, or multilobulated mass that was hypervascular and variable enhancement after
injection. Calcification was present in 40% of cases. McLarney et al. (e436) noted the appearance of the
fibrolamellar variant as a lobulated heterogeneous mass with a central scar in an otherwise normal liver and
cautioned that it not be confused with an FNH of the liver. Recently, PET/CT scan has been reported to be
helpful for preoperative staging, selection of appropriate site for biopsy, identification of occult metastatic
disease, follow-up for residual or recurrent disease, and assessment of response to chemotherapy in HCC and
other pediatric abdominal neoplasms (e461). Sevmis et al. recommend mandatory serial AFP screening and
combined imaging studies in the follow-up of children with chronic liver disease (e617). Imaging studies may also
be helpful in differentiating metastatic tumors from primary malignant liver tumors, in that the former are more
likely to show hypoechogenicity on abdominal ultrasound examination, while the latter are more likely to show
vascular invasion and contrast enhancement on CT scan (189).
Staging
Multiple staging systems have been proposed for HCC. Lu et al. found the TNM staging system to be superior to
the Okuda, Cancer of the Liver Italian Program (CLIP), and the Chinese University Prognostic Index (CUPI)
staging systems for prognostication in HCC patients undergoing curative resection (106). On the other hand,
Seo et al. found the CLIP system to have better predictive power than the TNM and Barcelona Clinic Liver
Cancer (BCLC) staging systems (164). In yet another study comparing seven prognostic staging systems
(including CLIP score, BCLC staging, the Groupe d'Etude et de Traitment du Carcinome Hépatocellulaire
[GETCH] classification, CUPI grade, the Japan Integrated Staging [JIS] score, modified JIS [mJIS] score, and
Tokyo score), Kondo et al. found the JIS score to be the best system in patients undergoing hepatectomy for
HCC (92). More recently, in pediatric cases, the PRETEXT system devised for hepatoblastomas (44) has gained
popularity.
Treatment and Outcome
The usual strategy for pediatric HCC is the combination of surgery and neoadjuvant chemotherapy. However,
the relative chemoresistance of HCC makes surgery essential (44) (e332). Unfortunately, resectability at the time
of diagnosis is possible in only 10% to 30% of cases (e121). Neoadjuvant chemotherapy may improve tumor
resectability (e785). The most frequent chemotherapy regimen used in children is doxorubicin and cisplatin (44)
(e485), although its effects are potent especially in resectable disease. For tumors still not resectable after
chemotherapy, locoregional ablative therapies such as transarterial chemoembolization have been used
(e332,e414). Tumor size and serum AFP level, alone or in combination, are reportedly useful in predicting the
presence or absence of vascular invasion before hepatectomy for HCC (159). Liver transplantation may be
helpful when resection is impossible and transplantation should be considered as soon as possible in these
patients (e81,e183,e332). The presence of extrahepatic disease, nodal involvement, macroscopic vascular
invasion, and/or distant metastases are obvious contraindications to transplantation. The experience with liver
transplantation for HCC is still scarce in children. Although Sevmis et al. claim excellent results with both
cadaveric and living-donor transplants (e617), Otte et al. have observed relatively poor results, similar to those in
adults with HCC, except in a few highly selective series (135). In a recent study, patients with larger (3 to 5 cm)
tumors, high serum AFP levels (>455 ng/mL), or a high MELD score (of 20 or more) had poor posttransplantation
survival (74).
The prognosis of HCC is poor with an overall survival rate at 3 years below 25% (44) (e121,e330), notoriously
worse than that of hepatoblastoma despite similar multidisciplinary approaches. Major prognostic factors are the
presence
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of metastatic disease and the extent of disease, especially surgical resectability. In the SIOPEL-1 study that
included 40 children with HCC, 33% were associated with cirrhosis, multifocal tumors were common (56%), as
were metastases (31%), and extrahepatic tumor extension, vascular invasion, or both in 39%. Preoperative
chemotherapy achieved a partial response in 49% of patients, complete tumor resection was achieved in 36% of
patients, whereas 51% never became operable. Overall survival at 5 years was 28%, and eventfree survival was
17%. Most deaths resulted from tumor progression. Resectability, presence of metastases, and high PRETEXT
score predicted poor outcomes (44). In a Korean study of 16 pediatric HCC, estimated 5-year survival rate of all
patients was 27.3%, but 62.5% for patients who underwent complete tumor resection versus 0% for those who
underwent palliative resection or no operation (e785). The statistically significant prognostic factors were tumor
stage, presence of metastasis, and complete tumor resection.
Childhood hepatoblastoma and HCC differ with respect to age (18 months versus 10.2 years), sex (females
versus males), HBsAg status (none versus 64%), tumor stage (low versus high), tendency to rupture (36%
versus 9%), chemosensitivity (more for hepatoblastoma), and tumor respectability (91% versus 45%), with
considerably worse survival for HCC than hepatoblastoma (36). Postovsky et al. have reported a case of
combined hepatoblastoma and HCC, where the HCC component recurred more than 5 years after initial
diagnosis, suggesting that prolonged follow-up may be required for these tumors (e543).
Traditionally, patients with the fibrolamellar variant of HCC (FL-HCC) have been considered to have a somewhat
better prognosis (e659). However, this is probably true in adults due to lack of association of this histologic form
with cirrhosis. In children, FL-HCC may be biologically similar in behavior to classic HCC. Controversy exists
whether FL-HCC has a better prognosis than classic HCC. Although some series have shown a better survival
for FLHCC than usual HCC (51) (e57,e84,e148,e368), this is due to a larger number of FL-HCC patients with
localized and resectable tumors in these studies. Others, including recent studies of children and young adults
with FL-HCC, have not shown favorable outcomes, with no difference in the rate or surgical respectability (44,
84) (e268,e332,e456). In a study of 46 children with HCC, Katzenstein et al. found 10 cases (22%) of FL-HCC.
Although the median survival was longer in patients with FL-HCC than for patients with typical HCC, the 5-year
survival rate was similar for both groups. There was also no difference in the number of patients with advanced-
stage disease, the incidence of surgical resectability at diagnosis, or the response to treatment between patients
with FL-HCC and patients with typical HCC. Children with initially resectable HCC had a good prognosis
irrespective of histologic subtype, whereas outcomes were uniformly poor for children with advancedstage
disease (84).
Gross Appearance
Grossly, HCC may be single or multicentric masses, with involvement of both the right and left lobes in over 70%
of cases. The tumors weigh 800 to 1,500 g and vary in size from 2 to 25 cm. The lesions on cut section are tan
to red and soft to firm with areas of hemorrhage and necrosis (Figure 15-55B). The surrounding lever may exhibit
a micronodular or macronodular cirrhosis in up to 60% of cases, which is somewhat less than the 48% to 92%
incidence seen in adults with HCC (e121). The cirrhosis may be related to biliary atresia or hereditary
tyrosinemia, among other causes. The fibrolamellar variant is more often a single mass that is firm and gray.
Cirrhosis is less frequent (4%) in patients with the fibrolamellar variant (120) FNH may be present in or adjacent
to the HCC in about 4% of patients with the fibrolamellar variant (e56).
Histopathology
Microscopically, the “usual” HCC and the fibrolamellar variant present distinctly different features. The usual
HCC is composed of trabeculae 2 to 10 or more cell layers in thickness (Figure 15-55C, D). The larger
trabeculae may display central necrosis, imparting an acinar or pseudoglandular appearance. Individual cells are
larger than normal hepatocytes, with nuclear hyperchromasia, anisocytosis, multiple nucleoli, and frequent and
bizarre mitoses (e659). Large, multinucleated osteoclast-like giant cells or “tumor giant” cells (the so-called
epithelial syncytial giant cells) may also be seen (10). Bile pigment may be present within the cytoplasm of tumor
cells or within the canaliculi between cells. Vascular invasion may be prominent, and metastases to lung and
lymph nodes may occur. Children with malignant liver tumors, especially with HCC, may have extensive
angiogenesis that induces a rapid tumor growth and leads to a poor prognosis (175). Pathologic factors including
tumor size greater than 2 cm, multifocality, and vascular invasion have been reported to be independent
predictors of poor survival after resection (132).
The fibrolamellar variant was originally described in 1956 by Edmondson (e190). FL-HCC accounts for 1% of all
HCC but 13% to 22% of HCC in younger patients, as it preferentially develops in children and young adults (51,
84). It has not been linked with viral infection, or other risk factors for HCC, and patients usually have normal
serum AFP (44, 84). Histologically, FL-HCC is characterized by large, deeply eosinophilic (oncocytic)
hepatocytes embedded within lamellar fibrosis (Figure 15-56) (e56). Individual cells vary from polygonal to
spindle shaped and often contain discrete, pale eosinophilic bodies. Clusters of these cells are separated by
narrow to broad bands of laminated collagen. Although rare, the most common variant of FL-HCC shows areas
of glandular type differentiation with mucin production (182). Immunohistochemically, they may stain positive for
fibrinogen, hepar, ferritin, and alpha-1-antitrypsin, but are negative
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for HBsAg (e659). AFP staining has been noted in 21% of fibrolamellar cases and 40% of the usual HCC cases
(e56); the latter 27% cases are also positive for HBsAg.
FIGURE 15-56 ▪ Fibrolamellar hepatocellular carcinoma. A: Broad bands of “plump” collagen separate clusters
of large hepatocytes with prominent eosinophilic cytoplasm and large nucleoli (H&E stain, original magnification
60×). B: The hepatocytes contain abundant smooth to finely granular cytoplasm. Note the bile within the
canaliculi between hepatocytes (H&E stain, original magnification 200×).
It may, on occasion, be difficult to differentiate the macrotrabecular variant of hepatoblastoma from HCC (199).
Computerized image analysis has been claimed to help distinguish hepatoblastoma from HCC (185). Also,
hepatoblastoma is rarely multiple and vascular invasion is uncommon even in advanced stage tumors (189).
Prokurat et al. have even described a novel group of hepatocellular neoplasias in older children and
adolescents, with an intermediate histology between HCC and HB, and a distinctive β-catenin pattern, that they
term “transitional liver cell tumors” (142).
Recently, glypican-3 has been claimed to be a specific immunomarker for HCC and has been used to distinguish
HCC from benign hepatocellular mass lesions, particularly HCA. However, the diagnosis of HCC should not rely
entirely on positive glypican-3 immunostaining because focal immunoreactivity can be detected in a small subset
of cirrhotic nodules. Also, glypican-3 expression in HCC can also be focal and thus, the lack of glypican-3
staining does not exclude the diagnosis of HCC (189). Further, glypican-3 may also be positive in
hepatoblastomas (200). However, in a tissue microarray study of 4,387 tissue samples from 139 tumor
categories and 36 nonneoplastic and preneoplastic tissue types, glypican-3 expression (using a 10% cut-off
score) was detected in 9.2% of nonneoplastic liver samples (11/119), 16% of preneoplastic nodular liver lesions
(6/38), 63.6% of HCCs (140/220), and in several nonhepatic tumors including squamous cell carcinoma of the
lung (27/50 [54%]), testicular nonseminomatous germ cell tumors (32/62 [52%]), and liposarcoma (15/29 [52%])
(17). HCCs of higher histologic grade have been reported to have loss of E-cadherin, nonnuclear overexpression
of β-catenin, and overexpression of osteopontin, with overexpression of osteopontin independently correlating
with vascular invasion (93). Yamaoka et al. (196) found 17/17 pediatric HCCs to be positive for
nuclear/cytoplasmic β-catenin in all childhood HCCs and suggest that β-catenin immunohistochemistry may be
helpful in identifying malignancy in an otherwise borderline lesion. They also observed E-cadherin expression in
all malignant pediatric liver tumors, while cyclin D1 expression was significantly detected in tumors of advanced
stage, suggesting that cyclin D1, a gene downstream of beta-catenin, might play a role in tumor progression
(196). EGFR overexpression is also reported in a majority of HCCs, suggesting a role for EGFR antagonists in
therapy. However, the increased expression does not correlate with an increase in the EGFR gene copy number
(30). Klein et al. report that although HCCs in children are morphologically similar to those in adults, the former
are more likely to be CK7-positive (89).
Molecular Pathology
In contradistinction to many other childhood tumors (e.g., neuroblastomas, rhabdomyosarcomas, and
ganglioneuroblastomas), amplification or overexpression of the oncogenes N-MYC, ERB A, ERB B, N-RAS, or
Shb is not seen with HCC or hepatoblastoma (e423). Fibrolamellar carcinomas show fewer chromosomal
abnormalities compared with those reported in literature for conventional HCC. The most common abnormalities
in FL-HCC occur in chromosomes 7 and 8, and tumors with chromosomal changes appear to behave more
aggressively compared with cases with no cytogenetic abnormalities. However, chromosomal changes do not
correlate with age, gender, and tumor size (81). Terracciano et al. have reported the occurrence of FL-HCC in a
young girl with a prior resection of a HCA; although there was no genetic alteration in the adenoma, several
chromosomal aberrations were detected in the FL-HCC (e698).
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The β-catenin pathway has been implicated in HCC (e778). MicroRNA profiling may help identify patients with
HCC who are likely to develop metastases/recurrence (31). The uniqueness of FL-HCC extends to their
molecular findings, as they show no evidence for involvement of many of the major pathways and genes that are
dysregulated in typical HCC, including AFP, TP53 mutations, and β-catenin mutations. However, much of their
molecular biology remains poorly described and awaits future investigation (182). The molecular pathology of
HCCs in children is probably similar to that in adults. The topic has been the subject of excellent recent reviews
(53, 54, 73, 124, 137, 158, 187, 194) and is beyond the scope of detailed discussion in this text.
UNDIFFERENTIATED EMBRYONAL SARCOMA

UES is the fourth or fifth most common pediatric liver tumor (102) (e661). The term “undifferentiated (embryonal)
sarcoma” was given to this lesion by Stocker and Ishak in 1978, prior to which the tumor was known as
embryonal sarcoma, mesenchymoma, primary sarcoma, fibromyxosarcoma, or malignant mesenchymoma.
Pathogenesis
The histogenesis of undifferentiated sarcoma of the liver remains unresolved. Suggestions that UES is a
sarcomatoid variant of hepatoblastoma (e445) have not found acceptance. The observation of UES occurring in
association with mesenchymal hamartoma has suggested that the former may arise in a setting of the latter (see
discussion above, in the section on mesenchymal hamartoma). This concept of malignant transformation
occurring in a dysgenetic or hamartomatous lesion is similar to what has been described for other malignancies
such as adenocarcinomas arising in bronchogenic and choledochal cysts, Wilms tumor from perilobar
nephrogenicrests, and pleuropulmonary blastoma from presumed congenital lung cysts (not the case).
UES has been associated with the Li-Fraumeni syndrome (e369). An embryonal or congenital origin has been
considered unlikely by some authors, because UES has also been reported in adults. The histogenesis of UES
is probably from a mesenchymal lineage. There is no clear differentiation into rhabdomyosarcoma or
fibrosarcoma, although myogenic differentiation has been suggested in a few cases based on
immunohistochemical findings. The overlap of immunohistochemical staining patterns and ultrastructural features
shown by UES and hepatic rhabdomyosarcoma has led Parham et al. to suggest a common histogenesis,
perhaps from a multipotential mesenchymal stem cell (e513).
FIGURE 15-57 ▪ Undifferentiated embryonal sarcoma. A: Age distribution in 48 cases. B: The tumor mass
contains multiple cystic areas filled with gelatinous or hemorrhagic material.

UES occurs primarily in children aged 6 to 10 years (Figure 15-57A), with 88% of cases occurring in those under
15 years of age (e149). Others have reported a median age of 9.5 to 12 years, with 63% occurring in children 6
to 10 years of age (32, 138). In most reported series, there is an almost equal incidence in both sexes (96, 102).
In a large reviewed series of 113 cases in literature, 46 were males and 31 were females. There is no racial
predilection.
Abdominal pain or an abdominal mass is seen at presentation in 87% of cases. Unusual presentations include
dyspnea/cardiac murmur (due to extension of the tumor through the inferior vena cava into the right atrium and
ventricle), jaundice, chest pain, and fever (96) (e131,e661). The abdominal mass and pain are often
accompanied by anorexia, vomiting, lethargy, and malaise. Tumor rupture may lead to an acute abdomen (96)
(e297,e661). Sakellaridis et al. report a presentation mimicking acute appendicitis (e587). Laboratory findings
display a variety of abnormalities of SGOT, LDH, and alkaline phophatase, but serum AFP is uniformly negative
and serum bilirubin is rarely elevated (96) (e661).
UES appears predominantly as a solid lesion on sonographic studies. The tumor is isoechoic or hyperechoic
relative to the surrounding liver parenchyma. Cystic areas comprise an average of 19% of tumor volume.
Sonographic findings are usually in agreement with gross pathologic findings in terms of proportion of solid and
cystic components (32). Computed tomographic scans reveal predominantly
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water attenuation. As determined by unenhanced scans and bolus contrast-enhanced scans, an average of 88%
of the tumor volume shows water attenuation. Areas of intermediate or soft attenuation are also noted around the
periphery of all lesions. On MRI, the tumor appears predominantly hypointense relative to the liver on T1-
weighted images, with areas of high signal intensity present centrally, corresponding to areas of recent
hemorrhage. T2-weighted images show predominantly high signal intensity, cystic foci, internal debris, and
septations (32). A fibrous pseudocapsule of low signal intensity on T1-weighted and T2-weighted images is
sometimes seen (e545). MRI and CT may show a misleading cystlike appearance of an UES compared with
ultrasonography and pathologic findings in which the tumors are predominantly solid (>85% of tissue mass) (32)
(e286). This discrepancy with gross appearances following tumor resection probably results from the abundant
myxoid stroma in these tumors. A cystic appearance on imaging may lead to a misdiagnosis of hydatid disease,
especially in areas endemic for this parasitic infestation (e10,e117,e321,e620). On angiography, UES commonly
appears as an avascular or hypovascular hepatic mass (e573). Angiograms have shown that the tumor derives
its vascular supply from the hepatic arterial system. Angiography has been helpful in delineating hepatic vein
invasion and in vascular mapping for surgery (e341).
FIGURE 15-57 ▪ (continued) C: Loose and usually incomplete fibrous tissue separates the normal liver (right)
from the malignant tumor (left) (H&E stain, original magnification 30×). D: Entrapped and degenerating bile
ducts surrounded by anaplastic cells in a loose mesenchymal matrix lie next to the pseudocapsule separating the
tumor from the normal liver (left) (H&E stain, original magnification 40×). E: Bizarre tumor giant cells with large
and multiple nuclei are scattered throughout the lesion (H&E stain, original magnification 200×). F: Smooth
eosinophilic globules are present in the cytoplasm of small and large tumor cells (H&E stain, original
magnification 200×).
Pachera et al. have reviewed the clinicopathologic features of UES in adults based on 51 cases in literature. The
mean age of affected adults is 31 years (range 15 to 86 years), with a female preponderance (28 F, 19 M). The
right lobe is more commonly affected than the left lobe (59% versus 22%), with both lobes involved in 20% of
cases. Tumors often exceeded 10 cm in size, with an average weight of 1,400 g. Spontaneous rupture was
reported in only two cases. Results of liver function tests are usually normal, whereas high AFP levels have been
reported only in five adults, and raised CA-12 in one. In adults, the appearance of a cystic lesion on imaging
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has led to a mistaken diagnosis of a benign lesion with delay in diagnosis in 24% of cases (138)

Although UES was uniformly considered to have a very poor prognosis in the past, complete resection and
aggressive chemotherapy have changed the outcome favorably in recent years. In their original series, Stocker
and Ishak documented an average survival time of only 11 months. Patients who undergo an incomplete tumor
resection have a tendency toward poorer outcomes (96) (e385) and radical excision of the tumor provides the
only chance of cure. However, a complete resection was possible to achieve in only 65% (33 patients) of the
cases reported in the literature (138). Moon et al. observed a 50% to 90%reduction in tumor volume with
preoperative chemotherapy, rendering the tumors respectable (e452). Polychemotherapy has been practiced
with agents such as doxorubicin, cis-diaminodichloroplatinum, cyclophosphamide, dacarbazine, 5-FU, and
vincristine (e526). The Soft Tissue Sarcoma Italian and German Cooperative Groups treated 17 children with
UES using the same multimodal approach as for patients with other sarcomas including conservative surgery at
diagnosis, multiagent chemotherapy, and second-look operation in cases of residual disease, with additional
radiotherapy in 2 of 17 patients. Twelve patients were alive with follow-up ranging from 2.4 to 20 years (e71).
The tumor usually spreads by direct extension into adjacent organs and sometimes extends into the right atrium
via the inferior vena cava. Rupture of the tumor can occur and massive intraperitoneal spread has sometimes
been found (96). Metastases are rare, but have been reported in the lung, bone, pleura, and peritoneum (102)
(e4,e78).
Gross Appearance
UES is a large tumor with an average weight of more than 1,200 g (range 90 to over 4,000 g) (96) (e661). The
tumor ranges from 10 to 35 cm in diameter (32, 96). The mass is in the right lobe of the liver in 69% of cases, in
the left lobe in 14%, and involves both lobes in 17%. Pedunculated or exophytic tumors have been documented.
The tumor is well demarcated from the adjacent liver by a compressed incomplete fibrous pseudocapsule. The
cut section is variegated and soft. Myxoid gelatinous areas alternate with confluent areas of necrosis and
hemorrhage (Figure 15-57B). Foci of hemorrhage or necrosis are present in over 50% of the cases and may
constitute up to 80% of the tumor. The tumor is predominantly solid; the mean percentage of the solid component
is 83%. An average 17% of cross-sectional areas of the tumors are composed of empty cavities (32). These
cysts are up to 4 cm in diameter and contain gelatinous brown contents (96). Calcification is rare to absent (32)
(e573). The uninvolved liver is normal in appearance. Pathological features are similar in adults and children
(138).
Histopathology
Microscopically, the tumor is separated from the normal liver by an incomplete fibrous pseudocapsule of varying
thickness (Figure 15-57C). This tumor pseudocapsule and the tumor immediately adjacent to it may contain
remnants of normalappearing hepatocytes and bile ducts (Figure 15-57D). The bile ducts may extend 0.5 to 1.0
cm into the lesion and show hyperplastic or reactive epithelial changes that may even appear anaplastic. These
bile ducts are not present deeper in the tumor, nor within metastases, and are considered to represent entrapped
or residual bile ducts rather than neoplastic elements of the tumor. The major component of the tumor consists of
loose to dense foci of stellate or spindleshaped cells with ill-defined outlines in a myxoid stroma (Figure 15-57D).
Multinucleated cells with hyperchromatic nuclei are frequently scattered throughout the lesion (Figure 15-57E) or
may only be a minor component. These cells may contain eosinophilic globules that are PAS-positive and
diastase-resistant (Figure 15-57F); the globules may also be seen extracellularly. Histology may appear varied
due to differing proportions of myxoid stroma, cellularity, hemorrhage, and necrosis. There is marked disparity in
individual cell size and anisonuclsosis. Mitoses are abundant, with both atypical and bizarre mitotic forms.
Proliferation index ranges from 30% to 95% (87). Some densely cellular areas have small round cells with
hyperchromatic nuclei without nucleoli. Anaplastic malignant cells occur closer to the duct epithelium elements,
as mentioned above. Numerous reticular fibers surround small groups of cells, and focal collagenization and
hyalinization are present. Extramedullary hematopoiesis may be present. In a few tumors there are foci of direct
invasion into hepatic sinusoids. Eosinophilic hyaline globules are present both intracellularly and extracellularly.
These globules are PAS-positive and diastase-resistant. Patterns mimicking a sarcoma as a minor component of
the tumor have been recorded, including osteoidlike matrix (96, 102), “leiomyoblastic” (e244), and lipoblastic
differentiation (e147,e231). The neoplastic cells may resemble fibroblastic, histiocytoid, fibrohistiocytoid, and
myofibroblastic cells, occasionally suggesting a malignant fibrous histiocytoma, a tumor reported only in the liver
of adults. Following chemotherapy, resected pathologic specimens show central necrosis, fibrosis, and
dystrophic calcification (e452). Histologic dedifferentiation has been described following multiple recurrences
(e127). In a comparative study of 14 primary and two recurrent UES, recurrent tumors showed greater cellularity,
anaplasia, and pluripotential differentiation compared with primary tumors (197).
FNA cytology commonly yields a combination of polygonal and spindle cells. Polygonal cells are large with round
or lobulated nuclei and occasionally are multinucleated with one or several nucleoli and variable cytoplasm with
poorly defined borders. A few intracytoplasmic and extracytoplasmic eosinophilic globules are also observed
(e232,e362,e539). Similar cytologic findings have also been described in
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peritoneal washings (e19). Findings on FNAC have been considered distinctive from other childhood liver
tumors, allowing a confident preoperative diagnosis (e362,e646).
Immunohistochemistry shows evidence of widely divergent differentiation into mesenchymal and epithelial
phenotypes, suggesting that immunostains have no specific or diagnostic relevance, but, by using a panel of
antibodies, may help exclude other tumors. Variable positivity has been described for vimentin, BCL-2,
pancytokeratin, CD10, calponin, desmin, smooth-muscle actin, muscle-specific actin, p53, alpha-1-antitrypsin,
alpha-1-antichymotrypsin, desmin, CD56, and CD68 (96, 138, 139, 197). The tumors are usually negative for
myoglobin, myogenin, muscle-specific actin, h-caldesmon, S-100, ALK-1, nonspecific enolase (NSE),
carcinoembryonic antigen (CEA), F-VIII, and AFP (87, 102), although these could be anecdotally positive.
Aberrant cytokeratin expression has been explained on the basis of genetic deregulation rather than
differentiation (96).
Ultrastructurally, Agaram et al. have described the hallmark features to include dilated RERs and secondary
lysosomes with dense precipitates, which correlate with the eosinophilic globules seen on light microscopy.
Dilated mitochondria and mitochondrial-RER complexes are often seen. Other features include intracytoplasmic
fat droplets, scant actin microfilaments, and focal glycogen pools (4). Primitive fibroblasts, small mesenchymal
cells, and membrane-bound bodies that are alpha-1-antitrypsin or alpha-1-antichymotrypsin positive have been
described by others (e4).
Molecular Pathology
Leuschner et al. undertook DNA ploidy studies in five cases and found that four tumors were diploid and one was
hypodiploid (102). Chou et al. reported an aneuploid DNA stemline with high proliferative S phase in two patients
studied with flow cytometry (e130).
In the first description of the chromosomal changes in UES, Iliszko et al. reported near-triploid and near-
hexaploid clones with several chromosomal rearrangements (e302). Sowery et al. analyzed six cases of UES by
both conventional cytogenetics and CGH. Although CGH demonstrated several chromosomal gains and
deletions in each case, there was no specific abnormality seen in every case and no critical event important in
tumorigenesis could be identified. Patterns of chromosomal changes included gains of chromosome 1q (four
cases), 5p (four cases), 6q (four cases), 8p (three cases), and 12q (three cases), and losses of chromosome 9p
(two cases), 11p (two cases), and chromosome 14 (three cases) (170). Other cytogenetic abnormalities have
also been reported in UES, including near-triploid and near-hexaploid clones with several chromosomal
rearrangements (e302). A clonal telomeric association (a cytogenetic phenomenon in which chromosome ends
fuse to form dicentric, multicentric, and ring chromosomes) has been observed in UES (e597).
Mutation of TP53 gene but not the Wnt or telomerase pathways have been suggested to be involved in
pathogenesis (e389). In fact, Lack et al. (96) described a 9-year-old boy, who was a member of a kindred with
the family cancer syndrome (Li-Fraumeni syndrome), including a sister with soft tissue sarcoma of the wrist, a
father with osteogenic sarcoma of the jaw, a mother with soft tissue sarcoma of the pectoralis muscle, and a half-
brother with osteogenic sarcoma of the femur. Tawa et al. (e691) analyzed the expression of a multidrug-
resistance (mdrl) gene in a UES of the liver in a 4-year-old boy, and noted a 7- and 11-fold increase in the gene
expression level at the time of a first and second intracranial relapse. They suggested that acquired drug
resistance as seen in their patient may correlate with overexpression of the mdrl gene.
NESTED STROMAL EPITHELIAL TUMOR OF THE LIVER

Nested stromal epithelial tumor is a recently described primary pediatric hepatic neoplasm that has been variably
called “ossifying stromal-epithelial tumor,” “calcifying nested stromal-epithelial tumor,” “desmoplastic nested
spindle cell tumor,” and “nested stromal-epithelial tumor” (26, 67, 68, 115, 121) (e306). Less than 30 cases have
been described in literature at the time of writing this section.
Pathogenesis
The tumors are of uncertain histogenesis; a possible origin in a hepatic mesenchymal precursor cell with
primitive differentiation along the bile duct lineage has been suggested (67).
Clinical Features
Patients have ranged from 2 to 33 years of age; however, most tumors have been described in the first decade
of life. Makhlouf et al. noted that four of their nine cases had a history of calcified hepatic nodules since early
childhood (115). Ectopic ACTH production can lead to Cushing syndrome that abates following tumor excision.
Most patients, however, are asymptomatic and are discovered to have the tumor incidentally. Meir et al. report a
case that was associated with hydronephrosis. The hydronephrosis was discovered on antenatal ultrasound,
whereas the hepatic neoplasm was incidentally discovered on routine follow-up abdominal imaging at 2 years of
age (121). In Heerema-McKenney series, one 2-year-old patient subsequently developed nephroblastomatosis
and Wilms tumor of the kidney, while another patient had a history of omphalocele, bowel obstruction due to
postoperative adhesions, hypoplastic left kidney, and developmental delay (67).

Partial hepatectomy is probably curative, although local recurrences have been reported in a few patients. Local
recurrences are successfully treated with either surgery
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or radiofrequency ablation (115). Makhlouf et al. have suggested that this tumor is best considered a low-grade
malignancy. Long-term prognosis appears to be good; six of eight patients in one series were alive and well up to
22 years after surgery (115). However, Brodsky et al. have reported a case in a 17-year-old girl with aggressive
clinical behavior with multiple hepatic recurrences and an extrahepatic lymph node metastasis, suggesting that
close follow-up is essential in these patients (26).
Gross Appearance
Based on the reported cases, the tumors are well circumscribed but not encapsulated and range in size from 4 to
30 cm. The tumors are intrahepatic; a pedunculated mass has also been described. On cut surface they appear
multinodular, with a homogeneous, tan, granular-appearing cut surface. Variably sized foci of softening, cyst
formation, calcification, or gritty ossification may be observed.
FIGURE 15-58 ▪ Nested stromal epithelial tumor. A: The tumor is comprised of variably sized distinct nests of
epithelioid cells embedded in variably myo-fibroblastic to desmoplastic stroma (H&E, 100×). B, C: The tumor
cells in the nests are positive for cytokeratin (B: 100×) and WT-1 (C: 200×) immunostains.
Histopathology
Nested stromal-epithelial tumors have been described as nonhepatocytic, nonbiliary tumors with nests of
epithelial and spindle cells, an associated myofibroblastic stroma, as well as variable calcifications and
ossifications (67, 68). Architecturally, the tumor-liver interface is well-defined and the tumors consistently display
an organoid arrangement of cellular nests comprised of spindled and/or epithelioid cells surrounded by a variably
prominent collar of delicate myofibroblasts (Figure 15-58A). The stroma between the nests is usually
desmoplastic. The periphery of the tumor shows a (probably entrapped) bile duct component. Psammomatous
calcification may be sparse to prominent; when present, they are usually within or adjacent to cellular nests.
Focal osteoid formation or ossification is common. The cellular nests have rounded edges and are relatively
uniform in size in a given case; older children may show larger nests, suggesting that the tumor may grow slowly
with age. Focal
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neuroendocrine-appearing architecture has been described in cases with Cushing syndrome. The nests are
composed predominantly of plump to fusiform spindled cells with centrally placed or scattered epithelioid cells.
Epithelioid cells may predominate in cases with extensive calcification. Both spindle and epithelioid cells have
bland oval nuclei with well-defined nuclear membrane, stippled chromatin, and variably conspicuous nucleoli.
The cytoplasm is predominantly eosinophilic, with focal cells containing clear cytoplasm; epithelioid cells have
distinct cellular borders. Mitoses are rare to scattered. Delicate osteoid formation may be present between the
epithelioid nests. The desmoplastic stroma, a prominent feature in all four tumors, variably cellular, and
composed of cells with morphologic features of myofibroblasts is not seen. Hill et al. specifically mentioned the
lack of evidence of a ductal plate abnormality and lack of vascular invasion (68).
Immunohistochemically (Figures 15-58B, C), the tumor cells coexpress vimentin and cytokeratins, at least focally.
They also exhibit moderate to strong diffuse nuclear staining for WT-1, using either the C-terminal or N-terminal
antibodies (67, 68, 115, 121). There is variable staining for EMA, CD56, CD57, S-100, and other mesenchymal
markers. Synaptophysin and chromogranin stains are reportedly negative in all cases (67, 68). ACTH
immunohistochemistry may be positive in tumors associated with Cushing syndrome (154). The desmoplastic
stroma has been reported to prominently display collagen type IV and smooth muscle actin (68).
Hill et al. performed ultrastructural studies in three cases and observed bland spindled and polygonal cells with
focal basal lamina and focally well-developed cell junctions. Few mitochondria and sparse profiles of rough
endoplasmic reticulum were seen in the cytoplasm. The polygonal cells contained focal collections of
intermediate filaments and had interdigitating cell membranes. No neurosecretory granules were identified (68).
On the other hand, Brodsky et al. report an abundance of rough endoplasmic reticulum and mitochondria in a
tumor that behaved aggressively with intrahepatic recurrence and lymph node metastasis (26).
FIGURE 15-59 ▪ Embryonal rhabdomyosarcoma of the biliary tract. A: Age distribution. B: Ultrasonography
displays the dilated ducts proximal to the tumor mass.
FIGURE 15-59 (continued) C: The tumor occupies the major ducts within the porta hepatis (center) and extends
proximally along the intrahepatic ducts. D: The tumor cells form a “cambium” layer of rhabdomyoblasts between
the bile duct epithelium (top) and wall (bottom) (H&E stain, original magnification 75×).
Molecular Pathology
Molecular studies for Ewing sarcoma family transcripts and SYT-SSX fusion transcripts have been negative in
the cases studied (68, 115). Hill et al. found a normal karyotype in the single case that they evaluated (68).
Brodsky et al. report a cytogenetically complex tumor that later recurred and metastasized (26).
EMBRYONAL RHABDOMYOSARCOMA OF THE BILIARY TRACT

Although it is the most common sarcoma in the pediatric patient, rhabdomyosarcoma of the liver accounts for
only 0.8% of all rhabdomyosarcomas and 1.0% of all liver tumors. At the same time, rhabdomyosarcoma is the
most common malignant tumor of the biliary tree in childhood. It is difficult to diagnose and delayed diagnosis
influences the prognosis. The occurrence of rhabdomyosarcoma of the liver and biliary tree was first reported in
1875 (e767). Almost 85 cases of biliary rhabdomyosarcoma have been reported in the literature; 75% of patients
are under 5 years of age (85). The lesion is seen primarily (75% of cases) in children younger than 5 years of
age and rarely in those older than 15 years (Figure 15-59A) (e396,e540,e593)
Jaundice is seen as the presenting symptom in 60% to 80% of cases and may be accompanied by cholemia,
pale stools, and hepatomegaly, often confused with infectious hepatitis. Other symptoms include fever,
abdominal distension, nausea, and vomiting (e367). The jaundice is reflected in moderate elevations of
conjugated and unconjugated bilirubin, with total bilirubin of 1.5 to 9.0 mg/dL. ALP may also be elevated, along
with a mild rise in SGOT. Imaging studies, including CT, MRI, ultrasonography (Figure 15-59B), and
cholangiography may clearly demonstrate the site of obstruction within the intrahepatic ductal structures. Due to
its rarity, it may be misdiagnosed as a choledochal cyst (7) (e480,e702).
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Treatment is aimed at surgical resection, although complete resection is possible in only 20% to 40% of patients
because of extension of the tumor into the liver, regional metastasis, and local extension to the duodenum,
stomach, and pancreas. Recent preoperative therapy using standard protocols for embryonal
rhabdomyosarcoma has proved effective, with Pollono et al. (e540) reporting complete remission using a
multidrug protocol as the initial treatment of a 3-year-old girl after obtaining adequate transparietohepatic biliary
drainage. Long-term survival of approximately 20% in previous years has risen for patients treated with
preoperative chemotherapy and surgical “second-look” (e540,e570,e593).
Grossly, the tumor often presents as a botryoid, gelatinous mass occluding the lumen of the right and left or
common bile duct (Figure 15-59C). The ducts proximal to the lesion are frequently dilated, and the walls of the
duct containing the lesion are thickened. The tumor may extend into the liver as a soft lobulated mass.
Occasional cases arise in the intrahepatic bile ducts (85).
Microscopically, the botryoid masses within the bile ducts are covered by a layer of cuboidal epithelium (bile duct
epithelium) that may be inflamed or ulcerated. Beneath the epithelium lies a dense layer of tumor cells, the upper
portion of the cambium layer (Figure 15-59D). Cells within this area are small and hyperchromatic, with scant
cytoplasm. Deeper to the bile duct epithelium, the cells lie in a loose myxoid stroma and exhibit the typical
features of embryonal rhabdomyosarcoma with round, spindle, or straplike shapes; elongate nuclei: scant
acidophile cytoplasm; and frequent mitoses. As with other rhabdomyosarcomas, cross-striations may
occasionally be found, but immunohistochemistry studies are consistently positive for desmin, with myoglobin
and myosin identified in more differentiated cells (e659). The tumor is usually highly vascular, and areas of
recent and remote hemorrhage and acute and chronic inflammation may be found throughout the lesion. The
adjacent hepatic parenchyma is often compressed, and bile may be present within canaliculi and hepatocytes.
Nicol et al. have compared the clinicopathologic features of UES and hepatobiliary rhabdomyosarcoma (134).
Although similarities do exist between the two lesions, UES has a male:female ratio of 1:1, a median age of
occurrence of 10.5 years, and histology showing hyaline globules and diffuse anaplasia. Rhabdomyosarcoma,
on the other hand, has a male:female ratio of 1.8:1 with a median age of 3.4 years and routinely lacks diffuse
anaplasia and hyaline globules. Polyclonal desmin and muscle-specific actin are variably immunoreactive in both
tumors; however, myogenin and myogenic regulatory protein D1 (MyoD1) is mostly negative in UES, but positive
in rhabdomyosarcoma. With a median follow-up of 8 months, 11 of 18 patients with UES were still alive, whereas
the estimated 5-year survival for biliary tract rhabdomyosarcoma was 66%. Establishing the correct diagnosis of
these distinct clinical and pathologic entities is important, as surgery alone may be curative in UES, whereas
initial chemotherapy is often recommended for the treatment of biliary tract rhabdomyosarcoma (see Chapter 24).
ANGIOSARCOMA
Angiosarcoma of the liver accounts for less than 2.5% of liver tumors in children (see Table 15-14). Selby et al.
(162) studied 10 patients (six girls and four boys) ranging in age from 18 months to 7 years, and noted the
presence of three older cases at 13, 17, and 18 years (Figure 15-60A). There is a reported predominance in
females (female:male ratio of 2:1) and a mean age at presentation of near 4 years (162) (e486). This is in
contrast to infantile hemangioendothelioma, which almost always occurs in the 1st year of life. However, hepatic
angiosarcoma has also been reported in neonates (133). The most frequent presenting symptom is a rapidly
enlarging
P.735
abdominal mass, which may be accompanied by jaundice, diarrhea, abdominal pain, or vomiting. Congestive
heart failure commonly seen with hepatic hemangioendotheliomas is absent with hepatic angiosarcomas (e196).
An association with environmental exposure to Thorotrast, vinyl chloride, androgenic and anabolic steroids, oral
contraceptives, and diethylstilbestrol, as reported in adults, has not been observed in children (e467). There is
also no established syndromic or genetic association. Angiosarcoma arising in a child previously treated for
infantile hemangioendothelioma has been described but is unusual (11) (e346). Treatment, including resection,
radiation, transplantation, and a variety of chemotherapeutic agents, has been unsuccessful, and patients have
rarely survived for more than 2 years (11). Gunawardena
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et al. (e260), however, report the 44-month survival without recurrence of a 4-year-old girl following surgical
resection and postoperative chemotherapy with alternating cycles of ifosfamide and etoposide, cisplatinum and
adriamycin, and vincristine and actinomycin D and cyclophosphamide for 18 months.
FIGURE 15-60 ▪ Angiosarcoma. A: Age distribution in 10 cases. B: On CT, multiple hypodense nodules are
present in the liver. C: On cut section, the liver displays multiple areas of dense white tissue and areas of
hemorrhage. D: Foci of spindle cells and hemorrhage are scattered throughout the liver parenchyma (H&E stain,
original magnification 40×). E: Bizarre endothelial cells fill and greatly distend the sinusoids of the liver,
compressing and destroying hepatic trabeculae (H&E stain, original magnification 200×).
Hepatic angiosarcomas are often large multicentric lesions composed of well-demarcated, fleshy, tan nodules
approximately 7 cm in diameter displaying areas of hemorrhage and necrosis (Figure 15-60B, C).
Microscopically, the tumor is characterized by nodules of spindled cells in a whorled pattern (Figure 15-60D).
Larger nodules composed of malignant vascular channels may also be present. Tumor cells are large, with
hyperchromatic nuclei and frequent mitoses (Figure 15-60E). Intracytoplasmic and extracellular eosinophilic
globules that are PAS-positive are present in most cases. Dimashkieh et al. have observed that the histology of
pediatric hepatic angiosarcoma is distinct from adult angiosarcoma, with the former displaying hypercellular
whorls of sarcomatous cells, or “kaposiform” spindle cells, in addition to the general features of angiosarcoma
(50). Immunohistochemical stains are positive with vascular markers, alpha-1-antichymotrypsin, and Ulex
europaeus but negative for keratin and AFP (162). Metastases to lungs, pleura, bone, adrenals, mesentery, and
kidney have been described (11, 162).
OTHER NEOPLASMS SEEN IN THE LIVER

Metastatic lesions such as neuroblastoma, Wilms tumor, and lymphoma are the most common neoplasms seen
in the liver, but a variety of other primary neoplasms have been described. EBV-associated leiomyosarcoma has
been described following liver transplantation in two children—the first, a 9-year-old boy who developed a tumor
in his allografted liver 2 years after transplantation, and the second, in a 12-year-old girl, who, after
transplantation, developed the leiomyosarcoma in the retroperitoneum involving the superior mesenteric vein
(180). Malignant neoplasms that are rarely seen include malignant rhabdoid tumor, endodermal sinus (yolk sac)
tumor, and lymphoma (e659).
FIGURE 15-61 ▪ Cholecystitis and cholelithiases. A: Gallbladder with red finely granular mucosa. B: Chronic
cholecystitis with markedly thickened gallbladder wall and scattered chronic inflammatory cells (H&E, 40×).
GALL BLADDER
Congenital anomalies of the gallbladder include agenesis, duplication, bilobation, multiseptation, diverticula,
ectopia, and congenital fistula (173). Agenesis occurs as an isolated anomaly in the majority of cases and is an
incidental finding at autopsy in childhood. The gallbladder may be reduced to a fibrous cord or be diminutive in
EHBA. In the neonate, a small or hypoplastic extrahepatic biliary tree may reflect a low-flow state in severe
cholestatic liver disease. Alagille syndrome, A1AT, INH, and familial cholestatic syndromes are some of the
conditions in which gallbladder hypoplasia may be seen. In CF, the gallbladder may be small and contain viscid
mucus. Rarely, the bile ducts may be obstructed by biliary sludge.
The most common acquired disease of the gallbladder is cholelithiasis (Figure 15-61) (e659). This condition may
be a complication of hemolytic disease, including congenital spherocytosis, sickle cell disease, and thalassemia.
In most cases, the condition has been idiopathic. As in adults, there is a female preponderance in childhood
cases, and cholecystitis is often associated. Some other conditions predisposing to cholelithiasis include TPN,
biliary stasis, ileal disease, sepsis, prolonged fasting, inflammatory bowel disease, short gut syndrome, ileal
resection, PSC, prematurity, dehydration, immaturity of the hepatic glucuronyl transferase, ceftriaxone therapy,
CF, cirrhosis, Wilson disease, porphyria, biliary
P.737
dyskinesia, medications, and biliary tract anomalies, such as choledochal cyst. Cholelithiasis with cholesterol
stones is seen in obese adolescents, both male and female. Tumors of the gallbladder are extremely rare in
children; biliary rhabdomyosarcomas have been discussed above.
FIGURE 15-61 ▪ (continued) C: Cholesterolosis characterized by foamy macrophages in lamina propria (H&E,
400×). D-G: Cholelithiases vary from cholesterol choleliths (D, E), ebonized choleliths (F), and calcium choleliths
with milk-like bile (G).
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Chapter 16
The Pancreas
Mariko Suchi
ORGANOGENESIS AND EXOCRINE HISTOGENESIS

During week 4 of gestation, the ventral foregut gives rise to two pancreatic buds at the junction of the hepatic duct.
The dorsal primordium develops in the mesentery. The ventral pancreatic bud rotates with the gut and fuses with the
larger dorsal anlage and with the duodenum, and the fused primordia take up their normal position against the
posterior abdominal wall in the concavity of the duodenum. The ventral bud gives rise to the uncinate process and
the inferior portion of the head of the pancreas, and the dorsal primordium gives rise to the body, tail, and superior
portion of the head (Figure 16-1). The duct of the dorsal pancreas opens more proximally into the duodenum and the
ventral duct more distally, together with the common bile duct. Fusion of the primordia during the middle of week 6 of
gestation leads to fusion of the duct systems. The duct of the ventral pancreas becomes the main pancreatic duct of
Wirsung. The duct of the dorsal pancreas usually remains patent as the minor duct of Santorini.
During week 7 of gestation, simple, undifferentiated epithelial tubules grow into a loose mesenchyme. The epithelium
rapidly forms a ramifying duct system, from which buds of cuboidal cells form the first recognizable acinar units by
week 10; endocrine elements are also present by this time. The pancreas, from week 10 to term, continues to ramify
and gives rise to exocrine and endocrine elements. Ductal elements, especially the centroacinar cells, can be hard to
identify definitively from acinar and endocrine cells at this stage, but they express keratins 7 and 19 in addition to the
keratins 8 and 18, which are found on acinar and endocrine cells after week 16 (17). Mucin glycoprotein gene
messenger RNA (mRNA) studies reveal MUC6 in the pancreatic ducts from 13 weeks of gestation, and MUC3 is
detected transiently at 13 weeks only (e201). Pancreatic exocrine development, like pulmonary alveolar maturation,
is largely a postnatal event. The appearance of the pancreas at birth is one of underdeveloped acinar elements in a
mesenchymal stroma (Figure 16-2). Acinar tissue increases rapidly after birth. Imrie et al. (74) showed that the ratio
of acinar to connective tissue volume increases in a linear manner from 0.5 at week 32 to 2.0 at week 52 after
conception. In an assessment of the morphologic maturation of an infant pancreas, the length of postnatal survival is
more important than the gestational age. The embryology of the pancreas is reviewed in detail by Cubilla and
Fitzgerald (e46).
FIGURE 16-1 ▪ Pancreas, ducts, and derivation. The pancreatic duct of Wirsung (W) drains most of the pancreas,
joining the common bile duct (Cbd) proximal to the ampulla of Vater (AV). The accessory duct of Santorini (S) may
drain part of the gland through a separate opening. A portion of the head and the uncinate lobe (stippled area) are
derived from the ventral bud of the pancreas.
CONGENITAL ANOMALIES AND MALFORMATIONS

Agenesis and Hypoplasia
Agenesis of the pancreas refers to a complete absence of the gland, rather than a lack of the endocrine or exocrine
portions alone. Insulin promoter factor-1 (IPF1), a homeodomain protein, is important for pancreatic development in
the mouse, and its targeted disruption results in agenesis (e109). A homozygous point deletion in the IPF1 at
13q12.1 has been identified in a patient with pancreatic agenesis (165), and heterozygosity has been associated
with neonatal diabetes mellitus (164). The clinical presentation of pancreatic
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agenesis is that of diabetes mellitus and malabsorption (185,e49,e155). Congenitally absent pancreas may be
associated with diaphragmatic hernia (e138). Some infants have also lacked a gallbladder or intrahepatic bile ducts
(e58,e242).
FIGURE 16-2 ▪ Pancreatic development during gestation. A: At week 10 of gestation, the pancreas consists of
rudimentary buds projecting from a central tube set in a loose mesenchymal stroma. Endocrine cells are already
demonstrable. (Hematoxylin and eosin stain, original magnification ×155.) B: At week 17 of gestation, small lobules
of acini and islets are visible. C: By week 40 of gestation (in a child who lived 1 day after birth), the endocrine
elements lie centrally within the stalk of the lobule, and undeveloped acinar tissue is located peripherally. D: At the
age of 22 months, the remarkable development of acinar tissue is obvious. (B-D: Hematoxylin and eosin stain,
FIGURE 16-3 ▪ Small pancreas. A: Hypoplastic pancreas (2.7 g, 3-cm long) from a 5-month-old child with probable
tyrosinemia. B: Short pancreas (3-cm long) from a 2.5-year-old child with multiple spleens, intestinal malrotation, left
bronchopulmonary isomerism, and extrahepatic biliary atresia.
Functional pancreatic agenesis refers to organ failure without anatomic evidence of gland absence (e94). A
functionally hypoplastic pancreas may be familial and can also lead to exocrine and endocrine insufficiency (e260).
Severe pancreatic hypoplasia has been described in the Wolcott-Rallison syndrome (176).
Partial agenesis has been ascribed to failure of development of the dorsal or ventral primordium and may be familial
(196,e94). More commonly, the pancreas is misshapen and described as short, stubby, or globular. These variants
are not associated with hypofunction. Short pancreas may be an isolated finding, but it is seen with complex
congenital heart disease and as part of a wider malformation complex that includes congenital heart disease, multiple
spleens, and intestinal malrotation (e87). Figure 16-3 illustrates a short pancreas in association with syndromic
extrahepatic biliary atresia. It has also been observed in the complete trisomy 22 syndrome (e50). Pancreatic
anomalies, including agenesis, have been seen in some fetuses and infants with triploidy (e57).
FIGURE 16-4 ▪ Large pancreas. A: Congenital syphilis. An extensive, fine fibrosis distorts the pancreas. B:
Congenital leukemia. Acinar elements are widely separated by the leukemic infiltrate, which is granulocytic in this
instance. (A,B: Hematoxylin and eosin stain, original magnification × 100.)
Pancreatic Enlargement
Pancreatic weights are given in Appendix, Organ Weights. The noncystic pancreas is sometimes larger than usual or
hyperplastic. Some infants with an enlarged pancreas have the Beckwith-Wiedemann syndrome (e224). Marked fatty
replacement of the exocrine portion with preservation of the islets is found in the lipomatous pseudohypertrophy of
the Shwachman-Diamond syndrome (66, 81). Immune and nonimmune hydrops fetalis can lead to pancreatic
enlargement through extramedullary hematopoiesis, and infiltration with leukemia can cause massive pancreatic
enlargement (Figure 16-4). Down syndrome with congenital megakaryoblastic leukemia and pancreatic fibrosis has
been
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reported (e19). In congenital syphilis, pancreatomegaly is a consequence of extensive interstitial fibrosis and
inflammation (e178) (Figure 16-4).
Abnormalities of Position
The pancreas and the duodenum are retroperitoneal and separated from the posterior abdominal wall by an
avascular plane. Abnormalities of fixation or position are often associated with left-sided diaphragmatic hernias. A
“floating” pancreas on a mesentery, in the absence of a diaphragmatic hernia, has been reported (e117). Partial
situs inversus with normal cardiac situs but inversion of the abdominal viscera, including the pancreas, has been
seen with annular pancreas (e3).
Annular Pancreas
A ring of pancreatic tissue can encircle the second portion of the duodenum completely or partially (Figure 16-5).
Johnston described two forms, extramural and intramural (e107). In the extramural form, a flattened band of normal
pancreatic tissue can be separated from the duodenum. A duct originating anteriorly runs around the duodenum to
join the main pancreatic duct. In the intramural form, ectopic pancreatic tissue is located within the duodenal wall,
and small ducts drain directly into the duodenum. Duodenal obstruction is an associated malformation in this form of
annular pancreas, not simply a mechanical constriction (e60). Several mechanisms have been proposed to explain
the pathogenesis of the extramural form of annular pancreas. Two main hypotheses share the basis that the annular
pancreas forms from the ventral anlage. The presence of large amounts of pancreatic polypeptide in islets of
extramural annular pancreas supports this view (169,e215). Lecco postulated fixation of the tip of the single central
ventral bud before rotation with subsequent persistence of the ventral lobe around the duodenum (e135), and this
hypothesis is generally accepted. A recent report (e175), on the other hand, supports persistence and hypertrophy of
the left portion of the paired ventral bud, suggested by Baldwin (e13).
FIGURE 16-5 ▪ Annular pancreas. A,B: An annular pancreas completely surrounds the second portion of the
duodenum. An accessory spleen is present within the tail of the pancreas (trisomy 6).
As many as 20% of infants with annular pancreas are said to have trisomy 21 (104), and annular pancreas may be
associated with cardiac defects or other intestinal malformations, such as tracheoesophageal fistula, Meckel
diverticulum, absence of the gallbladder, and imperforate anus (e89,e116,e159,e200). Annular pancreas in a mother
and three of her four children has been reported (e103), and the documentation of other familial instances
(139,e41,e90,e146,e163) suggests an autosomal dominant inheritance or an involvement of an autosomal recessive
sexinfluenced gene (98).
Annular pancreas may present in the fetus with polyhydramnios or in the neonate with bile-stained vomiting if the
constriction is below the ampulla. In older children and adults, annular pancreas may become symptomatic if
duodenal ulceration or pancreatitis develops.
Ectopic Pancreas
Ectopic (heterotopic) pancreas is widely distributed, largely within the gastrointestinal tract. This condition has been
discovered in 2% to 15% of all autopsies (97, 147). Pancreatic tissue is most frequently found in the wall of the
duodenum, jejunum, or stomach (e182). It is not unusual to find a nodule in the stomach consisting of a centrally
ulcerated pit with localized thickening of the gastric wall (Figure 16-6).
Seifert (147) describes three types of ectopic pancreatic tissue. The first is similar to normal pancreas, with a full
complement of acinar, ductal, and islet constituents. The second is characterized by incomplete lobular arrangement,
few acini, many ducts, and an absence of endocrine elements. In the third type of ectopic pancreas, only proliferating
ducts are present, without acinar or endocrine elements. This form is usually interpreted as an “adenomyoma” or
“myoepithelial hamartoma” of the bowel wall (144). Immunohistochemistry of islet tissue in heterotopic pancreas has
demonstrated a normal distribution of all cell types (e85).
Pancreatic tissue can be seen in the hilum of the liver or within the liver substance (e160). Although on occasion
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a metaplastic process has been suggested for microscopic focus of exocrine pancreatic tissue in a posthepatitic
cirrhotic liver (e264), it is unlikely to be metaplastic when endocrine cells are seen. Pancreas has also been
described in the omentum, mesentery, Meckel diverticulum, vitelline duct, and umbilicus (e258). Ectopic pancreas
has been associated with duplication cysts of the gut and has been reported in fallopian tubes, abdominal lymph
nodes, and adjacent to the thyroid (99,e36,e167,e225). Intrasplenic islands of pancreas are found in trisomy 13-15
syndrome (59). Although heterotopic pancreas is often an incidental finding, it may present clinically as peptic
ulceration, massive hemorrhage, biliary obstruction, cholecystitis, pyloric obstruction, intestinal obstruction,
intussusception, or cystic degeneration (12). Hyperinsulinism (HI) associated with islet cell adenomatous hyperplasia
in the ectopic pancreas has been described (129,195,e193). Rarely, it may present with neoplastic transformation
(57). Solid-pseudopapillary neoplasms have been identified in ectopic pancreas (75,e61).
FIGURE 16-6 ▪ Ectopic pancreas. A: Umbilicated mucosal nodule of the pyloric area. B: A jejunal subserosal
nodule. C: An ectopic intramural (gastric) pancreas has ducts, exocrine acini, and endocrine component. D: Higher
magnification of C. Islands of pancreatic tissue are separated by smooth muscle bundles. (C,D: Hematoxylin and
eosin stain, original magni-fication, ×25 and ×100, respectively.)
Jaffe et al. (e104) indicated that mediastinal pancreatic pseudocysts often arise from below the diaphragm, but
isolated mediastinal pancreatic pseudocysts do exist (e258). Examination of intrapulmonary enteric cysts has
revealed pancreatic elements (e44).
Pancreatic Cysts and Cystic Pancreatic Dysplasia

Congenital cysts of the pancreas are usually incidental findings and are rarely symptomatic (32). Simple
epitheliumlined cysts may be solitary and unassociated with cystic diseases of other organs or complex malformation
syndromes, but a review of older cases reveals that underlying disorders were probably present, such as a trisomy
or
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tuberous sclerosis (59,88,e149,e177,e190) (Figures 16-7 and 16-8; eFigure 16-1). Dermoid cysts have been
described in children (e11). The abnormal pancreas of Beckwith-Wiedemann syndrome may contain cysts (162,e68)
(Figure 16-8). Lymphatic malformation (184) (Figure 16-9) and intestinal duplications that communicate with the
ductal system of the pancreas may present as “pancreatic cysts” (e7,e26,e225).
FIGURE 16-7 ▪ Trisomies. A: Trisomy 13. Poorly circumscribed areas of splenic stroma extend into the pancreas,
entrapping pancreatic ducts with mucoid lining cells. (Hematoxylin and eosin stain, original magnification ×200.) B:
Trisomy 18. Numerous pancreatic cysts lined by duct-type epithelium are scattered. (Hematoxylin and eosin stain
with immunohistochemistry for neuron-specific enolase, original magnification ×50.)
Polycystic kidney disease, of the autosomal dominant or recessive type, may involve the pancreatic ductal system,
but pancreatic involvement appears to be uncommon. Potter and Craig (e188) noted pancreatic cysts in only 2 of
370 cases. Pancreatic cysts are seen in the Meckel-Gruber syndrome (e199,e212) (eFigure 16-1).
Ivemark et al. (e100) and others (178,e45) have described a familial form of renal, hepatic, and pancreatic cystic
dysplasia, which is now generally included under the rubric of polycystic kidney and hepatic disease-1 (PKHD-1). Of
all the reported cases, only 50% are familial, and the condition is associated with anomalies in other organ systems
(100). Bernstein et al. (e23) emphasized that the triad is not unique but also occurs in trisomy 9, Meckel-Gruber
syndrome, Jeune syndrome, Saldino-Noonan syndrome, Elejalde syndrome, and glutaric aciduria type II, all of which
must be excluded first (e232,e270). Severe cystic involvement of the pancreas may occur in von Hippel-Lindau
disease, and polycystic pancreas can be the main or only manifestation of von Hippel-Lindau disease in young
patients who have no prior history of pancreatic disease (51,e99). Minor cystic dysplastic changes are sometimes
present in tuberous sclerosis.
Two reports have been published of microcystic cystadenomas of the head of the pancreas in children with
disseminated cytomegalovirus infection (e9,e37). The cystic dilation appears to be secondary to obstruction.
Cystic fibrosis in the older child can be associated with large, single or multilocular cysts (20,e39,e244). The
pancreatic pseudocyst, caused by rupture of a duct into the lesser sac or abdominal cavity, consists of a fibrous
inflammatory wall around an autodigested cavity. The entity is usually caused by trauma, surgery, or inflammation
(pancreatitis). The fibrous wall of the cyst has no epithelial lining.
Variations in the Pancreatic Ducts

After the dorsal and the ventral pancreatic anlage fuse, the dorsal duct becomes the accessory duct of Santorini, and
the ventral duct becomes the main draining duct of the pancreas, the duct of Wirsung. The fusion can occur at a
single point proximally or at two points, both proximally and more distally (170). The normal architecture varies
widely. The dorsal duct may regress completely, or the dorsal and ventral ducts may not communicate at all
(pancreas divisum) (e21).
In pancreas divisum, the pancreas is separated into two portions. The pancreatic ducts fail to fuse, and the dorsal
duct of Santorini drains most of the gland through
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the minor papilla. The ventral duct of Wirsung drains only the smaller pancreatic remnant. Because of the recent
advances in endoscopy, particularly endoscopic retrograde cholangiopancreatography, pancreas divisum is
diagnosed with greater frequency (163,e236). Some investigators believe that pancreas divisum is clinically
significant (e81), whereas others are more skeptical (40,e6,e236). The combination of acute pancreatitis and
pancreas divisum is treated by endoscopic retrograde cholangiopancreatographic sphincteroplasty (133,e137).
FIGURE 16-8 ▪ Pancreatic cysts. A: A large pancreas (120 g) presented as an abdominal mass in a newborn baby
with Beckwith-Wiedemann syndrome. Multiple cystic spaces are seen on cut surfaces. (Color version of Copyright
1990 from Beckwith-Wiedemann syndrome with unusual hepatic and pancreatic features: A case expanding the
phenotype by Steigman CK, Uri AK, Chatten J, et al. Pediatr Pathol 1990;10:593. Reproduced with permission of
Taylor & Francis Group, LLC., http://www.taylorandfrancis. com) B: Microphotograph of A. No normal pancreatic
tissue is identified. Numerous ectatic ducts, clusters of endocrine cells, and few acini are in loose fibrous connective
tissue. (Hematoxylin and eosin stain, original magnification ×50.) C: 15-year-old child with oral-facial-digital
syndrome type I. Cystically dilated pancreatic ducts with periductal fibrosis (Hematoxylin and eosin, original
magnification ×25.)
The anatomy of the pancreaticobiliary junction varies noticeably (e234). The pancreatic duct can join the common
bile duct within the duodenal wall, or it can enter the duodenum separately. The common channel can be short or
long. If it is longer than 2 cm, the ducts join outside the duodenal wall, and this construction has been implicated in
the pathogenesis of choledochal cyst (e265).
Stenosis of the ampulla may present with pancreatitis, but in the newborn it more commonly manifests as bile duct
perforation (e52).
Pancreatic Pathology in Trisomies

Lesions of the pancreas characteristic of trisomy 13 were illustrated long before the chromosomal defect was
identified (e212). Hashida et al. (59) demonstrated the multiple, poorly demarcated aggregates of splenic tissue in
the tail and the body of the pancreas (Figure 16-7). These splenic islands
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contain pancreatic acini, islets, and knots of ducts lined by tall columnar epithelium rich in goblet cells. Microcystic
changes may be focal or quite widespread with inspissation, features that suggest a duct obstruction as the
pathogenesis (e150). Ectopic splenic tissue in the gastric fundus and upper pole of the kidney has contained
pancreatic elements (e166).
FIGURE 16-9 ▪ Lymphatic malformation. A: Lymphatic malformation presented as a cystic pancreatic mass in a 12-
year-old child. (Courtesy of Pierre Russo, M.D., Philadelphia, Pennsylvania.) B: Lymphatic channels of variable
sizes are embedded within the pancreatic lobular septa and parenchyma. A small number of mononuclear cell
infiltrates are present in the walls. (Hematoxylin and eosin stain, original magnification ×25.)
The pancreas in trisomy 18 often exhibits lobular fibrosis (59) and focal fibrotic nodules in which clusters of ducts
and atrophic acini are found (e204). An area of cystadenomatous changes with back-to-back cysts is seen less
frequently (Figure 16-7). These cysts along with inflammatory aggregates suggest an obstructive etiology (59).
Annular pancreas may occur in as many as 8% of infants with trisomy 21 (e159). A short pancreas has been
described in trisomy 22, and pancreatic anomalies, including agenesis, are described in infants with triploidy (e57).
Marked enlargement of the somatostatin-producing D cells has been reported in the pancreas of triploid fetuses
(141).
EXOCRINE PANCREAS
Functional Development
Proteolytic activity is demonstrable in pancreatic homogenates from 500-g fetuses, and the levels of trypsin,
chymotrypsin, and lipases increase during intrauterine life, accelerating before term (e141). Fetal mRNA levels of
trypsinogen and lipase are much lower than adult levels (118). The presence of these enzymes suggests that the
fetus can use swallowed amniotic fluid for nutritional purposes (e247). Secretory trypsin inhibitors are demonstrable
in the fetal pancreas and in the gastrointestinal, urinary, and respiratory tracts by week 10 of gestation (e70).
Amylase is absent in the fetal pancreas, and salivary amylase predominates in amniotic fluid (e176,e247). Although
trypsin and chymotrypsin levels are near normal adult levels by birth, lipase and, in particular, amylase levels in the
intestinal tract remain low and increase slowly during the first year of life (103).
Zymogen granules are first evident in the developing pancreas by week 12 as elliptic and round structures (e132).
By week 20 of gestation, basal round granules predominate, and the complex basolateral cell interdigitations are
established (103) (eFigure 16-2). Developing ductal cells are filled with glycogen during the first 20 weeks of
gestation, after which the glycogen disappears, first from the larger ducts and then from progressively smaller ones.
Pancreatic secretion is influenced largely by neural cholinergic stimulation, cholecystokinin, pancreozymin, and
secretin. In the adult, pancreozymin is responsible for pancreatic enzyme release, and secretin promotes fluid and
electrolyte release. Lebenthal et al. (102, 103) demonstrated that the pancreas of a neonate is unresponsive to
exogenous cholecystokinin or secretin earlier before 1 month of age and not fully responsive before 2 years of age.
The functional aspects of the developing pancreas, with particular reference to the perinatal period, have been
summarized by Lee and Lebenthal (103).
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Abnormalities of the Exocrine Pancreas Without Fibrosis
Isolated Enzyme Deficiencies
Trypsinogen deficiency is rare. It is characterized by malabsorption, growth failure, anemia, and hypoproteinemia,
beginning in the neonatal period (e245,e246). An association with imperforate anus suggests that these cases are
actually instances of Johanson-Blizzard or Shwachman-Diamond syndrome (50). Congenital enterokinase deficiency
may mimic cystic fibrosis in that the infants fail to thrive and have diarrhea, hypoproteinemia, and edema (102).
Isolated congenital lipase deficiency presents with severe steatorrhea soon after birth without failure to thrive or
anemia; nonpancreatic lipases, such as lingual lipase, may be effective in preventing fatty acid deficiency (e64).
Because the maturation of amylase production is normally delayed, alleged cases of isolated amylase deficiency are
controversial (111). Lerner et al. (e139) established criteria that must be satisfied to document amylase deficiency.
The pancreatic morphology is not described in the cases of isolated enzyme deficiency, although Townes (e245)
described the pancreas of the sibling of a child with trypsinogen deficiency as having “immature acini,” and zymogen
granules were not observed.
Exocrine Atrophy Without Fibrosis

Exocrine atrophy is also called lipomatous atrophy or lipomatous pseudohypertrophy because the shape of the
gland is preserved but the acinar component is replaced by fat (66,e213). This appearance may be common to a
number of pathologic processes.
Shwachman-Diamond Syndrome
With an incidence estimated at 1 in 200,000 births, the Shwachman-Diamond syndrome is the most common cause
of pancreatic insufficiency after cystic fibrosis. The clinical condition is described in a number of reports
(14,e56,e145,e173,e213). Pancreatic exocrine insufficiency is accompanied by growth retardation, short stature,
bone marrow depression with neutropenia, and skeletal changes, predominantly metaphyseal dysostosis.
Cases with anal atresia, Hirschsprung disease, and possibly asphyxiating thoracic dystrophy have been described
(e34,e111,e145,e246). The marrow dysfunction, neutropenia in most instances, may be fixed or cyclical and is
associated with the development of myelodysplasia and later leukemias, usually acute myelogenous leukemia
(156,e5,e173,e267). The pancreatic insufficiency is invariable, may be profound, and appears shortly after birth; less
often, it is mild and improves and normalizes with age in about half the patients (14,e91,e145).
Bodian et al. (14) and Shwachman et al. (153) reviewed the cases for which histologic evidence of pancreatic
disease was obtained. Biopsies were occasionally performed. Postmortem reports on older children indicated that
replacement of the bulk of the pancreas by fatty tissue gives the appearance of a lipomatous pseudohypertrophy
(66). Acinar tissue is absent, without scarring or fibrosis, and the pancreatic ducts and endocrine elements are
preserved (Figure 16-10). The characteristic lipomatous pancreas can be demonstrated by magnetic resonance
imaging (e129).
FIGURE 16-10 ▪ Exocrine atrophy in Shwachman-Diamond syndrome. The acinar elements are almost completely
replaced by fat (lipomatous pseudohypertrophy), with only small aggregates of endocrine tissue left around ducts.
Shwachman-Diamond syndrome is an autosomal recessive disorder, and the genetic basis has recently been
reported (15). The gene involved ( SBDS) is an uncharacterized gene, and resides at 7q11. Its 1.6-kb transcript
encodes a predicted protein of 250 amino acids. A pseudogene copy ( SBDSP) with 97% nucleotide sequence
identity is in a locally duplicated genomic segment. Recurring mutations resulting from gene conversion (substitution
of genetic material from another gene) were found in 89% of unrelated individuals of Shwachman-Diamond
syndrome. The converted segments include pseudogene-like sequence changes that result in protein truncation. In a
study including 23 unrelated patients, molecular genetic and hematologic evaluations demonstrated a poor
genotype/phenotype correlation (e128).
Johanson-Blizzard Syndrome
First reported in 1971 (79), the Johanson-Blizzard syndrome comprises congenital aplasia of the ala nasi, deafness,
hypothyroidism, dwarfism, absence of permanent teeth, and malabsorption resulting from pancreatic insufficiency.
Subsequent reports described urogenital abnormalities (e180) and imperforate anus (50) associated with this
syndrome. Postmortem examination reveals a total absence of acini with complete replacement of the pancreas by
adipose tissue and a few remaining islets with connective tissue around the ducts (31,e79,e161). Mutations in the
gene UBR1 have been recently detected in affected individuals from 12 of 13 families (197). UBR1 encodes one of
E3 ubiquitin ligases of the N-end rule pathway, a conserved proteolytic system. The UBR1 protein substrate,
presumably impaired degradation of which causes Johanson-Blizzard syndrome, is not yet known.
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Immunofluorescence pattern for trypsinogen indicated that there was no primary defect of zymogen synthesis.
Hypoplasia of the exocrine pancreas in association with facial anomalies, micrognathia, posterior cleft palate, and
dental hypoplasia has been referred to as the Donlan syndrome (e53), but overlap with the Johanson-Blizzard
syndrome seems likely (e84).
Other Abnormalities
Pancreatic acinar replacement by fat was observed in a 13-month-old girl with clinical features of leprechaunism,
developmental delay, and abnormalities of gonadotropin regulation (e238). The pancreas contained multiple islets
and ductules embedded in a matrix of adipose tissue but without acinar tissue. The morphology resembled that of
children with the Shwachman-Diamond or Johanson-Blizzard syndrome, but no other features of these syndromes
were present.
Abnormalities of the Exocrine Pancreas with Fibrosis

Sideroblastic Anemia and Exocrine Pancreatic Insufficiency: Pearson Syndrome
Pearson et al. (128) described a syndrome characterized by pancreatic insufficiency, refractory sideroblastic anemia,
and variable neutropenia in which the marrow is normocellular but the cells are vacuolated.
Rötig et al. (e207) documented changes in the mitochondrial DNA, and a number of mitochondrial deletions have
since been reported. Morikawa et al. (117) described a neonate with Pearson syndrome and diabetes mellitus and
postulated a connection with Kearns-Sayre syndrome. The features of Kearns-Sayre syndrome, a mitochondrial
myopathy, may develop later in life in patients who survive the early manifestations of Pearson syndrome
(e134,e154). The most common deletion involves 4,977 base pairs of mitochondrial DNA, and Pearson syndrome
can be diagnosed by blood tests from Guthrie cards (179).
The pancreatic pathologic features differ from those of the Shwachman-Diamond or Johanson-Blizzard syndrome in
that acinar atrophy with fibrosis, not lipomatosis, is present (117, 128).
FIGURE 16-11 ▪ Neonatal hemochromatosis. A: Exocrine acinar cells contain brown refractile pigment. (Hematoxylin
and eosin stain, original magnification ×400.) B: The islets are devoid of iron, but acinar lobules have a marked
intracytoplasmic iron deposition. (Prussian blue stain, original magnification ×400.)
Neonatal Hemochromatosis
Acinar deposition of iron in adult primary hemochromatosis is associated with interlobular and intralobular fibrosis
(168). In neonatal hemochromatosis, hemosiderin deposition in the pancreatic acini is massive, although fibrosis is
mild, and the islets are generally spared, at least in the early stages (e27,e77,e122,e221) (Figure 16-11). Acinar iron
deposition in the absence of reticuloendothelial iron deposition is not pathognomonic of primary hemochromatosis; it
was also seen in some control cases (154). Two sibs with a neonatal hemochromatosis phenotype that included
pancreatic iron deposition also had hypertelorism and trichomalacia, a trichohepatoenteric syndrome (183).
Cystic Fibrosis
Fanconi et al. (45) and then Anderson (e10), while investigating causes of malabsorption, discovered in some of their
patients a condition they termed cystic fibrosis of the pancreas. Recognition of the pancreatic lesion preceded the
clinical delineation of the disease. Farber (e63) proposed mucous plugging of all secretory glands (mucoviscidosis)
to be the pathogenetic key.
Cystic fibrosis is caused by defects in the cystic fibrosis conductance regulator gene (CFTR), localized to 7q31.2
(138,e114,e205). Pancreatic insufficiency is not obligatory, and 15% of patients are clinically pancreas “sufficient.”
The degree of pancreatic disease varies widely at any age, although the disease is progressive and leads to
increasingly more severe changes with time. The final stage of cystic fibrosis is characterized by an obstruction of
the pancreatic ducts by viscous secretion leading to complete acinar atrophy accompanied by fibrosis and
lipomatosis (125,e56,e233).
The tissue alteration can be recognized even before 40 weeks of gestation (74,e233,e237). The ratio of acinar to
connective tissue volume is 0.5 at 32 weeks after conception,
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increasing to 2.0 at 52 weeks in normal controls (74). In the cystic fibrosis pancreas, the ratio of acinar to connective
tissue, low to begin with, decreases from 0.5 at 35 weeks to 0.3 at 52 weeks after conception. Further degeneration
of exocrine tissue supervenes postnatally.
The earliest visible lesions are eosinophilic concretions in acini and ductules, which may lead to acinar or ductular
dilation and flattening of the lining epithelium (125). These concretions generally stain with periodic acid-Schiff (PAS)
and contain calcium. The changes may be focal in preterm infants and in mildly affected cases. Postnatal
acceleration of the pathogenetic train of inspissation, obstruction, dilation, epithelial damage, atrophy, cell
destruction, and fibrosis with minimal inflammation leads to progressive acinar loss with replacement by fibrous
tissue (Figure 16-12; eFigure 16-3) and adipose tissue.
Even though acinar tissue may disappear, islet tissue is preserved until very late. The endocrine changes in cystic
fibrosis are considered later in this chapter, and the appearance of large cysts in cystic fibrosis has already been
discussed (e39).
FIGURE 16-12 ▪ Cystic fibrosis. A: The pancreas of this 12-year-old child is lipomatous with scattered cysts. B: An
8-year-old child with a strong family history of cystic fibrosis was asymptomatic and, at autopsy, had only cystic
dilation with inspissation of ducts, and acini with minimal fibrosis. (Hematoxylin and eosin stain, original magnification
×200.) C: A 10-year-old child with advanced acinar atrophy and fatty replacement has periductal endocrine
overgrowth. (Hematoxylin and eosin stain, original magnification ×100.)
Inspissation and Other Changes of Pancreatic Ducts
Baggenstoss (e12) described the widespread inspissation of secretions in centroacinar cell-lined ductules in patients
with uremia. The finding is not restricted to uremia but is also seen in children dying with acidosis, dehydration,
cardiac failure, or sepsis (e212). Inspissation may also be seen in the pancreas of children who have experienced
prolonged hyperalimentation, but they usually have many of the other conditions listed.
Striking oncocytic changes of the ducts and centroacinar cells have been observed in an infant with mitochondrial
myopathy, lactic acidosis, and ‘ragged red’ muscle fibers (MELAR syndrome). Centroacinar hyperplasia of an
impressive degree was observed in a young adult with HIV infection (eFigure 16-4).
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Pancreatitis in Childhood
In contrast to pancreatitis in adulthood frequently related to alcohol intake, the common causes of pancreatitis in
children are trauma, multisystemic diseases, drug induced, infections, and biliary tract diseases while what are
included in the “multisystem” category vary among the studies (36,188,e144,e254). Pancreatitis can also be seen in
children with branched-chain organic acidemias, methylmalonic acidemia, isovaleric acidemia, maple syrup urine
disease (83), and the Pearson syndrome. When all causes are excluded and the etiology remains undetermined, the
pancreatitis has been traditionally labeled idiopathic. This group accounts for 8% to 34%, and leads the “cause” of
childhood pancreatitis in some series (36,e254). Except for patients with cystic fibrosis, hereditary pancreatitis, and
pancreatitis secondary to congenital structural or metabolic abnormalities, most children have a single, self-limited
episode of pancreatitis, and few cases progress to chronicity (188). On the other hand, children with recurrent or so-
called idiopathic chronic pancreatitis may possess mutations and sequence variations in modifier genes (8) as
discussed below.
Acute pancreatitis of any cause varies from interstitial edema to necrotizing hemorrhagic inflammation depending on
the severity and time point of the process. Fat necrosis is the characteristic finding, and results from sequential
reactions including release of fatty acids from triglyceride esters by lipase and combination of the fatty acids with
calcium (saponification). The formed insoluble salts produce grossly visible chalky white to yellow areas.
Microscopically, outlines of degenerated fat cells with basophilic calcium depos-its are seen along with acute
inflammatory infiltrate (Figure 16-13).
FIGURE 16-13 ▪ Acute pancreatitis. A: Traumatic pancreatitis in a 7-year-old child showing fat necrosis with
saponification (arrow) and acute inflammatory infiltrate. (Hematoxylin and eosin stain, original magnification ×100.)
B: Cytomegalovirus pancreatitis with inclusions in acinar, ductal, and endocrine cells (arrow heads). (Hematoxylin
and eosin stain, original magnification ×200; courtesy of James E. Dimmick, M.D., Vancouver, British Columbia.)
Recurrent pancreatitis and chronic pancreatitis produce fibroinflammatory changes of the parenchyma (94). The
pancreas shows extensive fibrosis, acinar atrophy characterized by reduced number and size of acini, and variable
dilatation of the ducts with calcification (Figure 16-14). The endocrine islets are generally relatively spared and
embedded in the fibrotic tissue. They may appear fused and enlarged, but in end-stage diseases, they eventually
disappear.
Traumatic Pancreatitis
Blunt trauma is recognized as a cause of immediate or delayed pancreatitis in children (Figure 16-13) and an
important cause of pancreatic pseudocyst. A pancreatic pseudocyst may occur as the result of child abuse, but
bicycle injuries are the most common cause in children (e18,e28).
Infectious Pancreatitis
The pancreas may be the seat of any disseminated infection, such as herpes simplex, cytomegalovirus infection
(Figure 16-13), or bacterial sepsis, but it is unusual to encounter clinically relevant pancreatitis (78). In some
instances, the late consequences of a previous infectious pancreatitis are serious.
Rubella may cause an interstitial pancreatitis as part of the expanded rubella syndrome, and pancreatic insufficiency
or even diabetes mellitus can ensue (e33,e55,e105). Mumps is a classic cause of pancreatitis in late childhood
(e220). Coxsackievirus may selectively involve the exocrine or
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endocrine components of the pancreas (78). Parainfluenza 3 pancreatitis, confirmed by immunohistochemical
labeling, produced multinucleated giant cells in the pancreas of a child with severe combined immunodeficiency
(186). Chronic coxsackievirus B interstitial pancreatitis in the absence of a meningoencephalomyocarditis and acute
pancreatitis in a child with α1-antitrypsin deficiency have been reported (e113). In most cases of pancreatitis in
children with acquired immunodeficiency syndrome (AIDS), the disease is mild and opportunistic infections are
absent, although nonspecific inflammatory changes such as focal lymphoplasmacytic aggregates are common (85);
in one child with AIDS and clinical malabsorption, chronic pancreatitis was noted at autopsy without an identified
opportunistic organism (e243). Escherichia coli pancreatitis usually accompanies septicemia, and congenital syphilis
usually causes a pancreatitis in which ductular obliteration, acinar loss, and exuberant interstitial fibrosis with
concentric perivascular accentuation are noted (e27,e69,e178) (Figure 16-4). Gummas are rare (e194).
Inflammatory Pancreatitis
Pancreatitis can accompany childhood collagen vascular diseases, such as lupus, but it may be hard to distinguish
between the effects of disease and those of treatment, particularly drugs (155). Pancreatitis has been described in
Henoch-Schönlein purpura, Reye syndrome, hemolyticuremic syndrome, and Crohn disease (e30,e74,e127,e211).
Immunodysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is a rare X-linked recessive
disorder of immune regulation manifesting with neonatal onset diabetes mellitus, severe enteropathy, eczema,
anemia, thrombocytopenia, and hypothyroidism. The disorder had been known by alternative names including X-
linked polyendocrinopathy, immune dysfunction, and diarrhea and X-linked autoimmunity and allergic dysregulation.
A mutant mouse strain, scurfy (sf) resembles IPEX (e76), and the disease-causing gene Foxp3 encoding scurfin was
identified (e32). Subsequently, the human IPEX locus was mapped to Xp11.23-q13.3 (10), and mutations have been
identified in the human orthologue ( JM2, FOXP3) (22,e20,e257). Patients with protein-truncating mutations have
been reported to demonstrate an absence of FOXP3-nuclear positive lymphocytes in their small and large intestines
(61). Postmortem pancreatic histology is almost always abnormal. Findings include mild-to-dense lymphocytic
infiltrate, acinar loss with fibrosis (chronic sclerosing pancreatitis), dilated ducts, and cystic changes
(132,191,e168,e203) (Figure 16-14). Islets of endocrine cells are decreased or absent in most cases with severe
diabetes mellitus (105,e108,e203).
FIGURE 16-14 ▪ Chronic pancreatitis. A: Obstructive pancreatitis with fibrosis, duct ectasia, acinar atrophy, and
relative abundance of endocrine islets. Several pancreatic stones were also recovered. (Hematoxylin and eosin
stain, original magnification ×50) B: Pancreas of a 79-day-old infant (born at 30 weeks of gestation) with
immunodysregulation, polyendocrinopathy, and enteropathy, X-linked inheritance syndrome (IPEX) shows diffuse
mononuclear cell infiltrate, fibrosis, and acinar loss. Residual endocrine islets are present.
Obstructive Pancreatitis
Some of the causes of obstructive pancreatitis in children have already been mentioned in the section on congenital
malformations, such as annular pancreas, pancreas divisum, gastric duplications that connect to the pancreatic duct
system, and anomalies of the pancreaticobiliary junction (e7). Gallstone pancreatitis in children occurs only in the
presence of a predisposing condition, such as myelomeningocele or hyperalimentation (e8). Cystic fibrosis is the
prototype of a chronic obstructive pancreatitis.
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Drug-induced Pancreatitis
Unlike drug-induced hepatitis detected by elevated transaminases that are part of the routine metabolic profile,
pancreatitis may be often ignored among adverse drug reactions. This is also because of the difficulty in pointing to
a drug as the cause of pancreatitis. A recent review categorizes the effects of drugs on pancreas (180) (Table 16-1)
and incorporates voluminous data published by Biour et al. (13). Class I is reserved for medications implicated in
greater than 20 reported cases of acute pancreatitis with at least one documented re-exposure. Eleven drugs listed
in this class have been linked to pancreatitis in individuals less than 15 years of age. Class II comprises medications
implicated in more than 10 cases of acute pancreatitis with or without positive rechallenge. Class III includes all
medications reported to be associated with pancreatitis, too numerous to be listed in this chapter. In a study among
hospitalized children, valproic acid was the most common drug associated with acute pancreatitis, followed by
asparaginase (188).
Hereditary Pancreatitis
Classic hereditary pancreatitis follows an autosomal dominant inheritance pattern with incomplete penetrance and a
highly variable disease expression (65,e43). Attacks of acute pancreatitis usually begin in childhood, but age of
onset ranges from infancy to the fifth or sixth decade of life (101,e112,e206). The disorder is relatively rare, but is
most commonly caused by one of the two mutations (R122H and N29I) of the cationic trypsinogen gene (PRSS1)
located at 7q35 (56,190,e256). In the early literature, the mutation nomenclature was based on the chymotrypsin
numbering system (87, 189). The genetic numbering system, which designates the initiator methionine as position 1,
has been subsequently adopted (171). Cationic trypsinogen is one of the three isoforms of the digestive proenzyme
trypsinogen, and represents approximately twothird of total trypsinogen in the pancreatic juice. Activation of
trypsinogen to trypsin normally occurs in the duodenum by the brush-border localized enterokinase and also by
autoactivation by trypsin. The mutations either increase stability or increase autoactivation of trypsin (145, 189).
Pseudocysts of the pancreas develop in about 10% of patients with hereditary pancreatitis, and pancreatic
insufficiency and diabetes mellitus are late occurrences (e206).
Table 16-1 ▪ MEDICATIONS ASSOCIATED WITH PANCREATITIS
Class I Class II
Didanosine Rifampin
Asparaginase Lamivudine
Azathioprine Octreotide
Valproic acid Carbamazepine
Pentavalent antimonials Acetaminophen
Pentamidine Phenformina
Mercaptopurine Interferon Alpha-2b
Mesalamine Enalapril
Various estrogens Hydrochlorothiazide
Opiates Cisplatin
Tetracycline Erythromycin
Cytarabine Cyclopenthiazidea
Steroids
Sulfamethoxazole/trimethoprim
Sulfasalazine
Furosemide
Sulindac
Listed from top by the order of number of reported cases.
aDrugs not currently FDA approved in the United States. Table modified from
Trivedi CD, Pitchumoni, CS. Drug-induced pancreatitis: an update. J Clin
Gastroenterol 2005;39:709.
Genetic Risk Factors in so-called Idiopathic Chronic Pancreatitis

Identification of genetic alteration in hereditary pancreatitis has provided a significant tool to investigate other
pathogenic factors of acute and chronic pancreatitis. Subsequent studies revealed a different PRSS1 mutation
detected almost exclusively in patients without a family history of chronic pancreatitis (e38,e261). In addition, Witt et
al. (192) reported a close linkage of sequence alterations in SPINK1 to idiopathic chronic pancreatitis. SPINK1
encodes a natural antagonist of trypsinogens, serine protease inhibitor, Kazal type I, also known as pancreatic
secretory trypsin inhibitor, which binds reversibly to trypsin and inhibits its activity. Sequence variations of CFTR,
the gene responsible for cystic fibrosis, are also associated with idiopathic chronic pancreatitis (e42,e216). A recent
review provides the role of CFTR compound heterozygosity and mild-to-variable mutations in idiopathic chronic
pancreatitis (28). Transheterozygous status with sequence variations in different genes in certain individuals
demonstrates additive effects of these modifier genes on pancreatitis risk (8, 172). Idiopathic chronic pancreatitis has
been a traditional clinical diagnosis describing the lack of an identifiable cause. As more genes and/or more
mutations are identified that cause or predispose to chronic pancreatitis, the number of patients with “idiopathic”
disease is reduced. At the same time, criteria of hereditary pancreatitis have been changing over the years (173),
and the spectrum of disease associated with the CTFR mutant genes keeps expanding (8). The differentiation
between hereditary and idiopathic chronic pancreatitis becomes difficult.
Exocrine Tumors
Primary pancreatic tumors are rare in children. The scarcity of cases and evolving nomenclature hinder us from
studying these tumors and comparing current cases and remote published cases. Three epithelial pancreatic tumors,
pancreatoblastoma, acinar cell carcinoma, and solid-pseudopapillary neoplasm, appear in a recent review of
malignant pancreatic neoplasms in childhood and adolescence (152), and are discussed here. Ductal
adenocarcinoma, the common tumor type in adults, has been reported in children mostly in the older literature
(e130,e239). As the pediatric pancreatic neoplasms have been better characterized, this category has become
exceedingly
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rare (23, 152). Tumors and masses that may occur, but not specifically, in pancreas, include vascular lesions (184),
lymphomas, and other childhood sarcomas (e.g., rhabdomyosarcoma). Tumors of endocrine cell origin will be
discussed later.
Pancreatoblastoma
Pancreatoblastoma (e93), also called pancreaticoblastoma, is an epithelial neoplasm that exhibits multiple lines of
differentiation including acinar differentiation, often with a lesser degree of endocrine and ductal differentiation, and
is associated with squamoid corpuscles (68, 90). A distinct mesenchymal component can also be seen. Some view
this as the infantile or “blastomatous” form of acinar cell carcinoma. In support of this interpretation, a considerable
overlap exists among pancreatoblastoma and acinar cell carcinoma (24). Pancreatoblastomas are usually large,
solitary masses (Figure 16-15), ranging from 1.5 to 20 cm with a mean of 10.6 cm (92) and partially encapsulated.
Microscopically, they are highly cellular, and the epithelial tumor cells are arranged in solid sheets and as small acini.
The acinar differentiation is demonstrated by immunohistochemical positivity for pancreatic enzymes such as trypsin
and chymotrypsin (119) and the presence of zymogen granules by electron microscopy (e110). The tumor usually
has a lobular pattern, separated by stromal bands that may be hypercellular. The squamoid corpuscle is the
characteristic feature of pancreatoblastoma (Figure 16-15). The structures may
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be loose aggregates of larger spindle cells, or more frankly squamous, with keratinization. The cells forming
squamoid corpuscles are not immunoreactive to antibody against cytokeratin 7, while acinar and solid areas are
positively labeled (123) (eFigure 16-5). Alpha- fetoprotein production has been reported (24,e164), a character
shared with cases of acinar cell carcinoma in childhood. It is common to detect endocrine and ductal differentiation
by immunohistochemistry as a minor component of the tumor (92). The data on genetic alterations in
pancreatoblastoma are limited, but allelic loss of chromosome 11p has been described (3,e115). Abnormalities
involving adenomatous polyposis coli (APC)/b-catenin pathway are demonstrated (3) (eFigure 16-5).
Pancreatoblastomas in children are usually detected before developing metastatic diseases and are curable by
surgery (152). Marked responses to preoperative chemotherapy have been described (e250). This is in contrast to
pancreatoblastomas in adults that are, in most instances, fatal.
FIGURE 16-15 ▪ Pancreatoblastoma. A: A 9.3-cm tumor removed with the tail of pancreas and the spleen from a 4-
year-old girl. (Courtesy of James F. Southern, M.D., Milwaukee, Wisconsin.) B: Tumor consists of a mixture of areas
with acinar arrangement and squamoid corpuscles. C: A squamoid corpuscle with central necrosis is on the lower
right corner. A stromal band separates the areas of acinar differentiation with pinpoint lumina and cells showing
darker cytoplasm and basally located nuclei. (B,C: hematoxylin and eosin stain, original magnifications, ×50 and
×200, respectively.)
Pancreatoblastoma has been described in association with the Beckwith-Wiedemann syndrome (41,120,e123). It is
probably important to distinguish the adenomatous endocrine nodules of some infants with Beckwith-Wiedemann
syndrome from pancreatoblastoma.
Acinar Cell Carcinoma

Acinar cell carcinoma is a malignant epithelial neoplasm that shows features of exocrine enzyme production by the
neoplastic cells. By definition, endocrine and ductal components are minimal and do not exceed 25% of the
neoplastic cells (67). The histology and the clinical behavior in the pediatric population of acinar cell carcinoma are
very similar to pancreatoblastoma, and in some cases the pathologic distinction between the two can be difficult (24,
152). Acinar cell carcinomas are usually large, solid, circumscribed tumors that sometimes show necrosis and cystic
degeneration (91). They are microscopically highly cellular lesions that characteristically lack the desmoplastic
stroma commonly seen with the ductal carcinomas (Figure 16-16, eFigure 16-6). The tumor cells show solid,
trabecular, or glandular growth patterns as well as acinar formation. The cytoplasm tends to be abundant and
granular. Immunoreactivity for trypsin, lipase, and chymotrypsin and ultrastructural demonstration of zymogen
granules confirm acinar differentiation (67). As both acinar cell carcinoma and pancreatoblastoma share a densely
cellular morphology, can exhibit a minor endocrine component, and demonstrate acinar differentiation, it is
sometimes impossible to decide whether a tumor is a “squamoid corpuscle-free” pancreatoblastoma or an acinar cell
carcinoma (24). It is therefore advised and practical to define pancreatoblastoma as a tumor with the characteristic
squamoid corpuscles and is distinguished from acinar cell carcinoma (90). Genetic alterations in acinar cell
carcinomas also points toward the similarity between the two tumors (2).
FIGURE 16-16 ▪ Acinar cell carcinoma. The tumor is highly cellular with virtually no stroma. The tumor cells are
arranged in solid sheets and nests with small luminal spaces. (Hematoxylin and eosin, original magnification ×100.)
Solid-Pseudopapillary Neoplasm
This tumor is also known as solid-pseudopapillary tumor, papillary cystic tumor of the pancreas, solid and papillary
epithelial neoplasm, and papillary-cystic neoplasm, but solid-pseudopapillary neoplasm is currently the preferred
term (69). Most cases are found in females in the second and the third decades of life (137), but a few male patients
have been reported (21,e148).
The tumors are generally large, both solid and cystic (Figure 16-17), and located anywhere in the pancreas. Solid
sheets of epithelial cells may have an endocrine appearance, with uniform cells that have sharply defined cell
borders. Perivascular pseudopapillae are interspersed with cystic degenerated areas. PAS-positive globules may be
present in the cytoplasm (Figure 16-17). Despite recent studies, the line of differentiation of solid-pseudopapillary
neoplasm is still unknown (93,96,119,e48). Consistently positive markers by immunohistochemistry are CD56,
vimentin, α1-antitrypsin, nuclear b-catenin, and CD10 (1, 124) (eFigure 16-7). Some tumors show positive labeling by
synaptophysin, but chromogranin is negative. The presence of progesterone receptors is frequently reported
(e136,e268), while there are conflicting results on estrogen receptors (e71). The prognosis for patients with this
neoplasm is excellent (93); most are cured by excision, but 10% to 15% have recurred locally (137) or metastasized
(e48).
ENDOCRINE PANCREAS
Histogenesis, Maturation, and Morphology
During weeks 6 and 7 of intrauterine life, the dorsal and ventral pancreatic buds fuse. A simple epithelial tube of
endodermal origin grows into the mesenchyme and gives rise to the endocrine and exocrine pancreas. It has been
demonstrated in the rat that disaggregated and presumably single islet cells can regenerate new islets in culture that
differentiate
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into insulin- and glucagon-producing cells (e187). Pax4 and Pax6 are required for normal islet cell development in
mice (e208,e231).
FIGURE 16-17 ▪ Solid-pseudopapillary neoplasm. A: A 2.5-cm tumor removed from the tail of the pancreas in a 14-
year-old girl (Courtesy of Marta E. Guttenburg, M.D., Philadelphia, Pennsylvania.) B: Despite the grossly
circumscribed appearance, microscopic infiltrative growth is common, especially into the adjacent non-neoplastic
pancreas. (Hematoxylin and eosin stain, original magnification ×50.) C: The basic architecture is solid cellular nests
with small vessels. Some cells show cytoplasmic vacuolization. (Hematoxylin and eosin stain, original magnification
×100.) Inset: Eosinophilic “hyaline globules” are periodic acid-Schiff positive and diastase resistant, and typically
found in the cytoplasm. (Periodic acid-Schiff with diastase stain, original magnification ×400.) D: The tumor cells
situated away from the vessels degenerate, resulting in pseudopapillae. (Hematoxylin and eosin stain, original
Microdissection studies in the mouse have shown that both the exocrine and endocrine cells of the pancreas
develop from foregut endoderm; expression of both acinar enzyme and islet hormone genes is detected. Without
mesenchyme, the primordial cells develop into endocrine cells only, but in the presence of mesenchyme, ducts and
acini also form (e231).
Robb (e202) described the budding of islet cells from the pancreatic duct into the adjacent mesenchyme, visible after
10.5 weeks of gestation. The endocrine cells then lose their connection with the duct and become vascularized by a
central capillary. In the study of Stefan and colleagues (e229),
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glicentin-containing cells were the first to appear, becoming detectable at week 8 of gestation in the wall of the
developing duct. This pattern virtually disappeared by week 12 or 13 and was replaced by one in which the cells
were reactive for adult glucagon and glicentin. By week 9, primitive islets were found to contain insulin (B) cells,
somatostatin (D) cells, and glucagon (A) cells; pancreatic peptide (PP) cells were found only in the region of the duct
of Wirsung, presumably in the ventral lobe (e40).
Between weeks 16 and 20 of gestation, the bipolar islets of Robb have insulin at one pole and somatostatin or
glucagon at the other, except in the ventral lobe, in which PP predominates (e229). A central core of insulin cells
develops in the mantle islet, surrounded by somatostatin and glucagon cells in the body and the tail and PP cells in
the ventral head. The mature islets exhibit a trabecular arrangement, in which inner cells contain insulin and more
peripheral cells contain glucagon or somatostatin; this arrangement is thought to be important in paracrine cell-to-cell
control (e179). Gap junctions mediate cellto-cell communication for the biosynthesis, storage, and release of insulin
and other hormones, and connexin CX43 is expressed in islets (110).
FIGURE 16-18 ▪ Distribution of endocrine cells, which are revealed by immunohistochemistry for chromogranin A.
Respective survival dates are as follows: A: Stillbirth at 25 weeks. B: Forty weeks of gestation, 8 days of postnatal
survival. C: Twenty-two months. D: Eight years. (A-D: original magnifications ×100.)
Fetoscopy between weeks 19 and 21 of gestation has shown that levels of insulin, glucagon, and PP are similar in
fetal and maternal blood (e4).
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The distribution of the various cell types is not homogeneous within the pancreas, nor are they stable from fetal to
adult life. A portion of the head of the pancreas, ostensibly derived from the ventral primordium, is rich in islets
containing PP (136,e147). The number of somatostatin-containing D-cells is greater in the fetal and neonatal period
than later in life, reaching a peak between week 17 of gestation and 5 months of age (e198,e229). The distribution of
endocrine cells in the fetus and neonate is characterized by a greater number of single cells and small clusters of
cells outside the islets (76) (Figure 16-18).
The endocrine tissue in the developing pancreas lies centrally within the lobule, close to the ductal system from
which it buds. The larger and better-formed islets of Langerhans form the stalk of the lobule, and smaller aggregates
and single cells bud off within the more peripheral centroacinar tissues, resulting in the characteristic distribution of
the endocrine tissue in late fetal life. At term, the smaller peripheral clusters of endocrine cells may be numerous,
and the extrainsular endocrine cells may constitute much of the endocrine component of the newborn pancreas. This
is often confused with the diffuse form of congenital HI, formerly diffuse nesidioblastosis, by the uninitiated.
A large body of literature has been published on the quantification of the endocrine content of the pancreas at
various ages. The endocrine content of the pancreas can be expressed as a ratio of endocrine to acinar tissue. This
is easy to quantify in a mature pancreas, in which confluent acinar tissue is present around islets. It is much more
problematic in the pancreas of an infant, which consists mostly of mesenchyme and in which many of the largest
islets are “septal” (Figure 16-18).
FIGURE 16-19 ▪ Histologic features of pancreas from individuals less than 2 years of age. A: Abundant
islets/endocrine tissue is seen in the pancreas of a 5-month-old normoglycemic infant, especially in the head.
(Hematoxylin and eosin stain, original magnification ×100.) B: Endocrine cells budding off a duct (nesidioblastosis) in
an 8-day-old infant born at 40 weeks of gestation (Immunohistochemistry for chromogranin A, original magnification
×200.) C: Very large septal islets, such as this one in a 22-month-old child, are not uncommon. A small cluster of
endocrine cells is budding off a duct (nesidioblastosis) in the lower right corner (arrow). (Hematoxylin and eosin
The amount of endocrine tissue present at birth, in a rough compilation of available estimates, is 10%; this
decreases during acinar development to 5% by 6 months of age, and then gradually to the adult volumes of 1% to
2% (76,77,116,e82,e262). Figure 16-19 illustrates histologic features of normal pancreas in children often confused
to be abnormal, including abundant endocrine tissue, endocrine cell clusters budding off ducts, and large septal
islets.
The amount of endocrine tissue is not fixed. Ductal obstruction, with chronic pancreatitis and fibrosis, may be
associated with a resurgence of endocrine development. This situation can mimic endocrine neoplasia (e17).
Because volumetric determinations of endocrine mass are usually expressed relative to the acinar mass and
because a decrease in exocrine tissue produces a relative increase in the mass of islet cells, the observer should
determine whether an apparent excess of endocrine cells is absolute or secondary to acinar loss.
Abnormalities of the Endocrine Pancreas

Islet Hypertrophy
Islet size varies with age. Jaffe et al. (76) found that before the age of 2 months, only 3.5% of all islets are larger than
200 μm in diameter. Single septal islets at any age can be much larger, measuring as much as 700 mm in diameter
(Figure 16-19). The term islet hypertrophy should be used to describe a generalized phenomenon in which the
percentage of islets larger than 200 mm is excessive for a particular age.
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Borchard and Müntefering (16) provide values for islet size at different stages of gestation. True islet hypertrophy is
seen in infants of diabetic mothers and, less commonly, in infants with erythroblastosis fetalis (16,71,e255).
Endocrine Aplasia and Hypoplasia

Combined exocrine and endocrine hypoplasia was mentioned previously (e58,e94,e138,e155,e242). A child with
metabolic acidosis and diabetes shortly after birth had a pancreas in which exocrine elements appeared normal but
no islets were seen on conventional histologic examination; a sibling had a similar clinical story (e51). A congenital
absence of glucagon cells has been reported (but not illustrated), and an infant with normoinsulinemic hypoglycemia
had few glucagon-containing cells (e16,e78). A generalized paucity of somatostatin cells has been implicated in
some cases of neonatal hypoglycemia (e25). Some growth-retarded fetuses have been previously reported to show
endocrine paucity (e249), but a more recent study demonstrated no differences between intrauterine growth
retardation and control fetuses in insulin-positive areas or islet organization (11).
Infant of the Diabetic Mother

Maternal insulin and glucagon do not cross the placenta, although maternally derived or animal-derived antigen-
antibody complexes of insulin do (113). The changes in the endocrine pancreas of a child born to a diabetic mother
are a response to maternal hyperglycemia (e2,e106) and anti- insulin antibodies (113). The various changes in the
pancreas of the diabetic offspring are ascribed to variations in the severity of maternal disease, the stringency of the
therapeutic control, and the presence or absence of diabetic vascular complications (113,182,e88). Most infants of
diabetic mothers have islet hypertrophy (>10% of islets larger than 200 mm), an increased total islet cell volume,
pleomorphism of B-cell nuclei, eosinophilic insulitis (Figure 16-20), and periinsular or intrainsular fibrosis (71). Similar
features may be present in the pancreata of infants born to prediabetic mothers.
FIGURE 16-20 ▪ Infant of diabetic mother and nonimmune hydrops fetalis. A: Eosinophilic insulitis in an infant of a
diabetic mother. (Hematoxylin and eosin stain, original magnification ×200.) B: In this pancreas from an infant with
nonimmune hydrops, an islet is associated with extramedullary hematopoiesis. (Hematoxylin and eosin stain, original
Hultquist and Olding (71) stated that the pancreas of the infant whose mother is diabetic weighs less than that of a
normal infant when corrected for total body weight. This is particularly true of the infants of mothers with the most
severe diabetic complications. After week 34 of gestation, infants with a birth weight of 2.25 kg or more have an
excess islet cell volume. This correlation was stronger for the offspring of mothers with uncomplicated diabetes
because they had the largest babies. No difference was detected between islet cell volume in normal infants and the
volume in the offspring of mothers with severely complicated diabetes. Borchard and Müntefering (16) claimed that
an increased mean islet diameter is more characteristic of the diabetic infant than an increased number of islets.
The islet cell increase is largely the consequence of expansion of the B-cell mass from 40% of endocrine cells to
63.8% (182). This expansion is observed in the dorsal lobederived pancreatic polypeptide poor portion of the
pancreas
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(e.g., tail) (182) as well as in the pancreatic polypeptide rich (ventral lobe-derived) region of the pancreas. Milner et
al. (116) demonstrated that A-cell and PP-cell increases accompany B-cell hyperplasia in a diabetic pregnancy, with
the A-cell increase occurring only in the pancreatic polypeptide poor and the PP-cell increase in the pancreatic
polypeptiderich regions. Pleomorphism of the B-cell nuclei is seen with the increase in endocrine volume and is also
marked in the pancreata of the infants of mothers with complicated diabetes.
Wellman and Volk (e255) reviewed the issue of mesenchymal inflammatory infiltrate. Eosinophilic periinsulitis, the
most characteristic finding, occurs in about 50% of infants of diabetic mothers, whether or not the mother is receiving
insulin. The infiltrate is rich in eosinophilic myelocytes, may contain Charcot-Leyden crystals, and is said to
disappear within days of birth. Charcot-Leyden crystals can be seen in the macerated pancreas. Klöppel (e119)
suggested that the infiltrate is a local reaction to insulin-containing immune complex. Fibrosis within and around islets
is seen in association with hypertrophy and eosinophilia, but it is also described as an early in utero finding
independent of eosinophilic accumulation (71,e170).
Other, less constant findings in the islets of infants of diabetic mothers include an increase in the mitotic rate,
degranulation of B-cells, islet edema, hydropic swelling of islet cells, ribbon-like transformation of islet cells, necrosis,
and thickened extrainsular and intrainsular capillaries. Lymphocytic infiltration is not specific to these children.
A suggestion by Van Assche and Gepts (182) was that an intact hypothalamic-hypophyseal axis is required for the
development of pathologic pancreatic changes because they are not seen in the anencephalic offspring of diabetic
mothers.
It has been predicted that the prenatally affected islets of infants of diabetic mothers become insufficient through the
stress of postnatal life and that infants of diabetic mothers are more likely to develop diabetes mellitus. Several
epidemiological data show that consequences extend to adult life and even to the next generation through the
maternal line (63). Family histories secured from consecutive pregnant diabetic women (e151) indicated that patients
with gestational diabetes are more likely to have a mother with diabetes than gravidas with normal carbohydrate
metabolism. The studies on Pima Indians (e47,e185) have shown that, besides a genetic transmission of diabetes,
the diabetic intrauterine milieu can also induce a diabetogenic tendency in the offspring.
Hydrops Fetalis
The accumulation of fluid in the fetus results from various congenital and acquired/maternal conditions. Immune
hydrops is caused by blood-group incompatibility, mostly of ABO and certain Rh types, between mother and child.
The endocrine pancreas of Rh-positive infants born to Rhnegative mother with anti-Rh antibody has been described
to partly resemble that of infants of diabetic mother but with some differences. The amount of endocrine tissue is
increased in the tail of the pancreas (181), and it is parallel to the increased number of endocrine cells per islet. In
contrast to infants of diabetic mothers, the proportion of B-cells and the contribution of the different cell types are
unchanged. Milner et al. (115) report that the increased volume fraction of B, A, PP, and D cells is seen only in the
pancreatic polypeptiderich (ventral lobe) part of the pancreas.
Prevention of Rh immunization in at-risk mothers has reduced the incidence of this disorder, and nonimmune
hydrops has become more prevalent. The causes of nonimmune hydrops are manifold. Excess endocrine tissue and
islet cell hyperplasia (without morphometric confirmation) have been described in nonimmune hydrops fetalis (e165).
An islet associated with extramedullary hematopoiesis is depicted in Figure 16-20.
Diabetes Mellitus
Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin
secretion, insulin action, or both. Deficient insulin action is due to diminished tissue responses to insulin at one or
more points in the complex pathways of hormone action. The recently revised classification reflects our
understanding of the pathogenesis of diabetes mellitus (48). Four main forms are in the classification: type 1
diabetes mellitus, type 2 diabetes mellitus, other specific types, and gestational diabetes mellitus. Patients with any
form of diabetes may require insulin treatment at some stage of their disease. Such use of insulin does not, of itself,
classify the patient. The third category, other specific types, accounts for less than 10% of all diabetic patients, and
includes diabetes mellitus caused by monogenetic defects of B-cell function and insulin action, diseases of the
exocrine pancreas, endocrinopathies, drugs, infections, uncommon immune-mediated forms, and other genetic
syndromes. Some in this category are described elsewhere in this chapter. Provided below are descriptions of type 1
and type 2 diabetes, which are the two principal types of diabetes, followed by neonatal diabetes and maturity-onset
diabetes of the young (MODY) of which new insights have been delineated recently.
Type 1 Diabetes Mellitus

Type 1 diabetes mellitus is associated with an absolute insulin deficiency caused by destruction of insulin-producing
B cells of the pancreas. This form used to be designated as insulin-dependent diabetes mellitus or juvenile onset
diabetes mellitus, and, until recently, was considered the most prevalent type in children. It results from a cellular-
mediated autoimmune process (7). Antibodies detected in 70% to 80% of patients are autoantibodies to islet cells,
insulin, glutamic acid decarboxylase, and tyrosine phosphatase IA-2 and IA-2 b (130). The rate of B-cell destruction
is variable, but when it is rapid as seen in infants and children, severe hyperglycemia and/or ketoacidosis may be the
first manifestation. Type 1 diabetes mellitus has a complex pattern of genetic associations, and putative susceptibility
genes have been mapped.
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The most important is the class II MHC (HLA) locus at 6p21 (109). It is influenced by the DRB genes (e95), with
linkage to the DQA and B genes. These HLA-DR/DQ alleles can be either predisposing or protective (e192).
FIGURE 16-21 ▪ Type 1 diabetes mellitus of recent onset. An active insulitis is present within and around an islet.
Residual endocrine cells were demonstrable in this islet. (Hematoxylin and eosin stain, original magnification ×200.)
Morphologic changes in the pancreas of diabetic individuals are not consistent, and they rarely contribute to
diagnosis. The pancreas of classic type 1 diabetes may show a reduction in the number and the size of islets and
insulitis (49, 95) (Figure 16-21). Insulitis is characterized by islets infiltrated primarily by T-lymphocytes (112) and is
confined to those islets in the recent-onset diabetic that still contain B cells (e67). Other early features include
cellular vacuolation and nuclear pleomorphism (e72). Later in the course of the disease, insulitis is no longer seen
and B-cells become sparse (49). Interlobular fibrosis is a feature in some diabetics. Trophic changes of the exocrine
cells can be marked, with diffuse or, in the early stages, patchy, focal acinar atrophy (142,e120) (eFigure 16-8).
FIGURE 16-22 ▪ Islet amyloid deposition. A,B: A 4.5-year-old boy with dwarfism, genital hypertrophy, and diabetes
mellitus had amyloid in the islets. (A: Hematoxylin and eosin stain, original magnification ×200, B: Thioflavine T
Type 2 Diabetes Mellitus

Patients with type 2 diabetes have insulin resistance and usually relative (rather than absolute) insulin deficiency.
Relative insulin deficiency implies an inadequate secretory response by the pancreatic B-cells to compensate for
insulin resistance. The specific etiologies are not known, but autoimmune destruction of B-cells does not occur.
Patients should not have any of the other causes of diabetes listed under other specific types (48). Nevertheless,
environmental factors, such as a sedentary life style and dietary habits, play a role in the pathogenesis. Most
patients with this form of diabetes are obese, and the link between obesity and diabetes is mediated by insulin
resistance (84). The incidence of type 2 diabetes mellitus, previously considered to be a disease of adult, has
recently risen remarkably in children in different geographic areas of the world (5,e62,e118,e186,e248). Although
type 2 diabetes is often associated with a strong genetic predisposition, more so than type 1 diabetes (e15, e172),
its genetics are complex and not clearly defined.
The pancreas generally shows no change other than islet amyloid deposition (148), which becomes more frequent
with age in both diabetics and nondiabetics (e120). Islet amyloid is formed from islet amyloid polypeptide, which is
secreted with insulin, and is seen in longstanding adult cases of type 2 diabetes (26). Animal studies have shown
some evidence of a direct role for amyloid in the pathogenesis of type 2 diabetes (70,e253). Figure 16-22 shows
amyloid replacement of islets in a 4.5-year-old boy with diabetes, but also with dwarfism and genital hypertrophy. B-
cell mass is
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suggested to be decreased in type 2 diabetes mellitus, but this remains controversial (19,26,148,e83,e197,e230).
Neonatal Diabetes Mellitus

Neonatal diabetes is rare, with a reported incidence of 1 in 400,000 live births (151), and may be defined as insulin-
requiring hyperglycemia that is diagnosed within the first three months of life (54, 131). It is a heterogeneous group
of disorders, therefore, not part of the etiology-based classification recently proposed (48), but is a useful category
when faced with infants with diabetes mellitus of neonatal onset.
The disease may be transient, remitting by 18 months of age but relapsing during adolescence in a significant
proportion of patients (151,e252). Most patients are full-term, but growth-retarded, infants. Three inter-related
genetic mechanisms have been ascribed to more than 50% of transient neonatal diabetes mellitus (174): paternal
uniparental isodisomy of chromosome 6, paternal duplication of 6p24, and a methylation defect at a CpG island
overlapping exon 1 of ZAC (zinc finger protein associated with apoptosis and cell cycle arrest)/ HYMAI (imprinted in
hydatidiform mole). These observations suggest that transient neonatal diabetes may result from overexpression of
an imprinted gene on 6p24 and displaying paternal expression. Pancreatic morphology has not been described
specifically for this form.
Permanent neonatal diabetes requires lifelong therapy, and a variety of causes and associations have been
identified. They include pancreatic hypoplasia or aplasia (e49,e58,e138,e155,e242), Wolcott-Rallison syndrome
(18,e263), homozygous inactivating mutations of insulin promoter factor 1 (IPF1) affecting proteins involved in
pancreas formation (165), homozygous or compound heterozygous glucokinase mutations with complete enzymatic
loss (e174), and immunodysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome (191,e20,e257).
Pancreatic findings at autopsy from patients with Wolcott-Rallison syndrome and IPEX syndrome are described
elsewhere in the chapter. The most common genetic cause, accounting for 34% to 64% of permanent neonatal
diabetes mellitus, is activating mutations in the KCNJ11, which encodes Kir6.2, a subunit of the adenosine
triphosphate (ATP)-sensitive potassium channel of the B cell (54,e66,e152,e251). This finding has a significant
impact on management because many patients can be switched from insulin to oral sulfonylureas (e181). Most
recently, activating mutations in ABCC8 encoding the other component of the potassium channel have been reported
to cause neonatal diabetes (9). The channel abnormality is also responsible for congenital HI discussed later.
Maturity-Onset Diabetes of the Young (MODY)

MODY is characterized by autosomal dominant inheritance and early-onset diabetes mellitus (60). Early onset is
defined as at least one to two members of the family being diagnosed before age 25. These patients have
monogenetic defects resulting in B-cell dysfunction, are not known to be insulin resistant, and do not need to be
obese to develop diabetes. Although an earlier definition of MODY included early-onset type 2 diabetes and
autosomal dominant inheritance (60), MODY is under other specific types in the recent classification (48), and not
part of type 2 diabetes mellitus. Nevertheless, childhood type 2 diabetes can be confused with MODY as both have
insulin secretion, and are not usually insulin dependent or prone to ketoacidosis. Of 112 nontype 1 children reported
in a survey performed in the United Kingdom, 25 had type 2 diabetes and 20 had MODY (e59). The rest was
secondary and unclassifiable due to incomplete data. In contrast to type 2 diabetes, MODY patients were younger
(10.8 vs. 12.8 years), less likely to be overweight, and none were from ethnic minority groups. Fifty-six percent of
type 2 patients were of ethnic minority groups.
To date, six genetic defects that cause MODY have been uncovered, five of which correspond to transcription
factors expressed in pancreatic B-cells: hepatic nuclear factor (HNF)-4α (193), HNF-1α (194), IPF1 (164), HNF-1β
(64), and neurogenic differentiation 1/B-cell E-box transactivation 2 (108) for MODY 1, 3, 4, 5, and 6, respectively.
Loss-offunction mutations of glucokinase that catalyzes the first step in glucose metabolism cause MODY 2 (46). In
addition to their effects on B-cell function, deficiency of some of these transcription factors affects function of other
organ systems (e101). Patients with HNF-1a mutations have decreased renal reabsorption of glucose and glycosuria
(e158). The deficiency of HNF-4α affects triglyceride and apolipoprotein biosynthesis (e218). Families with HNF-1β
mutations presenting with renal cysts and diabetes have been described (e24,e124).
Hyperinsulinism
HI is the most common cause of hypoglycemia in infants and children (158). Clinically transient forms of HI are seen
in neonates born to diabetic mothers, infants with birth asphyxia and/or small for gestational age (158), and
Beckwith-Wiedemann infants (43). The histologic features of pancreas in some of these conditions are mentioned
elsewhere in this chapter.
Persistent HI in children, unlike adults, is rarely due to islet cell adenoma (insulinoma) (Table 16-2), but most often
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represents a congenital genetic disorder. The incidence of congenital HI in the general population ranges from one
in 27,000 to 50,000 live births (53). In communities with high rates of consanguinity, the incidence may be as high as
one in 2,500 live births (e153). As described below, the disorder is quite heterogeneous. Insulin levels are not
usually dramatically elevated, but rather there is inadequate suppression of insulin secretion at low plasma
concentrations of glucose (i.e., HI rather than hyperinsulinemia) (159). The diagnosis is based on evidence of the
effects of excess insulin, which includes inappropriate suppression of lipolysis and ketogenesis and an
inappropriately positive glycemic response to glucagon at times of hypoglycemia (e65,e227). Uncontrolled
hypoglycemia may lead to seizures or permanent brain damage, and immediate medical intervention is required.
Table 16-2 ▪ FINDINGS IN PANCREAS RESECTED FROM CHILDREN WITH HI
Histology of Pancreas Percentage of Children Affected

(%)
Diffuse endocrine abnormality (large nuclei in islets) 45
Adenomatosis/adenomatous hyperplasia (focal, multifocal, or 45

generalized)
Adenoma 1
Normal 5
Equivocal/difficult to classify 4
Note: All known and unknown underlying genetic abnormality inclusive.
The Children's Hospital of Philadelphia (1983-2005, n = 159).
Infants with congenital HI were once believed to have abnormal pancreatic development associated with persistence
of packets of islet cells (B-cells) budding off ducts, termed nesidioblastosis (e131,e269). Observations based on
immunohistochemical investigations have shown that nesidioblastosis, as defined above, is a common feature of the
pancreas in normoglycemic neonates and infants (76,e196,e262), and nesidioblastosis by itself is no longer
considered the underlying histologic basis of congenital HI (114,134,159,e195). A supplemental discussion on
nesidioblastosis and HI is provided at the end.
Congenital HI is caused by a number of genetic abnormalities in the pathways regulating insulin secretion by
pancreatic islets (Table 16-3). A standardized nomenclature system has been proposed to facilitate communication
among investigators/clinicians and identification of the precise clinical, biochemical, genetic, and physiological
characteristics of each specific disease (53). The use of HI is recommended as a general term (instead of
hyperinsulinemic hypoglycemia). If the genetic etiology is known, the mutated gene is added to the name, such as
KATP-HI for HI due to mutations in the ATP-sensitive potassium channel genes and HI-GCK for HI due to mutations
of glucokinase gene. When other clinical or histological characteristics are known, these should be stated, such as
hyperinsulinismhyperammonemia syndrome (HI/HA) for HI/HA and focal HI for focal disease (see below). This
proposal has been conceptually accepted, but many variations of the term are still seen in publications.
Hyperinsulinism with B-cell ATP-Sensitive Potassium Channel Abnormalities (KATP-HI)

As a group, the ATP-sensitive potassium channel abnormalities are by far the most common cause of congenital HI.
Three genetic mechanisms, recessive mutation, paternally inherited mutation with somatic loss of maternal 11p15,
and dominant mutation, are known to cause insulin dysregulation. Children often present with severe hypoglycemia
in the newborn period, with the exception of the rare dominantly inherited form. Because the channel is impaired, that
is, channelopathy, they often do not respond to medical therapy with a channel agonist diazoxide, requiring further
treatment including, but not limited to, pancreatectomy. Each one of the three is discussed separately along with its
histology.
HI caused by recessive ATP-sensitive potassium channel mutations and associated diffuse histologic abnormality
(diffuse HI): One of the most common forms of HI is associated with recessive mutations in one of the two adjacent
genes on chromosome 11p that comprise the B-cell ATP-sensitive potassium channel: the high-affinity sulfonylurea
receptor 1 (ABCC8, formerly SUR1) (175,e171) and its regulated ion pore, potassium inward rectifier 6.2 (KCNJ11,
formerly Kir6.2) (121,e240). Loss-of-function mutations cause closure of the channel, leading to depolarization of the
membrane and activation of a voltage-gated calcium channel that results in exocytosis of insulin granules.
The pancreatic histology of this form is characterized by the presence of abnormally large islet cell nuclei that are
distributed throughout the pancreas. Frequency and easiness of finding enlarged endocrine cell nuclei vary from
case to case. Large nuclei are empirically determined to be nuclei four times that of the nearby acinar cell nuclei
(135) or nuclei occupying an area more than three times larger than the surrounding endocrine nuclei (e235) (Figure
16-23). A morphometric analysis revealed that the mean nuclear radius of the 50 largest nuclei of this type is
significantly larger than the mean nuclear radius measured in islets that are present outside or away from the
adenomatous (focal) lesions in the focal form described next (149). The cytologic changes also include “bizarre”
crescent-shaped or ovoid nuclei with occasional nuclear pseudoinclusions (intranuclear cytoplasmic invagination). It
is important to note that not all islets contain these abnormal nuclei, and the number of islets with characteristic
nuclei may be small. Ductuloinsular complex composed of endocrine cells in the epithelium of the ducts and in
connection with the endocrine cell clusters may be present (Figure 16-23). The B-cell proliferation rate is not higher
when the fraction of Ki-67 positive B-cells was compared to a control group (149). Moreover, the mean total
endocrine area and the volume density of B-cells are not increased in this form (76,134,e196,e262). The major
source of confusion is the lack of familiarity with the histologic features of the normal newborn and infant pancreas,
in which endocrine tissue is abundant, and smaller peripheral clusters of endocrine cells may constitute much of the
endocrine component (Figures 16-18 and 16-19). These are the features that were previously interpreted as
excessive “nesidioblastosis.”
HI caused by paternally inherited ATP-sensitive potassium channel mutations together with somatic loss of
maternal 11p15 and associated focal histologic abnormality (focal HI): An equally common and histologically distinct
form of HI results from a paternally inherited mutation in the channel genes (ABCC8 and KCNJ11) together with a
sporadic somatic loss of the maternal 11p15 that contains
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imprinted genes involved in cell proliferation. The unbalanced expression of the imprinted growth factor (IGF2) and
tumor-suppressor genes (H19 and CDKN1C) leads to adenomatous hyperplasia; the expression of the mutated
paternal gene causes unregulated insulin secretion from the hyperplastic lesion (33). p57kip2 is the product of one of
the imprinted genes ( CDKN1C) that are normally expressed from the maternal allele and is lost in endocrine cells
within the adenomatous lesions. The loss of p57kip2 expression can be visualized by immunohistochemical labeling
(86, 166) (eFigure 16-9). Identification of this form of disease has major clinical implications because this form, if
detected, can be surgically excised and cured without near-total pancreatectomy (4, 35).
Table 16-3 ▪ GENETICS AND PATHOLOGY OF CONGENITAL HI
Genetic
Abnormality Examples of
Clinical Suggested
Gene Protein Information Pathologic Findings Nomenclature
ABCC8 SUR1 Recessive Diffuse changes with HI-SUR1

mutation large islet cell nuclei Diffuse
HI
KCNJ11 Kir6.2 Recessive Diffuse changes with HI-Kir6.2

mutation large islet cell nuclei Diffuse
HI
ABCC8 SUR1 Paternally Focal changes with HI-SUR1

inherited mutation adenomatous Focal HI
and loss of hyperplasia/adenomatosis
maternal 11p KATP-
HI
KCNJ11 Kir6.2 Paternally Focal changes with HI-Kir6.2

inherited mutation adenomatous Focal HI
and loss of hyperplasia/adenomatosis
maternal 11p
ABCC8 SUR1 Dominant Anecdotal Large islet cell dominant

mutation nuclei SUR1-HI
KCNJ11 Kir6.2 Dominant No pancreatectomy dominant

mutation Kir6.2-HI
GCK glucokinase Dominant Anecdotal HI-GCK

mutation
Normal or large islet size

and infrequent large islet
cell nuclei
GLUD1 glutamate Dominant Anecdotal HI-

dehydrogenase mutation GLUD1
Hyperammonemia HI/HA
HADH SCHADH Recessive No pancreatectomy HI-
(SCHADH) mutation SCHADH
Not known Dominantly No pancreatectomy EIHI

inherited
Physical exercise
induced
SUR1, sulfonylurea receptor 1; Kir6.2, inward rectifier 6.2; SCHADH, short-chain L-3-hydroxylacyl coenzyme
A dehydrogenase.
The pancreatic histology is characterized by a lesion formed by the confluence of hyperplastic but apparently
normally structured islets occupying greater than 40% of the cross-sectional area of pancreatic lobules (76) (Figure
16-24). The lesion pushes the exocrine elements aside or haphazardly incorporates them. There is recapitulation of
islet structure, with peripherally located A- and D-cells and B-cells aggregating more centrally. Other histologic terms
frequently used and accepted are adenomatosis and adenomatous hyperplasia. In contrast to insulinomas, the
lesions are difficult to identify grossly (Figure 16-24) because they do not distort the normal lobular architecture. The
boundary between the uninvolved portion of the pancreas and the lesion may be sharp (Figure 16-24), but may also
be vague and ill defined (eFigure 16-10). The lesions are generally small, thus their designation as focal HI. In one
series, 24 of 35 lesions were less than 1 cm in the greatest dimension (161). However, the lesion may be multifocal
and/or occupy a large portion of the pancreas to even the entire pancreas (76, 167). The genetic pathogenesis of
the latter is the same as smaller typical lesions, as demonstrated by the loss of expression of p57kip2 (166). In these
rare cases, the designation focal lesion or focal HI causes confusion, yet using the word diffuse is equally
troublesome. A better terminology is being sought and generalized adenomatosis may be an option (76). Cases with
ectopic pancreatic tissue harboring this type of lesions
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have also been described (129,e193). Although there are large nuclei in the confluent islet tissue of the
adenomatous focal lesions, the islets in uninvolved portions of pancreas are reported to have a “resting” appearance
with B-cells showing little cytoplasm and nucleus (135). B-cell nuclear crowding expressed as the number of B-cell
nuclei/1,000 μm2 of B-cell cytoplasm is higher in islets outside the lesion of this focal form as compared to islets of
the diffuse form described above (150). This difference may be subtle and is not appreciated by other retrospective
studies without morphometric measurements (e102,e222,e235).
FIGURE 16-23 ▪ ATP-sensitive potassium channel HI, diffuse form. A: Quantity of endocrine component is not
significantly different from pancreas of normoglycemic individuals of similar age (2 months). (Immunohistochemistry
for insulin. Original magnification ×50.) B: On a low-to-medium power field, a few large and hyperchromatic
endocrine cell nuclei can be spotted. (Hematoxylin and eosin stain, original magnification ×200.). C: Enlarged islet
cell nuclei are defined as those occupying an area at least three times larger than the neighboring endocrine nuclei,
for diagnostic purposes. (Hematoxylin and eosin stain, original magnification ×400.) D: Ductuloinsular aggregates
may be present in some cases, but they are seen too seldom to use as a diagnostic criterion. (Immunohistochemistry
for chromogranin A, original magnification ×200.)
In a limited number of institutions, intraoperative frozen sections are performed to identify patients with the focal form
and further guide the extent of pancreatic resection (135, 167). The presence of islet cell nuclear abnormalities (e.g.,
enlarged more than three times, “bizarre” shaped)
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suggests the recessively inherited diffuse form, and a near-total pancreatectomy follows. The absence of nuclear
changes in islets is indicative of the focal form, and a search for a focal lesion continues until the lesion is identified.
Examples of difficult cases are those with an ill-defined border of the focal form, with generalized adenomatosis, and
with infrequently encountered and/or localized islet cell nuclear abnormality (167). Most recently, preoperative
diagnosis of patients with the focal form is aided by fluorine-18 L-3,4-dihydroxyphenylalanine ([18F]-DOPA) positron
emission tomography (58, 127). This technique localizes the lesion within the pancreas and can detect even an
extrapancreatic ectopic lesion (129,e97).
FIGURE 16-24 ▪ ATP-sensitive potassium channel HI with loss of maternal 11p15 (focal form). A: Much of the lobule
in the right lower half is occupied by endocrine tissue (adenomatosis). (Hematoxylin and eosin stain, original
magnification ×200.) B: Islets outside the adenomatous lesion contain nuclei of normal size. C: There may be large
endocrine cell nuclei within the adenomatosis. (B,C: Hematoxylin and eosin stain, original magnification ×400.) D:
The adenomatous lesion is difficult to distinguish from the neighboring pancreas grossly. E: Immunohistochemistry
confirms the abundance (>40%) of endocrine elements within the lobules. (Immunohistochemistry for chromogranin
A, original magnification ×200.) (D,E: Copyright 2004 from A multidisciplinary approach to the focal form of
congenital HI by partial pancreatectomy by Adzick NS, Thornton PS, Stanley CA, et al. J Pediatr Surg 2004;39:270-
275. Reproduced with permission of Elsevier.)
Hyperinsuminism caused by dominant ATP-sensitive potassium channel mutations: Rare dominantly expressed
ABCC8 and KCNJ11 mutations have been described (72,e142,e241)
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with a milder clinical presentation. Pancreatic pathology is anecdotally reported to be similar to the recessively
inherited form (72,e241).
Hyperinsulinism Caused by Defects of Other Genes
Abnormalities in three other genes are associated with generally milder forms of HI that usually respond to medical
therapy with diazoxide. Patients with these disorders tend not to have their pancreas resected, and therefore,
histologic descriptions are scarce.
Glucokinase, a hexokinase with a low affinity for glucose, controls the rate-limiting step of B-cell glucose metabolism
and is responsible for glucose-mediated regulation of insulin secretion. The gene, GCK, is at 7p13-15. The enzyme
with a gain-of-function mutation has a higher affinity for glucose, so that glycolysis and inappropriate insulin
secretion take place at relatively low blood concentration of glucose (52). Several dominantly inherited mutations of
the glucokinase gene have been described with variable clinical presentations (34). Some are mild and can be
controlled by diazoxide while others may present with extremely severe HI that cannot be managed by diazoxide.
Pathologic descriptions remain anecdotal. The pancreas of one case was reported to be normal (e75) while a
systematic study of another case demonstrated moderately enlarged islet cell nuclei and increased average size of
islet profiles compared to the control and cases caused by recessive ATP-sensitive potassium channel mutations
(30). Of note, mutations in glucokinase that decrease enzymatic activity result in MODY 2 (46) as mentioned earlier
in the chapter.
Another autosomal dominant form of HI is caused by gain-of-function mutations of the glutamate dehydrogenase
gene, GLUD1 (160), located at 10q23.3. Glutamate dehydrogenase is a mitochondrial enzyme, and catalyzes the
reaction converting glutamate to a-ketoglutarate, a substrate for the TCA cycle. This form of HI is known as the
HI/HA and is distinguished by persistently elevated plasma ammonia concentrations to three to five times normal, as
a result of the enzymatic abnormality being expressed in the liver as well as in the pancreas (e226). A
pancreatectomy specimen has been described (not illustrated) as showing “unusual islet cells arranged in ribbon
pattern (islet cell dysplasia)” (187).
The most recently described metabolic abnormality resulting in HI is short-chain L-3-hydroxylacyl coenzyme A
dehydrogenase (SCHADH) deficiency (27,e162). Each proband had a homozygous mutation, and the patients were
medically managed.
There is an additional form of dominant HI that is physical exercise induced (126,e156,e157). Two families, Finnish
and German, have been reported. The patients have abnormal insulin response to infusion of pyruvate, but the
specific defect has not been elucidated. Sequence analysis of genes encoding monocarboxylate transporters did not
identify sequence variants that cosegregate with the phenotype in the families.
Adenoma
Adenomas are rare in the pediatric population (Table 16-2). When HI manifests as a noncongenital manner after 6 to
12 months of age, an insulinoma needs to be considered. Adenomas are generally well demarcated (Figure 16-25),
and differ from the lesions of adenomatosis in that they do not have intermixed acinar elements and do not
recapitulate mini-islets. An adenoma does not contain all the cell types in normal proportions, although more than
one cell type may be represented. Most lesions previously described as adenomas in the pediatric literature
represent adenomatosis (adenomatous hyperplasia) when the illustrations are critically reviewed (e29).
Hyperinsulinism and Nesidioblastosis

To explain the origin of islet cell tumors of the pancreas, Laidlaw referred to the continuity of duct epithelium and islet
cells observed in normal pancreas, speculating islet tumor formation to be an exaggeration of normal processes
involving totipotent “nesidioblasts” (e131). He proposed a condition, termed nesidioblastosis, wherein islet cell
proliferation from ducts becomes disseminated, as the putative cause for hypoglycemia. Yakovac investigated the
histopathology of then idiopathic hypoglycemia of infancy and reported that a defining feature was B-cell
nesidioblastosis (e269). Nesidioblastosis was thereafter used to support the hypothesis that neonatal hypoglycemia-
HI is a developmental problem. The word nesidioblastosis subsequently acquired a clinical connotation essentially
equating it to the disorder presenting with persistent hypoglycemia, particularly in neonates (114). The term has also
been used frequently to describe the diverse histopathology found among pancreata obtained from patients with HI.
Microscopic anatomy included in nesidioblastosis has been the presence of endocrine cells closely associated with
ducts, small clusters of endocrine cells scattered throughout the exocrine pancreas, large islets with or without
hypertrophic islet cell nuclei, and a proposed but not proven increase in endocrine cell mass. As a result, it has
become increasingly difficult to ascribe specific meaning when authors provide statements such as “the pancreas
showed nesidioblastosis.” Other terms, for example, islet cell dysplasia and nesidiodysplasia, were subsequently put
forward to compromise over objections raised in the use of nesidioblastosis, which is seen in normal developing
pancreas. Pathologists who recognized the frequent association of endocrine cells and ducts in fetal and pediatric
material were among the first to challenge conventional wisdom regarding the pathologic basis of congenital HI
(76,e195,e262). Then followed endocrinologists, biochemists, and geneticists, who further delineated an
understanding that HI is a functional defect in insulin regulation, one not simply due to islet development. Several
genetic abnormalities are now described underlying congenital HI, and the pancreatic histology varies according to
these alterations and subtypes of HI. The use of nesidioblastosis as a histologic finding in HI is therefore confusing,
and it does not provide or characterize distinct anatomic or
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clinical information. Moreover, lumping various histologic changes to a single term hinders objective description of
pathology, which is the key in recognizing different pathological processes.
FIGURE 16-25 ▪ Islet cell adenoma from a 10-year-old boy. The tumor is composed of a monotonous population of
endocrine cells arranged in trabeculae and cords, and has a relatively sharp border. (A,B: Hematoxylin and eosin
stain, original magnification ×25 and ×200, respectively.)
Pancreatic Islets in Shock

Bernstein (e22) described three newborns dying shortly after birth in whom renal tubular and selective pancreatic
islet necrosis was found. Asphyxia was implicated. Seemayer et al. (146) found the same pattern in only 10% of
infants with other severe manifestations of shock.
Viral Infections
Nonselective involvement of the pancreas is seen in disseminated herpesvirus, cytomegalovirus, varicella-zoster
virus, and rubella virus infections (78). Selective damage to islet cells has been seen in coxsackievirus B infection
(78,e73), although caution should be exercised in distinguishing viral effects from the changes of shock, described
earlier. The onset of diabetes after coxsackievirus B infection has been documented, although direct evidence for
virally induced diabetes is lacking (e271).
Malformation Syndromes
Beckwith-Wiedemann Syndrome
Beckwith-Wiedemann syndrome is a congenital overgrowth syndrome that is clinically and genetically
heterogeneous. A number of complex genetic and epigenetic abnormalities resulting in dysregulation of imprinted
growth regulatory genes clustered at 11p15 have been demonstrated (38,e140). Phenotypical features include
macrosomia, macroglossia, omphalocele, visceromegaly (80), and, in about one-third to half of cases, hypoglycemia
that is attributed to HI (37, 43). The hypoglycemia is transient in the majority of infants and resolves within the first
few days of life. In about 5% of children, the HI persists and extends beyond the neonatal period, requiring either
continuous feeding, medical therapy, or partial pancreatectomy in rare cases.
The available pancreatic histology is, therefore, usually limited to the severe cases in which the patient has died or
had partial pancreatectomy. The pancreatic parenchyma is composed of small endocrine cell clusters, well-formed
islets, and large, confluent, and complex islet-like aggregates of endocrine cells in a background of a relatively
narrow rim of exocrine acini (77) (Figure 16-26). The endocrine cells often show large cytoplasm and large nuclei.
Focal areas of necrosis may be found in the larger endocrine nodules. When immunohistochemical reaction is
applied, the isletlike aggregates recapitulate islet topography with the insulin-positive B-cells residing in the center
and the non-B-cell being at the periphery of the “macroislets” (e228) (eFigure 16-11). A lack of segregation of
pancreatic polypeptide-rich islets to the head of pancreas (ventral pancreas origin) has been described. In a
Beckwith-Wiedemann patient with a Meckel diverticulum, the heterotopic pancreas within the diverticulum showed
numerous enlarged islets or isletlike aggregates, some with a diameter of up to 1,600 μm,
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comprising approximately 15% of the pancreatic tissue (e209). The abundance of endocrine tissue forming irregular
nodules and aggregates is reminiscent of the appearances seen in adenomatous hyperplasia (adenomatosis, focal
HI) associated with paternally inherited ATP-sensitive potassium channel mutations together with loss of maternal
11p. The difference is, however, that the abnormality is present throughout the pancreas in Beckwith-Wiedemann
syndrome (Figure 16-26). In one case with mosaic paternal uniparental disomy for 11p15, p57kip2 protein was readily
identified within the large islets, which is in contrast to the loss of p57kip2 expression in the B-cells within the
adenomatous lesions of focal HI (73).
FIGURE 16-26 ▪ Beckwith-Wiedemann syndrome. A,B: Much of the pancreatic lobule is formed of complex islet-like
aggregates of endocrine cells. Exocrine acini are poorly developed. (A: Hematoxylin and eosin stain, B:
Immunohistochemistry for chromogranin A, original magnification ×100.)
A pancreas examined at 11 months of age at the time of death of a patient with Beckwith-Wiedemann syndrome
shows significantly more acinar differentiation and proliferation as compared to the partial pancreatectomy specimen
at one month of age (47). Another report by Sotelo-Avila and Gooch (e223) describes islet cell hyperplasia in five
children who died of their disease-associated tumors, even though the earlier hypoglycemia had been transient.
Steigman et al. (162) reported a 2-day-old autopsy case with an enlarged and solely cystic pancreas containing
numerous irregularly shaped ectatic ducts with sparse islands of endocrine tissue and exocrine acini (Figure 16-8).
The pancreatic histology seen in Beckwith-Wiedemann syndrome may not be uniform as the underlying genetic and
epigenetic abnormalities are highly variable.
Beckwith-Wiedemann syndrome with hemihypertrophy is associated with a striking tendency toward the
development of embryonal tumors in a number of organs, and pancreatoblastoma is one of them (41,e123,e189).
Perlman Syndrome
Beckwith-Wiedemann syndrome and the syndrome of renal hamartomas, nephroblastomatosis, and fetal gigantism
overlap to some degree. One-half of the cases are said to have islet cell hyperplasia (62,e183), hypoglycemia occurs
(e80), and HI may be responsible. We have seen a large pancreas associated with Perlman syndrome (Figure 16-
27).
Wilcott-Rallison Syndrome
Wilcott-Rallison syndrome is an autosomal recessive disorder that is characterized by permanent neonatal insulin
requiring diabetes mellitus and multiple epiphyseal dysplasia (e263). Other features include osteopenia, mental and
growth retardation, hepatic and kidney dysfunction, cardiac abnormalities, exocrine pancreatic dysfunction, and
neutropenia (42,e214). The syndrome results from mutations in the gene encoding the eukaryotic initiation factor 2-a
kinase 3 (EIF2AK3, also called PERK) (18, 39). The transmembrane kinase EIF2AK3 is localized in the endoplasmic
reticulum and phosphorylates EIF2A (e217), preventing B-cell death and relieve endoplasmic reticulum stress by
reducing the number of unfolded proteins in the endoplasmic reticulum (198,e126). Autopsy of one case revealed a
markedly hypoplastic pancreas with only a narrow cord of tissue (176). Histology showed a reduction of acinar tissue
and increased
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interstitial fibrosis. The islets appeared prominent with more glucagon staining cells than insulin staining cells.
FIGURE 16-27 ▪ Perlman syndrome. A: An 11-day-old infant delivered at 30 weeks of gestation presented with
hypoglycemia and constellations of malformations consistent with Perlman syndrome. The pancreas was large and
weighed 20 g. (Courtesy of Ralph A. Franciosi, M.D., Milwaukee, Wisconsin.) B: The pancreatic lobules appear
disorganized, and are composed of irregularly shaped cords and islands of endocrine cells and poorly developed
acini.
Leprechaunism
Donohue (e54) described infants with a characteristic facies, hirsutism, enlarged genitalia, decreased muscle and
subcutaneous tissues, and “dysendocrinism.” An autosomal recessive defect in the insulin receptor gene (INSR) has
been documented in some patients (44), and leprechaunism is listed in the recent diabetes classification under
genetic defects in insulin action (48). On the other hand, intermittent hypoglycemia with HI has been described, and
in a selective review of the literature, Rosenberg et al. (140) found that islet hyperplasia was reported in 67% of the
cases at autopsy. An unusual case described by Szilagyi et al. (e238) had the features of lipomatous pseudoatrophy
with preserved islets and is mentioned earlier in the chapter.
Other Disorders Affecting Endocrine Pancreas

Cystic Fibrosis
In the pancreas, cystic fibrosis primarily affects the exocrine component causing pancreatic insufficiency. However,
diabetes mellitus has been recognized as a complication that commonly develops around 20 years of age. The
prevalence increases with age, and, with improved survival and prospective screening by glucose tolerance test,
approaches to 30% (107,e133,e272).
Several studies have been published focusing on islet changes in cystic fibrosis (106,157,e1,e98,e125). Although
qualitative and quantitative methods differed among the studies and the pathology was always accompanied by
exocrine and interstitial alterations, a decrease in the fraction of insulin-positive B-cells in islets was generally
demonstrated in advanced cystic fibrosis. A “qualitative” islet number (e98) and the volume density of endocrine
tissue to pancreatic tissue (106) were decreased in cystic fibrosis patients as compared to the control groups.
Endocrine cell composition was not significantly different between pancreas showing predominantly fibrotic pattern
and lipoatrophic pattern (106).
In diabetic young adults, islets were described as having a disorganized and lobulated appearance with thin fibrous
septa enclosing the capillaries and subdividing the islets (157). Large amount of amyloid deposition was also
demonstrated (29).
In contrast, an early endocrine increase has been also mentioned (e125). Multiple foci of neoformation of islets
illustrated by islet cells arising from and around the ductal lumen were reported in all 11cystic fibrosis patients (age:
3 months to 7 years) as compared to less frequent encounters in the control subjects (e31). Neoislet formation from
a small duct was present in nondiabetic children, but not in nondiabetic and diabetic young adults in a different
series (e98).
The morphologic alterations may provide the basis for the glucose intolerance and overt diabetes mellitus eventually
developing in some with cystic fibrosis. However, not all patients with advanced cystic fibrosis become diabetic.
Other late complications of cystic fibrosis such as liver damage and peripheral insulin resistance might contribute to
the changes in glucose metabolism seen in cystic fibrosis patients (106, 177).
Hereditary Tyrosinemia Type I

Tyrosinemia may be associated with glycosuria and refractory hypoglycemia. The pancreas in some infants has
been shown to contain many large islets (e184,e219), but this is not a constant feature in this disease (143). The
variability
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likely comes from the difficulty in accurately assessing islet hypertrophy in infant (true increase in the percentage of
islets larger than 200 mm for a given age). Mitotic activity within islets and hyalinization of islets have been seen in
some cases (e86).
Ataxia-Telangiectasia
The familial disease with cerebellar ataxia, oculocutaneous telangiectasia, and immune disorder with IgA deficiency
is associated with insulin-resistant diabetes mellitus (25). Islet cell hyperplasia may be impressive; however, the
marked nuclear cytomegaly is not confined to islets but is a systemic manifestation of the disease (e14).
Sudden Infant Death Syndrome (SIDS)

In a retrospective review of infants with SIDS, examination of the pancreas did not divulge endocrine pathology
(e143,e169). Hisaoka et al. (e92) claimed to have found “endocrine cell dysplasia” in 2 of 15 infants with SIDS, but
no glucose or insulin determinations were available to support an etiologic connection. In a review of 112 pancreata
from victims of sudden infant death, Klensang et al. (89) found no morphologic or morphometric differences between
them and 19 controls. It is not unreasonable to assume that some infants or even older children with HI may present
as instances of sudden death (6), but the diagnosis must rely on laboratory documentation of the HI. The
morphologic features of normal infant pancreas may appear abnormal or similar to a form of HI if qualitative and
quantitative differences from the adult pancreas are not taken into consideration (55, 77).
Endocrine Tumors in Childhood

Most of the descriptions of endocrine adenomas in childhood appear to represent adenomatosis (76,e29,e121). True
adenomas (Figure 16-25) have traditionally been classified according to the hormone produced. Adenomas may be
part of the multiple endocrine neoplasia type I syndrome; even in this syndrome, functioning adenomas of the
pancreas are unusual in childhood. Adenomas that produce gastrin and the Zollinger-Ellison syndrome are described
in the adolescent pancreas (e259). Carney et al. (e35) described the occurrence of pheochromocytoma(s) or
pancreatic islet cell tumor(s) or both, in two or more members of three unrelated families with a pattern consistent
with autosomal dominant inheritance, and suggested a syndrome different from multiple endocrine neoplasia.
Another 18-year-old with pheochromocytoma and a nonfunctioning islet cell adenoma of the pancreas has also been
described (e273). Some of these patients may have had von Hippel-Lindau disease, which is associated with islet
cell tumors, adenomatosis, pancreatic cysts and, in some instances, pheochromocytoma (122,e96,e191).
Malignant islet cell tumors are diagnosed on the basis of distant metastasis to distinguish them from multiple
adenomas. Rare examples in childhood have been noted: a metastasizing insulinoma in a 14-year-old (e266) and a
corticotropin-producing tumor of the head of the pancreas (e210). Judson et al. (82) reported a well-differentiated
pancreatic endocrine neoplasm of an 18-year-old that metastasized to the breast with intraductal spread. Overall,
malignant, metastasizing tumors of the pediatric endocrine pancreas are extremely uncommon (e266).
ACKNOWLEDGMENT
The author wishes to acknowledge the significant contribution to this chapter as portions were adapted from the
previous edition, authored by Dr. Ronald Jaffe, Professor, University of Pittsburgh School of Medicine, and former
Pathologist-in-Chief, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania. His generosity in providing
additional illustrative materials for figures, constructive advice, and encouragement is deeply appreciated.
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Chapter 17
The Kidney and Lower Urinary Tract
Aliya N. Husain
Theodore J. Pysher
Rapid advances in the field of genetics and molecular biology are leading to a better understanding of normal
embryology, congenital malformations, glomerular and tubulointerstitial diseases, and neoplasia of the kidney
and lower urinary tract. Approximately one-third of all congenital malformations are found in the urogenital
system, many of which are part of complex multisystem anomalies with cumulative effects that are lethal in the
neonatal period (e33,e35,e37,e88). Almost 80% of congenital uropathies seen in second-trimester fetuses are
associated with other anomalies—both chromosomal and nonchromosomal, either syndromic or in casual
combination (e53). Malformations of the bladder are often accompanied by major anomalies of the male and
female genital tract because of the inter-related embryologic development of these organ systems. Glomerular
diseases, reflux nephropathy, and infections are important causes of morbidity in childhood. Although cancer of
the kidney is relatively uncommon in the pediatric age group, 5-year survival from Wilms tumor has increased
from 73% in patients diagnosed in 1975 to 1977 to 92% in the period 1996 to 2002 (81), thus establishing a
successful model for national multicenter study groups.
EMBRYOLOGY
Functionally, the urinary and the genital systems can be divided into two entirely separate systems; however,
embryologically and anatomically they are intimately interwoven. Both develop from a common mesodermal ridge
(intermediate mesoderm) along the posterior wall of the abdominal cavity, and initially the excretory ducts of both
systems enter a common cavity, the cloaca. In humans, three separate but overlapping renal systems form. The
pronephros, which is the most caudal and nonfunctional, regresses completely by the end of the 4th week of
gestation, during which time the first excretory tubules of the mesonephros appear that may function for a short
period. While the caudal tubules are still differentiating, the cranial tubules and glomeruli show degenerative
changes, and by the end of the second month, most have disappeared. In the male, a few of the caudal tubules
and the mesonephric duct persist and participate in the formation of the genital system, but they disappear in the
female, leaving a few vestigial structures only (156).
The metanephros, or permanent kidney, appears in the fifth week at the level of the upper sacral segment, with
its blood supply coming from the lateral sacral branches of the aorta. By the eighth week, it “ascends” to the
lumbar region, mainly secondary to differential growth of the embryo, and derives its blood supply from
progressively higher levels of the aorta. In the pelvic ectopic kidney, the renal arteries arise from a lower level of
the aorta or from the iliac arteries. The nephrons develop from the caudal end of the nephrogenic cord (now
termed the metanephric blastema), while the renal excretory system (collecting duct, calyces, pelvis, and ureter)
develops from the ureteric bud, which is an outgrowth of the mesonephric duct close to its entrance into the
cloaca. The proximal tip or the ampulla of the ureteric bud grows dorsally and cranially, pushes the metanephric
blastema, and undergoes a series of dichotomous branching, the ampulla of each of which ultimately induces
nephron formation. Each division proceeds more rapidly at the poles, so that the kidney acquires its
characteristic shape. The first few generations of branches coalesce to form the renal pelvis and calyces (e240).
Nephrons form from condensation of the metanephric blastema, which develops a cyst-like cavity, elongates,
and folds back to become S-shaped. One end fuses with the ampulla that induced it, while at the other end a
mesh of capillaries develops and invaginates the nephrogenic vesicle to form a glomerulus. The upper and
middle limbs of the nephrogenic vesicle elongate and differentiate into the proximal and distal convoluted tubules
and the loop of Henle.
The process of nephrogenesis can be divided into four periods (e240,e241). From 7 to 14 weeks of gestation,
the ureteric bud branches dichotomously for six to eight generations, with each branch inducing the formation of
a new nephron. From 14 to 22 weeks, nephron arcades are formed, with the innermost nephron formed first
(juxtamedullary nephron) (eFigure 17-1). From 22 to 36 weeks, no branching of the ureteric bud occurs. The
ampulla extends to the subcapsular cortex to induce four to seven nephrons (eFigure 17-2). Thus, the nephrons
formed last are subcapsular (the nephrogenic zone seen in sections of fetal kidneys)
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(Figure 17-1). From 36 weeks of gestation through birth and up to maturity, the nephrons grow, but no new
nephrons are formed. In extremely premature infants, nephrogenesis continues after birth until the kidney
reaches maturity.
FIGURE 17-1 ▪ Early third-trimester kidney with subcapsular nephrogenic zone. (Hematoxylin and eosin stain,
Evidence from studies by Potter (e251) indicates that nephrons in the developing metanephros may begin
functioning as early as the eleventh or 12th week after conception. In fact, it has been suggested that the
formation of a tubule fluid is essential to ensure the normal development of the renal pelvis and calyces.
Molecular Regulation of Kidney Development

As with most organs, differentiation of the kidney involves epithelial-mesenchymal interactions. Epithelium of the
ureteric bud from the mesonephros interacts with mesenchyme of the metanephric blastema. The mesenchyme
expresses WT-1, a transcription factor that makes this tissue competent to respond to induction by the ureteric
bud. WT-1 also regulates production of glial-derived neurotrophic factor (GDNF) and hepatocyte growth factor
(HGF or scatter factor) by the mesenchyme, and these proteins stimulate branching and growth of the ureteric
buds. The tyrosine kinase receptors RET, for GDNF, and MET, for HGF, are synthesized by the epithelium of
the ureteric buds, establishing signaling pathways between the two tissues. In turn, the buds induce the
mesenchyme via fibroblast growth factor 2 and bone morphogenetic protein 7. Both these growth factors block
apoptosis and stimulate proliferation in the metanephric mesenchyme while maintaining production of WT1.
Conversion of the mesenchyme to an epithelium for the nephron formation is also mediated by the ureteric buds,
in part through modification of the extracellular matrix. Thus, fibronectin, collagen I, and collagen III are replaced
with laminin and type IV collagen, characteristic of an epithelial basal lamina. In addition, the cell adhesion
molecules, syndecan and E-cadherin, which are essential for condensation of the mesenchyme into an
epithelium, are synthesized. Regulatory genes for conversion of the mesenchyme into an epithelium appear to
involve PAX2 and WNT4 (156).
Table 17-1 ▪ RENAL MALFORMATIONS SEEN IN PEDIATRIC AUTOPSIES
No. of Cases
Anomaly Series 1* Series 2+ Total (%)
Renal agenesis, bilateral 16 13 29 (12)
Renal agenesis, unilateral 10 6 16 (6.6)
Renal dysplasia, bilateral 45 33 78 (32)
Renal dysplasia, unilateral 4 5 9 (2.1)
Renal dysplasia, unilateral, with contralateral renal agenesis 9 3 12 (5)
Autosomal recessive polycystic kidney disease 5 10 15 (6.2)
Autosomal dominant polycystic kidney disease 1 1 2 (0.8)
Renal fusion 20 12 32 (13.2)
Renal ectopia 4 1 5 (2.1)
Congenital hydronephrosis, bilateral 6 5 11 (4.5)
Congenital hydronephrosis, unilateral 4 8 12 (5)
Ureteral duplication 10 5 15 (6.2)
Renal hypoplasia 1 3 4 (1.7)
Other 0 6 6 (2.5)
Total 135 111 246 (∽100)
Series 1 * compiled from 1,442 consecutive autopsies performed at Minneapolis Children's Medical
Center from 1977 to 1987, including stillborn nfants and children younger than 1 year of age.
Series 2+ compiled from 1,242 pediatric autopsies performed at Loyola University Medical Center from
1978 to 1998, including stillbirths and children up to 18 years (Unpublished data from Aliya N. Husain,
M.D.).
CONGENITAL MALFORMATIONS OF THE KIDNEY

If all malformations are considered, ranging from incidental findings with no clinical significance to major lethal
anomalies, it is estimated that congenital abnormalities of the kidney and urinary tract are present in 10% of all
newborns (93, 98). Worldwide, a substantial percentage of children develop chronic kidney disease early in life,
with congenital renal disorders such as obstructive uropathy and aplasia/hypoplasia/dysplasia being responsible
for almost one-half of all cases (188). Table 17-1 lists the relative frequency of
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malformations seen in two series of pediatric autopsies—one from a children's hospital and the other from a
tertiary care university medical center. Forty-one (38%) of the 107 renal malformations in series No. 2 were
associated with major malformations of at least one other organ system.
FIGURE 17-2 ▪ Oligohydramnios (Potter sequence) is associated with lowset and deformed ears, beaked nose,
receding chin, and lower limb positional deformity.
A wide variety of renal malformations result in the oligohydramnios (Potter) sequence (i.e., characteristic facies,
including low-set ears, beaked nose, prominent epicanthic folds, receding chin, limb deformities, growth
retardation, and pulmonary hypoplasia) (Figure 17-2). These abnormalities are the result of decreased amniotic
fluid rather than renal malformations per se. When these findings are associated with renal agenesis, the term
Potter syndrome (as initially described by Potter in 1946) is used. Renal findings in children with oligohydramnios
sequence are listed in Table 17-2. It has become clear that this sequence can result from even a relatively short
duration of oligohydramnios, including persistent leakage of amniotic fluid (e147).
Table 17-2 ▪ RENAL FINDINGS IN CHILDREN WITH OLIGOHYDRAMNIOS SEQUENCE
No. of Cases
Renal Abnormality Series 1 Series 2 Total (%)
Bilateral renal agenesis 16 32 48 (30)
Bilateral cystic dysplasia 17 30 47 (29)
Unilateral agenesis with contralateral dysplasia 9 8 17 (10)
Obstructive uropathy 13 — 13 (8)
Autosomal recessive polycystic kidney disease 4 2 6 (4)
Renal ectopia and fusion 1 1 2 (1)
Autosomal dominant polycystic kidney disease 1 1 2 (1)
Other 13 13 26 (16)
Total 74 87 161 (∽100)
Most urogenital abnormalities are now diagnosed antenatally on high-resolution ultrasound scans. This has
enabled recognition of those that are not compatible with survival and these can be managed with termination of
pregnancy (82). Congenital anomalies of the kidney and urinary tract are responsible for approximately 40% of
cases of childhood end-stage renal failure in the United States (150). The classification of congenital and
developmental anomalies of the kidney given in Table 17-3 includes cystic diseases because many of these are
inheritable disorders or are secondary to malformations of the kidney parenchyma and lower urinary tract (20).
Renal Ectopia
Permanent malposition of one or both kidneys is seen in 2% of pediatric autopsies (Table 17-1). The incidence is
even higher in perinatal autopsies because renal ectopia is commonly associated with multiple other
malformations. The ectopic kidney(s) may be located in the pelvis (most common), on the other side (crossed
renal ectopia) with or without fusion, or even in the thorax (rare) (e223,177). Prenatal ultrasonographic diagnosis
of pelvic kidney is possible, usually after 24 weeks of gestation (e210). Although renal function is normal in the
neonatal period in patients with renal ectopia without other associated malformations, hydronephrosis eventually
develops in 56% secondary to obstruction, reflux, or malrotation (28). Pseudocrossed renal ectopia occurs when
an enlarging retroperitoneal mass displaces the kidney to the contralateral side of the abdomen.
Renal Fusion
Renal fusion, often with ectopia, was seen in 32 (1.2%) of 2,684 pediatric autopsies (Table 17-1). The most
common fusion anomaly is horseshoe kidney, in which both kidneys are normally lateralized but have fused
lower poles (Figure 17-3) and are located in a lower than normal position. The incidence of horseshoe kidney is
reported to be 1 in 600 in the general population (156). One-third of persons with this
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condition have associated congenital malformations of other organs, including Turner syndrome (18, 33, 68);
two-third have a major urologic complication, most of which require surgery, although newer techniques such as
laparoscopic robotic-assisted management allow for minimally invasive procedures (31). Individuals with
horseshoe kidney are at higher risk for the development of stones (56, 61) and tumors (50, 121, 161), including
carcinoids (151). The association of extrarenal Wilms tumor (WT) with horseshoe kidney has led to the theory
that there is a nexus between the fusion of the metanephric blastema during weeks 6 and 7 of intrauterine life
and that “ectopic” metanephric blastema cells may give rise to extrarenal Wilms tumor (e163). Rare cases of
renaladrenal fusion have been described, which may present as a renal mass in the upper pole (49).
Table 17-3 ▪ CLASSIFICATION OF MALFORMATIONS OF THE KIDNEY, INCLUDING CYSTIC

DISEASES
I. Renal position and form
A. Ectopia
B. Fusion
II. Renal quantity
A. Agenesis (bilateral, unilateral)
B. Hypoplasia
C. Renal tubular dysgenesis
D. Renomegaly
E. Duplication
F. Supernumerary
III. Hydronephrosis
IV. Renal dysplasia/cystic diseases (gross and/or microscopic)
A. Renal dysplasia
1. Sporadic (bilateral, unilateral)
2. Hereditary
3. With malformation syndromes
B. Polycystic kidney disease
1. Autosomal recessive (infantile type)

2. Autosomal dominant (adult type)
C. Medullary cysts
1. Medullary sponge kidney
2. Medullary cystic diseases
D. Cortical cysts
1. Glomerulocystic disease (s)
2. Simple cysts
3. Microcysts associated with syndromes
E. Renal cysts with hereditary syndromes
Renal Agenesis/Hypoplasia
Inadequate renal tissue can be considered as a continuum, ranging from renal agenesis to subtle congenital
nephron deficits. Renal agenesis (i.e., the complete absence of one or both kidneys) is commonly accompanied
by other malformations of the genitourinary tract and various lower body defects, which has led to the theory that
it is part of a field defect (e250). Although the exact cause of human renal agenesis/hypoplasia remains
unknown, recent literature supports that one or more genetic mutations result in molecular dysregulation of
nephrogenesis. Homozygous null mice for c-Ret (e276), Gdnf (e215,e248,e270), and Gfrα-1 (e49) all exhibit
bilateral renal agenesis due to the inhibition of ureteric bud growth and branching morphogenesis. Pax2 plays an
integral role in the initiation and maintenance of the Ret/Gdnf pathway by not only activating the ligand of the
pathway, but by also enhancing the expression of the pathway receptor Ret (36). Since an exhaustive review is
beyond the scope of this chapter, one can focus on Pax2, one of the earliest genes expressed widely during fetal
kidney development in the nephric duct, the metanephric mesenchyme, the ureteric bud, and in the S-shaped
body. Early failure in the first two developmental stages (e.g., homozygous inactivation of Pax2) precludes
formation of metanephric kidneys and causes bilateral renal agenesis, incompatible with life. Interference with
the later stages affects the extent of branching morphogenesis (e.g., heterozygous Pax2 mutations). Although the
resulting nephron deficits are compatible with life, they may be moderately severe and account for up to 40% of
the children in dialysis and transplant units around the world. Finally, the effect of Pax2 on apoptosis in the
branching ureteric bud seems to imply a quantitative process that is finely tuned. Modest changes in this
program could account for subtle nephron deficits in “normal” humans and increased risk of hypertension or
susceptibility to acquired renal disease later in life (44, 139).
FIGURE 17-3 ▪ Horseshoe kidney with fused lower poles.
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Bilateral Renal Agenesis
Uniformly fatal, bilateral renal agenesis, although less common than unilateral renal agenesis (URA), is seen
more frequently in pediatric autopsies (1.1% of total autopsies in Table 17-1). The incidence of bilateral renal
agenesis varies from 0.1/1,000 to 0.3/1,000 births (e310). It accounts for one-third of births with the
oligohydramnios sequence (Table 17-2). The male-to-female ratio is 2.5:1. It is usually associated with severe
oligohydramnios (Potter syndrome), intrauterine growth restriction, extrarenal anomalies, and malpresentation.
The ureters and renal arteries are also absent, and the urinary bladder is hypoplastic or absent. The adrenals
are disc-shaped secondary to lack of molding from the kidneys (eFigure 17-3). Forty percent of affected infants
are stillborn, and the remainder die in the immediate postnatal period, generally of pulmonary hypoplasia (e249).
Bilateral renal agenesis is usually sporadic, although familial cases have been described (e228,e263,e274).
Hereditary renal adysplasia (agenesis/dysplasia syndrome) is manifested as various combinations of unilateral or
bilateral agenesis, unilateral or bilateral renal dysplasia, or dysplasia of one kidney and agenesis of the other, for
which autosomal dominant inheritance with varying expression has been suggested (e44,e213). An increased
prevalence of congenital renal anomalies was identified in the relatives of index patients with bilateral renal
agenesis/adysplasia (14.7%) compared to controls (2.2%), with a recurrence risk of 6.2 for first-degree relatives
(163).
Other reported malformations associated with bilateral renal agenesis include congenital pulmonary airway
malformation type 2 (cystic adenomatoid malformation), leftheart hypoplasia (e50), sirenomelia (3), and urorectal
septum malformation sequence (e325). A case of sirenomelia, limb reduction defects, cardiovascular
malformations, and renal agenesis has been reported in a fetus born to an insulin-dependent diabetic mother
(e197).
Bilateral renal agenesis has been described in mice homozygous for a trap mutation in the gene encoding
heparan sulfate 2-sulfotransferase (Hs2st) (e45). Analysis of kidney development in Hs2st mutants reveals that
the gene is not required for two early events—ureteric bud outgrowth from the Wolffian duct and initial induction
of Pax2 expression in the metanephric mesenchyme. It is required, however, for mesenchymal condensation
around the ureteric bud and initiation of branching morphogenesis. It is possible that the Hs2st mutant phenotype
is a consequence of compromised interactions between growth factors and their signal transducing receptors.
Unilateral Renal Agenesis

URA is a common developmental defect in humans, occurring at a frequency of approximately 1 in 500 to 1,000
births (168). Although compatible with normal life, URA is still seen twice as often in pediatric autopsies (0.6%)
than in the general population (0.3%) owing to its frequent association with other complex malformations (e266).
The male-tofemale ratio is about 1:1. Long-term follow-up of patients with URA has shown that these patients are
at higher risk for the development of proteinuria, hypertension, and renal insufficiency (e8).
Malformations of the genitalia are commonly associated with URA. These include ipsilateral absence of fallopian
tube, unicornuate and bicornuate uterus (e134), cysts of the epididymis and seminal vesicle (e206), agenesis of
vas deferens, cystic dysplasia of testis (e330) and rete testis (e326), ectopia of vas deferens (e75), and urorectal
septum malformation sequence (ambiguous genitalia with absence of perineal and anal openings) (e325). Single
cases of various chromosomal anomalies have been reported in patients with URA associated with complex
anomalies (e79, e 84,e175,e327,e328).
Cystic dysplasia of the testis appears to be associated consistently with renal malformations, most frequently
ipsilateral renal agenesis; both conditions could be explained by failure of development of the ureteric bud (e40).
Blind-ending ureteric bud remnant has been described in association with URA and renal dysplasia (e198).
Renal Hypoplasia
Renal hypoplasia, defined as histologically normal kidneys with a weight that is less than two standard deviations
below the norm, is extremely rare. In the older literature, any small kidney was labeled hypoplastic, irrespective
of the cause. Currently, small kidneys that are also dysplastic are considered with the dysplastic group, and
those with scarring, inflammation, and hypertensive changes are end-stage kidneys assigned to the “underlying
disease” category. Segmental renal hypoplasia (so-called Ask-Upmark kidney), which may be unilateral or
bilateral and is characterized by localized atrophic scarring, is now thought to be secondary to vesicoureteral
reflux and a form of reflux nephropathy.
In true renal hypoplasia, the absolute number of nephrons is reduced, possibly as a consequence of inadequate
branching of the ureteric ducts that results in a decreased number (<5) of reniculi (e28,e34); however, the renal
shape is normal. Unilateral hypoplasia is a sporadic condition, only rarely associated with lower urinary tract
anomalies; patients present with hypertension and are predisposed to reflux nephropathy (e28). Bilateral
hypoplasia results in renal insufficiency and early death in severe cases; less severe cases manifest growth
retardation, chronic renal insufficiency, and mental retardation (e28).
Bilateral oligomeganephronic renal hypoplasia is a nonfamilial form of congenitally small kidneys characterized
by slowly progressive renal insufficiency. The absolute number of nephrons is reduced. The glomeruli and
tubules that are present are larger than normal. Infection, dysplasia, and obstructive uropathy are absent.
Although several causes, including toxic factors, renal infection, vascular insufficiency, and disseminated
intravascular coagulation,
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have been mentioned, it is not known what factors arrest the development of the metanephric renal blastema,
presumably between weeks 14 and 20 of fetal life (e43). Abnormalities of chromosome 4 (e5) and mutations in
hepatocyte nuclear factor-1beta (HNF-1beta) (157) and PAX2 (158) have been reported in patients with
oligomeganephronia.
Significantly decreased glomerular number without decreased renal weight, thought to be due to impaired renal
development in utero, has been reported in adult patients with primary hypertension (86). Considering how
common primary hypertension is worldwide, this form of renal “hypoplasia” may be the most frequent renal
“malformation.”
Renal Tubular Dysgenesis

Familial renal tubular dysgenesis (RTD) is a rare autosomal recessive congenital disorder of renal tubular
development associated with late-onset oligohydramnios, Potter sequence, skull ossification defects, and
neonatal respiratory and renal failure with normal kidneys on ultrasonography (e1,e2,e255) that makes prenatal
diagnosis difficult (142). In some cases, only hypoplasia of the proximal convoluted tubules is reported (167),
while in others, microdissection has demonstrated marked shortening of all the nephron segments, from the
glomeruli to the collecting tubules, rather than an isolated abnormality of the proximal convoluted tubules (e10).
Despite the lack of normal proximal tubules, the major site of water resorption in the kidney, the principal clinical
manifestations are caused by fetal and neonatal oliguria. Increased accumulation of renin has been
demonstrated in the affected kidneys of three patients, which may reflect local vasoconstriction leading to
reduced glomerular perfusion (e31).
The clinical, pathologic, and radiologic features of familial RTD and skull ossification defect are similar to the
phenotype associated with fetal exposure to angiotensinconverting enzyme inhibitors, which suggests an
abnormality of the renin-angiotensin-aldosterone system (e178,94). Mutations in the genes for the renin-
angiotensin system have been reported with RTD (60, 182). Tubular lesions similar to those of RTD have been
described in twin-twin transfusion syndromes, acardia, unilateral lesion in renal artery stenosis, end-stage renal
disease of various causes (e182), in utero exposure to nonsteroidal antiinflammatory drugs (NSAIDs) (e161) and
piroxicam (e314), fetal renal vein thrombosis (e239), and severe congenital liver disease resembling neonatal
hemochromatosis (e14). A similar staining pattern with various lectins suggests that at least some of the
conditions studied are the result of renal ischemia, which could be acquired in utero or in early or later postnatal
life (e182).
Renomegaly
The most common form of renal enlargement is compensatory hypertrophy, in which a single functioning kidney
may reach twice the normal size and can be detected in utero. Bilateral renomegaly secondary to an increased
number or size of normally developed nephrons is seen in growth-related disorders (172) such as Beckwith-
Wiedemann syndrome (e252,e265,e291), hemihypertrophy, Perlman syndrome (e244), and congenital nephrosis
of the Finnish type (e148).
Renal Duplication (Duplex Kidney)
Duplex kidneys are the most common anomalies of the upper urinary tract in childhood with an estimated
incidence of 0.8% (39). The term renal duplication denotes the presence of two separate pelves in the same
kidney accompanied by complete or partial duplication of the ureter (e227) (Figure 17-4). These kidneys usually
have greater than normal number of reniculi. The anatomical and functional divisions between upper and lower
moieties of duplex kidney are extremely variable. The underlying pathological condition associated with a lower
moiety is usually massive vesicoureteral reflux to the lower collecting system and only rare obstruction. The
nonfunctioning upper moiety is usually associated with obstructive ectopic ureter (with or without ureterocele)
(39). Bilateral duplex kidneys have recently been reported in a boy with a mutation in the X-chromosomal gene
(L1CAM) for cell adhesion molecule L1 (100).
Supernumerary Kidney
Supernumerary kidney is one of the rarest of renal malformations with some 80 cases reported so far (39). In
addition to the normal two kidneys, an additional, usually small kidney is present within the renal fascia caudal to
and completely
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separate from the ipsilateral kidney (92). An ectopic ureterocele or common distal ureter may be associated with
this condition (e51).
FIGURE 17-4 ▪ Renal duplication (duplex kidney) with two separate pelves in the same kidney and more than the
normal number of reniculi.
Hydronephrosis
Hydronephrosis may be congenital or acquired, unilateral or bilateral, and mild to severe. Renal dysplasia may or
may not be present. Hydronephrosis is readily seen on antenatal ultrasonography but does not necessarily imply
obstruction. Although most cases will resolve spontaneously (17), the probability of a significant pathology is
related to the degree of pyelectasis, as seen on the third trimester ultrasound study. Criteria of obstruction are
difficult to define with precision, but two that are well-accepted are size of the renal pelvis (>15mm) and relative
renal function (77, 183).
Hydronephrosis is the most common cause of an abdominal mass of genitourinary tract origin in neonates
(e167,e211,e318). It is most frequently caused by obstruction of the ureteral-pelvic junction, which leads to
dilation of the renal pelvis and calyceal system. Depending on the severity and timing of the obstruction, the
appearance of the renal parenchyma varies from relatively normal to markedly atrophic, with fibrosis and a scant
chronic inflammatory infiltrate.
The specimen most commonly seen in surgical pathology is a portion of the ureteral-pelvic junction that shows
remarkably little pathology on microscopic examination. When end-stage nonfunctioning hydronephrotic kidneys
are removed, marked dilation of the pelvis and calyces with only microscopic foci of residual renal parenchyma
can be seen (Figures 17-5 and 17-6). Neonatal hydronephrotic kidneys seen at autopsies usually have a
histologically normal, although grossly compressed, cortex and medulla.
Hydronephrosis is often associated with renal dysplasia, so that the definition of these two entities often
overlaps. Also, because urinary tract obstruction is a common underlying condition, it is best to consider them as
the opposite ends of a spectrum. When severe obstruction occurs early in fetal development, it results in renal
dysplasia; when it occurs late, one sees hydronephrosis; when it develops in between, both hydronephrosis and
dysplasia are apparent to varying degrees. Hydronephrosis associated with reflux disease is discussed later in
this chapter in the section on tubulointerstitial diseases.
FIGURE 17-5 ▪ Nephrectomy specimen from a 5-year-old who presented with a unilateral renal mass. The
kidney appears to be one large cystic structure.
FIGURE 17-6 ▪ Cut section of the kidney in Figure 17-5 shows a markedly dilated pelvis and calyces and very
little residual renal parenchyma.
Although the vast majority of cases of hydronephrosis are sporadic, some syndromic associations have been
reported (47,e247,e304,187). Hydronephrosis should also be distinguished from the rare disorder of
megacalycosis (Puigvert disease), which is characterized by calyceal dilation, an increased number of calyces,
hypoplasia of the pyramids of Malpighi, a normal renal pelvis, and, most importantly, normal renal function
(e111).
RENAL DYSPLASIA/CYSTIC DISEASES

Cystic diseases of the kidney are a heterogeneous group of congenital (sporadic and genetic) and acquired
disorders characterized by multiple cysts. In view of our better understanding of the genetic basis for some of the
cystic renal diseases, the original “Potter classification” into types I to IV is no longer widely used (e242). There
is no universally accepted classification for cystic diseases. Also, some authors classify renal dysplasia under
abnormal renal differentiation or developmental defects (e106,93,97), whereas others place it with cystic
diseases (e257,e283).
The term multicystic is reserved for a category of renal dysplasia characterized by multiple unilateral or bilateral
cysts, while polycystic is conventionally used for hereditary autosomal recessive and autosomal dominant kidney
diseases.
Renal Dysplasia
Multicystic dysplastic kidneys (probably more accurately called dysgenetic kidneys) are the most common type of
malformed kidneys seen in pediatric autopsies (Table 17-1),
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with bilateral dysplasia accounting for 32% and unilateral dysplasia (with or without contralateral agenesis) for
7.1% of patients with renal malformations. It may involve one or both kidneys or part of one kidney, with or
without enlargement of the affected kidney and with or without grossly visible cysts. Renal dysplasia is one of the
most common causes of an abdominal mass in children younger than 1 year (e129), although it may present in
older children and adulthood (84).
FIGURE 17-7 ▪ Massively enlarged cystic dysplastic kidneys.
The most common form of dysplasia is sporadic; however, a genetic contribution to its cause is being
increasingly recognized (159). It is associated with obstruction of the ureteropelvic junction and bilaterally
enlarged distorted kidneys (Potter type IIA) that are no longer reniform (Figure 17-7). Cysts of varying sizes can
be appreciated through the capsule, and on sectioning are seen to be irregularly distributed throughout the
parenchyma, with no identifiable cortex or medulla left (Figure 17-8).
The microscopic hallmark is the presence of immature dysplastic-appearing tubules surrounded by collarettes of
condensed mesenchyme (Figure 17-9) that stains with periodic acid-Schiff. The tubules are lined by a single
layer of cuboidal epithelium that often appears to be excessive, so that it is folded and may fill the lumen (Figure
17-10). The cells are not differentiated and tend to have a relatively high nuclear-to-cytoplasmic ratio (thus the
term dysplasia). The basement membrane may be thick and eosinophilic. A myxoid, moderately cellular
condensation of spindle cells is seen around the tubules. The remaining connective tissue is loose and contains
many blood vessels, lymphatics, and peripheral nerves. Islands of immature-appearing cartilage can be identified
in a majority of cases (depending on the number of sections examined), but their presence is not required for the
diagnosis of dysplasia (Figure 17-11). Cysts of varying sizes with a markedly flattened lining epithelium or no
identifiable lining are formed by the dilated, dysplastic tubules (positive for keratin and negative for CD31). Cysts
can occur in any part of the nephron. Varying numbers of normal glomeruli and tubules can be identified between
the dysplastic areas.
FIGURE 17-8 ▪ Cut surface of cystic dysplastic kidney with multiple small, variably sized cysts involving both
cortex and medulla.
FIGURE 17-9 ▪ Cystic dysplastic kidney with disorganized renal parenchyma in which immature tubules are
surrounded by collarettes of condensed mesenchyme. (Hematoxylin and eosin stain, original magnification ×40.)
The terms renal adysplasia and hypoplastic dysplasia are used to describe small kidneys with extensive
dysplasia
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(Figure 17-12) that are totally nonfunctioning or minimally functioning, respectively. The essential histologic
features are the same regardless of the size of the kidney or the extent of involvement.
FIGURE 17-10 ▪ Dysplastic tubules with an excessive amount of lining epithelium, which is thrown into papillary
folds. (Hematoxylin and eosin stain, original magnification ×40.)
FIGURE 17-11 ▪ Disorganized renal parenchyma and island of immature cartilage in cystic dysplastic kidney
disease. (Hematoxylin and eosin stain, original magnification ×100.)
The vast majority of sporadic multicystic dysplastic kidneys are associated with congenital urinary tract
obstruction, which is often at the ureteropelvic junction but may occur at any level. Several animal models for
renal dysplasia after gestational ureteral obstruction have been described (97). An ultrasonographically guided
fetal kidney biopsy may rarely be used to detect the histologic features distinctive of dysplasia (e46,e114).
Occasionally, open renal biopsies are performed in patients with renal insufficiency and lower urinary tract
anomalies, which most often show renal dysplasia (e41). Grading of dysplasia has been described based on
renal glomerular count and degree of dysplasia and correlated with lung development in patients with Potter
sequence (e282).
In patients who survive the immediate postnatal period, clinical complications include hypertension, febrile
urinary tract infection, vesicoureteral reflux, progressive scarring, and renal failure (8, 128). In 3% to 5% of cases
of dysplastic kidney, nodular renal blastema is also present (e68), and although Wilms tumor developing in
dysplastic kidney has been reported (e219), a systematic review of 26 published studies with follow-up showed
no increased risk of development of WT (119).
A multitude of genetic diseases, malformation syndromes, and chromosomal disorders have been described in
which renal dysplasia is a major or a minor component. An excellent tabulation of these can be found at the end
of Chapter 22 in Potter Pathology of the Fetus, Infant and Child (93). A brief summary is provided in Table 17-4.
Numerous genetic defects involving various transcription factors (WT-1, PAX-2, EYA-1, HNR-1b) growth factors
(increased expression of TGFβ1 and increased β-catenin/SMAD1 signaling), survival factors (BCL2 and PAX-2
are upregulated in cystic epithelia in dysplastic kidneys), and adhesion molecules (KAL-1, glypican-3, FRAS1
and FREM-2) are being described in syndromic and nonsyndromic cases of renal dysplasia (97).
FIGURE 17-12 ▪ Cystic dysplastic kidneys, shown bisected in the middle of the picture, are smaller than the
adrenals above (Potter type IIB).
Winyard et al. (191) have shown that apoptosis is prominent in undifferentiated cells around dysplastic tubules,
which perhaps explains the tendency of these organs to regress. In contrast, apoptosis was rare in dysplastic
epithelia thought to be ureteric bud malformations. A high rate of proliferation has been demonstrated postnatally
in dysplastic tubules, and PAX2, a potentially oncogenic transcription factor, is expressed in these epithelia
(192). In contrast, both cell proliferation and PAX2 are downregulated during normal maturation of human
collecting ducts. Ectopic expression of BCL2, which encodes a protein that prevents apoptosis during renal
mesenchymal to epithelial conversion, has been observed in dysplastic kidney epithelia. Thus, dysplastic cyst
formation may be understood in terms of aberrant temporal and spatial expression of master genes that are
tightly regulated in normal human nephrogenesis.
Failure of normal insulinlike growth factor (IGF) and IGFbinding protein gene expression in the development of
multicystic renal dysplasia suggests a role for the IGF system in the progressive histopathologic changes of this
disorder (e204). Tubular epithelial production of platelet-derived growth factor A (PDGF-A) may induce
collagenous matrix production by adjacent fibroblasts, and marked upregulation of PDGF-A by interstitial cells
may be responsible for sustainable fibrogenic effects in the fetal kidney that contribute to renal maldevelopment
(e194).
Polycystic Kidney Disease

According to current concepts, the term polycystic kidneys should be used to describe only two forms of
inherited disease, autosomal recessive (ARPKD) and autosomal dominant (ADPKD) polycystic kidney disease,
and not for any other disease in which multiple renal cysts are present. Despite different patterns of inheritance,
clinical presentation and
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typical appearance of the kidneys, these two diseases have some similarities. Both diseases are caused by
mutations in proteins located in primary cilia resulting in renal concentrating defect and both are characterized by
increased rates of tubular epithelial proliferation and apoptosis (181). Elucidation of the pathogenic mechanisms
of PKDs has been aided by the availability of several animal models. Rodent models have arisen by
spontaneous mutation, random mutagenesis, transgenic technologies, or gene-specific targeting. Many of the
proteins encoded by these mutated genes are expressed in the primary cilium or the centrosome—indicating the
importance of the ciliary-centrosomal axis to normal tubular epithelial cell differentiation—or at sites of cell-cell or
cell-matrix adhesion. Cytogenesis results from loss-offunction mutations in these genes or from loss-of function
or gain-of-function mutations in genes that encode downstream signaling molecules and transcription factors in
the cystogenic pathway (180).
Table 17-4 ▪ SYNDROMES AND DISEASES ASSOCIATED WITH RENAL DYSPLASIA
Chromosomal Congenital
Name Heredity Defect/Locus Major Features Hepatic

Fibrosis
Meckel-Gruber syndrome AR 17q22-q24 Posterior Yes

(e392) encephalocele,
Polydactyly,
microcephaly
Zellweger syndrome (e402) AR UK Peroxisomal Yes

deficiency,
cerebrohepatorenal
syndrome
Ivemark (II) syndromea AR UK Renal, pancreatic, Yes

(e246,e292,e363, hepatic dysplasia
e409,e505)
Jeune syndrome AR UK Asphyxiating thoracic Yes

(e65,e83,e316) dystrophy
Carnitine AR UK Myopathy No
palmitoyltransferase
deficiency (e369)
Short-rib Polydactyly AR UK Lethal skeletal Yes

syndrome (e355) dysplasias, multiple
anomalies
Hereditary renal adysplasia AD, XL UK URA with No

(e169) contralateral
dysplasia
Nail-patella syndrome AD LMX1b Hypoplastic nails, No

(e134) mutation absent patellae,
glomerular changes
Tuberous sclerosis complex AD TSC1:9q34 Tumors of skin, No

(e254,e514,e553) TSC2:16p13.3 brain, heart, and
kidney
von Hippel-Lindau AD VHL gene Retinal and CNS No

syndrome (e364) carci mutations hemangioblastomas,
noma, pheochromocytoma, renal cysts-clear cell
pancreatic islet cell tumors carcinoma
Beckwith-Wiedemann Usually 11p15.5 Organomegaly, No

syndrome (e307,e394) sporadic alterations nephroblastoma
tosis, WT
DiGeorge syndrome (e453) Sporadic DGS1 Hypoplasia of thymus No

del(22q11) and aortic arch
DGS2 del(10p) defects
Prune-belly sequence Sporadic UK Deficient abdominal No

wall musculature,
urinary tract dilation,
cryptorchism
Trisomy 13, 18, 21 (e368) Riskfactor: 13,18,21 No

advanced
maternal
age
Fetal alcohol syndrome In utero UK CNS dysfunction, No

(e534) exposure growth deficits
Diabetic embryopathy (e81) In utero UK Macrosomia, No

exposure congenital
malformations,
stillbirth
aNot to be confused with asplenia and cardiac malformations, also known as Ivemark (I) syndrome.
AR, autosomal recessive; AD, autosomal dominant; XL, X-linked; UK, unknown; CNS, central nervous
system.
Autosomal Recessive Polycystic Kidney Disease
ARPKD is rare, with an incidence of 1 in 20,000 live births and extreme variability in its severity. The gene
abnormality has been mapped to the short arm of chromosome 6 named polycystic kidney and hepatic disease
(PKHD1) gene because of the consistent hepatic involvement. The PKHD1 gene (6p12.2) and related protein
named polyductin or fibrocystin are highly expressed in the epithelial cells of the collecting ducts and to a lesser
extent in the biliary ducts and pancreas. Analogous to autosomal dominant PKD, polyductin (fibrocystin) localizes
in the primary cilia of renal epithelial cells (20). Almost
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every vertebrate cell has a specialized cell surface projection called a primary cilium. Although these structures
were first described more than a century ago, the full scope of their functions remains poorly understood. There
is emerging evidence that in addition to their well-established roles in sight, smell, and mechanosensation,
primary cilia are key participants in intercellular signaling. This new appreciation of primary cilia as cellular
antennae that sense a wide variety of signals could help explain why ciliary defects underlie such a wide range
of human disorders, including retinal degeneration, polycystic kidney disease, Bardet-Biedl syndrome, and
neural tube defects (170).
In ARPKD, nephrogenesis proceeds normally, and the earliest abnormality involves the medullary ducts.
Oligohydramnios occurs subsequently (usually before 20 to 21 weeks of gestation). These observations suggest
that in severe fetal ARPKD, medullary collecting duct dilation occurs first and is successively followed by cortical
collecting duct dilation, increased renal echogenicity, and diminution of urine production (e1 19).
Thirty to 50% of patients present with oligohydramnios (Potter sequence): massively enlarged, symmetric,
reniform kidneys (Figure 17-13); and pulmonary hypoplasia. Death occurs in the perinatal period. The gross and
microscopic hallmark is the presence of tubular cysts with a diameter of 1 to 2 mm arranged radially. The cysts
are uniformly distributed and can be appreciated through the capsule of the markedly enlarged kidneys, which
retain their shape (Figure 17-14). On cut section, the cortex and the medulla are often unrecognizable. The cysts
represent tubular dilation of presumably normally formed collecting ducts; normal glomeruli and tubules are seen
between the cysts (Figure 17-15). In the medulla, the cysts are more rounded. Significant fibrosis, inflammation,
and obstruction are absent.
FIGURE 17-13 ▪ Autosomal recessive polycystic kidney disease with massively enlarged symmetric reniform
kidneys.
FIGURE 17-14 ▪ Cysts of autosomal recessive polycystic kidney disease can be appreciated on the cortical
surface. The cut section shows radially oriented cysts in the cortex and more rounded cysts in the medulla.
In cases with a later presentation, the degree of renal enlargement is less and the cystic change is less diffuse.
However, all forms of ARPKD are associated with congenital hepatic fibrosis, more recently termed ductal plate
malformation (see Chapter 15). Dilation of the interlobular bile ducts is associated with a variable degree of
portal fibrosis (e78).
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The lobular architecture of the liver is preserved, but all portal areas are expanded and contain tortuous, slightly
dilated ducts at the periphery with blood vessels in the middle (Figure 17-16). Stereologic studies have indicated
that what appear as ducts on histologic section are in fact cisterns communicating with each other (e158). Similar
hepatic changes are seen in Meckel, Zellweger, and Jeune syndromes, medullary cystic disease complex, and
tuberous sclerosis (e183).
FIGURE 17-15 ▪ Radially arranged cysts of autosomal recessive polycystic kidney disease. Normal glomeruli and
tubules are seen between the cysts. (Hematoxylin and eosin stain, original magnification ×40.)
FIGURE 17-16 ▪ Ductal plate malformation of the liver with expanded portal area, peripheral tortuous dilated bile
ducts, and blood vessels in the middle. (Hematoxylin and eosin stain, original magnification ×40.)
Mutational analysis of ARPKD presenting as infants and congenital hepatic fibrosis presenting in later childhood
or adulthood with minimal or no renal disease has defined a broader spectrum of ARPKD. Congenital hepatic
fibrosis with minimal kidney involvement can result from missense mutations in PKHD1 (2).
The clinical course of children with ARPKD who survive the neonatal period is variable and appears to be age
dependent; however, the long-term prognosis in the majority of cases is better throughout childhood and youth
than is often stated with a mean life expectancy of 27 years (64). Early detection and appropriate management of
renal failure and systemic portal hypertension are important.
Autosomal Dominant Polycystic Kidney Disease

Commonly referred to as adult PKD because the vast majority of cases become symptomatic in the fourth and
into the fifth decade of life, ADPKD is more common than ARPKD, with an incidence between 1/200 and 1/1,000
of the general population. ADPKD accounts for about 10% of adult kidney transplant recipients but is rare in
children.
ADPKD has two disease loci, PKD1 and PKD2 that encode the membrane glycoproteins, polycystin-1 and -2,
which have been localized to the primary cilia of renal epithelial cells (181). The primary cilia are finger-like
projections on the surface of all kidney cells, except acid-base transporting intercalated cells in the collecting
duct. Cilia have been proposed to serve as mechano- or chemosensors, responding to and interacting with the
microenvironment. Abnormal cilia structure or function or both may lead to abnormalities in cell proliferation and
tubular differentiation, ultimately leading to cyst formation (101).
FIGURE 17-17 ▪ Low-power photomicrographs illustrate the differences between cystic dysplastic kidney (left),
autosomal recessive polycystic kidney disease (middle), and autosomal dominant polycystic kidney disease
(right). (Hematoxylin and eosin stain, original magnification ×40.)
Approximately 85% of affected families have mutations in PKD1 gene (e245), which has been mapped to
chromosome 16p13.3 (e258) and the remaining 15% have mutations in PKD2, which has been localized to
chromosome 4q13-23 (e170,e212,e246). Affected persons in these families appear to have a phenotype similar
to that in PKD1 families, but the onset of cystic disease, hypertension, and renal insufficiency is delayed. A third
gene, PKD3, is suspected in a few families but has been identified on chromosome 2p (20).
Autosomal dominant polycystic kidney disease diagnosed in utero or in the first year of life is reportedly
associated with more severe renal cystic disease (e119). Although the majority of ADPKD infants survive, they
tend to have more significant hypertension and a more rapid decline in renal function than do their affected adult
relatives (e55). The kidneys vary in size from normal to enlarged, and rounded cysts range in size from
microscopic (in asymptomatic children with disease detected on screening performed because of a positive
family history) to about 3 cm in diameter. Some infants present with unilateral renal cysts. In contrast to the cysts
seen in ARPKD, these cysts occur in any part of the nephron and are present in both the cortex and the medulla
(Figure 17-17, eFigure 17-4).
Medullary Cysts
Cysts in the medulla can occur as part of several cystic kidney diseases (e.g., multicystic dysplasia, ARPKD, and
ADPKD). The term medullary cystic disease encompasses two clinically and pathologically distinct entities.
Medullary Sponge Kidney

Also referred to as precalyceal canalicular ectasia, medullary sponge kidney is a generally sporadic disease
with an equal sex distribution. It most commonly presents in adults, although some cases have been described in
children and
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even infants. Medullary sponge kidney is a developmental disorder characterized by ectatic and cystic
malformation of the papillary collecting ducts in the renal medulla; the condition is most often bilateral, but it may
involve only one kidney or only one or several reniculi. Medullary sponge kidney remains symptomless unless
complicated by urinary tract infection, renal stones, or hematuria—hence its presentation in later life. It is best
diagnosed by intravenous pyelography, which shows dilated medullary tubules and the so-called papillary blush
or bouquet of flowers.
Juvenile Nephronophthisis-Medullary Cystic Kidney Disease Complex

Originally described as two separate diseases, juvenile nephronophthisis (JNPH) and medullary cystic kidney
disease (MCKD) are now considered to be part of the same complex, with similar clinicopathologic features.
They are both inherited progressive tubulointerstitial diseases characterized by medullary cyst formation and
secondary glomerular sclerosis. The main differences are that JNPH has a younger onset (average age, 11.5
years) with recessive inheritance and MCKD has an adult onset (average age, 28.5 years) with dominant
inheritance (e139). JNPH, linked to mutations in more than one gene, occurring at different ages, has been
mapped to chromosome 2q (juvenile form, NHPH1, with nephrocystin as a gene product), chromosome 9q
(infantile form, NHPH2), chromosome 3q (adolescent form, NHPH3), and chromosome 1p36 (juvenile form,
NHPH4), accounting for 70% of all cases of the JNPH-MCKD group (20).
MCKD usually occurs in the third to fourth decades of life, sharing the same clinical renal presentation with
JNPH, except for the growth retardation and extrarenal malformations, which are absent in MCKD, and for the
later age of occurrence of uremia. Two forms are recognized: MCKD1 and MCKD2, which have been mapped to
chromosomes 1q and 16p, respectively. In approximately 15% of the cases of JNPH-MCKD complex, no family
history is found, possibly representing a new mutation (20).
Polyuria, polydipsia, salt wasting, anemia, and growth retardation precede end-stage renal disease. Cysts 1 to
15 mm in diameter, located primarily at the corticomedullary junction, are seen in only 70% of the patients (e139).
The remaining patients have no cysts. All cases have in common a chronic sclerosing tubulointerstitial disease,
which is usually more severe than the cystic component (e135,e181). JNPHMCKD complex is a major cause of
end-stage renal disease in children, accounting for 10% to 25% of these patients. Associations with retinitis
pigmentosa, hepatic fibrosis, skeletal defects, and central nervous system abnormalities have been described
with familial JNPH but are typically absent in medullary cystic disease (e139).
Cortical Cysts
Glomerulocystic kidney disease (GCKD) and glomerulocystic kidney (GCK) are associated with cortical cysts.
FIGURE 17-18 ▪ GCKD with cystic dilation of Bowman spaces; the medulla is uninvolved. (Hematoxylin and
eosin stain, original magnification ×40.)
GCKD, first described by Taxy and Filmer in 1976 (e302), is characterized histologically by cystic dilation of
Bowman spaces and atrophy of the glomerular tufts (Figure 17-18). The term disease is suitable only for the
familial autosomal dominant or sporadic GCK. It is now recognized that GCK is not a single disease entity but
can be divided into five categories: (i) familial, (ii) associated with heritable diseases, (iii) syndromic,
nonhereditary, (iv) sporadic, and (v) acquired GCK (98).
Most GCKD cases are transmitted according to an autosomal-dominant mode of inheritance, but the responsible
gene has not been mapped yet to a specific locus, which, however, is not linked to the PKD1 and PKD2 loci,
although a higher incidence has been noted among members with ADPKD. This disease is usually discovered in
infants more often within the context of a familial history of ADPKD and less often as sporadic GCKD of young
infants, although presentations occurring in older children and adults have also been observed of both familial
and sporadic type with the latter reflecting the occurrence of new mutations (20).
Ultrasonographically, minute cysts, smaller than those occurring in autosomal-dominant polycystic kidney
disease, are seen in the echogenic renal cortex. No cysts are observed in the renal medulla. Kidneys in GCKD of
ADPKD phenotype are bilaterally enlarged and diffusely cystic, in which the main microscopic finding is
represented by glomerular cysts, but asymmetric onset of this disease has also been seen. Kidneys in sporadic
GCKD of non-ADPKD phenotype may be seen with either clustered or diffuse cysts. Kidneys in familial-dominant
GCKD of older patients are normal in size, although occasionally they have been observed of enlarged size.
Finally, familial hypoplastic GCKD is probably a different type of GCKD, in which kidneys are smaller than normal
and often associated with medullocalyceal abnormalities. Familial hypoplastic GCKD is associated with
mutations in the hepatocyte nuclear factor-1 -b gene (HNF1B or TCF2); its gene locus is at 17cen-q21.3 and is
also found in some families with maturity-onset diabetes of the young, type V,
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which is the result of heterozygous mutations of the same HNF1B gene (193).
Glomerular cysts in all types of GCKD are less than 1 cm in size and located in the cortex from the subcapsular
zone to the inner cortex (eFigure 17-5), histologically similar to glomerular cysts seen in other disease conditions.
Familial and sporadic GCKD of young infants may also show renal medullary dysplasia and biliary dysgenesis
(“ductal plate malformation”) in the liver (20).
GCK may be a major component of heritable syndromes such as tuberous sclerosis, orofaciodigital syndrome,
brachymesomelia-renal syndrome, trisomy 13, and short-rib Polydactyly syndrome. This category also includes
glomerular cysts in several syndromes, namely Jeune syndrome and familial JNPH, better known for chronic
progressive tubulointerstitial disease. Glomerular cysts occur as a minor component (i.e., scattered cortical
cysts) in several other syndromes, among them Zellweger syndrome, in which cysts are typically present but
usually inconsequential, only occasionally serious enough to affect renal function. In all the syndromes, the cysts
are inconsistently expressed (e29).
Acquired GCK disease has been described, following hemolytic-uremic syndrome (e3,e306) and in progressive
systemic sclerosis (e281).
Simple Cysts
Simple cortical cysts, or retention cysts, which are very common in adults, are rarely seen in children. They are
important because they may present as an abdominal mass, or their appearance ultrasonographic or radiologic
images may raise the diagnostic consideration of cystic WT. Simple cysts arise from the cortex, are unilocular,
contain yellow clear fluid, and are lined by a single layer of cuboidal epithelium.
Cysts Associated with Syndromes

Cysts of the cortex (sometimes referred to as pluricystic kidney), usually asymptomatic, have been described as
a minor component of multiple malformation syndromes, both inheritable and noninheritable, including tuberous
sclerosis, von Hippel-Lindau disease, Meckel-Gruber syndrome, orofaciodigital syndrome-type I, trisomies 9, 13,
18, 21, short-rib-polydactyly syndrome, Jeune asphyxiating thoracic dystrophy syndrome, Zellweger
cerebrohepatorenal syndrome, VATER association, lissencephaly, renal-hepatic-pancreatic dysplasia, glutaric
aciduria type II, Ellis-van Creveld syndrome, Elejalde syndrome, Peutz-Jeghers syndrome, Robert syndrome,
phocomelia syndrome or pseudothalidomide syndrome), and Bardet-Biedl syndrome (20). In the following
diseases, the renal cysts are histologically distinct from dysplasia.
Tuberous Sclerosis
Tuberous sclerosis complex is an autosomal dominant systemic malformation syndrome, linked to TSC1- and
TSC2-suppressor genes, mapped on chromosome 9q and chromosome 16p, respectively, with the former
encoding hamartin and the latter, which accounts for two-third of the mutations, encoding tuberin (20). It is
characterized by hamartomatous proliferations of skin, brain, kidney, eye, bone, liver, and lung. In addition to the
well-recognized association with renal angiomyolipomas, which occur in 40% to 80% of patients with tuberous
sclerosis (e23), characteristic cortical cysts are present in about 50% of patients. The extent of involvement
varies; small cysts may be diagnosed on imaging, or “polycystic kidneys” may lead to renal failure. The cysts
vary in size and are lined by hyperplastic epithelium, which is often multilayered and papillary, with abundant
eosinophilic granular cytoplasm (Figure 17-19). Solid nodules of these cells may also form. Mitotic activity
evident in these cells may be related to the increased risk for neoplasia (e32). The histologic findings are so
characteristic as to be virtually diagnostic of tuberous sclerosis when seen in an early biopsy performed before
the onset of other stigmata of the disease (e30).
FIGURE 17-19 ▪ Renal cysts of tuberous sclerosis lined by characteristic hyperplastic epithelium with abundant
eosinophilic granular cytoplasm. (Hematoxylin and eosin stain, original magnification ×200.) (Courtesy of Dr.
John Hicks, Houston, TX).
Von Hippel-Lindau Disease

Von Hippel-Lindau disease is an autosomal dominant multisystem (pre)neoplastic disorder genetically linked to a
germline mutation of a tumor-suppressor gene (VHL gene) located on chromosome 3p. It is characterized by
retinal angiomas, cerebellar hemangioblastomas, and cysts and tumors of the pancreas (microcystic adenoma),
kidneys, and, less frequently, other abdominal organs. Renal cysts lined by hyperplastic epithelium with clear
cytoplasm and a “hobnail” appearance are associated with a markedly increased risk for the development of
renal cell carcinoma (RCC); multifocal cystic adenocarcinomas develop in 45% to 50% of patients beyond the
third decade of life (e104,e145,e259). Less frequently, the cysts are numerous enough to simulate ADPKD.
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Meckel-Gruber Syndrome
Meckel-Gruber syndrome has an autosomal recessive inheritance; is mapped to the long arms of chromosomes
17, 11, and 8; and occurs in 1/10,000 births (20,e268). It is characterized by posterior encephalocele, cystic
kidneys, congenital hepatic fibrosis (ductal plate malformation), and Polydactyly; additional features constituting
several variants have been described. The kidneys are always bilaterally involved, although they may
occasionally be variably involved. Round cysts arise from any part of the nephron, with microcysts seen in the
subcapsular area and larger cysts in the medulla. The cysts are lined by a single layer of low-to-high cuboidal
epithelium and are separated by loose, immature mesenchyme that may bulge into the cysts. Metaplastic
cartilage is not usually present.
The pathogenesis of renal cysts in Meckel syndrome remains unknown. Study of midterm fetuses has shown
that the kidneys are already enlarged by 11 to 20 weeks of gestation (110). Nephrogenesis is more or less
normal at the periphery of the kidney. It appears that the nephrons are formed normally and the tubules and
ducts are secondarily converted to cysts.
GLOMERULAR DISEASES
Metanephric blastema condenses around the end of the ureteric bud at about day 32 of development, and
elongation, branching, and subsequent fusion of proximal generations of the bud give rise to the pelvicalyceal
system and collecting ducts. The first glomerulotubular structures appear during week 8 as a result of the
interaction of subcortical blastema with the ampullary ends of the collecting ducts, and glomerulogenesis
continues until gestational week 36 when the neogenic (nephrogenic) zone disappears and nine to eleven
generations of glomeruli are present (e19). The number of glomeruli in human kidneys varies from 250,000 to 1.8
million. This marked interindividual difference may be genetically programmed or due to perinatal factors such as
low birthweight (estimated relation: 250,000 glomeruli per kilogram at birth), and may predispose persons with
lesser numbers of glomeruli to renal failure in adulthood (104). Immature (fetal) glomeruli are characterized by
their small size and prominent corona of visceral epithelial cells, and normally this corona disappears during the
first year (e19). Mean glomerular diameter increases from 112µm at birth to 167 µm at 15 years (e214), and
enlarged (hypertrophied) glomeruli suggest a compensatory response to reduced nephron mass (e277). The
thicknesses of the glomerular capillary wall and the lamina densa increase from 169 ± 30nm and 98 ± 23 nm,
respectively, at birth to 285 ± 39 nm and 219 ± 42nm, respectively, at 11 years (e312). The molecular structure
of the glomerular basement membrane also changes with age. Collagen al or al and a2 (IV) synthesized by
podocytes, endothelial cells, and mesangial cells of immature glomeruli is replaced by collagen and a3 and a4
and a5 (IV) produced exclusively by podocytes (1).
FIGURE 17-20 ▪ Needle biopsy specimen of kidney viewed through a dissecting microscope. Glomeruli appear
as red dots in the central region, and vasa recta in the outer medulla as linear striations at either end. (Original
magnification, 5×.)
The pathologist most often encounters glomerular diseases in renal biopsy specimens collected with biopsy guns
having needles of 18 gauge or less, and may be asked to examine the gross specimen for the presence of
glomeruli with a magnifying lens or dissecting microscope (Figure 17-20). The presence of renal cortex may be
inferred if one sees capsule and fat at one end of the biopsy specimen and architecture consistent with medulla
at the other, but the macroscopic recognition of glomeruli requires sufficient blood flow within glomerular
capillaries, and this may be reduced by disease. Definitive identification of glomeruli may rarely require rapid
frozen section, or the pathologist may be asked to perform a rapid frozen section to determine if crescents are
present. In either case, the tissue submitted for frozen section can also be utilized for immunofluorescent (IF)
studies. Whenever possible, tissue should be sampled for light, IF, and electron microscopy (EM), even if all
those studies are not initially requested, and with the smaller-gauge biopsy needles now used by pediatric
nephrologists, two or three cores are usually required. The specimen submitted for light microscopy (LM) should
contain as much cortex as possible along with the corticomedullary junction, whereas only cortical tissue is
ordinarily required in the specimens submitted for IF and EM.
Our understanding of pediatric renal pathology has been greatly facilitated by contributions from two
multiinstitutional collaborative studies, the International Study of Kidney Disease in Children (ISKDC) and the
Southwest Pediatric Nephrology Study Group (SPNSG), which have resulted in several seminal publications that
are cited at the end of the chapter. The terms most commonly used to describe the lesions encountered in renal
biopsy specimens are listed in Table 17-5. Children with renal disease usually present with proteinuria or
hematuria, alone or in combination, with or without associated systemic disease.
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Less commonly, patients present with a nephritic syndrome that includes proteinuria, hematuria, red blood cell
and white blood cell casts, and decreased plasma levels of complement components, or with acute renal failure,
renal concentration defects or chronic renal failure without known antecedent disease. Isolated proteinuria and
hematuria do not usually warrant biopsy study, and most children with nephrotic syndrome responsive to steroid
therapy or acute glomerulonephritis attributable to streptococcal disease do not undergo biopsy unless the
course is atypical or the response to therapy is suboptimal. Typically, the glomeruli in patients with isolated
proteinuria or hematuria are optically normal or show focal and segmental glomerulosclerosis (Figure 17-21A) or
mesangial hypercellularity (Figure 17-21B). Diffuse and global mesangial hypercellularity with thickening of
capillary walls and obliteration of capillary loops resulting in accentuation of the lobular architecture of the
glomerulus (Figure 17-21C) or the presence of crescents, proliferations of parietal epithelial cells and
inflammatory cells in Bowman space (Figure 17-2ID) are usually associated with a nephritic syndrome or acute
renal failure. IF and electron microscopic studies are usually necessary to arrive at a more precise diagnosis. A
granular pattern of immunofluorescence—along capillary loops (Figure 17-22A), within mesangia (Figure 17-
22B), or both (Figure 17-22C)—indicates immune complex deposition; and the site and the composition of the
immunoreactant(s) depend on the disease. Crescents stain brightly for fibrinogen (Figure 17-22D). Linear
staining along the capillary wall may indicate antiglomerular basement membrane disease (usually only IgG) or
dense deposit disease (usually only C3) (Figure 17-22D). The histologic, IF, and ultrastructural lesions for
specific diseases are described later, but a careful inventory of the lesions in all renal compartments—glomeruli,
tubules, interstitium, and vessels—and correlation of the morphologic findings with the clinical history and the
results of renal function tests and serologic studies are necessary for the proper clinicopathologic interpretation
of renal biopsy specimens from patients of any age.
Table 17-5 ▪ TERMS USED IN DESCRIBING GLOMERULAR LESIONS

Focal Involvement of <50% of all glomeruli
Diffuse Involvement of >80% of all glomeruli
Segmental Involvement of <50% of a glomerulus
Global Involvement of <50% of a glomerulus
Hyalinosis Accumulation of eosinophilic, PAS-positive, silver-negative, structureless

material that stains red with trichrome stains (glycoproteins and lipids)
Sclerosis Accumulation of eosinophilic, PAS-positive, silver-positive structureless

material that stains blue or green with trichrome stains (collagen IV)
Fibrosis Accumulation of eosinophilic, PAS-negative, silver-negative fibrillar material

that stains blue or green with trichrome stains (collagen I, III)
Mesangial proliferation More than three mesangial cells per peripheral mesangial area
Mesangiocapillary A combination of mesangial proliferation and capillary wall thickening

(membranoproliferative)
glomerulonephritis
Adhesion (senechia) Attachment of part or all of the circumference of a glomerular tuft to Bow
man capsule. Adhesions may be fibrous or fibrinous.
Crescent A proliferation of glomerular epithelial cells and inflammatory cells that fills
part (segmental) or all (circumferential) of Bowman space. Crescents may
be cellular, fibrocellular, or fibrous.
PAS, periodic acid-Schiff stain
Glomerulopathies that Usually Present with Proteinuria or Nephrotic Syndrome

The incidence of idiopathic nephrotic syndrome is two to seven per 100,000 children, 95% of whom respond to
steroid therapy, although 60% to 80% of these will experience one or more relapses (45). The distribution of
lesions in untreated children with nephrotic syndrome in the ISKDC, conducted from January 1967 through June
1974, was minimal change disease (MCD), 76.4%; membranoproliferative glomerulonephritis (MPGN), 7.5%;
focal segmental glomerulosclerosis (FSGS), 6.9%; mesangial proliferative glomerulonephritis, 4.6%; focal global
glomerulosclerosis, 1.7%; membranous glomerulonephritis (MGN), 1.5%; and chronic or unclassified
glomerulonephritis, 1.4%. Patients with MCD were younger than those with FSGS or MPGN (80% versus 50%
versus 3% under 6 years old at diagnosis), showed a different sex ratio (male-to-female ratios of 60:40, 70:30,
and 36:64), and presented less frequently with hypertension (21%, 49%, and 51%) or hematuria (23%, 48%, and
59%) in addition to nephrotic syndrome (e232). A response to prednisone at 8 weeks was seen in 93% of
patients with MCD, 75% with focal global glomerulosclerosis, 30% with FSGS, 56% with diffuse mesangial
hypercellularity, 7% with MPGN, and none with MGN (e254). MCD is under-represented in current biopsy
practice because most patients with this lesion respond to steroid therapy and do not undergo biopsy; however,
even allowing for the changes in biopsy practice, the
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incidence of FSGS in children appears to be increasing in all ethnic groups (e39,52), becomes more apparent in
patients over 6 years of age at presentation (e296), and appears to be more common and more aggressive in
African-American (e151,e290) and possibly Japanese (e329) children. Familial FSGS has been attributed to
mutations that alter the membrane (podocin), cytoskeleton (a-actinin-4, CD2-AP), extracellular matrix adhesion
molecules (β4 integrin), sialylated surface proteins, or nuclear proteins (WT1, LMX1B, SMARCAL1) of the
visceral epithelial cell (38, 45, 145), though many of these patients do not present until adulthood and there is
considerable variability in the clinical severity and response to therapy, especially among heterozygotes, implying
that other genes or nongenetic triggers may be involved (27). Altered expression or distribution of these and
other podocyte proteins have been found in studies of nonfamilial FSGS (e.g., dystroglycans reduced in MCD
but not FSGS (27), and reduced podocin (63) and synaptopodin (174) in FSGS), suggesting that reorganization
of podocyte proteins in response to injury is a key step in the pathogenesis of proteinuria (e16,146) Microarray
analysis of RNA from renal biopsy specimens of 10 children with FSGS compared with five controls found a
“gene expression fingerprint” of 429 genes, many of which had not been previously implicated in the
pathogenesis of FSGS (162). FSGS, often with lesser (non-nephrotic) levels of proteinuria, is also the lesion
seen in cyanotic congenital heart disease, sickle cell anemia, massive obesity, HIV, and other viral infections
(parvovirus,
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SV40, and some cases of hepatitis C). Nephrotic syndrome in the first year of life may be associated with lesions
that occur in older children but is more often caused by one of two lesions unique to this age group—congenital
nephrotic syndrome of the finnish type (CNF) and diffuse mesangial sclerosis (DMS) that are discussed later.
Medical complications of nephrotic syndrome include acute infections and thromboembolic disease related to the
nephrotic state, and long-term effects on bones, growth, and the cardiovascular system related to the disease
and its treatment (45).
FIGURE 17-21 ▪ Glomerular lesions observed in pediatric renal biopsy specimens. A: FSGS with the sclerotic tuft
in the 11 o'clock position adherent to Bowman's capsule, and segmental proliferation of visceral epithelial cells at
the 2 o'clock to 4 o'clock position. B: Mesangial proliferation is defined as more than three mesangial cell nuclei
per peripheral mesangial focus. C: Mesangiocapillary or membroproliferative glomerulonephritis shows both
mesangial proliferation and thickening of capillary loops. D: In crescentic glomerulonephritis, a segmental, or in
this case, circumferential proliferation of epithelial and inflammatory cells in Bowman space compresses the
underlying glomerular tuft. (A and D: Hematoxylin and eosin, B and C: Periodic acid-Schiff stain, original
magnifications ×400.)
FIGURE 17-22 ▪ Immunofluorescence patterns observed in pediatric renal biopsy specimens. A: Granular
staining along capillary loops. B: Confluent granular staining in mesangia. C: Combination of capillary and
mesangial granular staining. D: Linear staining for C3 along the capillary wall and bright rings with mesangia in
dense deposit disease (type II membranoproliferative glomerulonephritis). [Fluorescein isothiocyanate-
conjugated anti-IgG (A-C) or anti-C3 (D), original magnifications 400×].
Minimal Change Disease, Focal Segmental Glomerulosclerosis, and Diffuse Mesangial Hypercellularity
By definition, MCD should show no significant abnormalities by LM; FSGS should show segmental tuft sclerosis
with adhesion to Bowman's capsule in a minority of glomeruli (Figure 17-21A), and diffuse mesangial
hypercellularity should show three or more mesangial cells in most tufts of most glomeruli (Figure 17-2IB). Slight
segmental increases in mesangial matrix and cellularity and focal interstitial fibrosis are within the spectrum of
“minimal change,” but segmental proliferation of visceral epithelial cells (Figure 17-21A, 2 o'clock to 4 o'clock
position) may be the earliest lesion of FSGS. D'Agati et al. (38) have subdivided FSGS into five categories:
FSGS, NOS, and cellular, perihilar, tip and collapsing variants. NOS is the most common form seen in children
and adults, and the collapsing variant confers a more guarded prognosis in children as well as adults (169). The
classic ultrastructural findings in patients with the nephrotic syndrome include diffuse retraction of foot processes
of visceral epithelial cells, microvillous transformation along the cell membrane, and vacuolization and lipid
droplets within
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visceral epithelial cell cytoplasm, the prognostic significance of which is not certain (e269). Most authors
consider any segmental glomerulosclerosis significant, but rare globally sclerotic or hyalinized glomeruli are
occasionally seen in otherwise normal infant kidneys. It has been speculated that these may represent the
residua of a population of large glomeruli that develop early in gestation, serve some unknown function during
fetal life, and involute shortly after birth (e19). Emery and MacDonald found hyalinized glomeruli in the kidneys of
75 of 200 (38%) infants and children up to 15 years of age (0.5% to 30% of glomeruli in affected kidneys, but in
most cases the range was 1% to 2%) and noted that rare sclerotic glomeruli were present in many of the kidneys
that had no such glomeruli in the selected field (e95). Kohaut et al. found focal segmental hyalinosis in 9 of 29
autopsy specimens from children without apparent renal dysfunction and focal global sclerosis in 22, but the
percentages of involved glomeruli were 0.7% for the segmental lesion and 1.9% for the global lesion (e177).
Thus, a rare globally sclerotic glomerulus might be within normal limits but should initiate a search of serial
sections through the block for a segmentally sclerotic glomerulus. Examination of serial sections is also
recommended if focal tubular atrophy, interstitial fibrosis, enlarged glomeruli, segmental hyalinosis, segmentally
positive immunofluorescence, collagen in glomeruli by EM, or an incomplete therapeutic response is found
(e225).
Arguing that FSGS is a lesion with prognostic significance, but not a single disease, McAdams et al. classified
biopsy material from 134 children with nephrotic syndrome as MCD (normal light and fluorescent microscopy,
diffuse foot process retraction by EM), mesangial proliferation (at least two to three cells in most mesangia by LM
and diffuse foot process retraction and thinning of the glomerular basement membrane by EM), or “primary”
FSGS (segmental tuft sclerosis by LM and preservation of foot processes by EM). FSGS with foot process
retraction (“fusion”) was considered a “secondary” lesion in MCD or diffuse mesangial hypercellularity. Thus
defined, the mean age at onset of MCD was 8.6 years; the racial distribution was similar to that of the region in
which the hospital was located, and “secondary” FSGS developed in 41% of cases. Progression to end-stage
renal disease occurred in 14% of all patients with MCD but in 30% of those with “secondary” FSGS, and FSGS
recurred in two of eight transplants. The mean age at onset of mesangial proliferation was 7.0 years; African-
American patients were under-represented, and “secondary” FSGS developed in 55% of cases. Progression to
end-stage renal disease occurred in 13% of all patients with mesangial proliferation but 23% of those with
“secondary”, and FSGS recurred in 5 of 12 transplants. The mean age at onset of “primary” FSGS was 13
years; the proportion of African-American patients was more than twice that in the region in which the hospital
was located, and FSGS was by definition present in all cases. Progression to end-stage renal disease occurred
in 34% of these patients, but this lesion did not recur in any of nine transplants (e207).
IF microscopy in this group of diseases is usually negative or reveals only segmentally variable, non-pattern
staining for IgM with or without C3 or C1q, bright staining for C1q, or, rarely, a pattern suggestive of IgA
nephropathy. The reader is referred to the exhaustive reviews by Nadasdy et al (e225) and Olson and Schwartz
(e238) for a discussion of the significance of IgM nephropathy. In the above- report of pathologic findings of 134
children with nephrotic syndrome described earlier, McAdams et al. concluded that there was insufficient
evidence to consider IgM nephropathy or C1q nephropathy, discussed below, valid categories of childhood
nephrotic syndrome (e207). However, in a review of biopsies of 121 children with steroid-resistant or dependent
nephrotic syndrome and 331 with nonnephrotic proteinuria and/or hematuria, Zeis et al. (198) found mesangial
IgM in 20 of the 85 nephrotic syndrome biopsies and 44 of the 331 nonnephrotic proteinuria biopsies, and noted
evolution to FSGS in six of the former (30%) and seven of the latter (16%), compared to 4.6% and 0% for the
IgM-negative biopsies in those groups.
Jennette et al. described a proliferative glomerulonephritis with mesangial granular C1q as the dominant or
codominant immunoreactant in 15 adolescents and young adults who presented with proteinuria or nephrotic
syndrome (e155). In a report of 20 children (<18 years old at presentation) with C1q nephropathy, Lau et al.
noted that 40% presented with nephrotic syndrome and another 30% with nephrotic range proteinuria, that 55%
were boys and 60% were African-Americans, that the most common histologic finding was FSGS (40%) or MCD
(30%), and that renal survival was best predicted by nephrotic syndrome at presentation (49% at 5 years for
those with and 78% for those without nephrotic syndrome) (96) Markowitz et al. described 19 cases of C1q
nephropathy in a series of 8,909 native kidney biopsies and noted that it was a disease of children and young
adults (age range: 3 to 42 years, mean: 24.2 years) with a female and African-American preponderance. Renal
biopsies showed FSGS in 17 and MCD in two patients, always with codeposits of IgG and many with codeposits
of IgM (84%), C3 (53%), or IgA (32%), and these authors concluded that C1q nephropathy fell within the
spectrum of MCD/FSGS (106). Rarely in children with nephrotic syndrome and MCD by LM, one finds a
fluorescent antibody pattern characteristic of IgA nephropathy. These patients may have coexistent MCD and
mild IgA nephropathy, and they usually respond to steroid therapy for MCD. In contrast, most patients with IgA
nephropathy who present with nephrotic syndrome show segmentally proliferative, necrotizing, or sclerotic
lesions by LM; do not respond to steroid therapy; and have a guarded prognosis (e6).
Membranous Glomerulonephritis
MGN is seen in 1.5% of children (e232) and 18.5% of adolescents with nephrotic syndrome. In children, the age
at onset is usually 8 to 16 years and the sex ratio is equal. Most patients
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have microscopic hematuria in addition to proteinuria, but macroscopic hematuria is uncommon. Thirty-five
percent of cases of MGN in children are secondary to systemic diseases, whereas the incidence of secondary
MGN in adults is 23% (e107). Kleinknecht et al. found that more than 50% of children with secondary MGN had
an underlying infectious disease, such as hepatitis B or congenital syphilis, and that another 27% of cases were
secondary to lupus or another autoimmune disorder. However, the proportion of both “secondary” MGN and of
MGN due to hepatitis B is decreasing as a result of the availability of hepatitis B vaccine. Drugs and neoplasia
were very uncommon causes of secondary MGN in that series (e174). Following the description in 2002 of a
remarkable case of antenatal MGN due to maternal antibodies directed against neutral endopeptidase, a
podocyte and tubular brush border protein, which was present in the fetus but not the mother (40), Ronco and
coworkers have reported other cases of MGN in early life attributable to alloimmunization (153). Primary MGN in
children and adults appears to be an autoimmune disease against a podocyte or a basement membrane antigen
(14).
FIGURE 17-23 ▪ AB: MGN with diffuse thickening of capillary walls that in some stages exhibit short “spikes”
extending from the outer surface of the capillary. CD: On Ehrenreich and Churg stage I small electron-dense
deposits are present along the outer aspect of the basement membrane, but in stage III, larger deposits are
incorporated into the basement membrane. (Periodic acid-Schiff stain, original magnification ×400. B: Jones
mefhenamine silver stain, original magnification ×600. C,D: lead citrate and uranyl acetate.)
Histologically, glomeruli in MGN appear large and have uniformly thickened capillary walls but patent capillary
lumens (Figure 17-23A). The diagnostic “spikes” seen on silver stains (Figure 17-23B) represent notches along
the outer aspect of the normally argyrophilic basement membrane due to immune complexes that do not take up
the silver. Spikes cannot be detected when the deposits are small or sparse (Figure 17-23C) or when they have
been fully incorporated into the basement membrane (Figure 17-23D). Mesangial hypercellularity, glomerular
lobulation, and segmental inflammation, necrosis, or sclerosis are more
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common in secondary MGN (164). Glomerulosclerosis indicates advanced disease and interstitial fibrosis and
tubular atrophy correlate with the degree of proteinuria and stage of disease (e199). However, FSGS (43) or
interstitial fibrosis in the absence of glomerulosclerosis or tubular atrophy (e321) may portend an unfavorable
course.
IF microscopy reveals granular staining along capillary walls (Figure 17-22A) and occasionally also within
mesangia (Figure 17-22C). IgG and C3 are very commonly present, but a “full house” of immunoreactants
suggests lupus or another systemic disease. Mesangial deposits also suggest systemic disease but are seen in
31% of children with idiopathic MGN (e66). Ehrenreich and Churg described four stages in MGN: stage I, small
subepithelial deposits (Figure 17-23C); stage II, larger and more numerous deposits bordered by projections of
the lamina densa; stage III, incorporation of deposits into the lamina densa (Figure 17-23D); and stage IV, a
thickened and irregular basement membrane without recognizable deposits (e89). Patients may present at any
stage and may have deposits characteristic of more than one stage. Foot process retraction is typically extensive
in all stages. The SPNSG found that younger children tend to have more advanced disease (stage III or IV) and
that lower stages were associated with a shorter clinical duration of disease before biopsy (e66).
Diabetic Nephropathy
Diabetic nephropathy develops in 40% to 50% of patients with insulin-dependent diabetes mellitus. Long-
standing disease, poor metabolic control, smoking, male sex, non-Caucasian race, and other genetic factors
predispose patients to the development of nephropathy (127). It is unusual for clinical nephropathy to develop in
less than 10 years, but mesangial expansion and basement membrane thickening begin to appear within 2 to 5
years, even before the onset of microalbuminuria (e98). Ellis and Pysher found diffuse intercapillary
glomerulosclerosis in 11 children and nodular intercapillary glomerulosclerosis (Kimmelstiel-Wilson lesion) in one
child, all of whom had had insulin-dependent diabetes mellitus for only 4 to 10 years (e94). This and the other
glomerular lesions of diabetic nephropathy, hyalinosis fibrin caps and capsular drops, and hyaline
arteriolosclerosis, may also be seen in kidney biopsy specimens from massively obese adolescents (Figure 17-
24). IF microscopy shows a characteristic linear staining along the glomerular capillary walls and tubular
basement membranes for IgG and albumin, and hyalinotic lesions often stain with IgM and C3. The earliest and
most characteristic ultrastructural lesion is thickening of the lamina densa of the glomerular basement membrane,
but with time the width of the membrane varies as thinner areas develop as a result of microaneurysms and the
deposition of neomembrane (e313). Other ultrastructural findings include increased mesangial matrix, variable
effacement of foot processes, and subendothelial accumulations of electron-dense material that correspond to
fibrin caps and should not be confused with the deposits seen in immune complex diseases.
FIGURE 17-24 ▪ Obesity-related glomerulonephritis. Nodular mesangial sclerosis, hyaline caps, capsular drops,
and arteriosclerosis, all features of diabetic nephropathy, are also present in this adolescent with obesity-related
nephropathy. (Periodic acid-Schiff stain, original magnification ×400.)
Nephrotic Syndrome in the First Year of Life

The term congenital nephrotic syndrome is used to describe either the clinical occurrence of nephrotic syndrome
in the first 3 months of life, regardless of etiology, or a specific disease that was first recognized and is most
common in Finland, commonly referred to as Congenital Nephrotic Syndrome of the Finnish type (CNF) (e121).
In 1998, this autosomal recessive disorder was mapped to the NPHS1 gene at 19q13.1 that encodes nephrin, a
185-kDA transmembrane protein in the slit diaphragm of podocytes (e168). In 2000, autosomal recessive steroid-
resistant nephrotic syndrome (SRNS), a disorder of older children and adults, was mapped to the NPHS2 gene
that encodes podocin, another component of the slit diaphragm (24). In a review of patients with onset of
proteinuria before 3 months of age and histologic and ultrastructural findings consistent with CNF, Koziell et al.
(91) found that this phenotype could result from either NPHS1 or NPHS2 mutations but that triallelic mutations
involving both genes resulted in congenital nephrosis with an FSGS phenotype. It is of interest that decreased
expression of podocin (63), but not nephrin (74) has been described in children with nonfamilial nephrotic
syndrome. A second disorder, DMS, is also unique to this age group, but approximately 5% of children with
MCD, 5% with focal glomerulosclerosis, and 5% with MGN present in the first year of life (e123). Nephrosis or
proteinuria in infants has been reported in conjunction with several genetic disorders (Denys-Drash, Frasier,
Galloway-Mowat syndrome, Lowe and nail-patella syndromes (129), other disorders of neuronal migration
(e144), and type I carbohydrate-deficient glycoprotein syndrome (e308), infections (cytomegalovirus, hepatitis B
and C,
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HIV, malaria, rubella, syphilis, and toxoplasmosis), infantile systemic lupus erythematosus (SLE), mercury
toxicity, hemolytic-uremic syndrome (HUS), WT, drug reactions, and, as described above, alloimmunization to
podocyte proteins. However, it is likely that some patients in the reports of “secondary” congenital nephrosis may
have had unrecognized NPHS1 or NPHS2 mutations (129). Sibley and colleagues, first in a study of 48 infants
from their own institution (e285) and then in a literature review that included 502 infants (e286), showed that
except for patients with DMS, which progressed to end-stage renal disease regardless of the patient's age at
onset, patients in whom a lesion presented after 3 months of age had a much better outcome than those in whom
the same lesion presented before 3 months of age, regardless of the type of lesion.
FIGURE 17-25 ▪ Congenital nephropathies. A: Tubular ectasia, interstitial inflammation, and variable mesangial
hypercellularity are nonspecific features seen in CNF. B: Increased mesangial matrix and segmental tuft sclerosis
are seen in the early stages of DMS in this newborn infant with Denys-Drash syndrome. (A: Hematoxylin and
eosin, original magnification 200×, B: Periodic acid-Schiff stain, original magnification 400×.)
CNF is most common in Finland, where it occurs in 1/8,000 births, but many non-Finnish familial and sporadic
cases have been reported (e231). Affected infants are typically small for gestational age and are born at 35 to 38
weeks of gestation with deformations of the skull, hips, knees, and elbows, which are ascribed to the markedly
enlarged placenta that weighs more than 25% of the infant's birth weight. Other abnormalities (small nose with
low bridge, widely separated cranial sutures, large fontanelles, delayed ossification) may be secondary to
hypothyroidism as a consequence of urinary loss of thyroid-binding globulin (e286). Proteinuria in utero also
leads to increased levels of a-fetoprotein in the amniotic fluid and maternal serum. Although proteinuria is
present at birth in CNF, renal function is usually normal during the first 6 months, and no extrarenal disorders are
present. In contrast, congenital nephrotic syndrome due to other causes typically presents later in the first year of
life with less massive proteinuria, extrarenal manifestations are evident in congenital infections and syndromes
with urogenital or neurologic components, and the rate of renal deterioration is much faster with DMS or
interstitial nephritis (e144). The histologic hallmark of CNF is patchy dilation of the proximal tubules (Figure 17-
25A), but this may not be present in biopsy specimens, especially those obtained before 6 months of age (e144),
and is neither sensitive nor specific for CNF (e121). Glomeruli may show mesangial hypercellularity or crescents,
and larger than normal glomeruli appear to be too closely spaced, but no glomerular lesion is diagnostic by light,
IF, or EM (e121,e286). An interstitial lymphoid or myeloid infiltrate may be present. Proteinuria recurs in 25% of
patients after transplantation, all of whom in one report had the same Fin-major NPHS1 mutation, and may be
due to the development of antinephrin antibodies (131).
DMS usually presents between 3 and 11 months, somewhat later than CNF, but the characteristic lesion has
been reported in an 18-week fetus (e293). Mesangial sclerosis begins as an increase in fibrillar matrix but not
cellularity (Figure 17-25B), and it progresses to transform the entire tuft into a shrunken hyalinized ball
surrounded by a rim of visceral epithelium within a prominent Bowman space that may contain crescents (e286).
A zonal distribution of small simplified glomeruli and undifferentiated tubules beneath the capsule, and relatively
normal glomeruli but dilated tubules near the medulla may be present (e121). Immunofluorescence studies may
be negative or show mesangial staining for IgM, C3, and C1q in intact glomeruli, and IgM and C3 outline the
sclerotic glomeruli. By EM, endothelial and especially mesangial cells appear hypertrophic, and there is a
marked increase in mesangial matrix (e121).
Habib et al. reported DMS as the usual renal lesion in patients with the Denys-Drash syndrome (e126). Initially,
only genetic males with pseudohermaphroditism, nephropathy, and WT were included in this syndrome;
however, since the recognition of patients who do not express the full syndrome, females with the full syndrome
and patients with the characteristic nephropathy who also have either genital abnormalities or WT have been
included (e67). The genital abnormality in Denys-Drash syndrome is either ambiguous genitalia or normal female
genitalia with an XY karyotype, and children
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in whom WTs develop generally manifest bilateral tumors at a mean age of 18 months (e77). Several mutations
in the WTsuppressor gene, WT1, have been reported in patients with Denys-Drash syndrome (e67). Moorthy et
al. suggested that some patients previously reported to have Denys-Drash syndrome had, in fact, the Frasier
syndrome of streak gonads and male pseudohermaphroditism associated with XY gonadal dysgenesis and
nephrotic syndrome progressing to end-stage renal disease (e216). Patients with Frasier syndrome are at risk for
gonadoblastoma but not WT (e216), and the glomerular lesion in Frasier syndrome is FSGS. Frasier syndrome is
due to a mutation in intron 9 of the WT1 gene, but the tumor risk is much less than in Denys-Drash syndrome
because Frasier patients have one normal copy of WT1 (e15). WT1 mutations were found in four often patients
with DMS who did not have evidence of a urogenital abnormality or WT (“isolated diffuse mesangial sclerosis”),
but in two of these patients, the mutations were different from those described in Denys-Drash syndrome (e153).
WT1 mutations characteristic of Frasier or Denys-Drash syndrome were found in three of 32 girls with SRNS, but
in none of 54 males with SRNS or 114 males and females with steroid-dependent nephrotic syndrome (9).
Nail-Patella Syndrome, Collagen Type III Glomerulopathy, Pierson Syndrome

Nail-patella syndrome may be a cause of proteinuria in infancy, childhood, or adulthood. The cardinal features of
this condition are dysplasia of the nails and absent or hypoplastic patellas, but most patients also have iliac
horns and dysplasia of the elbows. A nephropathy develops in some kindreds. LM may show patchy tubular
atrophy and interstitial fibrosis, but the glomeruli are normal or show only irregular thickening of the capillary wall
or mesangial expansion or segmental or global sclerosis. Immunofluorescence studies are usually negative. EM
reveals prominent thickening of the glomerular basement membrane, which has a mottled appearance caused by
irregular but sharply defined electronlucent areas containing fibers that have the periodicity of collagen,
especially if the grids have been stained with phosphotungstic acid. Progression to renal failure occurs in about
30% of patients with renal disease, but the course in an individual patient is unpredictable (65). Nail-patella
syndrome is due to mutations in the LIM-homeodomain protein LMX1B at 9q34 that regulates transcription of
collagen IV subtypes a3 and a4 (e85), and mutation analysis has shown correlation of mutations in this domain
with proteinuria but not to the extra-renal manifestations of the disease (22). Collagen type III glomerulopathy
presents with progressive proteinuria in late infancy to adulthood and most affected children go on to renal
failure. Glomeruli are markedly enlarged and show expanded mesangia and thick capillary walls, and by EM the
mesangia and the subendothelial space are electron lucent or mottled. Collagen fibers can be demonstrated with
phosphotungstic acid staining, but patients do not have the extra-renal manifestations of nail-patella syndrome
(65). Pierson syndrome, congenital nephrotic syndrome and microcoria, is due to mutations of LAMB2 on
chromosome 3p that encodes laminin β2 that anchors the podocyte foot process to the basement membrane.
Pathologically, it may show DMS or glomerular hypercellularity with variable thickening, thinning, rarefaction and
lamination of the glomerular basement membrane on EM (65).
Glomerulopathies that Usually Present with Hematuria with or without Proteinuria

The most important diseases in this category in children include the primary IgA nephropathies—Berger disease
and Henoch-Schönlein purpura (HSP) nephritis, and the basement membrane nephropathies—Alport syndrome
and thin glomerular basement membrane disease. Hematuria is a well-known complication of hypercalciuria, but
no specific pathologic lesion is associated with this condition (e298), and the histopathologic abnormalities in
loin-pain hematuria syndrome are nonspecific. In all these conditions, the finding of red cell casts or hemosiderin
in tubular epithelial cells lends support to a diagnosis of hematuria originating in the kidney rather than in the
lower urinary tract. The primary IgA nephropathies are defined by the presence of IgA as the dominant or
codominant immunoreactant in the absence of clinical or laboratory features of systemic lupus erythematosis.
Characteristic but not always pathognomonic ultrastructural lesions are observed in many cases of Alport
syndrome, and an ultrastructural lesion defines thin glomerular basement membrane disease.
IgA Nephropathy (Berger Disease)

IgA nephropathy was described by Berger and Hinglais in 1968 (e27), but the association of recurrent hematuria
and focal glomerulonephritis, often in patients with a recent history of upper respiratory infection, had been
recognized many years earlier. Today, IgA nephropathy is the most common glomerulopathy worldwide,
accounting for 5% to 10% of cases of glomerular disease in North America, 15% to 30% in Europe, and up to
50% in Japan (69). These wide regional variations in incidence may be due, in part, to differences in
ascertainment. For example, children of school age in Japan undergo an annual screening urinalysis, and three
quarter of cases of IgA nephropathy in Japan are detected when only microscopic hematuria is present. In
Europe and North America, 75% of children with IgA nephropathy present with gross hematuria (e146). In the
SPNSG compilation of 83 children from the United States with IgA nephropathy who were 18 years of age or
younger at first presentation, 59 were boys (71%) and the mean age at clinical presentation was 9.9 years
(range, 3 to 17.3 years) (e142). In the 91 patients less than 15 years of age reported by Levy and coworkers in
France, 63 were boys (69%), and the age range was 3.3 to 14 years (e191). Many cases of IgA
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nephropathy occur within a few days after an upper respiratory or a gastrointestinal infection; in contrast, several
weeks usually separate the antecedent infection from the onset of postinfectious glomerulonephritis. Serum
levels of IgA are significantly elevated in up to 50% of adults but in only 8% to 16% of children with IgA
nephropathy (e146), but serum levels of IgA or of IgA-fibronectin cannot be used in lieu of renal biopsy for the
diagnosis of these conditions (69). Recent studies have implicated aberrant glycosylation of the hinge region of
the IgA1 molecule in the pathogenesis of IgA nephropathy. The abnormal molecule is not cleared by the
reticuloendothelial system, elicits the formation of complement fixing IgG-IgA complexes, and can bind to
mesangial cells and activate complement via lectin pathways. The IgGIgA complexes show promise as
biomarkers (124).
Classically, IgA nephropathy is characterized by focal segmental to global mesangial hypercellularity by LM
(Figure 17-21B), confluent granular mesangial deposits that stain more brightly for IgA than for other
immunoglobulins by IF microscopy (Figure 17-22B), and electron-dense deposits within and especially along the
periphery of mesangia and adjacent to mesangial cells. However, the histologic picture is quite variable. In the
SPNSG study, the initial biopsy specimens from 28 of the 83 (34%) patients showed mesangial hypercellularity
(focal and segmental in 24 and mild to moderate in 23), but the specimens from 25 patients (30%) were
histologically normal (50% of the biopsies from girls and 22% of those from boys), and those from 30 patients
(36%) showed segmental necrosis, collapse, sclerosis (focal segmental sclerosis with or without proliferation in
18 patients), synechiae, or crescents (>50% of glomeruli involved in two patients) (e142). In an earlier report of
62 of these patients, this group found global sclerosis of 10% or more glomeruli in 10% of the biopsy specimens,
more often but not exclusively in specimens that also had segmental lesions. Tubulointerstitial lesions were seen
in 38% of specimens with normal glomeruli, 60% of those with segmental mesangial hypercellularity, and 70% of
those with the more severe segmental lesions (e220). Levy et al. found histologically normal glomeruli in 26 of 91
specimens (29%), diffuse mesangial hypercellularity in 3 (3%), segmental mesangial hypercellularity in 41 (45%),
and more severe segmental lesions in 21 (23%) (e191).
Mesangial deposits with IgA as the predominant immunoreactant define this disease, but 23% of biopsy
specimens in the SPNSG series (e220) and 16% to 22% in the report of Levy et al. (e191) also showed capillary
wall deposits by immunofluorescence. In addition, IgG was present in 52% (e142) to 60% (e191) of specimens,
C3 in 76% (e142) to 87% (e191) IgM in 19%, C4 in 15% and C1q in 13% (e142). In addition to IgA nephropathy
and HSP nephritis, the differential diagnosis of dominant or codominant mesangial IgA deposits includes lupus
nephritis (LN), C1q nephropathy, HIV-associated glomerulonephritis, poststaphylococcal glomerulonephritis, and
combinations of IgA nephropathy with MCD, MGN, and ANCA-associated glomerulonephritis (69). Mesangial
electron-dense deposits were detected in 56 of 58 specimens in the SPNSG series, and 16% also showed small,
usually juxtamesangial subendothelial deposits, 10% showed subepithelial deposits, and 9% showed
intramembranous deposits (e220). Variations in the contour, caliber, or consistency of the glomerular basement
membrane were noted in 40% of biopsy specimens in the SPNSG series, almost exclusively in those with
relatively severe mesangial hypercellularity or segmental necrotic or sclerotic lesions by LM (e220). In an
ultrastructural study of 34 patients with IgA nephropathy, Vogler et al. noted focal and segmental attenuation,
splitting, duplication, paramesangial microaneurysms, and subepithelial protrusions of the glomerular basement
membrane—features that were more marked in specimens with relatively severe lesions by LM and capillary wall
deposits by IF microscopy (e311).
The SPNSG found a correlation of proteinuria and episodic gross hematuria with the more severe histologic and
ultrastructural lesions (e142). Levy et al. found that patients with normal glomeruli by LM presented with
macroscopic hematuria that might recur with upper respiratory infections but that proteinuria or elevation of blood
urea nitrogen or creatinine levels was transient or absent in these patients, and renal failure, nephrotic
syndrome, or hypertension did not develop. Patients with mesangial hypercellularity had a similar presentation
and course, but more significant proteinuria was noted at presentation or developed later. In contrast, end-stage
disease developed in 6 of 21 patients with more significant segmental lesions, including five of ten in whom more
than 50% of glomeruli displayed such lesions; moderate renal failure and hypertension developed in two
patients, severe hypertension in two, and significant proteinuria in three (e191). In a review of biopsy specimens
from 65 children in whom IgA nephropathy was diagnosed during a 15-year period, Welch et al. noted that renal
failure developed in only five, all of whom were severely hypertensive and three of whom were nephrotic at
presentation; and the biopsy specimens from these five patients showed crescents in 50% to 90% of glomeruli
(e322). A recent study of 250 adults and children with IgA nephropathy followed for a median of 5 years found
that mesangial and intracapillary hypercellularity, segmental glomerulosclerosis, and tubular atrophy and
interstitial fibrosis independently predicted renal outcome, but crescents did not, possibly because patients with
more severe disease were excluded. (30)
Henoch-Schönlein Purpura Nephritis

HSP is a clinical syndrome involving the skin, joints, gastrointestinal tract, and, in 20% to 50% of cases, the
kidneys. The heart, lungs, central nervous system, and muscles may also be affected. Although the incidence
peaks at 4 to 5 years, older children and adolescents seem to be at higher risk for the development of renal
disease. There are no specific laboratory tests for HSP, but titers for galactose-deficient IgA are elevated if renal
disease is present, and, serologic studies
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are useful in excluding the other leading causes of rash and renal disease in children—lupus and microscopic
polyarteritis nodosa (polyangiitis) (e6). Renal involvement in HSP is heralded by asymptomatic gross or
microscopic hematuria, but proteinuria may also be present, and in some series from referral centers, a nephrotic
syndrome is present in 50% of patients with renal disease. Renal failure develops in up to 20% of patients in
these series, but the course appears to be less ominous in unselected patients (e127). After a mean follow-up of
23 years, Goldstein and coworkers found no evidence of renal disease in 82% of children with HSP who had
presented with hematuria with or without proteinuria, but hypertension and impairment of renal function
developed in 44% of those who had presented with an acute nephritic or nephrotic syndrome (e109).
The renal lesions in HSP are similar to those in IgA nephropathy by light, IF and EM, but the glomerular disease
tends to be more severe and to more often include crescents, and the proportion of glomeruli with crescents is
the basis for the ISKDC classification of HSP nephritis (69). In a summary of three series, children with minimal
(ISKDC grade I) or purely mesangial proliferative (ISKDC grade II) lesions had a 3% chance of developing
chronic renal insufficiency or dying within 6 years, while for children with crescents in 50% or more of glomeruli
or membranoproliferative-like lesions (ISKDC grades IV, V, or VI) the risk was 35% (69). The skin lesion in HSP
is a leukocytoclastic vasculitis, and deposits of IgA are seen in the walls of blood vessels in biopsies of fresh
purpuric lesions, but they are not seen in skin specimens from patients with IgA nephropathy and may disappear
in later stages of HSP (e191). As in IgA nephropathy, the outcome is worse in patients with severe segmental
lesions and crescents (e127).
Alport Syndrome
Alport syndrome includes various combinations of lesions of the kidney, inner ear, eye, skin, smooth muscle,
platelets, and granulocytes that are caused by mutations in genes coding for type IV collagen (e116). Proceeding
from the observation that the antiglomerular basement membrane antibodies from patients with Goodpasture
syndrome did not stain glomeruli from patients with Alport syndrome, it was learned that type IV collagen in all
basement membranes is made up of a triple helix of two alpha-1 chains and one alpha-2 chain, but that with
maturation in certain basement membranes this structure is replaced by a triple helix composed of various
combinations of four other chains, alpha-3 through alpha-6, and that the genes for these chains are arranged in
head-to-head pairs on chromosome 13 (COL4A1 and COL4A2), chromosome 2 (COL4A3 and COL4A4), and the
X chromosome (COL4A5 and COL4A6) (508). Approximately 80% of cases of Alport syndrome are X-linked
secondary to mutations in the gene at Xq22 that encodes the alpha-5 chain of type IV collagen; and other
patients have autosomal recessive or, less frequently, autosomal dominant disease secondary to mutations in
the alpha-3 and alpha-4 genes on chromosome 2 (e189). The distribution of the alpha-3 through alpha-6 isomers
in the body accounts for the organs involved in Alport syndrome.
Most authorities recommend that several criteria be met before a diagnosis of Alport syndrome is assigned to an
individual or a family. Persistent unexplained hematuria; a history of nephritis, unexplained hematuria, or gradual
progression to end-stage renal disease in a first-degree relative; bilateral sensorineural hearing loss in the
2,000- to 8,000-Hz range; anterior lenticonus or other characteristic ocular lesions; macrothrombocytopenia or
granulocyte inclusions; widespread ultrastructural alterations in the glomerular basement membrane or
immunohistochemical evidence of complete or partial loss of the Alport epitope in glomerular or epidermal
basement membranes (Figure 17-26A,B); or demonstration of a mutation in one of the type IV collagen genes
listed above are examples of such criteria, but none alone is considered necessary or sufficient for a diagnosis of
Alport syndrome (e116). Hematuria is demonstrable by 5 years of age in affected boys with X-linked Alport
syndrome and in homozygotes and many heterozygotes of either sex with autosomal recessive disease, but renal
disease may not be evident until adulthood in autosomal dominant disease (e102). The progression to end-stage
renal disease is rapid in persons with autosomal recessive disease, often occurring between 5 and 15 years of
age, and these patients typically are deaf but have no ocular abnormalities (e102). The progression to end-stage
renal disease in X-linked Alport syndrome is more variable but roughly similar within kindreds, which show a
bimodal distribution of the mean age at which end-stage renal disease develops in affected members. Hearing
loss is universal, and ocular lesions are confined to “juvenile” kindreds, which have a mean age at onset of
endstage renal disease of less than 31 years. In contrast, only half of affected patients in “adult” kindreds (in
whom endstage renal disease occurs later) have hearing loss (e116). In a study of 195 families with X-linked
Alport syndrome, a genotype-phenotype correlation could be demonstrated with males (90% chance of
developing end-stage renal disease before age 30 with large rearrangements compared to a 50% chance with
missense mutations) (80), but not females (79).
Histologic findings in children under 10 may be minimal, and at any age they are nonspecific. The number of fetal
glomeruli may be increased, and there may be variable degrees of segmental or global mesangial
hypercellularity, thickening of capillary walls, tuft sclerosis, patchy tubular atrophy, red cell casts or hemosiderin
in tubular epithelial cells, and aggregates of foam cells in the interstitium (e116). Results of the standard
immunofluorescence studies are negative, an important finding in ruling out IgA nephropathy or an immune
complex glomerulonephritis, and in many (but not all) kindreds, the glomerular or epidermal basement membrane
fails to stain with fluorescein-tagged antiglomerular basement membrane antibodies obtained from patients with
Goodpasture syndrome or monoclonal antibodies to collagen IV chains (Figure 17-26C,D).
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The characteristic ultrastructural abnormalities of Alport syndrome include irregular thinning, thickening, splitting,
and a wavy intersecting lamellation known as the basket weave pattern (Figure 17-26A), often with 50-nm-
diameter electron-dense granules between lamellae. However, renal specimens obtained early in life may show
no abnormalities, and the most common observation in children is thinning of the basement membrane to less
than 150nm (Figure 17-26B) (65). The immunofluorescence studies and variable ultrastructural findings are
consistent with the hypothesis that affected basement membranes contain an abnormal type IV collagen in which
the chain that normally contains the product of the mutated gene is defective or absent, and the structural and
functional consequences of this abnormal collagen are progressive (e165).
FIGURE 17-26 ▪ Basement membrane nephropathies. A: Marked thinning, fraying, intersecting lamination, and
granularity of the glomerular capillary basement membrane are characteristic of hereditary nephritis (Alport
syndrome), but are not seen in all cases. B: Diffuse thinning of the capillary basement membrane is seen in
familial hematuria, and may be the only ultrastructural lesion in Alport syndrome. C,D: Staining for collagen IVa5
is seen along the glomerular capillary basement membrane and, to a lesser extent, Bowman capsule in control
(C) but not patient (D) glomeruli. (A,B: Lead citrate and uranyl acetate, C,D: Fluorescein isothiocyanate-
conjugated anti-collagen IVa5, original magnification 400×.)
Thin Glomerular Basement Membrane Disease

The only significant abnormality in the renal biopsy specimens of 20% to 25% of children or adults evaluated for
isolated hematuria is thinning of the glomerular basement membrane on ultrastructural study (Figure 17-26B)
(e166) The term benign familial hematuria is applied when this lesion is the only abnormality, the family has a
history of isolated hematuria that follows an autosomal dominant pattern, and affected persons do not manifest
the progressive renal disease or extrarenal manifestations of Alport syndrome (e116). The term thin glomerular
basement membrane disease describes a pathologic finding that may be familial or sporadic and may be
associated with a benign or progressive
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course (e166). Heterozygous mutations in the COL4A3 and COL4A4 genes have been found in some, but not
all, children from kindreds with benign familial hematuria (143). Furthermore, thinning of the glomerular basement
membrane may be the only abnormality in a renal specimen from a child with Alport syndrome, even after a
careful search for proteinuria and extra-renal lesions in the patient and abnormal collagen distribution in the
biopsy, underscoring the need for close clinical follow-up and consideration of type IV collagen mutation analysis
(65). Diffuse thinning of the glomerular basement membrane has also been described in the mesangial
proliferative form of childhood nephrotic syndrome (e207) and other glomerulopathies associated with mesangial
proliferation (e69).
Almost by definition, renal specimens with thin glomerular basement membrane disease are normal by light and
IF microscopy. Focal immature or globally sclerotic glomeruli, areas of tubular atrophy, and variable staining for
immunoglobulin or complement components along the glomerular capillary loop have been reported (e166);
however, global sclerosis of 25% or more glomeruli was a feature in 9 of 16 patients with thin basement
membrane disease who were subsequently reclassified as Alport syndrome variants on the basis of collagen
IVa3, a4, and a5 expression and ultrastructural study (97). The cardinal finding in this group of diseases is
diffuse thinning of the glomerular basement membrane by EM (Figure 17-26B). The widths of the lamina densa
and capillary wall increase throughout childhood, but reported measurements of these structures have shown
considerable interlaboratory variation, emphasizing the need for each facility to establish its own reference range
(65). The mean thicknesses of the glomerular basement membranes in published reports of thin basement
membrane disease have ranged from 150 to 300nm, but most pediatric series have used a cut-off of 250nm (65).
Dische has suggested that a threshold of glomerular basement membrane thickness may exist below which
hematuria occurs with increased frequency, and that physiologic variation in basement membrane thickness may
account for some cases of asymptomatic microscopic hematuria in children of school age (e82).
Loin Pain-Hematuria Syndrome

Gross hematuria is accompanied by loin pain in many adults with IgA nephropathy, but such pain is encountered
less often in children with IgA nephropathy (e150). Loin pain-hematuria syndrome refers to gross or microscopic
hematuria accompanied by unilateral or bilateral loin pain, often severe enough that opiates are required. Most
patients are women between 20 and 40 years of age, but adolescent boys and girls and an occasional younger
child are included in reported series. Subtle biochemical or renovascular lesions have been described in some
reports, and several reports have included patients with psychiatric disorders or an altered perception of pain
(e47). By LM, a renal biopsy specimen may show a slight segmental proliferation of mesangial cells, mild
interstitial fibrosis, especially near the corticomedullary junction, and minimal thickening of the walls of
intracortical arteries and arterioles. Immunofluorescence often reveals linear or flecklike staining for C3 in the
walls of arterioles, but not in the glomeruli. However, such staining in arterioles is not uncommon in specimens
from patients with hematuria of many causes. EM shows no electron-dense deposits or diagnostic abnormality of
the glomerular capillary basement membrane. Since none of these findings have any diagnostic specificity, the
pathologist's role in the evaluation of these patients is to confirm, if possible, the renal origin of hematuria and
exclude other conditions that might present with hematuria.
Glomerulopathies that Usually Present with a Nephritic Syndrome of Hypertension, Impaired

Renal Function, Hypocomplementemia, and Cellular Casts, Protein, and Blood in the Urine
The most important causes of the “nephritic syndrome” in children include postinfectious glomerulonephritis,
MPGN, and LN. These three conditions constitute the differential diagnosis of mesangiocapillary or endocapillary
proliferative glomerulonephritis because they can produce a similar histologic lesion characterized by marked
glomerular hypercellularity with accentuation of the lobular architecture and thickened capillary walls (Figure 17-
21C). However, variations related to the stage or age of the lesion do exist. A similar clinical picture can be seen
in crescentic glomerulonephritides (Figure 17-21D). Although crescents can occasionally be seen in almost any
glomerulopathy, the major differential diagnosis of crescentic glomerulonephritis in children and adolescents
includes immune complex diseases, such as postinfectious glomerulonephritis and LN, dense deposit disease,
and antiglomerular basement membrane disease, which are discussed in this section; the small-vessel
vasculitides, which are discussed in the section on renovascular diseases; and IgA nephropathy and HSP
nephritis, discussed previously (e76).
Postinfectious Glomerulonephritis
The incidence of acute glomerulonephritis following throat or skin infections with group A streptococci in the
United States and Europe has been declining for nearly 50 years, but poststreptococcal glomerulonephritis is still
a relatively common disease worldwide, especially in tropical countries (116). Renal biopsies are usually
obtained only if gross hematuria persists beyond 1 month; hypocomplementemia persists beyond 6 weeks;
hypertension persists beyond 2 months; progressive deterioration of renal function or evidence of extrarenal
disease is present; nephritis occurred within 48 hours of pharyngitis; age is less than 2 years, or there is a family
history of renal disease (e141). Infections with organisms other than group A streptococci can produce
morphologic features similar to that seen in
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poststreptococcal acute glomerulonephritis (hence the more generic term postinfectious glomerulonephritis), but
many of these organisms can also elicit other forms of glomerular disease (116).
Histologically, poststreptococcal glomerulonephritis evolves over several weeks from an endocapillary
proliferative (mesangiocapillary) (Figure 17-21C) and exudative (increased neutrophils within tufts)
glomerulonephritis to a mesangial proliferative glomerulonephritis (Figure 17-21B) with patent capillary loops and
normally thin capillary walls (e192). The immunofluorescence pattern also evolves from a coarse capillary
granular (Figure 17-22A) staining for IgG and C3, with lesser amounts of other immunoreactants, to a mesangial
granular (Figure 17-22B) staining for C3, typically without staining for other immunoreactants. Sorger et al.
observed the capillary granular pattern, which they termed starry sky, in 13 of 42 patients, typically in specimens
obtained within 2 weeks of the onset of symptoms, and noted the mesangial pattern in 19 patients who
underwent biopsy later (e289). These authors also noted a third immunofluorescence pattern—confluent lumpy
staining along capillary loops and lesser staining within and around mesangia—that they termed garland that
was observed in both early and later biopsy specimens and tended to occur in older patients (median age, 21
years) and those who presented with significant proteinuria or the nephrotic syndrome (e288). Ultrastructurally,
patients with the capillary wall (starry sky) pattern by immunofluorescence showed domed electron-dense
deposits on the epithelial side of the basement membrane over which the foot processes of visceral epithelial
cells characteristically arch (Figure 17-27). These subepithelial “humps” can be sparse to numerous and were
flattened and focally confluent in specimens that showed the garland pattern by immunofluorescence (e288). An
association between atypical humps and unfavorable outcome had also been noted in other reports (e140,e192).
Basement membrane deposits may persist for years in some patients (e13), but few if any humps are typically
seen in later biopsy specimens from children (e192). Thus, the absence of humps in a later specimen does not
exclude the diagnosis of postinfectious glomerulonephritis, and because structures consistent with humps, have
been described in other conditions, the finding of a rare hump, typically above the junction of the capillary loop
and mesangium, suggests (70), but does not necessarily establish this diagnosis.
FIGURE 17-27 ▪ Postinfectious glomerulonephritis. The foot processes of an epithelial cell arch over large
subepithelial “humps.” (Lead citrate and uranyl acetate).
Nearly all children with well-documented acute poststreptococcal glomerulonephritis recover completely (e253).
However, Lewy et al. reported persistent clinical abnormalities in 5 of 46 children, and follow-up renal biopsies
after 735 to 2,753 days demonstrated persistent mesangial hypercellularity in three of five patients,
glomerulosclerosis in three of five, and tubular injury in four of five. The patients who died in the acute phase of
disease or who developed persistent clinical abnormalities initially manifested markedly reduced renal function
and prominent cellular proliferation, exudation of leukocytes, and crescent formation. However, other patients
with equally marked reduction in renal function and equally severe glomerular lesions recovered completely.
Patients with milder clinical disease had uniformly good outcomes, and this led these authors to conclude that it
is unlikely that chronic glomerulonephritis such as MPGN evolves from mild or unrecognized acute
poststreptococcal glomerulonephritis (10, e192).
Not unexpectedly, nonstreptococcal postinfectious glomerulonephritides manifest a more varied morphology.
Staphylococcal infections often show a predominance of mesangial deposits with IgA as the dominant
immunoreactant. The glomerulonephritis associated with subacute bacterial endocarditis may be diffuse and
proliferative, but the classic lesion is a focal and segmental fibrinoid necrosis or thrombosis that evolves to
similarly distributed sclerotic lesions in glomeruli by LM, but diffuse global, predominantly mesangial and
subendothelial deposits by immunofluorescence and EM. Acute bacterial endocarditis can produce a variety of
renal lesions ranging from a proliferative glomerulonephritis, often with crescents, to interstitial inflammation to
infarction, and glomeruli show mesangial and intramembranous deposits as well as subepithelial humps that
seem to persist longer than those in poststreptococcal glomerulonephritis. The glomerulonephritis associated
with infected ventriculoatrial shunts is similar to that seen in acute poststreptococcal glomerulonephritis,
including the presence of increased numbers of neutrophils, but typically shows mesangial and subendothelial
rather than subepithelial deposits (116).
Membranoproliferative Glomerulonephritis
Type I MPGN was initially described in children by West et al. (e324) and Gotoff et al. (e113) in 1965, but it also
occurs in adults, and the median age at onset is 21 years. Type II MPGN, or dense deposit disease, was first
described by Berger and Galle in 1963 (e26) and is more common in
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children than adults, with a median age at onset of 11.5 years. The designation type III MPGN has been applied
to several lesions over the years (e48,e173,e301) but is now generally reserved for the disorder with disruption
of the glomerular basement membrane described by Strife et al. (e301) This lesion is probably lumped with type I
disease in most reports, but the frequencies of types I, II, and III MPGN in children in range from 44% to 54%,
20% to 32%, and 14% to 36%, respectively (e323). In addition to these idiopathic forms of MPGN, a glomerular
lesion essentially identical to MPGN type I is seen in the nephritis associated with infected ventriculoperitoneal
shunts, hepatitis C, sickle cell disease, and α1-antitrypsin deficiency (201).
Up to 70% of children with idiopathic MPGN present with nephrotic syndrome (e124), but a persistent nephrotic
syndrome is a poor prognostic sign (e323). Most patients have hematuria that is often gross, but asymptomatic
proteinuria or hematuria was the only sign at presentation in 65% of patients with type III and 22% of those with
type I MPGN (e152). Extrarenal abnormalities, especially partial lipodystrophy and densities in the retinal
epithelium, are seen in patients with type II MPGN (e6). Decreased levels of the third component of complement
(C3) are seen in all forms of MPGN, and recent studies suggest that MPGN II and, possibly, MPGN I are due to
dysregulation of the complement cascade due to mutations in the genes for factor H or another regulatory
protein, or stabilization of C3 convertase against these regulatory proteins by C3 nephritic factor. (99) Evidence
for a genetic basis for MPGN types I and III includes an increased incidence of the HLA haplotypes B8, DR3,
SC01, and GL02; and partial defects of the complement system, rare familial cases, and the low frequency of the
disease in African-Americans (e323).
Histologically, type I MPGN shows uniformly enlarged and hypercellular glomeruli with expanded and
hypercellular mesangia (Figure 17-21C), compressed capillary lumens, and thickened capillary walls with
segmental double contours (“tram tracks”) on silver stains. Increased numbers of neutrophils are seen in
glomeruli in 25% of cases (e157) and crescents in 10% (e169). Hyaline “thrombi,” large eosinophilic globules in
glomerular capillaries, raise the question of cryoglobulinemia and hepatitis C (e72). The interstitium shows
edema, lymphocytic infiltrates, and patchy fibrosis. Type II disease shows more variable cellularity but more
uniformly thickened capillary walls, and type III MPGN generally has a less pronounced and more variable
cellularity. Immunofluorescence microscopy in type I disease shows coarse granular staining along capillary
loops and the periphery of expanded mesangia, the “peripheral pattern” for C3 and, less often, IgM, IgG, C1q,
and IgA. Type II MPGN shows a linear or a ribbonlike staining of capillary walls and hollow rings in mesangia for
C3 (Figure 17-22D), and, less intensely and less often, for other immunoreactants (e284). Type III MPGN shows
finely granular to confluent capillary wall and central mesangial staining forC3(e301).
FIGURE 17-28 ▪ A: Type I MPGN with subendothelial electron-dense deposits and interposed mesangial cell
cytoplasm. B: Type II MPGN (dense deposit disease) is defined by irregular ribbons of electron-dense material
along the glomerular capillary basement membrane (A,B, lead citrate and uranyl acetate stain, original
magnifications ×3,000.)
The three types of idiopathic MPGN are defined by their ultrastructure. In type I, the lamina densa of the
glomerular capillary wall is normal, but numerous electron-dense deposits and cytoplasmic processes
(interposition) are seen in the subendothelial space (Figure 17-28A). The two lines of the histologic “tram track”
are the original lamina densa and the new membrane deposited between the interposed material and the
endothelial cell (e323). Mesangial deposits are infrequent, but subepithelial deposits are seen in 30% to 50% of
cases (200). Type II MPGN is characterized by extensive ribbonlike densities in the glomerular basement
membrane (Figure 17-28B), mesangia, and, in some cases, tubular basement membranes, and similar deposits
have been observed in extrarenal locations (202). Type III MPGN shows a thickened basement membrane with
subendothelial and subepithelial deposits that are less electron-dense than those in MPGN I, and silver
impregnation reveals a frayed and laminated basement membrane (e301).
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If MPGN is untreated, renal failure develops within 10 years in 50% of children, and within 20 years in 80% to
90% (e6). Crescents, sclerotic glomeruli, extensive double contours, and tubulointerstitial disease have been
associated with a poor outcome in type I MPGN (199). In the study of Habib et al., 18 of 44 children with type II
MPGN progressed to end-stage renal disease within 10 years, and end-stage renal disease developed in ten of
these children within 2 years. Factors that seemed to predict a poor outcome included nephrotic syndrome,
macroscopic hematuria, and decreased renal function at the time of presentation (e122). In contrast, only 2 of the
16 children with type II MPGN studied by the SPNSG had a rapidly progressive course, and only six evidenced
progressive disease after a mean follow-up of 10 years. Pathologic rather than clinical features best predicted
progressive disease in that report, and these included a mesangiocapillary pattern, mesangial sclerosis, and
electron-dense deposits in mesangia (e63).
Lupus Nephritis
Dubois estimated that 20% to 25% of all cases of SLE present in childhood or adolescence (e86). The most
common presenting complaints in children with SLE are arthritis, arthralgia, rash and fever, but renal, cardiac,
and central nervous system involvement becomes evident as the disease progresses (e101,e108,e171,e208),
and urinary or renal function abnormalities develop in 60% to 80% of children with SLE, usually within 2 years
from the onset of disease (133). Most patients are girls, but the female predominance may be less striking in
children under 12 than in adolescents (e171). The frequency of SLE is increased in Hispanic, Asian, and African-
American children (e188), and the course of LN is more severe in Hispanics and African-Americans, possibly
because of socioeconomic as well as biological factors (13, 32, 90). Renal involvement in SLE is heralded by
hematuria, proteinuria, and hypertension, and these findings may prompt a renal biopsy before the diagnosis of
SLE has been made. In children with an established diagnosis, renal biopsy may be performed to characterize
the extent of renal disease or response to therapy.
LN is generally categorized by some variation on the World Health Organization (WHO) classification originally
formulated in 1974 coupled with an indication of the activity and the chronicity of disease. The 2004 International
Society of Nephrology/Renal Pathology Society (ISN/RPS) Classification maintains the emphasis on the
appearance of glomeruli but incorporates information from IF and EM and includes subdesignations for activity
and chronicity. In Class I, minimal mesangial LN, glomeruli are normal by LM but have mesangial immune
deposits by IF (such findings qualified for Class IIa in the original WHO classification, in which Class I glomeruli
were normal by LM, IF, and EM, or Class Ib in the 1982 modification). In Class II in the ISN/RPS scheme,
mesangial proliferative LN, glomeruli show mesangial hypercellularity or matrix expansion without histologic
alterations of capillary 1974 WHO Class IIb, 1982 WHO Class IIa or IIb), and the ISN/RPS classification allows
very rare small subendothelial or subepithelial deposits by IF or EM in Class II. Class III, focal LN, and Class IV,
diffuse LN, show focal (<50% of glomeruli) or diffuse glomerulonephritis, respectively, typically with
subendothelial immune deposits by IF and EM, and the lesions may be active (A) or chronic (C) (or both—A/C),
and, in Class IV, segmental (IV-S) or global (IV-G) to indicate whether the majority (>50%) of affected glomeruli
show segmental or global involvement. Class V, membranous LN, is diagnosed, alone or in combination with
class III or IV, when there are subepithelial immune deposits or their sequelae over greater than 50% of the
capillary wall; and Class VI indicates global sclerosis of 90% or more of glomeruli without evidence of activity
(189). Lesions indicative of active disease include endocapillary hypercellularity, leukocyte infiltration,
subendothelial hyaline material, fibrinoid necrosis, karyorrhexis, cellular crescents and interstitial inflammation.
Lesions indicative of chronic disease include glomerulosclerosis, fibrous crescents, tubular atrophy and
interstitial fibrosis (e11).
The incidence of the various categories in published reports depends on the population studied, the indications
for biopsy, and the specific criteria used for classification, but after pooling data from several large pediatric
series and using the modified WHO classification, Lehman and Mouradian found mild or no glomerulitis in 26%,
focal proliferative LN in 25%, diffuse proliferative LN in 42%, and membranous LN in 6% (e188). Applying the
INS/RPS criteria to a group of 39 children, Marks et al. found class I in 2%, class II in 13%, class III in 15%, class
IV in 51%, and class V in 20% with 12% of cases overlapping between classes III or IV and class V (107). In a
series of 25 children with LN, Zappitelli et al. noted good correlation with clinical and laboratory parameters for
biopsies obtained at the time of diagnosis, but not for follow-up biopsies (197). Electron-dense deposits with
curvilinear patterning, so-called fingerprint deposits, and tubuloreticular aggregates in the cytoplasm of
glomerular endothelial cells (Figure 17-29) are characteristic of LN and easiest to find in class IV disease.
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Immunofluorescence microscopy reveals IgG in nearly all cases of LN, regardless of WHO class, and IgM and
IgA in most (coexpression of these three immunoreactants is referred to as a “full house”). C3 is detected in most
and C1q or C4 in many cases. Unlike IgA staining in IgA nephropathy, that in LN is generally less intense than
IgG staining. In patients without an established diagnosis, a “full house” of immunoreactants, numerous
mesangial deposits in an otherwise typical MGN, immune deposits along tubular basement membranes or in
tubular nuclei, “fingerprint deposits,” or tubuloreticular aggregates raise the possibility of SLE.
FIGURE 17-29 ▪ LN may show tubuloreticular aggregates within endothelial cells (Lead citrate and uranyl
acetate stain, original magnification ×5,000.)
Crescentic Glomerulonephritis
During the influenza pandemic of 1919, Goodpasture described the development of hemoptysis and renal failure
in an 18-year-old young man (e112), and the eponym Goodpasture syndrome was applied to the combination of
pulmonary hemorrhage and glomerulonephritis by Stanton and Tange in 1958 (e297). Linear staining for
immunoglobulin along the glomerular basement membrane was described in 1964 (e272), the role of
antiglomerular basement membrane antibody in the pathogenesis of this form of glomerulonephritis was
elucidated in 1967 (e190), and the recommendation to limit the term Goodpasture syndrome to a pulmonary-
renal syndrome caused by antiglomerular basement membrane antibodies was made in 1971 (e203).
Antiglomerular basement membrane disease accounts for only 6% (e154) to 15% (e63) of crescentic
glomerulonephritis in children; immune complex diseases account for 50% (e154) to 70% (e63) of cases; and the
small-vessel vasculitides associated with antineutrophil cytoplasmic antibodies (ANCA) account for 20% (e63) to
35% (e154) (see discussion of systemic vasculitides in the section on renovascular diseases). In patients of all
ages, 95% of biopsy specimens from patients with antiglomerular basement membrane disease contain some
crescents, and an average of 70% of glomeruli are involved. Comparable figures for other glomerulopathies are
90% of specimens and 48% of glomeruli for antineutrophil cytoplasmic antibody-associated vasculitides, 40% of
specimens and 31% of glomeruli for classes III and IV LN, 53% of specimens and 24% of glomeruli for HSP
nephritis, 27% of specimens and 24% of glomeruli for IgA nephropathy, 25% of specimens and 17% of glomeruli
in poststreptococcal glomerulonephritis, 20% of specimens and 21% of glomeruli in type I MPGN, 12% of
specimens and 17% of glomeruli in membranous LN, and 5% of specimens and 17% of glomeruli in MGN (e154).
The presence of crescents portends a worse prognosis regardless of underlying disease, with the possible
exception of poststreptococcal glomerulonephritis in children, in which some studies show no worsening of
outcome (e63); but others find that most patients with this lesion progress to chronic renal insufficiency or end-
stage renal disease (e295).
Crescents are initially cellular (Figure 17-21D) and resolve or organize into fibrocellular or fibrous forms. The
constituent cells are predominantly macrophages or epithelial cells, and the proportion of each appears to be a
function of the age of the lesion and the cause of the glomerulonephritis. Epithelial cells predominate in older
lesions and in those caused by immune complex diseases (e154). Glomerular tufts beneath crescents may be
compressed, necrotic, or sclerotic, but the better-preserved tufts in antiglomerular basement membrane disease
and the pauciimmune glomerulonephritis secondary to small vessel vasculitis are generally normal, whereas in
immune complex glomerulonephritis, they may show mesangial hypercellularity and thickening of the capillary
wall. Extensive disruptions of the capillary wall and Bowman capsule may be seen in the vicinity of crescents,
and the interstitium can show a mixed cellular infiltrate of varying intensity and distribution or patchy tubular
atrophy and interstitial fibrosis in cases of longer duration. Crescents stain brightly with labeled antibody to fibrin
in all forms of crescentic glomerulonephritis, but the staining pattern observed in the underlying glomerulus
depends on the primary disease. In antiglomerular basement disease, there is linear staining along glomerular
capillary walls for IgG and usually C3, but only rarely for IgA or IgM. In immune complex-mediated diseases, there
is granular staining characteristic of the underlying disease and in vasculitis-related crescentic
glomerulonephritis there is absent or very weak staining. By EM, all forms of crescentic glomerulonephritis can
show endothelial cell swelling, expansion of the subendothelial space, disruptions of the glomerular basement
membrane and Bowman capsule, and effacement of the foot processes of visceral epithelial cells, and in immune
complex-mediated disease, there are dense deposits. The SPNSG found a correlation between large gaps in the
glomerular basement membrane and fibrocellular or fibrous as opposed to cellular crescents, and between these
findings and a poor clinical outcome (e63).
TUBULOINTERSTITIAL DISEASES
The renal tubule, consisting of the proximal convoluted tubule, loop of Henle, and distal convoluted tubule, is
derived from the metanephric blastema through a process of elongation between the developing glomerulus and
the collecting duct. The renal interstitium is composed of extracellular matrix and two or three types of interstitial
cells whose function is poorly understood. Ordinarily, the interstitium is inapparent in the cortex, comprising less
than 10% of the volume, but it occupies progressively more volume as one proceeds through the medulla to the
papillary tip (e162). In addition to the diseases that primarily affect the tubules and interstitium, tubular injury and
atrophy and interstitial inflammation and fibrosis are components of many glomerular and vascular diseases, and
an increasing interstitial volume, which reflects tubular loss and interstitial fibrosis, is the best morphologic
correlate of deteriorating renal function and progressive renal failure (e36).
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Acute Tubular Necrosis
Isolated acute tubular necrosis (ATN) is seen infrequently in biopsy specimens because a biopsy is not
performed if the diagnosis can be established clinically. However, it is not unusual to see ATN in conjunction with
other lesions, especially in allograft biopsies performed because of a sudden decline in renal function. The two
classic categories of ATN are ischemic and toxic (e61). The former, also known as acute vasomotor
nephropathy, follows renal hypoperfusion of any cause, and in children, it most often occurs in conditions
associated with massive fluid shifts, such as shock, sepsis, and trauma. Toxic ATN is defined as dose-
dependent toxic renal injury, and in children, it is most often caused by an antibiotic, such as an aminoglycoside
or amphotericin-B, or an antineoplastic agent, such as cisplatin or ifosfamide. However, clinically many patients
have risk factors for both types, and though the two types of ATN differ in the extent and the location of injury
along the tubule, it can be difficult to make this distinction on a biopsy specimen. In renal biopsy specimens, one
initially sees swelling of tubular epithelial cells and loss of the brush border in proximal tubules (best appreciated
in sections stained with periodic acid-Schiff). Cell death is indicated by nuclear dropout, hypereosinophilia, and
apoptosis; and the cells exfoliate into the lumen along with proteinaceous material (Figure 17-30). Two key
histopathologic clues to ATN are mitotic figures in tubular epithelial cells, rarely seen if there is not tubular injury,
and ectasia of tubular lumens. One may also see casts and refractile crystals in distal tubules, mild interstitial
edema, mononuclear cell infiltration, and accumulation of nucleated cells in the vasa recta (e236).
Interstitial Nephritis
Inflammation of the renal interstitium is known as interstitial nephritis or tubulointerstitial nephritis because
extension of inflammatory cells into the epithelium of tubules (tubulitis) and associated tubular injury are
frequently present. Such inflammation is most often caused by infection or a drug, but it is also the renal lesion in
obstructive and reflux uropathies and in several immunologically mediated metabolic and familial diseases, as
well a cellular rejection of a renal allograft. Acute interstitial nephritis is characterized by interstitial edema and an
infiltrate of activated lymphocytes, predominantly T cells, variably admixed with neutrophils and eosinophils. This
condition is rare in childhood, but a compilation of the data on 55 patients in reports from Pittsburgh (13 patients)
(e92), Tokyo (21 patients) (89), and Serbia (21 patients) (123) reveals that 45% of cases could be ascribed to
infections (predominantly streptococci in the report from Pittsburgh, Yersinia pseudotuberculosis in the report
from Tokyo, and hantavirus in the report from Serbia), 13% to drugs, 20% to the tubulointerstitial nephritis and
uveitis syndrome, and 22% were unclassified. Presenting symptoms included fatigue, fever, gastrointestinal
disturbances and weight loss, laboratory studies documented acute renal failure with low urinary specific gravity
or glucosuria suggesting tubular dysfunction, and in many of the cases the diagnosis was initially made on the
renal biopsy. Pyelonephritis is a subset of interstitial nephritis, caused by hematogenous or ascending bacterial
infection, in which the collecting system is involved in addition to the interstitium. Chronic interstitial nephritis is
characterized by interstitial fibrosis, tubular atrophy, and an infiltrate of small lymphocytes. Plasma cells,
macrophages, and granulomas may be seen in acute or chronic interstitial nephritis (e65). Renal biopsy is
necessary to establish the diagnosis of interstitial nephritis, but because the histologic response is not specific,
clinical and laboratory findings must be correlated to determine a cause.
FIGURE 17-30▪ATN is characterized by variable ectasia of lumina and necrosis and desquamation of epithelial
cells. (Jones methenamine silver, original magnification 40×.)
Interstitial Nephritis Caused by Infectious Agents

It seems that almost any organism can cause acute interstitial nephritis. Historically, β-hemolytic streptococci
were the most important bacteria and measles virus was the most important virus associated with this condition
(e160). However, these are examples of reactive interstitial nephritis in which organisms do not directly infect the
kidney, and other organisms with a similar pathogenesis include Brucella, Legionella, Yersinia, Epstein-Barr
virus, HIV, Leishmania donovani, and Toxoplasma gondii. In contrast, Escherichia coli, Staphylococcus aureus,
invasive streptococci, Leptospira, fungi, mycobacteria, rickettsiae, cytomegalovirus, herpes simplex virus, Asian
and European hantaviruses, BK polyomavirus, and adenovirus do infect the kidney. E. coli may cause acute
pyelonephritis (see later) or, in rare circumstances, malakoplakia of the kidney. Bacteria and fungi can be
demonstrated with histologic stains; cytomegalovirus, herpes simplex virus, BK polyomavirus, and adenovirus
produce characteristic inclusions and can be identified with specific immunohistochemical stains or molecular
probes.
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Rickettsiae require specific IF staining and hantavirus infection must be diagnosed serologically (e54,e65).
Acute pyelonephritis is a clinical diagnosis made in an infant with fever and evidence of a urinary tract infection
or in an older child with significant bacteriuria, systemic symptoms, or renal tenderness. Hematogenous infection
may occur in the first 2 months of life, but thereafter acute pyelonephritis results from ascending infection. The
usual pathogen is E. coli, but Klebsiella, Proteus, Pseudomonas, Enterobacter, and Enterococcus species,
group B or group A streptococci, and coagulase-negative staphylococci can also cause urinary tract infections
(UTIs) in infants and children (e136). The pathologic features of acute pyelonephritis include patchy interstitial
infiltrates of neutrophils that may form abscesses and eventually involve tubules with the formation of white blood
cell casts in the urine. However, because prompt clinical diagnosis and treatment typically lead to resolution,
these lesions are only rarely encountered in pathology specimens.
Chronic pyelonephritis is characterized by inflammation and fibrosis that involves the pelvicalyceal system in
addition to the interstitium, features shared with the analgesic and sickle cell nephropathies but not other forms
of interstitial nephritis. Failure to make that distinction in autopsy series in the past led to an overestimate of the
likelihood that urinary tract infection would progress to chronic renal disease (e260). The relationship between
vesicoureteral reflux and renal scarring has also been called into question by cortical imaging studies. Scans
performed with technetium 99mTc dimercaptosuccinic acid (DMSA) have a sensitivity of 89% and a specificity of
100% for histologically confirmed pyelonephritis in studies in piglets (which have a collecting system similar to
that of humans), and results are positive in 50% to 85% of children with a clinical diagnosis of acute
pyelonephritis. Fifty percent of these lesions resolve on imaging studies in 4 to 6 months, and the remainder
appear to develop into segmental parenchymal scars. Vesicoureteral reflux was present in 25% to 40% of
children with acute pyelonephritis and positive DMSA scan findings, but renal scarring occurred as often in
patients without as in patients with vesicoureteral reflux (e136). The fact that sites of renal scarring conformed to
the distribution of composite papillae, which have a more patulous pore than simple papillae, supports the notion
that scarring is caused by intrarenal reflux (e256). However, many children have parenchymal scarring at the
time of their first imaging study, which suggests that they had an earlier unrecognized infection or that damage
might have occurred in utero (e262). In kidneys that are small and scarred as a consequence of obstruction, the
severity of inflammation and scarring often varies markedly between renal lobules, and when dysplastic features
are also present, such as islands of cartilage and primitive collecting ducts, early intrauterine reflux seems
certain. Congenital or acquired intrarenal reflux, rather than segmental hypoplasia, is the explanation for the
segmental scarring in Ask-Upmark kidney (e7). Recognition of intrauterine vesicoureteral reflux and dysplasia is
important because children with such lesions are more likely to develop hypertension and end-stage renal
disease than are the renal scars acquired as a result of postnatal obstructive pyelonephritis (e136). Sclerotic
glomeruli are seen in scarred areas, and FSGS can develop in the remaining glomeruli as a result of
hyperperfusional injury.
Complications of acute pyelonephritis that are unusual in children include pyonephrosis and perinephric
abscess. In pyonephrosis, an acute inflammatory exudate fills the renal calyces and pelvis because of a high-
grade obstruction, and perinephric abscesses develop when the inflammation penetrates the renal capsule.
Complications of chronic urinary tract infection that are also uncommon in children are xanthogranulomatous
pyelonephritis and malakoplakia. Xanthogranulomatous pyelonephritis, which occurs in the setting of urinary
obstruction or stone disease, presents clinically in affected children with abdominal pain, fever, weight loss, and
anorexia, often with a palpable flank mass, and is characterized by orange-yellow foci or distinct masses that are
composed of foamy macrophages, neutrophils, lymphocytes, plasma cells, and multinucleated giant cells with
frequent calcification (19,e62,e128). Malakoplakia, in which yellow-brown nodules composed of foamy
macrophages contain round laminated Michaelis-Gutmann bodies, has been described in the urinary bladder
(165), kidney (72), colon (87), soft tissues and bone (35), and skin (e96) in children.
Drug-Induced Interstitial Nephritis

Many commonly used therapeutic agents can cause an acute interstitial nephritis. This allergic reaction is
generally unpredictable and may be associated with other systemic symptoms. Antibiotics, especially β-lactams
and NSAIDs, are most often implicated, but other antibacterial and antiviral agents, anticonvulsants, and diuretics
have produced interstitial nephritis (e65). Other drugs, including some of the above-listed agents, can produce
glomerular, tubular, or renovascular lesions. Only a minority of patients with druginduced interstitial nephritis
have eosinophilia of blood or urine, and eosinophils, if present, usually constitute 10% or fewer of the infiltrating
cells in biopsy specimens (e224). They are most often seen in reactions to antibiotics, especially methicillin, and
least often in reactions to NSAIDs and cimetidine. The majority of cells are T lymphocytes. Neutrophils and
basophils are rare. Epithelioid granulomas were initially observed in reactions associated with sulfonamides but
have subsequently been reported in association with numerous drugs (e65) and have been described in children
(179).
Immune-Mediated Tubulointerstitial Nephritis

Antitubular basement membrane antibodies are usually identified in the context of glomerular or interstitial
disease. However, Helczynski and Landing found antitubular basement membrane antibodies in 3 of 13 cases
originally classified as nephronophthisis/medullary cystic disease and
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suggested that they were the cause of the renal injury (e135). Similarly, tubulointerstitial nephritis with granular
deposits of immunoglobulin and complement components along tubular basement membranes or in the
interstitium is usually seen in conjunction with an immune complex disease that also affects glomeruli, most
commonly SLE; however, rare cases of apparent primary immune complex-mediated tubulointerstitial nephritis
have been reported in children and adults (e91,e172). The predominant cell in most interstitial nephritides is the
T-lymphocyte, but a primary abnormality of T-cells is suspected in tubulointerstitial nephritis with uveitis (66).
Because uveitis and interstitial nephritis are found in Sjögren syndrome, Behçet syndrome, sarcoidosis, and
several infections, these conditions must be excluded before a diagnosis of tubulointerstitial nephritis with uveitis
is made (e65).
Tubulointerstitial Nephritis in Hereditary Diseases

Tubulointerstitial nephritis is an important component of Alport syndrome (e116), Alagille syndrome
(arteriohepatic dysplasia) (e149), and Bardet-Biedl syndrome (mental retardation, pigmentary retinopathy,
Polydactyly, obesity, and hypogenitalism), formerly known as Laurence-Moon-Biedl syndrome (e271). The
nephronophthisis-medullary cystic disease complex is likely a combination of several diseases, including two
autosomal recessive conditions— familial infantile nephronophthisis, which maps to 9q22 to 31 (e229), and
familial JNPH, which maps to 2q13 (e230)— and an adult-onset autosomal dominant medullary cystic disease
that maps to 1q21 (e209). All forms are characterized by shrunken kidneys with cysts up to 2.0 cm at the
corticomedullary junction or in the medulla and extensive tubular atrophy and interstitial fibrosis with a variable
infiltrate of lymphocytes and plasma cells (e54). Colvin and Fang have summarized the reports of five families
with an HLA-linked familial interstitial nephritis and 10 infants with a progressive tubulointerstitial nephropathy
that may or may not have a genetic basis (e65).
RENOVASCULAR DISEASES
Hemolytic Uremic Syndrome
HUS is the most common cause of acute renal failure in childhood (e287). In 1925, Moschcowitz described a 16-
year-old girl with clinical and pathologic features of what we would now recognize as thrombotic
thrombocytopenic purpura (e217). In 1955, Gasser et al. introduced the term HUS to describe the disease they
reported in five children with hemolytic anemia, thrombocytopenia, and acute renal failure (e105). Subsequently,
Riley et al. reported the association of two outbreaks of hemorrhagic colitis with the rare E. coli O157:117
serotype (e261), and Karmali et al. recognized the association between toxins produced by E. coli and sporadic
cases of HUS (e164). Microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure constitute
the diagnostic criteria for HUS, and approximately 90% of pediatric cases are preceded by a diarrheal prodrome.
Enteropathogenic E. coli have been linked to 75% of cases of postdiarrheal HUS. These organisms
asymptomatically inhabit the intestines of cattle, and contaminated beef products are implicated in most
epidemics. However, most cases of HUS occur sporadically and may be acquired by drinking water or
consuming products contaminated by cattle feces and by person-to-person spread (e287). Damage to colonic
tissue is enhanced by an influx of neutrophils attracted by the release of cytokines from colonic epithelial cells
when the toxin binds to these cells. The toxin is transported in the plasma or on the surface of monocytes or
platelets and binds to receptors on susceptible cells, and is then internalized and causes the death of these
cells. Toxin binds to glomerular endothelial and mesangial cells and glomerular and tubular epithelial cells, which
release cytokines that upregulate the expression of receptors on endothelial cells. Cell death occurs as a result
of inhibition of protein synthesis or apoptosis, the endothelium becomes procoagulant, and a thrombotic
microangiopathy (TMA) ensues (111). Involvement of brain, liver, pancreas, heart, lung, skeletal muscle, skin,
parotid gland, and retina has been reported in HUS, but central nervous system dysfunction occurs in one-third
of cases, and central nervous system hemorrhage is the most common cause of death. Interindividual variations
in the presence or density of receptors, especially in the brain, may account for the seemingly unpredictable
extrarenal complications of HUS (e287).
Three major categories of pathologic lesions have been described in the kidney in HUS—cortical necrosis,
glomerular TMA, and arterial TMA (e125). Tubular and interstitial injury are most likely secondary, possibly
through endothelial injury in peritubular capillaries (141), and striking degrees of apoptosis have been described
in tubular epithelial cells (178). Cortical necrosis is discussed in a subsequent section of this chapter, but the
histologic lesion noted in the noninfarcted portions of the renal cortex in patients with cortical necrosis associated
with HUS is usually glomerular TMA (e9). In glomerular TMA obliteration of the capillary loops by a combination
of fibrin and platelet thrombi result in the fragmentation of red blood cells (Figure 17-31), and swelling, necrosis,
and detachment of endothelial cells and expansion of the subendothelial space by electron-lucent “fluff.”
Neomembrane beneath the endothelial cell and the normal lamina densa on the other side of the fluffy material
may impart a double contour to the capillary wall in histologic sections stained with silver methenamine. IF
microscopy shows granular deposits of fibrin-reactive antigen, apparently within capillary loops. Mesangia often
show a decreased amount of matrix (mesangiolysis) but are usually normocellular. Early arterial TMA is
characterized by narrowing of the lumens of interlobular (intracortical) arteries and arterioles by endothelial cell
swelling and fibrinoid
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mural necrosis, and later lesions show intimal fibrosis or laminar proliferation (“onion skinning”). Red blood cells
and fibrin thrombi may accumulate in the lumina and walls of affected vessels at any stage. Glomerular and
arterial TMA can be present in the same biopsy specimen, but usually one or the other predominates, and the
most common glomerular lesions seen with arterial TMA are collapse or retraction of the glomerular tuft and
“paralysis” (exaggerated congestion) of capillary loops, presumably reflecting obstructive lesions in afferent and
efferent arterioles, respectively. Habib et al. noted that in cases in which arterial TMA predominated, the
superficial glomeruli were collapsed, but glomerular TMA could be seen at deeper levels (e125).
FIGURE 17-31 ▪ Hemolytic uremic syndrome. Fibrin thrombi and fragmented red blood cells occlude glomerular
capillaries, and fibrin is seen in an areriole. (Hematoxylin and eosin, original magnification 400×.)
The prevalence of one or another pathologic lesion may be a function of the age of the patient or the evolution of
the disease. In the series of 70 consecutive patients reported by Habib et al., 55 were less than 28 months old
and 15 ranged in age from 3 to 16 years. Of the 55 infants, 45 had a preceding diarrheal illness, 42 had oliguria
(21 for longer than 7 days), and 18 had central nervous system symptoms. Nine of the ten patients with cortical
necrosis were infants (the tenth patient was 3 years old), and 26 of the 29 patients with predominantly glomerular
TMA were infants. Of the 15 older children, five had a preceding diarrheal illness, seven had oliguria (all
episodes lasted for 6 days or longer), none had central nervous system symptoms, and 10 of the 13 patients with
predominantly arterial TMA were older children. Two of the nine infants and all ten of the older children who did
not have a diarrheal prodrome had arterial TMA and progressed to renal failure, and four of the five older
children with a diarrheal prodrome had glomerular TMA or cortical necrosis and recovered. Oliguria and
hypertension occurred whether glomerular or arterial TMA predominated; anuria was observed only if glomerular
TMA was present (and patients with a greater proportion of glomeruli so involved had anuria of longer duration
and a worse outcome). Hypertension was more severe with arterial TMA regardless of the patient's age (e125).
Arterial TMA is the predominant lesion in adults with HUS (95). In a series of biopsy or autopsy specimens from
24 children, 6 months to 12 years of age at presentation, glomerular TMA was the predominant lesion in 8 of 15
specimens obtained within 16 days of hospitalization, and arterial TMA was seen in all nine obtained 17 days to
3 months after presentation, but not in any of the specimens obtained earlier (e9). In follow-up biopsy specimens
obtained after 1 year, patients who had had predominantly glomerular TMA showed varying degrees of
glomerulosclerosis but generally normal vessels, whereas those who had had predominantly arterial TMA
showed mainly vascular lesions (e125).
Atypical HUS may follow nonenteric infections, such as streptococcal pneumonia, and has been described as a
complication of other glomerulopathies, several drugs, pregnancy, bone marrow transplantation, neoplasms,
collagen vascular disorders, and HIV infection (e287). Many of these patients have mutations in the complement
regulatory proteins—membrane cofactor protein (MCP), complement factor H (CFH), and factor I (IF), and
patients with MCP mutations have a better prognosis and more favorable outcome following transplantation than
do those with CFH or IF mutations (85). Factor H deficiency is also found in some cases of familial HUS (e316)
and deficiency or inhibition of Factor 11 is implicated in dense deposit disease (99). HUS has been reported to
recur in transplants in up to 41% of patients (e133), but was not observed in any of 62 children whose primary
disease was Shiga-toxin associated HUS (51), suggesting that recurrences develop in patients with a genetic
predisposition to HUS.
Renal Involvement in Systemic Vasculitides

Renal involvement in vasculitis in children is seen most often in HSP, microscopic polyarteritis (polyangiitis),
Wegener granulomatosis, Churg-Strauss syndrome, and (macroscopic) polyarteritis nodosa, but kidney disease
can also occur in Kawasaki disease and Takayasu arteritis (e264). Jennette noted that no classification of the
systemic vasculitides has been universally accepted but outlined a functional approach to the diagnosis of those
that involve the kidney. Giant cell arteritis and Takayasu arteritis cause granulomatous inflammation in larger
arteries and may involve the aorta and main renal artery, with subsequent luminal narrowing and development of
hypertension. Macroscopic polyarteritis nodosa and Kawasaki disease cause segmental necrosis in medium-
sized vessels, including the interlobar and the arcuate arteries in the kidney, which can result in renal
hemorrhage, ischemia, or infarction. Small-vessel vasculitis is characterized by a focal and segmental
glomerulonephritis, often with segmental necrosis of the glomerular tuft and segmental cellular crescents. Such
glomerular lesions are further categorized by IF microscopy. If immunoglobulin and complement components are
deposited in glomeruli, the likely diagnosis
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is lupus or another immune complex-mediated vasculitis, Henoch-Shönlein purpura nephritis, or cryoglobulinemic
vasculitis. If immunoglobulin or complement components are not present, the so-called pauci-immune
glomerulonephritides include microscopic polyangiitis (microscopic polyarteritis nodosa), Wegener
granulomatosis, and Churg-Strauss syndrome (e54).
Macroscopic polyarteritis nodosa can occur in children, and Kawasaki disease is a childhood illness with a peak
incidence in the first year of life (e81,e264). Kawasaki disease preferentially involves the coronary arteries, but
mediumsized renal arteries are the next most frequently affected site (e226), and it can also cause renal artery
stenosis (48, 53). The early histologic lesion in renal arteries in both these diseases involves the media, but in
polyarteritis nodosa it is characterized by fibrinoid necrosis, whereas in Kawasaki disease, medial edema with
myocyte degeneration, subintimal edema, and leukocytic infiltration are seen (e154). Microscopic polyangiitis,
Wegener granulomatosis, and Churg-Strauss syndrome share many clinical features, and most affected patients
have ANCA antibodies. Recent studies suggest that these diseases develop as a result of molecular mimicry
when antibodies directed toward an epitope on an infectious agent, such as FimH on fimbriated bacteria, cross
react with lysosomal membrane protein-2 and cause pauciimmune focal necrotizing glomerulonephritis (83), and
there has been a single case report of pulmonary hemorrhage and renal disease in a newborn infant due to
transplacental passage of ANCA antibodies (11).
Ear, nose, and throat involvement is more common in Wegener granulomatosis, and Churg-Strauss syndrome is
characterized by allergic rhinitis or asthma and peripheral blood eosinophilia (e193). Histopathologically, all three
conditions produce segmental fibrinoid necrosis that can involve glomerular and alveolar capillaries, arterioles in
many organs, and venules in the skin and sinuses (e154). As noted above, the most common lesion in renal
biopsy specimens is segmental necrosis of the glomerular tuft with crescent formation, and fluorescent antibody
and ultrastructural studies show no immune complexes, a key negative that excludes the immune complex
vasculitides that may have similar histologic features. A minority of specimens show fibrinoid necrosis of
interlobular arteries, and the predominant tubulointerstitial lesion is periglomerular inflammation. Interstitial
eosinophils suggest, but are not diagnostic of, Churg-Strauss syndrome (e154).
Renal Vein Thrombosis

Renal vein thrombosis occurs in the fetus or the neonate under conditions of dehydration, sepsis, maternal
diabetes, birth asphyxia, or polycythemia (e138), and less frequently in the infant or the older child with nephrotic
syndrome (e60) or leukemic hyperleukocytosis (e222). It may present as a flank mass or with hematuria,
hypertension, or renal failure, and it can be readily diagnosed by renal ultrasonography (e138). Entrapment of
the left renal vein between the aorta and the superior mesenteric artery (nutcracker phenomenon) may be a
cause of renal congestion and postural proteinuria (e187). Grossly, the involved kidney is enlarged,
hemorrhagic, and friable. Histology reveals intense congestion of veins and capillaries in the interstitium,
interstitial edema or hemorrhage, variable degrees of necrosis of tubular epithelial cells, and margination of
leukocytes in glomerular capillaries.
Renal Artery Stenosis

Renal artery stenosis in children is most often caused by fibromuscular dysplasia (175). It has also been reported
in neuro-fibromatosis, Takayasu arteritis, Williams syndrome (e70), Kawasaki disease (48, 53), and Alagille
syndrome (e24). In addition, renal artery thrombosis has been reported in a dehydrated infant (e93).
Fibromuscular dysplasia, or renal artery dysplasia, typically affects girls and women in the second or the third
decade but can be seen in younger children. It is classified according to the layer of the vessel wall that is
affected, but nearly 90% of cases show medial fibroplasia with aneurysms in which a longitudinal section of the
artery shows thickened fibrotic ridges alternating with almost fullthickness defects resulting from a loss of smooth
muscle and elastic laminae, or perimedial fibroplasia in which much of the outer portion of the media is fibrotic
(e196). Renal artery stenosis in neurofibromatosis may be caused by compression by an encircling
neurofibroma, adventitial or intimal compression in larger vessels by a proliferation of Schwann cells, or
mesodermal dysplasia by nodular proliferations of smooth muscle cells in the intima or media of smaller vessels,
and hypertension in neurofibromatosis may also be due to proliferative lesions in the aorta causing coarctation,
or to an associated pheochromocytoma (e237).
Renal Cortical Necrosis and Papillary Necrosis

Renal cortical necrosis occurs in response to a sudden and sustained loss of renal perfusion caused by arterial
thrombosis, as in the HUS, or shock, as in acute blood loss, overwhelming sepsis, or severe perinatal asphyxia.
It is characterized by coagulative necrosis. Thrombi in glomeruli suggest that a TMA, such as disseminated
intravascular coagulation or HUS, may be present, but correlation with clinical information is usually necessary to
determine a cause.
Papillary necrosis in adults is usually a complication of analgesic abuse or diabetes, but it has been reported,
often in conjunction with renal cortical necrosis, in neonates following asphyxia or shock (e4), infants following
gastroenteritis (e59), and children with sickle cell disease (e202), disseminated candidiasis (e307), Wegener
granulomatosis (e317), and meningococcal sepsis (59). The affected papillary tips are grossly yellow and show
coagulative necrosis with a neutrophilic infiltrate at the junction with viable tissue, and necrotic papillae may
slough into the collecting system and cause acute obstruction.
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Radiation Nephritis
Radiation therapy can cause acute or chronic renal injury. Fibrinoid necrosis in arteries and arterioles
progresses to subintimal fibrosis, which may be the only clue that the associated glomerulosclerosis, tubular
atrophy, and interstitial fibrosis were caused by radiation (e447).
Bartter Syndrome
Bartter syndrome is an unusual secondary hyperaldosteronism in which patients have hypokalemic alkalosis with
hypercalciuria and hyperreninemia but normal or low blood pressure. The characteristic renal lesion is
hyperplasia of the juxtaglomerular apparatus in the hilum of the glomerulus, which is markedly enlarged and
shows more than the allowable eight cells (e237). Both sporadic and familial forms have been described. The
latter have an autosomal recessive pattern of inheritance and have been mapped to the region of the Na-K-2Cl
cotransporter gene at 15q15 to 21 (e17).
RENAL NEOPLASMS
During the past 40 years, cooperative groups have been remarkably successful at targeting pediatric renal
tumors that comprise only 7% of all childhood cancers. They have enabled the development of accurate
diagnostic criteria, stage and histology-based therapeutic stratifications, and appropriate surgical techniques. In
addition, they have demonstrated that irradiation in conjunction with several active chemotherapeutic agents are
effective. The overall result has been a dramatic improvement in the prognosis for most patients with WT (the
most common pediatric renal tumor), from approximately 8% at the beginning of the century to approximately
50% in 1960 to greater than 90% in 2000. Most children in European countries are registered as patients in the
International Society of Pediatric Oncology cooperative group protocols, which rely on the use of preoperative
neoadjuvant chemotherapy and the provision of postoperative chemotherapy based on pathologic response. In
contrast, the pediatric cooperative groups centered in North America have favored primary nephrectomy, with
postoperative chemotherapy based on pathologic analysis of untreated tumors. Although these two approaches
are difficult to compare, both have met with similar success in treating children with WT. During the 40 years of
its existence, the National Wilms Tumor Study (NWTS), currently enrolling 85% of all new cases diagnosed in
North America, has contributed greatly to the increase in long-term survivorship. The results of the NWTS clinical
trials are widely published, and many complete detailed reviews are available. The pathologist seeking
guidelines for managing pediatric renal tumor specimens is referred to any of the current recommendations by
Perlman (134, 135).
The classification of pediatric renal tumors and their relative percentages are given in Table 17-6. As more is
being discovered about the underlying genetic defects in these tumors, classifiers based on gene expression
provide diagnostic confidence and accuracy greater than that of pathologic analysis alone; however, these have
to be used in the appropriate histopathologic context (76). This chapter only summarizes the salient features of
these rare neoplasms, the study of which has answered many questions also relevant to other neoplasms.
Table 17-6 ▪ PRIMARY RENAL TUMORS OF CHILDHOOD
Tumors Relative
Percentage
WT, favorable histologya 80
Anaplastic WT 5
Mesoblastic nephroma 5
Clear-cell sarcoma 4
Rhabdoid tumor 2
Miscellaneous 4
Neuroblastoma
Peripheral neuroectodermal tumor
Synovial sarcoma
RCC
Angiomyolipoma
Lymphomab <1
aIncludes cystic,
partially differentiated nephroblastoma, and cystic nephroma, which
together comprise fewer than 5% of cases of favorable WT cases
bIncludes only cases presenting as renal

tumors. In many patients with
leukemia/lymphoma, renal lesions develop later.
Nephroblastoma (Wilms Tumor)

Nephroblastoma is one of the most common malignant, solid, extracranial tumor of childhood, with an incidence
of 1/10,000 white children (the incidence is higher among blacks and lower in Orientals) (e300). The estimated
yearly occurrence of WT is 400 to 500 cases in the United States with only 15 cases a year in the Chicago area
for instance (134, 135). It is slightly more common in girls (e56), in whom it tends to present at an older age
(mean age, 36 months in boys versus 42 months in girls) (e221). It is uncommon in neonates and infants and is
only occasionally reported in adults. Most cases of “adult WT” are primitive neuroectodermal tumors, synovial
sarcomas, or metanephric adenomas. Approximately 10% of nephroblastomas develop in association with one of
several well-characterized dysmorphic syndromes (e.g., WT, aniridia, genitourinary malformation, mental
retardation, the WAGR syndrome; Denys-Drash syndrome, a syndrome characterized by mesangial sclerosis,
pseudohermaphroditism; Beckwith-Wiedemann syndrome, characterized by hemihypertrophy, macroglossia,
omphalocele, and visceromegaly). Five percent of cases are bilateral, with the bilateral tumors more likely to be
associated with one of these syndromes as well as with nephrogenic rests. A positive family history is found in
2% of patients with WT (e42).
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Molecular and Cellular Biology
Advances in the genetics of renal tumors are driving diagnosis and therapy stratification (29). The genetics of
WT are very complex with multiple genetic events (some mutually exclusive) leading to tumor formation.
1. 1. WT1: The first genetic locus was identified in patients with the WAGR syndrome, which carries a 30% risk
of developing WT (135). Abnormalities involving WT1 are consistently found in the tumors of WAGR patients
as well as in patients with Denys-Drash syndrome, which carries a 90% risk of nephroblastoma. Different WT1
abnormalities are associated with these different syndromes. WT1 is an important regulatory molecule
involved in cell growth and development. It is expressed in a tissue-specific manner. In the developing embryo,
WT1 expression is found primarily in the urogenital system. In adult tissues, WT1 expression is found in the
urogenital system, central nervous system, and in tissues involved in hematopoiesis, including the bone
marrow and the lymph nodes. The WT1 gene is located in the p13 region of chromosome 11. WT1 encodes a
zinc finger DNA-binding transcription factor that is essential for normal genitourinary development, and
mutations or deletions are found in 15% of sporadic cases of WT. An additional one-third of all
nephroblastomas show loss of heterozygosity (LOH) at this locus. β-catenin is a cellular adhesion molecule
that promotes overexpression of the c-myc and cyclin D1. Activating mutations in the β-catenin gene (CTNNB1
on chromosome 3p22) have been detected in 15% of patients with WT. There is a strong correlation between
reduced expression of the WT1 gene and the β-catenin mutation (185). However, the majority of WT express
wild-type WT1, sometimes to high levels. Furthermore, in WT that express wild-type WT1, it is not known
whether the persistent expression of WT1 contributes to the development of the disease or is just a reflection
of tumor ontogeny (148). Identical WT1 mutations and LOH patterns have been reported in both
nephroblastomas and their associated nephrogenic rests. This suggests that WT1 inactivation may result in
the formation of a nephrogenic rest and that at least some nephroblastomas are the result of subsequent
genetic events occurring in a nephrogenic rest (135).
The uncontrolled growth of cancer cells can be due to the loss of function of tumor suppressors and/or the
activation of oncogenes. Although these are opposite functions that would be intuitively mutually exclusive for
a single protein, evidence is emerging that one protein can exhibit both properties under different cellular
conditions. An example of this is the oncogene Myc.1. The WT1 protein has a similar dual behavior depending
upon the cell type in which it is expressed. There is evidence that WT1 can behave either as a tumor
suppressor or an oncogene in the development of the malignancies (195). The presence or absence of
regulatory protein partners (e.g., Par-4, p53, EGFR, FGFR1) may account for the variable behavior of WT1 in
different malignancies. In addition to protein-protein interactions, other mechanisms that can affect the function
of WT1 include alternate splicing, usage of alternate promoters, and posttranslational modification of WT1
(113, 195). Adding to the complexity of WT1 is the fact that it has multiple layers of regulatory activity that are
both DNA- and RNA mediated (46).
2. 2. WT2: The second locus, 11p15 (WT2), is associated with Beckwith-Wiedemann syndrome, which carries a
5% risk of developing WT (173). The WT2 locus, comprising the two independent imprinted domains
IGF2/H19 and KIP2/LIT1, can undergo maternal deletion or alterations associated with imprinting (160). Some
functions of this gene are related to IGF-2, which encodes embryonal growth factor (185).
3. 3. FWT1 and FWT2: Approximately 2% of WT patients have a family history of WT. Familial WT cases
generally have an earlier age of onset and an increased frequency of bilateral disease, although there is
variability among WT families, with some families displaying later than average ages at diagnosis. Only a
minority of tumors carries detectable mutations in WT1, and it can be excluded as the predisposition gene in
most WT families. Two familial WT genes have been localized, FWT1 at 17q12-q21 and FWT2 at 19q13.4;
lack of linkage in some WT families to either of these loci implies the existence of at least one additional
familial WT gene (154).
4. 4. WTX: Recently, a gene located at Xq11.1, and named WTX, was shown to be inactivated in WTs (149).
WTX inactivation appears to follow a “one hit hypothesis” since males only have one copy, and females only
need to lose the copy on the active × chromosome. Onehit inactivation of a tumor-suppressor gene on the ×
chromosome is a departure from the traditional biallelic Knudson model and has been postulated but never
documented until this study. WTX appears to participate in the WNT signaling pathway and to promote the
ubiquitination and degradation of β-catenin (105, 126). Previously WTX mutations, both small deletions and
point mutations, were observed in 15/51 (29.4%) of WTs, and the mutation frequency was approximately
equal in males and females (149). This study also noted that WTX alterations were never seen in tumors
carrying mutations in either WT1 or CTNNB1. The implication of these data was that the etiology of
approximately 50% of WTs involved mutations in either WT1 or WTX. A more recent study assessed 125
tumors and showed that WTX alterations were approximately equally frequent in WTs with mutations in WT1
and/or CTNNB1 and in tumors with no mutation in either WT1 or CTNNB1, and that WTX mutations occurred
with about the same frequency as WT1 mutations. Thus, about one-third of tumors carry mutations at WT1,
CTNNB1, and/or WTX (155).
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5. 5. p53: The tumor-suppressor gene p53 on chromosome 17p is thought to play a role in a subset of patients
with WT. The high correlation of p53 mutations and anaplastic WT suggests that p53 alterations are required
for progression to the anaplastic phenotype (88, 132). They have also been shown to correlate with
recurrence/metastasis in tumors that are not anaplastic (73,e180).
6. 6. LOH 1p, 11p, and 16q: Grundy and Coppes (e117) studied the relationship of tumor-specific LOH and
phenotype in a total of 286 cases enrolled in the NWTS. LOH has been thought to represent a “second hit”
affecting a tumor-suppressor gene (loss of the remaining, normal allele following mutation or deletion of the
first allele). Preliminary analysis of their data suggests an association between LOH at 11p, age at diagnosis,
and histopathologic grade. A young age of the patient at diagnosis was observed for tumors with LOH at
11p13, and these tumors were less likely to have anaplastic histology (e117,e118). Despite a favorable
outcome for most patients with favorable histology WT, the LOH for chromosomes 1p and 16q is an adverse
prognostic factor (62). High telomerase expression is also an adverse prognostic factor in favorable histology
WT (42). The presence of LOH for chromosomes 1p and 16q will direct therapy stratification among favorable
histology Wilms tumor patients in the current COG renal study (AREN0532, AREN0533) (134).
7. 7. Other loci, including 1q, 2q, 7p, 9q, 14q, and 22, have also been implicated in the etiology of Wilms tumor
through studies of LOH, loss of imprinting, and constitutional chromosomal defects (155).
WT is believed to originate from the metanephric blastema and is histopathologically characterized by a triphasic
pattern of blastemal, epithelial, and stromal elements that can show a wide variety of patterns and differentiation.
LOH and clonality studies have shown that the different histologic components are of tumor origin (e331,67).
Gross Features
Nephroblastoma commonly presents as a solitary, more or less rounded mass arising from any part of the
kidney. The tumor origin is multicentric in 7% of cases (Figure 17-32), and 5% of cases are bilateral (135). The
tumor kidney specimen weight ranges from 60 to 6,350g, with a median of 550 g (e221). The bulging cut surface
is pale gray, soft, friable, and lobulated, and areas of hemorrhage, necrosis, and cyst formation are often
apparent (Figure 17-33). The tumor is sharply demarcated from the adjacent renal tissue by a pseudocapsule
(eFigure 17-6). The tumor may protrude into the calyces and sometimes the ureter, forming polypoid
excrescences resembling botryoid rhabdomyosarcoma. It often invades the renal vein, from which it may extend
up through the vena cava to the right atrium.
Adequate sampling is critical. One tissue block for each centimeter in the maximal dimension is recommended.
Evaluation of the renal pelvis and sinus, vein, capsule, and all lymph nodes is needed for staging. Beckwith and
Perlman's suggestions for handling pediatric renal tumors include the following: receiving the specimen intact,
avoiding frozen sections, not stripping the capsule, inking the surface, bivalving to demonstrate the relationship
of tumor to kidney and renal sinus, taking initial sections for diagnosis and special studies (cytogenetic,
molecular, and ultrastructural), fixing overnight in refrigerator, taking most of the sections from the periphery,
including any areas that appear different (eFigure 17-7), documenting the exact source of each section, and
generously sampling uninvolved
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kidney (e20). In addition submit sections that include the triangular interface between the intrarenal tumor
pseudocapsule, the extrarenal tumor pseudocapsule, and the renal capsule. Careful consideration must be given
to the renal sinus, which extends into the kidney following its medial contour and carries blood vessels and
nerves within its fat and connective tissue (134).
FIGURE 17-32 ▪ Multicentric WT, cut surface. The larger, dominant mass invaded the spleen, and a second
small round tumor is seen in the lower pole.
FIGURE 17-33 ▪ Cut surface of WT is bulging, soft, and friable and has a nodular variegated appearance with
areas of hemorrhage and necrosis. Normal kidney can be identified at the one pole.
Microscopic Features
The type of histologic pattern seen in WTs was of prognostic significance before the era of modern
chemotherapy. Within the same specimen, the pattern tends to be uniform; however, it varies greatly from tumor
to tumor. Classical triphasic WT is composed of blastemal, epithelial, and stromal components (Figure 17-34,
eFigure 17-8), but biphasic and monophasic tumors are not uncommon. When one component comprises more
than two-third of the tumor, the tumor is designated accordingly. The mixed type, in which no component
predominates, is most common (41%), followed by blastema-predominant (39%) and epithelium-predominant
(18%). Stroma-predominant WT is rare (1.4%) (e273). With adequate sampling, microscopic foci of all three
components can be recognized in most cases.
Metanephric blastema is the most primitive cell type in WT and is characterized by densely packed primitive cells
lacking identifiable features of differentiation by LM (Figure 17-35). Tumors with a diffuse blastemal pattern and
noncohesive, infiltrative margins are highly aggressive but usually respond to current therapy (e22). The
organoid blastemal patterns (serpentine, nodular, and basaloid) are characterized by regularly defined
aggregates of blastemal cells set in a myxoid mesenchymal background, without aggressive infiltration and with a
clearly demarcated edge, as is usual in all WT patterns.
The epithelial component of WT is most often of nephrogenic type, in which various stages of tubular and
glomeruloid differentiation are seen (Figures 17-36 and 17-37). Heterologous epithelial patterns include
mucinous, squamous, and neuroepithelial and neuroendocrine cells. Similarly, stromal patterns may be
nephrogenic (myxoid, fibrous, smooth muscle, and adipose cells) or heterologous (skeletal muscle, which is most
common, cartilage, and bone) (Figure 17-38).
FIGURE 17-34 ▪ Classic WT showing a triphasic pattern of blastema, tubules, and a glomerulus. (Hematoxylin
and eosin stain, original magni-fication ×200.)
FIGURE 17-35▪ Classic WT showing a predominantly blastemic appearance. (Hematoxylin and eosin stain,
Anaplastic nuclear change is the only marker of “unfavorable histology” in WT. Other pediatric renal tumors with
an unfavorable histology or in the high-risk category of the International Society of Pediatric Oncology (e273),
such as clear-cell sarcoma and rhabdoid tumor, are separate neoplastic entities and not variants of WT.
Anaplastic nuclear changes refer to extreme cytologic atypia, not minor variations in nuclear shape or size.
Anaplasia is defined as a threefold increase in nuclear diameter, hyperchromasia of the enlarged nuclei, and
multipolar mitotic figures (Figure 17-39A,B). These changes are severe enough to be detected when scanned
with a ten times objective.
Anaplastic nuclear changes are a marker of resistance to therapy and do not imply aggressiveness. All patients
are
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staged irrespective of presence or absence of anaplasia. Patients with anaplastic stage I WT generally do well
with conventional therapy (Table 17-7). Anaplasia is currently designated as focal when it is limited to one or a
few discretely demarcated foci within the primary tumor and limited to the kidney. An adverse prognosis for
anaplastic nuclear changes is associated only with stages II through IV tumors with diffuse anaplasia (e97).
FIGURE 17-36 ▪ Classic WT showing neoplastic tubules in a blastemal background. (Hematoxylin and eosin
FIGURE 17-37 ▪ WT showing a predominantly tubular or epithelial pattern. (Hematoxylin and eosin stain, original
It appears that therapy neither obscures nor produces anaplasia (e332). WT can be accurately staged in
nephrectomy specimens obtained following chemotherapy. Staging based on the extent of viable tumor cells is
directly related to outcome (e273). Post-therapy specimens may have extensive residual mature skeletal muscle
(eFigure 17-9).
Regional lymph node metastasis is the most common site of noncontiguous spread of a WT. Benign inclusions in
regional lymph nodes should not be misinterpreted as metastatic WT. Peritoneum, liver, and lung are the other
common metastatic sites. Peritoneal metastases with desmoplasia can simulate a desmoplastic small round cell
tumor. Bone marrow and skeletal metastases are rare; fewer than 2% of classic WTs metastasize to bone.
FIGURE 17-38 ▪ WT with area of skeletal muscle differentiation in the stromal component. (Hematoxylin and
eosin stain, original magnification ×400.)
FIGURE 17-39 ▪ WT showing anaplasia in the form of atypical multipolar mitotic figures; A: Within the
blastomatous component and B: within the epithelial component (Hematoxylin and eosin stain, original
magnification ×400.) (Courtesy of Dr. John Hicks.)
Bilateral Wilms tumors occur in 5% of patients and are designated as stage V disease, but the prognosis
depends on the substage, that is, the stage of the largest tumor and presence or absence of anaplasia. The
largest clinical experience is that of the NWTS (166). Several clinical and pathologic features characterize this
group of tumors; genitourinary tract anomalies (16%), younger age at diagnosis, presence of
nephroblastomatosis (67%), multicentricity (61%), and favorable histology (90%) are findings that tend to
differentiate the stage V cases from all others. The overall 3-year survival was 76% in the NWTS.
Immunohistochemistry, EM, and molecular cytogenetics are useful in those cases in which the diagnostic
material is limited, predominantly in differentiating Wilms tumor from other childhood small blue cell tumors (e.g.,
PNET, rhabdomyosarcoma, and neuroblastoma).
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Table 17-7 ▪ NATIONAL WILMS TUMOR STUDY STAGING DEFINITIONS
Stage Definition
I Tumor confined to kidney parenchyma and completely resected. Renal capsule intact, not
penetrated by tumor. No involvement of vessels of renal sinus. No biopsy before nephrectomy
(fine-needle aspiration biopsy is acceptable).
II Tumor extends beyond kidney parenchyma but is completely resected. Tumor penetration of
renal capsule into vessels of the renal sinus, including the renal vein, or localized spillage
confined to the flank. Specimen margins uninvolved by tumor.
III Residual nonhematogenous tumor confined to abdomen. Tumor in abdominal nodes, tumor
spillage involving peritoneum, peritoneal implants, tumor involvement of resection margin. IV
Hematogenous metastases or nodal deposits outside abdomen.
V Bilateral renal tumors. In such cases, whenever possible, the lesions on each side should be
staged individually, with a substage designation according to the highest individual tumor
stage (e.g., stage V, substage 1).
From Beckwith JB. Renal tumors. In: Stocker JT, Askin FB, eds. Pathology of solid tumors in children.
London: Chapman and Hall, 1998, with permission.
Cystic Variants of Nephroblastic Tumors

Cystic nephroma and cystic partially differentiated nephroblastoma are benign neoplasms currently considered to
be a part of the spectrum of nephroblastoma (103, 135). Both are well-circumscribed tumors composed entirely
of cystic spaces separated by delicate septa (Figure 17-40A). The solid component conforms to the contours of
the cystic spaces. In cystic nephroma (formerly known as unilateral multilocular cyst), it is composed of mature
cell types (Figure 17-40B), whereas in cystic partially differentiated nephroblastoma, the cystic septa contain
embryonal cell types without anaplasia (Figure 17-41A,B). If larger, solid, expansile regions are present, the
tumor should be considered a conventional WT.
Nephrogenic Rests and Nephroblastomatosis

Nephrogenic rests are abnormally persistent foci of embryonal cells with the potential of developing into WT.
They are encountered in 25% to 40% of WT and in 1% of routine postmortem examinations in infants (16, 135).
When these are multifocal or diffuse, the term nephroblastomatosis is used (Figure 17-42). They are classified
into perilobar (Figure 17-43) and intralobar types (eFigure 17-10), which exhibit different biologic behaviors and
have been shown to be genetically different, which may explain the ethnic differences in the epidemiology of WT
(25, 54). They consist of variable amounts of blastemal, epithelial, and stromal components but do not show the
same potential to evolve into WT. The rests may be dormant, sclerosing, or hyperplastic (eFigure 17-11) (e18).
The majority regresses and becomes fibrotic, and some give rise to WT, with the remnants of nephrogenic rest
visible at the periphery of the neoplasm.
FIGURE 17-40 ▪ Cystic nephroma (unilateral multilocular cyst) of the kidney. A: Gross picture of the cut surface
shows multiple thin-walled cysts. Only a small amount of residual kidney present at the top. B: Typical cysts are
lined by cuboidal or flat cells and a nondescript spindle cell stroma. (Hematoxylin and eosin stain, original
Perilobar rests occur in hemihypertrophy (Figure 17-44) and Beckwith-Wiedemann syndrome and also in
association with some sporadic tumors. Perilobar rests are occasionally seen in cystic renal dysplasia and are
rarely associated with mesoblastic nephroma. Intralobar rests are seen with the WAGR and Denys-Drash
syndromes (Figure 17-45). Hyperplastic rests and Wilms tumors comprise a morphologic
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continuum that cannot be distinguished cytologically. Hyperplastic perilobar rests tend to preserve the original
shape of the rest and have a distinct interface with the adjacent renal parenchyma, whereas the intralobar rest
intermingles with the adjacent kidney.
FIGURE 17-41▪ Cystic partially differentiated WT. Cysts have features very similar to those of cystic nephroma,
but the surrounding stroma has immature tubules and cartilage in (A), and islands of blastoma in (B).
(Hematoxylin and eosin stain, original magnification ×200.) (B: Courtesy of John Hicks. M.D. Houston, TX).
FIGURE 17-42▪ Perilobar nephroblastomatosis in an infant with massive enlargement of the kidneys. The
compact, uniform blastema with a nodular configuration and the discrete interface with the adjacent parenchyma
are characteristic features. (Hematoxylin and eosin stain, original magnification ×40.)
FIGURE 17-43▪ Typical appearance of perilobar nephrogenic rests. (Hematoxylin and eosin stain, original
FIGURE 17-44▪ Seven-year-old boy with hemihypertrophy that was diagnosed only after he was found to have a
large WT (Figure 17-36). (Courtesy of David Hatch, M.D., Loyola University Medical Center, Maywood, Illinois.)
FIGURE 17-45▪ Intralobar nephroblastomatosis with blastema and immature tubules blending into the
surrounding kidney. (Hematoxylin and eosin stain, original magnification ×100.)
OTHER CHILDHOOD NEOPLASMS

Congenital Mesoblastic Nephroma
Congenital mesoblastic nephroma (CMN) is a stromal neoplasm of infancy, composed of myofibroblasts, and is
most commonly diagnosed in the first 3 months of life. First described by Bolande et al. (e38), it was thought to
be a variant of WT but, based on the cytogenetic data available currently, is now considered to be distinct from
WT. The classic variant of CMN shows no consistent genetic abnormality and probably represents infantile
fibromatosis of the kidney. In contrast, the cellular variant is identical to infantile fibrosarcoma with the same t(12;
15)(p13;q25) chromosomal translocation resulting in ETV6-NTRK3 gene fusion (e176). CMN is the most
common renal tumor of infancy, with no sex predilection and only an occasional association with Beckwith
Weidemann syndrome. Most cases are detected because of an abdominal mass (eFigure 17-12), although
polyhydramnios, premature delivery, and nonimmunologic hydrops have been reported. Hypertension secondary
to renin production by entrapped renal elements, which may be immature and dysplastic, is not uncommon (135).
Grossly, the tumor is solitary, unilateral, characteristically whorled or trabeculated, and gray-white to yellow, with
a rather indistinct tumor-kidney interface and a softly bulging cut surface (Figure 17-46). Cysts, hemorrhage, and
necrosis are common and have no prognostic significance. The tumor tends to arise centrally within the kidney
and extensively involves the renal sinus; thus, the medial margin needs to be carefully sampled.
FIGURE 17-46▪ Cut surface of CMN shows yellow-white bulging cut surface with focal hemorrhage.
FIGURE 17-47▪ Classic pattern of CMN with intersecting bundles of uniform, bland spindle cells with minimal
atypia. (Hematoxylin and eosin stain, original magnification ×200.)
Microscopically, classic, cellular, and mixed patterns are recognized with frequencies of 24%, 66%, and 10%,
respectively, and a mean age at presentation of 7 days, 4 months, and 2 months, respectively. The classic
pattern, originally described by Bolande, is characterized by intersecting bundles of spindle cells with minimal
atypia and infrequent mitoses (Figure 17-47). At the periphery, the tumor infiltrates extensively into the renal
parenchyma, so that wide margins of excision are necessary (Figure 17-48). Dysplastic entrapped tubules and
islands of cartilage are often seen. The cellular mesoblastic nephroma has a distinct pushing border and is
characterized by dense cells, mitoses, and a “sarcomatous” appearance (Figure 17-49A,B). Areas of cellular
mesoblastic nephroma may be seen in an otherwise classic tumor, with eventual overgrowth of the former
evidenced by the finding of compressed remnants of the classic pattern at the periphery of a cellular mesoblastic
nephroma (e21).
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However, all mixed tumors studied so far have shown no ETV6-NTRK3 gene fusion (5). By
immunohistochemistry, both types of tumors react with antibodies directed toward myofibroblasts. Recurrences
and metastases occur in about 5% to 10% of patients, risk factors for which are cellular histology, stage III or
higher, and involvement of intrarenal or sinus vessels (55, 135).
FIGURE 17-48▪ CMN infiltrates and overgrows renal elements. (Hematoxylin and eosin stain, original
FIGURE 17-49▪ Cellular mesoblastic nephroma. A: Low power shows high cellularity with hemangiopericytoma-
like vessel and area of necrosis. B: High power shows mitoses and some pyknotic cells. (Hematoxylin and eosin
stain, original magnification: A, ×200; B, ×400.)
Clear-Cell Sarcoma of the Kidney

Although a rare tumor (∽20 cases occur in the United States every year), the diagnosis of clear-cell sarcoma of
the kidney (CCSK) is clinically important because it is a tumor with “unfavorable histology” that responds to
chemotherapeutic regimens containing doxorubicin, actinomycin, and vincristine. In addition, it has been called
the great masquerader because it can mimic, or be mimicked by every other pediatric renal neoplasm. The age
distribution is similar to that of Wilms tumor, with a peak incidence during the second year of life; however, the
age ranges from 2 months to 54 years (7). There is no association with any anomaly or syndrome.
CCSK is always unilateral, unicentric, and generally irregularly shaped with a distinct tumor-kidney junction
(e21). It has a variable color and a glistening gelatinous surface (Figure 17-50). Cysts are often present. It seems
to arise deep within the renal parenchyma.
Microscopically, various patterns can be seen; however, most tumors are quite monomorphous with a
characteristic tumor-kidney interface. At low magnification, this appears scalloped and irregular but usually
sharp. Under high magnification, the interface is less sharp because the tumor infiltrates between and around
renal structures (eFigure 17-13), which may show metaplastic changes. In the classic pattern, an evenly
distributed network of vascular septa with parallel capillary-sized vessels subdivides the tumor into cords and
nests that are six to 10 cells wide (Figure 17-51A). The tumor cells are polygonal, with indistinct cell-cell borders,
finely granular nuclear chromatin (hence the pale nuclei), and small or absent nucleoli (Figure 17-51B). Mitoses
are variable but less than in other renal malignancies. The cytoplasm usually consists of delicate tendrils
surrounding intercellular space (hence the “clear-cell” appearance), although it is important to recognize that not
all CCSKs are clear. Histologic variations include epithelioid (eFigure 17-14), spindle cell (eFigure 17-15),
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sclerosing, myxoid, cystic, pericytomatous, palisading, and monstrocellular patterns. However, most CCSKs
exhibit the classic pattern, and even in those with a variant pattern, some areas with the classic pattern can be
found.
FIGURE 17-50▪ CCSK showing distinct tumor-kidney junction, gelatinous nodular cut surface, and yellow areas
of necrosis. (Courtesy of Christopher Weber, M.D., Ph.D., University of Chicago, Chicago, IL).
FIGURE 17-51▪ A: CCSK showing a characteristic vascular or plexogenic pattern in which ill-defined groups of
uniform tumor cells are separated by a capillary network of vessels. (Hematoxylin and eosin stain, original
magnification ×100.) B: The nuclei often have an optically clear appearance, as in papillary carcinoma of the
thyroid. (Hematoxylin and eosin stain, original magnification ×300.)
One study of 14 CCSKs showed the genetic expression profile of CCSK to be highly distinctive as compared to
WT. The finding that many of the genes upregulated in CCSK are involved with neural differentiation,
development, or function may be an important reflection of the cell of origin of CCSK. Additionally, two pathways
activated in CCSK (Sonic hedgehog and phosphoinositide-3-kinase/Akt) have also been implicated in other
pediatric neural tumors. Lastly, potential therapeutic targets (nerve growth factor receptor, CD117 and epidermal
growth factor receptor) were identified that may prove to be useful in the treatment of CCSK (37). So far, three
cases of CCSK have been reported to have translocations att(10;17) (26).
CCSKs are strongly positive for vimentin (eFigure 17-16) and vascular markers highlight the typical distribution of
small vessels. Positivity for CD99 (Mic-2) (eFigure 17-17) and CD56 (eFigure 17-18) may also be seen. The
differential diagnosis includes WT, rhabdoid tumor, and cellular variant of mesoblastic nephroma.
Almost 40% of cases metastasize to bone, hence the original term bone-metastasizing renal tumor of childhood
(e201). It can also metastasize to other unusual sites, including skeletal muscle, orbit, brain, meninges, and
spinal cord. Late recurrences have been described as many as 5 to 8 years after diagnosis (e115,e179). In
contrast to WTs, even stage I CCSKs have relatively high recurrence rates, presumably because of occult
micrometastases at the time of diagnosis. Prognosis varies with stage at presentation: 97% 6-year survival for
stage I to 50% for stage IV (7).
Rhabdoid Tumor of the Kidney

The histogenesis of this rare (2.5% of NWTS cases) and highly malignant (80% mortality) tumor of the infantile
kidney remains unknown despite extensive study. What is known is that it is not related to WT or to any
syndrome except the familial rhabdoid tumor predisposition syndrome. The only known associations are with
hypercalcemia in a few cases and, in 15% of cases, primitive neuroectodermal tumor of the brain (e221). It is
seen in infants and children, with a 1.5:1 male-to-female ratio (e21). Metastases are generally widespread at the
time of diagnosis (e315,e319). Primary extrarenal rhabdoid tumors can be seen in both children and adults,
particularly in the central nervous system. The unifying feature at all sites is the presence of a mutation or
deletion of the INI1 gene located at chromosome 22q11.
Grossly, rhabdoid tumor is usually round, pale, soft, and unencapsulated. Satellite tumors may be present.
Microscopically, the pattern is monomorphous; sheets of large, loosely cohesive tumor cells are characterized by
intracytoplasmic hyaline inclusion and vesicular nuclei containing prominent central nucleoli (Figure 17-52).
These features may be focal and should be looked for carefull
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in any undifferentiated tumor of the kidney. The finding of rhabdoid features in other tumors, such as Wilms
tumor and mesoblastic nephroma, does not imply the same poor prognosis (e320).
FIGURE 17-52 ▪ Rhabdoid tumor of the kidney showing sheets of large, atypical mononuclear cells with a
prominent nucleolus in each nucleus. There are characteristic intracytoplasmic hyaline inclusions indenting some
of the nuclei. (Hematoxylin and eosin stain, original magnification ×300.)
FIGURE 17-53 ▪ Rhabdoid tumor of the kidney showing lack of immunoreactivity for BAF47. Note normally
positive endothelial cells and scattered lymphocytes. (Peroxidase-antiperoxidase stain, original magnification
×200.)
Immunohistochemically, the tumor cells are consistently positive for vimentin with frequent coexpression of
cytokeratin (eFigures 17-19 and 17-20), epithelial membrane antigen, desmin, and neurofilament. The staining
pattern is characteristically patchy and strong, with small clusters of positive cells in a background of nonreactive
tumor cells, seen in over 90% of cases. Other markers, including CD99 and CD56 (eFigure 17-21), have been
reported but are not found consistently (e56). They may represent nonspecific antibody entrapment by the
filamentous arrays seen ultrastructurally to correspond to the cytoplasmic inclusion (e21).
All rhabdoid tumors appear to contain mutations or deletions that inactivate the hSNF5/INI1 gene, whose role is
to alter the conformation of the DNA-histone complex so that transcription factors have access to target genes
(e309). Immunohistochemical staining using antibody to hSNF5/INI 1, B AF47 has been shown to be very
sensitive and highly specific for the detection of hSNF5/INI1 loss-of-function, which correlates well with the
biallelic inactivation of this tumor suppressor gene (23). (Figure 17-53)
Renal Cell Carcinoma

Malignant epithelial tumors are rare in children and in part appear to be intrinsically different than those in adults.
Clear-cell RCC is quite rare in the absence of a predisposing genetic condition such as von Hippel-Lindau
syndrome. Papillary RCC is the most common histologic subtype of RCC with the same pathologic appearance
and genetic changes as seen in adults. Foamy macrophages are present in over 80% of tumors. There is strong
positivity for cytokeratin 7 and epithelial membrane antigen (EMA). Typically, the stage at presentation is low,
surgery is the treatment of choice and any residual tumor is unresponsive to chemotherapy.
FIGURE 17-54▪Renal medullary carcinoma. Tumor cells have dark cytoplasm, clear nuclei, and very prominent
nucleoli. Note acute inflammation and sickled red blood cells. (Hematoxylin and eosin stain, original magnification
×200.)
Renal medullary carcinoma is a rare highly malignant tumor associated with sickle cell trait that occurs in
adolescents and young adults (e74). It appears to have a distinct molecular signature that clusters closely with
urothelial (transitional cell) carcinoma of the renal pelvis, rather than RCC (196). Presenting symptoms are flank
pain, hematuria, and a palpable abdominal mass. It is usually a lobulated neoplasm arising in the renal medulla.
Histologically, it often shows cribriform and reticular growth patterns, reminiscent of yolk sac tumor (eFigure 17-
22). The cells have dark cytoplasm, clear nuclei, and prominent nucleoli (Figure 17-54). Focally, rhabdoid
features (eFigure 17-23) and intracytoplasmic lumens may be present. The stroma is often prominently
desmoplastic and marked acute and chronic inflammation is characteristic (eFigure 17-24). It is widely metastatic
at diagnosis, is unresponsive to chemotherapy and radiotherapy, and has a mean survival of only four months.
There is overlap with rhabdoid tumor.
Translocation-associated renal tumors are defined by their genetic features (majority have translocations
involving the TFE3 gene located at Xp11.2 and a number of variant partner genes). These tumors have a nested
or a tubulopapillary pattern composed of cells with voluminous clear-toacidophilic cytoplasm and distinct cell
borders separated by thin fibrovascular septa (Figure 17-55). The tumor cells, in contrast to other RCCs, are
negative or only focally positive for EMA, cytokeratin CAM5.2 and vimentin, but show nuclear reactivity for TFE3
or TFEB proteins (eFigure 17-25) (57).
Other Rare Renal Tumors

Ossifying renal tumor of kidney is a rare neoplasm, characteristically seen in infant boys, that presents with
hematuria. A calcified mass in the renal pelvis grossly resembles calculi and is microscopically composed of
proliferating
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spindle cells admixed with partially calcified osteoid matrix. The prognosis is excellent, with no known
recurrences or metastases (e57,e156).
FIGURE 17-55 ▪ RCC with translocation at Xp11.2. There are distinct cell borders and abundant clear cytoplasm.
Metanephric tumors are rare benign tumors that have a pathologic spectrum from adenoma (most common of the
group, occurring mainly in females with a mean age of 41 years) to adenofibroma (containing both epithelial and
stromal components, occurring at a mean age of 82 months with 2:1 male-to-female ratio) to stromal tumor (mean
age of 2 years) (135). All are unencapsulated and show distinctive morphology (eFigures 17-26 and 17-27).
Primitive neuroectodermal tumor occasionally occurs in the kidney in older children and adults. It is a small blue
cell tumor, the diagnosis of which rests on the findings of pseudorosettes (Figure 17-56), CD99 and FLI-1
positivity, andt(11;22).
Angiomyolipomas and oncocytomas may occur in children, almost always in association with tuberous sclerosis.
Primary neuroblastoma and lymphoma of the kidney also occur rarely.
FIGURE 17-56 ▪ Primitive neuroendocrine tumor of kidney showing pseudorosettes and an entrapped tubule.
DISEASES OF THE URETERS, BLADDER, AND URETHRA

Congenital Malformations of the Ureter
Malformations of the ureter are common and often occur in combination (e.g., duplication and ectopia, or ectopia
and ureterocele with or without obstruction).
Ureteral Agenesis
Unilateral or bilateral ureteral agenesis is almost always seen with renal agenesis. Whenever a ureter is present,
some renal tissue can usually be identified.
Ureteral Duplication
The vast majority of cases of ureteral duplication are sporadic, although a few cases have been reported with
syndromic associations (e120,e275). All types of ureteral duplication are much more common in girls (male-to-
female ratio of 6:1) (171). Duplication of the upper part of the ureter and renal pelvis occurs in association with
premature branching of the ureteric bud; complete duplication occurs when two ureteric buds form. Partial
unilateral duplication is the most common form and is associated with duplication of the renal pelvis and a duplex
kidney. Often, the upper pole is smaller and dysplastic, with the associated ureter tortuous and dilated. In cases
with complete duplication, the upper pole ureter inserts ectopically.
Ureteral Ectopia
Ureteral ectopia is more common in girls, often associated with ureteral duplication, and frequently presents with
symptoms of urinary tract infection (e100). In boys, the ectopic orifice is located more often in the urinary tract
than in the seminal tract (e303). That part of the kidney drained by the ectopic ureter is often dysplastic. The
severity of the dysplasia is apparently determined by the position of the ectopic ureteric orifice; the more lateral
the ectopia, the more severe the degree of dysplasia and hypoplasia of the kidney (e278-e280). Nonfunctioning
renal segments are treated by laparoscopic nephroureterectomy of the upper pole (137), and functioning renal
segments are conservatively treated, usually with ureterovesical reimplantation (117).
Ureterocele
Ureterocele is a congenital cystic dilation of the intravesical portion of the distal ureter. It is commonly detected
by prenatal ultrasound (41). In childhood it usually presents with symptoms of vesicoureteral reflux and chronic
infection. The ureteral orifice may be normally positioned or ectopic at the neck of the bladder or ureter, in which
case it is most often associated with the upper pole of a duplex kidney (171).
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Ureteral Obstruction
Intrinsic ureteral obstruction, bilateral in 20% of cases, is most often seen at the ureteropelvic junction, causing
hydronephrosis (Figure 17-57), or associated with multicystic dysplasia, in which case the ureter(s) may be
atretic. The obstruction of the ureteropelvic junction may be caused by stenosis, valves, or functional constriction
(138). Extrinsic obstruction is seen in retrocaval ureters, and less often in retroiliac ureters. In obstruction of the
vesicoureteral junction, the presence of an additional smooth muscle collar surrounding the terminal ureter has
been demonstrated (e83). A number of experimental studies have shown that the younger the age of the patient
at the time of unilateral ureteral obstruction, the more severe the growth impairment of the ipsilateral kidney and
the greater the compensatory growth of the opposite kidney. Renal growth and development are impaired
through complex interactions between regulators of cell proliferation, cell destruction, and extracellular matrix
(e58).
Vesicoureteric Reflux
Vesicoureteric reflux is a congenital defect of the urinary tract that causes urine to flow retrogradely from the
bladder to the kidneys due to short intravesical ureter(s), poorly developed trigone, and ectopic abnormally large
ureteral orifice(s). It is associated with recurrent urinary tract infections, hypertension, and renal failure (third
most common cause in children). Prevention of recurrent UTIs is believed to significantly reduce the risk of reflux
nephropathy. However, despite medical and surgical therapy, the incidence of renal failure in these patients has
not decreased. Thus, it is proposed that the renal damage associated with reflux is congenital and arises from a
defect that affects both renal and urinary tract development (115).
FIGURE 17-57 ▪ Left hydronephrosis secondary to unilateral ureteropelvic junction obstruction.
Acquired Lesions of the Ureter

Inflammation and neoplasia primarily involving the ureter are rarely, if ever, seen by the pediatric pathologist.
Chronic inflammation and fibrosis may be seen in association with pyelonephritis and vesicoureteral reflux. The
ureter may be secondarily involved by rhabdomyosarcoma arising in the urinary bladder and other
retroperitoneal tumors. Transitional cell carcinoma of the ureter is seen only in adults.
Congenital Lesions of the Bladder and Urethra

Most patients with bilateral agenesis of the kidneys also have agenesis of the bladder, although a small,
contracted bladder may be identified in some. Agenesis is also seen as part of severe malformations (e.g.,
sirenomelia, caudal regression syndrome, and limb-body wall defects). Hypoplasia of the bladder is commonly
seen with bilateral multicystic dysplastic kidneys. Duplication of the bladder is uncommon and rarely occurs as
an isolated anomaly; rather, it is generally part of a complex malformation, as in the VACTERL (vertebrae, anal,
tracheoesophageal, renal, and limb) association (Chapter 4).
Bladder Exstrophy
Bladder exstrophy is an uncommon anomaly with an even sex ratio (120). Epispadias, exstrophy of the bladder,
and cloacal exstrophy are a spectrum of related malformations that result from a failure of the primitive streak
mesoderm to invade the anterior part of the cloacal membrane (156). The size of the exstrophic bladder varies
from patient to patient, ranging from a small hole in the anterior abdominal wall through which the bladder trigone
protrudes on straining at micturition to a large defect through which the entire posterior wall of the bladder is
exposed (Figure 17-58). The pubic symphysis remains open. Epispadias, in which the opening of the urethra is
on the upper surface of the penis, is present in males, and bifid clitoris in females. In cloacal exstrophy, the
bladder is separated into two halves by the central exstrophic bowel; other urogenital anomalies are also
present.
Even after surgical repair, bladder function is often not normal. Complications include vesicoureteral reflux,
cystitis cystica, squamous metaplasia, and an increased risk for the development of squamous cell carcinoma or
adenocarcinoma.
Obstructive Lesions
Obstruction of the urinary tract results in a series of changes referred to as obstructive uropathy is a leading
cause of renal
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failure in childhood and adolescence. It accounts for 16.3% of pediatric renal transplantations (15). The
obstruction can occur at multiple levels of the urinary tract, including the urethra, bladder outlet, and ureters.
Renal lesions in obstructive uropathy vary from bilateral hydronephrosis to severe and diffuse hypodysplasia in
which variably sized cysts mimic polycystic disease. A less severe renal change, characterized by the
conservation of normal renal structure and the presence of subcapsular cysts (Potter type IV), is seen less
frequently (e73).
FIGURE 17-58 ▪ Newborn boy with bladder exstrophy. (Courtesy of David Hatch, M.D., Loyola University
Medical Center, Maywood, Illinois.)
The causes of bladder outlet obstruction include posterior urethral valves in males, urethral stenosis or atresia,
and functional neck obstruction. Posterior urethral valves are the most common cause of lower urinary tract
obstruction in male infants with an incidence of 1 in 5,000 to 8,000 live births (15). Severe obstruction can be
detected in utero and treated by vesicoamniotic shunt placement, the efficacy of which decreases in the latter
part of gestation (Figure 17-59).
Prune Belly Syndrome

Also called triad syndrome or Eagle-Barett syndrome, prune belly syndrome is rare, with an incidence of
1/40,000 live births (21), and often fatal. It predominantly affects boys and consists of absence or hypoplasia of
the abdominal wall musculature, cryptorchism, and urinary tract anomalies. Findings include hypoplasia of the
prostate, urethral obstruction in many but not all cases, markedly distended bladder, megaureters, bilateral
hydronephrosis with atrophy, and often renal dysplasia and cyst formation. The condition is named for the
characteristically lax and wrinkled appearance of the abdominal wall (Figure 17-60). The earliest manifestations
are fetal ascites and Potter syndrome.
FIGURE 17-59▪ Severe obstruction secondary to posterior urethral valve was diagnosed in utero and a
vesicoamniotic shunt placed. A thick-walled, dilated bladder, bilateral hydroureters, and hydronephrosis were
found at autopsy in this newborn boy, who died of pulmonary hypoplasia.
The pathogenesis of prune belly syndrome is not known; it may simply arise from the effects of early urethral
obstruction (176) or else from a basic defect of the mesoderm from which the triad of abnormalities develop. In a
study of the urethra and genital tracts of 21 patients with prune belly syndrome and 23 patients with posterior
urethral valve, the seminal ducts and vesicles and prostatic glands were abnormal
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in the former group and normally developed in the latter (e299). According to the authors of the study, this major
difference stems from a primary defect of the intermediate and lateral plate mesoderm in prune belly syndrome,
affecting the embryogenesis not only of the mesonephric and paramesonephric ducts but also of the musculature
of the abdominal wall and urinary organs, and prune belly valves, when present, are intrinsic components of the
mesodermal defect of the urethra in prune belly syndrome.
FIGURE 17-60▪ Newborn boy with prune-belly syndrome; note the lax anterior abdominal wall and redundant
scrotal skin. (Courtesy of David Hatch, M.D., Loyola University Medical Center, Maywood, IL.)
Megacystic Microcolon Intestinal Hypoperistalsis Syndrome

Megacystic microcolon intestinal hypoperistalsis syndrome (MMIHS) is a rare congenital disorder characterized
by a massively enlarged urinary bladder without mechanical outlet obstruction, microcolon, and a hypoperistaltic
bowel with normal ganglion cells in a majority of cases (140). Absence of interstitial cells of Cajal, vacuolar
degeneration of smooth muscle, neuronal dysplastic changes associated with increased laminin and fibronectin,
and excessive smooth muscle cell glycogen storage with severely reduced contractile fibers displaced to the
extreme periphery of cells have been reported (140). A lack of functional a3 subunit of nicotinic acetylcholine
receptor has been demonstrated in most MMIHS tissues, which may prove to be the underlying pathogenesis of
this rare syndrome (147).
Urachal Remnants
The patent urachus, which connects the developing urinary bladder with the allantoic duct, normally becomes a
solid cord by month 4 of gestation. Patency may persist either completely, so that a fistula forms between the
umbilicus and the bladder, or partially, in which case a sinus forms that usually opens into the umbilicus (Figure
17-61). A cyst develops if the urachus remains partially patent anywhere along its length. Persistence of the
distal urachus where it joins the bladder produces a variably sized diverticulum. These remnants can be lined by
transitional, intestinal, or squamous epithelium. There is a membrane to an ostomy site. Symptoms and
complications include persistent umbilical discharge, infections, and development of carcinoma in adulthood.
FIGURE 17-61▪ Patent urachus with sinus opening into the umbilicus. (Courtesy of Preston Black, M.D., Loyola
University Medical Center, Maywood, IL.)
ACQUIRED LESIONS OF THE BLADDER AND URETHRA

Cystitis
Cystitis in children can be broadly classified into two main categories: specific, in which the cause is known (e.g.,
bacteria, fungi, viruses, drugs, or radiation), and nonspecific, in which the cause is unknown (e.g., proliferative,
interstitial, and eosinophilic cystitis and malakoplakia).
The etiology of UTIs is affected by underlying host factors such as age, diabetes, spinal cord injury, or
catheterization. Consequently, complicated UTI has a more diverse etiology than uncomplicated UTI, and
organisms that rarely cause disease in healthy patients can cause significant disease in hosts with anatomic,
metabolic, or immunologic underlying disease. Most uncomplicated UTIs in children are caused by
enterobacteria, mainly E coli 90% of which possess P fimbriae that allow the bacteria to adhere to the
uroepithelial cell lining. S. aureus is more commonly seen among children with indwelling catheters. Coagulase-
negative staphylococci and Candida app. are commonly associated with infections after instrumentation of the
urinary tract. In nosocomial UTI, half of whom have had prior instrumentation, the organisms are E coli (28%),
Candida spp. (18%), Enterococcus (13%), Gram-negative fermenters (13%), Enterobacter (10%), and
Pseudomonas (10%). Although there have been minimal changes in the predominant uropathogens over the
past decades, there have been significant changes in resistance patterns to antimicrobials that need to be
considered when determining the most appropriate empiric therapy (152).
Granulomatous Cystitis
Granulomatous cystitis may be idiopathic or associated with a specific infection (e.g., tuberculosis,
schistosomiasis, fungal infections in the immunocompromised). It may also be seen as part of chronic
granulomatous disease of childhood, which is a rare congenital abnormality of the phagocyte NADPH (reduced
nicotinamide adenine dinucleotide phosphate) oxidase system (e87,e294).
Cystitis Cystica and Glandularis

Cystitis cystica and cystitis glandularis are two forms of chronic proliferative cystitis, cystitis cystica being more
common, that are often seen together in patients with chronic inflammation of the bladder. Proliferative cystitis
eventually develops in patients with bladder exstrophy and may be a
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preneoplastic change, which would explain the increased risk for adenocarcinoma in these patients (125).
Cystoscopically, small rounded projections of the bladder mucosa are seen. Microscopically, cystic structures
are apparent in the submucosa, composed of transitional and glandular epithelium (eFigure 17-28). When the
lining resembles intestinal epithelium and the cysts become dilated with mucin, the condition is called cystitis
glandularis. Chronic inflammatory infiltrate is usually minimal. A case with a 15-cm botryoid-like polyp has been
described (4).
Interstitial Cystitis
Interstitial cystitis (IC) is a chronic noninfectious, probably inflammatory disorder of the bladder that primarily
affects female adults. Occasionally, it can be seen in adolescent girls (122). Classic IC is characterized by
frequency, nocturia, and suprapubic pain with ulceration (Hunner ulcer); in the nonclassic form, ulceration does
not occur. The etiology and the pathogenesis are still undetermined, and the pathologic diagnosis is essentially
one of exclusion. IC appears to be a syndrome with neural, immune, and endocrine components in which
activated mast cells play a central role (e90,e305). The bladder transitional cell epithelium is normally covered by
a mucin layer composed of glycosaminoglycans. This layer is thought to be almost impermeable, thereby
preventing urine solutes from diffusing into the subepithelial components of the bladder. IC might affect this layer
by increasing solute permeability, possibly leading to irritation, inflammation, and sensory-nerve sensitization of
the bladder. Potassium could be the main offending substance and its diffusion across the permeable transitional
epithelium the primary irritant; hence the development of the potassium sensitivity test for the diagnosis of IC
(136).
Eosinophilic Cystitis
Eosinophilic cystitis is a rare disorder. In children, it may be associated with parasites, food allergens, or drugs.
Associated risk factors include bronchial asthma, atopic diseases, and environmental allergens (184). It has also
been reported in association with chronic granulomatous disease (12). The bladder mucosa may be markedly
polypoid, so that embryonal rhabdomyosarcoma must be included in the differential diagnosis (144).
Histologically, extensive eosinophilic inflammation of the bladder wall is present.
Malakoplakia
Malakoplakia is a chronic inflammatory disease that was originally described in the urinary bladder but can
involve many other organs and soft tissues. It is rarely seen in children (109, 165). Histologically, it is
characterized by chronic inflammation, histiocytes, and poorly formed granulomas. A diagnostic feature is the
Michaelis-Guttmann body, which is a laminated calcospherite present in the cytoplasm or extracellularly (Figure
17-62). It stains with period acid-Schiff, iron, and von Kossa stains and may represent bacterial degradation
products (190).
FIGURE 17-62▪ Malakoplakia of the bladder in a child with a surgically repaired exstrophy. The inflammatory
infiltrate is composed of lymphocytes, plasma cells, and macrophages, some with purple stained
MichaelisGuttmann bodies. (Hematoxylin and eosin stain, original magnification ×200.)
Hemorrhagic Cystitis
Acute hemorrhagic cystitis may be infectious or sterile. BK virus has been shown to be the main cause of viral
hemorrhagic cystitis in bone marrow transplant patients (58). BK virus cystitis has also been reported in
nonimmunocompromised hosts (e267). In patients receiving hematopoietic stem cell transplantation older age at
transplant, allogeneic transplant, cyclophosphamide-containing conditioning, moderate-to-severe acute graft-
versus-host disease (GVHD) and hepatic GVHD were associated with higher risks of HC (34). Adenovirus type
11 is responsible for acute, self-limiting cystitis with the sudden onset of gross hematuria, dysuria, and urinary
frequency (e218). E. coli and occasionally Candida albicans may also cause hemorrhagic cystitis.
Cyclophosphamide therapy is complicated by ulceration of the bladder mucosa and massive hemorrhage into the
submucosa in 7% of patients receiving the drug (e185). During high-dose therapy, up to 35% of patients have
severe hemorrhagic cystitis (e52). Ifosfamide produces hemorrhagic cystitis even more commonly, which is its
main dose-limiting toxicity (e195). The cytokines, tumor necrosis factor-a and interleukin-1, nitric oxide, nitric
oxide synthetase, and platelet-activating factor have been shown to be involved in the pathogenesis of
hemorrhagic cystitis (e110,e292). The blood loss may be so severe that blood transfusions and even surgical
intervention are warranted. Marked cytologic atypia can be seen in the regenerating epithelium. The incidence of
urothelial neoplasms is increased in patients receiving long-term cyclophosphamide therapy. Mesna is a drug
that protects urothelium and prevents hemorrhagic cystitis and may even decrease the risk for urothelial
carcinoma (e25,e200).
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Tumors of the Bladder and Urethra
All tumors of the bladder and urethra are rare in children; benign tumors are even more infrequent. Benign
tumors in children described in case reports include polyps, papilloma, hemangioma (4,78,e186), neurofibroma
(e159), leiomyoma (114), paraganglioma (118), granular cell tumor (e243), nephrogenic adenoma (eFigure 17-
29) (186), and inverted papilloma (194).
Rare cases of transitional cell carcinoma, leiomyosarcoma and secondary involvement by leukemia, lymphoma,
and WT have been reported.
Inflammatory Myofibroblastic Tumor (Pseudosarcomatous tumor)

Inflammatory myofibroblastic tumor (IMT) is occasionally seen in the bladder. Myxoid, leiomyomatous, and
sclerosing matrix patterns are seen, with the myxoid type being most common (eFigures 17-30,17-31) (e143).
The proliferating cells stain with vimentin, muscle-specific actin, smooth muscle actin, desmin, and occasionally
keratin and, rarely, Epstein-Barr virus (e80,e103,108). Although this tumor was long considered a benign
proliferative response to inflammation, the frequent anaplastic lymphoma kinase gene alterations indicate that it
is a neoplastic process (112). Some of the pathologic aspects occurring in a few of these tumors that support this
view include local recurrence, development of multifocal noncontiguous tumors, infiltrative local growth, vascular
invasion, and, rarely, malignant transformation (e64,71). However, a recent review of 35 pediatric cases showed
no recurrence or metastasis (75). IMT should be regarded as a soft tissue-mesenchymal tumor with an
indeterminate or low potential for malignancy (see Chapter 24).
Rhabdomyosarcoma
Although the term rhabdomyosarcoma indicates a mesenchymal tumor derived from striated muscle,
rhabdomyosarcoma typically arises in sites lacking striated muscle. Approximately 250 new cases of
rhabdomyosarcoma are diagnosed in the United States each year (e184), 15% to 30% of which are found in the
genitourinary tract (e99,e205,e235). Thus, although rhabdomyosarcoma is the most common tumor of the lower
genitourinary tract in the first two decades of life (e12), only a handful of cases occurring in the bladder are seen
in the United States each year. The majority of cases are sporadic; however, rhabdomyosarcoma has an
association with the familial cancer syndromes, including Li-Fraumeni, Beckwith-Wiedemann, neurofibromatosis
type 1, and Gorlin syndrome (130). The mean age at diagnosis is 5 years, with a male-to-female ratio of 3:2
(e130,e131). Symptoms include hematuria, signs of bladder outlet obstruction (abdominal pain and distension,
dysuria) and, occasionally, abdominal mass (102).
The vast majority of cases of rhabdomyosarcoma of the bladder are of the botryoid subtype of embryonal
rhabdomyosarcoma. The gross configuration in most cases is grapelike. The tumor cells form a distinct layer with
a thickness of several cells, at least focally, near the epithelium. The superficial stroma next to the epithelium is
loose. The condensed tumor cells or cambian layer of Nicholson (e234) varies in thickness and extent (Figure
17-63). Some grossly grapelike lesions do not show the cambian layer under the epithelium. By this definition,
these would not be called botryoid rhabdomyosarcoma, but rather embryonal rhabdomyosarcoma (e233).
Cytogenetic and molecular features are discussed in the soft tissue chapter (Chapter 24).
FIGURE 17-63▪ Embryonal rhabdomyosarcoma as a polypoid urethral mass from an 8-week-old girl. The
concentration of small, primitive tumor cells beneath the mucosal surface is typical of sarcoma botryoides.
In the vast majority of cases, the stroma of the botryoid lesion consists of a very loosely cellular tissue with a
myxoid appearance. In the remainder, the stroma is more cellular. The appearance of the tumor cells also varies.
In about 50% of the cases, the tumor cells are small and primitive, show very little myogenesis, and often
demonstrate stellate cytoplasmic processes. In the remainder, the tumor cells are somewhat larger and more
definitive myogenesis is present, consistent with rhabdomyoblasts. The cytoplasm of the rhabdomyoblasts varies
from slight to abundant with cross-striations.
The importance of recognizing the botryoid subtype lies in the fact that these patients have a very good
prognosis (95% survival at 5 years); in contrast, patients with embryonal rhabdomyosarcoma have a 5-year
survival of 67%, and those with alveolar and undifferentiated sarcoma have 5-year survival rates of 54% and
47%, respectively (e233).
The goal of multimodality therapy is to improve outcome while preserving organ and function; therapy is
intensified according to a risk-based study design (e71,e132). Bladder rhabdomyosarcoma is responsive to
chemotherapy and radiotherapy. A complete loss of tumor cells was observed in 12 of 26 patients after induction
therapy. Cystectomy specimens showed diminished tumor cells with varying degrees of cellular maturation
(e137). There is lack of agreement concerning the significance of mature-appearing cells in posttreatment
biopsies.
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Chapter 18
The Female Reproductive System
Elizabeth J. Perlman
Michael K. Fritsch
Abnormalities confined to the genital tract are quite unusual in prepubertal girls and such disorders seldom come
to the attention of pediatricians or pediatric pathologists. Major developmental abnormalities affecting the
reproductive system are often eclipsed by concomitant urinary tract abnormalities, which are more immediately
clinically significant. Abnormal gonadal development is an important group of diseases that may also result in
abnormal development of secondary sexual characteristics. The most frequently acquired diseases of the female
genital tract include infections and neoplasms. Infections confined to the female reproductive tract are seldom life
threatening in childhood, yet they may result in reproductive sequelae during adulthood. Neoplasms are
dominated by those arising in primordial germ cells.
ANATOMY AND EMBRYOLOGY

The female reproductive tract consists of the gonads, a ductal system (Fallopian tubes, uterus, cervix, vagina)
and the external genitalia (clitoris, labia majora, labia minora, vestibule, mons pubis). The process of sexual
differentiation can be divided into various phases. Chromosomal (genetic) sex is determined by the XY or XX
genotype, with the potential for abnormal deletion or addition of sex chromosomal material such as in Turner and
Klinefelter syndromes. Gonadal sex refers to gonadal differentiation into testis or ovary and is predominantly
dependent on the expression of the sexdetermining region on Y gene (SRY). SRY begins a cascade of molecular
signals, resulting in male gonadal differentiation; the absence of these male-specific signals and the presence of
several female-specific signals result in normal female differentiation. Phenotypic sex refers to the differentiation
of the ductal system and of the external genitalia, a process that is regulated by production of the Müllerian
inhibitory substance/antiMüllerian hormone (MIS/AMH) by the Sertoli cells of the testis and steroid hormone
production by the testis (testosterone/dihydrotestosterone) and the ovary (estrogen/progesterone). Lastly, the
assigned or adopted sex is usually determined by the chromosomal, gonadal, and phenotypic sex at birth but
may be altered postnatally in a variety of the intersex disorders [also referred to as disorders of sex development
(DSD)]. These processes occur predominantly in utero, with the final phenotypic changes being initiated by the
onset of puberty.
Primordial Germ Cells

The primordial germ cells first appear in the wall of the yolk sac at about 3 to 4 weeks after fertilization and
migrate through the hindgut and mesonephric ridge to the genital ridge beginning about week 4 to 5, a process
mediated by a number of molecular factors (140, 154, 163). Germ cells that do not reach the genital ridge are
thought to disappear, but they have also been proposed as the cell of origin of extragonadal germ cell tumors.
The initial events in testicular and ovarian differentiation are independent of the presence or genotype of
primordial germ cells in the gonad (19). However, completion of appropriate ovarian development depends upon
the presence of meiotic germ cells. In the absence of meiotic germ cells, the ovarian structure degenerates
leaving streak ovaries (15, 166).
Early Gonadal Development

The gonadal ridge develops at the ventromedial aspect of the mesonephros by a proliferation of mesodermal
cells and thickening of the overlying coelomic epithelium at 4 to 5 weeks' gestation. Between 4.5 and 6 weeks,
the indifferent gonad is indistinguishable as male or female (Figure 18-1). Within the developing gonads, the
somatic cells express a number of genes that maintain the indifferent gonad and dictate sex-specific gonad
development. In mice, these include the homeobox gene Emx2, the polycomb group gene Cbx2, and the LIM
homeodomain gene Lhx9 (152). In both humans and mice, the Wilms tumor gene isoform WT1-KTS and the
steroidogenic factor 1 (previously referred to as SF-1 and now designated NR5A1) genes are important in
maintaining the indifferent gonad. Mutations in NR5A1 can lead to adrenal-gonadal failure, mostly affecting male
gonad development, as maintenance of NR5A1 expression is also critical
P.838
in testis development. The WT-KTS isoform is required for cell survival and proliferation within the bipotential
gonad in both males and females (15, 105, 152, 166, 169). Mutations are associated with three syndromes
characterized by gonadal dysgenesis [Wilms tumor/aniridia/gonadal dysgenesis/retardation—WAGR (OMIM
194072), Denys-Drash (OMIM 194080), and Frasier (OMIM 136680)] [reviewed in (81, 129) and at OMIM,
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=omim]. The phenotype of affected individuals includes genital
and kidney defects as well as an increased risk for the development of Wilms tumors. At about 7 weeks'
gestation, in XY embryos, a Y-linked genetic switch, SRY, is expressed by pre-Sertoli cells (15, 19). The
expression of SRY is necessary and sufficient to trigger male testis development; however, both the timing and
the level of SRY expression are critical for normal development. In an XY embryo, the lack of SRY results in
ovarian development. The SRY protein contains a highly conserved DNA-binding domain that allows specific
genes to be turned on or off (19). One such gene is SOX-9, which is thought to be a Sertoli cell differentiation
factor. Mutation in SOX-9 leads to the human dwarfism syndrome camptomelic dysplasia (OMIM 114290), which
is often associated with XY sex reversal (69). Duplication of SOX-9 causes XX female-to-male sex reversal.
NR5A1 expression in the developing testis regulates expression of several male-specific genes, including
MIS/AMH (106) (see Chapter 27).
FIGURE 18-1 ▪ The undifferentiated gonad lies adjacent to the mesonephros and the Wolffian and Müllerian
ducts. The mesonephros has a profound effect on normal gonadal differentiation.
Ovarian Differentiation
The ovary can be identified at 7 to 8 weeks' gestation by the absence of testicular cords. After their arrival in the
gonad, germ cells in the female continue to undergo active mitotic cell division, a process that continues until
birth. It is estimated that approximately 3 to 4 million germ cells are present in each ovary by 20 weeks' gestation,
and then the number decreases to about 0.5 to 1 million at term (102). The mechanism leading to the loss of
oocytes remains poorly understood. At approximately 12 weeks' gestation, the first germ cells begin to enter into
meiosis, a process first seen close to the medullary region (Figure 18-2). On entering meiosis, the primary oocyte
will arrest at the diplotene stage of the first meiotic prophase and become enclosed by follicular cells to form
primordial follicles. Follicular, or granulosa cells are in direct contact with the germ cells and are thought to play a
role in regulating the continued meiotic arrest in the germ cells. Oogenesis is a gradual process that is generally
complete by the third trimester, and there is no further increase in the number of primary oocytes thereafter
(Figure 18-3). However, this has recently been questioned, at least in the mouse, where germ or stem cell
replication may continue into adulthood (140). In addition to the germ cells, the ovary is populated by stromal
cells that continue to remodel during the early first trimester, resulting in the formation of the ovarian cortex and
the medulla. During the second trimester, a subepithelial collagenous connective tissue layer develops within the
ovarian cortex beneath the basement membrane. The interstitial (thecal) cells can be detected during the first
half of the second trimester, although estrogen production begins as early as 8 to 10 weeks' gestation. The
importance of the production of estrogen by the developing fetus remains somewhat controversial with some
evidence that significant estrogen production does not occur until following birth (166). The histology of the
developing ovary has been previously described in detail (107, 128).
FIGURE 18-2 ▪ Developing ovary at approximately 22 weeks' gestation showing formation of primordial follicles
containing oocytes arrested in meiosis I in the deep ovarian cortex and premeiotic oogonia in the superficial
cortex. Germ cell proliferation continues within the premeiotic oogonia until term.
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FIGURE 18-3 ▪ Developing ovary at term showing numerous primordial follicles, and early development of the
subepithelial stromal layer that will become more prominent with age. The number of germ cells will decrease
progressively with age.
While many of the molecular details of testis development have been clearly established, the details regulating
molecular ovarian development are still being explored. The NR0B1 gene, previously designated DAX-1, is a
dosage-sensitive gene locus on the X chromosome that encodes an orphan nuclear receptor protein. This gene
was initially thought to be a specific ovary-determining gene but has since been shown to be more essential for
normal testicular development and is not required for normal ovarian development. NR0B1 is expressed in the
normally developing ovary but turned off in the developing testis (15, 140, 152, 166). Mutations of NR0B1 lead to
hypogonadotropic hypogonadism with primary testicular defects and are associated with adrenal insufficiency.
Loss of function of NR0B1 in XX females does not alter normal ovarian development. Overexpression of NR0B1,
however, leads to ovarian development even if SRY is expressed. NR0B1 therefore appears to be more
important in normal testis development. Recent evidence suggests that intact WNT4 signaling is necessary for
normal development of the Müllerian duct, suppression of the interstitial cell lineage in the developing ovary, as
well as oocyte maintenance (151, 152, 166). WNT4 is expressed in the bipotential gonad of both sexes, but it
remains highly expressed only in the ovary. Without WNT4 expression in XX individuals, malespecific changes
occur that include the presence of steroid producing cells within the ovary, persistence of the Wolffian ducts, loss
of the Müllerian ducts, and the development of
a male-specific coelomic blood vessel to the ovary. In the absence of WNT4, germ cells can still enter meiosis,
but there is massive apoptosis of the germ cells prior to birth. WNT4 expression seems to provide a protective
niche in the ovarian cortex for female germ cell survival. Other genes proposed to be involved in ovary-specific
development include FOXL2, Pisrtl, and follistatin (Fst), although only FOXL2 has been shown to be important in
human ovarian development thus far (152, 166). Mutations in the forkhead transcription factor 2 (FOXL2) gene in
humans are associated with eyelid defects and premature ovarian failure [blepharophimosis, ptosis and
epicanthus inversus syndrome—BPES (OMIM 110100)]. FOXL2 is expressed by pregranulosa cells.
The ovary differs from the testis in that the presence of germ cells is essential for normal ovarian development. In
the developing testis, the male germ cells do not play a significant role in the structural development of the
organ. Female germ cells appear to follow an intrinsic clock to enter the first meiosis and arrest prior to
completion. Once female germ cells have entered meiosis, they have committed to the oocyte fate. During fetal
development, the oocyte becomes surrounded by a single layer of granulosa cells to form the primordial follicle.
Figla (factor in germ line a) is an oocytespecific basic helix-loop-helix transcription factor that is critical for
recruiting granulosa cells to form the primordial follicles. In the absence of Figla expression, primordial follicles do
not form and oocytes are rapidly depleted after birth. Figla is not expressed in male germ cells and does not
appear to directly regulate meiosis (reviewed in ref. 166).
At birth, the ovary is tan, flat, and elongated and measures about 1.3 × 0.5 × 0.3 cm. and weighs less than 0.3 g
(113). Before birth, some primordial follicles can develop further. The ovum enlarges and the surrounding
follicular cells become more cuboidal to columnar and thereby form a primary follicle. This may be followed by
stratification of the granulosa cells and increased granulosa cell proliferation, resulting in a preantral follicle
(Figure 18-4). The Graafian follicle demonstrates a cavity within the granulosa cell layer.
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The granulosa cells in these follicles have scant cytoplasm and often surround cavities filled with deeply
eosinophilic material, known as Call-Exner bodies that may result in microscopic structures resembling
gonadoblastoma or annular tubule-like profiles (83). These likely represent abnormal folliculogenesis. Thecal
cells, which differentiate from the stromal cells at the periphery of developing follicles, may be seen. Throughout
childhood, the ovaries enlarge to reach the size and the shape of an adult ovary (4 × 2 × 1 cm, 5 to 8 g). During
the prepubertal period, the number of oocytes and primordial follicles continues to decrease and the amount of
ovarian stroma increases. Like the testicular Leydig cells, ovarian hilus cells disappear during childhood and
reappear during puberty.
FIGURE 18-4 ▪ Primary follicle, preantral stage, showing a thick layer of granulosa cells surrounding the oocyte.
Several Call-Exner bodies containing acellular hyaline material are present.
The Ductal System

The development of the urinary and reproductive systems is highly interdependent, which explains the high
incidence of coexisting genital and urinary tract anomalies in several syndromes. Early in development (˜week 4)
paired mesonephric (Wolffian) ducts arise from the intermediate mesoderm and nephrogenic cord (Figure 18-5).
In the female embryo, the mesonephric duct is needed for induction of paramesonephric (Müllerian) duct
development from invaginations of the coelomic epithelium. The mesonephric and the paramesonephric ducts
are enclosed in a peritoneal fold that gives rise to the broad ligament of the uterus. In the male embryo AMH/MIS
produced by fetal Sertoli cells leads to regression of the ipsilateral paramesonephric duct between weeks 8 and
10. AMH/MIS is proposed to have multiple functions in women (115, 140). After the sensitivity of the
paramesonephric ducts to AMH/MIS has disappeared, the ovarian granulosa cells begin to produce AMH/MIS
with increasing levels that peak at about 10 years of age. AMH/MIS is thought to maintain meiotic arrest in the
oocyte of the developing follicle in prepubertal females (9).
FIGURE 18-5 ▪ Diagram indicating the gestational period of development of the ovary, ducts, and external
genitalia.
In the female embryo, the paramesonephric ducts fuse caudally before reaching the urogenital sinus, a process
that is completed by week 10 (Figure 18-5). The unfused paramesonephric ducts become Fallopian tubes and
the fused portions the uterus and the upper vagina. The distal tip of the Müllerian duct abuts the posterior wall of
the urogenital sinus within a patch of mesoderm. This point is the future site of the hymenal membrane. The
patch of mesodermal urogenital sinus epithelium begins to proliferate, forming a column of squamous cells called
the vaginal plate that eventually gives rise to the vaginal epithelium. The vaginal plate and the Müllerian duct
become patent by canalization early in the second trimester (by week 18). Mesonephric ducts in the female
embryo begin to regress if not stimulated by testosterone by about week 10; however, mesonephric remnants in
the broad ligament and lateral wall of the uterus and vagina can persist as Gartner ducts. By 13 weeks'
gestation, the body (corpus) of the uterus and the cervix begin to be distinguished. In the fetus and the newborn,
the cervix is twice as long as the corpus, whereas in the adult, the corpus is about two times longer than the
cervix. At birth, the cervix and uterus together measure about 4 cm in length. The effects of maternal hormones
(estrogens and progestins) result in a proliferative-to-weakly secretory endometrium at birth and cervical
squamous cell maturation. These changes rapidly disappear after birth. During childhood, the endometrium is
usually thin, with inactive glands in a spindled inactive stroma. The uterus reaches a plateau of growth in the
second year of life, until the premenarchal uterine growth increase. The final adult (nulliparous) uterus measures
7 to 8 cm in longest dimension and weighs between 40 and 80 g.
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The final maturation of the female reproductive tract is at the beginning of uterine bleeding (menarche), which
occurs between 11 and 15 years of age. Menarche appears to be occurring earlier in US females, which has
been proposed to be associated with the increased incidence of childhood obesity. The early menstrual cycles
are often anovulatory and can result in disordered proliferative endometrium. By midadolescence, regular
menstrual cycles should be occurring, along with the monthly histologic changes that are well described for the
adult female reproductive tract.
A number of genes have been reported as important in regulating normal internal female genital development.
WNT4 expression is essential for normal Müllerian development. A few patients have recently been described
with mutations in WNT4 that present with phenotypes similar to that seen in Mayer-Rokitansky-Kuster-Hauser
syndrome (Müllerian agenesis). In addition, spatial and temporal expression patterns of members of the HOXA
gene locus are essential for normal development of the Fallopian tubes (HOXA9), uterus (HOXA 10), cervix
(HOXA 11), and upper vagina (HOXA13). In mice, other transcription factors that have also been implicated in
normal Müllerian duct formation include Lim1, Pax2, Emx2, Wnt5a, Wnt7a, and p63 (a p53 homolog) (169).
Recently, it has been shown that expression of the Msx2 gene in mice is essential for normal vaginal
development and that alterations in Msx2 levels may account for some of the female reproductive tract
phenotypes seen with diethylstilbestrol exposure in utero (168). In the mouse, normal uterine gland development
(uterine adenogenesis) and branching involve several important genes including Lif, calcitonin, several Wnt-
signaling genes, matrix metalloproteinases, and their inhibitors, insulin-like growth factors, estrogen receptor a,
and prolactin (reviewed in ref. 169).
Female External Genitalia

The external female genitalia begin to form during the fourth week of embryonic development. The genital
tubercle forms ventral to the cloacal plate as two stromal elevations of the ectoderm. Lateral to the cloacal plate
on each side, two parallel folds develop, the labia majora and the minora. As the labioscrotal folds extend
cranially around the genital tubercle, they fuse and become the mons pubis (164). The role of estrogen in the
development of any and all of the female reproductive tract is unknown. Levels of maternal estrogens are high
throughout pregnancy, and local production by the fetal ovaries begins early, but is currently thought to be of
little consequence. While estrogen secretion in utero does not appear to be important for sex determination,
estrogen signaling does appear to be critical in the maintenance of the maturing ovary later during puberty.
Blocking estrogen signaling in puberty results in transdifferentiation of the ovary and expression of Sertoli cell
markers (27). Estrogen signaling appears to be critical in maintaining the mature ovarian structure by
suppressing testicular development during early adult development. If a female fetus is exposed to elevated
androgens before 10 to 12 weeks of gestation, the external genitalia may become ambiguous or resemble a
phenotypic male. The vagina will often open into the membranous portion of the urethra. If androgens become
elevated after week 20, the only effect will be an enlarged clitoris.
The entire vulva, with the exception of the vestibule, is lined by keratinized, stratified squamous epithelium. The
vestibule and the vagina are lined by nonkeratinizing squamous epithelium, which becomes glycogenated in
women of reproductive years, which should not be confused with koilocytotic change. The vaginal vestibule
contains the orifices of the paraurethral (Skene) glands, the major (Bartholin) and minor vestibular glands, and
the urethral meatus. The paired paraurethral glands are located on either side of the urethral meatus, are
composed of pseudostratified mucous-secreting columnar epithelium, and are drained by ducts lined by
transitional epithelium. The vestibular glands contain acini composed of simple columnar, mucous-secreting
epithelium. The major vestibular (Bartholin) glands are drained by ducts lined proximally by mucous-secreting
epithelium, and more distally by transitional epithelium with terminal lining by squamous epithelium on their exit
just external to the hymenal ring. The minor vestibular glands are located close to the surface and ring the
vestibule.
PREMATURE OVARIAN FAILURE

Premature ovarian failure can occur before or after the onset of menarche. Premature ovarian failure is defined
by a deficiency of sex steroid production, high gonadotropin levels, and amenorrhea in any woman less than 40
years old. Chromosomal and genetic abnormalities including gonadotropin ligand or receptor defects, cholesterol
desmolase deficiency, galactosemia, Turner syndrome, Fragile-X syndrome, Swyer syndrome, and
blepharophimosis syndrome (FOXL2 gene mutation) are all associated with premature ovarian failure (91). In
addition, infections, autoimmune disorders, and iatrogenic causes can lead to premature ovarian failure (91). In
particular, treatment of childhood malignancies can result in a variety of disorders of abnormal puberty including
premature puberty or hypogonadotrophic hypogonadism with premature ovarian failure (95).
STRUCTURAL ABNORMALITIES OF THE FEMALE REPRODUCTIVE ORGANS

The phenotypic abnormalities that can occur in the development of the female reproductive tract are numerous.
These may represent isolated poorly understood variations in normal development, or they may be associated
with major malformation syndromes; either may be related to chromosomal abnormalities or defects in known and
unknown genes. Additionally, teratogens have been associated with abnormalities of the reproductive tract. DSD
are discussed separately.
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The Ductal System
Müllerian duct anomalies are frequently associated with intersexual disorders, discussed below. In patients with
normal ovaries, Müllerian duct malformation occurs in about 0.5% of women and is frequently accompanied by
anomalies of the urinary tract (122). These Müllerian duct anomalies include lateral and vertical fusion defects,
hypoplasia, or absence of Fallopian tubes, uterus, or the upper vagina. Uterine abnormalities can be structurally
and morphologically divided into three categories. These include (a) complete failure of formation of the
Müllerian duct unilaterally (resulting in unicornuate uterus) or bilaterally (absent uterus), (b) arrested Müllerian
duct development (hypoplastic uterus), or (c) abnormal lateral fusion of the Müllerian ducts to varying extents,
resulting in paired uteri and cervices, accompanied by a vaginal septum (uterus didelphys), paired uteri and one
cervix (bicornuate uterus), or a uterine septum (septate uterus) (45). Affected patients may be asymptomatic or
may present with infertility, repeated abortions, breech delivery, preterm delivery, dyspareunia, or dysmenorrhea.
Because the Wolffian ducts are essential for inducing the Müllerian ducts, defects in Wolffian duct development
may likewise lead to uterine abnormalities. Isolated abnormalities of the Fallopian tubes (duplication or absence)
are rare. Abnormalities of the cervix are also very rare and include atresia or hypoplasia, which is due to failure
of canalization of the Müllerian ducts. The molecular events regulating the later events of Müllerian development
including lateral fusion remain largely unknown.
Absence of the vagina occurs in 1 in 4,000 to 5,000 women and is often associated with Müllerian anomalies. In
the Mayer-Rokitansky-Kuster-Hauser sequence, there is an absent vagina and often poorly formed uterus and
Fallopian tubes (37, 45, 48). These patients are genotypic (46, XX) and phenotypic (normal external genitalia)
females with normal endocrine status and they often present with amenorrhea. Some cases are associated with
upper urinary tract abnormalities and/or spine and skeletal abnormalities. MURCS (Müllerian, Renal, Cervical,
Somite) association appears to be an extreme presentation of these clustered anomalies (OMIM 601076) (48). It
has been suggested that this sequence may be due to abnormal development of the Wolffian duct, with resulting
abnormal Müllerian duct development. It has also been proposed that the etiology is related to abnormal HOXA
gene expression during Müllerian development; however, despite numerous studies looking for mutations in the
HOXA gene cluster none have been found to date in patients with Mayer-Rokitansky-Kuster-Hauser sequence.
The Mayer-Rokitansky-Kuster-Hauser sequence usually occurs in a sporadic manner, and therefore aberrant
HOXA gene expression may be related to temporally altered gene expression levels by exogenous factors. Other
anomalies of the vagina result from developmental defects involving the cloaca or urogenital sinus. As previously
discussed, the lower third of the vagina and the hymen are thought to be derived from ectoderm. If the urogenital
sinus develops normally, the vagina can be present as a blind pouch even in the absence of normal Müllerian
duct development. A variety of miscommunications between the urethra, rectum, and vagina have been
described (42). Vaginal obstruction can result from absence of communication between the introitus and the
vaginal canal, related to defects in vertical fusion. Etiologies include imperforate hymen, atresia of the lower
vagina, or a transverse vaginal septum (failure of vertical canalization of the vaginal plate at the site of fusion
between the urogenital sinus and the Müllerian ducts). Imperforate hymen, formed where the urogenital sinus
and canalized, fused sinuvaginal bulbs meet, is the most common cause, and if there is complete obstruction this
leads to a marked dilatation of the vagina and the uterus (hydrometrocolpos—retention of secreted mucous). If
the person is asymptomatic until the onset of menses, the obstruction can result in hematocolpos.
The External Genitalia

Abnormalities in the development of the perineum, labia, and clitoris are not uncommon. The normal variations
and structural abnormalities of the vulva have been extensively reviewed (141). Complete absence of the
external genitalia is rare and occurs as part of malformation syndromes such as sirenomelia, limb-body wall
defects, Robinow syndrome, multiple pterygia syndrome, Fryns syndrome, or CHARGE association. Vulvar
duplication is rare and is associated with multiple congenital anomalies. Abnormalities of the clitoris and labia
may cause problems in assigning the correct sex at birth. Clitoral hypertrophy may resemble male hypospadias.
Exposure of the female fetus to excess male hormones, such as in congenital adrenal hyperplasia (reviewed in
ref. 90), a maternal or fetal virilizing tumor, or maternal medication, can result in clitoral hypertrophy and may
result in partial fusion of the posterior portion of the labia. Complete absence of the clitoris is rare. Many of the
molecular events involved in early genital tubercle (anlage for the penis and clitoris) development, especially
related to penile development, have recently been reviewed (165).
DISORDERS OF SEX DEVELOPMENT (INTERSEX DISORDERS)

Intersex disorders are characterized by congenital conditions associated with abnormal development of the
gonads or secondary sex organs, or both, either with normal or abnormal sex chromosomes (Table 18-1) (44, 63,
78). In patients with normal sex chromosomes, these disorders are primarily associated with excess androgens in
women and abnormal androgen or MIS action in males. Patients with abnormal sex chromosomes are divided
into those with and without sexual ambiguity. These disorders are often associated with reproductive failure, and
certain groups are at increased risk for the development of gonadal neoplasms. Classification schemes have
been delineated based primarily on phenotype
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and genotype, including pseudohermaphroditism, mixed gonadal dysgenesis, true hermaphroditism, and pure
gonadal dysgenesis. However, it is important to note that many of these conditions reflect a spectrum of
phenotypic changes within a single genotype, or a spectrum of genotypic changes within a single phenotype.
Therefore, disorders of sexual development are discussed by underlying etiology, when known. A new
consensus statement regarding intersex disorders, now referred to as DSD, was recently published (78). This
includes numerous changes in nomenclature provided in Table 18-1.
Female Pseudohermaphroditism (46,XX DSD)

Female pseudohermaphroditism results from excessive androgen exposure in 46,XX females with two ovaries
and normal Müllerian duct development. The process is characterized by a spectrum of virilization of the external
genitalia varying from clitoral enlargement, if androgen exposure begins after the 12th week of fetal life, to
complete masculinization of the external genitalia if the process begins very early in fetal life. The source of
androgen excess is most commonly related to enzymatic defects in the biosynthetic pathways of steroid hormone
production from cholesterol. Glucocorticoids, mineralocorticoids, and sex hormones are synthesized through
common intermediates; therefore, a defect in one enzyme can lead to overproduction of other products (90, 93).
Table 18-1 ▪ CLASSIFICATION OF DSD

I Nomal sex chromosomes
A. Female pseudohermaphroditism (excess androgens in females)—(46,XX DSD)
1. Adrenogenital syndrome (21-hydroxylase deficiency, 11-|3-hydroxylase deficiency)

2. Maternal ingestion of androgenic hormones
3. Maternal virilization
B. Male pseudohermaphroditism (deficient androgens in males)—(46,XY DSD)
1. Testicular regression syndrome

2. Gonadotropin-Leydig cell defects
3. Steroid enzyme deficiencies (testosterone/dihydrotestosterone)
4. Androgen insensitivity syndromes
5. Persistent Müllerian duct syndrome
II Abnormal sex chromosomes
A. Sexual ambiguity frequently present
1. Mixed gonadal dysgenesis—(45,X/46,XY MGD)

2. True hermaphroditism—(ovotesticular DSD)
B. Sexual ambiguity infrequently present
1. Pure gonadal dysgenesis—(46,XY complete gonadal dysgenesis)

2. Klinefelter syndrome—(47,XXY)
3. Turner syndrome—(45, X)
4. XX male syndrome—(46,XX testicular DSD)
Shown in bold is the new nomenclature. From Lee PA, Houk CP, Ahmed SF, et al. Consensus
statement on management of intersex disorders. International Consensus Conference on Intersex.
Pediatrics 2006;118:e488-e500.
Adrenogenital syndrome (congenital adrenal hyperplasia) is the most common cause of female
pseudohermaphroditism and is most commonly an autosomal recessive disorder. 21-Hydroxylase deficiency
(OMIM 201910) accounts for more than 90% of cases of congenital adrenal hyperplasia and occurs in about
1:50,000 births. Lack of 21-hydroxylase prevents conversion of progesterone to 11-deoxycorticosterone and 17-
hydroxyprogesterone to 11-deoxycortisol, thereby resulting in deficiencies of Cortisol and aldosterone, which can
be life threatening in the neonatal period. As a result, adrenocorticotropic hormone levels are high and the
biosynthetic intermediates shift the equilibrium reaction toward overproduction of androgenic sex steroids, in
particular testosterone. Estrogen levels do not increase because conversion of testosterone to estrogen is
dependent on the presence of aromatase that is only found in specific target organs. The degree of virilization
varies depending on the severity of the enzymatic defect and the timing of the onset of the endocrine effects.
Another enzyme deficiency in this pathway leading to a similar phenotype involves 11-β-hydroxylase (OMIM
202010). Some girls with congenital adrenal hyperplasia can present late with delayed menarche,
oligomenorrhea, hirsutism, and polycystic ovaries (90).
Other causes for female pseudohermaphroditism include maternal ingestion of synthetic androgens or progestins
during pregnancy or the presence of a maternal virilizing tumor such as a primary or metastatic ovarian tumor
and the luteoma of pregnancy, a hyperplastic lesion of the thecalutein or stroma-lutein cells. The degree of
masculinization in the latter is usually mild, suggesting that the luteoma does not become functional until the
second half of gestation. The fetal gonads and Müllerian duct structures are unaffected, and normal secondary
female sex characteristics, ovulation, and menstruation develop at puberty.
Disorders of Sex Development Associated with Abnormal Sex Chromosomes

Abnormalities in the sex chromosomes include deletions, additions, and mosaicisms, all of which can result in a
wide variety of effects on gonadal development and thereby phenotypic expression. The gonads in these
patients can range from a streak gonad to a relatively normal ovary or testis (11). As a result, sexual ambiguity
may or may not be present. Interestingly, within a single genotypic abnormality and genetic background, the
phenotype may vary widely, likely due to small differences in genetic expression within the very restricted time
periods characterized by many of the steps of gonadal differentiation (11).
Mixed gonadal dysgenesis (45,X/46,XY MGD; OMIM 233420) refers to patients with at least one testis, persistent
Müllerian duct structures, and incomplete development of Wolffian duct structures. The external genitalia are
usually ambiguous with abnormalities of the labioscrotal swellings. The disorder is heterogeneous, with the most
frequent karyotypes being 45,X/46,XY or 46,XY. Many 45,X/46,XY
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individuals are phenotypically normal males, and only a small percentage present with mixed gonadal dysgenesis
(121). About two-third of patients with mixed gonadal dysgenesis are raised as females. The gonads may have a
wide variety of appearances depending upon the chromosomal complement present. They often demonstrate a
disorganized arrangement of testicular and ovarian elements that do not resemble either gonadal type (Figure
18-6).
FIGURE 18-6 ▪ Dysgenetic gonad removed from a 3-year-old child with ambiguous genitalia. Ovarian-like stroma
is intermixed with abnormally developed sex-cord like structures containing primitive germ cells.
True hermaphrodites (ovotesticular DSD; OMIM 235600) are quite rare and contain both fully developed ovarian
and testicular tissues either separately or combined as an ovotestis. The external phenotype can be either male
or female, but usually the genitalia are ambiguous. Phenotypic men usually have incomplete virilization,
gynecomastia, and monthly hematuria secondary to menstruation into a persistent urogenital sinus. The most
common karyotypes include 46,XX (50% to 70%), 46,XY or mosaic 46,XX/46,XY, 46,XY/47,XXY, 45,X/46,XY (78,
93). The testis is usually in the scrotum or labia, and the ovary is always abdominal. The ovotestis can be
anywhere along the descent pathway but is most commonly abdominal. The ovotestis is frequently arranged with
the ovarian and testicular tissues immediately adjacent to each other, usually with a sharp demarcation (155)
(Figure 18-7). By reproductive age, the ovarian portion of an ovotestis usually appears to be normal, complete
with follicles, corpora lutea, and corpora albicans. The testicular portion, however, is usually abnormal, with loss
of germ cells and tubular sclerosis. The type of ductal organ that develops adjacent to a gonad often depends on
the type of gonad present at that site. In true hermaphrodites, an epididymis or a vas deferens is next to a testes
and a Fallopian tube is adjacent to an ovary. A Müllerian or a Wolffian structure develops adjacent to an
ovotestis but not both. A variety of uterine anomalies can be present, although most true hermaphrodites with a
uterus menstruate. The underlying defect leading to true hermaphroditism is not yet completely established.
FIGURE 18-7 ▪ Ovotestis removed from a 6-year-old child with ambiguous genitalia and an undescended left
testicle. Peripheral lymphocyte karyotype 46,XY. The gonad demonstrates two distinct regions: the area on the
right composed of ovarian stroma and numerous primordial follicles, and the area on the left lower corner shows
normally developed seminiferous tubules with reduced numbers of germ cells.
Pure or complete gonadal dysgenesis or XY sex reversal (46,XY complete gonadal dysgenesis) includes a
number of conditions and is characterized by phenotypic females with Müllerian ductal structures (uterus and
Fallopian tubes) and streak gonads. The most common karyotype is 46,XY (Swyer syndrome; OMIM 306100),
and the defect is due to an X-linked recessive mutation or deletion of the SRY gene on the short arm of the Y
chromosome (35, 97, 104). Patients with Swyer syndrome have a female phenotype with a uterus and Fallopian
tubes, but the karyotype is 46,XY and two dysgenetic gonads in the abdomen. In the rare patients with 46,XX
gonadal dysgenesis or XX sex reversal (46,XX testicular DSD; OMIM 278850), the disorder is usually an
autosomal recessive one, with or without an abnormality of the X chromosome (72, 132). An autosomal dominant
form also exists (OMIM 154230). The gonad is that of a streak, with ovarian-like stroma and no oocytes. Other
genetic defects leading to 46,XX testicular DSD include a translocation of the SRY gene (OMIM 480000) or a
duplication of SOX9 gene (OMIM 608160) that can both result in a male phenotype (78).
Turner syndrome occurs in about 1 in 3,000 live female births and is most commonly due to mosaic or nonmosaic
45,X karyotype. It is estimated that the vast majority of fetuses with nonmosaic 45,X karyotype spontaneously
abort. Common features of this syndrome include phenotypic females with short stature, webbing of the neck
(cystic hygroma in utero), congenital lymphedema of the hands and feet, preductal coarctation of the aorta,
ventricular septal defects, micrognathia, renal anomalies (horseshoe kidney, hydronephrosis secondary to
ureteropelvic obstruction), congenital nevi, short fourth metacarpal, and streak gonads (by adulthood) (47). Not
all of these features appear in every patient, and other occasional anomalies have also been described. Most
recently, haploinsufficiency for the short
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stature homeobox gene (SHOX), located on the X and Y chromosomes, has been proposed to account for some
of the phenotypic findings in Turner syndrome patients (reviewed in ref. 47). The fetal ovary in a patient with
Turner syndrome is normal histologically until about 16 to 18 weeks' gestation. Following entry into meiosis,
however, without the second X chromosome the germ cells disappear, resulting in a streak ovary by adulthood.
These patients have primary amenorrhea by adolescence. The internal genitalia are normal female. Germ cell
tumors in pure Turner syndrome are very rare due to the absence of germ cells and the absence of the Y
chromosome. Epithelial ovarian tumors can arise in these ovaries but at a rate no greater than that in normal
females.
Tumors Associated with Gonadal Dysgenesis

Patients with dysgenetic gonads are at risk for the development of ovarian neoplasms. More than 50% of these
neoplasms are gonadoblastomas, which are benign tumors, found only in patients with Y chromosomal material
and dysgenetic gonads (Figure 18-8) (131). The frequency of occurrence of gonadoblastoma in the
abovementioned clinical populations correlates with the frequency of the Y chromosome; gonadoblastoma is
seen in approximately 30% of patients with mixed gonadal dysgenesis, less than 3% of individuals with true
hermaphroditism, and over 50% of patients with 46,XY pure gonadal dysgenesis (23). Patients with 46,XX pure
gonadal dysgenesis and patients with Turner syndrome only very rarely develop gonadal tumors; however, hilus
cell hyperplasia and hilus cell tumors have been reported (132). The significance of the development of
gonadoblastoma is that it is associated with a very high frequency of concurrent or future development of a
malignant germ cell tumor, most commonly dysgerminoma (146). The risk of developing a germ cell malignancy
with various forms of DSD has recently been summarized (23, 39) and the groups with the highest risks include
patients with gonadal dysgenesis (+Y), partial androgen insensitivity syndrome (nonscrotal gonad), Frasier
syndrome or Denys-Drash syndrome (those with +Y). Dysgerminoma has been discovered as early as 6 months
of age (146), and patients as old as 60 years of age have been seen by the authors. Because of the high
incidence of gonadoblastoma in women with XY gonadal dysgenesis, a susceptibility locus on the Y chromosome
has been proposed, located on the proximal long arm (23, 126, 147). The pathology of gonadoblastoma is
described later.
FIGURE 18-8 ▪ Small, streak gonad from a 46,XY phenotypic female that is composed of wavy ovarian-type
stroma and no primordial follicles. Deep within the cortex, well-defined nests composed of a mixture of germ cells
and granulose-like cells, consistent with gonadoblastoma.
ACQUIRED ABNORMALITIES AND OTHER LESIONS

Infections
Infections of the lower genitourinary tract account for the majority of genital lesions in premenarchal girls. These
infections are most commonly due to a variety of bacterial organisms that do not penetrate the mucosa and are
not related to a specific disease. Infections common in the sexually active, such as Gardnerella vaginalis and
molluscum contagiosum, are quite rare in young children and should raise the suspicion of sexual abuse
(Chapter 7). Specific infections of the vulvovagina include human papillomavirus (HPV), herpes simplex virus,
syphilis, and molluscum contagiosum.
Human Papillomaviruses:
Condyloma acuminata are sexually transmitted lesions caused by papilloma viridae, most commonly the HPV
types 6 or 11, although HPV 2 may also be seen (49). These lesions may involve the vulva, vagina, cervix,
urethra, and perianal skin. Vulvar and vaginal lesions are commonly papillary and are almost always multiple;
cervical lesions are often flat, white lesions surrounded by hyperemic mucosa. Uncommonly, the involved
epithelium may extend into the endocervical glands and, therefore, have an endophytic appearance. Most
lesions are asymptomatic unless secondarily infected. Histologically, parakeratosis, acanthosis, hyperkeratosis,
and dyskeratosis are evident (Figure 18-9). The typical koilocytic cells with perinuclear cytoplasmic halos
surrounding irregularly contoured (“raisinoid”) nuclei may be seen in the more superficial or intermediate layers.
Although intranuclear and cytoplasmic inclusions are not found by light microscopy, electron microscopy has
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shown intranuclear viral particles. Condyloma acuminata are commonly associated with pregnancy, and may
result in laryngeal papillomas of infants exposed during delivery. Regression may occur after pregnancy;
however, the clinical course may be quite protracted without treatment. In rare cases, progression to carcinoma
in situ, verrucous carcinoma, and squamous carcinoma of the vulva may occur. Condyloma acuminata may at
times be difficult to distinguish from vulvar intraepithelial neoplasia. The presence of a flat, macular growth
pattern, abnormal mitoses, atypical nuclei, marked variation in nuclear size and shape, and hyperchromasia are
all characteristics of vulvar intraepithelial neoplasia. The modes of transmission of HPV include perinatal,
autoinoculation, heteroinoculation, and sexual abuse (49, 94). Cervical infections with HPV types
16,18,31,33,35, and 45 are associated with the development of intraepithelial neoplasia (7). Only a small number
of sexually active teenagers have dysplastic cells on cervicovaginal cytologies requiring colposcopically directed
cervical biopsies. Invasive cervical carcinoma has rarely been reported in teenagers (65). The FDA approved an
HPV vaccine in June 2006 for women aged 9 to 26 years that is effective against several high-risk subtypes of
HPV (123). Despite clear evidence of effectiveness in reducing infection by high-risk HPV, the proposed
mandatory use of this vaccine in young girls (prior to the beginning of sexual activity) has raised an ethical
debate.
FIGURE 18-9 ▪ Condyloma acuminatum showing parakeratosis, ancanthosis, and numerous koilocytic cells with
nuclear irregularity and prominent perinuclear vacuolization. No dysplasia is present.
Herpes:
Patients with genital infection with herpes simplex virus types I or II present with dysuria and vulvar pain, often
accompanied by generalized malaise and fever. The clinical picture is dominated by the appearance of vesicles
and shallow ulcers that are often secondarily infected. Only two thirds of culture-positive women show diagnostic
genital lesions. Histologically, the ulcers typically demonstrate
extension deep into the epidermis, with the characteristic intranuclear inclusions present at the periphery of the
lesion. Late in the evolution of the ulcer, the infected cells undergo karyorrhexis and lysis, and therefore, infected
cells may not be identifiable in biopsy material. Cytologic evaluation of scrapings from a fresh ulcer or freshly
opened vesicle will usually show the characteristic viral cytopathic effects. Recurrent episodes of herpetic vulvitis
are common; however, these episodes decrease in frequency over time whether or not acyclovir is given.
Anogenital herpes in children raises the concern of sexual abuse, but is not definitive evidence (61). Varicella
infection of the lower genital tract is rare and most commonly detected in postmenopausal women.
Syphilis:
The primary lesion of syphilis is the chancre, a painless, shallow ulcer with raised edges that usually presents
within 10 to 90 days of initial contact. These lesions often occur on inconspicuous surfaces, such as the cervix,
and in about 50% of patients, the primary lesion is never seen (60). Histologically, the chancre is characterized
by ulceration of the epidermis with acute and chronic inflammation within the dermis. There is a marked
perivascular inflammatory response with a large number of plasma cells. The lack of specificity of these findings
raises the importance of considering syphilis in the differential diagnosis of inflammatory lesions.
Lymphadenopathy may develop 3 to 4 days after the chancre appears. If the primary stage is left untreated, the
secondary stage of the disease will become evident within 6 weeks to 6 months when the patient will show
elevated plaques measuring up to 3 cm, especially on the vulva. These plaques are known as condylomata lata
and demonstrate marked acanthosis, epithelial hyperplasia, and hyperkeratosis. The inflammatory response
within the dermis is similar to that seen in the chancre. Both the chancre and the condyloma lata are rich in
spirochetes, which may be detected by the Dieterle or Warthin-Starry silver stains. However, these stains may
be negative even with active infection. Serologic studies should be performed if syphilis is considered clinically or
pathologically; even these studies may be negative for weeks after the presentation of the primary chancre.
Other methods used for detecting spirochetes include dark field examination of serum expressed from the base
of the ulcer or by a fluorescent-conjugated antibody technique. These methods are more sensitive and specific
than the silver stain on paraffin-embedded tissue (60).
Molluscum Contagiosum:
Molluscum contagiosum is usually an asymptomatic infection caused by a moderately contagious virus often
passed through sexual contact. The lesions are generally multiple, small, smooth 3- to 6-mm papules with a
central umbilication. Diagnosis rarely requires biopsy. Cytologic identification of the typical intracytoplasmic
inclusion bodies (molluscum bodies) within scrapings or in biopsy material is adequate to confirm the diagnosis
(Figure 18-10).
Chlamydia Trachomatis:
The most common sexually transmitted disease in adolescent girls is Chlamydia trachomatis (119).
Approximately 22% of urban adolescent girls
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have endocervical cultures positive for this organism, and the majority are asymptomatic (10). The organism
most commonly infects the columnar and immature squamous cells of the endocervix; however, salpingitis and
endometritis may be seen, which often leads to infertility (10). Cell culture is the optimum diagnostic test and has
an accuracy rate of about 90%. Infected patients show lymphocytic inflammation and reactive epithelial changes
by cytology. Some observers have reported cytoplasmic inclusion bodies in infected cells; however, others
interpret these bodies as nonspecific cytoplasmic vacuoles. Therefore, the finding of lymphocytes, reactive
epithelial cells, often dyskeratotic cells, and vacuolization of metaplastic cells should be considered suggestive
but not diagnostic for chlamydial infection. Chlamydial infection of the vulva or vagina may result in
lymphogranuloma venereum, a skin lesion characterized first by painless skin erosion, followed by lymphadenitis
involving superficial groin lymph nodes, which may ulcerate and rupture. Over time, the chronic inflammatory
process and chronic lymphatic obstruction may result in stricture, fibrosis, and nonpitting edema of the vagina
and rectum.
FIGURE 18-10 ▪ Molluscum contagiosum with numerous epidermal cells containing large intracytoplasmic
inclusion bodies, the so-called molluscum bodies, which are typically found in the lower cells of the stratum
malpighii. The molluscum body compresses the nucleus, which appears as a thin crescent at the periphery of the
cell.
Miscellaneous Infectious Diseases

Cytomegalovirus, Epstein-Barr virus, Candida, and other fungal and bacterial infections may cause acute or
chronic inflammatory lesions of the lower genital tract, most
commonly without ulceration. Chancroid is a rare genital ulcer caused by Haemophilus ducreyi, which is
identified by culture alone. Histologically, chancroid shows a granulomatous inflammation with Gram-negative
organisms. Tuberculosis of the vulva is rare and is usually associated with the disease at other sites. Sexual
transmission is most uncommon in the absence of immunosuppression. Enterobius vermicularis (pinworm) may
cause a severe vulvovaginal pruritus in infected children. Granuloma inguinale is caused by
Calymmatobacterium granulomatous, a Gram-negative encapsulated rod. Primary lesions may present
anywhere in the lower genitourinary tract as painless papules or necrotizing ulcers. The diagnosis depends on
the identification of large, vacuolated histiocytes containing the characteristic cytoplasmic encapsulated bacilli
called Donovan bodies, demonstrated by Warthin-Starry or Giemsa stains.
Noninfectious Inflammatory Diseases

Behçet Syndrome:
Behçet syndrome is characterized by the triad of recurrent oral ulcers, vulvar ulcers, and various ophthalmologic
inflammations (OMIM 109650). Acne, cutaneous nodules, thrombophlebitis, encephalopathy, and colitis may also
be present. The vulvar ulcers, seen in postmenarchal females, may be deep and characteristically relapse.
Histologic examination reveals chronic inflammation and necrotizing vasculitis, which is considered a cardinal
finding. Vascular endothelial cell swelling may result in arteriolar occlusions and venous thrombosis. Behçet
disease is presumed to be autoimmune in etiology (125). Healing of the ulcers may result in severe scarring.
Childhood Behçet may run a less severe course (71).
Crohn Disease:
Vulvar involvement by Crohn disease is rare and characterized by ulcerations that are often multiple, deep, and
secondarily infected. The diagnosis may be difficult, particularly if this is the presenting site of the disease (130).
Histology demonstrates noncaseating granulomatous inflammation with extensive granulation tissue within the
dermis (Chapter 14).
Lichen Sclerosus:
Lichen sclerosus is a dermatosis of unknown etiology characterized pathologically by thinning of the epithelial
layer, blunting or loss of the rete ridges, and a homogeneously collagenized or edematous subepithelial layer in
the dermis with a band of chronic inflammatory cells beneath (Chapter 25). There is an absence of melanosomes
and disappearance of the melanocytes, resulting in a hypopigmented patch that may be pruritic. The microscopic
findings may vary considerably, depending on the age of the lesions, excoriation, and treatment. Lichen
sclerosus is not limited to the elderly population and may be seen in the reproductive years, and has been
reported in children as young as 18 months of age (OMIM 151590) (111). In children, symptoms include dysuria,
painful defecation, and rectal bleeding (12). This may lead to anal fissures and genital and perianal ulcers, which
may be confused with sexual abuse. Although lichen sclerosus is sometimes associated with vulvar squamous
carcinoma, it is not considered to be a premalignant condition.
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Bullous Diseases (See Chapter 25):
The vulva may be involved with virtually any dermatologic disease; however, some of the bullous diseases may
have their first manifestations in the vulva and in childhood. Darier-White disease (keratosis follicularis) is an
autosomal dominant skin disorder that frequently involves the vulva (OMIM 124200). Patients present anytime
after late childhood with crusted, hyperkeratotic papules that often appear darker than the surrounding skin.
Histologically, these papules show acantholysis of the suprabasal epithelial cells resulting in clefts that extend
from the basal layer through the granular layer. Corps ronds, nuclear grains, and dyskeratotic cells can be found
in the granular layer. Hyperkeratosis, acanthosis, and papillomatosis are seen, along with keratotic plugs.
Inflammation is minimal unless the lesions are secondarily infected. The affected gene is ATP2A2, which
encodes a sarco/endoplasmic reticulum Ca2+-ATPase. Hailey-Hailey disease (familial benign pemphigus or
benign chronic pemphigus) is an autosomal dominant disease that may also be sporadic (OMIM 169600). Onset
often occurs during adolescence, and several cases confined exclusively to the vulva have been reported. The
lesions are characterized by clusters of acantholytic vesicles resulting in suprabasalar lacunae. Unlike Darier
disease, vesicles and bullae are found. Acantholysis is more prominent than in Darier disease, and the basal
cells maintain their orientation to the basement membrane. Minimal dyskeratosis is seen. Mutations in the
ATP2C1 gene have been identified in several kindreds. ATP2C1 encodes a human homolog of an ATP pump in
yeast that accumulates calcium into the Golgi. Benign chronic bullous disease of childhood (linear IgA bullous
dermatosis) commonly involves the genital region of children. It presents as clusters of annular pruritic lesions
that evolve into tense bullae, which may then ulcerate. Patients may have fever and anorexia, and a preceding
infection is identified in 50% the cases. These lesions may be mistaken for evidence of child abuse. Biopsy
reveals subepithelial vesicles that may contain granulocytes and eosinophils, and epidermal microabscesses
may occur. The diagnosis depends on the identification of linear deposition of IgA in the basement membrane,
which may react against the bullous pemphigoid antigens 180 or 230, members of the dermoepidermal adhesion
complex (158). The differential diagnosis includes dermatitis herpetiformis (which shows granular IgA deposition)
and bullous pemphigoid (which shows linear IgG basement membrane deposits to the above BP180, BP230
antigens) (40, 158).
Vulvar involvement may be seen in Stevens-Johnson syndrome, the severe form of erythema multiforme. This
disease may be associated with herpes virus or mycoplasma infection, drug therapy, malignancy, or radiotherapy
and is characterized by involvement of the mouth, eyes, and skin with associated fever and other systemic
symptoms. The histologic features include necrotic keratinocytes, cellular edema, and intraepithelial vesicles.
The dermis shows a prominent chronic inflammatory infiltrate with extravasated red blood cells. Recent reports
suggest that activation of Fas on keratinocytes by FasL secreted by peripheral blood mononuclear cells
represents the initial step leading to diffuse apoptotic cell death of epidermal cells (1).
TUMORS OF THE FEMALE GENITAL TRACT

Benign Cystic Lesions
Bartholin Cyst:
Bartholin glands produce a clear mucoid secretion that continually lubricates the vestibular surface. The ducts of
Bartholin glands are prone to obstruction, resulting in cystic dilatation of the duct and secondary infection. The
epithelium lining the cyst may be squamous, transitional, or low cuboidal mucinous, and is immunoreactive for
carcinoembryonic antigen.
Mucous Cysts:
Vulvar mucous cysts are lined by tall-tocuboidal Alcian blue positive mucous-secreting epithelium. Squamous
metaplasia may be present. Mucous cysts likely arise from the urogenital sinus epithelium, and they lack both
myoepithelial cells and muscle fibers.
Gartner Duct Cysts:

Commonly seen are remnants of the mesonephric duct within the lateral wall of the vagina known as Gartner
duct cysts. They are thin-walled cysts lined by low cuboidal epithelium that may or may not be ciliated. Smooth
muscle may be present in the submucosal region.
Cysts of the Canal of Nuck (Peritoneal Lined Cysts):

These cysts are found in the superior aspect of the labia majora or inguinal canal and are believed to arise from
inclusions of the peritoneum at the inferior insertion of the round ligament into the labia majora, analogous to the
hydrocele of the spermatic cord. They may get quite large and must be distinguished from an inguinal hernia.
Müllerian Cyst:
Of uncertain genesis, Müllerian cysts can be located anywhere within the vagina and are lined by any of the
epithelia of the Müllerian duct, including mucinous, endocervical, endometrial, and ciliated tubal types.
Squamous metaplasia may also be observed. The majority of vaginal cysts represent Müllerian remnant cysts or
epidermal inclusion cysts (37).
Benign Solid Tumors

Hidradenoma Papilliferum:
Papillary hidradenoma is a benign tumor of apocrine sweat gland origin that presents as an asymptomatic small
mass in the labia majora or the lateral labia minora. This tumor has not been described before puberty and
almost all cases have occurred in white women (8, 157). Histologically, the papillary hidradenoma is composed
of tubules and acini lined by cuboidal epithelial cells with an outer layer of myoepithelial cells. These cells may
simulate a well-differentiated adenocarcinoma, and entrapped epithelial cells may create a pseudoinfiltrative
appearance. Mitotic figures are rare, and only mild nuclear pleomorphism is present (153). Therapy requires only
local excision.
Müllerian or Mesonephric Papilloma:

Several tumors that are composed of complex, arborizing papillae with a
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fibrovascular core supporting bland-appearing epithelial cells (92) have been reported within the vagina of young
girls. In some areas, the tumor may appear as either a solid mass or may contain glandular lumina and
eosinophilic hyaline globules. Their embryologic origin remains uncertain (139).
Fibroepithelial Polyp (Mesodermal Stromal Polyp):

Fibroepithelial polyps are uncommon hamartomatous polypoid masses of the vagina that are of interest to
pathologists because of their inclination to show bizarre stromal cells that may be confused with embryonal
rhabdomyosarcoma (100, 114). The age at presentation ranges from 16 to 75 years and while the majority of the
lesions arise in the vagina, they may be found in the cervix and the vulva (100). The lesions are usually
asymptomatic and discovered incidentally. They resemble an acrochordon and microscopically show a stratified
squamous epithelium covering an edematous stroma with variable numbers of fibroblasts. The lesions often
demonstrate marked hypercellularity, pleomorphism, mitotic counts of more than ten mitoses per ten high-power
fields, and atypical mitoses. The immunolocalization of steroid receptors in these bizarre cells and the frequent
relationship to pregnancy raise the possibility that these may be hormonally induced.
Miscellaneous Lesions:
As with virtually all other soft tissue neoplasms, capillary and cavernous hemangiomas and lymphangiomas may
occur in the lower female genitourinary tract and are similar to those in other anatomic sites. These lesions
should be distinguished from entities such as Kaposi sarcoma (which may have a hemangioma-like appearance)
and bacillary angiomatosis. Angiokeratomas are variants of hemangiomas that occur almost exclusively in the
scrotum and the vulva. Histologically, the dilated vascular channels are separated by strands of squamous
epithelial cells growing down from the overlying epithelium. This may be accompanied by various degrees of
acanthosis and papillomatosis. Neurofibromas, leiomyomas, granular cell tumors, hemangiopericytomas,
inflammatory pseudotumors, Langerhan cell histiocytosis, and alveolar soft part sarcoma have likewise been
described.
MALIGNANCIES OF THE LOWER GENITAL TRACT

Malignancies of the lower female genital tract are rare, with an incidence of about 0.5 cases per million female
children per year (74). The majority of these malignancies (more than 80%) are sarcomas, most commonly
rhabdomyosarcoma; approximately 10% are carcinomas and 5% extragonadal germ cell tumors. These
malignancies are discussed below in greater detail.
Rhabdomyosarcoma
Embryonal rhabdomyosarcoma is the most common malignancy of the lower genital tract in girls. Although these
lesions may arise in the vulva and the uterus, by far the most common genital site is the vagina. Most patients
present before the age of 5 years, with a peak incidence between 1 and 2 years (24, 54, 79). Patients often
present with vaginal bleeding or discharge, a palpable abdominal mass, or gross protrusion of a polypoid mass
at the introitus. The site of origin is often the anterior vaginal wall, with extension into the bladder and the rectum.
The initial size of the tumor has little prognostic significance (see Chapters 17 and 24).
FIGURE 18-11 ▪ Embryonal rhabdomyosarcoma of the genitourinary tract may be deceptively hypocellular, with
inapparent cytoplasm.
The most common histologic appearance of female genital rhabdomyosarcomas is that of the botryoid embryonal
subtype, with round-to-spindled cells of varying size in a loose, myxoid stroma (Figure 18-11). Eosinophilic
cytoplasm may or may not be apparent, and cytoplasmic cross-striations may occasionally be seen. The tumor
cells often crowd around blood vessels, and a cambium layer may be present with condensation of tumor cells
beneath the vaginal epithelium. The myxoid stroma may in some cases be rather hypocellular, resulting in a
tumor mass that may resemble a benign polyp. Rhabdomyosarcomas of the cervix provide a greater diagnostic
challenge histologically owing to the presence of islands of mature metaplastic cartilage in more than 40% of the
cases (29). This histologic manifestation appears to be unique to cervical rhabdomyosarcomas for unknown
reasons. An uncommon histologic pattern in the childhood genital tract is the diffuse form of embryonal
rhabdomyosarcoma. The differential diagnosis of rhabdomyosarcoma in the female genital tract includes
fibroepithelial polyps, Müllerian papillomas, and rhabdomyomas.
The clinical presentation and prognosis varies with the site of involvement. The majority of patients with vaginal
rhabdomyosarcomas present before the age of 5, and the lesions are localized. These tumors are treated by
chemotherapy, most commonly vincristine, dactinomycin, cyclophosphamide, and often adriamycin, followed by
resection (88). Given this therapy, among 26 patients with localized vaginal tumors treated according to the
Intergroup Rhabdomyosarcoma Study (IRS) protocols, six relapsed, five of whom were successfully salvaged;
the remaining patients were cured (53). There is now a broad consensus that primary chemotherapy after an
initial biopsy is the recommended therapeutic plan, followed
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by local excision (2, 86). Rhabdomyosarcomas of the uterus and cervix are rare and have been considered to be
distinct from those of the vagina, with a mean age of presentation of greater than 14 years and a seemingly
better prognosis (54). However, recent studies relying on more conservative management using preoperative
chemotherapy and radiotherapy has resulted in increased survival (26, 179).
FIGURE 18-12 ▪ EST of the vagina may demonstrate the entire histologic spectrum, similar to that seen in the
ovary and testis. Many lack the characteristic Schiller-Duval bodies and other architectural features, such as the
tumor illustrated. The histologic clues are the reticular pattern, the course chromatin, and the occasional
cytoplasmic pink globules.
Endodermal Sinus Tumor

The lower female genital tract is one of the more common sites for extragonadal, nonsacral endodermal sinus
(yolk sac) tumor. This is presumably due to abnormal migration of primordial germ cells early in gestation;
however, this does not explain the predilection for the vagina. These tumors present in children younger than 3
years of age, with the peak incidence between 8 and 11 months of age (25, 172). Presenting symptoms usually
include bloody vaginal discharge and a polypoid tumor filling the vagina. Microscopically, these tumors show the
same histologic patterns as those seen in the sacral region, infantile testis, and ovary (Figure 18-12). Although
reports before 1970 indicate an aggressive tumor with poor outcome, current chemotherapeutic regimens
containing cyclophosphamide, vincristine, and actinomycin have resulted in a 95% disease-free survival (25).
Several reports of children treated only with chemotherapy with complete response suggest that surgery may not
always be required (76).
TUMORS OF THE OVARY

Benign Cystic Lesions of the Ovary
The most common ovarian lesions detected radiographically are cysts derived from follicles at different stages of
maturation in prepubertal girls (32). Congenital ovarian cysts may be diagnosed in utero by ultrasound and are
associated with resolution; however, their evolution is variable and may require intervention (120, 176).
Complications such as torsion and rupture usually occur in cysts larger than 5 cm in diameter. Cysts that develop
in utero are most often lined by luteinized cells, whereas those in older children are more often lined by
granulosa cells. Premature infants born before the 30th week of gestation may have multiple follicular cysts
associated with estradiol production. These cysts are secondary to elevated follicle-stimulating hormone (FSH)
and luteinizing hormone (LH) secretion, and they may be associated with relative insensitivity of the
hypothalamus and anterior pituitary to negative feedback by estradiol (133).
Follicular cysts are commonly found in the ovaries of prepubertal females as an incidental finding. Rarely,
multiple follicular cysts may be the cause of pseudoprecocious puberty (138), although more often they are the
result of central causes of pseudoprecocity (110). As many as 75% of girls with juvenile hypothyroidism have
multicystic ovaries, and, rarely, the ovarian enlargement may be the presenting sign leading to a diagnosis of
hypothyroidism. Clinically, affected patients may show varying degrees of sexual precocity and galactorrhea due
to increased secretion of pituitary gonadotropins and prolactin. Treatment with thyroxin results in regression of
the ovarian cysts as well as the other symptoms (135).
Multiple follicular cysts should be distinguished from polycystic ovary syndrome (PCOS), which involves 3% to
8% of the female population. PCOS is responsible for 25% of cases of primary amenorrhea and is the most
common cause of delayed puberty and heavy anovulatory bleeding in adolescent females (16). It is characterized
by inappropriate gonadotropin secretion, hyperandrogenemia, increased peripheral conversion of androgens to
estrogens, chronic anovulation, and sclerocystic ovaries. The diagnostic criteria for PCOS were established in
2004 by the Rotterdam criteria (145), although this has been heavily debated subsequently. Affected patients
often have a history of premenarcheal obesity, secondary amenorrhea or oligomenorrhea, infertility, and
hirsutism. These features may occur alone or in any combination and the clinical spectrum is broad (6). The
unopposed estrogenic stimulation may cause menometrorrhagia and endometrial hyperplasia. Currently, the
underlying etiology of PCOS is widely debated; however, the resulting clinical manifestations are known to be
heavily impacted by environmental factors such as diet. While several genes have been linked with PCOS, the
evidence supporting this linkage is weak (34). Grossly, the ovaries of PCOS are enlarged two- to fivefold and
have smooth or nodular white surfaces, with multiple cysts located beneath the thickened cortex. Histologically,
multiple follicle cysts, atretic follicles, a prominent theca interna with luteinization, and medullary stromal
overgrowth are the principal histologic features. The superficial cortex is fibrotic and hypocellular (62). Maturing
follicles up to midantral stage and atretic follicles showing
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prominent luteinization of the theca interna may be twice as numerous as in normal ovaries. Primordial follicles
are often decreased in number (160). It is important to remember that these findings are not specific and may
accompany adrenal lesions such as Cushing syndrome, congenital adrenal hyperplasia, virilizing adrenal tumors,
primary hypothalamic disorders, ovarian lesions that produce excessive quantities of estrogens or androgens
and hypothyroidism. Long-term sequelae of PCOS include infertility, endometrial carcinoma, an increased risk for
cardiovascular disease due to type II diabetes mellitus, dyslipidemia, and systolic hypertension.
NON-NEOPLASTIC OVARIAN TUMORS

Endometriosis, or endometrioma, of the ovary may cause an adnexal mass in reproductive age females.
Pigmented foci on an otherwise normal ovary or a large solitary hemorrhagic cyst are the gross findings.
Histologic findings include columnar-to-plump cuboidal epithelium with condensed subepithelial stroma,
hemosiderin-laden macrophages, fibrosis, and inflammation. Rare cases may also contain smooth muscle.
Patients with uterine anomalies may develop severe endometriosis (127). Acute torsion of the adnexal structures
is an uncommon event but is a surgical emergency. The severity of the symptoms varies widely and includes
fever, nausea or vomiting, and abdominal pain (159). The correct diagnosis is rarely made preoperatively. The
preoperative radiograph commonly shows a pelvic mass with a cystic or solid texture, often with thin internal
septae, and may simulate an ovarian mass. Because the sigmoid colon cushions and secures the left adnexa,
the right adnexa is more often affected than the left. The Fallopian tube and ovary have usually undergone
hemorrhagic necrosis by the time of the surgery, often with secondary calcification. As a result, the cause of the
torsion is not clear in most cases. Massive ovarian edema is an unusual clinical entity most often occurring in
adolescence. It is characterized by marked enlargement of one or both ovaries due to marked accumulation of
edema fluid in the ovarian stroma. Massive ovarian edema may result from partial or intermittent torsion of the
mesovarium, interfering with venous and lymphatic drainage, but not with arterial blood flow (18, 167).
OVARIAN NEOPLASMS
Ovarian neoplasms account for approximately 1% of all childhood cancers. Although the most common ovarian
cancers in adults are epithelial, the distribution of histologic tumor types differs in children, with the majority being
derived from primordial germ cells (Table 18-2). Ovarian tumors are most frequently found from 10 to 14 years of
age, suggesting that hormonal factors may play a role in many. The most common symptoms at presentation
include abdominal pain that often simulates acute appendicitis, resulting in emergency laparotomy. Recent
advances in the management of these tumors have resulted in increased cure rates as well as preservation of
future fertility. Examples include new disease-specific chemotherapeutic regimens as well as the advent of
surgical staging (Table 18-3) (177).
Table 18-2 ▪ RELATIVE FREQUENCY OF OVARIAN NEOPLASMS IN CHILDREN AND

ADOLESCENTS
Histogenetic Category No. No. (%)
Germ cell 205 (58)
Coelomic epithelium 67 (19)
Sex cord-stromal 62 (18)

Supportive stroma and miscellaneous 19 (5)
Total 353 (100)
Ovarian Germ Cell Tumors

The ovary is the site of approximately 30% of all germ cell tumors. Ovarian germ cell tumors in children show a
higher frequency of malignancy than those in adults (98). The vast majority are diagnosed in postpubertal
adolescents; malignant germ cell tumors in the ovaries of very young children are exceedingly rare. Ovarian
germ cell tumors show a biologic and clinical heterogeneity not seen in their testicular or extragonadal
counterparts, with at least four distinct subgroups. These include mature teratomas, immature teratomas,
malignant germ cell tumors, and germ cell tumors arising in dysgenetic gonads.
Mature Teratoma:
Teratomas are defined as neoplasms containing a haphazard growth of one or more types of
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tissue derived from the three embryonic layers (ectoderm, mesoderm, and endoderm). Mature ovarian teratomas
represent 40% to 60% of all childhood ovarian neoplasms, and patients with these tumors most commonly
present at 13 to 15 years of age (30, 161). There is a 10% incidence of bilaterality. Mature teratomas can be
subdivided into those that are predominately cystic and those that are predominately solid. Cystic teratomas
characteristically contain the copious hair and sebaceous material characteristic of those in adults; however, this
type is less common in children (Figure 18-13). Immature elements are rarely found in predominately cystic
teratomas, and the malignant potential of cystic teratomas in children is minimal unless the child has a
constitutional genetic abnormality resulting in increased risk of development of neoplasms (such as Li-Fraumeni
syndrome). The solid mature teratoma, which is more common in children, may show a closer biologic
relationship to immature teratomas than to cystic mature teratomas, and it should be carefully sectioned to
exclude immature elements. Neuroglial tissue is the predominant component in these lesions.
Table 18-3 ▪ FEDERATION OF INTERNATIONAL GYNECOLOGISTS AND OBSTETRICIANS

(FIGO) STAGING OF OVARIAN CARCINOMA
I. Tumor limited to ovaries
A.Tumor limited to one ovary; no ascites
1. Capsule intact
2. Capsule ruptured or tumor present on the external surface
B. Tumor limited to both ovaries; no ascites
1. Capsule intact
2. Capsule ruptured or tumor present on the external surface
C. Tumor limited to ovaries; ascites present or positive peritoneal washings

II. Tumor involving ovaries with pelvic extension
A. Extension and/or metastases to uterus and/or tubes; no ascites
B. Extension to other pelvic tissues; no ascites
C. Tumor either IIA or IIB with ascites or positive peritoneal washings
III Tumor involving ovaries with intraperitoneal metastases outside the pelvis and/or positive
retroperitoneal lymph nodes
IV Distant metastasis, including parenchymal liver metastasis
FIGURE 18-13 ▪ Mature teratoma demonstrating multiple large and small cysts separated by heterogeneous
solid nodules.
The development of a somatic malignancy within a teratoma is a rare event in childhood. This malignant
transformation is thought to occur within differentiated teratomatous elements rather than from totipotent
embryonal cells. Within childhood ovarian teratomas, 7/246 tumors developed somatic malignancies (13). The
types of nongerm cell malignancies most commonly encountered were epithelial, glial, and embryonal. Such
nongerm cell malignancies are associated with a worse prognosis owing to poor response to therapy. In the past,
these events were referred to as teratocarcinoma or malignant teratoma, terms that are confusing and best
avoided.
Ovarian mature teratomas have been the most thoroughly studied biologically owing to their abundant numbers.
More than 325 cases have been cytogenetically analyzed, demonstrating 95% to be karyotypically normal and
the remainder to show nonrecurrent numeric abnormalities (80, 143). Studies of molecular loci show that the
majority of mature ovarian teratomas have entered, but have not completed, meiosis (64, 143). These studies
suggest that mature ovarian teratomas arise from germ cells arrested in meiosis I.
Immature Teratoma:
Immature teratomas are the third most common germ cell tumor seen in the adolescent female ovary. These are
considered to be of intermediate malignancy, a concept that is controversial. Although immature teratomas have
rarely been reported to metastasize, those that do so almost invariably contain endodermal sinus tumor (EST)
components in the original tumor. This raises the possibility that the metastasizing component is the EST, which
subsequently undergoes differentiation. Immature teratomas are predominately unilateral solid tumors that may
be quite large and are most often confined to the ovary. Immature teratomas can be graded histologically
according to the quantity of immature elements, most commonly the quantity of immature neuroectoderm (Figure
18-14) (99). Many variants of this grading system have been proposed; however, the differences between these
systems are not substantive. Grade 1 lesions are those with immature tissue limited to rare low magnification
fields, with not more than one field in any one slide. Grade 2 lesions contain immature neuroectoderm not
exceeding three low power fields (10X objective, with a 4X to 10X ocular for a total magnification of 40X to 100X
per slide). Grade 3 tumors show extensive immature neural epithelium in more than three low power fields per
slide. This grading system is based on the presence of immature neuroectoderm; the significance of immaturity
of non-neural elements (fetal muscle, cartilage, or kidney) is somewhat controversial. In practice, this seldom
presents
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difficulties because the immature elements are almost invariably accompanied by immature neural elements. A
common approach is to consider immature non-neural elements of any quantity as grade I. This grading system
has been most successfully applied to ovarian immature teratomas, in which the grade correlates with metastatic
potential as well as with behavior (101). The treatment of choice is unilateral oophorectomy. The reported
prognosis of immature ovarian teratomas in adults depends on the histologic grade of the tumor, the size of the
tumor, the age of the patient, and the stage at presentation. Analysis of the outcome of 41 pediatric ovarian
immature teratomas treated by surgery alone (19 grade 1, 13 grade 2, and 9 grade 3), 10 of which also showed
small foci of EST, suggests that pediatric tumors have a much better prognosis than adults. This study
documents only one recurrence of a grade 1 lesion that contained EST in the initial tumor (85). Other recent
reports also suggest that low-stage immature ovarian teratomas do not require chemotherapy (28, 82).
FIGURE 18-14 ▪ Immature teratoma of the ovary with several immature neuroepithelial tubules composed of
proliferating, primitive cells.
In the pediatric age group, the most significant pathologic event that occurs within an immature teratoma is the
development of a malignant component, most commonly EST. Such occurrences may be multifocal and may be
very difficult to confidently identify (Figure 18-15). A valuable indicator of this event is an elevated serum a-
fetoprotein (AFP) level. Most observers consider elevated AFP in “pure” immature teratomas to represent
unrecognized, small foci of EST. However, some reports of carefully examined tumors have suggested that
immature neural tissue or intestinal tissue may be a source of elevated levels of AFP. This is supported by the
immunoreactivity of these tissue types with AFP (108). The judgment of most experienced observers has been
that although these tissue elements may, in a minority of cases, explain a small, stable increase in serum AFP, a
large or rapidly increasing elevation in a patient without liver failure must be assumed to represent the presence
of EST (52).
FIGURE 18-15 ▪ Teratoma of the ovary with a microscopic focus of EST. The reticular pattern and the enlarged
nuclei with course chromatin provide histologic clues. The serum AFP was moderately elevated before surgery.
FIGURE 18-16 ▪ Gliomatosis peritonei characterized by nodules of mature glial tissue in the omentum.
Cytogenetic studies show a higher frequency of chromosomal abnormalities in immature teratomas (60%) when
compared with mature teratomas; however, no consistent abnormalities have been identified (103, 118, 143).
Most immature ovarian teratomas are diploid; however, occasional tumors are aneuploid in the triploid to
tetraploid range. Most of these high-level aneuploid tumors harbor foci of EST (4).
Gliomatosis Peritonei: Gliomatosis peritonei is a rare condition that occurs almost exclusively in the setting of
solid ovarian mature or immature teratoma. It is characterized by small gray-white nodules of mature glial tissues
on peritoneal surfaces. The pathogenesis of these nodules has been debated. They may arise from small
capsular ruptures of the ovarian mass, resulting in implantation on the peritoneal surfaces. Alternatively, they
may represent independent lesions arising within the subcoelomic mesenchyme. Studies comparing the DNA of
the glial implants with DNA of the associated ovarian teratomas and normal tissues demonstrate that gliomatosis
peritonei is genetically unrelated to the associated teratoma, supporting the second hypothesis (41) (Figure 18-
16). Mature nodules, whether peritoneal or in lymph nodes, may require additional surgery but have no adverse
prognostic significance and do not impact the staging of the ovarian lesion (50, 109). It is important that these
peritoneal nodules be adequately examined to exclude foci of immaturity.
Ovarian Dysgerminoma:
Dysgerminoma is the most common malignant germ cell tumor in the ovary, comprising 48% of such lesions. It is
the most common ovarian malignancy in children and adolescents, and it is the pathologic and biologic
equivalent of the testicular seminoma. There is a 10% to 15% incidence of bilaterality. Most dysgerminomas are
pure and are composed of aggregates or nests of uniform neoplastic cells with distinct, nonoverlapping cellular
borders (Figure 18-17). Germinomas often show a lymphocytic infiltrate and occasionally multinucleated giant
cells.
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Although anaplastic variants of germinomas (seminomas and dysgerminomas) have been rarely reported, these
foci may represent areas of solid embryonal carcinoma. Synciotrophoblastic cells may be scattered individually
throughout germinomas and may be responsible for human chorionic gonadotropin (hCG) production, but unless
they are accompanied by cytotrophoblastic cells, these cells do not represent choriocarcinoma and have no
effect on prognosis (148). Immunohistochemically, the majority of germinomas are positive for placental-like
alkaline phosphatase (PLAP), a cell surface glycoprotein. While PLAP is a valuable marker for germinomas, it
may also be present focally in embryonal carcinomas and ESTs as well as in a wide variety of somatic tumors
(17, 84). A more specific antibody has been reported recently that strongly and specifically recognizes
germinomas, embryonal carcinomas, and intratubular germ cell neoplasia, namely OCT4. This protein is highly
expressed in pluripotent stem cells and has been demonstrated to be useful in the distinction of metastatic GCT
from other tumor types (22). It is negative in ESTs.
FIGURE 18-17 ▪ Dysgerminomas are often arranged in nests of cells with well-defined cytoplasmic membranes
and rounded nuclei with prominent nucleoli. Lymphocytes and occasional multinucleate cells may be seen in the
stroma.
The majority of patients with dysgerminomas (70% to 80%) present as stage I (162). Dysgerminomas are
exquisitely radiosensitive, and the 5-year survival rate with radiotherapy ranges from 90% in stage I disease to
60% to 90% in patients with more advanced disease. Properly evaluated patients with stage Ia ovarian pure
dysgerminoma who desire fertility can be safely treated without radiotherapy by unilateral oophorectomy after
careful lymph node sampling alone (82). The tumor subsequently recurs in 17% of patients, but more than 90%
of these patients may be successfully treated with chemotherapy. Bilateral dysgerminoma may be treated with
bilateral oophorectomy and chemotherapy, with the uterus left in situ for future embryo transfer.
Endodermal Sinus Tumor: The second most common histologic subtype (22%) of malignant ovarian germ cell
tumor in children is EST, also called yolk sac tumor. Grossly, these lesions are most often tan to white and
mucoid in appearance, often with small cystic regions (Figure 18-18). EST has only been reliably distinguished
from other patterns of malignant germ cell tumor for the last two decades. Therefore, caution is advised when
evaluating earlier studies of malignant ovarian germ cell tumors; these studies often equated EST with
embryonal carcinoma and underappreciated the presence of EST within immature teratomas. The histology and
cytology of ESTs vary widely, often causing difficulty in diagnosis. For detailed description of the protean
manifestations of EST, many excellent reviews are available (144, 150). Several histologic subtypes of EST
have been described; most tumors contain several subtypes, and none of these subtypes have prognostic
implication (Figures 18-19, 18-20 and 18-21). The prototypic Schiller-Duvall bodies of EST (Figure 18-21) are
present in 50% to 75% of tumors. ESTs are commonly associated with highly elevated serum AFP levels, which
may be monitored clinically for recurrence and/or metastasis (136). Aggressive monitoring of serum AFP levels
constitutes one of the important improvements in the
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management of patients with germ cell tumor, particularly those with EST. The AFP should fall into the normal
range 5 to 7 weeks following surgery if resection of the tumor is complete. Rarely bilateral, ESTs are rapidly
growing, yet most present as stage Ia tumors.
FIGURE 18-18 ▪ Ovarian ESTs are often white, mucoid appearing, with microcysts.
FIGURE 18-19 ▪ ESTs often show a mixture of histologic types. The reticular pattern shows a network of
communicating spaces.
FIGURE 18-20 ▪ Solid regions may be seen within ESTs; however, they are usually a minority component. The
cells may contain large intracellular vacuoles. Hyaline bodies are occasionally seen.
Other Histologic Types of Nongerminomatous Tumors:

Pure embryonal carcinomas are rare ovarian neoplasms (4%) that should be differentiated from the more
common EST. These are more commonly seen as a minor component of a mixed germ cell tumor. Like ESTs,
embryonal carcinomas may show papillary, glandular, and solid areas. The cells are large, epithelioid, and often
anaplastic with large nucleoli, abundant mitotic activity, hemorrhage and necrosis (Figure 18-22). Embryonal
carcinomas show immunoreactivity for cytokeratin, but not for epithelial membrane antigen, a feature that may
help to distinguish embryonal carcinoma from other epithelial neoplasms (149). A minority of embryonal
carcinomas show focal, weak immunoreactivity for PLAP, as well as for AFP (73, 84). It has been noted that
embryonal carcinomas, but not other germ cell tumor histologic types, show immunopositivity for CD30, a marker
more conventionally utilized for Hodgkin lymphoma (77). CD30 is a member of the tumor necrosis factor receptor
superfamily whose expression protects against apoptosis (56). Embryonal carcinomas are reliably positive for
OCT4 (5).
FIGURE 18-21 ▪ Schiller-Duval bodies are present in many ESTs. These bodies are composed of a central
vascular core lined by tumor cells, a space, and then an outer rim of tumor cells.
FIGURE 18-22 ▪ Embryonal carcinoma is composed of large, overlapping nuclei with very large, prominent
nucleoli and prominent individual cell necrosis. The cytoplasm is characteristically amphophilic.
Ovarian choriocarcinoma is rarely seen as the sole histologic type, but may constitute a minor component within
a mixed germ cell tumor. Choriocarcinomas are composed of both medium-sized cytotrophoblastic and
multinucleate syncytiotrophoblastic cells with frequent evidence of hemorrhage (Figure 18-23).
Immunohistochemical stains for hCG identify syncytiotrophoblastic cells, with unreliable staining of
cytotrophoblasts. The prognosis of nongerminomatous germ cell tumors prior to the chemotherapy era was
dismal. With the advent of bleomycin, etoposide, and cisplatin protocols, survival rates of 70% to 90% have been
reported (46, 67).
FIGURE 18-23 ▪ Choriocarcinoma is composed of both multinucleate syncytiotrophoblastic cells and

cytotrophoblastic cells. This histologic subtype can often be found associated with areas of hemorrhage.
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Genetic studies of malignant ovarian germ cell tumors involving normal gonads show no difference from their
testicular counterparts. Most malignant ovarian germ cell tumors are aneuploid or near-tetraploid. Most contain
the i(12p) by classic cytogenetics and amplification of 12p by comparative genomic hybridization (3, 58, 118). As
previously mentioned, ESTs frequently develop in the context of immature teratomas. The biologic changes
associated with this histologic transformation have not been adequately studied; however, ploidy analyses have
suggested a genetic change is associated with the histologic transformation (4). The absence of the i(12p) in
immature teratomas and the presence of the i(12p) in ESTs associated with immature teratomas suggest that
one genetic change may be the acquisition of the i(12p) (59, 118). Recent studies have demonstrated c-kit
mutation in a substantial minority of ovarian dysgerminomas, similar to findings seen in testicular germinomas
(57).
Gonadoblastoma:
Gonadoblastoma is a rare tumor that arises in the dysgenetic gonads of phenotypic females having Y
chromosomal determinants, as discussed earlier (14, 131). Gonadoblastomas are usually quite small and
recognizable only on microscopic examination. Histologically, gonadoblastomas are characterized by nests
containing both germ cells and stromal cells of granulosa-Sertoli cell type (Figures 18-24 and 18-25). These
nests may be separated by stroma that often contains Leydig cells. Gonadoblastomas often show extensive
hyalinization. A common feature is the presence of laminated calcific concretions. Numerous calcifications
identified within a dysgerminoma should suggest the possibility that the patient may have gonadal dysgenesis
and may be at a high risk for developing a contralateral dysgerminoma. While dysgerminoma is the most
common histologic subtype of malignancy following gonadoblastoma, EST and embryonal carcinoma are also
reported. Recently, TSPY has been reported as a candidate gene involved in the development of
gonadoblastoma. The TSPY protein is expressed, along with PLAP and OCT4, in germ cells within
gonadoblastoma (68).
FIGURE 18-24 ▪ Gonadoblastomas are seen as small nodules within streak gonads that contain eosinophilic
hyaline bodies composed of basement membrane material. Calcifications are frequent.
FIGURE 18-25 ▪ Nodules of gonadoblastoma contain both germ cells and stromal cells, in varying proportions.
Serologic Markers:
Serum and CSF concentrations of AFP and hCG are useful as markers of certain types of germ cell tumors. AFP
is expressed at high levels by over 85% of ESTs (66) and at lower levels in other histologic types. The
predominant utility is for monitoring for recurrence or metastasis in AFP-secreting tumors. The half-life of AFP is
5 to 7 days. Other neoplastic and non-neoplastic disorders may result in elevation of AFP, for example, hepatitis,
cirrhosis, and other malignancies. The beta subunit of hCG is secreted by the syncytiotrophoblastic cells of the
placenta and thus is characteristically markedly elevated in choriocarcinomas. However, virtually all histologic
subtypes of malignant germ cell tumors may show rare or scattered syncytiotrophoblastic cells that may result in
mildly elevated hCG but do not indicate a worse prognosis. Elevations above 100 ng/mL are unusual and
suggest the true presence of choriocarcinoma. The half-life of hCG is approximately 20 to 30 hours.
Hematologic Malignancies Associated with Germ Cell Tumors:

The association between germ cell tumors and hematologic malignancy is well established but uncommon. The
vast majority of germ cell tumors that subsequently develop hematologic malignancies are malignant mediastinal
germ cell tumors in men (21, 33, 70, 87, 96). The associated hematologic abnormalities have included
erythroleukemia, histiocytic sarcoma, acute nonlymphocytic leukemia, myelodysplasia, and systemic mast cell
disease. Three ovarian germ cell tumors have been reported in association with hematologic abnormalities, two
of these were
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in 46,XY phenotypic females. The median interval between the diagnosis of the germ cell tumor and that of the
hematologic malignancy is 6 months, much shorter than the 25- to 60-month interval commonly seen in
chemotherapy-related hematologic malignancies. It has been proposed that the germ cells tumor may provide the
stem line of the hematologic malignancy. This is supported by the presence of the i(12p) in both the germ cell
tumor and the hematopoietic malignancy in several cases (20). Primary resections of germ cell tumors that show
proliferating hematopoietic cells should raise the suspicion of an associated hematologic abnormality; however,
more often this process is not detected at the initial resection.
Ovarian Sex Cord-Stromal Tumors

In addition to germ cells, the ovary is populated by granulosa cells, theca cells, interstitial (hilus) cells, and
stromal fibroblasts. Each of these components may give rise to a neoplasm, and such lesions are grouped
together as sex cord-stromal tumors. They account for 10% of all ovarian neoplasms in children and
adolescents. Most are composed of ovarian cell types, but some contain only elements of testicular type (such
as Sertoli cell tumors). Precocious puberty and virilization are the major clinical manifestations. Before 9 years of
age, most sex cord-stromal tumors are feminizing, and after 9 years of age, there is a predominance of virilizing
neoplasms. On occasion, it may be difficult or impossible to identify accurately a lesion as a stromal tumor or to
determine to which of the stromal tumor categories a tumor belongs. Younger age and early-stage disease are
important predictors for improved survival in patients with ovarian sex cord stromal tumors (180).
Immunohistochemistry has been used to verify the diagnosis of sex cord-stromal tumors. Inhibin, calretinin, and
CD99 are useful markers that positively mark the majority of sex cord-stromal tumors, while most are negative for
epithelial membrane antigen (51, 116).
Juvenile granulosa cell tumors constitute the most common type of functioning ovarian neoplasm. Although
these are commonly composed almost entirely of granulosa cells, they may also contain theca cells or fibroblasts
singly or in any combination. Fewer than 10% of all granulosa cell tumors are diagnosed in the first two decades
of life (142). The majority (80% to 85%) of granulosa cell tumors in children are histologically distinct from the
adult-type granulosa cell tumors described later. These juvenile granulosa cell tumors typically present with
precocious pseudopuberty or virilization in a child younger than 10 years old, although infants and elderly
patients have been reported (156, 170). A few cases have been associated with Ollier disease, Potter syndrome,
and other forms of dysmorphisms (170). Grossly, juvenile granulosa cell tumors are solid with fibrous bands
separating yellow nodules and are usually confined to the ovary (Figure 18-26). Microscopically, juvenile
granulosa cell tumors have a rather complex growth pattern with nodules of incompletely luteinized cells. Follicles
may be present that are irregular and resemble large Graafian follicles (Figure 18-27). The luminal contents
contain mucicarminepositive secretions. The stromal cells resemble fibroblasts or have polygonal outlines and
pale cytoplasm containing abundant lipid (Figure 18-28). Luteinization is commonly greater and the nuclei are
more hyperchromatic and immature than those in the adult-type granulosa cell tumors. Call-Exner bodies are not
a feature of these tumors and the nuclear grooves typical of adult granulosa cell tumors are infrequent. Some
mitotic activity and nuclear atypia is often present and is more pronounced than that seen in adult-type granulosa
cell tumors, resulting in their frequent misdiagnosis as malignant germ cell tumors (Figure 18-28). Although
juvenile granulosa cell tumors commonly present as stage 1, it is not uncommon for these tumors to rupture
(170). Lowstage tumors have an excellent prognosis; however, the outcome for patients with nonconftned tumors
may be poor (156). Although the adult granulosa cell tumor has a local recurrence of 20%, 92% of patients with
juvenile granulosa cell tumors were free of disease following surgical removal
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(156). However, those that recur tend to do so within 3 years and have a rapid course (170). There is no known
benefit from adjuvant chemotherapy or radiotherapy.
FIGURE 18-26 ▪ Juvenile granulosa cell tumor in a 7-year-old girl who presented with precocious puberty and a
pelvic mass.
FIGURE 18-27 ▪ Juvenile granulosa cell tumor of the ovary showing a smaller cyst lined by clear cells and
resembling a large, irregular Graafian follicle.
FIGURE 18-28 ▪ Juvenile granulosa cell tumor of the ovary with ovoid-toelliptical cells with pale eosinophilic
cytoplasm and polygonal cells with clear cytoplasm are found in the nodules. Nuclear atypia and mitoses are
often present.
Adult-type granulosa cell tumors may also occur in the first two decades of life. These tumors are characterized
by rounded follicles of varying sizes, minimal luteinization, and rare mitoses. The most differentiated forms show
Call-Exner bodies, which consist of granulosa cells in a radial arrangement around a small cystic cavity
containing a central rounded mass of eosinophilic material (142). Adult granulosa cell tumors may demonstrate a
wide range of histologic appearances, which are commonly mixed within a single specimen, often making their
diagnosis a challenge. Different patterns include microfollicular, macrofollicular, trabecular, insular solid-tubular,
diffuse (sarcomatous), and luteinized. Some show branching columns of cells, whereas others are more diffuse;
all show characteristic nuclear grooves (Figure 18-29). These tumors are commonly estrogenic and may cause
endometrial hyperplasia and carcinoma in 5% to 25% of women of reproductive age.
FIGURE 18-29 ▪ Adult-type granulosa cell tumors at times show a predominately solid pattern, which may cause
diagnostic difficulty. However, the prominent nuclear groves are seen in all tumors.
FIGURE 18-30 ▪ Sex cord tumor with annular tubules is composed of rounded epithelial units containing cells
with abundant eosinophilic cytoplasm that surrounds multiple hyaline bodies.
Sex cord tumors with annular tubules (SCTAT) is a distinctive ovarian neoplasm with morphologic features
intermediate between granulosa cell tumor and Sertoli cell tumor. SCTATs demonstrate multifocal cortical
stromal tumors that contain epithelial nests with single or multiple hyaline bodies, representing annular tubules
(Figure 18-30). These bodies may resemble the hyaline bodies seen in gonadoblastomas; however, the cells of
the annular tubules have more abundant, pale, and vacuolated cytoplasm and lack germ cells. Hyperestrinism is
common. Approximately one third of patients with SCTAT have Peutz-Jeghers syndrome (43, 175).
Sclerosing stromal tumors are rare neoplasms that most commonly present in younger women (14 to 19 years of
age) with irregular menses and abdominal pain (124). Grossly, the tumors are unilateral, firm, and gray to white;
areas of edema, necrosis, and cystic degeneration are common (Figure 18-31). Microscopic features include
spindled cells arranged in lobules separated by edematous stroma. Polygonal cells are scattered among the
spindle cells and may have
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signet ring-like features; however, these cells contain oil red O-positive lipid and not mucin (Figure 18-32). None
of these tumors has behaved in a malignant fashion.
FIGURE 18-31 ▪ Sclerosing stromal tumors show pseudolobulation at low power due to areas with differing
cellularity, collagen, and edema.
FIGURE 18-32 ▪ Sclerosing stromal tumors are composed of an admixture of spindle cells forming collagen and
lipid-laden theca-like cells with shrunken nuclei.
Sertoli cell tumors of the ovary are exceedingly rare. The predominant pattern is tubular, with a minority of
tumors demonstrating retiform or diffuse patterns. The tumors are most often virilizing, but may produce
estrogen.
Sertoli-Leydig cell tumors contain various proportions of Sertoli, Leydig, and indifferent stromal cells, often
similar to that seen in various phases of testicular development. These represent only 0.5% of ovarian
neoplasms and are the most commonly virilizing of all ovarian tumors (173, 174). Half have symptoms of
androgen excess or virilization, but a few have estrogenic manifestations. Survival is excellent, with tumor-related
deaths in only 5%, likely due to the fact that they are stage I at presentation in over 97% of patients (174, 178).
Sertoli-Leydig cell tumors have been classified on the basis of degree of differentiation and the presence or
absence of heterologous elements. The well-differentiated or pure Sertoli cell tumors are the least common.
These are composed of well-developed tubules or solid cords of cells with or without Leydig cells. Intermediate
tumors comprise poorly formed tubules in a cellular stroma with a nodular or a diffuse configuration (Figure 18-
33). Poorly differentiated or sarcomatoid tumors are composed of undifferentiated mesenchyme, poorly formed
cords of Sertoli cells, and a paucity of Leydig cells. Sertoli-Leydig cell tumors with heterologous elements
comprise 25% of all Sertoli-Leydig cell tumors and may contain cysts or glands with intestinal-type mucosa,
carcinoid tumor, rhabdomyoblasts, cartilage, or neuroblastoma (112). These seem to be more common in Sertoli-
Leydig cell tumors of younger patients and may be difficult to distinguish from immature teratomas. In recent
years, the retiform pattern has received increased attention due to their younger age at presentation (median
age of 15 years) and their often lack of androgenic manifestations, increasing the risk of misdiagnosis.
Occasional retiform Sertoli Leydig cell tumors may be associated with elevated serum levels of AFP.
FIGURE 18-33 ▪ Sertoli-Leydig cell tumor composed of irregular poorly formed tubules and cords lined by cells
containing moderate amounts of cytoplasm; occasional clusters of Leydig cells demonstrating atypia and
eosinophilic cytoplasm are seen.
Rare Tumors: A few examples of mixed germ cell-sex cord-stromal tumors have been described, but are quite
rare (75). Other abdominal soft tissue tumors have presented in the ovary, including desmoplastic small round
cell tumor, peripheral neuroectodermal tumors, and alveolar soft part sarcoma, to name a few.
Epithelial Neoplasms
Ovarian epithelial neoplasms arise from the surface epithelium of the ovary and, therefore, may express the
multipotential nature of the embryonic coelomic epithelium, including mucous, serous, endometrioid, and
mesenchymal appearances. These tumors comprise about 15% of ovarian neoplasms of patients younger than
20 years of age, and in this population, they occur after menarche and are virtually exclusively mucinous or
serous, with mucinous tumors comprising the majority (77%) (57). The majority are unilateral and benign;
approximately 15% are malignant. The pathologic categorization of these epithelial lesions is based on an
evaluation of epithelial proliferative changes. These subgroups are benign, atypically proliferating (“borderline”
tumors), and malignant. The intermediate group is defined as showing greater proliferation (including epithelial
budding and nuclear stratification, mitotic activity, and nuclear atypia) but showing no destructive invasion of the
stromal component. The criteria for inclusion into the intermediate group, and the nomenclature used, remain
controversial.
Serous neoplasms are usually composed of multiple cysts with watery and clear contents. Characteristic is the
presence of nodular papillary excrescences scattered over the lining of the cysts. These may be few and barely
visible or numerous. Microscopically, the cysts are lined by papillary processes covered by a single layer of
columnar-to-cuboidal cells
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(Figure 18-34). Intermediate neoplasms show more extensive and complex papillary patterns with stratification of
the epithelial lining. The neoplastic cells show loss of polarity, nuclear pleomorphism, and increased mitotic
activity. Most important, and required for the diagnosis of carcinoma, is the presence of stromal invasion.
Ovarian borderline serous tumors show an increased risk of recurrence, and histologic features have been
proposed and disputed that may define a subset of tumors with a greater risk of aggressive behavior
(micropapillary serous carcinoma) (38, 134, 137).
FIGURE 18-34 ▪ Serous cystadenomas are lined by an epithelium resembling that of either the Fallopian tube or
the surface epithelium of the ovary, and therefore may be ciliated or nonciliated.
Mucinous neoplasms are also usually multicystic tumors containing thick mucinous material. The lining of the
cysts is smooth and glistening, with infrequent papillary excrescences. Microscopically, the cysts are lined by
columnar nonciliated cells with faintly basophilic cytoplasm and the small, basally oriented nuclei (Figure 18-
35A). Some tumors show scattered goblet cells. The supportive stroma is cellular and commonly has a thecal
and even luteal appearance. Intermediate neoplasms are characterized by stratification of epithelial cells, loss of
nuclear polarity, nuclear pleomorphism, and frequent mitoses (Figure 18-35B). The assessment of the clinical
behavior of ovarian mucinous tumors demonstrates the importance of stromal invasion in predicting a poor
prognosis and defining the category of ovarian mucinous carcinoma (31, 55, 117).
FIGURE 18-35 ▪ Well-differentiated mucinous tumors most commonly show epithelium similar to that seen in the
endocervix, consisting of a single row of mucin-filled column cells with basal nuclei (A). Borderline mucinous
lesions show multiple fine papillary processes with stratification of cells and loss of polarity (B).
Small cell carcinomas of the ovary are often confused histologically with granulosa cell tumors and/or germ cell
tumors. These are highly aggressive tumors and should not be treated conservatively. They are often associated
with paraendocrine hypercalcemia. The patients range from 9 to 43 (average: 23.9) years of age, and the tumors
often present at a high stage (51, 171). The tumor is composed of poorly differentiated small cells with scant
cytoplasm and nuclei with clumped chromatin. Therefore, it must be distinguished from other primary and
metastatic small cell tumors that may involve the ovary, particularly in young patients. The cells may be diffuse or
arranged in nests or cords. Rounded follicles containing eosinophilic fluid and lined by neoplastic cells have
been demonstrated in over 75% of tumors. Mitotic figures are abundant. In one series of 15 cases, the tumors
were reliably positive for p53, WT1, and EMA. Of concern, the majority were positive for calretinin and 4/15 were
positive for CD56. All cases were negative with CK5/6, chromogranin, CD99, NB84, desmin, a-inhibin, and TTF-
1. Therefore, the combination of EMA and WT-1 nuclear positivity, the latter usually intense and diffuse, may be
of positive diagnostic value (89). Long-term survival is rare once the tumor has spread beyond the ovary, and
even stage 1A tumors have a survival rate of only 30%. Recent studies suggest that newer aggressive,
multiagent therapeutic regimens may improve survival somewhat (36). The cell lineage remains unknown. The
age
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distribution, the presence of follicle formation, and calretinin positivity suggest the possibility of a sex cord origin
(89).
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176. Zachariou Z, Roth H, Boos R, et al. Three years' experience with large ovarian cysts diagnosed in
utero. J Pediatr Surg 1989;24:478-482.
177. Zaloudek C, Kurman RJ. Recent advances in the pathology of ovarian cancer. Clin Obstet Gynaecol
1983;10:155-185.
178. Zaloudek C, Norris HJ. Sertoli-Leydig tumors of the ovary: a clinicopathologic study of 64 intermediate
and poorly differentiated neoplasms. Am J Surg Pathol 1984;8:405-418.
179. Zeisler H, Mayerhofer K, Joura EA, et al. Embryonal rhabdomyosarcoma of the uterine cervix: case
report and review of the literature. Gynecol Oncol 1998;69:78-83.
180. Zhang M, Cheung MK, Shin JY, et al. Prognostic factors responsible for survival in sex cord stromal
tumors of the ovary: an analysis of 376 women. Gynecol Oncol 2007;104:396-400.
Chapter 19
The Male Reproductive System, Including Intersex Disorders
Hikmat A. Al-Ahmadie
THE TESTIS
Testicular Development and Disorders
Normal sexual development and differentiation is the result of a complex process of genetic, molecular, and
endocrine mechanisms that are necessary for the development of the genitourinary system including the kidneys
as well as the adrenals. Chromosomal sex (genotype) is established at fertilization, and from the bipotential
gonads the male genotype (46,XY) leads to the development of the testis through a series of sex chromosome-
linked and autosomal genes. The testis in turn secretes essential hormones for the development of the external
male genitalia (phenotype) (36). A normal 46,XX female has ovarian and müllerian duct development because
the Y chromosome is instrumental in the suppression of the female reproductive tract. Of the genes involved in
the formation of the bipotential gonads, WT1 (Wilms tumor gene), NR5A1 (nuclear receptor subfamily 5, which
encodes the steroidogenic factor-1, SF-1), and LIM1 are perhaps the most important (36, 52) These events are
triggered by SRY (sex-determining region of the Y) located on the distal tip of the short arm of the Y chromosome
(e77,25,31,52,e323,60).
The SRY gene encodes a protein that acts on the HMG (high mobility group) DNA-binding domain in somatic
cells in the urogenital ridge to differentiate into Sertoli cells, the first differentiated cell type of the testis
(36,e317,e541). Although SRYis the essential determinant of testicular development, various autosomal genes,
downstream from SYR, are involved in this process including WT1, SF-1, DAX-1 (dosage-sensitive sex reversal,
adrenal hypoplasia critical region, on chromosome X, gene 1), and SOX9 (SRY-box 9)
(e208,e223,e270,e354,e418,70,e419).
Embryologically, the urogenital ridges appear at around the 4th week of gestation and are initially devoid of germ
cells. By the 5th week, primordial germ cells migrate to the genital ridge and are arranged into the seminiferous
cords.
Up to the 6th week, both male and female gonads appear relatively similar. By the 7th week of gestation, the
testes are formed with recognizable short, straight cellular tubules and are functioning with the synthesis of
antimüllerian hormone (AMH, müllerian-inhibiting substance, müllerian-inhibiting factor) by the Sertoli cells, which
develop from the somatic sex cord cells; and subsequently testosterone by the Leydig cells, which develop from
the intercordal gonadal mesenchyme in the 8th week (6,9,e78,e355). The intercordal mesenchyme is composed
of cells that migrate from the mesonephric stroma that eventually differentiate into testicular stromal cells and
blood vessels in addition to Leydig cells. The produced hormones are essential as AMH plays a major role in the
process of regression of the müllerian ducts and upper vagina, whereas testosterone is crucial for the
differentiation of the wolffian ducts, epididymis, vas deferens, and seminal vesicles. The rete testis develops from
the mesonephric remnants in proximity to the seminiferous cords. Development of a dense fibrous tunica
albuginea in the 8th week of gestation is definitive for testis formation. Testosterone synthesis peaks at 12 to 16
weeks of gestation, allowing for male secondary sexual development concurrent with the appearance of AMH
(75,e593). The remaining structures of the male genital system are derived from the urogenital sinus through the
differentiation of the endoderm-derived epithelium into prostate, urethra, bulbourethral, and periurethral glands.
In contrast, the wolffian duct derivatives are of mesodermal origin (20). The differentiation of the wolffian duct
occurs under the influence of testosterone secreted by the ipsilateral testis. The differentiation of the urogenital
sinus into male external genitalia occurs under the influence of dihydrotestosterone (DHT), which derives from
testosterone by enzymatic conversion by 5a-reductase. Two additional hormones FSH (follicle-stimulating
hormone) and LH (luteinizing hormone) play important roles in the development of the male genital system mainly
in the last months of gestation, regulating androgen production and Sertoli cell activity (e138,e144).
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The actions and the timing of these hormones are closely and precisely coordinated during development
(20,e228,e588). After birth, the testis continues to develop until puberty with changes affecting all testicular
components until puberty.
Congenital and Developmental Anomalies

A congenital or a developmental anomaly in the testis may be an insolated finding or it may be associated with a
sexual maldevelopment syndrome that may or may not have an underlying cytogenetic defect. The most common
presentation of these conditions is an “undescended” testis or an ambiguous external genitalia. Monorchidism, or
the presence of a single testicle, may be due to a testicular regression syndrome (TRS) (discussed later),
vascular injury, cryptorchidism, or another congenital anomaly.
Polyorchidism, or duplication of the testis, is a rare condition, with about 100 cases reported in the literature.
This condition usually manifests as triorchidism but bilateral duplication has been also reported (42,e291).
Clinically, two masses within the hemiscrotum, inguinal swelling and an undescended testis, or pain, are the
usual presentations. Associated conditions include those related to anomalies in the processus vaginalis (hernia,
hydrocele), testicular maldescent, intermittent torsion, epididymitis and varicocele, as well as malignant
neoplasms (e72,e620). The duplicate testis develops due to a division or duplication in the genital ridge and is
suggested to result from an abnormal division of the urogenital ridge before the 8th week of gestation (e85). The
most common variant is that of a supernumerary testis sharing an epididymis with the primary testis, but other
variants include a vascular pedicle with no epididymis, an independent blind epididymis, or a shared vas
deferens (e85,e537). The duplicate testis is smaller than a normal testis and its torsion is more frequent. The
microscopic appearance ranges from normal testicular tissue with intact spermatogenesis to disorganized
seminiferous tubules with diminished spermatogenesis. Rare associated chromosomal anomalies have been
reported (e72).
Cystic dysplasia of the testis is a rare congenital malformation first reported by Leissring and Oppenheimer in
1973 (e304), presenting as testicular enlargement due to cystic dilation of the rete testis. It is thought to result
from an embryologic defect around the 5th week of gestation preventing the connection of the rete testis
(afferent seminiferous tubule derived) with the efferent tubules (mesonephric or wolffian derived). The failure of
such connection leads to degeneration of the mediastinum testis into small cysts with progressive dilation
compressing and replacing the adjacent testicular parenchyma. Ipsilateral renal agenesis and multicystic
dysplasia of the kidney are the most common associated anomaly (e141,e322). Histopathologic features include
multiple, anastomosing, irregular cystic spaces of varying sizes and shapes predominantly located in the region
of the mediastinum testis but also displacing the testicular parenchyma, which becomes subsequently
compressed under the tunica albuginea. The cystic lining is flat epithelial resembling that of the rete testis
(e184). Spontaneous regression of this lesion has been recently reported after conservative management in one
case (e555).
Prepubertal macroorchidism is an idiopathic condition in most cases but may be associated with McCune-
Albright syndrome, juvenile hypothyroidism, and fragile X syndrome (e524,e586). Histopathologic findings include
some enlargement of the seminiferous tubules and thickening of tubular basement membrane. The tubules
contain Sertoli cells and scattered Leydig cells are usually present in the interstitium. Focal mild interstitial
fibrosis, occasional tubules containing only Sertoli cells, and focal paucity of Leydig cells are some of the
pathological findings. Some have attributed the testicular enlargement in the absence of other findings to
interstitial edema and obstruction. In one report, continuous splenogonadal fusion was reported in association
with macroorchidism (e528).
Vascular anomalies of the testis are rare and include angiomatous malformations and congenital
lymphangiectasis. Angiomatous malformation is a rare cause of testicular enlargement and consists of numerous
thin-walled vascular channels (e505). Congenital lymphangiectasis has been reported with bilateral
cryptorchidism and with Noonan syndrome, manifested by numerous ectatic and irregular lymphatic channels
with frequent anastomoses and seminiferous tubules with decreased diameter, immature Sertoli cells, reduced
spermatogenesis, and peritubular fibrosis (e397). Similar finding, however, have also been reported in the testes
of infants without other abnormalities at autopsy (e393).
Cryptorchidism or undescended testis, where either one or both testes fail to migrate to the base of the scrotum,
is one of the most common genitourinary disorders in male children, affecting 4% to 5% of full-term and 9% to
30% of premature males at birth (28,e231,86). Based on many recently published series, there has been an
increase in the incidence of cryptorchidism over recent decades in North America and Europe (87). The
cryptorchid testis can be found in any position along its usual line of descent including intra-abdominally;
however, approximately 80% will be located in the inguinal region, just outside the inguinal canal and 20% to
27% of cryptorchid testes are impalpable (e86,e273,e564). Rarely, ectopic locations outside the normal line of
descent have been reported that included the perineum, base of penis, abdominal wall, pubic region, and upper
thigh (e230). The majority of undescended testes will achieve full spontaneous descent by 1 year of age,
predominantly within the first 3 months, and only 0.8% of infants would have incomplete descent 12 months after
birth. Spontaneous testicular descent after the first year is very unlikely (19,e342,86,91). A number of studies
have reported an increased rate of undescended testis with low birth weight, maternal pre-eclampsia, mild
gestational diabetes, and breech presentation (e249,e591). Bilateral testicular maldescent was reported in as
high as 39% of babies with cryptorchidism, which was also more likely to occur in babies with low birth weights.
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In addition to the presence of a patent processus vaginalis in most cryptorchid patients, other abnormalities can
occur especially those pertaining to the genitourinary tract, which includes dysgenetic testis, duplication of the
ureter, hypospadias, renal dysplasia, and inguinal hernia (e111,e163,93). Of the most common associations with
cryptorchidism are malformations in the paratesticular structures such as the epididymis and its attachment to the
vas deferens and gubernaculum (e1,3,e151). In a study by Favorito et al. (e150), the reported incidence of
epididymal abnormalities in normal male fetuses was only 4% compared with 35% in patients with
cryptorchidism. The most common of these anomalies is detachment between the epididymis and the testis
followed by separation of the epididymis from the vas deferens and the long looping epididymis (e206).
Outside the genitourinary tract, abnormalities may include gastroschisis, omphalocele, imperforate anus, cardiac
anomalies, lower limb anomalies, and caudal spinal malformations (e109,e277,e300). It has been reported that
the etiologies of cryptorchidism and hypospadias are partly shared and the presence of both conditions
simultaneously is associated with increased risk for ambiguous external genitalia and intersex disorders
(e8,e255,93).
Multiple causes for testicular maldescent have been suggested including an abnormal differentiation of the male
sexual organs, midline abnormalities, anatomical anomalies of the gubernaculum testis, hormonal dysfunction
affecting the hypothalamo-pituitary-testicular axis (hypogonadotropic hypogonadism), mechanical impairment
(insufficient intraabdominal pressure, short spermatic cord, underdeveloped processus vaginalis), and heredity
(e109,e232).
The prepubertal undescended testis is usually smaller than the contralateral one and the difference becomes
even more significant with progressive lesions (e86). Generally, there is correlation between the age at the time
of orchiopexy or orchiectomy and the severity of the histologic alterations in the testis. The isolated cryptorchid
testis in the prepubertal child has only subtle quantitative abnormalities such as decreased percentage of tubules
containing germ cells (low tubular fertility index), decreased mean tubular diameter, apparent tubular loss, and
early interstitial fibrosis (e83,e361,67) (Figure 19-1). The postpubertal and adult cryptorchid testis usually
exhibits abnormalities in all testicular structures characterized by tubular sclerosis, maturational arrest in
spermatogenesis, and interstitial fibrosis (e432). Tubules with immature Sertoli cells only (Sertoli cell nodules)
may also be found (Figure 19-2), and areas of Leydig cell hyperplasia are frequent (e396,e416). Granular
transformation and degeneration of Sertoli cells have been
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reported in the cryptorchid testis, as well as in other testicular disorders (e392,78). Similar histopathologic
features were also identified in retractile testis, indicating that these conditions may share some causal
relationships and that they may require similar management approach (e100,e207). Some changes in the rete
testis have been reported particularly in postpubertal cryptorchid testis including adenomatous features or
dysgenesis with hypoplastic changes (e106,e395).
FIGURE 19-1 ▪ Cryptorchid testis. Atrophic seminiferous tubules characterized by irregular contour, thickened
basement membrane, and lack of spermatogenesis with Sertoli-only pattern. Loose fibrous interstitial stroma is
evident.
FIGURE 19-2▪A: Sertoli cell nodule in a cryptorchid testis. A well-circumscribed, nodular proliferation of immature
Sertoli cells filling the seminiferous tubules with central hyaline foci. The surrounding testicular tissue is atrophic.
Note the presence of Leydig cells in the interstitial area. B: We have encountered similar proliferation adjacent to
a MGCT in an adolescent.
The relationship between cryptorchidism and male infertility has been extensively studied, and links between the
two conditions have been made in a number of series. Cryptorchidism was reported as the cause of infertility in
up to 9% of cases (e80). Biopsies from cryptorchid testes may reveal lack of germ cells as early as 18 months of
age, the incidence of which increases with advanced age and with bilaterality (15). Similar trends were observed
even in patients who underwent orchiopexy, implying that the actual development of germ cells in cryptorchidism
might also be impaired (15, 37, 59). Some authors, however, cast some doubt on the level of certainty of this
causative relationship between cryptorchidism and infertility and advocate that while it is certain that untreated
men with bilateral abdominal testes will be infertile, the levels of fertility are unpredictable in other less severe
scenarios (unilateral cryptorchidism, inguinal testes, orchiopexy) (e563).
Another important association with cryptorchidism is the increased risk of developing testicular germ cell tumors
(GCT), especially seminoma, compared with normally descended testes. It is estimated that the risk of testicular
cancer in cryptorchid males is four times higher than that of the general population and approximately 10% of
testicular cancer patients had cryptorchidism. The unrepaired cryptorchid testis has a 7% to 35% likelihood of
developing a malignant germ cell tumor, especially seminoma (4,5,e50,e163,e183,e438,e542). It has been
shown that the risk of developing a malignancy increases with an abdominal testis compared with an inguinal
testis and also with those treated with orchiopexy postpubertal (15,e434,e598). In the latter case, the tubules are
arrested in maturation.
Disorders of Sex Development (Intersex Disorders)

The term intersex disorders has been largely used to refer primarily to a clinical scenario of an infant born with
external genitalia sufficiently ambiguous that sex assignment is not possible. However, the mechanisms
underlying such a clinical scenario are variable and some disorders in sex development do not necessarily
present with genital ambiguity. It has been recently recommended to use the term disorders of sex development
(DSD) to replace such terms as intersex, hermaphrodite, and pseudohermaphrodite (38, 76). DSD represents a
congenital condition characterized by discordance between phenotypic sex and chromosomal sex and in which
development of chromosomal, gonadal, or anatomic sex is atypical. Recent advances in molecular biology have
enabled us to further our understanding of the processes involving normal sexual differentiation and the inborn
errors that result in sexual ambiguity, which has led to improvement in diagnosing and managing patients with
DSD (49,e324,76). It is estimated that two-third of sexually ambiguous neonates are female
pseudohermaphrodites with congenital adrenal hyperplasia (e607). The remaining DSD are associated with
some abnormality in male gonadal development or persistence of the müllerian tract with or without an abnormal
constitutional karyotype.
Currently, the classification of DSD can be organized by broader categories in which the intersexual disorders
are divided into “abnormalities of genital differentiation,” due largely to the abnormal production or sensitivity of a
single hormone, or “abnormalities in sex determination,” due to abnormal gonadal differentiation, usually
testicular, with or without chromosomal aberration (see Table 19-1) (e369,76) (see Chapter 18).
Disorders of Genital Differentiation

These disorders are generally associated with a normal chromosomal composition and normal gonads. This
includes female and male pseudohermaphroditism.
Female pseudohermaphroditism occurs as a result of relative androgen excess in utero in an individual with
two ovaries and a 46,XX genotype. The elevated levels of androgen present during embryogenesis usually result
in genital ambiguity and may result in a male phenotype. The most common cause is the adrenogenital
syndrome (AGS, congenital adrenal hyperplasia). The manifestations of AGS in genotypically female patients
are related to defects in the biosynthetic pathways of mineralocorticoid, glucocorticoid, and sex steroids (e81). In
males with AGS, usually there is no evidence of genital ambiguity, but they may have an enlarged phallus. They
may also develop clinically detectable bilateral testicular nodules during childhood or young adulthood that may
be confused with true Leydig cell tumors (LCTs) and are designated as testicular tumors of the AGS (e468,80)
(see Chapters 18 and 21).
A rare condition, placental aromatase deficiency, causes maternal virilization during pregnancy and
pseudohermaphroditism of the female fetus. Due to mutations in the aromatase gene CYP19 and the resulting
lack of aromatase activity, fetal androstenedione cannot be converted to estrogen by the placenta and instead is
converted to testosterone peripherally, resulting in virilization of both fetus and mother (e248,e512).
Other conditions that might be associated with female pseudohermaphroditism are related to maternal factors
such as maternal ingestion of synthetic progestins or androgens or the presence of maternal virilizing
lesions during pregnancy including a luteoma of pregnancy (e166,e343,76,e585).
Male pseudohermaphroditism represents a heterogeneous group of intersex conditions that occur in
individuals with normal 46,XY karyotype and either identifiable testes or evidence that testes were present during
fetal development
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but the external genitalia are usually female or ambiguous (7,e386). The responsible defect may be: (a) at the
gonad level, leading to disorders of testosterone biosynthesis and metabolism or testosterone receptor
abnormalities; or deficiency in MIS gene, or (b) at the end organ level, where the developing tissues are
unresponsive to androgen stimulation leading to an abnormal phenotype. Other less well-defined causes may
also be responsible.
Table 19-1 ▪DISORDERS OF SEXUAL DEVELOPMENT
Disorders of Female Adrenogenital syndrome

genital pseudohermaphroditism
differentiation (female intersex)
—21 α-hydroxylase deficiency

—11 β-hydroxylase deficiency
Placental aromatase defect
Maternal ingestion of progestins or androgens
Maternal virilizing lesions
Male Testicular regression syndrome

pseudohermaphroditism
(male intersex) Leydig cell deficiency (Defective hCG-LH
receptor)
Defects in testosterone synthesis:
a. Testosterone and adrenocorticoid insufficiency
—Defect in cholesterol synthesis (Smith-Lemli-

Opitz syndrome)
—Congenital lipoid adrenal hyperplasia (defect

StAR gene)
Congenital adrenal hyperplasia (3 β-hydroxylase

dehydrogenase deficiency, 17 α-hydroxylase
deficiency)
b. Testosterone insufficiency only
—17 20-desmolase deficiency
—17 β-hydroxysteroid (17-ketosteroid reductase)

dehydrogenase deficiency
Defect in mullerian inhibiting system End-organ

defects:
a. Androgen receptor disorders (androgen

insensitivity syndromes)
—Complete testicular feminisation (androgen

receptor insufficiency)
—Partial androgen receptor insufficiency

b. Disorder of peripheral testosterone metabolism
—5-α-reductase type 2 deficiency
Disorders of Klinefelter syndrome (47 XXY)

sex
determination
Turner syndrome and Turner-like (45 XO and X

mosaicism)
XX male and XY female syndrome (sex reversal)
Pure gonadal dysgenesis (bilateral)
Defect in the Wilms tumour suppressor (WT1)

gene
—Denys-Drash syndrome
—Frasier syndrome
Mixed gonadal dysgenesis (Turner-like,

dysgenetic male pseudohermaphroditism,
gonadoblastoma)
True hermaphroditism
Adapted with modification from Robboy SJ, Jaubert F Neoplasms and pathology of sexual
developmental disorders (intersex). Pathology 2007:39(11:147-163.
Gonadal defects responsible for male pseudohermaphroditism include TRS, agenesis or deficiency of the Leydig
cells, defects in specific enzymes in the pathway of testosterone or DHT biosynthesis or receptors to these
hormones, or a defect in elaboration or action of MIS.
Testicular regression syndrome (TRS, congenital anorchia, vanishing testis) is a condition in which a testis is
thought to have once existed but has atrophied and disappeared during early development (34, 47). The testis is
clinically impalpable and no normal testicular tissue can be identified following exploration. Generally, congenital
absence of the testis, or testicular agenesis, is an uncommon anomaly as it was detected in less than 1% of
testis both in fetuses and cryptorchid patients (e151). This condition results from the irreversible destruction of
one or both testes during fetal life in an XY individual, resulting in variable hormonal deficiencies and
developmental anomalies based on the stage at which testicular damage occurred (e333,62). Unilateral
testicular destruction does not result in TRS. By histopathologic examination, the testis may be completely
absent or represented by only a microscopic remnant. In addition to having no gonadal tissue, pathologic
findings include a collection of vascularized fibroconnective tissue (85%), hemorrhage or hemosiderin deposition
(70%), calcification (60%) or
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giant cells near the residual vas deferens or epididymis, the expected site of the gonad (e87,34,47,62,e526). The
vas deferens ends blindly and a small and circumscribed nodule of tissue may be located in the retroperitoneum,
the iliac fossa, or in the scrotum. By definition, no evidence of preserved remnants of seminiferous tubules
should be present.
The clinical presentation of individuals with TRS is variable and is reflective of the specific stage of fetal
development during which the testes were damaged. Generally, at one end of the spectrum, when gonadal
regression occurs early in embryonic life before the testes release androgenic or antimullerian hormones, the
testes are absent and the phenotype is female. At the other end, regression occurring later and through fetal life
would allow for a male phenotype with infantile to nearly normal male genitalia and differentiated wollfian-derived
structures. Affected individuals commonly have ambiguous genitalia. A number of etiologies have been proposed
for TRS including inherited genetic defect, intrauterine infection, and infarction (e227,76).
It is presumed that testicular regression develops late in the fetal period after the mullerian structures regressed
under the influence of the mullerian inhibitory substance and the male gonads and genitalia developed under the
influence of the androgens. Despite the familial occurrences of TRS suggesting a genetic etiology, no specific
genes have been identified to be associated with it and in particular those related to the opening reading frame
sequence of SRY (e417). Unlike cryptorchidism, there is no increased risk of gonadal neoplasia, because there
is little, if any, residual gonadal tissue.
Leydig cell deficiency (agenesis or hypoplasia) is a rare condition of male pseudohermaphroditism thought to
be due to a defect in the human chorionic gonadotropin-LH receptor, primary agenesis or hypoplasia of the
Leydig cells, or an abnormal LH receptor molecule (e26,e478,e497,e514). Affected individuals are genotypically
males (46,XY) with female phenotype and unremarkable or ambiguous external genitalia. Bilateral, slightly small
to normal-size cryptorchid testes are present with fully or partially developed epididymides and vasa deferentia,
indicating that testosterone production by Leydig cells was intact early in embryonic development. The testes
exhibit interstitial fibrosis, but no mature Leydig cells are present and no testosterone production is noted. LH
levels are elevated in affected individuals. Tubules with Sertoli cells are found and mullerian structures are
typically absent, indicating appropriate testicular production of MIS by Sertoli cells during fetal life (76,e487).
Familial occurrence of this condition has been reported and a number of mutations in the transmembrane domain
of LH receptor gene, resulting in Leydig cell deficiency, have been identified (44,e307,e469,e618).
Defects in testosterone synthesis may be due to inborn errors of the enzymes involved in testosterone
biosynthesis in the testis or the adrenal gland that may result in subnormal levels of testosterone and DHT
during embryogenesis (relative estrogen excess) resulting in female or ambiguous external genitalia (e324,e388).
These defects may involve cholesterol synthesis (mutations in 7-dehydrocholesterol reductase gene) as in
Smith-Lemli-Opitz syndrome (e401,e615) or mutations in the steroidogenic enzymes responsible for the
conversion of cholesterol to testosterone and DHT, which include: (a) steroidogenic acute regulatory protein
(StAR) gene responsible for congenital lipoid adrenal hyperplasia (e34,e59,e97,e534), (b) 17a-hydroxylase
(e65,e133) and 3β-hydroxylase dehydrogenase (e371,e517) responsible for congenital adrenal hyperplasia, and
(c) 17-ketosteroid reductase (e426).
The degree to which the external genitalia develop depends upon the type and the severity of the defect. The
microscopic features of testes in patients with these conditions vary and may show large clusters of Leydig cells
surrounding seminiferous tubules. Germ cells (spermatogonia) are often normal in children but disappear by
puberty resulting in Sertolionly syndrome. Some germ cells, however, can persist and rarely develop into
intratubular germ cell neoplasia (e280). Mullerian-derived structures are absent but wolffian duct structures may
be present (76).
Defect in mullerian inhibiting system or the persistent müllerian duct syndrome (PMDS), also referred to
as hernia uteri inguinale, is a rare form of male pseudohermaphroditism characterized by the presence of
mullerian duct structures in 46,XY phenotypic males. The age at diagnosis ranges from a neonate to the fourth
decade. Most patients have unilateral or bilateral cryptorchid testes, normal or almost normal male external
genitalia, and an inguinal hernia containing a prolapsed infantile uterus and fallopian tubes (8,e253,e334,e472).
The testes may be histologically normal and the wolffian duct structures are developed with the vas deferens
embedded in the wall of the upper vaginal structure in most cases. Inguinal hernias occur in almost 40% of cases
(e583). Malignant testicular tumors such as intratubular germ cell neoplasia and seminoma have been rarely
reported in cases of adult PMDS patients with uncorrected cryptorchid testis (e29,e258,e338,e611). More
recently, rare examples of clear-cell adenocarcinoma of the müllerian duct and uterine adenosarcoma in a boy
with PMDS have been reported (e511,e554). PMDS has been reported with a familial occurrence and rarely in
identical male twins (e44,e149,e234,e359,e605).
PMDS is currently considered a heterogeneous group of disorders caused by at least two different defects in the
mullerian inhibiting system. The most common is a defect in the MIS (mullerian inhibiting substance) gene, also
known as AMH (anti-mullerian hormone) gene preventing it from producing any biologically functional MIS. The
second defect involves an abnormal AMH type II receptor resulting in end-organ insensitivity to MIS despite the
presence of biologically active MIS. In other patients, an abnormality in the timing of MIS secretion may exist
(e45,e234,e252).
End-Organ Defects
As mentioned earlier, responsiveness to androgen is required to the normal development of the external genitalia
and
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wolffian duct-derived structures. The presence of the enzyme 5a-reductase in the anlage of the prostate and
external genitalia is also required for the conversion of testosterone to DHT. An absent or unstable androgen
receptor in 46,XY individuals leads to impaired development of both wolffian duct-derived structures as well as
external genitalia. If only 5a-reductase is absent or defective, abnormalities confined to the external genitalia and
prostate will be observed.
FIGURE 19-3 ▪ Testicular feminization. A: In this example of complete testicular feminization, a fully developed
female phenotype is evident. B: The karyotype is that of a male (Contributed by Dr. Jerome Taxy, Chicago,
Illinois).
Androgen receptor disorders (androgen insensitivity syndromes) result in variable phenotypes ranging from a
female phenotype with intra-abdominal testes to ambiguous genitalia to a male phenotype with minimal clinical
abnormalities.
Complete testicular feminization due to complete androgen insensitivity (e.g., testicular feminization,
Goldberg-Maxwell-Morris syndrome, hairless women, androgen receptor insufficiency) is the most common form
of male pseudohermaphroditism occurring in 1 of 20,000 newborns (e6,e386,e422,e486,e601). It is caused by
failure of androgen receptor binding despite its production and secretion by the fetal testis. The underlying
mechanisms have been identified as mutations in the androgen receptor gene including point mutations resulting
in amino acid substitutions or premature stopcodons, frame shift mutations by nucleotide insertions or deletions,
complete or partial gene deletion, or intronic mutations affecting the splicing of the androgen receptor RNA (e63).
Due to the presence of phenotypically female external genitalia (Figure 19-3), the condition is rarely diagnosed
before puberty unless an inguinal hernia or labial mass is encountered or unless the disorder is known to be
familial (e14,e589). Primary amenorrhea is the most common complaint leading to evaluation and subsequent
diagnosis. The wolffian tract involutes resulting in cystic epididymides that are usually not connected to the
testes. The vasa differentia, seminal vesicles, and prostate are absent. As a rule, both the cervix and the uterine
corpus are absent. A fragment of fallopian tube may be found in up to one-third of cases (76,e486).
The testes are cryptorchid and may be intra-abdominal or inguinal, or in the labia majora and 50% are found in
inguinal hernias. Overall, the testes in androgen insensitivity syndrome are histologically similar to the
cryptorchid testis except that the tubules are less mature with possible spermatogonia but no spermatogenesis.
Leydig cells are absent or replaced by collagenized interstitial tissue in portions of the gonad, whereas sheets of
Leydig cells may be found near the hilus and nerves. Ovarian-like stroma replaces the testicular interstitium.
Hamartomas and Sertoli cell adenomas were reported in the majority of cases in the postpubertal testis
(e381,e463,e486,e514). These hamartomatous nodules are multiple, bilateral, tan, yellow, or white in
appearance with bulging cut surface and may be composed of immature Sertoli cells, germ cells, Leydig cells,
ovarian-type
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stroma, nonspecific fibrous stroma, and smooth muscle. The typical size varies from 1 to 10 mm, but may
occasionally be up to 40 mm (e486). Sertoli cell adenomas consist of nodules of predominantly or exclusively
packed seminiferous tubules with immature Sertoli cells that are 3 cm in average size but range up to 25 cm
(e486). A rare example of a testicular tumor resembling the sex cord with annular tubules has been reported
(e457). GCT, particularly seminoma and less commonly intratubular germ cell neoplasia, can sometimes be
encountered in patients with this syndrome and, rarely, sex cord-stromal tumors have been reported
(e158,76,e404,e619). The development of malignant gonadal tumors in patients with testicular feminization
usually occurs later in adulthood (e332).
Partial androgen insensitivity syndrome due to partial androgen receptor insufficiency accounts for 10% of
all cases of androgen insensitivity (e487) and encompasses several different phenotypes, ranging from
individuals with a predominantly female appearance to persons with ambiguous genitalia, or individuals with a
predominantly male phenotype (e63). Affected patients typically present at birth with genital ambiguity but severe
hypospadias, micropenis, bifid scrotum, and bilateral cryptorchidism are also common. Alternatively, the external
genital phenotype may be predominantly female with partial labial fusion and clitoromegaly (e610). The
underlying mechanism involves a qualitative defect in the androgen receptor (e195,e440,e498). Additionally, a
number of syndromes and conditions are characterized by partial androgen insensitivity including Reifenstein,
Lubs, Gilbert-Dreyfus, Rosewater and the infertile male syndromes, and Kennedy disease
(e196,e295,e430,e614).
A disorder of peripheral testosterone metabolism is caused by mutation in the enzyme 5a-reductase type 2,
which is responsible for converting testosterone to DHT to exert its effect on differentiating the urogenital sinus
into external male genitalia and prostate (e19,e235,e613). Affected males usually have female to ambiguous
external genitalia at birth (e153,e519,e522). The penis is small (clitoris-like) and lacks a urethral orifice. A blind
vaginal pouch and inguinal or labial testes may be observed. Wolffian-derived structures are normal but no
mullerian-derived structures are present. Due to activation of type 1 isoenzyme, some virilization occurs at
puberty demonstrated by penile enlargement, scrotal rugation and hyperpigmentation, and testicular enlargement
and descent. Microscopic findings of testicular tissue may include spermatogenesis, tubular atrophy, no
spermatogenesis, or Leydig cell hyperplasia. The prostate remains rudimentary and the seminal vesicles remain
underdeveloped (76).
Disorders of Sexual Determination

These disorders are generally associated with sex chromosome abnormalities resulting in abnormal gonad
formation. Affected individuals characteristically have additions, deletions, or mosaicism of the sex chromosomes
and the appearance of the gonads is variable, ranging from a streak gonad to a nearly normal female or male
both grossly and microscopically.
Mixed gonadal dysgenesis (MGD) is one of the most frequent causes of male sexual ambiguity in individuals
usually with a 45,X/46,XY or 46,XY karyotype. In one series, MGD was the diagnosis in approximately 8% of
children with intersex conditions (e53). It represents a heterogeneous group of abnormalities characterized by
persistent mullerian duct structures, a dysgenetic testis and a contralateral streak gonad (e15,7,e353,e369). The
phenotypical heterogeneity of MGD is attributed to the presence of a variety of different genetic abnormalities
causing the syndrome mostly related to deletions of both the short and the long arms of chromosome Y
(e92,e224,e551).
The loss of testicular functions leads to incomplete inhibition of mullerian development, incomplete differentiation
of wolffian duct structures, and incomplete male development of the external genitalia. Testicular maldescent
may also occur (e369,e474) and some patients have phenotypical features of a Turner-like syndrome
(e275,e551). An infantile or rudimentary uterus and at least one fallopian tube are found on the side with the
uncommitted streak gonad. An intra-abdominal or inguinal cryptorchid testis or fibrous streak dysgenetic testis
without an accompanying fallopian tube is present on the contralateral side. Organs of wolffian duct derivation
may be present with variable frequency. An epididymis is identified in two-third of cases and is usually present on
the side where there is a testis. The vas deferens is encountered less frequently and the seminal vesicle is
identified only rarely.
The gonad may be a testis or a streak gonad (Figure 19-4). Streak gonads may show partial differentiation into
testicular phenotype, or may exhibit features toward ovarian differentiation with the characteristic ovarian type
stroma and rare primordial follicles. However, no true ovary is present, which requires the presence of
differentiation with follicles in at least the antral stage (76). A unilateral macroscopic testis is found in 60% of
cases, whereas bilateral testes may be seen in about 15% of cases usually with an asynchronous degree of
maturity.
The testicular architecture is consistently abnormal in these individuals as the region of the tunica albuginea or
cortex contains widely spaced seminiferous tubules with ovarian-like stroma or immature primary sex cords
indeterminate between female and male structures. The medullary region may contain normal seminiferous
tubules and interstitium, but in some cases it is difficult to distinguish between female and male structures.
Occasional narrow closed seminiferous tubules are lined by Sertoli cells and in other examples the germ cells
may be seen directly lining the basement membrane of the seminiferous tubule without the Sertoli cell layer that
usually normally surrounds them. Leydig cells may be present in small clusters of varying size. Occasionally,
broad zones of the cortex may exhibit a degree of differentiation toward streak-like ovary, even displaying rare
primordial follicles. By puberty, the germ cells present in
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a streak gonad may degenerate and disappear, resulting in a gonad composed exclusively of fibrous tissue and
a few rete tubules. The hilar region of the streak gonad or streak testis is populated by hilus cells and rete or
mesonephric tubules. Hyperplasia of the hilus cells in response to pituitary gonadotropins may result in clinical
virilization. Tumors develop in about 10% of those with MGD and the dysgenetic gonads of which 25% to 30%
are malignant, the most common of which is gonadoblastoma accounting for 75% to 80% of all germ cell
neoplasms in this disorder. However, other GCT have been also reported including germinoma, yolk sac tumor
(YST), teratoma, embryonal carcinoma (EC), and choriocarcinoma (e102,e165,e427,e474). These tumors may
be unilateral or bilateral. Occasionally, Sertoli cell tumor and SCT-like proliferations of sex-cord elements have
been also reported (e102,e399). Early gonadal resection is recommended in order to avoid the development of
an invasive germ cell tumor and to avoid the consequences of onset of virilization in a patient who has been
raised as a female.
FIGURE 19-4 ▪ Mixed gonadal dysgenesis in a 3-year-old patient with a female phenotype. A: On one side, an
intra-abdominal streak gonad was present consisting of vascularized fibrous stroma. B: The contralateral side
contained an immature cryptorchid testis with predominant Sertoli cells and only occasional spermatogonia. C:
Bilaterally, structures of both wolffian (vas deferens) and mullerian (fallopian tube) origins were present.
Pure gonadal dysgenesis (PGD) refers to phenotypically female individuals with streak gonads and internal
genitalia that include mullerian structures (uterus and fallopian tubes). It occurs with both 46,XX and 46,XY
karyotypes and has both familial and sporadic patterns of inheritance (e199,e336). The stroma of the gonads
has an ovarian-like appearance (e451) and primary amenorrhea is the usual clinical presentation. PGD patients
with 46,XX karyotype only rarely develop gonadal tumors, examples of which include GCT and mucinous
epithelial tumors (e298,e370,e383). Some have hilus cell hyperplasia and hilus cell tumors with the usual
associated virilizing effects.
PGD patients with 46,XY karyotype are at higher risk for gonadoblastoma and other GCTs that may develop in
10% to 25% of cases and can be unilateral or bilateral (e139,285, e486,e487,82,e451,88).
True hermaphroditism (TH) is a disorder of gonadal differentiation defined by the concurrence of both ovarian
and testicular tissue, with coexistent ovarian follicles (not just connective tissue stroma) and seminiferous tubules
(not just Leydig cells). The gonads may be ovary and testis separately or combined in an ovotestis (76, 95).
Affected individuals may have either a female or a male phenotype with variable degrees of sexual ambiguity.
The clinical manifestations are variable and depend on the gonadal tissue present and the age
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at the time of diagnosis. TH is a rare condition both in North American and Europe but is more commonly
encountered in Africa, especially in South Africa (e198,30,45,e581).
The architecture and the distribution of gonadal tissues in TH take several forms with asymmetry of the gonads
in the majority of cases. An ovotestis represents the most frequently encountered type of gonad in this condition
(1,45,e581,95). Patterns of gonadal development include an ovary on one side and a testis on the other (30% of
cases), or an ovary on one side and a contralateral ovotestis (30%). Bilateral ovotestes are found in 20% or
more of true hermaphrodites and a testis-ovotestis combination is found in 10% of cases. In the majority of cases
(80%), the ovarian and the testicular tissues are arranged in an end-to-end fashion with a distinct line
demarcating the two tissues. The ovary, which is the second most common gonad in TH, preferentially develops
on the left side whereas the testis, which is the least common gonad encountered in TH, develops preferentially
on the right (76). The location of the gonad is influenced by the type and the quantity of gonadal tissue present.
Increasing amounts of ovarian tissue increase the probability that the gonad will be in an ovarian position, and as
a result it is very unlikely for female gonadal tissue (either ovary or ovotestis) to be situated in the inguinal canal
or in the labioscrotal fold. The position of the testis is less constant as most reside in the scrotum but can be
encountered in the inguinal region or in the normal ovarian position. The nature of the genital structure adjacent
to a gonad in TH follows that of the ipsilateral gonad, which is characterized by having a fallopian tube adjacent
to an ovary and an epididymis or vas deferens adjacent to a testis. Either a mullerian (more commonly) or
wolffian structure, but not both, is adjacent to an ovotestis.
In young patients, the microscopic appearance of the gonadal tissue is often normal with the ovarian tissue
containing numerous follicles, whereas the testicular parenchyma has normal appearing seminiferous tubules
with spermatogonia. In older patients, ovarian tissue with structures indicative of ovulation (follicles, corpora
lutea, and corpora albicantia) may be seen, but the testicular tissue (in testis or ovotestis) is usually abnormal
with incomplete development, lack of spermatogenesis, loss of germ cells, and tubular sclerosis. Scrotal testes in
these patients show less severe changes, sometimes showing faulty spermatogenesis (76).
The prevalence of gonadal neoplasms, mainly gonadoblastoma and other types of malignant germ cell
neoplasms, is estimated at 2% to 3% of cases (e272,50,e487,e545). A rare case of juvenile granulosa cell tumor
(JGCT) in this setting has been reported (e547).
The causes of TH are probably as varied as the karyotypic expressions and genetic aberrations appear to play a
key role in its development. Patients with a “Y” chromosome have a 2- to 3-fold increased frequency of having a
testis as opposed to an ovotestis, and nearly 75% of true hermaphrodites with an ovary and an ovotestis have a
46,XX karyotype. A 46,XX/46,XY karyotype represents true genetic chimerism, whereas the 46,XX karyotype is
very likely to represent a crossing over of the X and Y chromosome during first meiotic division in the primary
spermatocyte, or the presence of hidden mosaicism for SRY (30,e121,e413,e449,e488,e558). There are
examples where the patients were 46,XX and lacked the SRY gene in usual cells examined (leukocytes) but cells
from the gonad itself demonstrated SRY (e243). Autosomal dominant mutations that mimic SRYhave been
suggested as one possibility where SRY was absent (e459,e523). The 46,XY karyotype probably contains a
hidden 46,XX cell line or that SRY, if present, may act at a time too late to stimulate the development of a testis,
hence permitting ovarian tissue to develop.
Klinefelter syndrome (KS) is one of the most common causes of prepubertal delay and primary hypogonadism
in males, occurring in about 1 of every 500 to 1 of every 1,000 live newborn males and accounting for about 3%
of infertile males (e55,46,69,76,e608). In the majority of cases, the karyotype is 47,XXY, which usually results
from nondisjunction occurring during meiosis of either paternal or maternal gametes. The clinical picture varies
depending on the age when the diagnosis is first suspected. Men with KS present with sequels of androgen
deficiency like infertility, low testosterone, erectile dysfunction, and low bone mineral density. They typically are
tall men with narrow shoulders, broad hips, sparse body hair, gynecomastia, small testicles, and azoospermia.
Infants with KS may have normal external male genitalia at birth, which may cause a delay in its discovery.
However, in some individuals, other findings may be indicative of this syndrome such as hypospadia, micropenis,
and small, soft testes or cryptorchidism. In adults with KS, the testes are small and rarely exceed 2 cm in greatest
dimension. Histologically, the seminiferous tubules may show some degenerative changes during fetal life, which
increases with age to the point that by late childhood the primary spermatogonia are greatly decreased in
number. This degenerative process may dramatically accelerate shortly before the expected time of puberty (e9).
In adults, the testes are largely atrophic with hyalinized seminiferous tubules and prominence of Leydig cells.
Some tubules may be preserved, but lined only by Sertoli cells. Functionally, the Leydig cells are abnormal, as
evidenced by low levels of serum testosterone with elevated levels of serum LH and FSH.
A variety of neoplasms have been associated with KS including both gonadal and extragonadal GCTs. Most
extragonadal tumors occur in the mediastinum as teratoma and EC (e5,e48,e118,e296,e350), but rare examples
of primary intrapelvic seminoma have been reported (e293). In the testis, seminoma, teratoma, and EC have
been encountered (e340,e462,e532). LCTs are rare (e408,e525). Men with KS are at a higher risk of developing
breast carcinoma than men without KS. (e179,84). Additionally, various hematological malignancies have been
reported in individuals with KS, including acute leukemia, chronic myeloid leukemia, and malignant lymphoma
(e43,e159,e365,e405,e506).
Turner syndrome is a disorder of sexual differentiation that is discussed in detail elsewhere in the book,
whereas
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Turner-like mosaicism (45,X/46,XY) is part of the mixed gonadal dysgenesis discussed earlier (see Chapter 18).
XX Male and XY Female Syndrome (Sex Reversal)

The XX male syndrome is one of the rarest of all sex chromosome anomalies, occurring in about 1 of 24,000
newborn males and is characterized by a nearly normal but infertile phenotypical male with a 46,XX karyotype
(e473,e549). Genotypically, XX males share many characteristics of men with KS as both groups have a
generally masculine appearance, normal or near-normal external genitalia and azoospermia with associated
small testes, prominent Leydig cells, and tubules lined only by Sertoli cells. XX males, however, are generally
shorter in height, and the frequency of hypospadias and gynecomastia is higher. Prenatal diagnosis of this
syndrome is currently possible due to the increasing application of prenatal ultrasonography and genetic
analyses (e178). In some cases, the mechanism underlying this disorder has been identified as translocation of
the SR Y gene from the Y chromosome to the X chromosome during meiosis (e178,e578).
Rare cases of male-to-female sex reversal have been identified in which phenotypically female individuals with
46,XY karyotype are identified, with some of these cases involving duplication and translocations of the short
arm of the “Y” chromosome (e41,e435,e584).
Defects in the Wilms Tumor (WT1) Suppressor Gene

Syndromic male pseudohermaphroditism with gonadal dysgenesis and other genitourinary tract anomalies is
intertwined with several genes that are active in male sexual differentiation, and one of these is WT1 on
chromosome 11p13 (e580). The product of WT1 is a zinc finger transcriptional factor for many growth factor
genes and is expressed in the developing kidney, especially in the condensing mesenchyme, and elsewhere in
the genital ridge, in particular the Sertoli cells of the fetal gonad and mesothelium (e219,e424,73). Constitutional
mutations in WT1 are found in the following syndromes: Denys-Drash syndrome (90% or more of cases), Frasier
syndrome, and WAGR syndrome (e23,e38,13,e313).
Patients with Denys-Drash syndrome have the clinical triad of early renal failure secondary to diffuse
mesangial sclerosis, Wilms tumor in most cases (20% bilateral), and male pseudohermaphroditism in children
with a 46,XY karyotype (i.e., dysgenetic testes, cryptorchidism, and severe hypospadias with micropenis)
(e240,e378). Multiple gonadal abnormalities are reported in association with this syndrome and include normal
ovaries with signs of early ovarian failures, normal mullerian and wolffian ducts, and normal to dysgenetic testes
(e241,e242). Gonadoblastoma(s) are known to develop in the dysgenetic gonads of a child with a 46,XY
karyotype. Constitutional heterozygosity in WT1 represents missense point mutations within exons 8 and 9 in this
gene (e58,e108).
Frasier syndrome is rare and results from a mutated WT1 gene, specifically mutation of intron 9 resulting from
abnormal splicing that leads to an unbalanced ratio of the WT1 isoforms needed for normal development of the
glomeruli and gonads (e38,e431). It is phenotypically similar to Denys-Drash syndrome with male
pseudohermaphroditism (normal female external genitalia, streak gonads, and XY karyotype) and progressive
nephropathy (e282,e543). The nephropathy is usually focal segmental glomerulosclerosis, which differs from the
diffuse mesangial sclerosis seen in Denys-Dash syndrome. As a rule, Wilms tumors do not develop in the Frasier
syndrome patients because the loss of KTS-positive isoform of the WT1 protein retains its tumor suppressor
function (e389), but gonadoblastoma develops in the dysgenetic testis in the child with a 46,XY karyotype
(e494,e600). Mutations in the donor splice site in intron 9 of WT1 distinguish Frasier syndrome from Denys-
Drash syndrome (e38).
WAGR syndrome, which includes Wilms tumor, aniridia, genitourinary anomalies, and mental retardation, is the
phenotypic expression of the constitutional chromosomal deletion within the short arm of one copy of
chromosome 11p13 that includes both WT1 and PAX6 (e173,e219,e475).
Acquired Abnormalities and Other Lesions

Torsion of the testis or its appendage occurs with a yearly incidence of approximately one in 4,000 males
younger than 25 years and accounts for 60% to 90% of cases of acute scrotal pain and swelling in men up to 18
years of age (e76,e254,e437,e471). Torsion of the testicular appendages may be more common than actual
torsion of the testis, especially in prepubertal males, whereas torsion of the testis is seen more often in
adolescence, at the time when acute epididymoorchitis also becomes an important consideration in the
differential diagnosis (e254,e471). Torsion of the testis in infancy is rare but is well documented, including its
detection in the prenatal period (e32,e262,e441). Testicular torsion should be suspected in every boy with
testicular pain and must be diagnosed quickly and accurately in order not to risk testicular viability. Imaging
studies, including color Doppler ultrasonography and scintigraphy, can be very helpful especially in clinically
equivocal cases (e382,e402,e471,603). Torsion usually occurs in the absence of any precipitating event (e400).
However, in 4% to 8% of cases, it may be associated with trauma (e499) or other possible predisposing factors
including an increase in testicular volume (often associated with puberty), testicular tumor, testicles with
horizontal lie, a history of cryptorchidism, and a spermatic cord with a long intrascrotal portion (e22).
The pathology of testicular torsion is testicular ischemia whose degree depends on the duration of torsion and
the degree of rotation of the spermatic cord. Ischemia can occur as soon as 4 hours after torsion and is almost
certain after 24 hours. It has been reported that testicular salvage can be
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achieved in approximately 90% of cases if treated within 6 hours from the onset of symptoms, but this rate falls to
50% after 12 hours and to less than 10% after 24 hours (e123). Greater degrees of rotation lead to a more rapid
onset of ischemia (e471). Intermittent torsion with spontaneous resolution within 2 hours or less is a known
clinical event (e114,e533).
FIGURE 19-5 ▪ Testicular torsion. A: The testis is congested with hemorrhagic cut section (Courtesy: Dr. Jerome
Taxy, Chicago, Illinois). B: Microscopically, ischemic changes and features of hemorrhagic infarction are evident
with sloughing of the seminiferous tubules and interstitial edema and hemorrhage.
Grossly, the testis is enlarged with a tense, bluish tunica albuginea and a dark, hemorrhagic appearance on
cross sections (Figure 19-5). The epididymis has a similar appearance, and a spiral twist may or may not be
seen in the spermatic cord. Little if any testicular parenchyma is appreciated through the hemorrhage. There is a
sequence of microscopic changes in the testis that precede the final acute stage of near-total hemorrhagic
infarction, starting with interstitial edema and hemorrhage and premature sloughing of germinal cells into the
tubular lumina followed by diffuse interstitial hemorrhage and necrosis of germinal cells except for some viable
seminiferous tubules beneath the tunica albuginea (e191,e192,e193,e360). Total necrosis of the testis is present
in almost all cases of continuous torsion after 24 hours. Torsion of the testicular appendage results in a
hemorrhagic cystic structure measuring up to 5 mm in diameter.
Two types of testicular torsion are recognized, with different ages at clinical presentation and anatomic location
of the torsion. Extravaginal torsion (neonatal torsion, torsion of the spermatic cord) involves the testis,
epididymis, and peritoneal coverings and results from spiraling on a vertical axis in the area of the external
inguinal ring. This type accounts for approximately 6% of all torsion cases in childhood and occurs predominantly
in neonates because the testis and the gubernaculum are free to rotate (e122). Most cases are unilateral, but
some may occur bilaterally and may present as neonatal testicular enlargement if it occurred in utero (e261).
Intravaginal torsion (adolescent torsion) occurs when the testis, usually accompanied by the epididymis, is
abnormally suspended and twists within the tunica vaginalis. This is caused by an abnormality of the processus
vaginalis in which the tunica vaginalis covers not only the testis and the epididymis but also the spermatic cord.
This creates a bell-clapper deformity, present in approximately 10% of all men (e74), which allows the testis to
rotate freely within the tunica vaginalis (e471). The peak age of incidence of this type of torsion is between 12
and 18 years of age, and it accounts for up to 90% of torsions in later childhood and adolescence (e471,e616).
Epididymoorchitis produces symptoms very similar to those of torsion and generally manifests in adolescence
(e471,e559). Acute scrotal pain on the basis of acute epididymoorchitis is found in 15% to 35% of cases in
various pediatric series with this clinical presentation. Epididymoorchitis is uncommon in prepubertal boys, but
has been reported in association with urinary tract infections with reflux or with an accompanying anorectal or
related anomaly (e406,e483). A Gram-negative organism, such as Salmonella or Escherichia coli, may be
identified as the causative pathogen (e123,e205). Tuberculous epididymoorchitis is reported in children in the
less developed regions of the world (e344). Viral orchitis, especially mumps orchitis, has diminished with
vaccination (e314,e331,e407). Testicular pain related to a vasculitis-associated orchitis has been reported in up
to 22% of boys with Henoch-Schönlein purpura (e46,e124,e201).
Testicular microliths and calcified nodules are being more frequently identified recently due to the increased
use of ultrasound as they produce hyperechogenic signals (e433) in up to 5% of healthy individuals.
Histologically, they are concentric calcifications and their presence has been linked to cryptorchidism, testicular
regression, silent torsion, and testicular GCT (e130,e168,e467).
Toxic injury to the testis in childhood can result in loss of germ cells, atrophy, and possible subfertility. Systemic
chemotherapy and radiation of the testes or central nervous
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system are significant causes of testicular damage in survivors of childhood malignancy and in children who
receive cyclophosphamide for renal diseases (e148,e290,e305,e502,e516). The prepubertal state of the testis
does not protect the gonad from the late effects of treatment (e453). Decreased or absent spermatogenesis with
Sertoli-only tubules, interstitial fibrosis, and testicular atrophy are the principal histologic findings. The effects of
these medications are related to their cumulative doses (e132,e414,e576). Decreased testicular size correlates
with decreased sperm production and inhibin B levels and increased levels of LH, FSH (e57,e516,e574). It has
been suggested that despite these histologic effects, there is some recovery of spermatogenesis following
aggressive chemotherapy when pharmacologic protection has been instituted (e287). Additionally, testicular
tissue cryopreservation in prepubertal boys before chemotherapy and radiotherapy is now possible (e33,e560).
Children with renal failure may experience a significant loss of spermatogonia per seminiferous tubule, which
tends to increase with age but is not seen in all children with renal failure (e70).
Neoplasms of the Testis

Prepubertal testicular tumors are rare with an incidence of only between 0.5 and 2 per 100,000 children,
accounting for approximately 1% to 2% of all pediatric solid neoplasms (40,e260,e303). They represent a diverse
heterogeneous group of tumors of germ cell and non-germ cell origins (Table 19-2). Some of these tumors are
associated with sexual maldevelopment syndromes with dysgenetic gonads, or cryptorchidism. As a result of the
rarity of such tumors, the Section of Urology of the American Academy of Pediatrics established the Prepubertal
Testicular Tumor Registry (PTTR) in order to compile clinical and pathological data from multiple institutions.
Data from this registry have been published in a number of excellent studies and review papers (e211,51,e482).
It was reported that approximately 30% of these tumors occur in the first year of life and about 7% occur in the
neonatal period (51,e482). In earlier studies, the age of distribution was reported to be bimodal, with a peak in
the first 5 years of life and a gradually increasing frequency in late adolescence (e2,e3,e194,e315).
Table 19-2 ▪TESTICULARTUMORS IN CHILDREN
Germ cell tumors Yolk sac tumor 856
Teratoma 439
Epidermoid cyst 48
Mixed germ cell tumora 51
Embryonal carcinoma 20
Seminoma 7
Dermoid cyst 5
Choriocarcinoma 1
Gonadal-stromal tumors Leydig cell tumor 27
Sertoli cell tumor 29
Juvenile granulosa cell tumor (JGCT) 12
Stromal tumors, unspecified 37
Gonadoblastoma 5
Paratesticular tumors Rhabdomyosarcoma 115
Other sarcomas 5
Other tumors, unspecified 26
Miscellaneous tumors 34
Total 1717
Compiled data from 12 series (e3, 10, e99, e152, e194, e260, e303, e306, 63, 72, e482, e538).
aSome of these tumors were designated teratocarcinoma in their original reports.
The majority of pediatric testicular tumors are of germ cell origin followed in frequency by gonadal stromal tumors
(e303,63,72,e482), whereas rhabdomyosarcoma (RMS) represents the most common tumor of the spermatic
cord and paratesticular soft tissues (e372). In the newborn, however, the most frequent testicular tumor is JGCT
(e89,33,48). A painless nontender scrotal mass is the presentation of the majority of prepubertal testicular
neoplasms although, less commonly, the presentation may be that of testicular pain or trauma
(e99,e303,e572,e573). Incidental testicular tumors during work-up for gynecomastia or precocious puberty have
been reported in up to 10% of patients in one institution (e572). Adequate work-up of a testicular mass is
important to determine its nature, first to select the appropriate management approach, and second to avoid
misdiagnosis as a non-neoplastic condition that can potentially mimic testicular and paratesticular tumors. It has
been reported that 5% to 23% of pediatric testicular tumors were misdiagnosed as torsion or hydrocele
(e99,e103,e260,63,e572). Other conditions to be included in the differential diagnosis of a scrotal mass include
hernia, hydrocele, hematoma/trauma, torsion, epididymitis, mumps orchitis, Henoch-Schönlein purpura, and
paratesticular tumors.
A staging scheme by the Pediatric Oncology Group applies to pediatric GCT and is distinct from the adult
counterpart (Table 19-3). The protocol for the examination of specimens from patients with malignant germ cell
and sex cord-stromal tumors of the testis, exclusive of paratesticular malignancies, is useful in selected pediatric
cases (89).
Table 19-3 ▪STAGING OF PEDIATRIC GCT BYTHE PEDIATRIC ONCOLOGY GROUP
Stage I Tumor limited to the testis.

No clinical, radiographic, or histologic evidence of disease beyond the testis.
Appropriate decline in serum AFP (AFP half-life = 5 days).
Stage II Microscopic disease located in scrotum or high in spermatic cord (<0.5 cm from proximal
end).
Retroperitoneal lymph node involvement (<2 cm). Serum AFP persistently elevated.
Stage III Retroperitoneal lymph node involvement (>2 cm). No visceral or extra-abdominal
involvement.
Stage Distant metastases.

IV
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Germ Cell Tumors
GCT are the most common primary tumors of the testis in the first two decades of life, 50% to 60% of which
occur in the first 2 years (e152,e327). Significant differences exist between prepubertal testicular GCT and their
adult (postpubertal) counterparts. While adult tumors usually comprise a mixed histology of seminomatous and
nonseminomatous components, are most often malignant, and are almost always associated with intratubular
germ cell neoplasia, prepubertal tumors typically contain only one histologic type (either teratoma or YST), can
be benign or malignant, do not usually occur in undescended testes, and lack the intratubular germ cell
neoplasia component (e260,54,74). These differences are also reflected in their respective genetic
abnormalities. Prepubertal GCT are diploid (teratoma) or aneuploid (YST). Postpubertal GCT are hypertriploid
(seminoma) or hypotriploid (nonseminoma) and consistently have one or more copies of the short arm of
chromosome 12 [i(12p)]or other forms of 12p amplification (e260,53,74). Staging of pediatric GCT is distinct from
that of the adult counterparts (Table 19-3) (22, 39).
The most current World Health Organization (WHO) classification of testicular GCT divides them into tumors of
one histological type, which includes seminoma, EC, YST, trophoblastic tumors and teratoma; and tumors with
more than one histological type, which can contain any combination of any proportions of the pure forms (22).
Consensus has now been reached concerning the prognostic factors that determine the outlook for patients with
metastatic disease (39, 71).
Despite the weak correlation of most etiologic factors with testicular GCT, it is generally believed that these
tumors are associated with abnormal conditions in fetal life. A number of contributing factors are recognized
including cryptorchidism, prior testicular GCT, family history of testicular GCT, and certain somatosexual
ambiguity syndromes. Most of these factors, however, are important only in postpubertal boys and adults
(e161,e577).
In GCT of the testis, generally three clinicopathologic entities are recognized: the teratomas—YSTs of the
infantile testis, the seminomas and nonseminomas of adolescents and adults, and the spermatocytic seminomas.
This chapter will focus on the former with highlights on the other entities as they relate to the pediatric
population.
Yolk Sac Tumor

Formerly known as endodermal sinus tumor, YST is characterized by numerous patterns that recapitulate the
yolk sac, allantois, and extraembryonic mesenchyme. The nosology has evolved from the concept of infantile
adenocarcinoma, orchioblastoma, EC, and infantile EC to the current concept of a neoplasm with morphologic
features recapitulating the extraembryonic yolk sac or endodermal sinus.
YST is the most common testicular GCT in childhood, accounting for as much as 75% of prepubertal testicular
GCT (e152,40,e599). Despite its occurrence in all races, it is much more common in Whites than in Blacks,
Native Americans, and Indians (e 107,e311) and may be more common in Orientals when compared to
Caucasians (e257). The median age at presentation is 16 to 19 months (40,e482,e552). YST is more commonly
present in the right testis and the most common presenting symptom is a painless scrotal mass (40). Other
possible complaints include a history of trauma, acute onset of pain, and hydrocele. At least one case has been
reported in an intra-abdominal testis (e1 13). Serum a-fetoprotein (AFP) levels are elevated in more than 90% of
tumors in a number of studies including those that are based on data from the PTTR (40,e482,e552). It is
important to remember that normal AFP ranges in young infants are higher than those in older patients. Usually,
YST is not hormonally active rendering precocious puberty an unlikely presentation. Most tumors are not
associated with cryptorchidism or a dysgenetic gonad. Intratubular germ cell neoplasia is not observed in the
adjacent testis in children with pure YST in contrast to its ubiquitous presence in testicular GCT in adolescents
and young adults (57). By ultrasonography, YST is typically solid hypoechoic and devoid of cystic structures,
which when present within the mass, argue against the diagnosis of YST (e210). Approximately 10% to 20% of
children with YST will present with metastases (29,40,e482), which can occur hematogenously or via lymphatic
drainage, unlike adult YST, which metastasizes predominantly through lymphatics. Hematogenous spread alone
can occur in up to 40% of cases (29). YST metastases to the lung occur in 20% of cases compared with 4% to
6% to the retroperitoneal lymph nodes (e66,29,e617). The lungs represent the most common site of metastasis
followed by retroperitoneal lymph nodes, liver, and bones (83). When metastases develop, they usually appear
within 14 months of initial presentation (29,e221,40).
Grossly, pure YST is solid and soft with a pale gray to pale yellow cut surface, which can sometimes be
gelatinous or mucoid (32, 40). Hemorrhage and necrosis may be observed in large tumors; however, their
presence (and/or that of cysts) should raise the possibility of a mixed GCT, especially in the adolescent (e544).
Microscopically, the histopathological appearance of YST is similar in both the pre- and postpubertal age groups.
Several patterns are recognized that are usually admixed in variable proportions (Figure 19-6). It is not unusual
for one pattern to predominate; however, it is rare that an entire tumor comprises a pure single histologic pattern
(32,40,e544). The most common histologic pattern is the microcystic or reticular pattern, which consists of
meshwork of vacuolated cells producing a honeycomb appearance, often with hyaline globules. Tumor cells are
usually small and may contain pale eosinophilic secretions (32,e550). The endodermal sinus pattern consists of
papillary structures known as Schiller-Duval bodies, which are considered the hallmark of YST, even though
they are not required for the diagnosis. These structures consist of a stalk of connective tissue with thin-walled
blood vessels lined on the surface by a layer of cuboidal cells with clear cytoplasm and prominent nucleoli. Other
recognized microscopic patterns include: solid, macrocystic, glandular-alveolar, papillary,
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myxomatous, polyvesicular vitelline, hepatoid and enteric patterns (Figure 19-6). Mitotic activity can be brisk in
any of these patterns. Hyaline globules may be seen especially in the hepatoid and enteric patterns (e239,e570).
By immunohistochemistry, expression of AFP is helpful but can be variable and sometimes weak. Its absence,
however, does not exclude the diagnosis of YST. Low molecular weight cytokeratin is usually strongly
expressed. A number of proteins, usually present in the fetal liver, may also be expressed in YST such as α-1-
antitrypsin, albumin, and ferritin (e237,e239). Genetic analysis of infantile YST did not identify a specific gene or
genes involved in its development. However, a number of recurrent genetic anomalies are known to occur
including losses of the short arm of chromosome 1 (particularly the 1p36 region), the long arm of chromosomes 6
(6q21-26) and 16 and gains in the long arms of chromosomes 1 and 20 (20q13), the short arm of chromosome 3
(3p21-pter), and the complete chromosome 22 (e225,64,71,e579,90).
FIGURE 19-6 ▪ Yolk sac tumor. A-D: Predominantly solid and focal glandular pattern (A), microcystic pattern (B),
tubular/macrocystic pattern (C), and hepatoid pattern (D). Note the myxoid and hypocellular background (C,D).
The treatment of choice for prepubertal YST is surgical excision (i.e., radical orchiectomy). Metastatic work-up is
required for adequate staging of tumor and serum AFP is important in establishing the preoperative diagnosis
and also as a follow-up postoperatively for possible tumor recurrence (e103,e210,e482).
Teratoma
Teratoma is a tumor composed of several types of tissue representing different germinal layers (endoderm,
mesoderm, and ectoderm), forming somatic-type tissue in various stages of maturation and differentiation (11) for
which the term mature or immature (fetal-like) apply. However, based on findings of genetic studies, it is now
recommended to consider teratoma as a single entity regardless of the degree of maturation and differentiation
of the tissue comprising it (22). Tumors consisting of ectoderm, mesoderm, or endoderm only are classified as
monodermal teratomas. In its pure form, teratoma comprises approximately 3% of testicular GCT in adults and up
to 38% of the prepubertal GCT (11,e597) with a reported incidence that ranges from 0.5 to 2.0
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cases per 100,000 boys (e66). Teratoma is the second most common testicular tumor in children and
adolescents following YST with a relative frequency ranging from 13% to 60% (e66,e67,10,29,e315,e482). About
65% of prepubertal teratomas occur in the first 2 years of life with a mean age of 20 months and represent 50%
of GCT seen in the first decade of life(e373).
Most patients present with a firm, irregular, nodular, and nontender scrotal mass that usually does not
transilluminate. Approximately 2% to 3% of prepubertal teratomas may be associated with or misdiagnosed as
hydroceles, especially if the tumor has a cystic component. Teratomas usually present as a unilateral scrotal
mass (e66), but rare examples of bilaterality in infancy and childhood have been reported
(e3,e216,e320,e548,85). Teratomas in undescended, intraabdominal testes may present with abdominal pain
due to torsion, as calcification or ossification on imaging studies, or as an abdominal mass (e12,e142). Prenatal
sonographic diagnosis might be possible in cases of fetal abdominal mass, especially when the testis cannot be
detected in the scrotum by the 8th month (e345,e508). It is speculated that the undescended testis did not cause
the neoplasia, but was induced by it (e379). Teratomas are hormonally inactive; hence, precocious puberty is not
a common presentation and serum AFP levels are helpful in distinguishing them from YST (29,e485).
FIGURE 19-7 ▪ Teratoma in a prepubertal testis. A: Grossly, the tumor has a heterogeneous multinodular
appearance with cystic and solid areas. (Courtesy: Dr. Jerome Taxy, Chicago, Illinois). B, C: Microscopically, a
mixture of mature structures derived from ectoderm, mesoderm, and endoderm is noted, characterized by
keratinizing squamous epithelium, ciliated respiratory type epithelium, and mature cartilage.
By imaging studies, teratomas are generally wellcircumscribed and heterogeneous masses and a cystic
component is commonly demonstrated (26). On gross examination, teratomas are usually nodular and firm with a
variably cystic and solid cut surface (Figure 19-7A). The cysts may be filled with keratinous material or clear
serous or mucoid fluid. The solid areas may contain translucent, gray-white nodules representing cartilage.
Rarely, hair or melanin-containing tissue may also be seen. Areas of immature tissue are mostly solid and may
have an encephaloid, hemorrhagic, or necrotic appearance.
Microscopically, mature elements resemble normal postnatal tissue and typically include structures derived from
the three germ layers (Figure 19-7B). Structures of ectodermal origin are usually manifested by nests of
squamous epithelium
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with or without cyst formation and keratinization. Neural tissue may be encountered as foci of neuroglia.
Structures of endodermal origin are represented by glandular epithelium of enteric or respiratory type. Other
glandular tissue such as pancreatic, mucus producing epithelium, prostate, and thyroid may be found.
Mesodermal elements are represented by cartilage, bone, adipose tissue, fibrous tissue, and, most commonly,
muscle. Attempts at organ formation are frequently identified with smooth muscle encircling glands of respiratory
or enteric morphology. Immature, fetal-type tissue may also consist of ectodermal, endodermal, and/or
mesodermal elements. It usually occurs as islands of immature neuroepithelium resembling that of the developing
embryonic neural tube. Immature tissue may also have an organoid arrangement with blastomatous and primitive
tubular structures resembling that of the developing kidney or lung. Embryonic skeletal muscle, cartilage, and
nonspecific cellular stroma may also be encountered (32,11,e337,e373). The so-called fetus infetu is an
expression of extreme maturation and organization of a teratoma or a form of pathologic monozygotic twinning
(e12). A number of somatic type malignancies have developed in pediatric testicular teratomas some of which
developed following irradiation for a testicular teratoma with metastases. These included Wilms tumor,
leiomyosarcoma, angiosarcoma, and RMS (e568,e569).
Infantile teratomas are diploid. Genetic studies (karyotyping and comparative genomic hybridization) have failed
to demonstrate chromosomal changes in these tumors. In contrast, teratomas in adult testes are hypotriploid and
have genetic changes similar to those seen in other components of adult GCT(e367,64,71,90).
The prognosis is excellent in children since teratomas are universally benign tumors, unlike their adult
counterpart (11,e211,e450). Pure teratoma of the prepubertal testis has not been reported to metastasize and
does not develop, at least in the overwhelming majority of cases, from the lesion recognized as intratubular germ
cell neoplasia, unclassified (89). Although orchiectomy has been considered the treatment of choice for
prepubertal testicular teratomas, recent studies with long-term follow-up have demonstrated the safety and
efficacy of testis-sparing surgery (77,e485).
FIGURE 19-8 ▪ Epidermoid cyst. A: The cyst is well circumscribed and completely intratesticular. It contains flaky
yellow-white keratinous material (Courtesy: Dr. Jerome Taxy, Chicago, Illinois). B: Microscopically, abundant
lamellated keratinous material is filling the cyst with a fibrous wall separating it from the adjacent testicular
parenchyma. No intratubular germ cell neoplasia is noted in the adjacent seminiferous tubules.
Epidermoid Cyst
Epidermoid cyst is a benign tumor of ectodermal origin, characterized by its keratin-producing epithelium and
lack of other germinal layer components, differentiating it from teratoma (e501). It accounts for less than 1% of all
testicular tumors and 3% to 14% of pediatric testicular tumors with 25% occurring in the first two decades of life
(e134,e303,63,e482,e513). The nosology and the pathogenesis are uncertain. A germ cell origin is most likely;
however, intratubular germ cell neoplasia is not an accompanying feature (e134,e328). This lesion usually
presents as a firm, well-defined intratesticular nodule, with or without symptoms. On ultrasonography, it appears
as a central hypoechoic mass with an echogenic rim (e214,e503). Grossly, the cyst is confined to the testicular
parenchyma and is filled with flaky yellow-white keratinous material (Figure 19-8A). Orderly, stratified squamous
epithelium, a dense fibrous tissue wall, focal calcifications, and acellular keratinous debris are the histologic
findings (Figure 19-8B). The cyst and the surrounding tissue should be examined carefully for teratomatous or
dermal adnexal elements, a testicular scar or intratubular germ cell neoplasia, the presence of which should lead
to reclassification of the lesion as a mature teratoma. A conservative surgical approach with simple enucleation
has been advocated for this benign lesion (e49,e134,26,56,e481).
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Intratubular Germ Cell Neoplasia, Unclassified Type
Intratubular germ cell neoplasia, unclassified type (IGCNU) is a microscopic precursor lesion composed of germ
cells within the seminiferous tubules with abundant clear cytoplasm, large irregular nuclei, and prominent nucleoli
(22). This term refers to the lesion initially described by Skakkebaek as “carcinoma in situ” as well as to other
“differentiated” forms of intratubular germ cell neoplasia (e135,e187,e368,e520). IGCNU is present in up to 4%
of cryptorchid testes, in up to 5% of contralateral gonads in patients with unilateral GCT, and in up to 1% of
biopsies from oligospermic infertile men (e51,e69,e160,e182,e222, e391,e423,e452,e477,e627). Additionally, it
can be found in virtually all cases adjacent to invasive GCT in adult testes when residual testicular parenchyma
is present (e135,e238). Contrastingly, the association with GCT arising in prepubertal testes is still a source of
controversy and its true incidence is difficult to assess (e226,54,57). It is generally believed, however, that
IGCNU is not associated with teratomas and pure YST in early childhood, in keeping with a different
pathogenesis for this subset of testicular GCT. Rarely, IGCNU has been described in association with
maldescended testes, intersex states and rare infantile YST and teratoma (e226,e283,e420,e466,e529,e530). In
one series, IGCNU was reported in four patients with gonadal dysgenesis (65). In 12 patients with androgen
insensitivity (testicular feminization), three were found to have unexpected IGCNU when no tumor was clinically
apparent (e380). In another study of 102 cases of various intersex states, the authors reported IGCNU in 0 of 23
patients with androgen insensitivity syndrome (testicular feminization), 3 of 38 with gonadal dysgenesis, 1 of 12
with TH, 1 of 22 with male pseudohermaphroditism, and 1 of 7 with multiple congenital anomalies and ambiguous
genitalia (e456).
IGCNU is not reliably detected in the prepubertal at-risk patients (e110,e409,e420). Conversely, the identification
of atypical germ cells in prepubertal biopsies does not correlate with tumor risk. Although abnormal germ cell
morphology has been described in prepubertal patients with cryptorchidism (e16,67), the findings are different
from IGCNU, and their significance is not established, unlike the known significance of IGCNU. One large study
found no intratubular germ cells adjacent to GCT in prepubertal children to be positive for PLAP or c-kit; five of
seven were positive for PCNA and p53 was present in the two examined cases. These results indicate that germ
cells adjacent to infantile GCT are proliferative but not neoplastic and offer additional evidence that intratubular
germ cells and GCT in prepubertal boys are different from those of adolescents and adults (e212). Similar
studies have reported morphologic and immunohistochemical features of normal prepubertal germ cells that
resemble those of IGCNU that can persist up to 1 year of life (e25). Therefore, little or no benefit is derived from
the routine biopsy of cryptorchid testes at the time of orchidopexy in prepubertal boys, and, if biopsy is to be
performed, it should be delayed until after puberty. The assessment of risk by testicular biopsy in most
prepubertal patients is not currently possible. An important exception to this general rule applies to prepubertal
patients with intersex syndromes in whom the reliable identification of IGCNU or gonadoblastoma can be
accomplished in early childhood (e325,e381,65,e456).
Microscopically, the seminiferous tubules are partially or completely filled by large cells with round nuclei, coarse
chromatin, mitoses, and abundant clear cytoplasm (Figure 19-9A). A PAS stain demonstrates abundant
glycogen. Immunohistochemical markers that are reliably positive include PLAP, CD117, and OCT4 (Figure 19-
9B,C) (e69,e93,e236,e250,e266,e330,e390,e536). In contrast to invasive GCT in adult testis, the presence of
i(12p) in IGCNU has not been universally confirmed with most investigators suggesting it is not present
(e410,74).
Embryonal Carcinoma
EC is a rare tumor in the first decade of life and has a peak incidence in the 15- to 34-year old age group
(e152,e594). Although very common in mixed GCT, occurring in greater than 80% of them, pure EC is rare with a
rate of approximately 2.5% (e374). An adolescent or young adult presents with an enlarging painful scrotal mass
or metastases in the regional lymph nodes, abdomen, or mediastinum. The testis contains a gray, focally
necrotic, and hemorrhagic mass. The tumor is often poorly demarcated and the cut surface bulges markedly.
Microscopically, sheets of large, pleomorphic undifferentiated cells with enlarged irregular and vesicular nuclei,
distinct nuclear membranes, prominent nucleoli, and frequent mitoses are seen (Figure 19-10). Tumor necrosis is
evident. Primitive gland formation and papillary structures with or without fibrovascular cores may be
encountered. The characteristic immunohistochemical profile is: cytokeratinpositive, CD30-positive, OCT4-
positive, PLAP-positive (focal), and epithelial membrane antigen (EMA) negative (24,e250,e566). EC shares
similar genetic abnormalities with other adult GCT. Tumor stage is the single most important prognostic indicator
and pure or predominant EC in a testicular tumor is associated with increased risk of advanced disease
(e64,e147,e376).
Seminoma
Seminoma is a malignant GCT composed of relatively uniform cells, typically with clear or dense collagen
containing cytoplasm, well-defined cell borders, and large regular nuclei with one or more prominent nucleoli; the
cells resemble primitive germ cells. There is almost always an associated lymphoid infiltrate and frequently a
granulomatous inflammatory response (89). While seminoma is the most common primary testicular tumor in
adults, it is rare in prepubertal boys but is found more frequently in late adolescence (e2,e147,e590). In pediatric
cases, the average age of presentation is 9.7 years (e590). It remains crucial to
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distinguish seminoma from other forms (nonseminomatous) of GCT because of different treatments.
FIGURE 19-9 ▪ Intratubular germ cell neoplasia, unclassified type. A: The lesion consists of large cells with
round-to-irregular nuclei, coarse chromatin, and occasional nucleoli. A mitotic figure is present. The cytoplasm is
abundant and clear. B,C: The presence of lesional cells can be further facilitated by membranous expression of
CD 117 and nuclear labeling by OCT4.
Grossly, a seminoma characteristically forms a gray, cream to pale pink, soft, homogeneous, lobulated, and well-
defined mass that may have irregular yellow foci of necrosis. The tumor may occasionally present as multiple
macroscopically distinct nodules. Microscopically, the uniform cells of seminoma are arranged in sheets, clusters,
or columns and associated with lymphocytic infiltrate of variable density. Pseudoglandular, tubular, and cribriform
morphologies have been reported, but the basic cell morphology of seminoma remains the same. The
immunoprofile of seminoma is typically reactivity with vimentin, PLAP, CD117, and OCT4 (24,e250).
FIGURE 19-10 ▪ Embryonal carcinoma. Tumor cells are large and pleomorphic with enlarged irregular and
vesicular nuclei, distinct nuclear membranes, prominent nucleoli, and frequent mitoses. Numerous apoptotic cells
are present. Necrosis is a common finding.
Choriocarcinoma almost never occurs in childhood (e147) in its pure form but maybe found as a component of
mixed GCT, especially in adolescents (e152). The pathologic features and treatment are similar to those of the
same tumor in adults. Metastatic choriocarcinoma rarely occurs in infants from a primary tumor in the mother.
Mixed Germ Cell Tumor

Mixed germ cell tumor (MGCT) includes tumors containing two or more GCT components (22, 89). The individual
components are microscopically identical to those seen in pure GCT. While accounting for up to 54% of tumors
in the adult testis (e375), MGCT is rarely seen in prepubertal children and becomes increasingly common in the
second
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and third decades of life (e2,e147,e337). In one study, it accounted for 3% of prepubertal testicular tumors
(e303). A common pattern is EC with one or more components of teratoma, seminoma, and YST, but virtually any
combination can be seen. For diagnosis, similar to tumors of the adult testis, these tumors should be termed
“mixed germ cell tumor, composed of…” followed by a tabulation of their percentage. The adjacent testis usually
exhibits intratubular germ cell neoplasia. The combination of EC and teratoma has been previously termed
teratocarcinoma, but it is currently preferable to include these under the MGCT category and list the components
separately.
Sex Cord-Stromal Tumors

Sex cord-stromal tumors resemble the specialized supportive structures of the male or the female gonad and
include LCTs, SCTs, granulosa cell tumors, and tumors of the theca/fibroma group (22). They account for 4% to
6% of tumors of the adult testis and up to 12% of prepubertal testicular tumors (41,e303,63,e482,e513). Sex
cord-stromal tumors are generally benign with only rare cases of SCTs reportedly behaving in a malignant
fashion (e279,e504). Additionally, a number of stromal tumors have been reported without evidence of
differentiation toward any of the specific entities mentioned here and are referred to as undifferentiated sex cord-
stromal tumors. Some of these tumors have occurred in pediatric age patients; some also developed metastasis
(e556).
Leydig Cell Tumor

LCT, also known as interstitial cell tumor, is rare in children, accounting for up to 8% of pediatric testicular
tumors and 14% of stromal tumors (e442,22). Generally, approximately 20% of LCT occur in the first decade of
life. In this patient population, the tumor is most common between 3 and 9 years with a mean age at presentation
of 7 years (e185,e269). Although it typically presents with a painless testicular mass, patients usually display
signs of precocious puberty and elevated levels of serum testosterone and androstenedione and
dehydroepiandrosterone (e61,e99,e612). Gynecomastia may be seen in 10% to 15% of patients
(e95,14,e202,e352). LCT may be seen in patients with KS and 5% to 10% may have a history of cryptorchid
testis (e269). Ultrasonography typically shows a well-defined hypoechoic small solid mass. Grossly, the tumor is
well circumscribed and may be encapsulated. The cut surface is homogeneous yellow-brown, with possible
areas of hyalinization and calcification. Microscopically, the tumor is composed of large polygonal or round cells
with abundant granular, eosinophilic cytoplasm, forming sheets, trabeculae, and cords that displace seminiferous
tubules (Figure 19-11). Nuclei may vary in size and shape but atypia and mitoses are not reliable for predicting
aggressive behavior. Two other cell types seen occasionally are small round cells with scanty cytoplasm and an
eccentric, hyperchromatic nucleus and clear cells resembling the adrenal zona fasciculata. Occasional spindling
of tumor cells may be present. Lipofuscin pigment may be seen in up to 15% of cases. Crystals of Reinke are
present only in 30% to 40% of cases (e269). Abundant smooth endoplasmic reticulum and lipid are identified
ultrastructurally. By immunohistochemistry, LCT stains strongly and diffusely with antibodies to vimentin, inhibin,
calretinin, and melan-A (A 103) and shows variable staining with cytokeratins, EMA, desmin, S-100 protein,
chromogranin, and synaptophysin (e71,e233,e346,e622). Immunostains for carcinoembryonic antigen and PLAP
are consistently negative. LCT in prepubertal patients is benign and can be adequately treated by either radical
orchiectomy or testis-sparing surgery (e210).
FIGURE 19-11 ▪ Leydig cell tumor. A: Sheets of polygonal or round cells with abundant granular, eosinophilic
cytoplasm are evident, displacing seminiferous tubules. B: The cytoplasm is typically finely granular and
eosinophilic with mild variation in nuclear size and shape (inset).
In contrast to LCT, Leydig cell hyperplasia occurs in neonates, is bilateral, and shows a transition between
nodular and diffuse Leydig cell proliferation (68). Seminiferous
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tubules intermingle with Leydig cells throughout, and spermatogenesis is evident in tubules adjacent to nodules.
An important differential diagnosis to consider is the testicular “tumor” of the AGS. These lesions are usually
discovered in early adult life in patients with congenital adrenal hyperplasia but in up to one-third of cases are
found in children as small nodules. Similar lesions are typically seen in Nelson syndrome (e244). This condition
consists of bilateral, dark brown nodules with pleomorphic pigmented cells and hyalinized fibrotic stroma.
Although small lesions may involve the testicular hilum only, larger nodules almost always involve the testicular
parenchyma (e362,80). Awareness of this entity is important since these lesions usually decrease in size
following corticosteroid therapy and may be managed conservatively but surgical removal, either by tumor
enucleation or orchiectomy, may become necessary in refractory cases (e27,e468).
Sertoli Cell Tumor

SCT is a sex cord-stromal tumor of the testis composed of cells with features of Sertoli cells at variable degrees
of development. This is a rare neoplasm accounting for less than 1 % of all testicular tumors and typically
occurring in adults and only exceptionally reported in males younger than 20 (96). Some variant forms, however,
are more common in infants and children, especially those with syndromic and/or genetic associations such as
androgen insensitivity syndrome (79), Carney syndrome (e602), and Peutz-Jeghers syndrome (94,96,e625).
Painless and slow testicular enlargement is a common presentation (e164,96). Elevated estradiol levels, sexual
precocity, and gynecomastia may occur in patients with SCT of the Peutz-Jeghers syndrome (e11,e625).
Although this tumor is usually unilateral, those associated with syndromic conditions may be multiple and bilateral
(e286,e625). By ultrasonography, SCT is generally hypoechoic but can demonstrate variable echogenicity with
possible cystic areas.
FIGURE 19-12 ▪ Sertoli cell tumor. A,B: This tumor exhibits variable morphology characterized by solid nests in a
dense fibrotic background, compressed tubular arrangement (trabecular) with a resemblance to a carcinoid.
In general, no imaging characteristics would allow distinction from a GCT. An exception is the large cell calcifying
variant (see below), which is characterized by large areas of calci-fications that can be readily suspected by
ultrasound, especially when this tumor presents as multiple and bilateral masses (e91,e174).
The gross appearance of the enlarged testis is variable, ranging from a firm, circumscribed, lobulated, gritty, tan
or yellow nodule to a multicystic mass. Foci of hemorrhage may be seen but necrosis is uncommon.
Microscopically, SCT may vary in appearance ranging from tubular arrangement to retiform or solid growth
pattern to cords of tumor cells (Figure 19-12). The intervening stroma is fibrotic and moderately to sparsely
cellular or hyalinized. Tumor cells have round, oval, or elongate nuclei. The chromatin pattern is vesicular;
nucleoli are not prominent and nuclear grooves or inclusions may be seen. The cytoplasm can be pale-
toeosinophilic, clear, or vacuolated due to lipids. Mild nuclear pleomorphism and atypia may be seen in the
minority of cases. By immunohistochemistry, SCT is consistently reactive with antibodies against vimentin and
cytokeratins with variable expression reported with antibodies against inhibin and S-100 (e233,e346,e546,96). It
is typically negative for placental alkaline phosphatase, a-fetoprotein, and EMA (e94). Electron microscopy
reveals the characteristic features of Sertoli cells: tubular structures with well-defined basement membrane,
complex cytoplasmic interdigitations, numerous intercellular junctions, prominent Golgi apparatus, large lipid
droplets, abundant smooth endoplasmic reticulum, and Charcot-Böttcher crystals in some examples.
In children, SCTs typically follow a benign course. However, metastatic potential does exist especially in older
children (e279,e504,e556). Radical orchiectomy is the preferred treatment (e210). In older boys when the tumor
is suspected of behaving in a malignant fashion, patients should undergo evaluation for metastatic disease. The
latter condition should be treated aggressively with a combination of chemotherapy,
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radiation therapy, and retroperitoneal lymph node dissection (e211,77).
FIGURE 19-12 ▪ (continued) C: Complex and anastomosing tubular structures “retiform”, or cord-like structures
in a sclerotic to hyalinized stroma. D,E: Tumor cells with round, oval, or elongate nuclei, vesicular chromatin
pattern, occasional grooves, and inconspicuous nucleoli. F: The cytoplasm is pale-to-eosinophilic but can also
exhibit prominent clearing or vacuolization.
Large cell calcifying SCT (LCCSCT), as mentioned above, is a unique variant of SCT that can be sporadic
(60%), but can also be part of Peutz-Jehgers and Carney syndromes (40%) (e286). This variant tends to occur
in young individuals with an average age of 16 years and can be bilateral in 40% of cases. Associated features
include multiple endocrine disorders manifested by precocious puberty, gynecomastia, acromegaly, bilateral
primary adrenocortical hyperplasia, and pituitary adenomas. Cardiac myxomas and mucocutaneous
pigmentations are reported features in Carney syndrome (e82,e623). Microscopically, this tumor consists of
nests, trabeculae, small clusters, and cords of large polygonal cells with abundant eosinophilic finely granular
cytoplasm embedded in a myxohyaline stroma, which typically contains large areas of calcifications (Figure 19-
13). The nuclei are round to oval with vesicular chromatin pattern and inconspicuous nucleoli (e623). Intratubular
spread of tumor cells is usually present.
Interestingly, multifocal intratubular proliferations of Sertoli cells distinct from those observed in LCCSCT have
been recently reported in patients with Peutz-Jeghers syndrome in two separate studies. Eleven of the 14
patients from both studies did not have an associated SCT (e567,e587).
Juvenile Granulosa Cell Tumor

JGCT is a rare tumor consisting of structures resembling Graafian follicles. Despite its rarity, this tumor is the
most frequent congenital testicular neoplasm and most common tumor in the first 6 months of life (33,48,e538).
The reported incidence ranges from 3% (e482) to 6.6% of all prepubertal testicular tumors (41), with 50% of
tumors occurring in the neonatal period and 90% within the first year of life (e90,18,48,66,e455,e565), making it
exceptional to observe this tumor after the first year of life. Most of these tumors present as painless testicular
mass and typically are not hormonally active (26,e513). JGCT can be associated with
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abnormal karyotypic mosaicism and structural abnormalities in chromosome Y, especially in patients with
ambiguous external genitalia (16). Other associated abnormalities include MGD and hypospadias. JGCT can
occasionally occur in the undescended testes of infants with intersex disorders (e455,e624). All the reported
cases have had a benign course.
FIGURE 19-13 ▪ Large cell calcifying SCT. A: Sheets and cords of large polygonal cells with abundant
eosinophilic cytoplasm in a background of loose fibrous to myxohyaline stroma. B: Adjacent large areas of
calcifications are present.
By ultrasonography, JGCT is a cystic and septate hypoechoic mass (e565). Grossly, solid and cystic patterns
are present and hemorrhage may be observed (Figure 19-14). The cysts are usually thin walled and filled with
viscous or gelatinous fluid. Microscopically, it is identical to its ovarian counterpart. The tumor consists of
variably prominent solid and follicular or cystic patterns. The lining of the cysts consists of several layers of cells
that resemble the granulosa (inner) or the theca cells (outer). The follicles contain basophilic or eosinophilic fluid
that stains with mucicarmine. In nonfollicular areas, tumor growth can be in the form of sheets, nodules, or
irregular clusters. Hyalinization may be prominent. The tumor cells have round-to-oval hyperchromatic nuclei with
occasional nucleoli, and the cytoplasm is moderate to large with pale-to-eosinophilic appearance (Figure 19-14).
Mitoses, which are often readily seen, and atypia do not adversely influence the favorable prognosis (68). By
immunohistochemistry, the granulosa-like cells stain positive for cytokeratins, vimentin, and S-100. The theca-
like cells are positive for vimentin, smooth muscle actin, and focally for desmin (e197,e428,e547). The main
differential diagnosis is with YST, which can be solved by applying the appropriate immunostains
(26,e341,e565).
FIGURE 19-14 ▪ Juvenile granulosa cell tumor. A: The tumor is relatively well circumscribed and has both solid
and cystic areas. The solid areas are tan-white to yellow and the cysts can be prominent. B: Solid and follicular
to cystic patterns are evident.
Gonadoblastoma
Gonadoblastoma belongs to the category of testicular tumors containing both germ cells and sex cord elements.
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It is composed of a mixture of seminoma-like, large germ cells and sex cord cells having features of immature
Sertoli cells and granulosa cells. Gonadoblastoma is most commonly seen in MGD associated with ambiguous
genitalia (e284,43,79,81). The risk of developing gonadoblastoma in this setting is estimated to be 15% to 25%
(e421). Surgical exploration of the cryptorchid testis often demonstrates persistence of female-type internal
genital structures stemming from failure of involution of the müllerian ductal system (79). Bilateral involvement by
gonadoblastoma occurs in about one-third of cases (81). Karyotypic analysis of the patients, regardless of sexual
phenotype, almost always reveals a Y chromosome, with 46XY and 45X/46XY occurring most commonly (79).
FIGURE 19-14 ▪ (continued) C: Areas with stromal hyalinization may predominate. D: Tumor cells have roundto-
oval hyperchromatic nuclei and moderate-to-large amount of pale-to-eosinophilic cytoplasm (Courtesy of Dr.
Jerome Taxy, Chicago, Illinois).
Grossly, gonadoblastoma usually forms solid, yellow-tan nodules with gritty calcifications. The tumor size ranges
from microscopic foci to large masses up to 8 cm (81). Microscopically, the nodules usually consist of well-
defined, rounded nests of large, pale seminoma-like cells admixed with small, dark, angular, sex cord cells that
may form a peripheral palisade around the cellular nests (Figure 19-15). Nodular foci of hyalinized basement
membrane can be seen in the center of these nests and at the periphery. The stromal cells may become
polygonal, resembling Leydig cells, mostly in postpubertal patients. Calcifications appear initially on this
basement membrane and may become quite prominent. By immunohistochemistry, the germ cells stain similar to
those in ITGNU including reactivity with PLAP, CD 117, and OCT4 (e93,e104,e251), while the stromal cells
express inhibinandWT-1 (e229).
Gonadoblastoma is a premalignant lesion from which invasive GCT can develop, most commonly as seminoma,
but any nonseminomatous GCT may occur (81). Excision of a gonad with gonadoblastoma prior to development
of an invasive lesion is curative.
A number of cases have been reported in which the tumors consisted of a combination of neoplastic germ cells
and neoplastic sex cord-stromal elements arranged in a diffuse pattern rather than the nested pattern of
gonadoblastoma (e56,e339,e458). These tumors have been designated germ cell-sex cord/gonadal stromal
tumors, unclassified. In some of these tumors, at least according to a recent report, the neoplastic nature of the
germ cells has been disputed (e571), suggesting that some of these tumors might in fact represent sex cord-
stromal tumors with entrapped germ cells rather than unclassified mixed germ cell sex cord-stromal tumors.
Miscellaneous Tumors of the Testis

Congenital neuroblastoma may present as a testicular mass without evidence of disseminated disease until
months later (e75,e264). A subset of patients with neuroblastoma originating below the diaphragm or with
advanced stage disease may be more prone to testicular metastases and in some cases tumor relapse can
occur in a scrotal location (e294,e518). It has been also recently suggested that neuroblastoma can arise de
novo from the paratesticular tissue, presumably from sympathetic tissue remnants, as part of a multicentric
disease rather than metastasis (e575).
Nephroblastoma (Wilms tumor) can metastasize to the testis from a renal primary (e30,e492,e562) but can also
occur as a paratesticular, scrotal, or inguinal tumor (e7,e24,e412).
Carcinoid tumor has been rarely reported in the testis, especially those of children (e47,e156). Its microscopic
features are identical to those reported in other sites with predominance of the trabecular and the insular
patterns.
Tumors of hematopoietic origin may involve the testis. Overall, leukemia and lymphoma account for 2% to 5% of
all pediatric testicular tumors. They are the most common metastatic malignancies to the testicle in children and
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account for the majority of the tumors that present bilaterally (e210,e306). Primary testicular lymphoma in
children is rare and typically occurs prepubertally at 3 to 10 years of age (e
157,e203,e215,e319,e366,e415,e436). Secondary testicular involvement in children may also occur in
approximately 5% of systemic lymphomas (e120,e263). Leukemic involvement of the testis is more commonly
seen in acute lymphocytic leukemia (ALL) but can also develop in myelogenous leukemias (e181,e595). In a
postmortem survey of children with leukemia and lymphoma, the overall incidence of testicular involvement was
found in 25 of 39 cases (65%). In the majority of those cases, ALL was also identified in other organs, supporting
the view that testicular infiltration is indicative of widespread disease (e465). Although testicular involvement by
ALL may be clinically evident in about 8% of cases (e535), microscopic involvement may be as high as 21%
(e271). The testis may also be the site of relapse after bone marrow remission is established and may signal a
systemic relapse (e278,e292,e387).
FIGURE 19-15 ▪ Gonadoblastoma. A: Nodules of tumor consisting of a mixture of nests of large and pale
seminoma-like cells admixed with sex cord cells with small, dark, angular nuclei. B: A focus with prominent
calcifications. C: Hyalinized nodules of basement membrane material are surrounded by tumor cells.
In clinically evident cases, the testis has a bulging, pale tan surface with diffuse and nodular pattern of infiltration.
Microscopically, there is diffuse interstitial infiltrate of small cells with scanty cytoplasm, surrounding and
infiltrating the seminiferous tubules (e28,e292). The diagnosis can be establishe by needle or open wedge
testicular biopsy (e210). Juvenile xanthogranuloma rarely occurs in the testis of infants.
EPIDIDYMIS, SPERMATIC CORD, AND PARATESTICULAR TISSUES

A number of congenital anomalies of the mesonephric duct system can occur. These anatomic defects are
usually identi-fied either at the time of orchidopexy for an undescended testis or during an investigation for
infertility. The spectrum of morphologic manifestations ranges from total absence of the epididymis, vas
deferens, seminal vesicles, and ejaculatory ducts to selective atresia, cysts, diverticula, and ectopias
(17,e326,e351). Complete absence of the vas deferens is the most common congenital obstructive abnormality
of the male mesonephric duct system and is found in 3.5% to 8% of males evaluated for infertility (17); cystic
fibrosis should be considered in the diagnosis. Absence of the seminal vesicles and ejaculatory ducts often
accompanies absence of the vas deferens. Epididymal abnormalities are present in 36% to 43% of
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patients with maldescent of the testis. These include agenesis; atresia of the head, body, or tail; and a loop or an
elongated epididymis (e176,e276,e288). This association is especially common in the presence of an
undescended testis and a complete hernia sac (23,e213). Seminal vesicle cysts may simulate prostatitis clinically
and are associated with ipsilateral upper urinary tract abnormalities (e476). Spermatic cord cysts may be of
mesothelial or mesonephric origin (e394). Those of mesothelial origin are typically unilocular and lined by flat
poorly cohesive epithelium with chronic inflammation and hyalinization in the wall, while mesonephric cysts are
multilocular and lined by cohesive attenuated or columnar epithelium and may contain spermatozoa if in
continuity with the sperm-excretory ducts. In approximately 10% of cases, the origin of spermatic cord cysts
maybe indeterminate due to the lack of recognizable epithelium.
Small glandular or tubular inclusions, which probably represent embryonal remnants of müllerian ducts, are
found in 0.53% to 6% of hernia sacs from males in the first two decades (12,e175,e443,e531,e596). The ciliated
low columnar epithelial cells have eosinophilic cytoplasm and basal nuclei and are surrounded by a mantle of
fibrous tissue without smooth muscle. Due to their morphologic overlap, it is important to distinguish these
embryonal remnants in hernia sacs from the true vas deferens or epididymis because of the implications for
fertility and potential medicolegal ramifications (e175,e557). These embryonal tubular structures have a smaller
diameter than the vas deferens, often lack smooth muscle, and do not increase in diameter with advancing age
(e443). Tortuous blood vessels and tangentially oriented, mesothelial-lined spaces are other pseudoglandular
structures observed in hernia sacs.
Abnormalities of the epididymis and spermatic cord structures can also occur as a result of prenatal conditions.
Exposure to diethylstilbestrol in utero is associated with epididymal cysts and varicoceles in addition to
hypotrophic testes and capsular induration (e177,e606). Congenital rubella, in addition to causing
cryptorchidism, may be associated with absence or obstruction of the vas deferens or epididymis (e447). Cystic
fibrosis is a condition that, in addition to other complications in other organs, contributes to infertility in adult
males. The disease may additionally be associated with aplasia or hypoplasia of the vas deferens or epididymis
(e220,e312,e357). Moreover, congenital bilateral absence of the vas deferens has been linked to defects in the
cystic fibrosis transmembrane conductance regulator (CFTR) gene(e96,e358,e510).
A number of heterotopic tissues may be encountered in the paratesticular region such as in splenogonadal
fusion, ectopic immature renal tissue, ectopic adrenal rests, and rarely ectopic prostatic tissue. These lesions
should be considered in the differential diagnosis of a scrotal mass. Splenogonadal fusion consists of a spectrum
of malformations of unknown etiology involving abnormal fusion between the spleen and the gonad or
mesonephric derivatives such as the epididymis and vas deferens (e35,e189,e256,e268,e289,e363). This entity
was first mentioned by German pathologist, E. Bostroem in 1883 and later described in detail by G. Pommer in
1889 (e268).
There are two main morphologic types of this malformation: the continuous and the discontinuous type,
depending on the presence or the absence of a structural connection between the regular spleen and the
ectopic splenic tissue that is fused to the gonad (e448). Splenogonadal fusion occurs much more frequently in
males than females and is almost always left sided (e42,e411). Patients, typically younger than 20 years, usually
present with left scrotal swelling, left inguinal hernia, or cryptorchidism (e84). In about one-third of reported
cases, other congenital defects are additionally present, predominantly in the continuous type. The most
common of these anomalies is cryptorchidism; other associated anomalies include bilateral absent legs,
imperforate anus, spina bifida, diaphragmatic hernia, and hypospadias (e112,e140). Microscopically, normal
splenic tissue and testicular tissue are identified, but atrophy or immaturity may be seen. The cordlike structure
present in the continuous type is composed of an admixture of splenic and fibrous tissues.
Nodules of ectopic immature renal tissue (e186,e347,e621), ectopic adrenocortical tissue (adrenal rests)
(e267,e335,e539), and rarely ectopic prostatic tissue (e582) may be found incidentally along the spermatic cord
and adjacent to the epididymis (Figure 19-16).

A variety of acquired disorders of the epididymis and spermatic cord may present as an “acute scrotum” and may
simulate testicular torsion or neoplasia. Acute nonspecific epididymitis in prepubertal and early adolescent boys
presents with slow onset of scrotal pain, erythema, and edema (e180). Although coexistent anatomic anomalies
with this condition are rare in the pediatric age group, further work-up maybe warranted if this condition recurs
(e79,e204). Infants have a higher rate of associated acute epididymitis and genitourinary malformations than
older children (e356,e515). Tuberculous epididymitis, probably due to hematogenous spread of infection, mimics
malignancy because of the combination of painless testicular swelling and an abnormal chest radiograph
(e73,e344). This condition should be included in the differential diagnosis of testicular swelling in endemic
regions. Epididymal sarcoidosis causes granulomatous epididymitis and scrotal swelling mimicking a neoplasm
(e146,e604). Spermatic vein thrombosis may simulate intermittent testicular torsion, with acute pain and swelling
of the scrotum, spermatic cord, and epididymis (e105).
Henoch-Schönlein purpura may cause acute scrotum and scrotal swelling in up to 38% of patients and usually
resolves spontaneously and does not require surgery (e101,e116,e209). If the scrotum is explored, edema,
petechiae, and purpura of the scrotum, epididymis, and testis are the morphologic manifestations. Scrotal
abscess formation has been reported following appendectomy performed both by laparoscopic and open
approach (e167,e553).
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FIGURE 19-16 ▪ Ectopic adrenal tissue. A: One example of adrenocortical tissue presented as a palpable
paratesticular nodule. The lesion is well circumscribed and encapsulated and exhibits zonation similar to the
normal adrenal cortex. B: Another example of ectopic adrenal tissue incidentally discovered in an inguinal hernia
sac.
Varicocele results from dilatation of veins in the pampiniform plexus of the spermatic cord and is found in 16% of
boys between the age of 10 to 15 years, being uncommon in boys younger than 10 years (e218). In order to
prevent the progressive and irreversible damage to the testis, surgical correction of varicocele should be
performed soon after diagnosis regardless of the degree of severity and the presence or absence of symptoms
(e68,e321). The pathogenesis is venous stasis and reflux with vascular insufficiency and consequent
progressive tubular damage. Hydrocele is a lower abdominal and scrotal cystic mass resulting from accumulation
of fluid in the processus vaginalis or tunica vaginalis (e461,e527).
Meconium periorchitis presents as a large solitary paratesticular mass or several small nodules along the
spermatic cord and is frequently associated with a hydrocele (e60,e127). It may be the rare initial manifestation
of cystic fibrosis or result from volvulus, intestinal atresia, or ischemia (e470). A rare case of scrotoschisis
associated with meconium periorchitis has been also reported (e98). For this condition to occur, it requires an in
utero perforation of the gastrointestinal tract, allowing meconium to leak into the peritoneal cavity and then into
the tunica vaginalis via the processus vaginalis. The perforation may resolve antenatally with the scrotal lesion
as the only clue and manifestation of the process. Scrotal and abdominal calcifications on plain films and
hyperechogenic areas on scrotal ultrasound are the imaging abnormalities (e470,e491). The gross appearance
is a yellowish green, gritty mass with focal dystrophic calcifications. Microscopically, the lesion consists of loose
myxoid to irregular fibrous connective tissue. Aggregates of macrophages with multinucleated giant cells
containing brown bile pigment or cholesterol clefts and scattered calcifications may be seen. The mass may
spontaneously resolve without surgery (e127). A case of barium peritonitis secondarily causing an acute scrotal
lesion in an infant has been reported (e190).
Tumors of the Paratesticular Structures

Mesothelial proliferations have been reported in the paratesticular region in the pediatric age group ranging from
mesothelial hyperplasia to malignant mesothelioma.
Nodular mesothelial hyperplasia is a benign reactive process that may mimic a malignant process. It is
typically incidentally encountered in hernia sacs, the majority of which occur in the pediatric age group (e480). It
could also be associated with hydrocele or hematocele. The persistent irritation may easily result in histology
that mimics malignant mesothelioma such as papillae and small tubules, solid nests and cords extending into the
underlying reactive connective tissue simulating invasion (e17). However, the overall morphology is devoid of
overtly malignant features.
Malignant mesothelioma of the paratesticular region is rare and even rarer in young patients with only 10%
occurring in patients younger than 25 years (e17,e247,e429). Grossly, the tumor typically presents as thickening
of the tunica vaginalis with multiple friable nodular lesions. The histopathologic features are variable with the
majority of tumors showing pure epithelial phenotype. The biphasic tumors contain a variable proportion of
sarcomatoid morphology, basically resembling their pleural and intra-abdominal peritoneal counterparts. The
entire spectrum of differentiation may be seen ranging from well-differentiated tumors characterized by
tubulopapillary architecture and variably invasive tubules to poorly differentiated tumors with solid sheets, cords,
and nests of highly infiltrative epithelioid cells with necrosis.
Rarely, tumors of the ovarian epithelial types have been reported in the paratesticular region of adolescent
patients. Their histopathologic features and classification are identical to their ovarian counterparts (e247,e624).
Desmoplastic small round cell tumor (DSRCT) consists of proliferation of small round cells with an epithelial
growth pattern and a desmoplastic stroma. Although typically
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affecting the pelvic and abdominal cavities of adolescent males, involvement of the paratesticular region is not
uncommon (e117,e171). It may present in the paratesticular soft tissue, serosal surfaces, and the epididymis
near the junction with the rete testis. Grossly, the tumor is firm and has a white-to-tan appearance.
Microscopically, it consists of nests and anastomosing cords of uniformly small blue cells with scant cytoplasm
embedded in a densely fibrotic stroma. Focal tubular formation may be seen. Mitoses are readily seen and tumor
necrosis is commonly present. By immunohistochemistry, the tumor typically exhibits dual reactivity with keratin
and desmin. Additionally, neuron-specific enolase (NSE), EMA, and vimentin are expressed. DSRCT is typically
nonreactive for muscle common actin, myogenin, and chromogranin and is variably reactive for MIC2 (CD99) and
p53 (27,e297). DSRCT is characterized by a specific chromosomal abnormality, t(11;22)(p13;q12), resulting in
the fusion of the Ewing sarcoma gene EWS on 22q12 and the Wilms tumor gene WT1 on 11p13 (e172). The
detection of this gene fusion EWS-WT1 is both a sensitive and a specific marker of DSRCT (27,e10) and its
EWS-WT1 chimeric protein product is expressed in over 90% of tumors. Most patients with DSRCT develop
metastasis to regional lymph nodes or to distant sites (e117).
Mesenchymal paratesticular tumors in infants and children are rare and include a variety of neoplasms such as
hemangioma, juvenile xanthogranuloma, and RMS.
Paratesticular RMS is one of the relatively common tumors that involve the scrotal contents that are not of germ
cell origin and is the most common paratesticular sarcoma in children (e308,55,e460). The mean age of
occurrence in one large study was 6.6 years. Most paratesticular RMS are of embryonal subtype including the
spindle cell variant (e308), although alveolar subtype has been also reported. Their histopathologic features are
identical to their soft tissue counterpart. Metastases have been reported from these tumors to retroperitoneal
lymph nodes and distant sites (e460).
Of the vascular tumors, cavernous hemangioma predominates and usually follows a benign course (e318,e425).
A case involving the testis of a stillborn has been reported (e540). Bilateral testicular hemangiomas in neonates
have been associated with cystic hygromas and may regress spontaneously (e505). Juvenile
xanthogranuloma presents as a hard, irregularly enlarged testis with a circumscribed yellow nodule on cut
surface (e484,e561).
Other tumors of the connective tissue surrounding the testicle include benign (leiomyoma, fibroma, lipoma,
calcifying fibrous tumor/pseudotumor) and malignant entities (leiomyosarcoma, fibrosarcoma, liposarcoma,
rhabdoid tumor, and malignant peripheral nerve sheath tumor). These tumors are extremely rare, being mostly
the subject of case reports, and their morphologic features are similar to their soft tissue counterparts
(2,e37,e155,e259,e385,e445).
Melanotic neuroectodermal tumor (retinal anlage tumor) is a rare melanin-containing tumor typically affecting
the facial and skull bones. This tumor, however, has been reported in the epididymis and is seen mainly in
infants (35). Grossly, these are circumscribed firm epididymal tumors with a white-to-gray cut surface that may
show darker areas of pigmentation. Microscopically, the tumor consists of two components: the melanin-
containing epithelioid cells arranged in cords, nests, or glandular structures, and the small neuroblast-like cells in
a variably cellular stroma. These two components are usually intermixed. This tumor generally follows a benign
clinical course but may recur locally. No distant metastases have been reported but regional lymph nodes
(inguinal, retroperitoneal) have been rarely involved (e126,e245).
Adenomatoid tumor, the second most common tumor of the epididymis and cord in adults, can rarely be found
in children as a scrotal nodule (e52,e398,e496). The mass is gray, dense, and homogeneous, with a mucoid cut
surface. Microscopically, tubules and cords of flattened or cuboidal eosinophilic mesothelial cells in a fibrous
stroma are characteristic of this tumor (e129,e377).
Papillary cystadenoma of the epididymis is a rare benign epithelial tumor of the epididymal ducts and, rarely,
the spermatic cord. This tumor can be associated with von Hippel-Lindau disease especially when it is bilateral,
which can happen in about 30% to 40% of cases. The tumor is solid and cystic with occasional papillary
formation and is composed of cuboidal-to-columnar cells with clear or vacuolated cytoplasm (e246,e446).
THE PENIS
Most congenital abnormalities of the penis are related to defects in urethral closure, such as hypospadia and
epispadia, and meatal stenosis. Hypospadia, with an incidence of 1:300 male newborns, is an anomaly involving
the ventral aspect of the penis in the form of an abnormal ventral opening of the urethral meatus, an abnormal
ventral curvature of the penis (chordee), and/or an abnormal distribution of the foreskin. The extent of the
malformation is variable as the ectopic urethral opening (meatus) can be located anywhere from the tip of the
glans penis, along the penile shaft and scrotum, to the perineum. The form and the extent of the malformed
urethral opening can be variable but is rarely stenotic (e40,e136). Epispadia refers to the congenital absence of
the dorsal aspect of the urethra, resulting in a urethral opening on the dorsum of the penis. The most frequent
location of the opening is penopubic but can be penile or glanular. The incidence of male epispadia is 1 in
117,000 live male births. Associated urinary incontinence is frequently observed with penopubic epispadias and
occasionally with penile type, but is not associated with glanular epispadias. Congenital anomalies that have
been associated with epispadias include diastasis of the pubic symphysis, bladder exstrophy, renal agenesis,
and ectopic pelvic kidney (e170).
Other rare malformations of the penis include penile agenesis, or aphallia, diphallia, accessory scrotum, and
transposition of
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the penis and scrotum (penoscrotal transposition) (e88,e145, e200,e281,e299,e403,e464,e521). Some of these
conditions may have familial predisposition and may be associated with other anomalies mostly in organs of the
genitourinary tract (e31,e439).
Cutaneous viral infections and balanitis xerotica obliterans (BXO) are the principal acquired penile lesions in
children and adolescents. The presentation of human papillomavirus (HPV) infection is variable ranging from
asymptomatic infection to condyloma acuminata to bowenoid papulosis. Although DNA from certain known
pathogenic HPV strains was detected in foreskins from newborns undergoing routine circumcision, there was no
correlation with their respective mothers who had abnormal cervicovaginal cytologic smears (e479). In young
children with clinically evident condyloma acuminata, a sexual etiology was determined in more than half of them,
and occasionally these patients were found to have a mother with extensive condylomata observed at the time of
childbirth (e507). It has been also noted that condylomata acuminata in young people are associated with the
same HPV types found in anogenital lesions in adults (e626). Bowenoid papulosis, histologically identical to
preinvasive squamous carcinoma, is usually a condition of young adults but may affect young children and is
associated with HPV-16 (e62).
BXO is a chronic dermatitis of unknown etiology most often involving the glans and prepuce but sometimes
extending into the urethra. BXO is relatively common in children and occurs in approximately 9% of all
circumcised foreskins and in 19% to 40% of circumcisions performed for phimosis (e36,e169,e274). It may be
seen in boys as young as 2 years of age and appears clinically as a thick, white plaque on the prepuce, with
occasional involvement of the glans and meatus. The gross pathologic findings are subtle ranging from loss of
skin wrinkling to change in skin color and texture (thick and white or thin and pink) compared with the adjacent
skin. The microscopic features are identical to those of lichen sclerosus et atrophicus, which are characterized
by a thick subepidermal zone of acellular eosinophilic hyaline material underlying the keratotic and atrophic
epidermis. Slight basal liquefaction is characteristic, with occasional formation of bullae or ulcers. A dense
bandlike or patchy lymphoid infiltrate is present toward the deep border of the hyalinized zone, and clusters of
plasma cells are sometimes seen.
Fournier disease is a form of necrotizing fascitis affecting the penis and has been reported in children only rarely
(e4). Staphylococcal and streptococcal infections are responsible for the condition.
Overall, neoplasms of the penis are exceptionally rare in the first two decades of life. Squamous cell carcinoma is
rare in children in the United States but has been reported in several adolescents who were not circumcised
during childhood (e39,e188,e364,e384). Rare examples of endodermal sinus tumor of the penis have been
reported, with histopathologic features identical to their testicular counterpart (e13,e265). Benign and malignant
mesenchymal tumors such as cavernous hemangioma, neurofibroma, dermatofibroma, glomus tumor, malignant
lymphoma, malignant peripheral nerve sheath tumor, embryonal RMS, and clear-cell sarcoma have been also
rarely reported in the penis (e21,e119,e128,e489,e493,e500). The histopathologic features of these tumors are
identical to their soft tissue counterparts. Malignant peripheral nerve sheath tumor in this site has been usually
reported in the clinical setting of von Recklinghausen neurofibromatosis.
THE PROSTATE
Congenital abnormalities of the prostate are rare. Hypoplasia and dilation of the prostate are consistent findings
in the prune-belly syndrome (e329,e444,e592). The epithelium of the prostatic glands and ducts, prostatic utricle,
and prostatic urethra can undergo squamous metaplasia, in response to maternal estrogenic stimulation, during
prenatal life. This histologic feature gradually disappears in the early postnatal months (e20). Focal hyperplasia
of glandular epithelium, cystic dilatation of tubules, and intraluminal secretions are other histologic changes that
are observed in the fetal and neonatal prostate. Congenital abnormalities of the prostate are rare. Hypoplasia
and dilation of the prostate are consistent findings in the prune-belly syndrome (57,e444,e592). Cysts of the
prostatic utricle (mullerian duct cyst) are an unusual cause of lower urinary tract obstruction and inflammation in
boys (e137,e309).
Fibroepithelial polyps of the urethra have been reported typically in males younger than 10 years but can also
occur in older men. These are benign growths that can cause a variety of symptoms in young boys including
obstructive uropathy, infection, and/or hematuria. They typically occur in the posterior urethra near the
verumontanum and consist of a fibrovascular core with loose stroma covered by urothelial lining
(e18,e125,e131,e162). Surface ulceration, reactive atypia, and squamous metaplasia may develop in these
polyps, which are considered developmental anomalies and are treated by simple transurethral resection.
Overall, lesions and tumors of the prostate are rare in infants and children. A few reports of periprostatic
abscesses or hematomas appeared in the literature in which a midline pelvic mass was present accompanied by
scrotal abscess and fever and caused lower urinary tract obstruction in infants (e217,e609). Staphylococcus
aureus and E. coli were implicated as causative organisms in these cases.
RMS is by far the most common neoplasm of the prostate in children and adolescents (e316). Approximately 5%
of all pediatric RMS primarily involve the prostate (e115). RMS can occur anytime from infancy to early adulthood
P.894
but has a peak incidence during the first 4 years of life (10) and a mean age of presentation of 5.3 years (e316).
Overall, genitourinary involvement by RMS was found to more commonly affect infants younger than 1 year
compared to older children (e454). Like soft tissue RMS, another peak of incidence may be observed during
adolescence at 15 to 19 years. The presenting symptoms include bladder outlet obstruction, hematuria,
incontinence, infection, and a pelvic or an abdominal mass (e143,348,e349). Large tumors can be difficult to
assign a prostatic or a bladder origin, especially since both structures are frequently involved. An association
between RMS in genitourinary sites and neurofibromatosis (NF-1) has been reported in one study (e154).
FIGURE 19-17 ▪ Embryonal RMS. A: The tumor involves the prostatic and the bladder region. B: The tumor has
variable cellularity with small hyperchromatic cells and scant cytoplasm in a loose stroma. C: An occasional giant
cell with abundant eosinophilic cytoplasm is depicted. By ultrastructural examination, the cytoplasm contains thin
and thick filaments with densities representing the z-bands (Courtesy of Dr. Jerome Taxy, Chicago, Illinois).
In the pretreatment clinical staging for pediatric RMS, prostatic or bladder involvement, unlike the favorable
overall genitourinary location, is regarded as unfavorable site of involvement and assigned a higher clinical stage
(58, 61). Regional lymph nodal metastasis (usually iliac and paraaortic) can occur in up to 20% of prostate and
bladder RMS, necessitating adequate nodal sampling for proper staging of the tumor (e301).
Microscopically, the majority of prostatic RMS is of the embryonal type and is considered of favorable histology
(Figure 19-17). It remains important, however, to identify the rare cases of alveolar RMS in this location due to its
unfavorable histologic subtype and the additional need for more aggressive chemotherapy. For further review,
please refer to the soft tissue section for detailed histopathologic, immunohistochemical and molecular and
genetic evaluation of RMS. Multimodality treatment combining surgery, chemotherapy, and radiation therapy is
currently applied to pediatric RMS and has g reatly improved the prognosis (61,e301,e316) (see Chapter 24).
Rare examples of other tumors occurring in the prostate or prostatic region have been the subject of case reports
only. These include a malignant rhabdoid tumor (21), an undifferentiated carcinoma with disseminated
metastasis (e509), non-Hodgkin lymphomas (e54,e310), a pheochromocytoma (92), a teratoma with
angiosarcoma component (e302), and a case of fibromatosis (e495). Conventional prostatic adenocarcinoma
was not detected before the fourth decade in a study of 152 young male patients (e490).
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Chapter 20
The Breast
Jeffrey Mueller
Rebecca Wilcox
Jerome B. Taxy
The breast is often regarded as a modified sweat gland. Dichotomous branching of ductal structures ending as
lobules and acini characterizes its embryology. Mammary gland development begins during the fourth week of
embryonic life as mammary crests, thickened symmetric ridges of ectoderm on the ventral wall extending from the
axillary to inguinal regions all of which involute except in the region of eventual breast development. Mammary
buds, the solid downgrowths of cuboidal ectoderm in the mammary crest, penetrate into the underlying
mesenchyme during the sixth week. The subsequent mammary buds develop into the lactiferous sinuses, which
are then canalized when induced by placental sex hormones. By term, approximately 15 to 20 lactiferous ducts
are formed. Progressive branching of this system eventually forms the ductal-lobular architecture.
Late in gestation, the nipples arise from the primitive epidermis in the form of shallow pits. These are depressed
in the newborn but soon elevate due to the proliferation of the surrounding fibrovascular connective tissue of the
areola. The mammary glands of the newborn, irrespective of gender, are rudimentary but responsive to maternal
gestational hormones. They are clinically palpable and histologically characterized by secretory ducts and
edematous stroma. Secretions known as “witch's milk” may be produced secondary to maternal hormones in the
fetal circulation or production of prolactin in the infant pituitary.
From birth until puberty, the mammary glands remain undeveloped. In pubertal females, elevated levels of
estrogens, progestogens, and growth hormone result in breast enlargement (thelarche) due to the accumulation
of fat and the development of the ductal lobular units. Thelarche is classified by imaging modalities, principally
ultrasound, into Tanner stages 1 to 5, with 1 being the least and 5 being the most developed. In males, breast
tissue remains hormonally unstimulated, histologically characterized by ducts without lobular development
throughout life (91, 92, 100).
Palpable breast lesions are infrequent. In addition, the pectoral soft tissues are not typically sampled during the
course of a pediatric autopsy. Histology and cytology samples are
therefore rare. This may result in a lack of familiarity with the morphology of the breast in children. Among those
lesions surgically excised, there is a predominance of benign lesions, with fibroadenoma and gynecomastia
together constituting 50% to 70% of all cases (14, 15, 35, 36, 47, 58, 65, 115, 134, 135, 138) (Table 20-1). Most
lesions of the breast in the first two decades of life are clustered in the adolescent age group (25). Neinstein and
associates (104) reported the presence of newly detected masses in 3.25% of female adolescents. Most masses
were unilateral, well circumscribed, and solitary at clinical presentation. Pettinato and colleagues (115) reviewed
the experiences of three institutions, focusing on breast lesions in children and adolescents other than
gynecomastia and fibroadenomas and many were, by their infrequent nature, diagnostic and therapeutic
dilemmas. West and associates (160) reported unilateral thelarche in 26 (35%) of 74 children and adolescents
with a symptomatic breast mass, so that the clinical approach should be a cautious one.
ANOMALIES
Congenital Absence of the Breast
The presence of a nipple may be accompanied by absent or hypoplastic breast tissue due to the failure of the
pectoral portion of the mammary ridge to develop (150). This rare lesion has been subdivided into several clinical
categories based on distribution, associated defects, and inheritance as an autosomal dominant, sex-linked
recessive, or incompletely defined familial trait (18, 105, 144, 150). Alopecia, saddle nose deformity,
underdeveloped or missing teeth, absence of pectoral muscles, and anhidrotic ectodermal dysplasia are some of
the accompanying defects (18). Some of these children have the clinical manifestations of Poland syndrome
(aplasia of the pectoral muscle). Agenesis of breast lobules has been reported in cystic fibrosis (53).
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Table 20-1 ▪ BREAST LESIONS IN CHILDREN AND ADOLESCENTS: REVIEW OF EIGHT

SERIES AND TWO CHILDREN'S HOSPITALS
Eight Series from Primary St. Louis Total

Literature (30, 79, 93, Children's Children's %
108, 121, 194, 239, Hospital Hospital 1989-
247) 1970-1989 1998
Fibroadenoma 524 13 166 703(55)
Gynecomastia 38 34 81 153(12)
Cysts/fibrocystic changes 77 4 27 108 (8)
Macromastia 26 9 52 87 (7)
Other 19 3a 26b 48(4)
Inflammationc 39 2 1 42 (3)
Papilloma/papillomatosisd 24 — 3 27 (2)
Hyperplasia, NOS 18 — 4 22(2)
Hemangioma 12 — 1 13(1)
Fat necrosis 10 — 1 11 (1)
Cystosarcoma phyllodes 7 — 4 11 (1)
Rhabdomyosarcoma 7 1 1 9(<1)
Accessory breast tissue 8 1 — 9(<1)
Lipoma 5 — 2 7 (< 1)
Supernumerary nipple 2 4 — 6(<1)
Fibrosis 2 4 — 6(<1)
Granular cell tumor 1 — 3 4(<1)
Carcinoma 3 — 1 4(<1)
Fibromatosis 1 — 1 2(<1)
Total 825 75 374 1,274 (-

100)
aIncludes tubular adenoma (1), plexiform neurofibroma (1), lymphangioma (1)
bIncludes tubular adenoma (10), lactating adenoma (5), keloid (2), neurofibroma (2), angiosarcoma (2),
nipple duct adenoma (1), lymphangioma (1), hamartoma (1), stromal sarcoma (1), giant cell
fibroblastoma (1)
cIncludes mastitis and abscess.
dIncludes juvenile papillomatosis and intraductal papilloma. NOS, not otherwise specified
Supernumerary Nipple and Accessory Breast Tissue

Supernumerary Nipple
Supernumerary nipple (polythelia, accessory nipple) occurs in 2.5% of systematically examined neonates with a
slight male predominance (98, 155). Fewer than 10% are bilateral, and fewer than 5% arise at sites other than
along the embryonic mammary line, such as the back, shoulder, posterior thigh, face, and neck. Familial
supernumerary nipples have been reported (20, 151). The clinical features are those of a small pigmented
macule with a tiny umbilication (95) that histologically exhibits components of a normal nipple with occasional
hyperplastic duct epithelium, capillary proliferation, pilosebaceous units with intraluminal keratinous material, and
bundles of smooth muscle. Lactiferous ducts and terminal duct-lobular units may be inconspicuous (62, 73, 75,
94, 153).
Accessory Breast Tissue

Accessory breast tissue is unusual (1% to 2% of the white population), occasionally familial (159), presenting as
a mass in the axilla or vulva. In the vulva, this may be inseparable from hidradenoma papilliferum (27). Both
supernumerary nipples and accessory breast tissue have been noted more commonly in the Native American
population (56). The mass is ill defined and composed of histologically unremarkable predominately breast ducts
and subareolar structures. A variety of histologic and pathologic findings have been described in accessory
breast tissue, including fibroadenomas and ductal carcinoma (31, 64, 78, 115).
Breast Asymmetry
Breast asymmetry is common between Tanner stages 2 and 4, and it may persist to a mild degree in as many as
25% of young adults (60, 145). Unilateral breast enlargement with its attendant asymmetry is seen in neonates
as a response to maternal and placental hormones. This condition often spontaneously corrects; if no underlying
endocrine abnormalities are present, the asymmetry is correctable by augmentation mammoplasty
BREAST LESIONS
Fibroproliferative (Fibrocystic) Disease
Fibrocystic Changes
The termfibrocystic change has been historically used to refer to the spectrum of cysts, fibrosis, and epithelial
proliferation reflective, in part, of hormonal-induced changes related to
P.899
the menstrual cycle (59). Although more common in adults, it is occasionally seen in middle-to-late adolescence.
Similar to adults, these are characterized by diffuse cysts and masses with size fluctuations correlating with the
menstrual cycle and often manifesting perimenstrual tenderness. Biopsy may not be warranted, as the clinical
presentation and physical exam without imaging is probably diagnostic. In children, solitary cysts are more
common than multiple cysts (23). The histopathologic changes include dense hypocellular to moderately cellular
stromal fibrosis, cystic dilation, apocrine metaplasia, adenosis, and usual ductal hyperplasia. Atypical ductal
hyperplasia is rare in young women and is associated with an increase in risk of subsequent ductal carcinoma
(42, 45, 63). Treatments include improved breast support, steroids, oral contraceptives, vitamin E, and
avoidance of caffeine (33).
Dense fibrosis, occasionally termedfibrous mastopathy, is a localized and ill-defined fibrous proliferation of the
breast stroma and may represent the fibrous end of the fibrocystic spectrum. It compromises and eventually
obliterates the lobular breast parenchyma. Although a hormonal etiology is suspected, grossly the lesions are
described as “stony-hard” and lack cysts, distinguishing them from typical fibrocystic changes as noted above.
Microscopically, the lesions are divided into three groups based on the relationship of acinar tissue to stroma.
Type I shows prominent acinar tissue with scant concentric collagen bundles encircling the epithelial units. Type
II shows partial replacement of acini with dense bundles of collagen in an uneven manner. Type III reveals almost
complete replacement of the acinar tissue. Type I is most commonly seen in the younger age group. Excisional
biopsy is the treatment of choice (99, 121, 122).
Papillary Duct Hyperplasia

Among epithelial proliferations, papillary duct hyperplasia is uncommon, occurring almost exclusively in females,
with a median age of 17 years, although these are occasionally reported in neonates and young children. The
most common presenting symptom is a mass, less commonly accompanied by clear or bloody nipple discharge.
Intraductal papilloma may be clinically considered. The size of the lesion varies but up to 5 cm has been
reported. The cut surface grossly shows numerous cysts with papillary excrescences. Histologic examination
shows three different patterns including true papilloma, sclerosing papilloma, and papillomatosis. All show fronds
of epithelial cells in single or multiple layers with a fibrovascular stroma. Myoepithelial hyperplasia may be
present. The sclerosing papilloma pattern may show a radial scar lesion with small clusters of epithelial cells
trapped within the sclerotic stroma, probably representing lesional regression but histologically mimicking
carcinoma (124, 161).
Diabetic Mastopathy
Diabetic mastopathy is a complication of uncertain etiology associated with longstanding type 1 diabetes. The
patients typically present in late adolescence with a firm mass in one or both breasts. The histologic findings are
typified by lymphocytic lobulitis and ductitis, perivasculitis, and dense keloidal fibrosis (43). Recurrences are
common (77, 147).
Juvenile Papillomatosis
Juvenile papillomatosis is a benign proliferative lesion that despite the name also occurs in adults. The patients
are typically postpubertal, with a firm, solitary mass at the periphery of the breast. Bilaterality or multifocality in
the same breast is rare. When performed, mammography shows an area of increased density with poorly defined
borders. A risk for breast carcinoma has been reported in maternal female family members of young patients with
juvenile papillomatosis. Grossly, the tumor is a firm, discrete mass, which ranges from 1 to 8 cm, with slightly
irregular borders (Figure 20-1). The cut surface shows numerous cysts, 1 mm to 2 cm giving rise to the term
Swiss-Cheese breast. The intercystic tissue shows yellow-white flecks, similar in appearance to that of comedo-
type necrosis. Although histologic examination shows a variety of benign proliferative changes typically
associated with conventional fibrocystic disease, e.g., apocrine metaplasia, usual ductal hyperplasia, papillomas
with involutional features, and cysts, there is nothing to suggest a common etiology or clinical association. Within
the dilated ducts and cysts are numerous lipid-laden macrophages, consistent with stasis (Figures 20-2, 20-3
and 20-4). Microcalcifications are sometimes present. The ductal proliferation is typically usual or florid
hyperplasia, occasionally associated with sclerosis producing a radial scar pattern. Atypical ductal hyperplasia
has been reported within juvenile papillomatosis in up to 40% of cases. In situ carcinoma is rare in young adults.
Since fibroadenoma is the common preoperative diagnosis, the excisional biopsy margins may be inadequate
and recurrences may ensue (55, 67, 129, 139, 140).
Infection and Fat Necrosis

Infections
Infections of the breast occur at any age during childhood, including the neonatal period. Adolescents may
develop
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mastitis or breast abscess due to foreign bodies, trauma, nipple piercing, and infection of an epidermal cyst.
Localized tenderness, induration, and erythema may be followed by a fluctuant mass indicating abscess
formation. Staphylococcus aureus is the most common organism involved, but the spectrum of organisms is
wide, including Mycobacteria (34). Fine-needle aspiration may be diagnostic, precluding surgical biopsy. The
histologic picture is variable, ranging from acute and chronic inflammation to abscess formation, granulomatous
inflammation, and squamous metaplasia. Larger abscesses may need formal incision and drainage.
FIGURE 20-1 ▪ Juvenile papillomatosis. Gross appearance (the so-called Swiss cheese disease) with multiple
small cysts in a densely fibrotic background.
FIGURE 20-2 ▪ Juvenile papillomatosis. Low-power section showing fibrosis, multiple cysts, and prominent
papillary duct hyperplasia. Note that the lesion extends to the inked margin, a frequent feature in this lesion often
clinically mistaken for fibroadenoma.
Fat Necrosis
Fat necrosis, possibly related to previous surgery or trauma, is a localized mass with or without tenderness.
Grossly the nodules have a gritty yellow surface. The histology shows chronic inflammation with lipophages,
fibrosis, and dystrophic calcifications (34, 45).
FIGURE 20-3 ▪ BJuvenile papillomatosis. Histologic features include apocrine metaplasia and papillary duct
hyperplasia.
FIGURE 20-4 ▪ BJuvenile papillomatosis. Dilated cysts containing foamy macrophages.
Gynecomastia and Juvenile Hypertrophy

Gynecomastia
Gynecomastia, idiopathic excessive development of the breast in males, is the most common breast abnormality
in adolescent boys and a frequent source of surgical specimens (Table 20-1). The etiology may be an imbalance
between estrogen and androgen, along with end-organ response factors, although many conditions are
associated with gynecomastia (Table 20-2), including hyperthyroidism, hypogonadism, and drugs such as
exogenous hormones that may be used by athletes for performance enhancement. When an underlying clinical
endocrine abnormality is present, the gynecomastia is typically bilateral with a female pattern of enlargement and
areolar hyperpigmentation. Pseudogynecomastia is breast enlargement that is caused by increases in other
tissues such as muscle enlargement, obesity, or diffuse neurofibromatosis in the pectoral region. In addition,
pseudoangiomatous stromal hyperplasia
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(PASH) has also been described as a mimic of gynecomastia. The central subareolar region is typically affected
but it may be eccentric, unilateral or bilateral, ranging from 2 to 10 cm. Nipple retraction is rare (Figure 20-5). The
swelling is often painful and may interfere with participation in sports activities, but resolves over time. Grossly
the tissue is rubbery, white, and ill-defined (Figure 20-6), mixed with mature yellow adipose tissue. Histologically,
gynecomastia and juvenile hypertrophy are similar. The changes center on ductal epithelial hyperplasia and a
cellular, myxoid stroma. In early phases, there is a ductal epithelial proliferation with papillary and/or cribriform
patterns and accompanying myoepithelial hyperplasia. The periductal stroma is cellular and edematous (Figures
20-7 and 20-8). With time, there may be less epithelial proliferation and more collagenous stroma (59, 81, 108,
126). Some lobular formation may be seen in Klinefelter syndrome.
Table 20-2 ▪ SPECIFIC CONDITIONS ASSOCIATED WITH GYNECOMASTIA IN CHILDREN AND
ADOLESCENTS
Adrenocortical adenoma and carcinoma
Choriocarcinoma and other gonadotropin-producing germ cell neoplasms
Congenital adrenal hyperplasia
Familial gynecomastia with aromatase excess
Fibrolamellar hepatocellular carcinoma with aromatase excess
Growth hormone therapy
Hepatoblastoma
Hypergonadotropic hypogonadism
Large-cell calcifying Sertoli cell tumor with and without Peutz-Jegher syndrome
Prolactinoma
Pseudohermaphroditism (5-á-reductase deficiency)
Spinal cord disorders
von Recklinghausen neurofibromatosis
X-linked mental retardation
Drugs
FIGURE 20-5 ▪ Gynecomastia in a prepubertal boy.
Juvenile Hypertrophy and Macromastia

Juvenile hypertrophy is restricted to the female breast as a spontaneous, rapid, massive growth, unilateral or
bilateral (Figure 20-9), probably secondary to increased sensitivity to gonadal hormones. Macromastia,
deforming, painful overgrowth of the female breast, may be synonymous with juvenile hypertrophy. The usual
age of onset is menarche. An association with Hashimoto thyroiditis, rheumatoid arthritis, and myasthenia gravis
has been noted and therefore an autoimmune etiology has been alleged. The treatment primarily involves
antiestrogen therapy and/or surgical reduction. Grossly, the tissue shows a homogeneous tan-yellow cut surface
without a discrete mass. Histologic sections reveal an irregular proliferation of ducts with hyperplasia in a
hypocellular edematous stroma. Pseudoangiomatous stromal hyperplasia may be present (29, 34, 45, 87, 137).
FIGURE 20-6 ▪ Gynecomastia. Cut surface of gross specimen shows illdefined dense white tissue and fat.
FIGURE 20-7 ▪ Gynecomastia. Ducts cuffed by an edematous stromal “halo” and surrounding hyalinized stroma.
No lobules are present.
Epithelial-Stromal Lesions
Fibroadenoma, juvenile fibroadenoma, and cystosarcoma phyllodes (phyllodes tumor) are biphasic,
fibroepithelial tumors that may be regarded as a clinicopathologic spectrum. While fibroadenoma in children and
adolescents may be morphologically similar to its counterpart in adults, juvenile fibroadenoma is a term not
employed in the adult population. Juvenile fibroadenoma has effectively subsumed the benign phyllodes tumor in
this age group, since the morphologic characteristics and clinical behavior are similar (23). The term
cystosarcoma phyllodes, or phyllodes tumor, in children is used in reference to at least a low-grade malignancy.
FIGURE 20-8 ▪ Neonatal breast tissue. Ducts and edematous stroma. Note the histologic similarity to
gynecomastia.
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FIGURE 20-9 ▪ Juvenile hypertrophy. Asymmetric enlargement of right breast. Histologically, the features are
similar to gynecomastia.
Fibroadenoma and Tubular Adenoma

Fibroadenoma is the most common breast mass in adolescent females and as such accounts for most of the
excisional breast biopsies in this age group (Table 20-1). The tumors come to attention in late adolescence (23)
with a slowly enlarging, painless mass (59). Fibroadenomas are more common in African-American patients (33)
and range from 2 to 5 cm. Physical exam reveals a firm, freely mobile, well-circumscribed mass. As these tumors
are estrogen sensitive, growth may accelerate during pregnancy. Grossly, tumors are well circumscribed and
have a rubbery, bulging and lobulated cut surface with a myxoid or a gelatinous appearance. Occasionally, cystic
areas may be apparent. Histologically, fibroadenomas exhibit a proliferation of epithelial, myoepithelial, and
stromal elements. Two acknowledged histologic patterns of fibroadenoma are recognized based on variations of
the stromal and epithelial components but are not mutually exclusive. In the intracanalicular pattern, the stroma
surrounds and compresses epitheliallined ducts into anastomosing strands with slit-like lumens (Figure 20-10).
Open acinar and ductal structures simulating normal breast tissue characterize the pericanalicular pattern
(Figure 20-11). The different architectural patterns are frequently admixed and have no known prognostic or
clinical significance. The epithelial component of fibroadenoma typically exhibits ductal hyperplasia of the usual
or florid types. The stroma may be more cellular than is seen in adults; however, mitotic figures are rare. Stromal
differentiation may rarely take the form of muscle, adipose tissue as well as osteochondroid metaplasia (148).
FIGURE 20-10 ▪ Fibroadenoma. This tumor shows an intracanalicular pattern with ductal structures compressed
by a bland stroma. The stromal cells have a spindle-to-stellate appearance.
FIGURE 20-11 ▪ Fibroadenoma. Pericanalicular pattern. Individual tubules have a clear zone of myoepithelial
cells adjacent to the basement membrane. Stromal cells are uniformly distributed and exhibit a myxoid
background adjacent to the ducts.
Tubular adenoma is a variant of fibroadenoma clinically and macroscopically. In one series, there was one
tubular adenoma for 16 fibroadenomas (36). The solitary, well-circumscribed tumor has a firm, tan, and
homogeneous surface (Figure 20-12). As in fibroadenoma, the interface with the adjacent parenchyma is
discrete (Figure 20-13). This may be regarded as a fibroadenoma with a minimal stromal component. Small
tubules are lined by an inner
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layer of columnar epithelial cells and an outer layer of myoepithelial cells which are indistinguishable from small
ductules (66, 85). There may be occasional large ducts, mild fibrosis, sparse mononuclear inflammation, and
even lactational changes. Emphasizing the connection to fibroadenoma is the occasional histologic admixture of
tubular adenoma and fibroadenoma. Multiple bilateral tubular adenomas and fibroadenomas have been reported
in adolescent identical twins (101). Lactating adenoma may represent a third variant of fibroadenoma. These
adenomas, consisting entirely of lactational change, are unusual and are related to pregnancy. An excessively
myxoid fibroadenoma should raise the possibility of Carney Syndrome.
FIGURE 20-12 ▪ Tubular adenoma. Circumscribed and lobulated light tan lesion. Grossly, fibroadenoma is
similar.
FIGURE 20-13 ▪ Tubular adenoma. Uniform proliferation of closely packed small tubules and elongated ducts.
This pattern is similar to the pericanalicular pattern of fibroadenoma. Patterns mixed with fibroadenoma are
common.
Juvenile Fibroadenoma
Juvenile or cellular fibroadenoma is a rapidly enlarging tumor, sometime referred to as giant fibroadenoma due
to the large size (>5 cm) it achieves. This variant is more common in African-Americans (59). Grossly, the larger
tumors have a multilobulated, bosselated cut surface (Figure 20-14). Histologic features differ from the typical
fibroadenoma by exhibiting a hypercellular stroma and more epithelial hyperplasia (Figure 20-15A,B).
Nevertheless, the degree of stromal cellularity separating one from the other has never been defined or
quantitated. In addition to pericanalicular and intracanalicular patterns similar to those encountered in routine
fibroadenoma, there is a “leaf-like” pattern, i.e., clefts lined by hyperplastic epithelium similar to that seen in
benign phyllodes tumor, suggesting that juvenile or cellular fibroadenomas may in fact represent a benign
phyllodes tumor (23). Distinguishing between juvenile fibroadenoma and benign phyllodes tumor is difficult if not
arbitrary relying in part on a more cellular and heterogeneously distributed stroma adjacent to the ducts in a
benign phyllodes tumor and a more uniform stromal distribution in juvenile fibroadenoma. The clinicopathologic
difference between the two is not certain. The stromal cells in juvenile fibroadenoma lack atypia and mitoses are
sparse (<1 to 3 per 10 high-power fields). There
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may also be proliferation of the myoepithelial cells. These lesions are benign with recurrent potential.
FIGURE 20-14 ▪ Juvenile fibroadenoma. Adolescent girl with a solitary 6cm mass. The cut surface demonstrates
depressed clefted spaces defining a lobulated pattern. The lesion has been incompletely removed. (Courtesy of
Wendy Recant, M.D.)
FIGURE 20-15 ▪ Juvenile fibroadenoma in a 16-year-old girl. A: This field shows a portion of a leaf-like duct with
mild hyperplasia (right side). B: The surrounding stroma has a cellular appearance with an occasional mitotic
figure, but no cytologic atypia. (Courtesy of Wendy Recant, M.D.)
Phyllodes Tumor (Cystosarcoma Phyllodes)

In adults, cystosarcoma phyllodes is regarded as a tumor with three subtypes based on the histopathologic
features of the spindle cell stroma that are presumed to correspond to biologic potential, that is, benign,
intermediate (low malignant potential), and malignant. The fibroepithelial pathology has a morphologic
relationship to fibroadenoma; the distinction is based on stromal cellularity and an irregular stromal distribution. In
children, phyllodes tumor is an even less common fibroepithelial neoplasm for which the benign variant in its
clinical, radiographic, and pathologic features has been absorbed into the entity and concept of juvenile
fibroadenoma. Recent evidence from cytogenetic and molecular studies supports the hypothesis that
fibroadenoma and phyllodes tumor are related neoplasms and that progression of the former to the latter may
result from a biologic course of multiple recurrences (38, 106).
If the benign variant is excluded, phyllodes tumor or cystosarcoma phyllodes in children may be best regarded as
at least a low-grade malignancy that accounts for only 1% of breast lesions in children and adolescents (23)
(Table 20-1). The clinical presentation is that of a discrete and palpable mass, often circumscribed on
mammography. Commonly, there is a history of rapid enlargement and some patients may report rapid growth of
a pre-existing lesion (112). Ulceration, fixation, nipple retraction, discharge, and skin discoloration are not typical
(7).
Grossly the mass has a tan to gray, bulging, and clefted firm cut surface (Figure 20-16). The sizes have been
reported in the range of 1 cm to more than 15 cm and while a tumor less than 4 cm is often cited as a favorable
feature, the size of the lesion does not reliably correlate with clinical behavior (90). The margin of the tumor may
be locally infiltrative histologically even though it grossly appears circumscribed; those with invasive rather than
pushing borders have an increased risk of local recurrence. Inking practices for fibroadenomas vary, so that the
unsuspected phyllodes tumor may not be appropriately managed initially (16, 109, 119). Histologically, the
appreciation of a sarcomatous stroma may be a sampling issue, given the potential for distributional irregularities
(Figure 20-17A,B). The typical low-grade spindle cell stroma seldom overgrows the epithelial component.
Stromal density and mitoses are particularly noticeable adjacent to the branching ducts (110).
FIGURE 20-16▪ Phyllodes tumor. Mastectomy specimen with a large firm white mass with areas of hemorrhage
and clefts on cut surface.
FIGURE 20-17 ▪ Phyllodes tumor. A: Low power of a phyllodes tumor shows cellular stroma crowding the
epithelial component. B: Higher-power view of densely cellular and myxoid stroma in which the stellate-shaped
cells show occasional mitotic figures (arrows).
As a low-grade neoplasm with recurrent potential, most phyllodes tumors have a favorable outcome with 10-year
survival close to 90% (61). No single criterion is reliable for predicting clinical behavior although the mitotic rate
may be a significant factor in the evolution of metastatic disease (23), especially if there are more than 3 mitoses
per 10 highpower fields. Intracytoplasmic inclusion bodies composed of actin may be seen in stromal cells,
similar to those seen in infantile digital fibromatosis (13, 68). In this regard, the spindle cells have the
immunophenotype of myofibroblasts (8).
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If the sarcomatous component has features of an undifferentiated sarcoma or alveolar rhabdomyosarcoma, it
should be anticipated that the tumor would behave accordingly regardless of the epithelial component.
Liposarcoma or chondrosarcoma may be more favorable (Figure 20-18) (72, 115, 116, 118). The overall
recurrence rate of cystosarcoma phyllodes is 7% to 15%; metastases are exceptionally rare (3, 7, 16, 79, 119,
152). One example of metastasis was in a 14-year-old girl who had a very aggressive spindle cell sarcoma that
metastasized to the skin, soft tissue, and lungs (69). Another child, a 12-year-old girl, died with metastatic
embryonal rhabdomyosarcoma (115).
FIGURE 20-18 ▪ Malignant stroma of a phyllodes tumor with lipoblasts indicating liposarcomatous differentiation.
Nipple Duct Adenoma and Hamartoma

Nipple Duct Adenoma
Nipple duct adenoma (florid papillomatosis, erosive adenomatosis) is a lesion of adults only rarely reported in
children (114, 115, 127). Arapidly enlarging unilateral subareolar mass with erosion of the overlying skin and a
nipple discharge are the clinical features. Three histologic patterns are seen: sclerosing papillomatosis,
papillomatosis, and adenosis. Syringocystadenoma papilliferum resembles the nipple duct adenoma except for
the fact that the former lesion exhibits an epithelial transition to the skin surface and a conspicuous plasma cell
infiltration.
Hamartoma
Hamartomas include two entities: hamartoma of the breast and myoid hamartoma. These lesions are uncommon
at any age. In a review of 5,834 breast biopsy specimens, hamartomas accounted for 1.2% of benign breast
lesions (21). Most hamartomas present in adults, but they are also seen in middle-to-late adolescence as a
sharply demarcated mass thought clinically to represent a fibroadenoma. Dense fibrous stroma or adipose tissue
is the dominant component, the lobular-ductular units in the fibrous stroma do not have any appreciable
abnormalities, and the histologic interpretation may be fibrous mastopathy or normal breast tissue (30, 32).
Hamartomas have been characterized as a breast within the breast (83). Fibroadenomas with heterologous
cartilage or bone have been interpreted appropriately in the past as hamartomas (74).
Myoid (muscular) hamartoma is even less common, also reported more often in adults, and is paradoxical for a
true maldevelopment (22, 84). These lesions are smaller than 1 cm and are composed of plump stromal cells
with a myogenic phenotype in association with small ducts.
Carcinoma
Carcinoma of the breast is exceedingly rare in children (111, 112). Invasive carcinoma was found in 32 patients
with 6 DCIS and 3 LCIS in a recent study using SEER data between 1973 and 2004 (61). No cases of carcinoma
were found in 113 breast tumors in children in one study, and there was only one carcinoma of the breast in 234
cases of carcinomas in children in another series (93, 115). Most pediatric carcinomas occur after age 15; the
adult cases begin rising after age 25 (5, 28, 49, 61, 130). Secretory carcinoma, one of the rarest types of breast
carcinoma, was initially termed juvenile breast carcinoma as initial studies found the average onset to be in
childhood. Recently, there has been an association with a balanced translocation, t(12;15), as seen in several
pediatric mesenchymal tumors (76). Grossly, the tumor is 1 to 2.5 cm in diameter, separate from the nipple,
circumscribed, gray white, and firm. Nodules or lobules of tumor are separated by prominent bands of connective
tissue (Figure 20-19). The presence of extracellular secretions imparts a micromulticystic appearance to the
individual tumor nodules. Intracytoplasmic vacuoles are noted in most cells that otherwise have only mild-to-
moderate cytologic atypia. Secretory carcinoma (juvenile carcinoma) is often cited as the principal type of breast
carcinoma in the first two decades. However, in the SEER study, secretory carcinoma
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accounted for less than 10% of carcinomas (61). This variant also occurs in boys and adults (89, 128, 132, 143).
Secretory carcinoma in children and adolescents has been reported in association with juvenile papillomatosis
(50, 107, 149). In the few cases available for follow-up, only one of the original seven cases recurred, and only a
few have metastasized to regional lymph nodes (89). This indolent clinical behavior has been confirmed in other
reports (47, 111, 142, 143). Other types of infiltrating ductal carcinomas in young women generally are
associated with a poor outcome with overall 10-year survival of 54% (24, 61, 162).
FIGURE 20-19 ▪ Secretory carcinoma. Thick fibrous bands separate the lobules of tumor cells showing abundant
eosinophilic cytoplasm and secretions with intracytoplasmic vacuoles. (Courtesy Thomas Kravszim, D.)
A spectrum of histologic variants of breast carcinoma have been reported (1, 2, 57, 89, 120, 123, 142, 157).
Carcinoma of the breast is a recognized type of second malignant neoplasm, certainly not the most common, that
occurs later in life but earlier than expected in long-term survivors of other childhood malignancies (12). In the
past decade, remarkable advances have been made in understanding the genetic basis of breast cancer, and
breast cancer family syndromes have been identified with inherited mutations of the p53 gene or BRCA genes
(26, 41, 86, 133, 146). Some breast carcinomas in young patients are related to these conditions, and other early
childhood neoplasms may be manifestations of an inherited proclivity to breast and other cancers (19, 52, 80,
113).
MESENCHYMAL LESIONS
Lipoma
Lipomas are uncommon and may be associated with lipoblastomatous foci in young children (115).
Fibromatosis
Desmoid-type fibromatosis of the breast, as in other soft tissues, is a firm, deceptively discrete mass that may
produce skin fixation and dimpling. About 20% of all cases are encountered in the second decade; the condition
is rare in infancy (115). The gross appearance is a gray-white, fibrous mass measuring 1 to 10 cm with ill-defined
edges. Irregular bundles of mitotically inactive spindle cells are arranged in broad sheets or bands with open,
thin-walled vessels and variable collagenization surrounding normal breast parenchyma. Myxoid foci,
calcification, and lymphoid aggregates may be seen. Local invasion of soft tissue beyond the grossly apparent
margin of the mass may account for the frequent recurrence rate of up to 20% and emphasizes the importance of
careful evaluation of the tissue margins (158). The differential diagnosis in children includes nodular fasciitis (6)
and pseudoangiomatous stromal hyperplasia (PASH), also a presumed myofibroblastic proliferation (71, 117).
Immunohistochemical staining for vimentin and focally for smooth muscle actin is consistent with a
myofibroblastic lesion; the delicate spindle cells are also reactive for CD34 (163). Fibromatosis has been
observed in gynecomastia, most frequently in children (97). β-catenin is a useful marker for desmoid tumors.
FIGURE 20-20 ▪ Perilobular hemangioma. Collection of thin vascular channels adjacent to duct.
Vascular Tumors
Vascular tumors are soft tissue tumors that may originate in or near the breast or skin (23). Infantile or capillary
hemangiomas are typically multilobular infiltrating masses exhibiting lobular architecture defined by fibrous
septation and composed of small capillaries with little-to-absent thrombosis and canalization. Over time, the
vascular spaces eventually regress or involute as evident by an increase in fibrotic stroma (23) (Figure 20-20).
Angiomatosis, rare in children, is a diffuse, benign vascular lesion that may be associated with platelet trapping
syndromes. Histologically, vascular channels are distributed throughout the breast parenchyma (102, 125). The
vascular spaces are lined by flat endothelium without atypia. Both hemangiomatous and lymphangiomatous
channels may be seen. Although angiosarcoma may be considered morphologically, primary mammary or soft
tissue angiosarcoma in children is even more rare than angiomatosis. Angiomatosis is not a precursor lesion and
if possible should be excised with negative margins. Angiosarcoma is exceedingly rare in the pediatric age group
mostly appearing as case reports. A recent review includes two cases in the breast (37). Both epithelioid and
spindle cell morphology in a vasoformative pattern are apparent (see Chapter 24).
Granular Cell Tumor

Granular cell tumor of the breast is a benign lesion that presents a diagnostic challenge because of its clinical,
mammographic, and macroscopic resemblance to infiltrating duct carcinoma (51, 88, 103, 156). These
uncommon tumors represent less than 1% of breast lesions in young persons. Both boys and girls are affected.
The lesion arises in the deep dermis and subcutaneous tissue, infiltrating and surrounding adnexal and breast
parenchymal structures. Grossly, it is a yellow, gritty, infiltrative lesion measuring 1 to 2 cm (Figure 20-21).
Microscopically, large polygonal cells with abundant pale granular cytoplasm and central vesicular
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nuclei with prominent nucleoli are arranged in sheets or cords (Figure 20-22). The granularity is PAS positive,
diastase resistant, and S100 positive. Electron microscopy demonstrates the cytoplasm filled with lysosome-like
structures (Figure 20-23).
FIGURE 20-21 ▪ Granular cell tumor. Pale yellow cut surface with infiltrative borders. The excision is incomplete.
Sarcoma
Sarcomas in or near the breast are rare and mostly related to phyllodes tumors (61). Other sarcomas are primary
lesions of the chest wall and are discussed in the soft tissue chapter. The tumors mostly behave in a low-grade
fashion, amendable to surgical excision with a 10-year survival close to 90% (61). Sarcomas, especially alveolar
rhabdomyosarcoma, are known to metastasize to the breast.
Hematopoietic Lesions
The entire range of Hodgkin and non-Hodgkin lymphomas and leukemias rarely present as primary in the breast
or even as a site of extramedullary relapse, perhaps more frequent in leukemias (39, 46, 82, 96, 136).
Lymphomatous involvement of the breast in children is seen mainly in the setting of small noncleaved cell
lymphoma or Burkitt's lymphoma with bilateral involvement. The latter seems to occur in young pregnant or
lactating women (9, 44, 70, 154). Neither diffuse large cell nor mucosa-associated lymphoid tissue (MALT)
lymphoma is seen in children (17). The breast is an uncommon but well-documented site for granulocytic
sarcoma as an initial manifestation or relapse of acute myeloid leukemia (4, 10, 11, 40, 54). An association has
been noted with the t(8;21) (q22;q22) translocation in cases of primary acute myeloid leukemia with M2
morphology in children (48, 131, 141). Histologically, complete immunohistochemical studies, including
myeloperoxidase, are required to demonstrate the discohesive infiltrate of malignant small round cells and
establish the diagnosis.
FIGURE 20-22 ▪ Granular cell tumor. Small breast ducts surrounded by cords of polygonal cells with poorly
defined cell borders, small round nuclei, and uniform finely granular cytoplasm.
FIGURE 20-23 ▪ Granular cell tumor. Electron microscopy reveals cytoplasm packed with numerous lysosome-
like structures, corresponding to the histologic cytoplasmic granularity.
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Chapter 21
The Pineal, Pituitary, Thyroid, and Adrenal Glands
Richard M. Conran
Ellen Chung
Louis P. Dehner
Hiroyuki Shimada
The opinions and assertions contained herein are the private views of the authors and are not to be construed as official or as representing the views of the Uniformed Services
University or the Department of Defense.
The endocrine system represents a diverse group of organs and cell types involved in the maintenance of a homeostatic environment. It is composed of the pineal gland, the
pituitary gland, the thyroid gland, the parathyroid glands, the adrenal glands, the islets of Langerhan, and a diffuse network of neuroendocrine cells distributed throughout the
respiratory and gastrointestinal tracts (eFigure 21-1). Endocrine function mediated through endocrine, autocrine, and paracrine signaling mechanisms is also seen in other organs
including the hypothalamus, heart, thymus, kidneys, adipose tissue, skin, gonads, and the placenta.
Disorders related to pituitary (growth abnormalities), thyroid, or adrenal dysfunction or sexual maldevelopment and obesity are many of the diagnoses made in large pediatric
endocrine clinics. Many endocrine disorders recognized in childhood often require life-long treatment and are also a substantial component of adult endocrine practice (e834,e405,
e643,e1122,e528,e529). The study of endocrine disorders has been facilitated through molecular techniques with the identification of genetic aberrations in the afferent and efferent
limbs of hormonal actions, with loss of a critical enzyme in the biosynthetic or biodegradative scheme, or with the absence of a hormonal receptor. Recent advances in imaging
techniques combined with molecular diagnostics have also led to recognition of new disorders and new diagnostic and prognostic criteria, and the identification of new familial
inherited syndromes (Table 21-1) (e643,e333,e723,e1174). This chapter incorporates these newer modalities while continuing to focus on the developmental, acquired, and
neoplastic disorders involving the pineal, pituitary, parathyroid, thyroid, and adrenal glands in the pediatric age population. Disorders of the pancreas, ovary, and testis are
discussed in Chapters 16, 18, and 19, respectively.
PINEAL GLAND
Anatomy and Physiology
The pineal gland is a small, cone-shaped, 50- to 150-mg tan-brown structure attached to the superior aspect of the posterior border of the third ventricle. It develops at
approximately 7 weeks' gestation from an evagination of the ependymal lining covering the caudal portion of the roof of the third ventricle (142,e980,e1108). Based on magnetic
resonance (MR) imaging studies, the pineal gland increases in size from birth through 2 years of age, at which time it remains constant in size through adolescence (e1158). No size
difference has been noted between male and female children. In children older than 2 years of age, the average pineal gland measures 6.5 × 4.8 × 4 mm (e1158).
At approximately 5 years of age, calcifications, in the form of corpora arenacea, develop. These calcifications increase with age giving the pineal gland a hyperdense appearance on
computed tomography (CT) imaging at puberty (e1307). Pineal calcifications are observed in 8% of children by age 10, 20% at puberty, and 40% by age 20 (e313,e1307).
Histologically, the pineal gland is composed of nests of cells in lobular profiles, with a resemblance to the “zellballens” of paraganglia, surrounded by connective tissue septa
containing blood vessels and nerve fibers (21, 78, 171). The pinealocytes, or chief cells, have basophilic cytoplasm with large irregular nuclei and prominent nucleoli and are
arranged in cords or follicles within the lobules. Randomly distributed throughout the pineal gland in perivascular areas and between pinealocytes is a second cell population of
astrocytes. In the late third-trimester fetus and neonate, two populations of pineal parenchymal cells are identified with the small cell population disappearing with advancing age
(e980). The pinealocytes are immunoreactive for synaptophysin (SYN), chromogranin (CHR), and neurofilament protein (NFP), and the interstitial astrocytes are immunoreactive for
S-100 and glial fibrillary acidic protein (GFAP) (142,e1051).
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Table 21-1 ▪ ENDOCRINE ORGANS INVOLVED IN SELECTED FAMILIAL TUMOR SYNDROMES
Other
Syndrome Inheritance Gene Pituitary Parathyroid Thyroid Adrenal Manifestations
Beckwith-Wiedemann CDKN1C/NSD1 ACN
Carney complex AD PRKAR1A Adenoma PTC ACN
Cowden PTEN FTC/PTC
Familial adenomatosis polyposis APC PTC

coli
Familial medullary thyroid RET MTC

carcinoma
Familial SDHD, SDHC, PHEO Paraganglioma

paragangliomapheochromocytoma SDHB
Hyperparathyroidism-jaw tumor AD HRPT2 Adenoma/carcinoma
Li-Fraumeni TP53 ACN
McCune-Albright GNAS Adenoma Hyperplasia Hyperplasia/adenoma
MEN 1 AD MEN1 Adenoma Adenoma/hyperplasia ACN Islet cell

neoplasia
MEN 2 AD RET Adenoma/hyperplasia MTC PHEO Paraganglioma

Neurofibromatosis type 1 AD NF1 PHEO Paraganglioma
Von Hippel-Lindau AD VHL PHEO Islet cell

neoplasia
Paraganglioma
ACN, adrenocortical neoplasm; AD, autosomal dominant; FTC, follicular thyroid carcinoma; MTC, medullary thyroid carcinoma; PHEO, pheochromocytoma; PTC, papillary
thyroid carcinoma.
Modified from Table 5.01. Eng C. Inherited tumor syndromes. Introduction. In: DeLellis RA, Lloyd RV, Heiz PU, eds. World Health Organization classification of tumors:
pathology and genetics. Tumors of endocrine organs. Lyons: IARC, 2004;210.
The major hormone produced by the pineal gland is the indoleamine, melatonin, which plays a role in circadian rhythm regulation and gonadal steroidogenesis. Destruction of the
pineal gland by a benign cyst or tumor has led to precocious puberty. Interference with the inhibitory effect of melatonin on gonadal steroidogenesis represents one mechanism
(e307). Other physiologic functions attributed to the pineal gland include a role in modulating the hypothalamicpituitary-gonadal axis, hormonal rhythms, the sleep cycle, and body
temperature (e298,e923,e1242). Melatonin levels have been reported elevated in some children with primary pineal tumors (106,e755,e1237). Melatonin levels may be useful in
determining the adequacy of pineal tumor resection when the level was increased before surgery (e1237). Other aspects of the anatomy and function of the pineal gland are
discussed in more detail by Reiter (142).
Imaging
The normal pineal gland is less than 1 cm in size and isoattenuating to brain. Pineal lesions may be detected on CT, if large, but the pineal gland is best evaluated on MR imaging,
particularly in the sagittal plane. Imaging helps to distinguish pineal region neoplasms from common pineal cysts. Pineal cysts are isodense to cerebrospinal fluid (CSF) on CT and
are not associated with hydrocephalus (e426). The normal adult pineal gland is often centrally calcified on CT, but this process usually does not begin until age 10 to 12 (e1307), so
the finding of calcification in the pineal gland of a child less than 10 years of age should be viewed with concern. Calcification may be seen at the periphery of the pineal gland in the
older child or adult (eFigure 21-2). On MR, pineal cysts are optimally visualized in the sagittal plane, are homogeneous, and parallel the signal of CSF. Cysts greater than 1 cm in
diameter that are heterogeneous may indicate the presence of hemorrhage (e371). Following intravenous administration of gadolinium chelate, a rim of compressed normal pineal
tissue typically enhances, but the cyst itself does not enhance (e82).
Pineal germ cell neoplasms appear on CT as solid masses often with dense calcifications (eFigure 21-3 A to C). On MR, the solid portion is isodense to brain on T1-weighted
images and hyperintense to brain on T2-weighted images, while calcifications are hypointense on both pulse sequences. Pineocytomas on CT appear solid and may contain
calcifications, but calcifications are less common in pineocytomas than in germ cell neoplasms (e347). Pineal tumors may compress the tectum and aqueduct of Sylvius causing
findings of hydrocephalus (eFigure 21-3). Pineocytomas are hypo- to isointense to brain on T1-weighted images and hyperintense to brain on T2-weighted images (eFigure 21-4).
Pineoblastomas are variable in their MR appearance since these tumors are aggressive, may be large and lobulated, and have areas of necrosis (Figure 21-1A) causing a
heterogeneous appearance. Pineal parenchymal tumors generally enhance markedly after intravenous gadolinium contrast administration (e82).
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Developmental Disorders
FIGURE 21-1 ▪ Pineoblastomain a 3-year-old girl. A: Sagittal postgadolinium T1-weighted image shows a markedly-enhancing, lobulated mass (arrowhead) in the pineal region
below the splenium of the corpus callosum (S). B: This large, tan-gray, infiltrative pineal tumor has a heterogeneous appearance with hemorrhage, necrosis and leptomeningeal
extension. (Used with permission, Dr. David Louis, Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts.) C: This pineal tumor is composed of sheets
of primitive round to slightly ovoid cells (H&E stain, original magnification 200×). (Courtesy of Dr. Joe Parisi, Mayo Clinic, Rochester, Minnesota.) D: The tumor cells have irregular,
hyperchromatic nuclei and scant cytoplasm. Mitotic figures were also present (H&E stain, original magnification 400×). (Courtesy of Dr. Joe Parisi, Mayo Clinic, Rochester,
Minnesota.) E: The tumor cells demonstrate immunoreactivity with synaptophysin (immunostain for synaptophysin, original magnification 400×). (Courtesy of Dr. Joe Parisi, Mayo
Clinic, Rochester, Minnesota.)
Pineal agenesis has been reported as a component of other midline central nervous system developmental syndromes with absence of the corpus callosum, such as in Aicardi
syndrome (151,e398,e918). The contrasting abnormality, pineal gland hyperplasia, has been reported in children with genital enlargement (151,e1166).
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Pineal cysts (glial cyst) are a relatively common radiological finding on MR and as an incidental finding in 25% to 40% of autopsies. There is a female predilection (eFigure 21-5)
(21,106,171,e348,e371,e475,e754,e787, e824,e852,e1108). A pineal cyst larger than 1 cm in diameter may cause symptoms (headache, vertigo, and visual disturbances) in an
adolescent or young adult (142,e348,e307). Symptomatic cysts have been treated by surgical excision (113,192,e892,e1132). Possible mechanisms for pineal cyst development
include persistence of the ependymallined pineal diverticulum, secondary cavitation within the pineal gland, or as sequelae to hemorrhage in the gland (142,e787,e978,e1108). An
ependymal lining often accompanied by reactive-appearing astrocytes are the microscopic features. (eFigure 21-6) Approximately 5% of children with hereditary retinoblastomas
have pineal cyst as a benign variant of trilateral retinoblastoma (141,e94). Cyst formation is also seen in pineal neoplasms (50,106,e585).
Acquired Disorders
Neoplasms of the pineal gland region account for 2% or less of all primary CNS tumors in children and are discussed in more detail by Burger and Scheithauer (21) and in
Chapter 10. Classically, there are three histogenetic categories: tumors of pineal parenchyma (true pinealomas) (eFigure 21-7), glialderived tumors, and germ cell neoplasms, which
account for 50% to 60% of cases. The germ cell tumors have a variety of patterns ranging from germinomas and teratomas to malignant mixed germ cell neoplasms (eFigures 21-8
to 21-10) (48,111,e1,e33,e264,e321,e346,e484,e582,e907). Imaging studies have not been found diagnostic in differentiating among these tumors and do not distinguish between a
pineal neoplasm and a glial cyst (e977).
Pineal parenchymal tumors (PPT) are represented by the pineocytoma, pineoblastoma, and pineal parenchymal tumor (PPT) of intermediate differentiation (19,21,79,105,
e135,e625,e798,e1064). Pineoblastomas, like germ cell tumors, preferentially occur in the first decade of life in contrast to pineocytomas, which are seen in the second decade and
into adulthood. Almost 60% of PPTs are pineoblastomas (mean age, 2 to 3 years) and another 10% are pineocytomas (mean age 10 to 12 years) in the pediatric population (48, 52).
The M:F sex incidence for pineoblastomas varies among series from 5:1 to 1:2 for children 16 or younger (48,79,e264).
Pineoblastoma, like the other central primitive neuroectodermal tumors (PNET), is a tan-gray, soft, infiltrative tumor with or without hemorrhage and necrosis and often extends into
the leptomeninges (21) (Figure 21-1B). Sheets of primitive round to slightly ovoid cells with irregular, hyperchromatic nuclei and scant cytoplasm are observed on histological
examination. Mitotic figures and apoptotic bodies are readily identified (Figure 21-1C, D, eFigures 21-11 and 21-12). Focal necrosis and Homer-Wright rosettes are present in some
cases. Infrequently, photoreceptor differentiation is indicated by the presence of Flexner-Wintersteiner-like rosettes. Tumor cells are immunoreactive for SYN (Figure 21-1E, eFigure
21-13), CHR and NFP to a lesser degree and to retinal S-antigen in about 50% of cases (21, 34).
Trilateral retinoblastoma syndrome is defined by the development of a midline intracranial malignancy, usually a pineoblastoma, in the setting of hereditary retinoblastoma
(21,141,e94,e72,e124,e502,e759,e955). The rhabdoid tumor predisposition syndrome with a germline mutation in the INI1 gene is also associated with primary pineal neoplasms
(19,e133). Astrocytomas, discussed in chapter 10, involving the pineal gland also occur throughout the first and second decades. Pineal astrocytomas have been reported in
association with tuberous sclerosis and neurofibromatosis type 1 (e277,e893,e264). Papillary tumors presumably arising from the ependymal lining, usually seen in adults, have also
been reported in children (19,e176,e364,e478).
Pineocytoma, unlike the pineoblastoma, has a lobular appearance like other examples of endocrine or neuroendocrine neoplasms, is well circumscribed and displaces surrounding
structures. The tumor cells are uniform with small central nuclei and conspicuous eosinophilic cytoplasm with an absence of pleomorphism, necrosis and mitotic figures in most
cases. Homer-Wright and Flexner-Wintersteiner rosettes and large GFAP-positive fibrillary areas, referred to as pineocytomatous rosettes, are observed in these tumors (eFigures
21-14A, B and 21-15). Like the pineoblastoma, tumor cells are immunoreactive for SYN, CHR, NFP and neuron specific enolase (NSE), in addition to retinal S-antigen in
approximately 30% of cases (eFigure 21-16) (34,e798,e1292). Neurosecretory granules are identified ultrastructurally in contrast to their usual absence in pineoblastomas (e820).
The PPT of intermediate differentiation shows histological features of both pineoblastoma and pineocytoma with variable mitotic activity, necrosis and NFP immunoreactivity.
Comparative genomic hybridization suggests that this tumor more closely resembles the pineoblastoma, but generally has the favorable prognosis of a pineocytoma
(e363,e987,e1292).
Prognosis of PPTs is dependent on stage, tumor volume, histological type, and NFP immunostaining (52,79,e171,e499,e1293). These tumors are assigned to the following grades:
pineocytoma (WHO grade 1), pineoblastoma (WHO grade 4) and the PPT of intermediate differentiation (WHO grade 2 or 3) (19, 52, 79, 105). Pineocytoma has a favorable survival
(85% to 90%, 5 years), whereas the pineoblastoma is below 25% (19,52,79,e1052,e1053). Among the pineoblastomas, those tumors with mutated Rb1 gene are more aggressive
with decreased survival rate, if possible, when compared to the sporadic pineoblastoma (e947).
Other neoplastic lesions involving the pineal gland include Langerhans cell histiocytosis (LCH) (e440), cavernous angioma (e631), lipoma (e1 118), craniopharyngioma (e1215), and
meningioma (e775). Pineal involvement with acute lymphocytic leukemia is reported (e657). Infections, vascular malformations, epidermoid cyst, hemorrhage, and
P.915
apoplexy are nonneoplastic lesions of the pineal gland in children (e216,e660,e743,e752).
PITUITARY GLAND
Anatomy and Physiology
Posterior to the optic chiasma, the pituitary gland extends by a narrow stalk from the hypothalamus into the sella turcica, a small concavity in the sphenoid bone (9,101,134,e317).
The pituitary gland is a small ovoid structure which is divided into a red-brown anterior lobe (adenohypophysis), a gray-white posterior lobe (neurohypophysis), and an indistinct
intermediate lobe. The adenohypophysis is subdivided into the pars distalis, pars intermedia, and pars tuberalis, with the pars distalis accounting for the bulk of the anterior lobe.
More prominent in the fetal pituitary gland, the pars intermedia is inconspicuous in adolescents and adults. The neurohypophysis is subdivided into the pars nervosa, the
infundibulum, and the median eminence. The infundibulum and pars tuberalis comprise the pituitary stalk (eFigure 21-17).
The pituitary gland weighs approximately 100 mg at birth and increases in weight during adolescence to its adult weight of 500 to 600 mg (e37,e206), with the adenohypophysis
accounting for 80% of the gland (9, 101). Some populations, however, demonstrate a weight less than 500 mg (e1022). The pituitary gland of the neonate is especially prominent
owing to its stimulation by maternal hormones, but it undergoes some involution in the postnatal period, followed by increased growth through the age of 3 years (e638). A notable
increase in the size of the gland occurs with menarche and pregnancy (e366,e1022). Generally, the pituitary gland in women after puberty weighs more than the gland in men
(e227,e330,e1022). Suprasellar extension of the pituitary gland during puberty has been reported as a normal variant (e587).
The pituitary gland receives its vascular supply from two hypophysial arteries that branch from the internal carotid arteries and give rise to two anastomosing networks of capillaries
that surround the stalk and adenohypophysis. The hypophyseal-portal circulation, which arises from the second capillary plexus, supplies the adenohypophysis (9,101,e317). A thin
diaphragm, arising from the dura, covers the opening to the sella turcica, but in the center of the diaphragm the pituitary stalk passes through an aperture. The pituitary gland is not
covered by meninges. The periosteal dura lines the sella turcica.
The adenohypophysis is composed of three cell types on histological examination: the chromophobes, acidophils, and basophils, accounting for 50%, 40%, and 10% of
adenohypophyseal cells, respectively (eFigure 21-18) (e518). Based on immunohistochemistry and ultrastructural observations, six distinct hormonally active cell types are
identifiable in the adult gland. The cell types and their respective hormones are the somatotrophs (growth hormone), lactotrophs (prolactin), corticotrophs (ACTH), gonadotrophs
(FSH/LH), and thyrotrophs (TSH), accounting for 40% to 50%, 10% to 30%, 10% to 20%, 5% to 10%, and 5% of the adenohypophyseal cells, respectively (101, 134). Stimulating
and inhibitory hypothalamic factors released into the hypophyseal-portal circulation regulate the release of ACTH, TSH, FSH, LH, growth hormone, and prolactin from the
adenohypophysis (eFigure 21-19). Mammosommatotrophs (prolactin/growth hormone) are uncommon. Immunostaining reveals CK 7 and 8 positivity in these cells in addition to their
respective hormones (10, 33).
The folliculostellate cells are agranular, immunostain for S-100, GFAP and vimentin (VIM) and extend between the other adenohypophyseal cells. They are thought to have a
paracrine regulatory function on the hormone-producing cells (101). Calcified concretions are an incidental finding in the anterior pituitary of ostensibly normal fetuses and neonates
(e441,e442).
The posterior pituitary (neurohypophysis) contains the axonal processes of neurosecretory neurons that originate in the supraoptic and paraventricular nuclei of the hypothalamus
and are GFAP positive (eFigure 21-19). Vasopressin and oxytocin, produced in the neurohypophysis are stored in secretory granules (Herring bodies) in the nerve endings (101).
The pituitary gland arises developmentally from two anlages (e634). Ectoderm from the roof of the oral stomatodeum gives rise to the adenohypophysis, whereas neuroectoderm
from the floor of the diencephalon is the progenitor of the neurohypophysis. During the 4th week of gestation, an outpouching of ectoderm from the roof of the stomatodeum
(primitive mouth cavity) grows dorsally toward the diencephalon as Rathke pouch. Along this route of migration, progenitor cells of the future adenohypophysis may lag behind as
potential sources of ectopic anterior pituitary (e518). Constriction and disappearance of Rathke pouch during the 5th to 6th gestational week separate the adenohypophysis from the
stomatodeum. Concurrently, the elongating Rathke pouch passes between the developing presphenoid and basisphenoid bones of the skull and joins with the infundibulum, a
diverticulum arising from the diencephalon as the future neurohypophysis. The first vestiges of the hypothalamic-hypophyseal portal circulation are seen at 7 weeks' gestation, and
the process is completed at 18 to 20 weeks' gestation.
Somatotrophs and corticotrophs are identified immunohistochemically in the adenohypophysis between the 5th and 12th gestational week; by 12 to 13 weeks' gestation, thyrotrophs
and gonadotrophs are seen, and at 13 to 16 weeks' gestation, lactotrophs first appear. During the sixth gestational month, innervation of the neurohypophysis with axonal processes
from the supraoptic and paraventricular nuclei takes place.
The differentiation of the oral ectoderm into the terminal anterior pituitary cell types, their hormones and receptors is under the control of a large complement of genes and
transcription factors (eFigure 21-20). Several excellent reviews
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discuss the role of these factors in pituitary organogenesis in more detail (10,33,44,110,e83,e646,e900,e1068,e1288). The physiology of the different cell types, their hormones and
the mechanisms of action of their respective hormones is beyond the scope of this chapter, but is detailed by others (9,33,101, 110,143,e1049,e1050,e1298).
Imaging
Due to its small size and location within the bony sella, the pituitary is best evaluated with dedicated MR imaging. The adenohypophysis is isointense to gray matter and has a flat
superior margin until puberty when the margin becomes slightly convex, especially in girls. Due to the fat content of the hormones elaborated there, the neurohypophysis is
hyperintense compared to brain on T1-weighted images, producing the posterior pituitary “bright spot.” The pituitary stalk (infundibulum) is normally midline and no larger than the
basilar artery on axial images (e82). Developmental lesions may be detected on imaging. Ectopia of the posterior pituitary is seen as an abnormal location of the posterior pituitary
bright spot along the infundibulum or near the infundibular recess of the third ventricle (eFigure 21-21). Rathke cleft cysts are well-circumscribed, round or lobulated, and isodense to
CSF on CT. The signal intensity of the cyst on MR is variable depending on the protein content of the fluid. They are generally iso-to slightly hyperintense to CSF on T1-weighted
images and iso to slightly hypointense to CSF on T2-weighted images (eFigure 21-22) (e82).
Inflammatory or infiltrative disorders are optimally demonstrated on MR images. Lymphocytic and granulomatous hypophysitis and LCH appear similar on imaging studies. The
hypothalamus and infundibulum appear enlarged. Generally, uniform enhancement is seen following intravenous administration of gadolinium (eFigures 21-23 to 21-26)
(e504,e746,e1187,e1188). Primary pituitary tumors are best evaluated with dedicated MR imaging with and without contrast material. Microadenomas do not distort the gland but
are hypointense to the normal gland on T1-weighted images and enhance less than the gland on early dynamic postgadolinium imaging (eFigure 21-27). Macroadenomas distort the
gland and the infundibulum and enhance uniformly and intensely (e866).
Anomalies in pituitary gland development are uncommon and outlined in Table 21-2 (133,e150,e227,e229,e590,e1055, e1111,e1 119). Agenesis, complete absence of the pituitary
gland is rare as an isolated finding. Isolated agenesis of the pituitary has been noted in infants of diabetic mothers as a presumed form of diabetic embryopathy. Pituitary dysfunction
in neural tube defects is well documented (e329). Agenesis is usually associated with other midline and craniofacial abnormalities (101,e53,e1067,e1157). In the presence of
pituitary agenesis, the thyroid gland, the adrenal glands, and gonads are expectedly diminutive. The posterior pituitary or neurohypophysis may be present.
Table 21-2 ▪ CONGENITAL AND DEVELOPMENTAL ANOMALIES OF THE PITUITARY GLAND
Agenesis
Hypoplasia
Ectopic pituitary
Duplication
Rathke cleft cysts
Pars intermedia cyst
Dermoid cyst
Empty sella syndrome
Hamartoma
Teratoma
Isolated growth hormone deficiency
Combined pituitary hormone deficiency
Cranial vault abnormalities involving sella turcica

Transphenoidal encephalocele
Persistent craniopharyngeal canal
Modified from Parks JS, Felner EI. Hypopituitarism. In: Kliegman RM, Behrman RE, Jenson HB, Stanton BF, eds. Nelson textbook of pediatrics, 18th ed. Philadelphia, PA:
Elsevier, 2007; Chapter 558.
Adenohypophyseal hypoplasia with congenital hypopituitarism is reported in the presence of mutations in the genes controlling early development such as in POUF1 (pit-1) (e150,
e168,e172,e373,e386,e608,e800,e916,e917,e965,e1107, e1130,e1179,e1251). The adenohypophysis is absent or markedly hypoplastic, with an intact neurohypophysis
(e53,e641). Vascular malformations leading to pituitary hypoplasia represent another etiologic consideration (e544,e1095).
Hypopituitarism, defined as diminution or absence of one or more anterior pituitary hormones, is estimated to occur in 1:4,000 to 10,000 live births. A number of genetic
syndromes, conditions with widespread structural abnormalities, and midline CNS anomalies involving the hypothalamus are associated conditions. Mutations in various genes in
pituitary development are present in approximately 13% of isolated pituitary hormone deficiency (IPHD) and 20% of combined pituitary hormone deficiency (CPHD) cases and other
structural malformations (Table 21-3) (95,133,e916,e965,e1179). Hypopituitarism may be a complication of traumatic brain injury (TBI) (e78,e324,e868).
Various CNS anomalies are associated with pituitary malformations: holoprosencephaly (associated with rudimentary neurohypophysis and central diabetes insipidus), septo-optic
dysplasia, and hypothalamic-hypophyseal dysgenesis in bilateral anophthalmia (e161,e162,e481,e492,e1165). Many of these anomalies with pituitary dysfunction are linked to
genetic mutations including deletions of a portion of chromosome 14 that codes for several genes including BMP4 and OTX2 that are associated with ocular and pituitary
development. Bilateral anopthalmia is seen in association with BMP4 mutations (e703,e884). Mutations in the OTX2 gene may be associated with CPHD, a hypoplastic pituitary
gland, ectopic neurohypophysis, and Chiari malformation. Other syndromes in which hypopituitarism is a feature include
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MELAS syndrome, Kallmann syndrome, Rieger syndrome, trisomy 18, trisomy 13, Pallister-Hall syndrome, neurofibromatosis, Fanconi anemia, and ataxia-telangiectasia (e458,
e538,e542,e618,e620,e994,e1020,e1087).
Table 21-3 ▪ MANIFESTATIONS ASSOCIATED WITH MUTATIONS IN SELECTED GENES INVOLVED IN PITUITARY DEVELOPMENT
Gene Function Manifestations
LHX3, Development and maintenance of adenohypophysis CPHD, IPHD, pituitary hypoplasia, ectopia of neurohypohysis, Arnold-Chiari I
LHX4 malformation
HESX1 Early development of pituitary gland CPHD, IPHD, pituitary hypoplasia, ectopia of neurohypohysis, septo-optic
dysplasia
POUF1 (pit Differentiation of the somatotrophs, lactotrophs and thyrotrophs Growth hormone, prolactin and TSH deficiency
1)
PROP 1 Differentiation of the sommatotrophs, lactotrophs, thyrotrophs and 30%-50% of cases of familial CPHD
gonadotrophs
Tpit Differentiation of corticotrophs ACTH deficiency
Gli2, GH3 Holoprosencephaly and panhypopituitarism, Hall-Pallister syndrome
PTX2 Reiger syndrome
CPHD, combined pituitary hormone deficiency; IPHD, isolated pituitary hormone deficiency; ACTH, adrenocorticotropic hormone; TSH, thyroidstimulating hormone.
Based on data from Lap-Yin Pang A, Martin MM, Martin ALA, et al. Molecular basis of diseases of the endocrine system. In: Coleman WB, Tsongalis GJ, eds. Molecular
pathology: the molecular basis of human disease. Amsterdam: Elsevier, 2009:435-463.
Anencephaly is characterized by the presence of an anterior pituitary tissue within the mass of cerebrovasculosa tissue (eFigure 21-28A, B). The presence of somatotrophs,
lactotrophs, and gonadotrophs is demonstrated by immunohistochemistry. Corticotrophs and thyrotrophs, present in the pituitary in the second trimester, disappear owing to lack of
hypothalamic stimulation during the third trimester (e317,e618). A distinct neurohypophysis is absent. The adrenal glands are hypoplastic at birth (e945) (eFigure 21-28C, D).
Ectopia of anterior pituitary type tissue is common and invariably an incidental finding, generally, in the roof of the nasopharynx or as a pharyngeal pituitary
(e147,e250,e515,e749,e782). Persistence of Rathke pouch in the roof of the oronasopharynx is the source of the pharyngeal pituitary gland which has been reported in a number of
conditions including the anencephalic fetus, spina bifida, trisomy 18, and Meckel syndrome (e619,e621-e623,e1257). Ectopia of the posterior pituitary has been associated with
mutations in the genes responsible for pituitary organogenesis (eFigure 21-21) (95,e822,e851,e1165). Ectopic pituitary adenomas (PAs) are documented in the suprasellar region,
clivus, nasopharynx, and paranasal sinuses mainly in adults, but also in children (e31,e247,e276,e428,e451,e463,e520 e616,e996,e1248).
Rathke cleft cyst, with the formation of microcysts in the pars intermedia, is usually well circumscribed and is seen in normal pituitary glands in 2% to 26% of autopsies (e1235).
Usually asymptomatic, fluid accumulation in these epithelial-lined cysts may be symptomatic on the basis of compression of intrasellar or suprasellar structures with growth
retardation in children (the so-called pituitary dwarfism) (eFigure 21-22) (e230,e1235). Central precocious puberty has also been observed (e7). The cyst is filled with thickened
mucoid secretions or dark fluid (eFigure 21-29). Ciliated columnar or low cuboidal epithelium lines the cyst (e152). Other cystic lesions in the region of the pituitary include the
craniopharyngioma and intrasellar arachnoid cyst (e1091). A distinguishing feature of the craniopharyngioma is mixed cystic and solid areas with the presence of palisading and
squamoid-type epithelium (Figure 21-2A, B). Because the craniopharyngioma and Rathke cleft cyst have a shared histogenesis, ciliated columnar epithelium may be seen on
occasion in a craniopharyngioma. Abscess formation and hypophysitis are rare complications in Rathke cleft cysts.
Pituitary duplication is a rare disorder that is ascribed to a duplication of the prechordal plate and anterior aspect of the notochord. Two distinct pituitaries, each with a stalk, are
the typical presentation (e634). This anomaly has been seen with partial twinning; the median cleft facial syndrome, precocious puberty, and fetal exposure to meclizine (teratogenic
effect) (e283,e462,e998,e1016,e1231). A midline hypothalamic mass of disorganized neurons is accompanied by other midline developmental anomalies including a duplicated sella,
cleft palate, hypertelorism, agenesis of corpus callosum, and vertebral anomalies (e634). Nasopharyngeal teratomas have been reported in association with pituitary duplication in
infancy (e462,e530,e853,e1075,e1 106).
Empty sella syndrome (ESS) is usually an incidental finding in young children in contrast to adults (e110,e183,e191,e325,e1272). The primary form of ESS results from a defect
in the diaphragm covering the opening to the sella turcica, and arachnoid tissue extends through the diaphragmatic defect. Increased CSF pressure leads to enlargement of the sella
turcica and compression of the pituitary gland along the floor of the sella turcica, giving the appearance of an empty sella turcica (eFigures 21-30 and 21-31) (e114). Pituitary
infarction, pituitary atrophy from
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a tumor or other mass lesion, or prior hypophysectomy account for secondary ESS (e477).
FIGURE 21-2 ▪ Craniopharyngioma. A: This gross brain image shows a suprasellar cystic lesion filled with a dark brown fluid containing cholesterol debris. B: This
adamantinomatous variant consists of ribbons of epithelial cells with pseudopalisaded nuclei at the periphery of the lobules surrounding cystic spaces. The inner cells in the more
solid areas have a loose, stellate appearance. The so-called wet keratin is seen as intermixed stacks of necrobiotic squames. This image is from a 7-year-old girl, who presented
with headaches and decreased visual acuity and was found to have a suprasellar mass (H&E stain).
Acquired Disorders
Inflammatory and infiltrative disorders are known to involve the pituitary gland including infections, noninfectious inflammatory conditions, and infiltrative processes. Examples of
these diseases are congenital syphilis, mycobacteriosis, lymphocytic-granulomatous hypophysitis, LCH, sarcoidosis, Wegener granulomatosis, iron overload, storage disorder,
Rosai-Dorfman disease (RDD), and Hurler syndrome (e105,e109,e127,e208,e228,e267,e424,e731, e1043,e1061,e1083,e1282). In addition to the PA and craniopharyngioma, the
most common neoplasms of the sellar, parasellar, and suprasellar regions in children, germ cell neoplasms of the types seen more often in the pineal gland (60% to 70% of all
primary intracranial germ cell tumors) also present in the suprasellar-sellar region (30% to 40% of cases). Visual field defects, diabetes insipidus, and panhypopituitarism are the
principal clinical manifestations of suprasellar germ cell tumors.
Lymphocytic hypophysitis, typically observed in young women in the postpartum period with hypopituitarism, is seen in children as young as 9 years old; however, the condition
is generally uncommon in children (e208,e406,e504,e747,e780). It is regarded as an autoimmune condition because of its association with Hashimoto or lymphocytic thyroiditis
(e960,e1113). The adenohypophysis (lymphocytic adenohypophysitis) and neurohypophysis (lymphocytic infundibulo-neurohypophysitis) may be involved, and generically, the
designation of lymphocytic hypophysitis is used to describe both conditions (e1043,e1227,e1260). The pituitary is enlarged with a firm consistency and contains an inflammatory
infiltrate of small lymphocytes commingled with plasma cells (eFigure 21-32A to C). Eosinophils and some macrophages are also seen. Fibrosis is common, but may be inapparent in
a small biopsy. Hypopituitarism, diabetes insipidus, and symptoms of a mass are the usual clinical manifestations in both children and adults (e1260). Because the pituitary and sella
are enlarged, a PA is often the clinical impression.
Granulomatous hypophysitis with epithelioid or caseous granulomas has the differential diagnosis of infection (tuberculosis), sarcoidosis, rupture of a Rathke cleft cyst, LCH and
idiopathic granulomatous hypophysitis (e535,e541,e650,e1002,e1151). Granulomas are not a feature of lymphocytic hypophysitis, although a nosologic and etiologic relationship
may exist between these idiopathic inflammatory disorders (e504).
Xanthogranulomatous inflammation (cholesterol granuloma) of the sellar region is an inflammatory reaction characterized by cholesterol clefts, lymphoplasmacytic infiltrates,
hemosiderin deposits, fibrosis, foreign body giant cells, histiocytes, and eosinophilic necrotic debris. Although this pattern of xanthogranulomatous inflammation may be seen in
association with an adamantinomatous craniopharyngioma, Paulus et al. (e377,e922) have observed this pattern in idiopathic cases, mainly in adolescents and young adults, which
is not on the basis of a craniopharyngioma (e922).
Vascular lesions with hypopituitarism are uncommon in children, but hemorrhagic infarction of a pituitary macroadenoma, referred to as pituitary apoplexy or pituitary tumor
apoplexy is one such example (Figure 21-3A, B) (109,e280, e369,e370,e700,e856,e953). Sheehan syndrome is associated with severe maternal intrapartum hypotension with
pituitary infarction in the postpartum period (e1191,e1192). Presumed ischemia of the pituitary in sickle cell crisis is associated with decreased growth hormone secretion and
impaired growth in affected children (e1115). Some cases of septo-optic
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dysplasia, classified as a developmental anomaly, are thought to represent a vascular disruption of the anterior cerebral artery (e162,e736,e838,e999). Vascular lesions due to stalk
transection may occur secondary to trauma (e640,e816,e1291).
FIGURE 21-3 ▪ Pituitary apoplexy. A: Saggital section of brain showing hemorrhage within a pituitary macroadenoma. B: Coronal section showing hemorrhagic infarction of a 2-cm
diameter well-circumscribed pituitary macroadenoma.
Nonneoplastic cysts identified in children on radiological studies, are not clinically evident unless the sella turcica is expanded, leading to hypopituitarism and diabetes insipidus
(e473,e829). Cystic dilatation of Rathke pouch remnants is common; however, these cysts are usually smaller than 5 mm in diameter (eFigures 21-22 and 21-29) (e858). Rathke
cleft cysts arise from the squamous epithelium of the Rathke cleft, and infrequently become enlarged with symptoms resembling a craniopharyngloma (e230,e550). Arachnoid and
dermoid cysts are also regarded by some as congenital defects (e218). An intrasellar arachnoid cyst must also be distinguished from a craniopharyngioma
(e226,e829,e1091,e1109).
Pituitary hyperplasia is a nonneoplastic proliferation of one of the functional adenohypophyseal cell types (e55,e516,e1048). It is a polyclonal proliferation leading to pituitary
enlargement and may produce a suprasellar mass (e988). In children, somatotroph hyperplasia is reported in the McCune-Albright syndrome (MAS) and gigantism
(e644,e664,e696,e836,e915,e1303). Pituitary hyperplasia has also been reported in primary hypothyroidism (e372,e514). During pregnancy the pituitary gland doubles in size due
to the proliferation of the lactotrophs (responsible for prolactin secretion) and decreases in size postpartum (101).
Pituitary adenoma (PA) is a monoclonal neoplasm of the adenohypophysis. As many as 10% of all PAs present in the first two decades of life (e242,e899). Tumors arising in the
sellar region account for approximately 11% of all CNS tumors with PAs comprising 7.5% and craniopharyngiomas (CRPs) 3.2% (25). Most PAs are diagnosed in the second decade
(90% or so of cases) and less than 10% before 10 years of age. Between the ages of 15 and 19 years, PAs are the most common CNS tumor and were twice as common in girls as
boys (9,109,115,187, e1175). Reports of adenomas occurring in children less than 4 years are uncommon. One of the youngest examples of a PA occurred in a 7-month-old infant
with Cushing disease and an ACTH-secreting PA (e720). Most PAs are sporadic, but they are one of the tumors observed in multiple endocrine neoplasia, type 1 (MEN 1), MAS,
familial acromegaly syndrome, and Carney complex (61,129,e394,e450,e487,e488, e788,e796,e966,e1090,e1 144,e1256,e1268). The three mutated genes associated with these
familial tumors are MEN 1 in MEN 1 syndrome, PRKAR1A in Carney complex and the gene for aryl hydrocarbon receptor-interacting protein in familial acromegaly syndrome
(10,e407,e722,e1131,e1148).
In terms of function, the majority of PAs in children are prolactinomas (53%), and the remaining tumors are ACTH-secreting tumors (31%), growth hormone secreting tumors (9%),
and endocrine-inactive (null cell tumors) (3%) (115,e245,54,188). ACTH-secreting adenomas are more common before puberty in contrast to prolactinomas and growth-hormone
secreting tumors which are more common after puberty.
The clinical manifestations of PAs in children are variable and have been thoroughly documented (e173,e581,e661,e678). Headaches and visual field defects are the most common
findings due to mass effect. In functional hormonally active tumors, girls with prolactinomas present with amenorrhea and galactorrhea, whereas, gynecomastia and hypogonadism
are seen in boys. Children with ACTH-secreting adenomas present with Cushing disease and children with somatotropin or growth hormone-secreting adenomas present with
gigantism.
Most prolactinomas, growth hormone-secreting tumors, and endocrine-inactive PAs are macroadenomas (tumor larger than 10 mm in diameter) (Figure 21-4A, B) in contrast
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to the ACTH-secreting adenomas, which are more often microadenomas (tumor smaller than 10 mm in diameter) (eFigures 21-33 and 21-34) (9, e (272). Macroadenomas are more
common than microadenomas in children, consistent with the finding that prolactinomas are more common than ACTH-secreting tumors. Pathologically, PAs
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are classified on the basis of five-tiered features: endocrine activity, imaging studies and operative findings, histology, immunohistochemistry, and ultrastructure (42,e645). PAs are
soft and grayish-red, measuring 2 cm or less in diameter. On the basis of imaging criteria, four grades of tumors are recognized: grade I (smaller than 1 cm in diameter); grade II
(intrasellar lesion larger than 1 cm in diameter or with suprasellar expansion without invasion); grade III (small or large locally invasive tumor with bony invasion of the sella turcica),
and grade IV (large invasive tumor involving bone, the hypothalamus or cavernous sinus) (9). Larger aggressive tumors are more likely to be cystic, hemorrhagic, and necrotic
(Figure 21-3A, B). One or more concurrent histological patterns, diffuse, trabecular, or papillary, may be evident. The degree of cellularity is variable from highly cellular to more
scantily cellular tumors with a hyaline or amyloid-like stroma. The tinctorial quality of the cytoplasm has given rise to the characterization of PAs as basophilic, acidophilic or
chromophobe with some limitations. The tumor cells are generally rounded with some spindling on occasion; the rounded nucleus is central or eccentric, and the tumor cells may
have plasmacytoid qualities in the presence of an eccentric nucleus and prominent basophilic, acidophilic, or amphophilic cytoplasm. Prolactinomas are typically composed of
chromophobes or slightly acidophilic cells, have a solid or papillary pattern, and have a hyalinized stroma with or without microcalcifications (Figure 21-4C, D, eFigure 21-35). PAs, in
general, do not have a capsule (eFigure 21-33). Electron microscopy and immunohistochemistry are adjuncts to the characterization of these tumors (Figure 21-4E, eFigure 21-36)
(e517,e518,e1034). In addition to specific hormonal immunostaining, PAs are positive for SYN, CHR, and NSE (33).
FIGURE 21-4 ▪ Pituitary adenoma. A: A pituitary adenoma is shown in this sagittal T1W MR image of an 11-year-old boy with a cystic expansile mass (macroadenoma) arising within
the sella turcica and extending upward (Courtesy of James Smirniotopoulos, M.D., Bethesda, Maryland). B: Saggital section of brain showing a pituitary macroadenoma,
prolactinoma, with a homogeneous cut surface. C: The normal architecture of the pituitary gland is replaced by a diffuse growth pattern of cells. The normal histological pattern of
acidophils, basophils, and chromophobes arranged in a cord-like pattern is replaced by a single population of cells with acidophilic cytoplasm (H&E stain, original magnification
200×). D: The tumor cells are large with irregular nuclei and acidophilic cytoplasm (H&E stain, original magnification 400×). E: Tumor cells are immunoreactive for prolactin in this
pituitary macroadenoma (immunostain for prolactin, original magnification 400×). (Images C-E, courtesy of Dr. Joe Parisi, Mayo Clinic, Rochester, Minnesota.)
In terms of clinical behavior, macroadenomas are more likely to be invasive rather than the smaller expansile microadenomas. It is debatable whether PAs in children are more
aggressive than their adult counterparts (e272,e340,e751). Invasive adenomas are characterized by extension into the dura, bone, and cavernous sinus; these features are
generally not documented in the pathological examination. There is some correlation between the proliferative activity as determined by MIB-2 nuclear immunostaining and the
observed invasiveness of the tumor (e188,e353,e770).
CRPs, thought to be derived from remnants of the Rathke pouch, account for 3% to 4% of primary CNS tumors in children (25). Generally, the tumor is found between the pharynx
and floor of the sella turcica and is suprasellar in most cases (e5,e10,e400,e1261) (Figure 21-2A, eFigures 21-37 to 21-39). They may be parasellar or ectopic in the region of the
pineal gland (e4,e327,e385,e606,e1033,e1215). The differential diagnosis includes PA, infection, inflammatory processes, vascular malformations, and Rathke cleft cyst (e400).
Imaging has been found helpful in distinguishing among CRPs, PAs and Rathke cleft cysts (e226,e483).
Craniopharyngiomas (CRPs) in children are diagnosed between the ages of 5 and 14 years and have an equal male to female ratio (25). Tumors occurring during infancy are
uncommon (e67,e74,e549,e573,e732). Compressive symptoms including pituitary dysfunction with retarded growth are the principal clinical manifestations (75,e456,e457,e1105).
Diabetes insipidus due to posterior pituitary involvement is infrequent.
A calcified cystic suprasellar mass is the characteristic appearance on CT and MR scans (eFigure 21-39). Surgical resection may be followed by a recurrence (21,e26,e288,
e368,e704,e1104,e1233). These tumors are typically characterized as a calcified suprasellar mass or cyst that measure 1 to 10 cm in diameter by imaging studies. The gross
specimen consists of fragments of the cyst, and has a yellowish to dark brown appearance. Fluid contents have a dark oily appearance with cholesterol crystals and fragments of
keratinous debris (e1109). In children, the histological features are adamantinomatous or ameloblastic in appearance (e385,e1033); the papillary squamous pattern is seen more
often in adults (9,10,e400,e815). Beta-catenin mutations are seen in the adamantinomatous pattern (9, 10). Epithelial lobules are arranged in a cloverleaf-like pattern (e71,e112).
Palisading of the cells adjacent to the randomly distributed fluid-filled cyst-like spaces is another characteristic feature. Aggregates of necrotic, keratinized cells, or “wet” keratin
accompanied by dystrophic calcification are other features (Figure 21-2B, eFigure 21-40). Fibrosis, chronic inflammation, and cholesterol clefts are observed in the solid areas
(e589). A xanthogranulomatous reaction is prominent in some cases, especially in the presence of a ruptured cyst. Although CRPs are regarded as clinically benign, adherence to
the hypothalamus and extension into the surrounding brain parenchyma are found in some cases. Cytokeratin expression has been used to distinguish CRP from Rathke cleft cyst
(e1290). An uncommon variant of CRP is one with adamantinomatous features together with elements of a PA in a socalled collision tumor (e1296). In many of these “collision
tumors,” the adenoma is nonfunctional; however, immunohistochemistry displays gonadotropin, prolactin, ACTH, and TSH staining (e423,e586,e844,e1296).
Other tumefactive lesions of the pituitary and sellar region include the ganglion cell tumor (the so-called gangliocytoma), LCH, granular cell tumor, RDD, and salivary gland
hamartoma. Gangliocytomas are regarded as neoplasm by most observers, but hamartoma, by others; they may be found in association with PAs, pituitary hyperplasia, or as a
distinct mass (e404,e1204). Towfighi et al. have classified these lesions as either a mixed adenoma-gangliocytoma or pure gangliocytoma (e1204).
LCH is well documented in the CNS with involvement of the brain parenchyma or the hypothalamic-pituitary axis, in which case there is central diabetes insipidus. The pituitary stalk
is thickened on imaging studies (e155,e701,e714,e746) (eFigures 21-24 to 21-26). Almost 15% of those with multisystem LCH have hypothalamic-pituitary involvement (e522).
There is limited documentation of the pathology in such cases because the diagnosis is usually established on the basis of a biopsy from a more accessible site. A mixture
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of Langerhans cells, characterized by large, convoluted and indented nuclei, that are CD1a positive, mixed with an infiltrate of lymphocytes, plasma cells and eosinophils is the
characteristic appearance of LCH (Figure 21-5 A, B, eFigure 21-41A to C).
FIGURE 21-5 ▪ Langerhans cell histiocytosis. A: Biopsy from the pituitary stalk in an adolescent with diabetes insipidus. The infiltrate is composed of foamy histiocytes that were
immunoreactive with CD1a, in a background of lymphocytes, eosinophils, and plasma cells (H&E stain, original magnification, 400×). (Courtesy of Dr. Joe Parisi, Mayo Clinic,
Rochester, Minnesota.) B: CD1a positivity in histiocytic cells in a patient with LCH (immunostain for CD1a, original magnification 400×). (Courtesy of Dr. Joe Parisi, Mayo Clinic,
Rochester, Minnesota.)
RDD has craniospinal manifestations in a minority of cases, including the sellar-suprasellar region (e1284). In 50% of such cases, the RDD is limited to this site with obvious
problems in diagnosis. Salivary gland rest or heterotopia is an incidental microscopic finding on the surface of the posterior pituitary (e464). Other neoplasms of presumed
salivary gland type, granular cell tumor of the pituitary and pituitary stalk (e438,e1046), leukemia, lymphoma, and metastatic involvement of the pituitary are restricted to adults in
most cases (e444,e470,e1056,e1296). Both primary and metastatic germ cell neoplasms also occur in the pituitary.
PARATHYROID GLANDS
Anatomy/Physiology
The parathyroid glands, usually four in number, are pinkish, oval 4-to 6-mm in diameter glands located in proximity to the thyroid gland or even embedded within the thyroid. The
inferior and superior parathyroid glands arise as endodermal outpouchings from the dorsal bulbar portion of the third and fourth pharyngeal pouches, respectively, during the fifth
gestational week (e6). Concurrently, the thymus arises from the ventral aspect of the third pharyngeal pouches. Both the thymus and inferior parathyroid glands initially migrate
together caudally with the heart. During the descent, the thymus and inferior parathyroid glands separate and the inferior parathyroid glands localize to the inferior aspect of the
thyroid gland (e1065).
In children, the combined parathyroid gland weight for all four glands increases with age from a mean weight of 5 to 10 mg each in the neonatal period to an adult combined weight
of 120 mg for adult males and 140 mg for adult females by age 30 (e34,e938,e1065). In children younger than 10 years old, the mean weight of all four glands is less than 60 mg
(e416). In individuals between the ages of 11 and 20 years, the mean weight of all four glands has been recorded as less than 100 mg, but more recently a study of parathyroid
gland weight in children between the ages of 9 and 19 years indicated individual gland weights can range between 10 and 80 mg (e410).
The parathyroid glands in children tend to be solid and cellular with minimal fat. A connective tissue capsule encloses the gland. Chief cells arranged in sheets are the predominant
cell type. Blood vessels are intermixed among the parenchymal cells, and small delicate capillaries are present between the cells. The polyhedral chief cells have a small central
nucleus and clear cytoplasm. Oxyphil cells are not observed
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generally until puberty, if at all. Adipocytes within the gland initially appear around puberty with fatty infiltration gradually accounting for 25% to 30% of total gland weight after age 18
(41,e18,e416).
Calcium homeostasis is regulated by the interaction of parathormone (PTH), calcitonin, and vitamin D (eFigure 21-42) (24,46,e1003). In response to hypocalcemia, PTH is released
from the chief cells, which is accompanied by an increase in PTH mRNA within hours of the onset of hypocalcemia. Hyperplasia of chief cells occurs within weeks. In contrast,
hypercalcemia inhibits the release of PTH by activation of the chief cell calcium receptor. Serum phosphate levels, independent of vitamin D3, also affect PTH release
(24,46,e1003). The anatomy and physiology of the parathyroid glands is discussed in more detail by Lloyd et al. (102).
Imaging
The parathyroid glands are small and difficult to appreciate on imaging studies when not enlarged. Patients with hyperparathyroidism (HPT) are best evaluated with ultrasonography
(US) and/or radionuclide scintigraphy. In children, high resolution US should be the first line imaging modality (e339). Enlarged parathyroid glands in the neck are typically identified
posterior to the thyroid gland. As parathyroid glands are best identified based on proximity to the thyroid gland, ectopic glands pose a diagnostic challenge. Radionuclide
scintigraphy is more accurate than US (87% versus 80%), particularly for ectopic glands (e574). The combination of nuclear scintigraphic studies and US provides the highest
accuracy for preoperative localization of hyperfunctioning glands.
Nuclear medicine studies utilize99mTc sestamibi, which localizes to hyperfunctioning parathyroid glands as well as the thyroid gland and salivary glands. Sestamibi scans can be
performed in several ways. In dual isotope, single phase imaging, the patient is administered labeled sestamibi and 123I or 99m Tc pertechnetate, which are taken up by the thyroid
gland. The images are subtracted to show the activity only in the hyperfunctioning parathyroid glands (eFigure 21-43). Alternatively, a single isotope, dual phase technique may be
employed. Sestamibi washes out of the thyroid and salivary glands faster than the parathyroid glands, so delayed images show relatively greater uptake in the hyperfunctioning
parathyroid gland (e339). SPECT imaging in addition to planar imaging helps to localize the abnormality in the anterior-posterior plane. Further, the fusion of SPECT imaging to x-
ray based CT adds additional anatomic information that aids in precise localization of the parathyroid gland, which may be particularly useful in recurrences after surgery (e20).
Parathyroid adenomas may also be demonstrated on CT and MR, but the accuracy of these studies for preoperative localization is no greater than for US. On CT, adenomas are
usually well-defined and they enhance intensely following intravenous contrast administration (eFigure 21-44). On MR, adenomas are generally of intermediate signal on T1-
weighted images and high signal intensity on T2-weighted images and enhance intensely following intravenous administration of gadolinium chelate (Figure 21-6A) (e574). Prior to
the advent of laboratory screening, patients with undiagnosed, prolonged HPT developed characteristic findings on bone radiographs as well as nephrocalcinosis and
nephrolithiasis, but these findings are now rarely encountered (eFigures 21-45 and 21-46).
Supernumerary parathyroid glands are found in up to 16% of the population, with an additional single gland the most common presentation (e17,e414,e415,e954).
Supernumerary glands, with as many as 12 glands, may be of normal size or rudimentary. Parathyroid adenomas and carcinoma have been reported in ectopic parathyroid glands in
children (eFigure 21-44) (2,e989,e1059).
Ectopic parathyroid tissue or a normally formed gland is relatively common within the thyroid or thymus (e100,e468,e956). Ectopic parathyroid tissue has also been observed as
scattered small nests in the soft tissues of the neck and mediastinum owing to aberrant migration or premature separation of parathyroid primordial during fetal development (e1247).
Not surprisingly, nests of parathyroid tissue may be accompanied by equally diminutive nests of thymic tissue. Aberrant parathyroid and thymus are known to present as a recurrent
lateral neck mass in children (e275). Heterotopic parathyroid tissue has also been observed at remote sites, including the vagina (e663).
Agenesis-hypoplasia of the parathyroids, due to a defect in pharyngeal pouch development or defective neural crest migration, is uncommon as an isolated finding with associated
congenital hypoparathyroidism (e596). Agenesis-aplasia is more frequently associated with other syndromes, having been reported in the 22q11.2 deletion syndrome (DiGeorge
anomaly, DiGeorge syndrome), Smith-Lemli-Opitz syndrome type II, X-linked recessive hypoparathyroidism, Kenny syndrome, Kearns-Sayre syndrome, and trisomy 18
(e266,e349,e439,e1201,e1275). The parathyroid glands may be absent in as many as 50% of patients with 22q11.2 deletion syndrome (e995). Anomalies of the aortic arch, thymus,
thyroid, and C-cells in addition to abnormal facial development are also observed (e505,e506,e507,e524,e1017,e1180). Parathyroid hemorrhage is reported in osteogenesis
imperfecta and refractory hypocalcemia (e628) (see Chapter 3).
Cyst(s) of the parathyroid are rare in children and usually asymptomatic; these cysts may represent cystic degeneration of an adenoma, or due to a presumed aberration in
development (e186,e336,e1246,e779). Other cysts in the neck may contain both parathyroid and thymic tissue as developmental cysts of the third pharyngeal pouch (e198).
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FIGURE 21-6 ▪ Parathyroid adenoma. A: Axial T2-weighted MR image shows the hyperintense parathyroid adenoma (arrowhead) posterior to the thyroid gland (arrows) in a 14-
year-old girl. B: Parathyroid adenoma in a child with primary hyperparathyroidism is seen as a solitary enlargement of the left inferior gland. (Courtesy of Robert Dufour, M.D.,
Washington, DC.) C: Parathyroid adenoma in a 16-year-old girl who presented with flank pain due to nephrolithiasis, elevated serum calcium, decreased phosphate and increased
parathormone levels. The parathyroid gland was enlarged and red-brown on gross examination. D: The enlarged parathyroid gland shows a hypercellular parenchyma composed of
chief cells without intraglandular fat on low power. Necrosis was absent (H&E stain). E: The parathyroid gland is composed of a monotonous population of chief cells with no
intraglandular fat. Mitotic figures were absent (H&E stain).
Acquired Disorders
Hypercalcemia in childhood may be a manifestation of increased PTH secretion by an adenoma or hyperplasia (primary HPT) or PTH-related peptide-induced hypercalcemia of
malignancy, mutations involving the calcium-sensing receptor gene (CASK) [familial hypocalciuric hypercal cemia (FHH), neonatal HPT] or PTH receptor, conditions associated with
vitamin D excess (sarcoidosis, tuberculosis, granulomatous disorders), medications, immobilization, and other endocrine disorders (46,47,145,e166,e682). Anorexia, fatigue,
constipation, weight loss, weakness, and mental status changes are some of the clinical manifestations. Metastatic calcifications in various organs may result
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in organ damage if the hypercalcemia is not recognized (145).
Table 21-4 ▪ PRIMARY HYPERPARATHYROIDISM ETIOLOGY IN CHILDHOOD
Parathyroid adenoma
Sporadic (nonsyndromic)
Hyperparathyroidism-jaw tumor syndrome
Parathyroid hyperplasia
Sporadic (nonsyndromic)
Neonatal hyperparathyroidism
Familial isolated hyperparathyroidism
MEN 1
MEN 2a
Parathyroid carcinoma
MEN, multiple endocrine neoplasia,
Based on data from DeLellis RA, Mazzaglia P, Mangray S. Primary hyperparathyroidism: a current perspective. Arch Pathol Lab Med. 2008;132:1251-1262.
Primary HPT is uncommon in children with an incidence of two to five cases: 100,000 (Table 21-4) (83). Most children are older than 10 years at diagnosis and there is a male
predilection in contrast to the female predilection in adults (Figure 21-6B) (69,83,e123,e136,e262,e417,e500,e692,e729, e756,e886,e970). A solitary adenoma is the etiology in 80%
to 90% of cases. The serum calcium level is usually elevated to greater than 12 mg/dL (e1214).
In neonatal HPT and MEN syndromes, four gland hyperplasia is the common finding (eFigure 21-47). Other heredofamilial settings of HPT are HPT with or without fibro-osseous
tumor of the jaws, MEN 1 and MEN, type 2a (MEN 2a) (e34, e202,e299,e471,e486,e523,e525,e647-e649) (Chapter 27).
Multiple endocrine neoplasia 1, an autosomal dominant disorder, is characterized by parathyroid gland hyperplasia, PA, pancreatic endocrine tumors, extrapancreatic
neuroendocrine tumors, adrenocortical neoplasms, angiofibromas and lipomas (43). The MEN 1 gene, a tumor suppressor gene, has been mapped to chromosome 11q13 where it
encodes the protein, menin, that is involved in transcriptional regulation, genome stability, and cell division (95,e345,e498,e608, e637,e766,e767,e912,e1078,e1079). In addition to
parathyroid gland hyperplasia, medullary thyroid carcinoma (MTC) and pheochromocytoma (PHEO) are the other associated tumors of MEN 2a, an autosomal dominant disorder
with mutations in the RET gene (10q11.2) that encodes for a tyrosine kinase receptor. HPT-jaw tumor (HPT-JT) syndrome, an autosomal dominant disorder, is associated with
inactivating mutations in the tumor suppressor gene HRPT2 (1q25-32) that encodes for the protein, parafibromin (e85,e547,e1170,e1253). Solitary or multiple enlarged parathyroid
glands are accompanied by fibro-osseous lesions of the mandible or maxilla (95). Parathyroid carcinoma is reportedly more common in this syndrome. Renal cysts, hamartomas,
renal cell carcinoma, and Wilms tumor are other accompanying lesions and tumors (e243,e413,e1181). Isolated familial HPT, distinct from HPT-JT syndrome, is a rare disorder
without other associated endocrinopathies with germline mutations involving the CASR, MEN 1, and HRPT2 genes. All four glands show chief cell hyperplasia (e599,e913).
Osteopenia, subperiosteal phalangeal bone resorption, bone cyst formation, and genu valgum are some of the skeletal anomalies in long-standing unrecognized HPT
(e58,e102,e802) (eFigures 21-46 and 21-48). Hypercalciuria and nephrolithiasis are frequent manifestations of primary HPT in childhood (69,e698). Pulmonary calcinosis has also
been observed (e1200). Measurement of intact serum PTH distinguishes primary HPT from other causes of hypercalcemia in most cases; however, cases of HPT with apparent
normal PTH levels have been reported (e92). Preoperative US and radionuclide scan may be helpful in the localization of an enlarged gland but is more limited in a case of a small
adenoma or multigland hyperplasia (e939,e954). Intraoperative PTH testing has an important role in the differentiation of a solitary adenoma from multiglandular hyperplasia in
primary HPT (83,e580,e939,e954).
Neonatal primary HPT is an uncommon disorder associated with FHH (e73,e390). Hypotonia, failure to thrive, and respiratory distress are the clinical manifestations (e185,e500).
Severe hypercalcemia and elevated PTH levels are present (e393). Osteopenia, subperiosteal bone resorption, and multiple pathological fractures of long bones are some of the
overlapping skeletal findings with osteogenesis imperfecta. FHH, an autosomal dominant condition, has an estimated prevalence of 1:15,000 to 30,000 individuals. It is usually
asymptomatic with hypercalcemia, normal PTH levels, and decreased urine calcium excretion (69). Mutations in the CASR gene, which encodes for the calcium sensing receptor in
the parathyroid gland and renal tubular epithelium, are found in FHH and neonatal primary HPT (e282,e393,e485,e944,e1249,e1250).
Secondary HPT is a multiglandular hyperplasia of the parathyroid to hypocalcemia (102,e1007,e1027). Chronic renal failure is the major cause, with malabsorption, vitamin D
deficiency, and X-linked hypophosphatemic rickets as other less common causes (e629,e705,e773,e1030,e1138). Secondary HPT is also a feature of mucolipidoses type II and
maternal hypoparathyroidism (e32,e1042). Multiglandular hyperplasia is additionally seen in tertiary HPT, an uncommon entity in children, which is characterized by hypercalcemia
after renal function is restored after renal transplantation in children who had secondary HPT (e765,e828).
Parathyroid adenomas account for 80% of parathyroid tumors in primary HPT in children which is somewhat lower than the adult experience once familial HPT and other inherited
endocrinopathies are included. Several different genetic alterations involving parathyroid hormone, RET, MEN I, PRAD 1, p53, HRPT2, and G protein genes have been identified as
different pathogenetic mechanisms (24, 41, 43). Clonal analysis of sporadic parathyroid adenomas reveals a monoclonal
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cell population in contrast to the polyclonal population seen in diffuse hyperplasia, except for some cases of hyperplasia in secondary HPT due to chronic renal failure
(24,e1079,e1193). It is usually not possible to distinguish the single gland adenoma from hyperplasia morphologically without the benefit of a second parathyroid gland to examine
by frozen section. This differentiation can be accomplished with an intraoperative determination of PTH level which normalizes within a few minutes in the case of a single gland
adenoma whereas it will initially fall and return to elevated levels in the presence of hyperplasia (83,e512,e938,e983,e1159). An enlarged gland may be hidden in the thymus,
thyroid, or around the esophagus (e239,e417).
Parathyroid adenomas and hyperplasia have similar gross features with a reddish-brown appearance, weight in excess of 60 mg and dimension of 1 to 2 cm in greatest diameter
(Figure 21-6B, C). Any parathyroid gland weighing more than 40 mg in a child should be considered abnormal (41). In one study, the mean weight of an adenoma in a child was 597
mg with a range between 170 and 1550 mg (69). A nodular or diffuse pattern of chief cells with minimal interstitial fat interspersed is the usual microscopic finding (e970) (Figure 21-
6D, E, eFigure 21-49). Cellular pleomorphism, necrosis, and increased mitotic activity are usually not present, but some mitotic activity should not be viewed with any undue
concern. A well-formed capsule is usually not present, but the adenomatous portion of the gland is distinguishable from remnants of compressed and suppressed parathyroid gland
at the periphery if present. The chief cells often contain glycogen which is demonstrable by a periodic acid-Schiff stain with diastase digestion and for PTH and CHR by
immunohistochemistry. Normal glands demonstrate greater immunoreactivity to PTH compared to hyperplastic glands and adenomas (35). Adenomas involving two glands are
uncommon and have been reported in children with an increased frequency in the HPT-JT syndrome (43,69,e262,e1160,e1211).
Parathyroid carcinoma is a rare cause of primary HPT in adults and even more so in children (e460,e784,e795). Screening for germline HRPT2 mutations should be undertaken in
any child with either a personal or family history of parathyroid carcinoma (e599). Unlike the smaller adenoma, the carcinoma is a neoplasm that infiltrates into the soft tissues of the
neck and has vascular and capsular invasion (e1289,e795).
Hypocalcemia in children is multifactorial. It is due to decreased PTH production (hypoparathyroidism), PTH receptor defects, pseudohypoparathyroidism as in Albright hereditary
osteodystrophy, mitochondrial DNA mutations as in the Kearns-Sayre syndrome, dietary imbalances in vitamin D, calcium and magnesium, or increased inorganic phosphate
consumption (47,102,e35,e1276). Hypocalcemia is also observed with pancreatitis, sepsis, increased serum phosphate levels, renal failure, and antineoplastic therapy. Impaired
renal and bone response to PTH accounts for the hypocalcemia seen in premature infants (e655).
Hypoparathyroidism is due to a developmental anomaly of the parathyroid glands as in 22q11.2 microdeletion syndrome and 10p13 deletion as well as autoimmune disorders,
infiltrative disorders, prior thyroidectomy, or parathyroidectomy (102,e25,e153,e1210). Clinically, children are either asymptomatic or present with paresthesias, tetany, muscle
cramps, or seizures. Polyglandular autoimmune syndrome, type I, is an autosomal recessive multisystem autoimmune disorder due to a mutation in the autoimmune regulatory gene
(AIRE) on chromosome 21q22.3. It presents during infancy, childhood, or adolescence with hypoparathyroidism in 80% to 85% of patients, hypoadrenalism (Addison disease), and
chronic mucocutaneous candidiasis (80,e15). Parathyroid autotransplantation is effective in preventing the hypoparathyroidism associated with total thyroidectomy (e1102).
THYROID GLAND
The thyroid gland, a bilobed structure connected by an isthmus of thyroid tissue at the level of the trachea, is located in the mid-anterior neckline and is adherent to the larynx and
trachea (103,e383). The weight of the thyroid varies with sex and age through the fetal, infantile, and childhood periods of life. There are also differences in thyroid weight on the
basis of geography in the United States and elsewhere. As an approximation, the thyroid gland at birth weighs 1 to 2 g; by 2 years of age, it approaches 3 g; at 4 years of age, 4 to 5
g; and by 15 years of age or so, 15 to 20 g, which is near the adult weight of the gland.
As some measure of its importance, the thyroid gland is the first endocrine organ to develop as a proliferation of endodermal cells on the floor of the pharynx at approximately 3
weeks' gestation. Two small lateral and a larger median anlagen from the foramen cecum at the base are formed. Through a process of elongated cephalad embryonic growth rather
than active descent, the thyroid diverticulum resides between the first pharyngeal pouches. A thyroglossal duct as an attenuated canal is maintained until 6 to 7 weeks' gestation, at
which time it normally disappears as an intact structure, but its remnants are retained to become one of the most common anomalies of the neck, the thyroglossal duct cyst (TDC).
The endodermal cells differentiate into follicular cells in the eighth gestational week. Diminutive follicles without colloid are identifiable by 8 to 9 weeks' gestation. Well-defined
follicles containing colloid are observed by the end of the first trimester.
With the incorporation of the ultimobranchial body into the thyroid, the follicles acquire C-cells and solid cell nests in the interstitium (e189,e443,e763). A remnant of the thyroglossal
duct is the pyramidal lobe, a narrow ribbon of thyroid tissue, which is attached to the isthmus and is present in 40% to 65% of individuals (e128,e1004). More detailed discussions of
the embryology of the thyroid gland are found elsewhere (136,179,e292,e1199). In addition to POU1F1, several distinct genes, TITF1, TTIF2,PAX8, TSH,
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and TSHR are involved in its development and migration (17, 94, 136, 179, 184).
Thyroid follicles are the basic morphologic and functional unit of the thyroid gland, and comprise the majority of the thyroid parenchyma. The follicular cells are responsible for the
synthesis of thyroid hormone. Both the growth and synthetic function of the thyroid gland are under the control of thyroid-stimulating hormone (TSH) synthesized by the thyrotrophs
of the anterior pituitary gland; this hormone mediates its action by cyclic AMP following attachment to receptor sites on the follicular cell membrane. Through a classic feedback
mechanism, peripheral levels of thyroxine (T4) have a positive or negative effect on hypothalamic thyrotropin-releasing hormone with the release or not of TSH from the pituitary
thyrotrophs (103,e1163) (eFigure 21-50). Excess TSH as a response to low T4 in congenital hypothyroidism is the mechanism by which hyperplasia of the thyroid gland is mediated.
Stimulation or activation of the follicular cells by TSH results in the production of thyroid hormone from thyroglobulin. Several enzymes, localized to the follicular cell, are required for
thyroid hormone synthesis and loss of one of these enzymes on the basis of an autosomal recessive defect leads to dyshormonogenic goiter (Figure 21-7). The physiology and
biochemistry of the thyroid gland in the context of the various inherited disorders with clinical manifestations of congenital hypothyroidism or hereditary hyperthyroidism have been
reviewed by others (17,103,136,e292,e1223).
FIGURE 21-7 ▪ Dyshormonogenic goiter. This image is a section through the thyroid gland of an individual who presented with a dyshormonogenic goiter. The thyroid parenchyma
has a nodular pattern with retrogressive and hyperplastic changes including hemorrhage and fibrosis. (From Lloyd RV, Douglas BR, Young WF. Endocrine diseases. Atlas of
nontumorpathology. Washington, DC: American Registry of Pathology. Originally published in Atlas of tumor pathology, tumors of the thyroid gland, Fascicle 5, Third Series.
Washington, DC: Armed Forces Institute of Pathology).
The C-cell (parafollicular cell) is the other hormonally active cell of the thyroid, representing less than 0.5% of the total epithelial population. These neuroendocrine cells are
identifiable immunohistochemically by their reactivity for CHR, SYN, and calcitonin (103). Like the dominant follicular cell, the C-cell is enclosed within the basement membrane of
the follicle, but at the periphery of the follicle without any contact with the colloid. Unlike the endodermally-derived follicular cell, the C-cell progenitor migrates from the vagal or
cephalic region of the neural crest to the fourth and fifth pharyngeal pouches, one of whose derivatives is the ultimobranchial body (103,e1278). The greatest number of C-cells is
found in the upper two-thirds of the lateral lobes of the thyroid, along the central axis (103). The neonatal gland contains a tenfold number of C-cells as compared to the adult thyroid
as the number of C-cells diminishes with age (e1283). A paucity of C-cells in the thyroid is reported in DiGeorge anomaly (syndrome) on the presumed basis of a developmental field
defect in the formation of pharyngeal pouch derivatives (e179). Hyperplasia of C-cells in children is divided into physiologic hyperplasia, seen in neonates, after a
hemithyroidectomy, in the presence of autoimmune (Hashimoto) thyroiditis and in association with MEN 2a or 2b and neoplastic hyperplasia (103). Hyperplasia is defined as the
presence of 50 or more C-cells in one 10× magnification field. Medullary thyroid carcinoma (MTC) MTC in MEN 2a, MEN, type 2b (MEN 2b) and familial (non-MEN) MTC (FMTC)
are the consequences of RET gene germline mutations and are characterized by the presence of multifocal C-cell hyperplasia and often with multifocal MTCs (59,81,103,e1267).
Solid cell nests, a remnant of the ultimobranchial body, are the third cell type identified in the thyroid gland. They are localized to the upper and middle third of the thyroid gland and
have a parafollicular or intrafollicular location. These solid, squamoid appearing cells are immunoreactive with low molecular weight keratin and carcinoembryonic antigen. Cells with
follicular or C-cell differentiation are present within these nests and may account for the rare mixed follicular-medullary carcinoma.
More detailed comprehensive reviews of the functional and morphologic aspects of the thyroid gland have been detailed by others (39,93,96,103184,e36,e39,e850).
Imaging
Imaging studies are an integral component of the diagnostic evaluation of a child with an enlarged thyroid or other massproducing process in the neck. US is a basic modality and
provides for a confident diagnosis of a TDC, which appears as midline or paramedian cyst with or without debris when complicated by infection or hemorrhage (e574) (Figure 21-8A,
B). TDCs are commonly near the hyoid bone. Branchial cleft cysts have a similar imaging appearance, but are positioned away from the midline (eFigure 21-51).
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FIGURE 21-8 ▪ Thyroglossal duct cyst in an adult complicated by papillary thyroid carcinoma. A: Axial contrast-enhanced CT image shows a midline cyst (arrowhead). B: Axial CT
image caudal to (a) shows markedlyenhancing, midline mass (arrowhead).
Ultrasound is also useful in depicting thyroid nodules in patients with thyroid dysfunction or goiter. Complex cases may require MR (e574). CT is less desirable for the evaluation of
thyroid lesions because the use of iodinated contrast will preclude later radioactive thyroid ablation therapy if necessary for several weeks (e574). Thyroid carcinomas appear well-
defined and heterogeneous on US, CT, or MR. Papillary thyroid carcinoma (PTC) is more likely to contain cystic-appearing, necrotic areas compared to follicular thyroid carcinoma
(FTC) (e574) (Figure 21-9A). Most MTCs are solid and may contain coarse calcifications (Figure 21-9B).
Radionuclide scintigraphy with99mTc pertechnetate or123I is very useful in the evaluation of thyroid dysfunction and nodules or in localization of ectopic thyroid tissue (eFigure 21-
52A to C). Nodules with decreased radiotracer uptake (“cold” nodules) are more likely to be malignant than nodules that are inapparent or take up more of a radiopharmaceutical
agent than normal thyroid (“hot” or hyperfunctioning nodule). When ectopic thyroid tissue is identified, it is important to evaluate the neck base for an orthotopic thyroid gland (Figure
21-10A, B, eFigure 21-52A to C).
FIGURE 21-9 ▪ Papillary thyroid carcinoma in 15-year-old girl. A: Transverse sonographic image showing a heterogeneous mass (M) within the homogeneous thyroid gland (T). B:
Medullary thyroid carcinoma in an 8-year-old girl with family history of MEN 2a. Axial contrast-enhanced CT shows a mass within the left thyroid lobe which enhances less than the
surrounding thyroid gland (arrowhead).
Dysmorphism of the thyroid gland is a structural phenomenon with several morphologic expressions from absence or incomplete formation of a normal gland, failure in the normal
anatomic localization of the gland, or persistence of embryologic remnants with a branching lobular pattern of immature follicles rather than the dense formation of individual follicles.
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The recessively inherited defects in the enzymes responsible for thyroid hormone synthesis are another developmental disorder but are not characterized by a primary structural
anomaly of the thyroid gland (eFigure 21-53); however, the elevated TSH levels lead to multinodular hyperplasia with the formation of the so-called dyshormonogenic goiter (Figure
21-7) (e36,e39,e1 19). Clinically, these various developmental disorders present with congenital hypothyroidism, a mass at the base of the tongue or in the neck, or congenital
hypothyroidism with development of a goiter (e403). Many of these developmental anomalies also affect first-degree relatives indicating a familial component (e12,e203,e204,e702).
FIGURE 21-10 ▪ Ectopic thyroid gland in the trachea of an adult. A: Lateral tomogram shows an ovoid mass within the tracheal air column (arrowhead). B: Axial CT image shows
markedly enhancing eccentric mass in the trachea (arrowhead) and normal thyroid lobes in the orthotopic location (curved arrows).
Dysgenesis of the thyroid is a generic designation for various anatomic developmental anomalies that include complete failure in gland formation (agenesis), decreased amount of
thyroid tissue (hypoplasia), absence of a lobe (hemiagenesis), or ectopic location. Dysgenesis is an important etiology of congenital hypothyroidism whose incidence in the United
States is 1:3,000 to 5,000 live births (e446,e117,e449,e534, e626,e652,e679,e1202,e1243). Most causes of congenital hypothyroidism are due to dysgenesis or one of the inherited
defects in thyroid hormone synthesis (e104,e122,e192,e193, e338,e343,e388,e445,e545,e801,e837,e940,e941,e992,e993, e1128,e1212,e1219,e1243) (Table 21-5). Congenital
hypothyroidism has been also been observed in Williams and Down syndromes (e122,e445,e1128).
Congenital hypothyroidism in 80% to 85% of cases is associated with one of several types of dysgenesis. The prevalence of hypothyroidism in the neonatal period is 1:4,000 live
births for thyroid dysgenesis in contrast to dyshormonogenesis in 1:30,000 live births, transient hypothyroidism 1:40,000 live births and central hypothyroidism, hypothalamic/pituitary
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defect in 1:100,000 live births (17, 93). In one study of 230 children with congenital hypothyroidism, scintigraphy revealed the following findings: ectopia in 61%, goiter in 18%,
agenesis in 16%, normal in 4% and hemiagenesis in less than 1% (e303). In another series of 800,000 neonates with increased TSH and normally positioned thyroid glands, an
enlarged gland or goiter was observed in 55% of cases, a normal gland in 29% of cases, and hypoplasia in 16% of cases (e403). If the thyroid gland is anatomically orthotopic in the
presence of congenital hypothyroidism, a defect exists in thyroid hormone biosynthesis with the development of a dyshormonogenic nodular goiter or an inability of the gland to
respond to TSH (e287,e403,e1217,e1223). Dysgenesis is more common in females than in males (3:1) and is sporadic in most cases (85% of all cases) (136). Affected infants with
agenesis/hypoplasia have permanently elevated levels of TSH and low levels of circulating thyroid hormone. A number of mutations have been identified in the genes responsible for
thyroid development, including PAY8, TITF-1 (thyroid transcription factor 1), TITF-2 (thyroid transcription factor 2), and TSHR (TSH receptor) and are pathogenetically involved in
thyroid dysgenesis (17,94,136,140,179,184,e107,e117,e205,e253,e305,e341,e495, e496,e566,e635,e651,e658,e739,e741,e740,e757,e794,e883, e1173,e1196,e1198,e1232).
These genetic defects and their association with other diseases are reviewed elsewhere (136).
Table 21-5 ▪ ETIOLOGIC CLASSIFICATION OF CONGENITAL HYPOTHYRODISM
I. Primary Hypothyroidism
A. Dysgenesis (85%) (1:4,000)
Idiopathic or genetic (TITF-1, TITF-2, FOXE1, PAX-8, and TSHR defects)
Agenesis
Hemiagenesis
Hypoplasia
Ectopia
Lingual thyroid (90% of thyroid ectopia) (1:10,000)
B. Dyshormonogenesis (10-15%) (1:30,000)

Iodide transport (sodium-iodide symporter defect (MS gene)
Iodide organification and coupling defect
Thyroid peroxidase defect (TPO gene) (Pendred defect)
Thyroid oxidase 2 defect (DUOX1/THOX1 DUOX/THOX2 genes)
Defect in thyroglobulin synthesis or transport (Tgigene)
Iodotyrosine deiodinase defect (DEHAL1 gene)
C. Other (5%)
II. Secondary/Tertiary Hypothyroidism (Hypothalamic-Pituitary-Thyroid Axis Dysfunction) (1:100,000)
Genetic defects involving LHX3, LHX4, PROP 1, POUF1, HESX1, TRHR, TSHB
III. Peripheral Thyroid Hormone Resistance
Genetic defects involving MCT8, THRB
IV. Transient Hypothyroidism (1:40,000)
Maternal antithyroid antibodies, goitrogenic drugs, iodine deficiency
Based on data from Peter, F, Muzsnai A. Congenital disorders of the thyroid: hypo/hyper. Endocrinol Metab Clin North Am. 2009;38:491-507; LaFranchi S. Section 2:
Disorders of the thyroid gland. In: Kliegman RM, Behrman RE, Jenson HB, Stanton BF, eds., Nelson textbook of pediatrics, 18th ed. Philadelphia, PA: Elsevier, 2007;
Bettendorf M, Thyroid disorders in children from birth to adolescence. Eur J Nucl Med Mol Imaging. 2002;29(Suppl 2):S439-S446.
Hemiagenesis is another form of dysgenesis with failure in the formation of the left lobe in most cases. This anomaly occurs in less than 0.5% of the population and is more
common in females (e1072). Thyroid function is within normal limits (e750,785).
FIGURE 21-11 ▪ Lingual thyroid. A: Saggital section through the tongue, which shows a smooth, ovoid, 2 cm diameter mass in the posterior third of the tongue (arrow). Small and
large cysts with adjacent red-brown thyroid tissue are present in the mass. Incidental finding at autopsy in a 69-year-old man who died from cerebral hemorrhage. (From Turk JL,
Fletcher CDM, eds. Endocrine system. Royal College of Surgeons of England Slide Atlas of Pathology, 1985. Originally published by Gower Medical Publishing, Ltd. Reprinted with
permission of Elsevier Inc. and C.D.M. Fletcher, M.D.). B: This section of tongue shows the presence of thyroid follicles between the muscle fibers (arrows). This was an incidental
finding at autopsy in a stillborn infant (H&E stain, original magnification 100×).
Ectopia of the thyroid gland, which also has a female predominance, is more thoroughly documented on a morphologic basis than the other types of dysgenesis, as judged by the
descriptions in the literature (eFigure 21-54). The lingual thyroid occurs at the base of the tongue in approximately 1:10,000 individuals and is detected in most cases during a
diagnostic evaluation for congenital hypothyroidism or as an incidentally discovered mass (Figure 21-11A, eFigure 21-52) (e21,e89). The lingual thyroid accounts for approximately
90% of all thyroid ectopias (e742,e887). Most lingual thyroids are accompanied by an orthotopic thyroid (e532); however, a minority of lingual thyroids constitute the only site of
thyroid tissue (e532). Some cases classified as agenesis have a lingual remnant. Ectopic thyroid tissue including dual ectopia (location at different sites) and the exclusion of its
occurrence in a teratoma, has been documented in the submandibular region, trachea, heart, mediastinum, and various intra-abdominal sites (e22, e43,
e273,e304,e461,e663,e742). The presence of thyroid follicles in lymph nodes as so-called lateral aberrant thyroid represents metastatic thyroid carcinoma in many cases (103).
Thyroid neoplasia arising in ectopic thyroid, usually in a TDC, is recognized in children (e490,e1006,e1176).
Ectopic thyroid may be represented by individual microfollicles or small foci of multiple microfollicles or solid nests of follicular cells without apparent colloid formation. The follicles
are interspersed between bundles of skeletal muscle in the tongue or within the tissues of the other ectopic sites (Figure 21-11B, eFigure 21-55). In some instances, the epithelial
structures are not readily identifiable as thyroid
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tissue and may require immunohistochemical staining for thyroglobulin or thyroid transcription factor 1. In addition to the immature or nonfunctioning appearance of the ectopic
follicles, the ectopia is also hypoplastic because the total tissue volume of thyroid is less than normal for the age and sex of the patient.
FIGURE 21-12 ▪ Thyroglosssal duct cyst. A: This midline cyst was filled with tan-white mucoid fluid on gross examination. Fibrosis of soft tissue adjacent to the cyst was present. B:
This composite image shows the resected hyoid bone on the left with entrapped thyroid follicles (arrow). The area within the rectangle is magnified on the right side and shows a
cuboidal epithelium (arrowheads) lining the cystic spaces. A thyroid follicle is also present in this image (arrow). Lymphoid aggregates not shown were also present (H&E stain).
Another form of thyroid dysgenesis is an enlarged lobe composed of immature lobules of fetal-appearing follicles separated by an immature mesenchyme. Nodules of immature
cartilage or other heterologous tissues present within the lobule may suggest the interpretation of a teratoma.
Thyroglossal duct cyst (TDC) is the consequence in the failure of the thyroglossal duct to undergo complete obliteration and regression during fetal life (54,e898). Approximately
15% of all neck masses in children are TDCs with the clinical presentation of a midline anterior neck mass overlying the hyoid bone (54,100,e167,e295,e716). Rather than a midline
location, 10% to 25% of TDCs are found laterally, usually on the left side, and a minority occur at the base of the tongue, floor of the mouth, or within the thyroid itself. The TDC
differential includes branchial cleft cyst, lymphoepithelial cyst, lymphadenopathy including lymphoma, epidermal inclusion cyst, and other thyroid malformations
(100,e716,e387,e930,e1077, e1155,e1169). Most cysts are diagnosed at or before 5 years of age but are recognized throughout life (54,100,e24,e716). A familial association has
been reported (e627). A rare presentation of TDC is sudden death due to asphyxiation (54,e337,e467,e578,e659). Infected cysts may lead to fistula formation to the skin surface or
pharynx (54).
The pathologic findings of TDC vary from case to case with a dominant cyst or several smaller cysts in the soft tissues superior, inferior, or anterior to the hyoid bone (e318,e1185)
(Figure 21-8A, eFigure 21-56). The dominant cyst usually measures 1 to 2 cm; however it may be in excess of 4 to 5 cm in diameter. The contents may have a mucoid or purulent
appearance. TDCs are known to become infected. In some cases, it may be difficult to identify any cysts, but rather a firm, ill-defined fibrotic area that represents prior episodes of
chronic inflammation is present in the soft tissues (Figure 21-12A). Thyroid tissue is generally not appreciated in the gross examination and can be difficult to identify even
microscopically. Individual follicles or larger islands of well-formed follicles are found in less than 50% of cases. Cuboidal to stratified columnar epithelium with cilia lines the cysts in
50% or more of cases (Figure 21-12B). Nonkeratinizing squamous epithelium is present in 25% of cases. The type of epithelium may vary from one cystic structure to another in any
one specimen. The background stroma varies from a mucoid to a dense fibrotic appearance. Lymphoid aggregates adjacent to the cyst or cysts and the ciliated respiratory-type
epithelium have a resemblance to a branchial cleft cyst; however, the branchial cleft cyst typically occurs in the lateral portion of the neck. Psammomatous calcification may be found
in TDCs without accompanying PTC (e65). Fine-needle aspiration biopsy (FNAB) had a positive predictive value of almost 70% in cases of TDC (e1077). Follicular adenomas and
PTCs are reported in 1% to 4% of TDCs (Figure 21-8B) (54,e30,e134,e490,e904,e921, e929,e1006,e1176).
Branchial apparatus-associated anomalies are represented principally by the branchial cleft cyst (eFigures 21-51 and 21-57) (e198,e713,e1019,e1089,e1241). A similar lesion,
the lymphoepithelial cyst, is recognized in the thyroid (e46). The cyst is accompanied by chronic lymphocytic thyroiditis (CLT) in most cases. A bronchogenic cyst has also been
reported in the thyroid. Another type of branchial anomaly is the cyst or sinus from the oropharynx and/or hypopharynx
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with extension into the thyroid with the complication of recurrent acute thyroiditis.
Heterotopias in the thyroid gland include parathyroid, salivary gland, and thymic tissue (e13,e748) (eFigures 21-58 and 21-59).
Acquired Disorders
Persistent diffuse or nodular enlargement of the thyroid gland, regardless of its underlying nature, is referred to clinically as a goiter without any specific pathologic implications.
Through a variety of noninvasive and invasive techniques, including FNAB, an attempt is generally made to ascertain whether the pathological process is inflammatory, hyperplastic,
or neoplastic in nature before a decision is made about the need for surgical intervention (e737,e1271,e1297,e1244). US is helpful in the characterization of a nodule or nodules as
predominantly cystic, cystic and solid, or solid (e302,e1297).
Thyroid nodules are detected in 1% to 1.5% of children with the entire range of pathology from developmental to neoplastic processes [congenital hypothyroidism due to
dyshormonogenesis or ectopia, hemiagenesis, TDC, simple goiter, cystic lesions, nodular hyperplasia, follicular adenoma, Graves disease, and chronic lymphocytic (Hashimoto)
thyroiditis] (45,59,71,77,123,e556,e1279). Nodular hyperplasia (adenomatous hyperplasia) with a dominant nodule, followed by follicular adenoma, is the most common cause of a
thyroid nodule(s) in children (185). Studies have suggested that approximately 20% to 25% of solitary thyroid nodules are malignant with the overwhelming majority representing
PTCs (123, 145). Management of the solitary thyroid nodule is reviewed elsewhere (45, 123, 185, 190). Several studies have addressed the efficacy of ultrasound-guided FNAB of
the thyroid in the pediatric age group with comparable results to those in adults with a diagnostic accuracy in excess of 85% in most cases (e27,e40,e519,e556). Others have
reported a lower diagnostic accuracy rate (e1277).
One of the most common referrals to a pediatric endocrinologist is an enlarged thyroid gland (goiter) (e281). Most cases of a diffusely enlarged thyroid gland (nontoxic goiter) on
physical examination in children are due to autoimmuneassociated inflammatory conditions of the thyroid: CLT, juvenile lymphocytic thyroiditis, juvenile variant of Hashimoto
thyroiditis, autoimmune thyroiditis, and diffuse toxic hyperplasia (Graves disease) (e281,e289,e301,e355,e560,e967).
Chronic lymphocytic thyroiditis (CLT), which accounts for 40% of goiters in adolescents, affects females more commonly than males with a male: female ratio of 1:2 to 1:4,
compared to a 1:10 male:female ratio in adults (17,93,e281,e289). The mean age at diagnosis is 11 to 12 years (range: 1 to 19 years) (e289,e1010).
Rather than a smooth, enlarged gland, in most cases, nodularity may be present in 25% to 30% of cases. Most children (50% to 70%) are euthyroid, or asymptomatic with laboratory
values in the hypothyroid range, whereas 20% to 40% are clinically hypothyroid. Thyrotoxicosis is present in less than 5% of cases (e355,e964). Thyroid peroxidase (TPO)
antibodies are present in 80% to 90% of cases, and antithyroglobulin antibodies in 50% to 60% of cases (e309). Several mechanisms including T-cell mediated cytotoxicity,
cytokine-mediated, and antibody-dependent cell-mediated cytotoxicity directed against follicular epithelial cells are implicated in the pathogenesis of CLT (eFigure 21-60)
(e309,e559,e656,e745,e786).
Most cases of CLT in children are sporadic, but there is an increased association of CLT with HLA haplotypes, DR3, DR4, and DR5 (17, 93). HLA-DR2 and HLA-DQ1 apparently
have a protective effect against autoimmune thyroid disease (103). Polyglandular autoimmune syndrome type I, due to a defect in the autoimmune regulatory gene on chromosome
21q22.3, is defined in part by the presence of CLT; polyglandular autoimmune syndrome type II and type III are uncommon in the pediatric population (80). Systemic lupus
erythematosus, chronic juvenile arthritis, Sjögren syndrome, celiac disease, vitilgo, alopecia, mixed connective tissue disease, Bannayan-Riley-Ruvalcaba syndrome and type I
diabetes mellitus may be accompanied by CLT as part of an autoimmune diathesis (e42,e66,e120,e169,e217,e384,e445,
e501,e537,e576,e609,e639,e665,e666,e687,e797,e891,e962, e963,e433,e630,e814,e821). Approximately 4% of children with type I diabetes mellitus have CLT (e963). Trisomy 21
syndrome, Klinefelter syndrome, and Turner syndrome are three chromosomal disorders associated with CLT (e217,e445,e962). Approximately 25% of young individuals with
Turner syndrome have antithyroid antibodies and 10% have enlarged thyroids (e962,e217).
The pathological diagnosis of CLT is more often established by FNAB than by histological examination. Surgical resection is reserved for specific clinical circumstances, such as a
possible thyroid neoplasm (e867,e895,e1063). The thyroid is symmetrically enlarged and weighs more than 25 to 30 g. A pale, vaguely nodular, tannish-gray appearance with a
resemblance to lymph nodal tissue is noted on cross section after fixation (Figure 21-13A). On occasion, one or the other lateral lobe or the pyramidal lobe is larger with the loss of
symmetry. Any areas of discrete firmness, sclerosis, or nodularity may indicate the presence of PTC or scarring as in the fibrosing stage of CLT. Microscopically, lymphoid follicles
with reactive germinal centers are interspersed throughout the gland with destructive replacement of parenchyma (Figure 21-13B, eFigure 21-61B, C). An intermixture of mature
plasma cells is also apparent in a predominant population of B- and T-lymphocytes. The follicles are typically small and uniform, although some larger follicles with papillary
infoldings may be seen. Some of the intact thyroid follicles may contain intrafollicular histiocytes and giant cells as evidence of so-called palpation thyroiditis or the presence of giant
cells and lymphoid aggregates where follicles once resided. The diminutive follicles are lined by cuboidal or flattened epithelial cells or by epithelial cells with optically clear nuclei
and grooves as seen in PTC. The diagnosis of PTC is made in the presence of a discrete lesion(s). Classic Hürthle
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or oncocytic follicular cells as a diffuse finding are uncommon in CLT in children and, in this respect, do not fulfill the classic morphologic definition of Hashimoto thyroiditis (eFigure
21-62). However, CLT and Hashimoto thyroiditis are pathogenetically identical forms of autoimmune thyroiditis in all other respects. Mizukami et al. found no morphologic difference
in the types of chronic thyroiditis between adults and children younger than 10 years old (e827).
FIGURE 21-13 ▪ Chronic lymphocytic thyroiditis. A: This specimen shows the characteristic diffuse thyroid gland enlargement seen in chronic lymphocytic thyroiditis on gross
examination. A vaguely nodular pattern corresponding to the presence of lymphoid follicles is seen in this cut section. B: Chronic lymphocytic thyroiditis in this low power
magnification image shows prominent lymphoid aggregates interspersed between the thyroid follicles. Plasma cells and lymphocytes were present in the interstitium (H&E stain).
The fibrosing or end-stage of CLT with marked loss and atrophy of follicles, fibrosis with a finely nodular pattern and a diminution of the lymphocytic infiltrate is infrequently
encountered in children. As noted earlier, the morphologic diagnosis of CLT is usually based on FNAB (e256,e951). A mixture of individual and small nonpapillary groups of benign-
appearing follicular epithelial cells in a background of many dispersed small lymphocytes, some plasma cells, and histiocytes is the cytological finding. Hürthle cells are infrequent,
and even less common are papillary profiles of cells, whose presence should raise the possibility of PTC. Approximately 30% of cases of CLT in children had distinct nodules and
3% had a PTC (e44,e256,e688).
Other types of thyroiditis, infections and noninfections types occur in children infrequently (e75,e494,e735,e971,e1076). Abscess of the thyroid has been reported in children, and
opportunistic infections are seen in the immunocompromised setting (e432). Recurrent acute suppurative thyroiditis with or without abscess formation should suggest the presence
of a branchial pouch anomaly such as a pyriform sinus cyst or TDC remnant (54,e224,e452,e825,e826,e848,e1024). Most cultures demonstrate a mixed flora containing a
Streptococcus species (e164). Common features of acute suppurative thyroiditis include a painful/tender neck mass associated with fever. Involvement of the left lobe is more
common. A left hemithyroidectomy may need to be performed for recurrent infections (e224). An infectious etiology should be excluded in granulomatous thyroiditis in a child
because subacute giant cell or deQuervain thyroiditis is extremely rare in childhood.
Hyperplasia of the thyroid gland is either diffuse or multinodular in appearance. Diffuse hyperplasia is often associated with hyperthyroidism or thyrotoxicosis. The so-called simple
goiter is defined clinically as diffuse or nodular enlargement of the thyroid gland without obvious evidence of hyperthyroidism (e376,e380). Children with a simple goiter are
predominantly young adolescent females and do not experience any further gland enlargement. A small percentage, however, may develop CLT (e562).
The simple or colloid goiter is a more or less symmetrically enlarged thyroid gland with a diffuse or multinodular appearance (eFigure 21-63). The follicles vary in size with one or
more colloid-filled macrofollicles lined by a flattened layer of epithelial cells (eFigure 21-64). Formation of colloid cysts occurs in some cases. Multiple variably sized follicular nodules
with or without dense fibrous bands, cystic degeneration, hemorrhage, and nonspecific chronic inflammation are some of the contrasting gross and microscopic features of nodular
or adenomatous hyperplasia (e52). The follicles of the adenomatous nodules may be quite uniform to the extent that on the basis of a small nodule, it may be difficult to differentiate
a follicular adenoma from a dominant nodule in isolation from the other pathological findings. Alternatively, an individual nodule may have cystic changes with hemorrhage,
histiocytes, hyaline-type fibrosis, and calcifications. Papillary profiles are a source of concern in areas of degeneration, but the follicular cells do not have the requisite nuclear
features of PTC. On the other hand, PTC does arise infrequently in children and adolescents within one or more of the adenomatous nodules. A peripheral hyperplastic nodule may
be found in the surrounding soft tissues and even embedded in skeletal muscle as an example of a sequestered nodule.
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Multinodular hyperplasia is the pathological finding associated with the dyshormonogenic goiter (Figure 21-7). One example is Pendred syndrome with a goiter and hearing loss in
adolescence due to a defect in the PDS gene (SLC26A4 gene) on chromosome 7 that encodes for the protein, pendrin, which is involved in iodide transport across the cell
membranes whose absence results in decreased organification of iodide with disruption in thyroid hormone synthesis (e973,e1013). The follicular nodules of a dyshormonogenic
goiter tend to be cellular with the formation of microfollicles, trabecular profiles, and papillary formations with (eFigure 21-65) cellular pleomorphism, nuclear hyperchromatism, and
mitotic figures. Some of these features in a dyshormonogenic goiter can be worrisome with the addition of apparent angioinvasion at the periphery of the nodules. The thyroid has
especially atypical histological features in the presence of deiodinase deficiency. Well-differentiated thyroid carcinoma has been reported in dyshormonogenic goiters, but it is
difficult to judge whether the risk of malignancy is increased in these glands (e29,e36,e316,e791).
Diffuse hyperplasia with clinical hyperthyroidism (Graves disease) is an autoimmune disorder of the thyroid, with some overlapping immunological and pathological findings
with CLT. Hyperthyroidism also occurs infrequently on the basis of “toxic” nodular hyperplasia, functioning follicular adenoma, autosomal dominant nonimmune hyperthyroidism, and
congenital hyperthyroidism (e1194,e1195,e1197). The latter two disorders have been reported with activating germline mutations in the TSH-receptor gene (e284,e389,e448).
Sporadic congenital hyperthyroidism occurs in the presence of maternal autoimmune thyroid disease with the transplacental passage of maternal thyroid-stimulating
immunoglobulins. Only 1% of neonates whose mothers have active Graves disease during pregnancy have evidence of hyperthyroidism at birth (e948,e1306). Most cases of
hyperthyroidisim in children are on the basis of Graves disease (17,e533,e932,e933). Other etiologies of hyperthyroidism in children have been tabulated by LaFranchi (93, 94).
A screening study of school-age population children between 11 and 18 years of age revealed that almost 4% had clinical or laboratory evidence of “thyroid abnormalities” and
approximately 5% of those with abnormalities had hyperthyroidism (e968). This figure compares with other studies in which 10% to 15% of all pediatric thyroid disease is diagnosed
as hyperthyroidism (e690). Juvenile hyperthyroidism typically presents in girls (6:1, female-to-male ratio) who are usually 11 years of age and older (11 to 18 years) and have diffuse
enlargement of the thyroid (95% of cases) or less often have a dominant “toxic” or autonomous nodule (17). Hyperthyroidism occurs in families and is associated with MAS with
activating mutations in the stimulatory G protein (e1305,e1306). Germline mutations in the TSH receptor account for cases of toxic multinodular goiter and toxic thyroid adenoma.
Graves disease is characterized by hyperthyroidism, ophthalmopathy (exopthalmos), and dermopathy (pretibial myxedema) in the pediatric population. It has its peak incidence in
adolescence (11 to 15 years of age) and is three to five times more common in girls (17). The pathogenesis of Graves disease involves T- and B-cell dysregulation leading to the
production of several anti-TSH receptor antibodies, thyroid-stimulating immunoglobulin, thyroid growth-stimulating immunoglobulin and TSH-binding inhibitor immunoglobulin
(eFigure 21-60) (e140-e142). Thyroid-stimulating immunoglobulin mimics TSH and binds to the follicular cell TSH receptor leading to hypersecretion of thyroid hormones. The
thyroid growth-stimulating immunoglobulin also binds to the TSH receptor and stimulates follicular cell hyperplasia with the development of increased serum levels of thyroxine or
triiodothyronine and decreased TSH. The presence of anti-TSH receptor antibodies confirms the diagnosis of Graves disease versus other causes of hyperthyroidism. Total or
subtotal thyroidectomy is performed in those cases of medical failure or intolerance. The clinical management of Graves disease in children is the subject of continued study and
controversy (e447,e972,e991,e1305,e1125,e1153,e1116,e1240,e1280).
Pathologically, the thyroid gland is symmetrically enlarged without apparent nodules in most cases (Figure 21-14A, eFigure 21-66A) (e194). A red-brown color without an
appreciation of translucent colloid is noted on cut surface. The weight of the gland is generally more than 25 to 30 g, but this varies somewhat with the age of the patient. In the
unsuppressed gland, the follicular cells have a tall columnar appearance. Crowding of these cells leads to intrafollicular papillary infoldings on histological examination (Figure 21-
14B, eFigure 21-66B). The colloid has a pale watery appearance and is absent in some follicles. Those follicles with colloid often show peripheral scalloping of the colloid. These
latter findings are usually attenuated with preoperative suppression to diminish the function and vascularity of the gland (eFigure 21-67). Epithelial hyperplasia, through the action of
TSH, leading to more prominent intrafollicular papillary infoldings is seen in the gland treated by thiouracil. Iodine administration before surgery results in the accumulation of colloid
and the formation of macrofollicles. Rather than cuboidal to columnar epithelium lining the intrafollicular papillae, flattened epithelial cells cover the slender papillae. Marked follicular
cell pleomorphism can be seen in pretreated glands.
Lymphocytic infiltrates in the interstitium and lymphoid nodules with reactive germinal centers are prominent in some glands. Without the clinical history of Graves disease, a
diagnosis of CLT may be the preferred interpretation based on histological examination. The intrafollicular papillae may cause concern about PTC; however, the follicular cells lack
the typical cytomorphology of a PTC. At least in the pediatric age population, PTC is rarely found in the midst of diffuse toxic hyperplasia.
Neoplasms
The 2004 World Health Organization classification of thyroid tumors contains a number of histological types but the
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overwhelming majority of differentiated carcinomas of the thyroid in children are PTC. Institutional referral patterns may affect the proportion of MTC in children with RET mutations
in affected kindreds with MEN 2a or MEN 2b. Almost 30% of children with differentiated carcinomas at St. Louis Children's Hospital are MTCs because of MEN 2 referrals to the
institution. FTC and MTC comprise less than 10% of thyroid carcinomas in the experience of most other institutions. Undifferentiated (anaplastic) carcinomas are rare in children in
contrast to adults (e248,e1004).
FIGURE 21-14 ▪ Graves disease. A: This image shows diffuse symmetrical enlargement of the thyroid gland from a patient with Graves disease. The parenchyma has a deep red
color due to increased vascularity within the gland. (Reprinted with permission from Lloyd RV, Douglas BR, Young WF. Endocrine diseases. Atlas of nontumorpathology.
Washington, DC: American Registry of Pathology.) B: This section of thyroid gland from a patient with untreated Graves disease shows follicles with hyperplastic epithelium and
papillary infoldings. Pale watery colloid and an interstitial lymphocytic infiltrate (not pictured) were observed. The papillary infoldings (inset) lack the optically clear nuclei seen in
papillary thyroid carcinoma (H&E stain).
Differentiated carcinomas of the thyroid gland account for only 1% to 3% of all malignant neoplasms in the pediatric age group in North America (e314,e790,e1012). It has an annual
incidence of 2.4:100,000 children, less than 19 years of age (66). The most common histological type is PTC representing approximately 85% to 90% of all thyroid malignancies in
children. The follicular variant of PTC accounts for approximately 25% of PTCs (42,e57,e185,e138,e207, e274,e351,e354,e418,e421,e469,e497,e548,e564,e671,e686,
e769,e830,e1028,e1080,e1097,e1097,e1137,e1208,e1254, e1304). Other than PTC, FTC, and MTC, follicular adenomas, hemangiomas, lymphangiomas, teratomas, and plexiform
neurofibromas are the other types of tumors involving the thyroid in children. FTC and follicular adenoma have been observed in patients with congenital goitrous hypothyroidism
(e29,e36). Follicular adenoma is a relatively frequent cause of a solitary thyroid nodule in children (71). RDD, LCH, and hematolymphoid malignancies are examples of infiltrative
processes involving the thyroid in children (e306,e434,e684,e926,e1023,e1152,e1182,e1209). There is also the spindle-epithelial tumor with thymus-like differentiation that presents
in the thyroid.
Most carcinomas of the thyroid in children are diagnosed between 13 and 16 years of age, but individual cases have been reported throughout childhood, even in the newborn
(e175,e342,e791,e819). The female:male incidence is approximately 1:1 in carcinomas diagnosed prior to adolescence, but with a 3-6:1 female predominance during adolescence.
Many recent studies have looked at the molecular events underlying the development of thyroid cancer (59,e258, e300,e1071,e1114). A number of somatic mutations involving the
RET gene have been identified in sporadic PTC (42,59,e328,e721,e874,e1071); RET/PTC1 and the RET/PTC3 gene arrangements are found in a variable proportion of PTCs in
children (e258,e360). In children not exposed to radiation, the RET/PTC1 rearrangement is more frequent than the RET/PTC3 rearrangement, which is more common in radiation-
induced thyroid cancer (42,185,e360,e721). The “classic” papillary pattern is associated with RET/PTC1 rearrangement whereas the RET/PTC3 is found more often in the follicular
variant of PTC. In terms of behavior, PTCs with the RET/PTC3 gene rearrangement appear to have a somewhat more aggressive course than PTCs with the RET/PTC1
rearrangement (e694). Mutations involving BRAF are uncommon in PTCs in children less than 15 years of age at diagnosis (59,e924). FTC and follicular adenoma are associated
with RAS and PAX8-PPARγ (peroxisome proliferator-activated receptor gamma) mutations but these mutations do not distinguish between the two neoplasms (59,185,e367,e1114).
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MTCs, in contrast, demonstrate distinct mutations in the RET gene. Mutations in the RET proto-oncogene in the pericentromeric region of chromosome 10q11.2 have been identified
in three autosomal dominant syndromes, MEN 2a, MEN 2b, and FMTC (42,59,81,e772,e225,e299,e331,e332,e431,e942). The RETgene codes for a transmembrane receptor
tyrosine kinase that is involved in development of the kidney and nervous system. The gene spans 21 exons. Each of these syndromes involves mutations with different codons
(eFigure 21-68). MEN 2a and FMTC more frequently involve missense mutations in exons 10 and 11 involving codons 609,611,618,620, and 634. MEN 2b has a characteristic
mutation in exon 16 (codon 918) in 95% of cases and in codon 883 in exon 15 (42,e772).
PTC presents with a painless or a tender mass in the thyroid gland. Palpable cervical adenopathy at diagnosis is common since regional lymph node metastasis is present in 30% to
80% of children at diagnosis (45,132,185, e1080). Most cases of PTC are sporadic, but a family history should be sought since there are several familial-associated tumor
predisposition syndromes (e286). There is an increased incidence of PTC in children who have received radiation therapy for a prior neoplasm in the head and neck
(e125,e132,e358,e806,e1074,e1234). In one study, the average interval between the delivery of radiation and the diagnosis of carcinoma was 8.5 years, with approximately 75% of
patients exposed between 3.5 and 14 years before the development of the carcinoma (e99). An increased incidence of thyroid cancer with the signature RET/PTC gene
rearrangement was detected in children as early as 4 years after exposure to fallout from the Chernobyl nuclear reactor explosion in 1986 (e45,e391,e553,e554,e557,e870-
e873,e875, e876,e905,e909,e1037,e1208,e1252). Ten to thirty percent of children with PTC and no history of radiation exposure demonstrate RET/PTC gene rearrangement in
contrast to 50% to 70% in children with a history of radiation exposure (59). Nonneoplastic abnormalities such as multinodularity, fibrosis, and lymphocytic infiltrates have also been
reported in the thyroid gland after prior neck irradiation (e873).
PTC, as well as the follicular adenoma and carcinoma, is found in association with MAS (e249) and MEN 1 (parathyroid hyperplasia, islet cell hyperplasia, and PA) but not in MEN
2a or MEN 2b, in which MTC is the rule (e636). Other familial settings of non-MTC are Carney complex, familial adenomatous polyposis, and Cowden syndrome (42,e189,
e210,e211,e212,e213,e710,e721,e727,e931,e1140).
The gross features of PTC are variable from one or more solid, grayish-tan nodules; dense, poorly circumscribed foci of fibrous effacement of the normal gland; a cyst(s) with a
mural nodule or solid, reddish glistening nodule with a fibrous capsule (e254) (Figure 21-15A, B). Calcifications may be present. The classic papillary and follicular variants of PTC
usually present as a well-circumscribed, encapsulated tumor, whereas (e1004,e1186) the diffuse sclerosing variant is a poorly circumscribed focus of dense fibrosis replacing the
thyroid parenchyma and often extending into the surrounding soft tissues including the skeletal muscle. The sclerosing variant, though uncommon, is seen more often in children
than in adults. Among PTCs in children, the classic papillary type, follicular variant, solid type, mixed papillary and follicular pattern, and sclerosing variant were present in 11%,
35%, 30%, 17%, and 8% of cases, respectively and among adolescents, 26%, 28%, 24%, 20%, and 2% of cases, respectively (e1208).
The classic PTC is composed of branching fronds or papillae with fibrovascular stalks (Figure 21-15C, eFigure 21-69). Regardless of the particular histological pattern, the
pathologic diagnosis of PTC is based largely upon nuclear features including crowded and overlapping nuclei with an elongated cleaved appearance, often with prominent nuclear
grooves or folds, margination of chromatin with clearing of the nucleoplasm (“optically clear” or “Orphan Annie” nuclei) (e215) and cytoplasmic invaginations or nuclear
pseudoinclusions (Figure 21-15D, eFigure 21-70). Small concentric whorls of calcification (i.e., psammoma bodies) are present more commonly in the classic and sclerosing variants
of PTCs than in the other variants, especially the follicular variant (Figure 21-15E). Multifocal gross lesions, but more commonly multiple microscopic foci of PTC, are identified in the
ipsilateral and the contralateral lobe in 20% to 25% of cases. The latter finding is the rationale for subtotal-total thyroidectomy (e151,e472,e592,e1070, e1127,e1265).
Squamous metaplasia is a feature of PTC in the pediatric age group, which may cause some concern about the possibility of a higher grade thyroid carcinoma (42,e721). In other
cases, varying degrees of a desmoplastic stromal reaction may be encountered, which is so prominent as to superficially resemble the “amyloid stroma” of MTC; in other instances,
the fibrosis is associated with the infiltrative growth pattern of the sclerosing variant, which is associated with angiolymphatic invasion and numerous psammoma bodies. The
architecture of the follicular variant of PTC is exclusively follicular but diminutive intrafollicular micropapillae are seen with some frequency. Focal areas of classic PTC may be
present in some cases in other areas of the thyroid. The follicular variant of PTC is distinguished from the well-differentiated FTC by the presence of the characteristic nuclear
morphology of PTC. Capsular and vascular invasion are present in both PTC and FTC.
Lymphocytic infiltration of the surrounding thyroid is a common feature in PTC regardless of age, and its presence has been associated with an improved prognosis
(185,e730,e895). Regional lymph node metastasis is present in 30% to 80% or more of cases overall. Pulmonary metastasis is found in 6% to 8% of pediatric cases at diagnosis
(45,185,e672,e1029,e1224) although some series report a higher incidence (45).
The diagnosis of pediatric thyroid cancer is made for the most part on specific histopathological criteria.
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Immunohistochemistry is not usually necessary in most cases of non-MTC although the occasional solid PTC or FTC may require differentiation from MTC (e367). The tumor cells in
PTC are immunoreactive for cytokeratins, thyroglobulin, and TITF-1 (thyroid transcription factor-1) (42,e367,e721). Cytokeratin 19 is strongly expressed in PTCs. Staining for
RET/PTC rearrangements has also been utilized, but availability of sensitive antibodies is a limiting factor (42,e367,e721).
FIGURE 21-15 ▪ Papillary thyroid carcinoma. A: Section of thyroid gland from a young adult showing a solitary, tan 2.5-cm diameter well-circumscribed nodule. B: This young adult
had a history of radiation to his neck as a young child for tonsillar hypertrophy. On gross examination, two distinct well-circumscribed nodules with focal hemorrhage and necrosis
are seen. C: This low power image shows the typical papillary fronds with central fibrovascular core characteristic of papillary carcinoma (H&E stain). D: High power image of a
papillary frond showing the characteristic optically-clear nuclei and nuclear grooves characteristic of papillary carcinoma (H&E stain). E: Low power image of a papillary carcinoma.
Multiple psammoma bodies (foci of dystrophic calcification) are present in the background. Higher magnification (inset) show the characteristic concentric rings seen in a psammoma
body. Their presence strongly suggests a diagnosis of papillary carcinoma (H&E stain).
The prognosis in children with PTC is excellent despite the presence of local extrathyroidal spread (40% to 50% of
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cases) and lymph node metastasis (e175,e459,e563,e670, e865,e1056,e1302). The presence of invasion in the soft tissues of the neck from the primary or extranodal site
contributes substantially to the local morbidity of the disease.
The extent of disease and age at diagnosis are important prognostic features. Management is surgical resection in most cases with additional modalities in some cases (45).
Prognostically unfavorable histological variants of PTC are uncommon in children such as the tall cell, dedifferentiated, and poorly differentiated variants. Postoperative staging is
based on a combination of factors. The MACIS (metastasisage-completeness of resection-invasion-size) system has been found useful in children (45). In children less than 10
years of age, PTC is more locally aggressive and more likely to have pulmonary metastasis. Overall, the long-term survival rate for PTC is excellent in children with a 98% 10-year
survival, regardless of the pathologic stage.
Follicular neoplasms of the thyroid present several problems in pathological diagnosis without regard for age. One of the less consequential ones is the differentiation of a
follicular adenoma from a dominant nodule of multinodular or adenomatous hyperplasia. In some cases, it is a distinction without a difference in terms of prognosis. Follicular lesions
diagnosed pathologically as an adenoma have a delicate continuous or interrupted fibrous capsule separating the relatively monotonous follicular architecture to larger, more
variably sized follicles (eFigure 21-71). Follicular adenoma is a sporadically occurring tumor in most cases in children, but is reported in young individuals with Cowden syndrome
and pleuropulmonary blastoma familial tumor predisposition syndrome (e470,e1000).
FIGURE 21-16 ▪ Follicular thyroid carcinoma. A: This section of thyroid gland shows a well-circumscribed nodule with a thick irregular capsule within the thyroid parenchyma. The
neoplasm was composed of small well-defined follicles on histological examination. No well formed papillae or psammoma bodies were present. The nuclei did not have the optically-
clear appearance or nuclear grooves characteristic of the follicular variant of papillary carcinoma. B,C: Invasion of the adjacent capsule and blood vessels was present (H&E stain).
FTC is diagnosed pathologically, not on the basis of nuclear features which are often quite bland, but on the presence of a well-defined thickened, circumscribed fibrous capsule
with preferably more than one focus of transcapsular invasion as a “mushroom” of neoplastic follicles protruding through the capsule. Microvascular invasion in the capsule is
another diagnostic feature, but there should be adherence of tumor cells to the endothelium of the vessel or vessels and not free-floating tumor cells or pressing into a vascular
space with an interposed intact endothelium (Figure 21-16A to C, eFigure 21-72). It is common to identify groups of follicles pressing on capsular vascular spaces in a follicular
adenoma or dominant adenomatous nodule, which should not be interpreted as vascular invasion. An accurate diagnosis of an encapsulated, well-differentiated FTC often requires
extensive sampling of the fibrous capsule with well-oriented sections and the use of vascular endothelial markers or an elastic stain to confirm “bona fide” vascular invasion. The
distinctive nuclear features of PTC distinguish the follicular variant of PTC from FTC.
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FTC is immunoreactive for thyroglobulin, TITF-1, and low molecular weight cytokeratins (e367).
Moderate to poorly differentiated carcinomas of the follicular and papillary types are distinct yet uncommon neoplasms in children (e190,e479,e611,e642,e725,e997,
e1026,e1096,e1301). Some of these solid nested tumors are examples of insular-like carcinomas. These tumors present as well-defined neoplasms grossly with invasion often
appreciated during gross examination.
In general, intraoperative frozen section examination is often a frustrating exercise to resolve the differential diagnosis among the follicular variant of PTC, follicular adenoma,
dominant adenomatous nodule, and well-differentiated FTC. The characteristic optically clear nuclei of PTC are not seen in frozen sections or touch preparations, and although
nuclear grooves are helpful, they are insufficient alone for a specific diagnosis of PTC. Furthermore, the separation of the encapsulated, well-differentiated FTC from follicular
adenoma may require processing multiple blocks of tumor with capsule, and then multiple levels through individual blocks which is unsuitable for frozen section analysis.
MTC in children occurs almost exclusively in the familial setting with or without the other features of MEN 2a (MTC, diffuse parathyroid hyperplasia, and PHEO) or MEN 2b (MTC,
PHEO, intestinal ganglioneuromatosis, and mucosal neuromas) (e197,e345,e498,e543,e833,e890,e919,e1145).
Only 1% to 3% of differentiated thyroid carcinomas in children are MTCs except in some specialized medical centers (59,145,e1266). Sporadic MTCs, which are palpable, unifocal
neoplasms without C-cell hyperplasia, account for 80% to 90% of all MTCs in adults and children, however, are uncommon in children. The aggressive nature of MTC is evident in
adults who have regional lymph node metastasis in 50% or more of cases and distant metastasis (lung, liver) in 15% of cases at diagnosis (42,e772,e969,e1172). It is noteworthy
that approximately 20% of adults with apparent sporadic MTCs have germline RET mutations with its obvious familial implications (45).
Syndromic-associated MTCs in children are typically small, often microscopic, multifocal tumors in association with diffuse C-cell hyperplasia in the upper two-thirds of the lateral
lobes (e493). The small size of the tumor or tumors in syndromic MTC is in part a reflection of genetic screening of children in affected kindreds using molecular diagnostic
techniques (e680,e683,e719,e1101,e1218). Virtually all resected thyroids in the setting of MEN 2a, MEN 2b, and FMTC have microscopic multifocal C-cell hyperplasia, if not
microscopic or infrequently grossly visible tumors (e653).
On gross examination, MTCs present as a wellcircumscribed or infiltrative mass that is gray-white to tan in appearance. The individual tumors range from 1 mm or less to 4 to 5 cm
in diameter (Figure 21-17A, B). The several histological patterns include the common, compact solid-rounded nests to lobular, insular, or trabecular profiles. Whether the tumor cells
are rounded or spindled, the polygonal nuclei have finely dispersed chromatin and a prominent nucleolus. There are also small cell and even pigmented variants of MTC. Mitotic
activity and anaplasia are inapparent in most cases. Intersecting bands of fibrosis and/or an amyloid stroma are generally found in those tumors in excess of 2 cm in diameter (Figure
21-17C, eFigure 21-73).
One of the challenges in the pathological examination in syndromic cases is the differentiation between C-cell hyperplasia and microscopic MTC. The degree and extent of C-cell
hyperplasia can vary markedly from one prophylactic thyroidectomy to another. Foci of C-cell hyperplasia can be relatively inconspicuous without the assistance of
immunohistochemistry. In other cases, the C-cell hyperplasia is not only apparent, but extensive to the degree that there is concern about microscopic MTC. The hyperplasia is
recognized by a collection of C-cells partially filling the colloid space of the follicle and/or bulging into the perifollicular, interstitial space without breaching of the basement membrane
of the follicle (not always readily apparent). A microscopic MTC has a similar bulging growth from the follicle as C-cell hyperplasia, but more importantly, there is interstitial infiltration
and the displacement, if not the overgrowth, of contiguous follicles or coalescence of aggregates of enlarged, atypical cells. The tumor cells are larger than those of the surrounding
smaller hyperplastic C-cells, and the nucleoli are prominent in comparison to the inapparent or micronucleoli of the hyperplastic C-cells.
Immunohistochemical staining for calcitonin is helpful in the identification of inconspicuous foci of C-cell hyperplasia, or in the confirmation of the thyroid carcinoma as MTC (Figure
21-17D, eFigure 21-74). C-cells and MTC are also immunoreactive for SYN, CHR, and CEA, and nonreactive for thyroglobulin and TITF-1 as in PTC and FTC (103). In keeping with
the distribution of C-cells in the thyroid, hyperplasia and MTC have a predilection for the upper two-thirds of the lateral lobes. It is helpful to submit multiple sections from the superior
to the inferior pole of the resected gland.
Early prophylactic thyroidectomy with lymph node dissection and serum calcitonin levels in children with germline RET mutations is the recommended management (81,e1103).
Based on the specific RET codon involved, specific risk groups have been established with recommended surgical intervention dependent on the risk group (45,81,e1103,e1162). In
young children, most cases of MTC are associated with MEN 2b, and for this reason, thyroidectomy is recommended as soon as possible in MEN 2b RET-positive infants, whereas
in MEN 2a, surgery is recommended between 3 and 5 years of age (81). Prognosis is dependent on stage and postoperative calcitonin levels. Syndromic MTC with total
thyroidectomy and negative postoperative serum calcitonin levels at 6 and 12 months postsurgery have a recurrence rate of 5% and a 10-year survival rate of 98% (45).
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FIGURE 21-17 ▪ Medullary thyroid carcinoma. A: This image from a sporadic (nonsyndromic) medullary carcinoma shows a large tan-yellow, non-encapsulated mass that had a firm
gritty consistency on sectioning. B: Syndromic medullary carcinoma tends to be small and multifocal. This 0.4 cm in diameter tumor nodule (right) in a patient with MEN 2 is
demarcated from the red-brown thyroid parenchyma. A smaller nodule of medullary carcinoma (left) is also seen. C: This tumor on low power demonstrates the characteristic lobular
pattern. Nests of tumor cells with round nuclei were surrounded by bands of connective tissue. The round to polygonal tumor cells has an abundant eosinophillic or clear cytoplasm.
The nuclei are predominantly round to oval with coarse chromatin. The cells were immunoreactive for calcitonin (H&E stain). D: This section of thyroid gland from a child with MEN 2
shows a small focus of medullary carcinoma (arrow) and several foci of C-cell hyperplasia that were immunoreactive for calcitonin (immunostain for calcitonin).
Cervical-thyroidal teratoma (CTT) accounts for 3% or less of thyroid resections in children especially in the infancy period. Approximately 2% to 5% of germ cell neoplasms in
children present in the head and neck region, and the anterior portion of the neck including the thyroid gland is one of several specific sites in this anatomic region
(e200,e396,e604,e1255). There is an equal male to female ratio (68, 182, 1183). These tumors are typically congenital and are not subtle clinically given their size. Byard et al.
reported that 6 of 14 (43%) of cases were detected in stillborn infants or neonates who died within 2 days of birth (e182). Compression of the upper airway is the major complication
requiring early surgical intervention. A minority of cases are known to present beyond the infancy period. The mass fills the soft tissues of the anterior and lateral portions of the
neck. An attachment to the thyroid and its infiltration is not always demonstrable due to the size and extensive replacement of normal tissues. Grossly, these tumors are soft and
often cystic, measuring several centimeters in greatest dimension and microscopically are composed of a range of immature
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and some mature somatic elements. Immature neuroepithelium with primitive neural tubules and sheets of neuroblasts are often the dominant microscopic pattern and should not be
mistaken for neuroblastoma (NB) which can present in the cervical region but more lateral and with an exclusive neuroblastic appearance, usually poorly differentiated NB (e1238).
One confounding aspect is the presence of immature or mature teratoma in regional lymph nodes in some cases, which we have preferred to designate as “nodal gliomatosis” and
others have referred to nodal “deposits” rather than metastasis (e91,e293,e597,e1203). The excellent clinical outcome of CTT is usually not affected by the presence of nodal
deposits. As in sacrococcygeal teratomas, microscopic foci of endodermal sinus may be detected (e293,e669). If these foci of endodermal sinus tumor represent only a minor
component and are not in regional lymph nodes, the excellent prognosis may be affected in only a marginal fashion, but the decision about further management is complicated.
ADRENAL GLANDS
The adrenal glands are composed of an outer cortex and an inner medulla. Functionally, they are two separate endocrine organs, with the cortex responsible for steroid hormone
synthesis and the medulla for catecholamine production. In children and adolescents, the pyramidal-shaped right and crescentshaped left adrenal glands have an average combined
weight of 4 to 6 g, similar to adults with combined adrenal weights ranging between 2 and 8 g (87, 104). There is no difference in the weight of the adrenals between male and
female children. A coarse connective tissue capsule with attached periadrenal fat surrounds the gland. The fat in the vicinity of the adrenals has immature features of finely
vacuolated adipocytes in infants resembling those of a lipoblastoma. On sectioning, the adrenal cortex consists of a yellow subcapsular layer that corresponds to the zona
glomerulosa and zona fasciculata. A thin brown layer, the zona reticularis, separates the zona fasciculata from the gray-white central medulla. The adrenals receive their blood
supply from the inferior phrenic artery, aorta, and renal artery.
In the fetus, a prominent provisional (fetal) cortex is present. Adrenal weight ranges upward with the gestational age. There is rapid growth of the provisional cortex during the third
trimester. The average combined weight of the adrenals is about 2 g at 30 weeks' gestation compared to 6 g at birth in a term infant. Following birth, the provisional cortex involutes
and rapidly disappears, leaving the permanent cortex and central medulla (87) (see Appendices: Weights of Organs of 1- to 12- Month-old Girls, and Weights of Organs of 1- to 12-
Month-old Boys). The involuted remnant of the fetal cortex can be observed throughout the first 6 months of life. In the newborn, the adrenal gland on sectioning has a dark
redbrown appearance beneath a thin yellow cortical rim due to degeneration of the provisional cortex. The adrenal medulla, which makes up less than 1% of the fetal adrenal
compared with 10% of the adult adrenal, is generally not recognized on gross examination.
The definitive adrenal cortex is divided into three distinct zones. The zona glomerulosa, which accounts for approximately 10% of the adult adrenal gland, consists of islands of
haphazardly distributed cells beneath the connective tissue capsule. Individual cells contain small amounts of eosinophilic cytoplasm and have rounded nuclei. The zona fasciculata
located beneath the zona glomerulosa accounts for 70% to 80% of the adult adrenal cortex and consists of large polyhedral, lipid-laden cells arranged in columns 1- to 2-cells thick
separated by thin sinusoidal capillaries in the nonstressed adrenal gland. The nuclei are round, pale staining, and occasionally binucleated. The zona reticularis, which accounts for
less than 10% of the cortex, consists of anastomosing cords of small eosinophilic cells with deeply staining closely apposed nuclei. The adrenal medulla, which occupies the center
of the gland, consists of large, pale staining, polyhedral cells, known as chromaffin cells, which are arranged in cords and small islands. These cells, innervated by preganglionic
sympathetic nerve fibers, are modified postganglionic neurons.
In the fetus, the provisional (fetal) zone accounts for 70% to 80% of the total weight of the gland (eFigure 21-75) (e129,e130,e437). The fetal zone, composed of cords of large
eosinophilic cells surrounded by sinusoidal capillaries, is located beneath the permanent cortex. A distinct adrenal medulla is not identifiable in the fetal gland. Chromaffin cells,
however, are haphazardly scattered throughout the fetal cortex. In fact, neuroblastic cells from the neural crest migrate through the cortex as individual and small nests of primitive
appearing cells. These cells should not be interpreted as evidence of congenital NB.
Adrenal gland development is dependent on a number of factors. Steroidogenic factor (SF-1) encoded on chromosome 9q33, and DAX1, encoded on chromosome Xp21, are two
critical transcription factors required for adrenal gland development and steroidogenesis (95,188,e115,e715). Growth and maturation of the gland is also dependent on ACTH
stimulation.
The adrenal cortex is responsible for the synthesis of three classes of steroids, glucocorticoids, mineralocorticoids, and androgens. A series of cytochrome P450 enzymes are
involved in adrenal steroid synthesis (eFigure 21-76). The rate-limiting step is the transfer of cholesterol from the cytosol across the mitochondrial membrane. Several proteins,
including the steroidogenic acute regulatory protein (StaR) induced by ACTH, are involved in this rate-limiting step. In response to ACTH stimulation, cholesterol is metabolized
through a series of enzymatic steps in the zona fasciculata and reticularis into Cortisol, the major glucocorticoid (eFigure 21-77). Once secreted, Cortisol provides negative feedback
on the pituitary gland to inhibit further ACTH secretion. Aldosterone, synthesized in the zona glomerulosa, is a mineralocorticoid. Dihydroepiandrosteindione sulfate is the major
androgen and is primarily synthesized in the zona reticularis. During early fetal development, androgens of adrenal origin are responsible for differentiation of the male external
genitalia.
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The cathecholamines, epinephrine, and norepinephrine, are formed from tyrosine and secreted in response to sympathetic neural stimulation by the chromaffin cells. Extensive
reviews of adrenal steroidogenesis and catecholamine production are beyond the scope of this chapter but are widely available (12,87,96,e312,e1189).
Imaging
The visualization of the adrenal in infants is accomplished optimally by US. In older children, US is useful as a screening method for an adrenal mass. If an adrenal mass is
discovered, further imaging with MR or CT is indicated.
The normal adrenal glands of an infant on US are Y- or V-shaped on longitudinal images. The medulla is seen as an echogenic (bright) central line surrounded by the thin
hypoechoic (dark) cortex. A long straight adrenal gland may be seen in cases of renal agenesis or ectopia. In the presence of congenital adrenal hyperplasia, the adrenal glands are
enlarged with an abnormal undulating surface and/or replacement of the central echogenic line by a stippled pattern throughout the gland (e23). An adrenal mass in the neonate is a
cyst, hemorrhage, or congenital NB. In the case of adrenal hemorrhage, US provides the most useful modality in the follow-up period without the need for CT or MR (e823). The
echogenicity of the hemorrhage varies with the age of the hemorrhage and is usually heterogeneous. No flow is demonstrated to the mass on Doppler evaluation. On follow-up the
mass becomes smaller and more hypoechoic (dark) over time. The adrenal gland may become calcified, appearing as a dense focus with posterior acoustic shadowing (e823). NB
should be suspected if the mass fails to diminish in size on short interval follow-up. It should be noted that NB in neonates are more likely to be cystic than in an older child and these
cystic NBs may become smaller over time, but do not entirely resolve as a cystic hemorrhage. Calcifications may be identified in both lesions. The neonate may present with the
particular pattern of metastases of stage 4S disease with diffuse involvement of the liver, nodules of the skin, and infiltration of the bone marrow. Adrenal cysts have a varied
appearance on imaging depending on the presence or absence of complicating hemorrhage or infection. Simple cysts are anechoic (black) on US and of fluid attenuation on CT or
MR. Hemorrhage or infection causes a heterogeneous appearance to the internal structure of the cyst and the wall of the cyst may be calcified (Figure 21-18A to C).
FIGURE 21-18 ▪ A: Hemorrhagic adrenal cyst in an 18-year-old girl. Axial unenhanced CT image shows a mass in the right suprarenal region (arrowhead) which is denser than the
left kidney (LK) indicating acute hemorrhage. B: Coronal postgadolinium Tl-weighted image showing no enhancement of the cyst (arrow) above the enhancing right kidney. C: Image
of the sectioned resected specimen showing hemorrhage.
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FIGURE 21-19 ▪ Adrenal cortical tumor in 18-month-old girl with virilization. CT image without intravenous contrast material shows a mass in the region of the left adrenal fossa
(arrowhead) adjacent to the spleen (S).
If an adrenal mass is initially discovered by US, further imaging with CT or MR is necessary to characterize the nature of the mass. The imaging appearance of adrenal cortical
tumors on US, CT, and MR depends on their size (e920). Small tumors tend to appear homogeneous, whereas in larger tumors, central necrosis, calcification, or scar causes a
heterogeneous appearance (Figure 21-19, eFigure 21-78). Local spread or metastatic disease may be evident indicating an aggressive tumor. Extension into the vena cava should
be sought.
NB typically appears as a mass in excess of 2 cm in most cases (Figure 21-20). Calcifications are relatively frequent, which is particularly evident on CT but may also be seen on US
as echogenic foci possibly with posterior acoustic shadowing. Cystic areas from old hemorrhage or cystic or necrotic changes are anechoic (black) on US and of fluid attenuation on
CT and MR. After injection of contrast material, the tumor generally enhances heterogeneously. CT and MR are useful in evaluating the extent of disease. The primary tumor
frequently crosses the midline and surrounds the aorta and other vessels (Figure 21-21). Adjacent organ involvement may be seen and enlarged lymph nodes and liver metastases
may be identified (Figure 21-22). MR is particularly useful in demonstrating neural foraminal and spinal canal invasion (eFigure 21-79). PHEO appears as a soft-tissue mass on CT
with homogeneous, heterogeneous or rimlike enhancement postcontrast (e920). On MR, PHEOs are hypointense (dark) on T1-weighted images and hyperintense on T2-weighted
images. Postgadolinium images typically show intense enhancement with slow washout (Figure 21-23) (e1073). As lesions are frequently multiple, the radionuclide scan using131I-
MIBG may be helpful in the preoperative localization of lesions.
FIGURE 21-20 ▪ Neuroblastoma in 2-month-old boy. A: Longitudinal ultrasound image demonstrating a homogeneous mass (arrow) between the upper pole of the left kidney (LK)
and the spleen (SPL). B: The sectioned gross specimen shows a homogeneous yellowish-tan tumor and calcifications.
Agenesis, congenital adrenal hypoplasia (CAHP), congenital adrenal hyperplasia, and adrenal gland heterotopia are the major structural and biochemical disorders of a congenital
or developmental nature (Table 21-6). Except in the setting of anencephaly or other syndromes in which there is adenohypophyseal dysfunction or absence, bilateral adrenal
agenesis is rare (e319). Unilateral adrenal agenesis may be seen in combination with other malformational syndromes and in the setting of unilateral renal agenesis. Adrenal
fusion, characterized by the midline union of the adrenal glands giving the fused glands a horseshoe or butterfly shape (horseshoe adrenal gland), is rare and is associated with
other congenital anomalies (e624,e1150,e1294) (Figure 21-24A). Renal-adrenal fusion (accreta) and hepatic-adrenal and renal-adrenal union, characterized by
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intermingling of parenchymal cells of both organs, are uncommon in childhood (Figure 21-24B, eFigure 21-80) (e508). Alteration of adrenal shape occurs in the presence of renal
agenesis where the adrenal gland acquires a flat disk-shape in contrast to its normal triangular appearance (eFigure 21-81).
FIGURE 21-21 ▪ Neuroblastoma in 9-month-old boy. A: Contrastenhanced CT image of the abdomen demonstrates a mass (M) pushing the left kidney (LK) laterally and
surrounding the aorta (arrow). B: The sectioned gross specimen shows a multinodular, whitish-tan tumor with hemorrhage.
FIGURE 21-22 ▪ Congenital stage 4 neuroblastoma with Pepper syndrome. A: KUB shows marked enlargement of the liver pushing up on the hemidiaphragms and pushing the air-
filled bowel to the left lower quadrant. B: Contrast-enhanced CT scan of the abdomen shows a markedly enlarged liver diffusely infiltrated with small hypoattenuating masses (L).
Anterior to the right kidney (arrow) is an adrenal mass (arrowheads).
Ectopic adrenal tissue is usually observed in the abdominal cavity along the celiac axis or along the pathway of
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gonadal descent (e436). Intrarenal ectopia can simulate renal cell carcinoma or an invasive adrenal neoplasm (e1294). Adrenal ectopia is found in as many as 10% of orchiopexies
and in approximately 4% of inguinal herniorrhaphies (e605,e760,e799,e894) (see Chapter 19). Ectopic adrenal has also been observed in the lung, liver, brain, ovary, and placenta
as a rare isolated event (eFigure 21-82) (e11,e149,e510,e673, e1230,e1262,e1287). Most ectopic adrenal tissue, especially at distant sites, includes only adrenocortical tissue with
distinct cortical zonation in some instances. Small islands or nodules of adrenal cortical tissue can be found with some frequency in the fat surrounding the orthotopic adrenal gland.
True adrenal gland heterotopia, in which the adrenal gland is absent from its normal location and an adrenal gland with both cortex and medulla is identified, is usually present in the
vicinity of the celiac axis but has also been found at distant sites including the brain.
FIGURE 21-22 ▪ (continued) C: Massive hepatomegaly reflects the diffuse infiltration by neuroblastoma.
Wolman disease together with the related cholesterol ester storage disease is a heritable disorder characterized by an inborn error of acid lipase A deficiency (10q23.2-q23.3).
Vomiting, steatorrhea, failure to thrive, hepatosplenomegaly, and adrenomegaly with bilateral adrenal calcifications visible radiographically are seen in the neonatal period (Figure
21-25A) (e56,e412,e777). Cholesterol and triglycerides accumulate in the lysosomes of the liver, spleen, adrenal glands, gastrointestinal tract, hematopoietic organs, and brain. The
adrenal glands are symmetrically enlarged, yellow, and firm. There is a prominent yellow cortical rim and gray-white center (Figure 21-25B). The zona glomerulosa and outer zona
fasciculata are histologically unremarkable, but the fetal zone, zona reticularis and inner zona fasciculata have been replaced by haphazardly arranged foamy cells,
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which are accompanied by focal areas of necrosis and calcifications (Figure 21-25C). Cholesterol clefts may be identified as well (eFigure 21-83) (see Chapter 5).
FIGURE 21-23 ▪ Pheochromocytoma in a 12-year-old girl with hypertension. A: Axial contrast-enhanced CT image shows a heterogeneous mass (arrowhead) anterior to the right
kidney (RK). Also noted is a mass of the pancreatic tail which was a neuroendocrine tumor (arrow). B: Axial T2-weighted MR image demonstrates a heterogeneous,
predominantlyhyperintense mass in the right suprarenal fossa (arrowhead).
Table 21-6 ▪ CONGENITAL DISORDERS OF THE ADRENAL GLAND
Agenesis
Adrenal cytomegaly
Adrenal fusion
Congenital adrenal hypoplasia
Anencephalic form
Cytomegalic form (NROB1 gene defect, DAX1 mutation)
Miniature form
Congenital adrenal hyperplasia
21-hydroxylase deficiency (CYP21A2 gene defect)
11 β3-hydroylase deficiency (CYP11B1 gene defect)
17 α-hydroxylase deficiency (CYP17A1 gene defect)
3β-hydroxysteroid dehydrogenase deficiency (HSD3β2 gene defect)
Cholesterol desmolase deficiency (StAR protein defect) (congenital lipoid adrenal hyperplasia)
Ectopia
Metabolic disorders
Adrenoleukodystrophy
Wolman disease
FIGURE 21-24 ▪ Adrenal maldevelopment. A: Adrenal fusion. Midline fusion of two otherwise normal adrenal glands was found at autopsy in a newborn infant with multiple
malformations. B: Adrenal accreta. The adrenal gland (arrow) was firmly adherent to the adjacent kidney at autopsy. Both organs were separated by a joint band of connective
tissue on histological examination.
FIGURE 21-25 ▪ Wolman disease. A: Plain radiograph of the abdomen reveals triangular-shaped collections of mottled calcifications in the expected location of the adrenal glands.
B: Both adrenals are enlarged and deep yellow in color due to accumulation of cholesterol esters within the adrenal cortex in this autosomal-recessive inherited disease. Image from
www.humpath.com. (Reprinted with permission, Dr. Jean-Christophe Fournet, CHU Sainte-Justine, Montreal, Canada.)
FIGURE 21-25 ▪ (continued) C: Wolman disease was diagnosed in this 3-month-old boy with bilateral adrenal calcifications. The cortical cells of the zona reticularis and inner zona
fasciculata are swollen with vacuolated cytoplasm due to cholesterol ester accumulation. Dystrophic calcification is present in the foci of necrosis. (Reprinted with permission from
Lack EE, Tumors of the adrenal glands and extraadrenal paraganglia. AFIP atlas of tumor pathology, Fourth Series. Washington, DC: American Registry of Pathology).
FIGURE 21-26 ▪ X-linked adrenoleukodystrophy in a young male child. A: There was prominent atrophy of the adrenal cortex at autopsy. The inner cortical cells are enlarged with
abundant pale cytoplasm (H&E stain, original magnification 100×). B: Cortical nodules of ballooned cells with a waxy cytoplasm and faint striations are observed in this peroxisomal
disorder with defective fatty acid β-oxidation leading to accumulation of very long chain-saturated fatty acids within cells (H&E stain, original magnification 200×).
Adrenoleukodystrophy (ALD), a peroxisomal disorder with defective fatty acid β-oxidation leading to accumulation of very long chain-saturated fatty acids, is associated with
inflammatory demyelination of axons and loss of oligodendrocytes and atrophy of the adrenal glands (16,e108, e116,e220,e320,e401,e402,e567,e600,e610,e783,e843, e1092-
e1094). A neonatal autosomal recessive form that presents with hypotonia and seizures, and a childhood X-linked recessive form are recognized (e220,e610,e843) The adrenals are
atrophic and normal cortical zonation is absent (Figure 21-26 A, B, eFigure 21-84). The adrenal medulla appears unremarkable (e108). Cortical nodules of ballooned cells with waxy
cytoplasm are observed and between the nodules are macrophages with phagocytized lipid and mild fibrosis. Membrane-bound lipid vacuoles with cholesterol clefts are seen on
ultrastructural examination (eFigure 21-85) (e108) (see Chapter 5).
In the X-linked form of ALD [mutations in ABCD1 gene on Xp28 that codes for transporter protein (ALDP) in the peroxisome membrane], estimated to occur in 1:17,000 male infants
(hemizygotes and heterozygotes), the adrenals usually weigh 2 g or less (e108,e116). Quantitative (absent) and qualitative defects in ALDP lead to the accumulation of very long
chain saturated fatty acids (e108). There are more than 400 recognized mutations leading to variations in the clinical presentation. The morphologic appearance of the adrenal is
variable. The zona glomerulosa is recognized but decreased in thickness. The zona fasciculata and reticularis are markedly reduced in thickness so that the zona glomerulosa
occupies about half of the adrenal cortex thickness. The medulla is otherwise normal.
Adrenal cytomegaly is usually an incidental finding observed in the fetal cortex, focally or diffusely, in approximately 6% of normal adrenal glands (e61). It is more frequently seen
in stillborn, premature and newborn infants; however, it is also observed in older children. The cytomegalic cells contain large (two to three times the normal size),
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hyperchromatic, pleomorphic nuclei, often with prominent nucleoli and “pseudoinclusions” of cytoplasm and abundant vacuolated eosinophilic cytoplasm (Figure 21-27A, B). Mitoses
are absent. These cells have been reported in association with a number of malformational syndromes including trisomies 13 and 18 and in various perinatal-maternal conditions
including hemolytic disease of the newborn, nonimmune hydrops, eclampsia, intrauterine infection, sepsis, multifetal gestations, congenital lupus erythematosus, and
polyhydramnios (e59,e855). It has also been associated with Rh incompatibility and in utero fetal distress and as an incidental finding in approximately 1% of pediatric autopsies
(e357). Adrenal cytomegaly is one of the characteristic features of the Beckwith-Wiedemann syndrome (BWS), which (e365,e896) is a congenital disorder characterized by
exomphalos, macroglossia, and giantism. The estimated frequency is 1:13,000 live births. Dysregulation of several genes encoded on chromosome 11p15.5 is thought to be the
pathogenetic mechanism (104). Most cases are sporadic (85%); however, an autosomal dominant inheritance with variable expressivity is reported in familial cases
(e869,e888,e889). Wilms tumor, hepatoblastoma, adrenocortical neoplasms, NB, pancreaticoblastoma, and PHEO are some of the childhood neoplasms associated with BWS
(104). The adrenal glands in BWS are enlarged and may have a combined weight of 16 g or more. Grossly, the glands have cerebriform contours due to cortical hyperplasia (Figure
21-27C, eFigure 21-86). Large cells with bizarre nuclei (adrenal cytomegaly) are a prominent feature and are observed bilaterally. Diffuse sheets of such cells cause marked
expansion of the fetal zone (Figure 21-27D, eFigure 21-87). Hemorrhagic cysts may also occur with the formation of an abdominal mass in the neonate (e41,e778).
Congenital adrenal hypoplasia (CAHP) is an uncommon condition with an estimated incidence of 1:12,500 births (e69,e356,e603,e691). Three distinct histological patterns, so-
called cytomegalic, anencephalic, and miniature, are recognized (e180). A combined adrenal weight of less than 2 g in a term infant qualifies as hypoplasia. Use of a combined
adrenal weight-body weight ratio of less than 1:1000 improves diagnostic accuracy. Utilizing these criteria, CAHP is present in 2% or so of fetopsies and perinatal autopsies.
Prenatally, maternal plasma levels of dehydroepiandrosterone sulfate and estriol are useful in detecting CAHP in families at risk (e19,e935). Decreased maternal estriol levels are an
important diagnostic clue. X-linked, autosomal recessive, variable, or sporadic inheritances are reported (e121). Most infants with CAHP present with signs of adrenocortical
insufficiency and may present as sudden infant death syndrome (e88,e1014).
The cytomegalic type, which is the most common pattern, with X-linked inheritance is due to a deletion or inactivating mutation of NROB1 (Xp21.3-p21.2) that encodes for DAX-1
which is critical for adrenal gland development. Over 100 mutations involving the NROB1 gene have been reported accounting for the phenotypic variability (95,139,e3,
e8,e80,e503,e934,e936,e1 164,e1270). In one series, DAX-1 mutations were found in almost 60% of 46,XY phenotypic boys referred with adrenal hypoplasia and in all boys with
hypogonadotropic hypogonadism and a family history of adrenal failure (99,e144). The cytomegalic pattern has also been reported in association with other inheritance patterns
(e654). As part of a contiguous gene syndrome, a number of affected males also have glycerol kinase deficiency and Duchenne muscular dystrophy without any CNS anomalies
(e246). Grossly, the adrenals are small and lack a definitive cortex (Figure 21-28A). The fetal cortex has a disorganized architectural pattern consisting of clusters of large
adenocortical cells with variable nuclear hyperchromasia. The eosinophilic cytoplasm is vacuolated and intranuclear cytoplasmic inclusions may be seen in these cells (Figure 21-
28B).
The anencephalic type with autosomal recessive inheritance in many cases resembles the adrenal glands of anencephalic infants but in the absence of anencephaly (eFigure 21-
28C, D) (e437). The pituitary and CNS are either normal or may have developmental anomalies. Adrenal insufficiency is noted at birth and hypogonadism may develop at puberty
with survival beyond infancy. The small adrenal glands have a definitive, but attenuated cortex and the fetal zone is markedly diminished. An autosomal recessive pattern of
inheritance is often present.
The miniature type with a sporadic occurrence or autosomal recessive inheritance is seen in infants without any karyotypic abnormalities or developmental anomalies, although this
pattern has been reported in association with triploidy, and trisomies 13 and 18 (e577). Clinical manifestations are dependent upon the degree of hypoplasia. The miniature pattern
is associated with the onset of pregnancy-induced hypertension (e170). Grossly, the adrenals are small with a definitive cortex but a diminutive or absent fetal cortex. Otherwise
there is normal zonation and an absence of any cellular abnormalities (eFigure 21-88). Hereditary unresponsiveness to ACTH due to mutations involving the ACTH receptor gene
may mimic CAHP (95, 188).
Congenital adrenal hyperplasia (CAH), also known as adrenogenital syndrome, is a group of autosomal recessive disorders of adrenal steroid biosynthesis with similar
morphologic features (29,30,86,95,188,e412,e526,e708, e803-e805,e817,e864,e990,e1122,e1123,e1258,e1259,e1274). The incidence of CAH is 1:500 to 1:16,000 live births,
dependent on the population sampled, and is the most frequent cause of ambiguous genitalia in the neonate and/or of primary adrenal insufficiency in the pediatric population
(29,30,86,95,188, e579,e934,e1178). Approximately 90% to 95% of CAH cases are due to 21-hydroxylase deficiency and 5% to 8% due to 11 β-hydroxylase deficiency
(29,30,86,95,188,e803,e864,e214, e269,e427,e527,e536,e571,e598,e607,e662,e681,e697,e699, e937,e1120,e1121,e1178,e1273). Decreased Cortisol production interrupts the
normal feedback inhibition on the pituitary gland, leading to persistent ACTH secretion and continued synthesis of Cortisol precursors up to the level of the enzymatic
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defect. Symptoms and laboratory findings are dependent on which enzyme is absent. The diagnosis of CAH can be made prenatally by chorionic villus sampling during the first
trimester (29,30,86,e143,e209,e511,e846,e861-e863,e877-e879, e881,e885,e1124). Many states have neonatal screening programs for 17-hrydroxyprogesterone to detect 21-
hydroxylase deficiency. The administration of dexamethasone which crosses the placenta before 8 weeks gestation, has been
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helpful in preventing virilization of the external genitalia in utero (29, 30, 86). The clinical, laboratory and genetic features are beyond the scope of this chapter but are reviewed at
length by others (29,30,86,188,e412,e526,e708,e803-e805,e817, e864,e990,e1122,e1123,e1259,e1274).
FIGURE 21-27 ▪ Adrenal cytomegaly. A: Cytomegaly of the fetal adrenal cortex characterized by large cells with hyperchromatic nuclei is seen in this term infant with in utero fetal
demise due to a cord accident (H&E stain). B: Nuclear pseudoinclusion (cytoplasmic invagination into the nucleus) are seen in this image of adrenal cytomealy from a term infant
with in utero fetal demise (H&E stain). C: The adrenal glands from a 3-week-old infant with Beckwith-Wiedemann syndrome are hyperplastic with increased cortical nodularity and
redundant folds. (Reprinted with permission from Lack EE, Tumors of the adrenal glands and extraadrenal paraganglia. AFIP atlas of tumor pathology, 4th Series. Washington, DC:
American Registry of Pathology). D: There is marked cytomegaly with nuclear enlargement, hyperchromasia and nuclear “pseudoinclusions” in this section of fetal cortex from an
infant with Beckwith-Wiedemann syndrome on histological examination (H&E stain). (Reprinted with permission from Lack EE. Tumors of the adrenal glands and extraadrenal
paraganglia. AFIP atlas of tumor pathology, Fourth Series. Washington, DC: American Registry of Pathology).
FIGURE 21-28 ▪ Congenital adrenal hypoplasia. A: Section of bladder, kidneys, and adrenals from a 470 g preterm male infant. The combined adrenal weight was 0.147 g (versus
expected of 2.5 g). The brain and pituitary were normal on gross examination. A normal component of acidophilic cells was present in the pituitary. B: The adrenal glands consisted
of large cells with abundant eosinophillic cytoplasm. The nuclei were large and bizarre with eosinophillic inclusions similar to adrenal cytomegaly. (From James Arey, M.D., Luther
Youngs, M.D. Pediatric pathology: congenital malformations [Slide collection, 1966]. Washington, DC: The Armed Forces Institute of Pathology.)
The 21-hydroxylase deficiency (21-OHD) is the most common form of CAH (29,30,86,95,188,e880e269,e427, e527,e536,e579,e598,e1121,e1178,e1258,e1273). Clinical
manifestations vary with the severity of the enzymatic deficiency (e1120). The affected gene, CYP21A2, is located in the region of the major histocompatibility complex III on
chromosome 6p21.3 (29,30,86,95,e311,e427,e813,e990). Intergenic recombinations occur between CYP21A2 and the pseudogene CYP21A1P; these events account for most of
the mutations (80% of cases), and the remainder are deletions in CYP21 (29,30,86,e352,e709,e839,e1136,e1273). Three distinct clinical patterns of 21-OHD are recognized, classic
salt-wasting, simple virilizing (70% and 30% of classic subtypes, respectively), and nonclassic milder subtypes (29,30,86,95,188,e880,e70,e817). It is thought that these three types
represent a continuum from mild to severe rather than three distinct phenotypes. Greater than 50 CYP21A2 mutations have been described and these determine the particular
phenotypic expression (86). The incidence for the classic salt-wasting form is 1:10,000 to 1:16,000 live births, and the milder form may be as frequent as 1:500 to 1:1,000 individuals
which makes this condition one of the most common autosomal recessive disorders (29,30,86,188, e70,e860,e1273). In the classic form, failure to convert progesterone to the
mineralocorticoids, deoxycorticosterone and aldosterone, leads to decreased sodium reabsorption by the kidney, resulting in hyponatremia, hyperkalemia, acidosis, shock, and
death. Decreased glucocorticoid production due to the failure to convert 17-hydroxyprogesterone to 11-deoxycortisol leads to lack of negative feedback on the pituitary gland and
subsequent unimpeded ACTH secretion. The increased production of adrenal androgens causes virilization of the external genitalia, with fusion of the labioscrotal fold in the most
severe form in which case the female infant has a male-appearing external genitalia at birth. Hydrops of fetal stem villi has been reported in CAH (e392). Signs of androgen excess,
characterize the nonclassic form at puberty, with premature adrenarche, menstrual irregularities, acne, hirsutism, and sclerocystic ovaries (29, 30). Mineralocorticoid activity is
adequate. An attenuated pattern with biochemical abnormalities only is also recognized.
The 11-β-hydroxylase deficiency is the second most frequent pattern and accounts for 5% to 8% of cases
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(29,30,86,95,188,e214,e571,e662,e846,e937). The incidence is approximately 1:100,000 to 1:200,000 live births. Greater than 50 different inactivating mutations involving the 11 β-
hydroxylase gene (CYP11B1) on chromosome 8q21 are identified (29, 30, 86, 95, 188). Failure to convert 11-deoxycortisol to Cortisol and 11-deoxycorticosterone to corticosterone
results in increased mineralocorticoid activity, leading to hypernatremia, hypokalemia, and hypertension. Lack of negative feedback inhibition by Cortisol leads to increased
androgen production. Female pseudohermaphroditism and virilization of male and female infants postnatally are the other major manifestations (see Chapter 18).
The 17-a-hydroxylase deficiency (CYP17A1) accounts for approximately 1% of cases. Deficiency leads to failure to hydroxylate pregnenolone and progesterone, resulting in
decreased synthesis of androgens and Cortisol (29,30,86,95,188,e118,e397,e1041). Increased synthesis of corticosterone may cause hypertension. Affected females may present
at puberty with primary amenorrhea and males are incompletely masculinized (29,30,86,95,188, e118,e397,e1041).
The 3β-hydroxysteroid dehydrogenase deficiency and steroidogenic acute regulatory protein (StAR; cholesterol desmolase deficiency) are rare causes of CAH (29,30,86,
95,188,e9,e76,e139,e430,e570,e818,e911,e914,e1021, e1098). The 3β-hydroxysteroid dehydrogenase (HSD3β2) deficiency leads to salt wasting and female
pseudohermaphroditism and precocious masculinization in male infants. The so-called congenital lipoid adrenal hyperplasia is the only one of this group of disorders that is not
caused by a defective steroidogenic enzyme but is rather a defect in the StAR protein which is required for the transport of cholesterol to the inner mitochondrial membrane for
conversion to pregnenolone. Greater than 34 different mutations are present in the gene encoding for the StAR protein. Korean and Japanese populations are notably affected with
these mutations. These infants have adrenal insufficiency and a female phenotype (29,30,86,188,e1295).
Bilateral hyperplasia of the adrenal glands, with weights two to four times normal size, is the typical finding; however, normal-size glands are reported (104,e1041) (Figure 21-29A,
B). The external surfaces have a cerebriform appearance. Depletion of the lipid-rich cells of the zona fasciculata to compact eosinophilic cells, identical to those observed normally in
the zona reticularis, gives the glands a dark, tan-brown color on sectioning (Figure 21-29C, D, eFigure 21-89). The exception occurs in the adrenals of those individuals who have
been partially treated with steroids. The zone fasciculata under ACTH stimulation shows the greatest degree of hyperplasia among the three zones of the cortex. In contrast to the
other forms of CAH, cholesterol accumulated in the cytoplasm of the cortical cells imparts a bright yellow, nodular appearance to the cortex in congenital lipoid adrenal hyperplasia
(104,e422,e1167). Lipid-rich cells, cholesterol clefts, foreign body giant cells, and calcifications are the principal histological features. There is some resemblance in the latter
respect to the adrenals in Wolman disease. The presence of bilateral adrenal incidentaloma (unsuspected, nonhyperfunctional adrenal nodule), adrenal adenomas and the
development of adrenocortical carcinoma has been reported in association with CAH (88,89,e28,e111,e667, e1001,e1220).
Bilateral nodular hyperplasia of testicular adrenal rests [testicular adrenal rest tumors (TART)] is reported with some frequency in CAH in as many as 90% or more of adult males
(27,88,e62-e64,e103,e106,e236-e238,e240,e265, e411,e588,e706,e764,e849,e984,e985,e1126,e1133,e1134, e1133). Male infertility secondary to primary gonadal failure is
associated with TART. The testis has a firm multilobular, tan-brown appearance on cross-section and is commonly localized in the rete testis (eFigure 21-90). Confluent sheets of
polygonal cells with eosinophilic cytoplasm resembling adrenocortical tissue are present on microscopic examination (eFigure 21-91). These cells have the biochemical attributes of
adrenocortical cells. Morphological differentiation of TART from the Leydig cell tumor is difficult but TART tends to be bilateral in contrast to Leydig cell tumor. Reinke crystals are
absent in TART but present in up to 35% of Leydig cells tumors. These nodules can be locally resected in an attempt to preserve testicular parenchyma (e1126) (see Chapter 19).
Bilateral ovarian steroid cell tumors and malignant Leydig cell tumors have also been reported in the setting of CAH (see Chapters 18 and 19) (e86,e279,e1015).
Primary pigmented (micronodular) adrenocortical disease (PPAD) is associated with Cushing syndrome and 25% to 35% of cases have the manifestations of Carney complex
with myxomas, spotty skin pigmentation, and endocrine hyperactivity (89,104,e195,e196,e231,e362,e540, e1086,e1138,e1139,e1141,e1142,e1147). In addition to PPAD in 45% of
cases, growth hormone secreting PAs are present in about 10% of cases (e906). Two affected genetic loci in this autosomal recessive disorder have been mapped to chromosomes
2p16 and 17q22,24 (PRKAR1A) (89,104, e444,e614,e615,e753,e1147,e776,e1031). Pathologically, the adrenals are decreased, normal, or slightly increased in size. Multiple
pigmented nodules less than 4 mm in diameter occupy an otherwise atrophic appearing cortex with loss of normal zonation (eFigure 21-92). Nodules may be present in the
periadrenal fat (e613,e633). The enlarged cortical cells have an eosinophilic cytoplasm with abundant lipofuscin pigment (eFigure 21-93). These cells are immunoreactive for SYN
but fail to stain for CHR (e1143). In this respect, the cortical nodules of PPAD react in a similar manner to adrenocortical neoplasms.
Adrenocortical hyperplasia is also seen in BWS, MAS, and MEN 1. Cushing syndrome in the setting of MAS is associated with autonomously functioning multinodular hyperplasia
of the adrenals (e612). Cushing syndrome is present in 30% to 40% of cases of MEN 1 (e345).
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FIGURE 21-29 ▪ Congenital adrenal hyperplasia in a 7-week-old boy who had signs of intestinal obstruction. A: The kidneys and adrenal glands are shown, and the enlarged
adrenals (combined weight 16.8 g) have a convoluted cerebriform appearance due to the hyperplastic cortex. (From James Arey, M.D., Luther Youngs, M.D. Pediatric pathology:
congenital malformations Slide collection, 1966. Washington, DC: The Armed Forces Institute of Pathology.) B: This image of kidneys, adrenals and aorta is from another child with
congenital adrenal hyperoplasia showing enlarged cerebriform adrenals. C: The adrenal cortex is enlarged due to marked expansion of the zona fasciculata in congenital adrenal
hyperplasia. The cortex is predominantly characterized by a pattern of compact cells with focal collections of clear cells with lipid-rich cytoplasm interspersed (H&E stain). (Reprinted
with permission from Lloyd RV, Douglas BR, Young WF. Endocrine diseases. Atlas of nontumorpathology. Washington, DC: American Registry of Pathology.) D: High power image
showing lipid depletion of zona fasciculata cells with compact eosinophillic cytoplasm in congenital adrenal hyperplasia (H&E stain). (Reprinted with permission from Lloyd RV,
Douglas BR, Young WF. Endocrine diseases. Atlas of nontumor pathology. Washington, DC: American Registry of Pathology.)
Adrenocortical insufficiency can be congenital or acquired (188,e902,e934). ALD, CAHP, and CAH are the primary adrenal disorders with accompanying adrenal insufficiency.
Other inherited syndromes with adrenal insufficiency include Smith-Lemli-Opitz syndrome, Kearns-Sayre syndrome, and ACTH insensitivity syndrome. Infections, autoimmune
disorders, adrenal hemorrhage, and drugs represent acquired etiologies. In children autoimmune involvement can be isolated or part of an autoimmune syndrome. Autoimmune
polyglandular syndrome, type 1, a multisystem autoimmune disease, is associated with adrenal insufficiency and hypoparathyroidism. Pituitary and other CNS
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diseases, as discussed previously, are secondary causes. In one review, CAH was the most common etiology of adrenal insufficiency which accounted for about 70% of cases with
autoimmune adrenalitis as the second most common etiology (e944). These findings are similar to the study by Osuwannaratana et al. who observed that greater than 85% of cases
were examples of CAH with panhypopituitarism as the most common cause of secondary adrenal insufficiency (e902,e934).
Acquired Disorders
Adrenal cysts are relatively uncommon in children (104,e84,e174). There are four histopathogenetic types: epithelial, endothelial, pseudocystic, and parasitic (e98,e379). Adrenal
neoplasms can undergo cystic necrosis and simulate a large benign cyst, especially the cystic NB in the infant (e61,e261). Cystic cortical degeneration with microcysts may be seen
in stillborn infants exposed to substantial stress in utero. Adrenal cysts are also present in a number of syndromes including BWS, autosomal recessive polycystic kidney disease,
prune-belly syndrome, and Gorlin-Goltz syndrome (e16,e87,e234,e509,e551,e778,e842). Idiopathic adrenal cysts are reported from the neonatal period into adolescence (e156).
Bacterial, fungal, parasitic, and viral infections can involve the adrenal glands. Adrenal infections with or without necrosis are found in congenital intrauterine infections (Herpes
simplex with necrosis, cytomegalovirus with adrenalitis or necrosis), Varicella-zoster and congenital or acquired immunodeficiency disorders (eFigures 21-94 and 21-95) (e1011).
Histoplasmosis and tuberculosis are also recognized causes of Addison disease (e432,e454,e593,e1039). Paracoccidioidomycosis has been observed in children in South America
(e927) (see Chapter 6).
Adrenal hemorrhage occurs in a wide spectrum of lesions in children and adults (e1228). The hemorrhage may range from massive involvement of the gland to focal segmental
necrosis. Unilateral or bilateral involvement is dependent in some cases on the etiology. Fetal adrenal hemorrhage occurs with some frequency and has been observed as early as
the second trimester (e350,359). Although many lesions in the perinatal period are ascribed to birth trauma and in utero asphyxia, the etiology for most adrenal hemorrhages is
uncertain in many cases (e595). Trauma, sepsis, shock, underlying coagulopathy, arterial thrombosis secondary to umbilical artery catheterization, extracorporeal membrane
oxygenation (ECMO), neonatal stress, and renal vein thrombosis have been reported in association with neonatal adrenal hemorrhage (eFigure 21-96) (e48,e178,e552,e809,e897,
e943,e1066,e1100,e1117,e1177). Massive adrenal hemorrhage is one of several sources of an abdominal mass in an infant. Imaging studies are helpful in the diagnosis
(e219,e810). Resolution of a hemorrhage with progressive calcifications is well documented (eFigure 21-97). One should keep in mind the possibility of a NB (e310). Spontaneous
resolution after birth is a feature of the adrenal hemorrhage in contrast to an adrenal tumor (e1236). Transient adrenocortical insufficiency has been observed. Rarely, adrenal
abscess formation complicates adrenal hemorrhage (e60).
Trauma, adrenal tumors, stress, and infection are important considerations in the differential diagnosis of adrenal hemorrhage in older children and adolescents. The
traumaassociated adrenal hemorrhage is unilateral with a preference for the right adrenal gland (eFigure 21-98) (e1099). Child abuse must be considered in the differential
diagnosis of traumatic adrenal hemorrhage. Unilateral involvement of the right adrenal gland with adrenal medullary hemorrhage on histological examination has been found in child
abuse cases (e882). Bilateral adrenal hemorrhagic necrosis in the setting of sepsis with rapid onset of circulatory collapse, petechial rash (noninflammatory microangiopathy), and
coagulopathy is known as the Waterhouse-Friderichsen syndrome (WFS), and is most commonly associated with meningococcemia (e558,e695,e1222). Other common infectious
agents in children associated with WFS include group A β-hemolytic streptococci and Haemophilus influenza (e409,e419,e558). Congenital asplenia or splenic atrophy in the
setting of sickle cell anemia is a risk association for bacterial septicemia and development of WFS in children (e583,e726). Acute adrenal insufficiency is common (e480). Adrenal
hemorrhage in WFS begins in the adrenal reticular plexus and extends toward the capsule (Figure 21-30A). There is a loss of the adrenal cortical parenchyma with hemorrhage as
well as partial or complete cortical necrosis (Figure 21-30B). Subcapsular hematomas may form and extend into the periadrenal fat and surrounding tissues in severe cases.
Histological examination reveals compression of the sinusoidal capillaries with occasional rupture. Small fibrin thrombi may be seen in the sinusoidal capillaries as features of a
microangiopathy (e131).
Calcifications of the adrenal glands are found in several defined settings but most notably Wolman disease and resolving adrenal hemorrhage (eFigure 21-97) (104,
e56,e310,e412). There are also individual reports of adrenal calcifications in association with congenital nephrotic syndrome, as a sequela to congenital infections, congenital heart
disease and BWS (eFigure 21-94B) (104,e539,e546,e841, e903,e952,e1018,e1221).
Adrenal cortical neoplasms (ACNs) include the adrenocortical adenoma (AA) and carcinoma (ACC), and are rare in the pediatric age group (90,189,e137,e315,e474,e807,
e1032,e1146,e1171). The clinicopathological features of ACN in children, their behavior and epidemiology contrast nominally with the same neoplasms in adults. However, one of
the consistent observations is that these neoplasms in children, though having several morphologic features associated with ACC in adults, do not have the same unfavorable
prognostic implications, especially in children under 6 years of age (40, 90). Because these atypical histological features are commonly found
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in ACNs in children, they are disproportionately interpreted as ACCs. The incidence of cases classified as ACCs in children in the United States was 0.2 cases: 1,000,000
individuals less than 19 years of age (67). ACC accounts for less than 0.5% of all pediatric malignancies but is the third most common carcinoma in children exceeded by PTC and
salivary gland carcinoma (40,90,e575).
FIGURE 21-30 ▪ A,B: Waterhouse-Frederichsen syndrome in a young child who died of meningococcal sepsis is manifested by adrenal hemorrhage on gross examination and
diffuse hemorrhage throughout the cortex and medulla on histological examination (H&E stain).
There is a bimodal age distribution of ACNs and they are more common in females in the pediatric population. In a report of 256 cases from the International Pediatric Adrenocortical
Tumor Registry (IPATR), the male to female ratio was 1:1.6 (112). Two age distributions were noted by the IPATR, an infantile group with a peak incidence in the first year of life and
an adolescent group with a peak incidence between 9 and 16 years. A female predilection with a mean age at diagnosis of 4.6 years and nearly 50% of cases diagnosed in the first
4 years of life has been documented by others (40,e181,e237,e450,e681,e723,e857). The experience of Dehner and Hill is similar (40). In a review of 39 cases, ACN presented in
children between 7 days and 12 years of age with a mean age of 3 years and median of 2 years (40). Seventy-six percent of children were less than 4 years. The male:female ratio
was 1:2.5. Others have reported similar findings (e177,e233,e444,e676,e718). There are congenital examples of ACNs (90,e181,e555,e1040). One of the highest incidences of
purported ACCs in children (4.7 cases:1,000,000) has been identified in southern Brazil where a distinct germline p53 mutation has been found in the population, but whose other
features are not those of classic Li-Fraumeni syndrome (e177,e444,e781,e981,e982).
There are several syndromic associations with ACNs including BWS (hemihypertrophy, splanchnomegaly, macroglossia, and intraabdominal neoplasms), the Li-Fraumeni syndrome,
and Carney complex (40,90,e335,e365,e513, e555,e693,e712,e869,e888,e889,e976,e1129,e1147,e1269). Adrenal hyperplasia and ACN have been reported in MEN 1 syndrome,
MAS, and neurofibromatosis 1 (e187,e1239,e1263). Examples of ACN have been seen in the setting of CAH (e667,e1001). Bilateral ACNs, often seen in syndromicassociated
cases, and ectopic ACNs are uncommon (40, e601,e555).
Adrenocortical neoplasms in children account for 50% to 70% of cases of Cushing syndrome, in contrast to only 20% of cases in adults (e137,e315,e1161). Less often does an ACN
present with feminizing and masculinizing manifestations in children. Conn syndrome, due to an aldosteroneproducing ACN, is rare in childhood. Most tumors in Conn syndrome are
benign and characterized by their lipid-rich clear cells (e81,e291,e711).
The most important initial step in the pathologic examination of an ACN in childhood is weighing the tumor, since all other gross and histopathologic attributes of the tumor itself are
in a sense secondary, if the tumor is confined to the gland and does not have evidence of metastatic spread to regional lymph nodes or to more distant sites such as the liver and
lungs. In some cases, it may be difficult to judge whether a circumscribed mass in the adrenal is part of multinodular hyperplasia of the cortex or even a PHEO (eFigure 21-99)
(e49,e758).
Cortical neoplasms, not only in children but in adults, vary in size, weight, coloration, consistency (solid and/or cystic), presence or absence of hemorrhage, and presence or
absence of necrosis (Figures 21-31, 21-32 and 21-33, eFigure 21-100). A complete or incomplete fibrous capsule may be apparent at the periphery of the tumor, or the tumor
appears to compress the adjacent parenchyma without a capsule. As noted earlier, the size and weight, especially the latter, are closely correlated with the clinical outcome of an
ACN in a child. The cut surface in the absence of hemorrhage and necrosis often has a pale to bright yellow to yellow-brown appearance which may or may not be uniform
throughout because of cystic changes (Figure 21-31A, eFigure 21-100). Uncommonly, the tumor may have a brownish-black appearance due to lipofuscin accumulation in the
cytoplasm of tumor cells as in the case of the PPAD. A hemorrhagic mass may be difficult to differentiate from a NB.
A number of pathologic studies in the literature have examined every conceivable microscopic feature of ACN for their
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predictive prognostic value, but most of the studies have consisted principally of tumors in adults where cytologic atypia, mitotic activity, zonal necrosis and transecting fibrous bands
have predictive value in terms of outcome when several of these features are present in the ACN. Some of these same histological features are found with some frequency in ACNs
in children yet lack any significant correlation with prognosis. The classic pattern of an ACN is a neoplasm which is composed of clear or pale polygonal cells with abundant lipid-rich
cytoplasm or cells with more homogeneous eosinophilic cytoplasm, all arranged in short cords or trabecular profiles (Figure 21-31B, C, eFigure 21-101). The nuclei are uniform and
centrally positioned in the cell. The adjacent cortex is often compressed and atrophic (eFigure 21-102). Other histological patterns include diffuse, formless sheets of relatively
monotonous polygonal cells, alveolar pattern of loosely cohesive cells, glandular profiles, a yolk sac tumor-like pattern and delicate ribbons of cells in a hyaline myxoid stroma, either
as an exclusive pattern or component of the ACN (40,189,e184,e294,e344,e521,e792,e1264).
FIGURE 21-31 ▪ Adrenal adenoma. A: This adrenalectomy specimen that weighed 35 g was from a 17-year-old male who presented with hypertension. A 2-cm diameter yellow
cortical nodule surrounded by a thin rim of stretched uninvolved adrenocortical tissue is present. The remainder of the adrenal gland shows the typical zonation and was
unremarkable. B: Low power magnification demonstrating an adrenal adenoma with lipid-laden cells (balloon cells) arranged in small clusters that are surrounded by a thin delicate
vascular network. A rim of uninvolved (nonneoplastic) adrenocortical tissue compressed by the mass with focal hemorrhage is on the left (H&E stain). C: High power magnification
shows the typical clear lipidladen cells arranged in small clusters. Cellular and nuclear pleomorphism was not present. Mitotic figures were absent (H&E stain). This tumor would be
classified as an adrenocortical neoplasm, low risk in Dehner and Hill's proposed classification (see Table 21-7 reproduced from Dehner LP, Hill A. Adrenal cortical neoplasms in
children: Why so many carcinomas and yet so many survivors? Pediatr Dev Pathol 2009; 12:284-291).
If polymorphism is the theme for the various patterns in ACNs in children, then pleomorphism applies to the individual cellular features (Figures 21-32 and 21-33, eFigure 21-103). In
some ACNs, monomorphism is an appropriate characterization in the presence of uniform tumor cells. Especially prominent in cases of ACNs in children who are usually 4 years old
or less is pleomorphism in terms of individual cell size as bordering on tumor giant cells with bizarre nuclear configurations and intense hyperchromatism; these latter cell types when
present can constitute a minor or major component of a particular tumor (Figure 21-33).
By contrast, some tumors can have substantial mitotic activity yet are small (less 100 g) and confined to the gland. Other features of ACNs in children can include necrosis, either as
individual cells, or microfoci or macrofoci of necrosis which may have been appreciated in the gross examination. Intratumoral microinvasion of blood vessels and apparent
microscopic breaching of the capsule are additional histological findings (Figure 21-33).
It would appear from the preceding paragraph that an ACN with some of these features has attained the threshold for the pathologic diagnosis of ACC and for that reason many
studies of ACNs in children are represented by a majority of cases with a diagnosis of ACC (40). However, the paradox is that the prognosis for ACCs in children, especially those
under 5 years of age, are remarkably favorable with a 5-year event-free survival (EFS) of 70% to 80% (40).
Adrenocortical carcinoma indisputably occurs in children and these tumors usually weigh in excess of 400 g and in
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some cases may exceed 1 kg. These tumors have irregular contours, extensive areas of hemorrhage and/or necrosis and invasion beyond the capsule, into the surrounding soft
tissues and organs. The various histopathologic features associated with ACCs in adults are present throughout these tumors (40,189,e184,e294,e344,e521,e792,e1264). These
obviously malignant neoplasms are found in children beyond 10 years of age whose 5-year EFS ranges from 20% to 35%. These tumors metastasize to the liver (>90% of cases),
lungs (80%), retroperitoneal soft tissues and regional lymph
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nodes with less common spread to the bone and brain (189, e676,e946,e1239). Most children with “bona fide” ACCs are usually dead from tumor within 2 years of the diagnosis. As
another measure that putative ACCs in young children behave in a different fashion compared to adults are the observations that BWS-associated ACCs are not aggressive
neoplasms and that a congenital ACC is reported to have undergone spontaneous regression (e1038,e1060,e1226).
FIGURE 21-32 ▪ Adrenocortical neoplasm. A: This 250-g adrenal gland from an adolescent who presented with hypertension and signs of virilization had a homogenous tan
appearance on gross examination. A small portion of normal adrenal gland (arrow) is present. B: On histological examination there was prominent cellular pleomorphism with
enlarged atypical nuclei. Occasional mitotic figures were present. There was no capsular or vascular invasion. There were no signs of metastatic disease at the time of surgery. This
lesion was diagnosed as an atypical adenoma. This tumor would be classified as an adrenocortical neoplasm, intermediate risk in Dehner and Hill's proposed classification (see
Table 21-7 reproduced from Dehner LP, Hill A. Adrenal cortical neoplasms in children: Why so many carcinomas and yet so many survivors? Pediatr Dev Pathol 2009;12:284-291).
FIGURE 21-33 ▪ Adrenocortical neoplasm. A: A 2-year-old-male child with precocious puberty and accelerated bone age was found to have a 9.6 × 8 × 6.2 cm mass arising from the
right adrenal gland. There was no sign of metastatic disease at the time of surgery. The 215-g adrenal gland had extensive necrosis and calcification on gross examination. B: On
histological examination the tumor was composed of large, pleomorphic, eosinophillic cells. Nuclear enlargement with hyperchromasia, intranuclear inclusions and increased mitotic
rate were present. Capsular invasion (not shown) was also observed. This tumor was classified as an adrenocortical carcinoma. This tumor would be classified as an adrenocortical
neoplasm, intermediate risk in Dehner and Hill's proposed classification (see Table 21-7 reproduced from Dehner LP, Hill A. Adrenal cortical neoplasms in children: Why so many
carcinomas and yet so many survivors? Pediatr Dev Pathol 2009;12:284-291).
Table 21-7 ▪ PROPOSED RISK GROUPS FOR ADRENOCORTICAL NEOPASMS IN CHILDREN
Risk Group Criteria
Low Any cortical neoplasm confined to the adrenal gland and weighing less than 200 g.
Intermediate Any cortical neoplasm confined to the adrenal gland and weighing between 200 and 400 g.
Any cortical neoplasm weighing <400 g with microscopic invasion into surrounding soft tissues, completely resected, and no evidence of metastatic spread.
High Any cortical neoplasm weighing in excess of 400 g or with direct gross invasion into adjacent organs like the liver, spleen, or kidney or with metastatic
spread.
Reproduced with permission from Dehner LP, Hill DA. Adrenal cortical neoplasms in children: Why so many carcinomas and yet so many survivors? Pediatr Dev Pathol.
2009;12:289.
Three risk groups based on tumor localization and weight have been proposed as an alternative means of predicting the clinical behavior of a particular ACN in a child (40) (Table
21-7). Because of the unreliable correlation of histological features to prognosis of ACNs in children, some have gone so far as to eliminate histological features altogether in the
prognostic assessment. The three risk groups include those ACNs weighing less than 200 g and confined to the gland as “low risk” for malignant behavior and these tumors are
interpreted as adenomas (Figure 21-31, eFigures 21-101 and 21-102), to be contrasted with ACNs weighing in excess of 400 g with a high risk of malignant behavior and are
commonly associated with the various histopathologic features of ACCs in adults. The most problematic group consists of those ACNs that are confined to the gland but weigh
between 200 and 400 g; these neoplasms are designated “atypical” adenomas with uncertain malignant potential (Figures 21-32 and 21-33, eFigure 21-103). Some of these tumors
have invaded beyond the adrenal gland into adjacent tissues and/or have major vascular invasion, not simply microinvasion of vessels within the tumor itself; these tumors are
clearly behaving in a malignant fashion. Most of the “atypical” adenomas have a favorable outcome in our experience (40).
The immunophenotype of an ACN in a child is identical to its counterpart in the adult as the tumor cells are reactive with VIM, melan-A, inhibin, and calretinin. There are no
immunophenotypic differences between an adenoma and carcinoma (36,e50,e51,e374,e375,e395). ACNs, typically adenomas, may demonstrate immunoreactivity for cytokeratin;
carcinomas are usually nonreactive (36,e395). Ploidy analysis has limited value in the discrimination of an adenoma from a carcinoma. In one study of 50 ACNs in children, 21 of 29
patients (73%) with aneuploid tumors remained disease-free (e835,e1229).
The distinction between a cortical neoplasm and PHEO is not clear in every case, especially in a tumor with large, bizarre-appearing cells, granular basophilic cytoplasm, and a
nested growth pattern. The challenge is further heightened by the fact that the results of pertinent biochemical studies are usually not available to correlate with the pathological
findings. The tumor is more likely to be cystic and hemorrhagic, and the tumor cells are devoid of some of the tinctorial attributes that are useful in the differentiation of a cortical from
a medullary neoplasm. In these cases, immunohistochemistry is helpful with the differential diagnosis.
SYN and NSE are commonly immunoreactive in both PHEOs and adrenal cortical tumors, whereas CHR is nonreactive in adrenocortical tumors, but is consistently expressed in
PHEOs and paragangliomas (36,e90,e455,e811,e1058). Cytokeratin is typically not found in either PHEOs or paragangliomas with rare exceptions, but they are often
immunoreactive for VIM (36,e222,e221,e675). S-100 protein and HMB-45 staining is useful in the labeling of the sustentacular cells of PHEOs. It is necessary to acknowledge that
the results of bcl-2, cytokeratin, and VIM expression have not proven to discriminate between a cortical and medullary neoplasm in every case.
Peripheral NB Group Tumors. Classic or peripheral neuroblastic tumors are represented by the NB, ganglioneuroblastoma (GNB), and ganglioneuroma (GN) as a group of
histogenetically-related neoplasms of neural crest origin (eFigure 21-104). These tumors are histogenetically distinct from the central primitive neuroectodermal tumor (cPNET) and
Ewing sarcoma-primitive neuroectodermal tumor (EWSPNET) despite the presence of overlapping morphologic and immunophenotypic features (see Chapters 10 and 24).
Epidemiology: NB is the most common extracranial solid neoplasm of childhood and is surpassed in incidence only by the acute leukemias and primary brain tumors, principally
astrocytoma and medulloblastoma; SEER Program for 1975 to 2000 reported that NB accounted for 7.2% of all cancers among children younger than 15 years of age in the United
States, and the total incidence was 10.2 to 10.3:1,000,000 for males and 10.1 for females (155). The incidence rates by age are the following: 19.6:1,000,000 for ages 1 to 4 years,
2.9 for ages 5 to 9 years, and 0.7 for 10 to 14 years. The rates by race and ethnicity are the following: 10.8:1,000,000 for
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whites, 8.4 for blacks, and 7.5 for children in other racial/ethnic groups. In the United States, approximately 650 children are newly diagnosed each year (57). Based on the SEER
data, the 5-year relative survival rate is 65%, a figure which has remained more or less static for the past several decades.
In the past, NB was referred to as “enigmatic” because of its unpredictable behavior since these tumors manifest a wide range of clinical courses from an excellent prognosis due to
complete resectability, tumor involution, spontaneous regression and/or maturation or a fatal outcome due to tumor progression despite intensive treatment. Now NB is believed to
be biologically heterogeneous, and is composed of at least two subgroups, clinically favorable and unfavorable; these two subgroups have distinct molecular/genetic attributes
closely correlated with their clinical behaviors. Several epidemiological studies in the past have not identified any causal factors for NB; however, it may be necessary to analyze
neuroblastic tumors in each biological subgroup separately to elucidate any possible extrinsic factors.
Familial or hereditary NB, first recognized in 1945, is a rare entity (e (308) and has offered an opportunity to identify any hereditary NB predisposition genes: Maris et al. have
reported a hereditary NB predisposition gene (HNB1) on the distal short arm of chromosome 16p (16p12-13), (108) and Perri et al. have identified another gene on the distal short
arm of chromosome 4p (4p16) (135). Recently, activating mutations in the anaplastic lymphoma kinase (ALK) oncogene (2p23) have been found in hereditary NB cases as well as a
smaller subset of sporadic tumors (26,118,125,128, e561). The same gene has an important oncogenic role in anaplastic large cell lymphoma and inflammatory myofibroblastic
tumor. DICER1 has even been questioned as having a role.
Beckwith and Perrin used the term of NB in situ to describe an exclusively microscopic finding in neonatal and infant autopsies, histologically identical to NB, as an incidental finding
in or around the adrenal medulla. The incidence of these lesions has been calculated as 40 to 100 times that of clinically overt NBs (e95). Most NBs in situ during life are
asymptomatic (e453,e476). Since similar neuroblastic nodules are seen during the fetal development of the adrenal medulla, some have questioned the neoplastic potential if any of
NB in situ. It has not yet been demonstrated whether these lesions are clonal proliferations of genetically abnormal cells. Therefore, the premalignant or neoplastic nature of these
lesions remains unproven to date.
The anatomic sites of predilection for NB are related to the distribution of neural crest cells. They include the paravertebral region from the neck to the pelvis (3% to 5% of cases),
the adrenal medulla (35% of cases), the extra-adrenal retroperitoneum (30% to 35% of cases), and the posterior mediastinum (20% of cases) (20,e278,e840,e1025). Less common
primary sites include the cephalic, paratesticular, or para-ovarian tissues, and the inguinal region; one concern about these various sites is whether they represent a primary tumor
or metastasis (1, 23, 70, 182). Rarely, NB presents as apparent multifocal tumors (64,e1084). A primitive appearing neuronal tumor may occur as the only or predominant element of
a sacrococcygeal or ovarian teratoma, in which case, the neuroblastic cells usually have the characteristics of the central nervous system rather than the peripheral nervous system
or neural crest.
Occasionally, some difficulty is encountered in distinguishing an adrenal or perirenal NB from Wilms tumor (WT). Most WTs are well-demarcated intra-renal masses. Biologically
favorable perirenal NB usually grows outside of the kidney, while biologically unfavorable perirenal NB often shows a direct invasion into the renal parenchyma. The blastema-
predominant WT may frequently require immunohistochemical differentiation from NB: the blastemal cells of WT are positive for VIM and WT-1 and are negative for CHR and SYN.
In the case of EWS-PNET, the tumor may be positive for neuroendocrine markers as well as MIC2 (CD99) and FLI-1, but is negative for WT-1 and TH [tyrosine hydroxylase (TH)]
(131,e569).
Clinical Features: Signs and symptoms at presentation are related to the location of the primary tumor and the extent of disease. The most common presentation of NB is an
abdominal mass in which radiological imaging studies demonstrate a suprarenal or retroperitoneal mass with or without calcification (e145). Orbital metastasis also causes periorbital
ecchymosis and edema with the so-called raccoon or panda eyes. Invasion or circumscription of the kidney by a NB in the adrenal, retroperitoneum or the perihilar region can mimic
a WT. NB may cause renal artery stenosis due to compression leading to systemic hypertension.
Patients with localized disease are often asymptomatic. A localized NB may be discovered incidentally in a routine well-baby examination or by a caregiver. Metastatic spread is
seen in patients with “progressive” stage 4 disease and “regressive” stage 4S (S stands for “special”) disease (Table 21-8). Major metastatic sites in stage 4 disease include bone
marrow and bone. To find a metastatic nodule in the brain parenchyma is rare: CNS metastasis, when present, often show a form of diffuse meningeal spread. Lung metastasis at
initial diagnosis is also extremely rare (49). In stage 4S disease, liver, skin, and/or bone marrow (without bone destruction) are the sites of metastasis (e268). Congenital NB can be
diagnosed perinatally by US and placental examination. Most congenital NBs are stage 1 or 4S with an excellent clinical outcome (73,148,e268). It is interesting to note that
neuroblastic cells may be found in the fetal capillaries of the chorionic villi in the presence of a congenital NB, suggesting that the placenta as a source of dissemination (eFigure 21-
105) (126,e565,e1110,e1149). Another presentation is nonimmune fetal hydrops. Placental metastasis is often present in these cases (125,e572,e855). Spinal cord compression is
caused by a paravertebral tumor growing into the spinal canal through neural foramina (“dumbbell lesion”) or osteolytic metastasis with vertebral collapse (38,e958). Neurological
abnormalities include motor deficit, radicular or back pain, sphincter abnormalities, and sensory deficit.
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Table 21-8 ▪ INTERNATIONAL NEUROBLASTOMA STAGING SYSTEMA
Stage Localized tumor with complete gross excision, with or without microscopic residual disease; representative ipsilateral lymph nodes negative for tumor
1 microscopically (nodes attached to and removed with the primary tumor may be positive).
Stage Localized tumor with incomplete gross excision; representative ipsilateral nonadherent lymph nodes negative for tumor microscopically.
2A
Stage Localized tumor with or without complete gross excision, with ipsilateral nonadherent lymph nodes positive for tumor. Enlarged contralateral lymph nodes must be
2B negative microscopically.
Stage Unresectable unilateral tumor infiltrating across the midline,a with or without regional lymph node involvement; or localized unilateral tumor with contralateral
3 regional lymph node involvement; or midline tumor with bilateral extension by infiltration (unresectable) or by lymph node involvement.
Stage Any primary tumor with dissemination to distant lymph nodes, bone, bone marrow, liver, skin, and/or other organs (except as defined for stage 4S).
4
Stage Localized primary tumor (as defined for stage 1, 2A, or 2B), with dissemination limited to skin, liver and/or bone marrowb (limited to infants <1 year of age).
4S
Multifocal primary tumors (e.g., bilateral adrenal primary tumors) should be staged according to the greatest extent of disease, as defined previously, followed by a subscript
“M” (e.g., 3M).
aThe midline is defined as the vertebral column. Tumors originating on one side and “crossing the midline” must infiltrate to or beyond the opposite side of the vertebral
column.
bMarrow involvement in stage 4S should be minimal, that is, < 10% of total nucleated cells identified as malignant on bone marrow biopsy or on marrow aspirate. More
extensive marrow involvement would be considered to be stage 4. The MIBG scan (if done) should be negative in the marrow.
International Neuroblastoma Staging System. Reproduced with permission from Brodeur GM, Maris, JM. Neuroblastoma. In: Pizzo PA, Poplack DG, editors. Principles and
practice of pediatric oncology, 5th ed. Philadelphia, PA: Lippincott-Williams & Wilkins Publishers, 2006;1997:761-797
Other uncommon clinical manifestations of NB are listed in Table 21-9. A small proportion of cases may have a socalled paraneoplastic syndrome including the
opsoclonusmyoclonus-ataxia syndrome (Kinsbourne syndrome) with “dancing eyes” (rapid and irregular movement of the eyes) and/or myoclonus and ataxia of the limbs, trunk, and
eyelids (32, 55, 144). An immune-mediated pathogenesis is suggested by the presence of a prominent lymphocytic infiltrate and lymphoid follicle formation in the primary site along
with antineuronal antibodies. The prognosis in terms of tumor behavior itself is generally excellent, but cognitive and motor developmental delay and language deficit often persist
even after complete resection of the NB. Horner syndrome (ptosis, miosis, enophthalmos) and heterochromia (difference in color) of the iris may occur in the presence of a NB
involving the cervical sympathetic ganglia. Intractable diarrhea with hypokalemia and dehydration are the manifestations of vaso-active intestinal peptide-producing neuroblastic
tumor with differentiating neuroblasts or a GN (e255,e326,e584,e1047). Differentiating neuroblasts may also produce somatostatin and other neuropeptides (e959). Cushing
syndrome and systemic hypertension are other clinical presentations (51). Extremely rare cases of vilirizing adrenal GN with Leydig cells have been reported (e14,e420). The so-
called neuroblastic “leukemia” in the peripheral blood with extensive bone marrow involvement is an uncommon hematological event (e148,e925). Another hematopathological
finding is myelofibrosis in the absence of demonstrable metastatic NB in the bone marrow (e668,e674).
Table 21-9 ▪ UNCOMMON AND UNUSUAL CLINICAL MANIFESTATIONS OF NEUROBLASTOMA
Opsoclonus-myoclonus ataxia syndrome
Horner syndrome and heterochromia of iris
Intractable watery diarrhea with hypokalemia and dehydration (VIP secretion)
Cushing syndrome
Systemic hypertension
Virilizing, masculinization (ganglioneuroma)
Neuroblastoma “leukemia”
Myelofibrosis
Fetal hydrops with placental involvement
VIP, vasoactive intestinal peptide,
There are several distinct associations of NB with other disorders including neurofibromatosis, BWS, Hirschsprung disease, musculoskeletal and cardiovascular malformations, and
Turner syndrome (Table 21-10) (e96,e97,e126,e241, e244,e408,e808,e1009,e1281). Molecular studies of cases of familial NB have failed to provide any linkage with the genes
responsible for neurofibromatosis 1 and 2 (108,e761). The relationship of congenital NB to the syndrome of central failure of ventilation (incorrectly referred to as Ondine curse—the
curse actually involved the loss of all autonomic and perceptive function) often accompanied by Hirschsprung disease has been explained on the basis of a widespread abnormality
of neural crest cell development and migration (152,178,e252,e1005,e1135). An excess of thyroid carcinomas (histological type not stated) is reported in individuals who received
radiation therapy for NB; this excess persisted when the study was analyzed for radiation dose to the thyroid in other childhood neoplasms (e285). An unusual type of renal cell
carcinoma with oncocytoid features is reported as a second primary neoplasm in survivors of NB
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(13,53,e793). Biochemical Markers: NB is characterized biochemically by catecholamine synthesis with metabolites that are detected in the serum and urine; this property is utilized
in the initial diagnosis and clinical follow-up as a measure of therapeutic response (e435). The precursor amino acids for catecholamine synthesis are phenylalanine and tyrosine. A
series of enzymes, such as TH, DOPA decarboxylase, dopamine β-hydroxylase, and phenylethanolamine N-methyltransferase, are involved in the pathway of catecholamine
catabolism and production of norepinephrine and epinephrine. NB cells usually lack the last enzyme, phenylethanolamine N-methyltransferase, which is present in adrenal
chromaffin cells and PHEOs. Degradation of L-DOPA and dopamine by catechol-O-methyltransferase and norepinephrine by monoamine oxidase, are primarily responsible for
production of the metabolites, homovanillic acid (HVA) and vanillylmandelic acid (VMA). These two metabolites, VMA and HVA, are the most widely measured serum and urinary
products for the diagnosis of NB and GNB, because GN is not a biochemically active neoplasm in most cases. When the VMA/HVA ratio is less than 1, these tumors seem to have a
less favorable clinical outcome than those with a ratio of 1 or greater (e689). Elevated tissue levels of the neuropeptides, VIP and somatostatin, have been correlated with cellular
differentiation and low stage disease (e959). An elevated serum level of neuron-specific enolase is reported not only in NB, but also in other tumors such as EWS-PNET, small cell
neuroendocrine carcinoma, PHEO, acute lymphoblastic leukemia, and non-Hodgkin lymphoma (60). Although detecting NSE in serum is less specific for the diagnosis of NB, high
levels at diagnosis have been correlated with a poor clinical outcome in several studies; this marker has some value for monitoring of recurrent tumor (165,e1299,e1300). Elevated
serum ferritin levels are also observed in NB, Hodgkin lymphoma, leukemia, and carcinoma of the breast (60). Higher serum ferritin levels at diagnosis are associated with metastatic
NB and its poor prognosis (e113,e465,e466). Ferritin is not suitable for monitoring disease activity, since it becomes elevated from frequent blood transfusions during the clinical
course. High serum lactate dehydrogenase (LDH) has some prognostic value, although LDH is not tumor specific but elevated levels reflect tumor load and rapid cell turnover
(98,164,e113). Other tumor markers reported to correlate with disease stage and/or prognosis include serum CHR A levels (68) and serum neuropeptide Y levels (e632). Recently
detection of circulating MYCN DNA in serum has shown some promise in unmasking MYCN amplified NBs (31).
Table 21-10 ▪ ASSOCIATION OF NEUROBLASTOMA WITH OTHER DISORDERS
von Recklinghausen neurofibromatosis
Hirschsprung disease
Musculoskeletal and cardiovascular malformations
Turner syndrome
Central failure of ventilation (“Ondine curse”)
Increased incidence of thyroid carcinoma in irradiated neuroblastoma patients (in comparison with patients irradiated for other childhood neoplasms)
Renal cell carcinoma (oncocytoid variant)
Morphologic features: The bone marrow (BM) biopsy is one of the essential procedures in the staging of a newly diagnosed NB, but it is also important in the monitoring of disease
activity (163,e154,e845,e975). It is generally recognized that both BM needle and aspiration biopsies have their complimentary value (7). An adequate aspirate may be difficult to
obtain when the marrow is densely replaced by tumor or with fibrosis after therapy. Paratrabecular nests of metastatic NB are the characteristic findings in the involved biopsy, but
micrometastatic disease may require immunohistochemistry, flow cytometry, and even RT-PCR in an attempt to establish the presence of tumor cells in a posttreatment specimen
(14,84,154,e223,e334). TH, PGP9.5, and MAP2 immunostaining are useful with limitations on the basis of specificities and sensitivities for detecting the rare malignant cell.
Metastatic NB in BM from a newly diagnosed case typically demonstrates collections of poorly differentiated neuroblasts with only a hint of neuropil in the background. On the other
hand, differentiating neuroblasts, individually distributed or forming small clusters, with abundant neuropil are often seen in the BM after chemotherapy. Schwannian stroma is rarely
encountered in BM biopsies.
The International Neuroblastoma Pathology Committee (INPC) made recommendations in 1999 for terminology and morphologic criteria of neuroblastic tumors by adopting and
modifying the original Shimada classification (156, 157, 159). The recommendations were based on the hypothesis that these tumors provided one of the better models for analyzing
the biological relationship between molecular/genomic alterations and morphology. As outlined below, peripheral neuroblastic tumors are classified into four categories: (Table 21-
11) NB, GNB-intermixed, GNB-nodular, and ganglioneuroma (GN).
NBs are further subclassified into undifferentiated, poorly differentiated, and differentiating subtypes. Grossly (Figure 21-34A, B, eFigure 21-106), NB usually presents as a solid
circumscribed or multinodular mass, measuring 10 cm or less in greatest dimension, with considerable variation in appearance depending on the anatomic location, histological
subtype, and secondary changes. A deep reddish hemorrhagic appearance with or without scattered foci of glistening gray-white tissue is a common gross presentation for NB of
the undifferentiated or poorly differentiated subtype. Punctate or coarse calcifications or yellowish areas of coagulative necrosis are other relatively common macroscopic features in
these latter two subtypes. Cystic degeneration with or without hemorrhage is another feature; the cystic NB, commonly arising in the adrenal gland, may require extensive sampling
to identify microscopic foci of tumor. On the other hand, NB of the differentiating subtype is usually tan-yellow and less hemorrhagic with only limited areas of necrosis, if any.
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Table 21-11 ▪ CATEGORY AND SUBTYPES RECOMMENDED BY THE INTERNATIONAL NEUROBLASTOMA PATHOLOGY COMMITTEE
Category Subtype
Neuroblastoma (Schwannian stroma-poor)a Undifferentiated

Poorly differentiated
Differentiating
Ganglioneuroblastoma, intermixed (Schwannian stroma-rich)
Ganglioneuroma (Schwannian stroma-dominant) Maturing
Mature
Ganglioneuroblastoma, nodularb
(Schwannian stroma-dominant/stroma-rich and stroma-poor)
aMKI (mitosis-karyorrhexis index; Low, Intermediate, or High) is assigned along with subtype of each neuroblastic tumor.
bSubtype (undifferentiated, poorly differentiated, or differentiating) and MKI are assigned to the neuroblastomatous nodule of each ganglioneuroblastoma, nodular tumor.
From Peuchmaur M, d'Amore ESG, Joshi W, et al. Revision of the international neuroblastoma pathology classification: confirmation of favorable and unfavorable prognostic
subsets in ganglioneuroblastoma, nodular. Cancer. 2003;98:2274-2281; Shimada H, Ambros IM, Dehner LP, et al. Terminology and morphologic criteria of neuroblastic
tumors: Recommendation by the International Neuroblastoma Pathology Committee. Cancer. 1999;86:349-363
NBs are further defined as Schwannian stroma-poor, and composed of neuroblasts forming lobules which are completely or incompletely separated by delicate fibrovascular septa.
Putative Schwannian blasts may be detected as slender S-100 positive cells in the septal area (158). The typical neuroblast is round or slightly ovoid with a round to oval nucleus
with salt-and-pepper chromatin and scanty cytoplasm. With the formation of neurites, an eosinophilic fibrillary network or neuropil becomes apparent, but is not regarded as
“stroma.” Homer-Wright rosettes are arranged around a central tangle of neurofibrillary processes without a central lumen or canal. Differentiating neuroblasts, a transitional form of
neuroblastic differentiation toward ganglion cells, are characterized by synchronous changes in both the nucleus (enlarged, eccentrically located with vesicular chromatin pattern,
and a single prominent nucleolus) and cytoplasm (eosinophilic/amphophilic with a diameter usually two or more times larger than the nucleus). Neuritic processes or neuropil
becomes less prominent with ganglionic differentiation.
FIGURE 21-34 ▪ Neuroblastoma. A: Adrenal neuroblastoma (Schwannian stroma-poor), poorly differentiated subtype, measuring 5 cm × 4.5 cm in the greatest dimension, shows a
friable and hemorrhagic appearance. B: Adrenal neuroblastoma (Schwannian stroma-poor), differentiating subtype, measuring 6 cm × 4 cm in the greatest dimension, shows a soft
and less hemorrhagic appearance.
The undifferentiated subtype of NB is composed of undifferentiated neuroblasts without clearly identifiable neuropil or rosettes (Figure 21-35). In fact, there is very little to
differentiate these tumor cells from the nonneuroblastic round cell neoplasms of childhood without the assistance of immunohistochemistry and molecular/cytogenetic studies.
Preliminary data suggest that undifferentiated neuroblasts lack the potential for differentiation. S-100 protein staining demonstrates no or very few putative Schwannian blasts in the
septal areas of the tumor when septation is present. Some tumors in this subtype show a diffuse growth pattern without a lobular architecture.
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FIGURE 21-35 ▪ Neuroblastoma (Schwannian stroma-poor), undifferentiated subtype is composed of primitive cells without clearly recognizable neurite formation. Tumor cells in this
case often have one or few prominent nucleoli. Note that tumor cells are irregularly demarcated by thin fibrovas-cular septal tissue.
The poorly differentiated subtype is the most common pattern of NB in this group, and is diagnosed in most cases without difficulty since neuropil and/or Homer-Wright rosettes are
commonly present (Figure 21-36) (eFigure 21-107). Most tumor cells are typical neuroblasts, and less than 5% of the population is pursuing ganglionic differentiation. Lobular
formations of neuroblasts with thin fibrovascular septa are evident in many of these tumors. S-100 protein positive slender Schwann cells or putative Schwannian blasts are
detectable especially in the biologically favorable tumors of this subtype. It has been postulated that those Schwann cells/Schwannian blasts are recruited into the tumor by the
biologically favorable neuroblasts, rather than as end-stage product differentiation from the neural crest cells (e38).
NB, differentiating subtype contains 5% or more of tumor cells with the features of differentiating neuroblasts (Figure 21-37). These tumors also have a prominent neuropil. It is
thought that biologically favorable NBs of the poorly differentiated subtype can either regress or mature in the direction of the differentiating subtype. To date, among the biologically
favorable NBs, there is no clear distinction in molecular characteristics between tumors with a potential for regression and those with presumed potential for maturation. In fact,
during the process of tumor maturation from a poorly differentiated subtype to a differentiating subtype, the vast majority of neuroblasts undergo programmed cell death or apoptosis
before or after attaining a certain degree of neuroblastic differentiation.
FIGURE 21-36 ▪ Neuroblastoma (Schwannian stroma-poor), poorly differentiated subtype is the most common form of tumor in the neuroblastoma group. Neuroblastoma cells
produce neurites and can show rosette formations. Inset: Typical Homer-Wright rosette.
FIGURE 21-37 ▪ Neuroblastoma (Schwannian stroma-poor), differentiating subtype (containing more than 5% of the tumor cells showing an appearance of differentiating neuroblast
by definition) is often characterized by abundant neuropil formation. Tumor cells are irregularly separated by thin fibrovascular septa, but significant Schwannian stromal
development is not observed.
Some NBs have unique morphologic features including anaplastic appearing tumor cells which are characterized by the presence of enlarged, bizarre cells and atypical mitotic
figures (120,e259). There is a large cell type of NB with prominent nucleoli (175, 176). These rare tumors are known for their aggressive clinical course and often fatal outcome.
Ganglioneuroblastoma-intermixed is definned as a Schwannian stroma-rich tumor whose Schwannian component occupies more than 50% of the tumor area (Figure 21-38). The
histological features seem to imply that there is incomplete transition to a fully mature GN, but the process is not complete, as evidenced by the presence of scattered “residual”
microscopic foci or collections in neuroblasts in varying stages of differentiation with a background of neuropil. These neuroblasts, many with differentiating features to immature
ganglion cells, are in a process of either apoptosis or continuous maturation to mature ganglion cells. Individually distributed mature and maturing ganglion cells are also found in the
Schwannian stroma with the pattern of GN.
Ganglioneuroma is a Schwannian stroma-dominant neoplasm without any aggregates of neuroblasts in a neuropil background. The exclusive cellular elements are Schwann cells
with accompanying individually distributed or small
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groups of maturing/mature ganglion cells. Two subtypes, GN-maturing (Figure 21-39A) and GN-mature (Figure 21-39B), are included in this category. The GN-maturing, previously
designated as “GNB, well differentiated” in the original Shimada classification (159), contains scattered individual immature ganglion cells and/or neuroblasts in addition to mature
ganglion cells. The mature GN (GN-mature) is the fully mature peripheral neuroblastic tumor, and is composed of Schwannian stroma and mature ganglion cells which are
surrounded by satellite cells. Fully developed Schwannian stroma is seen in GNs and focally in the GNB-intermixed. Mature unmyelinated type of Schwann cells characteristically
forming multiple fascicles which are covered with perineurial cells are present. These areas of a mature GN without ganglion cells resemble a schwannoma. A wellformed capsule is
more characteristic of the schwannoma, whereas, the GN tends to blend into the adjacent soft tissues with some circumscription of the peripheral margins.
FIGURE 21-38 ▪ Ganglioneuroblastoma, intermixed (Schwannian stromarich) is characterized by an extensive Schwannian stromal development (S) occupying more than 50% of
tumor tissue. Pockets of naked neuropil (N) area containing tumor cells of various stages of neuronal differentiation are found. Tumor cells in those pockets are composed of a
mixture of differentiating neuroblasts and maturing ganglion cells with or without poorly differentiated neuroblasts.
FIGURE 21-39 ▪ A: Ganglioneuroma (Schwannian stroma-dominant), maturing is a tumor predominantly composed of Schwannian stromal tissue. Differentiating neuroblasts and
maturing/mature ganglion cells are distributed without clearly recognizable pockets of neuropil. B: Ganglioneuroma (Schwannian stroma-dominant), mature, is a completely mature
form in the neuroblastoma group. Fully mature ganglion cells are covered with satellite cells. Stroma component is well organized and shows multiple fascicular formations composed
of Schwann cells of unmyelinated type surrounded by perineurial cells.
Both the GNB-intermixed and GN have similar gross features with a firm consistency, and a cut surface with a tan-yellow, homogenous appearance with or without fibrous bands
(Figure 21-40).
Ganglioneuroblastoma-nodular is a composite tumor characterized by the presence of one or more grossly visible, often hemorrhagic/necrotic neuroblastic nodule(s) coexisting with
GNB-intermixed or GN (Figure 21-41A, B). There is typically an abrupt demarcation (pushing border or pseudo-capsular formation) between the neuroblastic nodule(s) and the
ganglioneuromatous component (GNB-intermixed or GN). Some neuroblastic nodules may not be clearly demarcated, but rather there is neuroblastic infiltration into the Schwannian
stromal component of GNB-intermixed or GN component. It is possible that some neuroblastic nodules are intratumoral metastasis into the ganglioneuromatous areas. Infrequently, a
neuroblastic nodule grows so large that the ganglioneuromatous component (GNB-intermixed or GN) can only be recognized microscopically, often at the periphery of the tumor, as
a narrow ribbon of GN. Neuroblastic nodules are usually evident in the gross examination of the primary tumor; however, they may be overlooked. For that reason, those primary
tumors with the features of GNB-intermixed or GN, but with metastatic NB
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to lymph node, bone or other sites are also included in the category of GNB-nodular (137).
FIGURE 21-40 ▪ Ganglioneuroblastoma, intermixed (Schwannian stromarich), in the mediastinum measuring 9 cm × 7 cm in the greatest dimension, is rubbery in consistency and
has no grossly visible nodule of neuroblastomatous growth. Tumors in both ganglioneuroblastoma, intermixed (Schwannian stroma-rich) and ganglioneuroma (Schwannian stroma-
dominant) category present a gross appearance similar to a Schwannoma.
As a component of the pathologic evaluation of a NB, the INPC has recommended a determination of the mitotic and karyorrhectic activities by the mitosis-karyorrhexis index (MKI)
which has been defined by three semiquantitative levels: low (<2% or < 100 mitotic and karyorrhectic cells per 5,000 neuroblasts), intermediate (2% to 4%) or 100 to 200 mitotic and
karyorrhectic cells per 5,000 neuroblasts), and high (>4% or >200 mitotic and karyorrhectic cells per 5,000 neuroblasts). The MKI is determined by counting the number of tumor
cells in mitosis and in the process of karyorrhexis, and should reflect an average for all tumor sections available. Cells in karyorrhexis, one of the apoptotic processes and individual
cell death due to severe genomic instability, are characterized by condensed and fragmented nuclear chromatin without nuclear membrane, usually accompanied by condensed
eosinophilic cytoplasm. Hyperchromatic nuclei without chromatin fragmentation are not included in the MKI count. It has been reported that increased mitotic and karyorrhectic
activity is correlated with MYCN amplification and excess production of MYCN protein (58, 161).
FIGURE 21-41 ▪ Ganglioneuroblastoma, nodular (composite, Schwannian stroma-dominant/stroma-rich and stroma-poor) arising the retroperitoneum. A: The tumor measuring 8 cm
× 7 cm in the greatest dimension, is tanyellow and rubbery in consistency, and contains a grossly visible hemorrhagic nodule. There are two (or multiple) distinct tumor types/clones
coexisting in the same tumor tissue of this category. B: As shown in this example, one tumor type (left side) has an appearance of neuroblastoma (Schwannian stroma-poor)
forming a grossly visible and often hemorrhagic nodule, and other (right side) has a feature of ganglioneuroma (Schwannian stroma-dominant).
Ultrastructure, Immunohistochemistry, Molecular Diagnostics: Ultrastructural features characteristic of neuroblastic cells include the presence of membrane-bound neurosecretory
granules in the cytoplasm and neuritic processes with typically arranged microtubules which are parallel to each other (e491,e1205,e1206). Rudimentary attachment structures are
found between adjacent neuroblasts. These features, however, are also detectable in EWS-PNETs. Nissl bodies composed of rough endoplasmic reticulum and free ribosomes are
found in the periphery of the cytoplasm of differentiating neuroblasts and ganglion cells. Neuromelanin can be detected in some of the differentiating neuroblasts (e429). Mature
Schwannian cells found in the GNB-intermixed and GN are the unmyelinated cells and contain multiple neurites in the individual cell bodies.
Immunohistochemically, neuroblastic cells are positive for NSE, NB84, PGP9.5, SYN, CHR, Leu 7 (CD57), a variety of NFPs, NCAMs, and other neural antigens (191,
e163,e201,e399,e812,e832,e901). TH is a useful marker for identifying neural crest cells, and is positive in both NB and PHEO/paraganglioma (74). In most cases, application of
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immunohistochemistry is more adjunctive to the histological examination, since the accumulative data of clinical and laboratory findings generally support a straightforward diagnosis
of NB. In our own practice (HS), PGP9.5, TH, MIC2 (CD99), desmin, MyoD1, myogenin, and lymphoid markers are applied as the immunohistochemical panel for distinguishing an
undifferentiated NB from the other malignant round cell neoplasms of childhood. Undifferentiated NBs are diffusely positive for PGP9.5, often sporadically positive for TH, and
negative for the other markers. While EWS-PNET is positive for PGP9.5 and MIC2 (CD99) but negative for TH, rhabdomyosarcoma is positive for desmin, MyoD1, and myogenin.
Hematolymphoid malignancies can be screened with CD43 and CD45 to be followed by additional specific lineage markers. When only VIM is immunoreactive, one should think in
terms of an undifferentiated NB or a hematolymphoid malignancy. Putative Schwannian blasts are positive for S-100 (use of monoclonal antibody against β-chain recommended)
and located in the thin fibrovascular septa demarcating groups/clusters of NB cells of especially the biologically favorable NB tumors (158). Ganglioneuro-blastoma and GN are
characterized by the S-100 positive Schwannian stromal development. Satellite cells around the fully mature ganglion cells are also positive for S-100.
Table 21-12 ▪ PROGNOSTIC DISTINCTION ACCORDING TO THE INTERNATIONAL NEUROBLASTOMA PATHOLOGY CLASSIFICATION
Age Favorable Histology Group Unfavorable Histology Group
Any Ganglioneuroma
(Schwannian stroma-dominant)
Maturing
Mature
Ganglioneuroblastoma, intermixed
(Schwannian stroma-rich)
Neuroblastoma
(Schwannian stroma-poor)
Undifferentiated and any MKI
Less than 1.5 Neuroblastoma Neuroblastoma

years (Schwannian stroma-poor) (Schwannian stroma-poor)
Poorly differentiated and low or intermediate MKI Poorly differentiated and high MKI
Differentiating and low or intermediate MKI Differentiating and high MKI
1.5 years up to Neuroblastoma Neuroblastoma

less than 5 years (Schwannian stroma-poor) (Schwannian stroma-poor)
Differentiating and low MKI Poorly differentiated and any MKI

Differentiating and intermediate or high MKI
Equal to or Neuroblastoma
greater than 5 (Schwannian stroma-poor)
years
Any subtype and any MKI
Ganglioneuroblastoma, nodular (composite, Schwannian stroma- Ganglioneuroblastoma, nodular (composite, Schwannian stroma-
rich/stroma-dominant and stroma-poor), favorable subseta rich/stroma-dominant and stroma-poor), unfavorable subseta
aAlltumors in the category of ganglioneuroblastoma, nodular were once classified into an unfavorable histology group according to the origina Shimada classification (159)
and the original International Neuroblastoma Pathology Classification (INPC) (156). However, the revised INPC distinguishes two prognostic subsets, favorable, and
unfavorable, by applying the same age-linked histopathology evaluation to the nodular (neuroblastoma) components of the tumors in this category (137). MKI, Mitosis-
karyorrhexis index.
From Peuchmaur M, d'Amore ESG, Joshi W, et al. Revision of the International Neuroblastoma Pathology Classification: confirmation of favorable and unfavorable
prognostic subsets in ganglioneuroblastoma, nodular. Cancer. 2003;98:2274-2281; Shimada H, Ambros IM, Dehner LP, et al. Terminology and morphologic criteria of
neuroblastic tumors: Recommendation by the International Neuroblastoma Pathology Committee. Cancer. 1999;86:349-363.
Frequently used molecular markers in a setting of differential diagnosis include EWS-ETS translocation (EWS-FLI1, EWS-ERG) and PAX-FKHR translocation. NBs are negative for
those translocations. Demonstrating EWS-ETS translocation by FISH and detecting its chimeric protein are diagnostic of EWS-PNET (8, 76). The presence of PAX-FKHR
translocation, detectable in around 80% alveolar rhabdomyosarcomas (130), is reported to indicate an aggressive clinical behavior of the disease (37).
International NB Pathology Classification: A morphological classification designed to be prognostically significant and biologically relevant, was established by the INPC in 1999
(156, 157) and revised in 2003 (137). This classification distinguishes two pathologic -prognostic groups: favorable histology (FH) and unfavorable histology (UH) group (Table 21-
12) (3, 5, 22, 56, 72, 97, 119, 121, 146, 162). This classification is age-linked and utilizes three morphologic indicators: status of Schwannian stromal development (stroma-poor,
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stroma-rich and stroma-dominant), grade of neuroblastic differentiation (undifferentiated, poorly differentiated, and differentiating), and MKI (low, intermediate, high). The histological
features should be evaluated on a resected specimen or biopsy before the initiation of chemotherapy/irradiation therapy. Metastatic sites except for bone marrow are eligible for
evaluation of all these histological features since a BM biopsy is not informative for MKI determination. FH NBs fall within the conceptual framework of an ageappropriate
maturational sequence starting with NB (Schwannian stroma-poor), poorly differentiated subtype (up to 1.5 years of age at diagnosis) to NB-differentiating subtype (up to 5 years of
age at diagnosis) to GNB-intermixed (Schwannian stroma-rich) to GN (Schwannian stroma-dominant). All GNs, the final end of tumor maturation, are thought to have had a
neuroblastic component in their early developmental stage. In this regard, most GNs are diagnosed in later childhood and into adulthood. FH neuroblastic tumors should have a low
(when diagnosed <5 years old at diagnosis) or an intermediate (when diagnosed <1.5 years old at diagnosis) MKI. In contrast, the histological features of UH neoplasms are
immature or inappropriate for the age at diagnosis, and include NB, undifferentiated subtype (at any age), poorly differentiated NB (>1.5 years of age), and all NB subtypes (>5
years of age). Among tumors in the NB category, those with high MKI (at any age), or an intermediate MKI (>1.5 years of age) are also assigned to the UH group.
Ganglioneuroblastoma-intermixed and GN, usually diagnosed in older children, are examples of FH tumors with an excellent prognosis (127). Tumors in the GNB-nodular
(composite, Schwannian stroma-rich/stroma-dominant and stroma-poor) category are further subclassified into two subsets, favorable and unfavorable, by application of the same
criteria of age-linked evaluation of histological features (grade of neuroblastic differentiation and MKI) to the nodular (NB-, Schwannian stroma-poor) component (137, 181).
Ganglioneuroblastoma-intermixed or GN are usually resected surgically. These tumors may encase the great vessels and/or organs, so that complete surgical excision with tumor-
free margins is often difficult and unnecessary in most cases since local recurrences are uncommon. When only a biopsy is available and shows features of GNB-intermixed or GN,
the pathologic diagnosis should be qualified by the comment, “the diagnosis is made based on review of limited material.” In this circumstance, careful reassessment of the primary
tumor site as well as a metastatic workup are recommended since there is the possibility of GNB-nodular in which case an unsampled neuroblastic nodule may exist. An unsampled
neuroblastic nodule, if present, is often hemorrhagic and necrotic or may show invasive growth into the surrounding tissues which may be apparent by imaging studies. Metastatic
foci may be demonstrable as well. Catecholamine determination is also advisable.
Histological changes after chemotherapy/irradiation therapy especially in the UH tumors do not provide any reliable information in predicting clinical outcome. These changes
include extensive necrosis, hemorrhage, hemosiderin deposition, fibrosis, and calcification along with varying degrees of tumor maturation, often presenting different histological
features from area to area, while, in the FH tumors, chemotherapy often seems to facilitate/expedite uniform tumor maturation without extensive necrosis, hemorrhage, and marked
hemosiderin deposition. According to the Children's Oncology Group (COG) Neuroblastoma Study, the INPC evaluation of either FH or UH, once determined based on the review of
prechemotherapy specimen, will not be altered during the clinical course of individual cases.
Molecular/Genetic Alterations: One of the more remarkable aspects of NB is the biological heterogeneity which is reflected in its tumorigenesis and diverse clinical behaviors.
Structural genetic alterations often detected in NB include genomic amplification of MYCN oncogene on chromosome 2p24 (e157,e158,e1069), allelic deletion of the short arm of
chromosome 1p36 (del 1p) (11,170,e158), allelic deletion of the long arm of chromosome 11q23 (del 11q) (11, 167, 169), and unbalanced gain of genetic material of the long arm of
chromosome 17q21 (17q-gain) (e146). Besides these alterations, allelic losses of genetic material on 3p, 4p, 9p, 14q, 16p, and 19q, as well as segmental gains of 1q, 5q, and 18q
have been detected in varying numbers of NBs (153). Most of these alterations are associated with unfavorable clinical behavior. Among these changes, 17q-gain is the most
frequent alteration in some two-thirds of NBs, and may be related to the tumorigenesis. Spitz et al. could not confirm the prognostic impact of 17q-gain in children with NB (168).
PHOX2B transcription factor (a homeobox gene functioning as an important regulator in development of normal autonomic nervous system) is mutated in a small proportion of NBs
(117, 177, 183).
MYCN amplification, one of the strongest indicators for aggressive tumor progression, is observed in 15% to 20% of all NBs; the result is excess MYCN protein production. MYCN-
MAX heterodimer formation in the tumor nuclei seems to prevent cellular differentiation, to promote cellular proliferation, and to effect genomic instability (e738,e1225). There is a
reproducible correlation between the molecular event of MYCN amplification and the morphologic features of a NB. Those tumors with amplified MYCN typically have
undifferentiated or poorly differentiated features with a high MKI reflecting increased cellular proliferation and apoptosis due to genomic instability (Figure 21-42) (58, 161). The
presence of prominent nucleoli in neuroblastic cells of undifferentiated or poorly differentiated tumor cells is reported to be an additional hallmark of MYCN amplification with a high
sensitivity and a relatively lower specificity (82, 172, 176).
MYCN status of the individual tumors is now tested by fluorescent in situ hybridization (FISH) analysis in many institutions. The International Neuroblastoma Risk Group (INRG)
Biology Committee has defined MYCN amplification by FISH analysis as “More than fourfold increase in the MYCN signal number compared with the reference probe located on the
chromosome 2q.” Furthermore, MYCN
P.967
gain has been defined as a signal increase but not up to the amplified status, whose clinical significance is yet to be determined (6). It is also noted that MYCN amplified status
usually remains unchanged after chemotherapy.
FIGURE 21-42 ▪ Neuroblastoma. Typical histological features of the MYCN amplified neuroblastoma include no Schwannian stroma development, no or limited neuroblastic
differentiation, and markedly increased mitotic and karyorrhectic activities (high MKI—mitosis-karyorrhexis index).
Activating mutations in the ALK oncogene appear to be responsible for many of the hereditary NBs and could also be relevant for some fraction of sporadic cases. Interestingly, 20%
to 25% of primary NBs present copy number increases at the ALK locus on 2p23, and elevated ALK gene-expression levels are reported in aggressive neuroblastic tumors
(26,118,125,128,186,e561). Genetic variation at chromosome 6p22 has been identified for NB susceptibility. Additionally, patients who are homozygous for the risk alleles at 6p22
are likely to have metastatic disease, MYCN amplification, and decrease relapse (107).
Gene expression-based analyses show that elevated levels of TrkA, CD44, and CAMTA1 correlate with favorable clinical outcome (62,160,e251,e854), while elevated levels of
expression of survivin, repp86, and PRAME are reported to correlate with adverse outcome (85, 114, 124). However, anyone of those candidates alone cannot sufficiently explain
the diverse clinical behaviors of NBs, and is not considered as an independent prognostic factor in clinical trials.
DNA ploidy patterns, diploid (“near-diploid”) or hyperdiploid (“near-triploid”), are reported to distinguish prognostic categories (92,166,e733,e734). A near-triploid DNA content due to
whole chromosomal gain (lack of structural chromosomal aberration) has been reported as a favorable prognostic indicator. In contrast, a near-diploid DNA content predicts a poor
clinical outcome for patients especially when they are infants.
Risk Grouping (INRG): Because of the biological complexity of tumors in the NB group, it is essential to establish a risk-group system for patient stratification and protocol
assignment in the clinical management. Those risk factors or so-called prognostic factors include age at diagnosis, clinical stage, histopathologic classification according to the
INPC, and molecular/genetic alterations.
Historically, 1 year of age at diagnosis has been utilized as a cutoff for predicting the prognosis. Recent analysis, however, demonstrates that the prognostic contribution of age to
clinical outcome is continuous in nature. There is a gradual worsening of prognosis with increasing age, and there is statistical support for any choice of age cutoff between 15 and
19 months for use of risk stratification (e689). Based on these results, the COG NB study is currently using two age cutoffs, 12 months (365 days) and 18 months (547 days), at
diagnosis in their risk-grouping scheme.
The International Neuroblastoma Staging System (INSS), a postsurgical staging system, has been used for prognostic purposes (Table 21-8) (e159,e160). Recently the INRG
proposed a pretreatment staging system, based on clinical criteria and image-defined risk factors (116). In order to facilitate the comparison of risk-based clinical trials conducted in
different regions and countries, the INRG defined four risk groups based on the combination of INRG stage (L1, L2, M, MS) (28, 116) age at diagnosis (cutoffs at 12 and 18 months),
histopathology (tumor category and grade of neuroblastic differentiation according to the recommendation by the INPC, MYCN status, 11q aberration, and ploidy: very low (>85% 5-
year EFS); low (>75% to <85% 5-year EFS); intermediate (>50% to <75% 5-year EFS), and high (<50% 5-year EFS) (186). In contrast, the COG NB studies distinguish three risk
groups for the purpose of patient stratification and protocol assignment based on the combination of INSS stage [1, 2, 3, 4, 4S] (e158,e160), age at diagnosis (cutoffs at 12 and 18
months), histopathology (INPC; FH versus UH), MYCN status, ploidy, 1pLOH, and unb11qLOH. Their projected 5-year EFS rates are greater than 95% for the low-risk patients with
surgery alone, greater than 90% for the intermediate-risk patients with surgery/biopsy and chemotherapy, and approximately 40% for the high-risk patients with intensive treatment
including bone marrow transplantation (e1088).
Mass Screening: A mass screening program for preclinical detection of NB by measuring catecholamine metabolites was initiated in Japan 35 years ago (e1044) and then
introduced to other countries including England, Germany, France, Austria, and Canada (149,e260,e602,e771). This program was based on the assumption that NB begins as a
nonaggressive disease and would eventually progress to a more aggressive disease, and secondly that one-time screening in early life could detect all or many of the NBs in their
nonaggressive state. In Japan, nationwide screening began in 1984 based on the significantly increased survival that could have been artificially raised due to increased incidence
of newly (and unnecessarily) diagnosed cases through the screening (147,e1045,e1154). Whereas controlled studies from Quebec (screened at the age of 3 weeks and 6 months)
(193,e594,e1168,e1285,e1286) and Germany (screened at the
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age of 1 year) (150,e1054) both reported similar and widely accepted results: (a) Screening almost doubled the incidence of NB and (b) cumulative mortality in the screened
population was not reduced compared with an appropriate control population. NB is composed of at least two distinct clinicalbiologic favorable and unfavorable behavior; tumors in
the former group, once established, typically favorable clinical course and do not progress into the latter group. It had also been known that certain NBs in the biologically favorable
group have the potential to spontaneously regress. However, the magnitude of such regression was not anticipated by the screening program. Screening failed to detect substantial
numbers of biologically unfavorable tumors before their progression. Beside these biological and clinical issues, mass screening for NB yielded many clinical, psychological, and
economic problems (the law of unanticipated consequences). In Japan, screening was finally terminated in 2004 because of the many pitfalls (180). Recently they reported a
Japanese experience of a screening program at 6 months of age, and introduced an on-going screening at 18 months of age (63, 65). Unfortunately their retrospective study
contains major methodological issues (e762), such as changes in diagnostic standards and treatment modalities over the study period.
PHEO is a relatively rare neoplasm whose annual incidence ranges from two to eight cases: 1,000,000 population. The term pheochromocytoma is derived from the brown color
observed when the tumor is immersed in a dichromate solution (Figure 21-43A, B) (e789). Approximately 5% to 10% of incidentally-discovered adrenal masses, mainly in adults, are
PHEOs (e728). Although 10% to 15% of cases present in the first two decades of life, some of the special clinical settings of PHEOs in children include the greater likelihood of a
syndromic association, estimated at 15% to 25% of tumors (to include BWS, von Hippel-Lindau syndrome, MEN 2a (50% of cases), MEN 2b, Carney complex, familial PHEO and
neurofibromatosis (1), bilaterality (commonly an association with one of the predisposing inherited disorders), and extraadrenal paraganglioma (25% to 40% of children) (Table 21-1)
(4,15,18,91,122,138,173,174,e2,e79,e93,e101,e235, e263,e290,e323,e361,e425,e482,e489,e707,e744,e859,e986, e1008,e1035,e1207,e1245).
FIGURE 21-43 ▪ Pheochromocytoma. A: This adrenal gland from a patient with episodic hypertension is replaced with a tan-white tumor with focal area of hemorrhage and necrosis
and fibrosis. The tumor cells were immunoreac-tive for chromogranin. B: Immersion of this tumor in a dichromate-containing fixative yielded a dark-brown color. This positive
chromaffin reaction is due to oxidation of the catecholamines, epinephrine and norepinephrine.
Genetic testing in 314 PHEOs in 56 patients with a family history and 258 patients with “sporadic” tumors, 27% had a hereditary tumor; among the 56 patients with a positive family
history, NF1 and germline mutations in VHL, RET, and SDHB (succinate dehydrogenase subunit B) and SDHD (succinate dehydrogenase subunit D) were identified (4). In patients
with apparent sporadic PHEOs, 11% had germline mutations in VHL, RET SDHB and SDHD. In a similar study of 271 patients with “sporadic” PHEOs, 24% had germline mutations
in VHL, RET, SDHB, and SDHD; younger age, multifocality, and extra-adrenal sites (paragangliomas) (122). Havekes et al. have recently reported that 40% of PHEOs (adrenal and
extra adrenal) had a hereditary basis (e482).
The WHO defines PHEO as a tumor of chromaffin cells of the adrenal medulla, and paraganglioma as a tumor arising from the extra-adrenal paraganglia; both tumors are neural
crest in origin (eFigure 21-104) (42,e724,e789). A number of studies, however, combine both adrenal medullary tumors and extra-adrenal tumors under the diagnosis
“pheochromocytoma.” When combined, 80% of these tumors arise in the adrenal medulla and the remainder in the extra-adrenal paraganglia (20%) (15,e482). In a review of 520
PHEOs, 50 PHEOs (9.6%) occurred in children less than 16 years of age (15). Among these 50 childhood cases, the male:female ratio was 2:1, bilaterality was present in 32% of
cases and extraadrenal location in 18% of cases. A hereditary syndrome was identified in 7% of cases. Local recurrence or metastasis after initial excision occurred in 12% of
children.
P.969
FIGURE 21-44 ▪ Pheochromocytoma. A: This adrenal gland demonstrates a central brown 3 cm diameter tumor replacing the adrenal medulla. The adrenal cortex (yellow) is seen
at the periphery of this tumor. This tumor was resected from a patient with a chief complaint of paroxysmal attacks of headache, blurred vision, tachycardia and diaphoresis. B: The
tumor cells are arranged in a characteristic alveolar “zellballen” or nesting pattern. They are surrounded by thin fibrovascular septate. The polyhedral tumor cells vary in shape and
size. Most cells have an eosinophillic granular cytoplasm. The ovoid nuclei have a dispersed stippled chromatin pattern with inconspicuous nucleolus. Mitotic figures were infrequent
in this tumor (H&E stain).
The major clinical signs and symptoms are related to the release of epinephrine with hypertension, paroxysms (headaches, palpitations and sweating) and either tachycardia
(epinephrine) or reflex bradycardia (norepinephrine) (18,e79,e232,e1156). Cerebral infarction, cardiomyopathy, and catecholamine crisis have been observed in children
(e270,e617). A female predilection is seen in some studies and others report a male preference (e322,e974). The average age at diagnosis in childhood is 12 to 15 years
(e290,e378,e928,e979).
Sporadic PHEO is typically a solitary, well-circumscribed mass with either a true capsule or a pseudocapsule related to the tumor expansion and compression of adjacent connective
tissue. Most tumors range in size from 3 to 5 cm, and the average weight is about 100 g (Figure 21-43A). Periadrenal brown fat is often seen. Because medullary tissue is
concentrated in the head and body of the adrenal, the smaller PHEO arises in the latter locations (Figure 21-44A). On cut section, the tumor is firm and gray or dark red or is
extensively hemorrhagic with cystic degeneration and friability. The chromaffin reaction is a manifestation of the catecholamines in the tissue and is produced by exposure of the
unfixed specimen to a dichromate solution, which leads to a deep brown coloration (Figure 21-43B).
Three principal histological patterns are found in PHEO: a trabecular pattern with anastomosing cords of cells, an alveolar or nesting pattern with “zellballen” formation, and a diffuse
or solid growth pattern (Figure 21-44B, eFigure 21-108). Spindle cells, angiomatoid foci, prominent interstitial and perivascular sclerosis, pseudopapillary formations and small
spaces filled with eosinophilic proteinaceous debris are focal or generalized features in any one tumor (e685). Nuclear pseudoinclusions and eosinophilic hyaline intracytoplasmic
globules are common. The individual tumor cells range from eosinophilic and granular to intense basophilia.
Immunohistochemistry has superseded many of the classic silver stains. These tumors are typically immunoreactive for VIM, CHR, VIP, and, infrequently HMB-45 (36,
e381,e768,e1213). Antibodies to S-100 protein stain the sustentacular cells. Electron microscopy reveals cells with interdigitating borders and poorly formed cells junctions.
Membrane-bound dense-core neurosecretory granules are prominent; these granules appear to be norepinephrine with a prominent eccentric electron-lucent space surrounding the
dense core.
The incidence of malignancy in childhood PHEOs is difficult to ascertain due to the inclusion of paragangliomas in many studies; however, it is estimated that 2% to 12% of these
tumors behave in a malignant fashion (91,e531). With inclusion of paragangliomas, the incidence is even higher, since paragangliomas especially in sites other than in the head and
neck region are more prone to malignant behavior. When paragangliomas are included in the assessment of prognosis, the incidence of malignancy approaches 50% in some series
inclusive of pediatric series (138,e257). Except for the presence of metastasis, no single histological feature of the tumor itself including local invasion is predictive of malignant
behavior (eFigure 21-109,173,174, e974,e1057,e1184). Risk factors for malignancy include the diagnosis of paraganglioma and tumor size greater than 6 cm.
P.970
However, tumor size is not always predictive of malignancy (e1085). The 5- and 10-year survival rates for malignant tumors is 78% and 31%, respectively (138). In aggregate, extra-
adrenal location, coarse nodularity, confluent necrosis, and absence of hyaline globules are features associated with malignancy (e717). An increased MIB-1 index (nuclear
immunopositivity) correlated with malignant behavior in some but not all studies (173,174,e165,e910,e1062,e1216).
Adrenal medullary hyperplasia is found in the setting of the MEN 2 syndromes as the presumed precursor of PHEOs. It has also been reported as an isolated finding in the
nonfamilial setting of hypertension and biochemical studies suggesting PHEO; however, no discrete adrenal medullary tumor is found at surgery (104,e961). Criteria for the
pathological diagnosis of adrenal medullary hyperplasia include an increase in adrenal weight which is accompanied by diffuse or nodular extension of the medulla into the alae
(e677,e908). Morphometric criteria include a decrease in the overall ratio of cortex to medulla (normal is 10:1) and an increase in the calculated medullary weight and volume
(eFigures 21-110 and 21-111).
Composite adrenal medullary neoplasms are rare entities that are composed in part of a PHEO, one of the three patterns of the neuroblastic tumors or a peripheral nerve
sheath neoplasm (e77,e382,e1189,e1190). Most of these tumors have been reported in adults, but have been infrequently observed in children.
Other tumor and tumor-like lesions of the adrenal gland include a variety of cysts of a presumed vascular nature. Extramedullary hematopoiesis in the setting of β-thalassemia may
present as an adrenal incidentaloma in childhood (e (949). Myelolipoma, one of the more common “incidentalomas” of the adrenal gland in adults, is extremely unusual in children
and slightly more frequent than the lipoma and leiomyoma (e47,e271,e296,e568,e847,e950,e1081,e1082). Hemangioma or hemangioendothelioma of the adrenal has been
observed in infancy (e297,e1112). One other unusual tumor of the adrenal gland that we have had an opportunity to study was an extrarenal Wilms tumor presenting in a 4-yearold
boy (e1036). Molberg et al. reported the occurrence of a primitive epithelial and mesenchymal neoplasm of the adrenal in an infant with virilizing signs, which was interpreted as an
adrenal blastoma (90,e831). Primitive neuroectodermal tumor family tumor has been reported in the adrenal gland of children (e591,e774). Though not an adrenal lesion per se,
subdiaphragmatic extralobar sequestration may simulate an adrenal tumor when it presents as a suprarenal mass (see Chapter 12) (e199,e957).
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Chapter 22
The Lymph Nodes, Spleen, and Thymus
Thomas L. McCurley
Mary M. Zutter
Andrea M. Sheehan
LYMPH NODES
The lymph node is a remarkable structure that serves as (a) a meeting place for antigen, antigen-presenting
cells, and naïve B- and T-cells to initiate the adaptive immune response, and (b) the site of clonal expansion and
differentiation of effector B- and T-lymphocytes (58). These processes so essential to normal immune function
also require a number of genetic events (DNA replication, class switching, somatic mutation, receptor editing,
etc.) that underlie most lymphomas (both Hodgkin and non-Hodgkin) (73).
Lymph Nodes (Normal Structure and Function)

The normal lymph node is a round or an ovoid encapsulated structure. It is usually small (2 to 3 mm) to modest
(∽1 cm) in size, but it may attain dramatic dimensions if the lymph draining into it is particularly rich in
immunogenic material. Macroscopically, normal lymph nodes are tan or creamy white in color, and the cut
surface may be homogeneous or vaguely nodular. In reactive lymph nodes, the hilum is visible on gross
examination. Microscopically, four anatomic compartments are present (Figure 22-1) (e314).
FIGURE 22-1 ▪ In this reactive lymph node with follicular hyperplasia, the germinal centers are widely spread,
vary in size, and are demarcated by a rim of small lymphocytes, the mantle zone. (Hematoxylin and eosin stain
4×.)
The most readily identified are the primary and secondary follicles, which are spherical collections of small and
large B- lymphocytes in the periphery of the lymph node. Although rich in B-cells, the follicles also contain T
helper cells and follicular dendritic cells (e205). Morphologically, the follicular center has a dark zone where
proliferation (clonal expansion) and somatic mutation occur, and an adjacent light zone that is the site of
selection of high-affinity B-cells and differentiation to plasma cells and memory B-cells (Figure 22-2).
Surrounding the follicle is a rim of uniformly sized small lymphocytes, the mantle, which is polarized toward the
subcapsular sinus and blends imperceptibly into the cortex. Like the follicles, the mantle is composed largely of
B-cells. Beyond the mantle and between the follicles is a T-cell-rich zone, the paracortex, which contains a
heterogeneous population of cells, including macrophages, interdigitating reticulum cells, scattered B-cells, and
abundant T-cells in various stages of activation (e313). High endothelial venules in this area serve as the site of
entry for naïve T-cells and B-cells (Figure 22-3). The medullary cords, not always evident in tissue sections as a
discrete zone, are located in the central portion of the lymph node and consist of elongated arrays of
lymphoplasmacytoid cells that surround the sinuses.
FIGURE 22-2 ▪ Polarization of the benign germinal center reflects the segregation of centrocytes to the light zone
and mitotically active centroblasts to the dark zone. (Hematoxylin and eosin stain 10×.)
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FIGURE 22-3 ▪ Interfollicular T-cell zone demonstrating high endothelial venules and dendritic cells.
(Hematoxylin and eosin stain 40×.)
The sinuses, endothelium-bounded spaces containing macrophages and antigen-presenting cells, form the
fourth anatomic compartment and converge on the hilum from multiple points along the subcapsular sinus.
Clinical Significance of Lymphadenopathy in Children

Palpable lymphadenopathy is more common in children and adolescents than in adults. The most common cause
of adenopathy in children is a benign lymphoid proliferation, and in many patients, some evidence of a self-
limiting infectious or inflammatory process can be found to explain the enlarged nodes (e234). The presence of
certain clinical factors suggests that biopsy may disclose a condition requiring specific treatment, including fevers
unresponsive to antibiotics, generalized adenopathy or massive localized adenopathy, mediastinal disease,
weight loss, peripheral blood cytopenias, and elevated serum levels of lactate dehydrogenase (e227). Slap et al.
(e288) defined three simple variables that identified lymph nodes which should be biopsied: (a) size greater than
2 cm in diameter, (b) abnormalities on chest x-ray, and (c) absence of symptoms of recent otolaryngologic
disease in patients with cervical adenopathy.
Table 22-1 ▪ APPROACH TO DIAGNOSIS AT THE TIME OF BIOPSY

Approach to Diagnosis in Patients with Lymphadenopathy
The availability of a broad array of new diagnostic techniques in hematopathology offers the opportunity for
making faster diagnoses with more precision on smaller samples using less invasive procedures such as fine-
needle aspiration (FNA) cytology and needle biopsy. The success of such efforts (if they are to be cost effective)
depends in part on the quality of communication between hematologists and hematopathologists so that the most
appropriate studies are ordered in a timely fashion.
The basic approach that we use in our practice is that we examine touch preps or smears on biopsies or
aspirates of lymph nodes immediately after staining (Table 22-1). Since the most common pediatric non-Hodgkin
hematopoietic lymphoid neoplasms are recognizable as malignant on touch preps/smears (anaplastic large cell
lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma, lymphoblastic lymphoma), these entities can be
quickly triaged to flow cytometric and cytogenetic analysis. Similarly, most reactive proliferations, metastatic
tumors, and those lymphomas with only a few dysplastic cells (as in Hodgkin lymphoma) would not benefit flow
cytometry but may require cultures, cytogenetics, and other studies can be triaged as well. This approach often
allows the definitive diagnosis of many malignancies within hours and prevents substantial waste in resources.
Immunophenotypic Studies of Lymph Node Biopsy Specimens

Flow cytometry is an essential part of the diagnosis and classification of non-Hodgkin lymphoma involving lymph
nodes. The rapid availability of results (1 to 3 hours) allows triage to appropriate ancillary genetic studies while
viable material is still available (32). In Hodgkin lymphoma and in non-Hodgkin lymphoma in which flow cytometry
is not
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performed, the wide variety of CD markers, which mark well in fixed tissue, allow immunohistochemical
characterization of most hematopoietic lymphoid neoplasms (Table 22-2).
Table 22-2 ▪ IMMUNOPHENOTYPIC MARKERS USED IN LYMPHOMA DIAGNOSIS
Cluster Antibody Antigen

Designation
General
markers
CD45 LCA Leukocyte common antigen (+ almost all NHL negative/DIM in most
acute leukemias, — in CHL, and many ALCL)
CC TdT Terminal deoxynucleotidyl transferase (+ >95% LBL 20% ANLL, — in all

NHL)
CD34 HPCA Human progenitor cell antibody (50% of blasts in ALL/ANLL) endothelial
cells
B-cell lineage markers
CD10 J5 CALLA; positive in follicle center cell NHL (+ >95% B-ALL, 100% BL,
25% of DLBL)
CD19 B4 Pan B-cell marker (present on all B-cell NHL including B-LBL)
CD20 L26 Mature B-cell marker (not present on most B-LBL)
CD23 Activated B-cells; follicular dendritic cells, low-affinity FcR for IgE
T-cell lineage markers
CD1a O10 Thymic T-cells, Langerhans cells
CD2 Leu5 T-cells; E-rosette receptor
CD3 Leu4 T-cells; TcR complex component NK cells (cytoplasm only)
CD4 Leu3 Helper/suppressor T-cells; MHC class II receptor,

monocyte/macrophages, 60% of ANLL
CD5 Leu1 Preferential T-cell marker, B-cell subset
CD7 Leu9 T-cells, some NK cells; FcR for IgM, 25% of ANLL
CD8 Leu2, T8 Cytotoxic T-cells; MHC class I receptor
CD16 Leu11 NK cells, granulocytes; IgG FcR III
CD43 MT1 Pan T-cell marker, blasts in ANLL, T and B-LBL
CD45ro UCHL1 T-cells, some macrophages
CD56 Leu7 NK and T-cells; N-CAM isoform, neuroendocrine tumors
CD57 T and NK cells
Monocyte/macrophage/accessory cell markers
CD1a O10 Some T-cells, Langerhans cells

CD15 LeuM1 Granulocytes, also positive in RS cell in classic Hodgkin lymphoma
CD21 1F8, B2 Dendritic cells, some B-cells; C3d/EBV receptor
CD68 KP1 Macrophages, monocytes, blasts in ANLL
S100 Langerhans cells, melanoma, other cell types
ALCL, anaplastic large cell lymphoma; ANLL, acute nonlymphocytic leukemia; BL, Burkitt lymphoma; B-
LBL, precursor B-cell lymphoblastic leukemia; CALLA, common acute lymphocytic leukemia antigen;
CHL, classic Hodgkin lymphoma; EBV, Epstein-Barr virus; Fcr, Fc receptor; MHC, major
histocompatibility complex; NHL, non-Hodgkin lymphoma; NK, natural killer; Tcr, T-cell receptor; T-LBL,
T-lymphoblastic lymphoma.
Cytogenetic Studies of Lymph Node Biopsy Specimens

For routine cytogenetic analysis, which is helpful in securing an accurate diagnosis in some cases, viable, fresh
tissue must be taken by sterile technique at the time of biopsy and placed into culture so that metaphase spreads
can be generated. In the appropriate clinical setting, some karyotypic abnormalities may be pathognomic for
certain types of malignancies (Table 22-3) and can therefore be used for diagnostic purposes (24,65,e178). In
other settings, especially precursor B-cell lymphoblastic lymphoma/acute lymphoblastic leukemia, the results of
cytogenetic studies can also be used for prognostic purposes (45,e248). The most common karyotypic changes
related to lymphoproliferative disorders in children include translocations of immunoglobulin and T-cell receptor
loci, which are frequently paired with loci involved in normal development and hematopoiesis (6,e153,68). A
major advance in diagnostic cytogenetics is the widespread availability of FISH studies for these translocations.
Two major advantages of FISH techniques over routine cytogenetics are (a) rapid turnaround (<24 hours) and
(b) they can be done on fixed tissues including touch preps or cytospin preps and in paraffin-embedded tissues
(Table 22-3) (12, 19).
Reactive Lymphadenopathy
The two major patterns of reactive lymphadenopathy are usually dominated either by follicular hyperplasia or
interfollicular expansion (immunoblastic, granulomatous, or histiocytic). Occasional reactive processes produce
an apparent diffuse alteration of architecture (Table 22-4) (e32). In practice, a single lymph node is constantly
exposed to a diversity of immunogens and therefore exhibits more than one pattern of response, but when the
degree of adenopathy is sufficient to warrant a biopsy, a single pattern usually dominates. Additional factors that
assist in the differential diagnosis include the age-specific nature of some disorders; certain reactive processes
affecting lymph nodes are rare in children (e.g., luetic lymphadenitis, Kimura disease), whereas others affect
them primarily (e.g., acute infectious mononucleosis, autoimmune lymphoproliferative disorder) (e280).
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Table 22-3 ▪ KARYOTYPIC AND GENETIC CHANGES ASSOCIATED WITH NON-HODGKIN

LYMPHOMA
Disease Abnormality Implicated Loci

Precursor B-cell LBL/B-cell ALL t(1;19)(q23;p13) pbx1-e2a
t(4;11)(p21;q23) AF4-MLL
Hyperdiploidy (>50 chr)
Hyperdiploidy (47;50 chr)
Hypodiploidy
B-cell large-cell lymphoma t(3q27;var) BCL-6

Burkitt lymphoma t(2;8) (p11;q24) IgL-lambda-myc
t(8;14) (q24; q32) c-myc-IgH
t(8;22) (q24;q11) c-myc-IgL-kappa
Precursor T-cell LBL/T-cell ALL t(1;14-15)(p12;q11) TAL1;TcR gamma

t(1;14)(p32;p14-15) TAL1;TcR delta
t(1;7)(p32;q34) TAL1;TcR beta
T-cell anaplastic large-cell lymphoma t(2;5)(p23;q35) NPM-ALK
ALL, acute lymphoblastic leukemia; IgH, immunoglobulin heavy chain; IgL, immunoglobulin light chain;
LBL, lymphoblastic leukemia; TcR, T-cell receptor.
Table 22-4 ▪ REACTIVE LYMPHADENITIS IN CHILDREN
Follicular hyperplasia
Nonspecific
HIV
Progressive transformation of follicular centers
Toxoplasmosis
Castleman disease/angiofollicular hyperplasia
Interfollicular reactions
Paracortical immunoblastic reactions
EBV
Hypersensitivity reactions (phenytoin)
Juvenile rheumatoid arthritis 1

Systemic lupus erythematosus 1,3
Kikuchi histiocytic necrotizing lymphadenitis 1,3
Autoimmune lymphoproliferative syndrome (ALPS) 1
Kawasaki disease
Granulomatous
Mycobacterial infection (MTB and atypical mycobacterial) 1,2
Cat-scratch disease 1,2,4
Fungal infection
Histiocytic
Sinus histocytosis
Lysosomal storage disorders
Hemophagocytic syndromes (Hemophagocytic lymphohistiocytosis)
Rosai-Dorfman
Dermatopathia
Langerhans cell histocytosis
Diffuse alteration of architecture
Sarcoidosis
Post-transplant lymphoproliferative disease
Other features
1. Follicular hyperplasia
2. Necrosis with neutrophils
3. Necrosis with apoptosis
4. Capsulitis
FOLLICULAR HYPERPLASIAS
Nonspecific Germinal Center Hyperplasia
Nonspecific germinal center hyperplasia is the most common of all benign histologic findings. The bulk of the
lymph node is composed of round or irregularly shaped germinal centers that vary in size. These contain small,
intermediate, and large mitotically active lymphocytes, tingible body macrophages, and apoptotic cells. The
hyperplastic follicles tend to remain in the cortical regions of the affected node, but in particularly robust cases,
the paracortex and the medulla may be compressed by the process (e60,e159). Immunophenotypic studies show
that the follicles contain a predominance of CD20+, CD10+, 6+ B-cells that do not stain for bcl2 (e205,e310).
Differential diagnostic considerations in children are few but include Castleman disease, HIV-related adenopathy,
and progressive transformation of germinal centers.
HIV-related Adenopathy
Most series treating the subject of HIV-related persistent generalized lymphadenopathy are based on a patient
population of homosexual young adult men at risk for HIV (e17,e37,e143). Reports of this condition in children at
risk for HIV because of maternal-fetal transmission or hemophilia present similar data (e41,e280,e336). A
spectrum of histologic findings may be seen in this context, with two clearly recognizable extremes. Florid
follicular hyperplasia, the earliest change of HIV-related persistent generalized lymphadenopathy, has many
features in common with nonspecific follicular hyperplasia, although the germinal centers are larger, often
serpiginous, and tend to fuse with focal follicular lysis (Figure 22-4) (e106,e228). Regressively
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transformed germinal centers typify late persistent generalized lymphadenopathy and are characterized by small
size, lymphoid depletion, and numerous dendritic cells, vessels, and amorphous eosinophilic deposits
(e143,e144,e159). The mantle is absent or very poorly formed, and the paracortex is proportionally rich in
histiocytes, plasma cells, and high endothelial venules because of the paucity of lymphocytes. The morphologic
features associated with persistent generalized lymphadenopathy (i.e., large and irregularly shaped follicles,
follicular lysis, follicular involution) are distinctive but not specific for HIV infection, and they have been seen in
5% to 10% of otherwise entirely unremarkable lymph nodes obtained as part of carcinoma staging before the
beginning of the AIDS era (e215,e287,e294).
FIGURE 22-4 ▪ Serpentine follicular center in a patient with HIV infection and a generalized adenopathy.
(Hematoxylin and eosin stain 4×.)
Progressively Transformed Germinal Centers

For unknown reasons, when clinically significant lymphadenopathy develops in some patients, the biopsy
specimen shows progressively transformed germinal centers (PTFC). Most patients are asymptomatic male
adolescents or young adults with isolated inguinal or cervical adenopathy (99,e225). The bulk of the lymph node,
which can be up to 5 cm in size, exhibits florid follicular hyperplasia; however, what makes this disorder
distinctive is the presence of scattered, very large (three to five times the size of surrounding germinal centers)
follicles that are dramatically expanded by an influx of small lymphocytes (Figure 22-5) with mantle cell-like
morphology and IgM+, IgD+ phenotype (e94,e224,e280). The borders of these “transformed” germinal centers
with the surrounding paracortex are blurred, and they may be encircled by wreaths of histiocytes (e94). Relative
to normal follicles, the transformed follicles exhibit a disrupted and dispersed dendritic cell network on CD21 or
CD23 staining (99). An important feature is the absence of variant Reed-Sternberg cells since the major concern
in differential diagnosis is lymphocyte predominant Hodgkin lymphoma that rarely may precede, follow, or be
concurrent with PTFC (e37,e129).
FIGURE 22-5 ▪ Progressively transformed follicular center (with disrupted follicle infiltrated by small mantle zone
lymphocytes) in a background of follicular hyperplasia. (Hematoxylin and eosin stain 4×.)
Toxoplasmosis
Acute toxoplasmosis, an infectious disease, is often accompanied by lymphadenopathy. This is usually limited to
the cervical lymph nodes, although occasionally patients with typical histology and serologic confirmation have
isolated inguinal or axillary lymph node enlargement (e103). Florid follicular hyperplasia dominates the histology
at low power and is invariably accompanied by patches of epithelioid histiocytes and parasinusoidal
accumulations of monocytoid B-cells. The histiocytic aggregates, randomly distributed throughout, abut and even
infiltrate the germinal centers (e70,e192,e283). Immunophenotypic studies play no role in confirming the
diagnosis but may be helpful in excluding conditions such as nodular lymphocyte-predominant Hodgkin
lymphoma, which may superficially resemble Toxoplasma-related lymphadenitis. The triad of florid follicular
hyperplasia, hyperplasia of parasinusoidal B-cells, and histocytic aggregates (variably encroaching on follicular
centers) has a high degree of sensitivity and specificity for diagnosis of toxoplasmosis (Figure 22-6) (34, 82).
FIGURE 22-6 ▪ Low power of toxoplasmosis demonstrating the triad of hyperplastic follicles, monocytoid B-cell
proliferation, and histiocytic aggregates encroaching of follicles. (Hematoxylin and eosin stain 4×.)
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FIGURE 22-7 ▪ Low power of hyaline vascular angiofollicular hyperplasia with “bag of marbles” small uniform
follicles evenly dispersed throughout the cortex and the medulla. (Hematoxylin and eosin stain 4×.)
Castleman Disease/Angiofollicular Hyperplasia

Most patients with hyaline vascular Castleman disease (HV-CD) present with cervical or mediastinal
involvement, or both (e142,e281). At low power, the architecture of HV-CD is nodular (Figure 22-7) with small
and involuted germinal centers throughout the node (bag of marbles). The mantles are expansive and composed
of small lymphocytes often in a laminated (orbiting) or “onion skin” pattern (e67,e101,e191). Multiple germinal
centers may be found within the boundaries of a single mantle zone, and in some sections, radially penetrating
hyalinized high endothelial venules are seen passing from the germinal centers into the adjacent paracortex
(lollypops) (Figure 22-8). The germinal centers are depleted of lymphocytes and contain both extracellular matrix
and abundant follicle dendritic cells (e159,e209). The cellular components of the interfollicular zone include
plasmacytoid monocytes, myoid cells, histiocytes, dendritic cells, and lymphocytes, which vary in proportion from
case to case (e209). Cuffing of sinuses by collagen may be prominent. Immunophenotypic studies lend little to
the diagnosis, although CD21 and CD23 highlight the dense aggregates of dendritic cells, which are in contrast
to the circumscribed but loose meshwork seen in normal germinal centers. Both HV-CD and PC-CD may show
monotypic plasma cells (usually lambda light chain) by immunohistochemistry (e218). Although differential
diagnostic considerations in adults include mantle cell lymphoma and follicle center cell lymphoma, these
malignancies are very rare in children. Partial nodal involvement by Hodgkin lymphoma may be overlooked if a
significant concomitant Castleman disease-like proliferation is present (e179) and small biopsy specimens of
mediastinal HV-CD may be mistaken for thymoma if involuted germinal centers are misinterpreted as Hassall
corpuscles.
FIGURE 22-8 ▪ The mantle zone has a laminated or “onion skin” appearance in the hyaline vascular type of
Castleman disease. Note the radially penetrating vessel. (Hematoxylin and eosin stain 10×.)
The plasma cell variant of Castleman disease (PV-CD) is usually a systemic disorder that has also been reported
in children (e142,e186). Patients present with fever and weight loss, and laboratory studies may show immune-
mediated cytopenia, an elevated erythrocyte sedimentation rate, and hypergammaglobulinemia (e40). When
localized, the adenopathy of PV-CD is typically axial (mediastinum or abdomen) like HV-CD. When the
adenopathy is multicentric, patients often have hepatosplenomegaly and symptoms fitting the POEMS
(polyneuropathy, organomegaly, endocrinopathy, M protein, skin changes) syndrome may be present
(8,e110,e182,e203). The low-power appearance of PV-CD is that of follicular hyperplasia with a marked
interfollicular plasmacytosis (e101,e152,e159,e209). The germinal centers are large and hypercellular, contain
dense eosinophilic material, and have a discrete, if thinned, mantle zone (e191,e323). As in the hyaline vascular
variant of Castleman disease (HV-CD), the secondary follicles may contain more than one germinal center within
the same mantle. The subcapsular and medullary sinuses remain patent, and extracapsular extension is
distinctly unusual. Rheumatic lymphadenitis, luetic lymphadenitis, immunocytoma, autoimmune
lymphoproliferative syndrome (ALPS), and HIV-related lymphadenopathy all are plausible diagnostic
considerations, and correlation with clinical and immunophenotypic studies is a successful means of excluding
these possibilities. PC-MCD in HIV-positive patients is usually associated with infection with human herpes virus
8 (e109). A subset of these patients may evolve into frank plasmablastic lymphoma (33).
INTERFOLLICULAR/PARACORTICAL REACTIONS—IMMUNOBLASTIC
Epstein-Barr Virus Infection (Infectious Mononucleosis)
Acute illness secondary to Epstein-Barr virus (EBV) infection (acute infectious mononucleosis) is common in
young children and is usually self-limited. In those few cases that culminate in biopsy, the clinical features are
often atypical—advanced
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age, the presence of “B” symptoms, a negative monospot test [uncommon except in very young children (<4
years) or early in infection], localized adenopathy, or persistent adenopathy, hepatosplenomegaly, or splenic
rupture (e174). Patients with X-linked lymphoproliferative disorder (Duncan syndrome) present with rapidly
progressive and usually fatal disease because of an inability to mount a successful immune response against
EBV-infected cells (e52).
FIGURE 22-9 ▪ The immunoblastic proliferation in acute infectious mononucleosis localizes to the paracortex and
may compress or distort residual germinal centers. (Hematoxylin and eosin stain 4×.)
At low power, architecture is obscured but generally preserved with moth-eaten follicles and prominent
paracortical expansion (Figure 22-9) by immunoblasts, plasma cells, and plasmacytoid lymphocytes (Figure 22-
10). Similar large cells pack the sinuses. Occasionally, large cells with a bilobed or multilobed nuclei and
prominent nucleoli are present, reminiscent of Reed-Sternberg cells (e5,e296,e306). Histiocytes may be
scattered singly or in small clusters, and increased numbers of capillaries and high endothelial venules also
contribute to the polymorphic appearance of the paracortex. Normal landmarks—germinal centers, subcapsular
and paratrabecular sinuses—are generally present but may be compressed or distorted by the immunoblastic
proliferation (e40,e286). In very early cases of EBV infection, monocytoid B-cell proliferations may be prominent
(5). Staining with CD20 and CD3 highlights the presence of a mixture of interfollicular B- and T-immunoblasts,
and a polytypic pattern of light-chain expression is always seen. The Reed-Sternberg-like cells are
characteristically CD20+, CD15-, and show variable reactivity for the activation antigen CD30 (e279) as well as
markers of EBV infection such as latent membrane protein (LMP) or EBV, RNA transcripts (EBER)
(e93,e210,e252). Difficult cases may exhibit sheetlike arrays of immunoblasts, a brisk mitotic rate, or extensive
necrosis and may closely mimic large cell lymphoma (e40,e226). In such cases, examination of the peripheral
blood smear for atypical lymphocytes, viral serology, and immunohistochemistry to better define architectural
preservation and establish the presence of EBV is helpful.
FIGURE 22-10 ▪ Small, intermediate and large cells fill the paracortex in acute infectious mononucleosis, often
with a predominance of immunoblasts. (Hematoxylin and eosin stain 40×.)
Non-EBV Viral Adenopathy

Lymphadenopathy may occur as a result of herpes simplex (e79) and cytomegalovirus infection (e311). Children
with some form of immune deficiency are the most frequently affected (e104,e112). Paracortical hyperplasia with
discrete foci of necrosis is the typical histologic finding in these cases (e194). In comparison with acute EBV-
related lymphadenopathy, the proportion of immunoblasts is less, and interfollicular areas are expanded by a
mixture of mature lymphocytes, plasma cells, histiocytes, plasmacytoid monocytes, and lesser numbers of
immunoblasts (e141). Viral inclusions that appear as smudged or hyperchromatic alterations of the nucleus
(herpes simplex virus) or very large eosinophilic structures within the nucleus (cytomegalovirus) can be identified
and may be most numerous adjacent to zones of necrosis (e303). Immunohistochemistry is helpful in excluding
neoplasia and can also document the presence of infected cells (e338). The viral inclusions in enlarged cells of
cytomegalovirus lymphadenitis are CD15+, a potential pitfall in the safe exclusion of Hodgkin lymphoma (e266).
Attention to clinical parameters will assist in discriminating viral lymphadenitis from other causes of necrotizing
lymphadenitis, which include KikuchiFujimoto disease and lupus erythematosus.
Hypersensitivity Reactions Emphasizing Phenytoin (Dilantin) Reactions

Hypersensitivity-related lymphadenopathy is quite rare and has been associated most commonly with phenytoin
(Dilantin) therapy (e2,e271) and vaccines (small pox, measles, tetanus) (e9,e12,e71). In phenytoin
hypersensitivity, the architecture of the lymph node is distorted by a paracortical proliferation of immunoblasts,
lymphocytes, plasma cells, and eosinophils. Germinal centers persist, and in some cases, a florid follicular
hyperplasia may accompany the immunoblastic reaction. Purely diffuse architectural effacement (e2) is rare. The
immunoblasts represent a mixture of CD20+ B-cells and CD3+ T-cells. Progression to lymphoma is well
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described (e2,e271). The pattern of paracortical expansion may be seen in T-cell lymphomas such as
angioimmunoblastic T-cell lymphoma (112), which is a major problem in differential diagnosis. A detailed history
as well as immunophenotypic studies is usually helpful.
Juvenile-onset Rheumatoid Arthritis Emphasizing Still Disease

Several different histologies have been described in juvenile rheumatoid arthritis including the classic findings
associated with adult RA of follicular hyperplasia, interfollicular plasma cells, and intrasinusoidal neutrophils
(e213). In Still disease, a pattern of paracortical immunoblastic proliferation mimicking lymphoma is occasionally
described as well as interfollicular necrosis similar to that seen in Kikuchi disease (64,67,e217,e312).
Systemic Lupus Erythematosus

Systemic lupus erythematosus is an autoimmune disease that affects both adolescents and young adults. When
lymphadenopathy occurs, it is typically peripheral and multifocal or generalized. Classically follicular hyperplasia
with patchy paracortical necrosis dominates the low-power appearance of the lymph node (e26,e189). The
germinal centers are well formed with a discrete mantle zone, and they are separated by an expanded paracortex
in which pockets of necrosis are randomly distributed (e77). The necrotic foci are composed of amorphous
eosinophilic material and apoptotic debris. Neutrophils and plasma cells are scarce. In addition, “hematoxylin
bodies” (round or oblong blue structures, 5 to 15 μm long, which stain with periodic acid-Schiff and the Feulgen
method) and vascular encrustations (Azzopardi effect) may be present (e98). Less commonly, necrosis
dominates the morphology, and follicles may be few and more widely spaced; in these circumstances, the lymph
node findings may resemble those of Kikuchi-Fujimoto disease (e68). Other patterns described in systemic lupus
erythematosus include follicular hyperplasia without necrosis resembling Castleman disease.
Histiocytic Necrotizing Lymphadenitis/Kikuchi-Fujimoto Disease

Kikuchi-Fujimoto disease (histiocytic necrotizing lymphadenitis) is uncommon in children. The median age is in
the third decade in most large series, although the age range is great (e15). Rare fatal cases in children have
been reported often associated hemophagocytic syndrome (e44). These nodes exhibit follicular hyperplasia, but
the histologic hallmark of this disease is zonal karyorrhexis with scant neutrophil response (e67,e68,e161). At
low power, the paracortex is distorted by pale-staining patchy zones of necrotic debris with a cellular rim
composed of apoptotic cells, histiocytes, small lymphocytes, plasmacytoid dendritic cells, and immunoblasts
(e81,e82,e254). The areas of necrosis may coalesce, but a serpiginous contour rarely develops. Beyond the
necrotic zone is a mottled paracortex, rich in small lymphocytes, immunoblasts, apoptotic debris and
plasmacytoid monocytes and high endothelial venules (Figure 22-11) (e307,e309). In children, the differential
diagnostic considerations for the early proliferative lesions of histiocytic necrotizing lymphadenitis include non-
Hodgkin lymphoma (e42). and mixed-cellularity Hodgkin lymphoma. Fully developed lesions may mimic Kawasaki
disease or herpetic lymphadenitis, and lupus-related lymphadenitis may be difficult or even impossible to
exclude. A third pattern is characterized by dominance of foamy histiocytes (e218). Immunophenotypic
characterization of the large cells at the periphery of karyorrhexis areas shows that they represent CD8 T-cells
and plasmacytoid dendritic cells (81). One report suggests that the sparsity of CD8 T-cells in SLE may be helpful
in differential diagnosis (54).
FIGURE 22-11 ▪ The border of necrosis shows apoptotic debris admixed with histiocytes small lymphocytes,
plasmacytoid dendritic cells, and immunoblasts in Kikuchi disease. (Hematoxylin and eosin stain 40×.)
Autoimmune Lymphoproliferative Syndrome

Lymphadenopathy secondary to loss of Fas or Fas ligand mediated apoptosis is a rare cause of non-neoplastic
lymphadenopathy, known as autoimmune lymphoproliferative disorder (ALPS). Patients with ALPS present
within the first 2 years of life with bulky generalized adenopathy and hepatosplenomegaly (e38,e73,e291,e292).
Enlarged lymph nodes show generally intact architecture with follicles ranging from floridly hyperplastic to small
involuting follicles with compressed mantle zones like those seen in hyalinized vascular Castleman disease. The
proliferation that occurs in the interfollicular areas consists of immunoblasts and transformed large cells with
scant-to-moderate cytoplasm (80,e291). Small lymphocytes, plasma cells, and histiocytes may be present. On
flow cytometry, CD2+, CD3+, CD4-, CD8- T-cells with an a-b T-cell receptor predominate. B-cells are
phenotypically normal (e63,e292). On tissue section, the immunoblasts in the interfollicular zones are virtually all
CD3+, double negative T-cells, with only a few showing
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reactivity for CD4, CD8, or CD20 (71,e247,e291). T-zone lymphoma may mimic ALPS, but the former typically
contains more small-to-intermediate cells and rarely has a CD4-, CD8- phenotype. Gene sequencing is
necessary to confirm the diagnosis of ALPS, which may be caused by mutations in Fas, Fas ligand, or the
caspase 10 gene (e73,138).
Kawasaki Disease
Kawasaki disease is endemic in Japan but rare in Western countries (e19). A slight male predominance has
been noted, and the peak incidence is in children 3 to 4 years old. Histologic descriptions are quite variable, and
it is clear that lymph node biopsy seldom yields findings on which a firm diagnosis of Kawasaki disease can be
made independent of clinical parameters. The main findings are patchy paracortical necrosis with phlebitis and
fibrin microthrombi (e111). Germinal centers are inconstantly present, as is an immunoblast-rich paracortical
expansion. If perinodal tissues are represented, an acute necrotizing arteritis similar to infantile polyarteritis
nodosa may be identified even in early phases; in established cases, a measure of luminal dilation is also
present in largercaliber vessels, with medial destruction (see Chapter 13).
INTERFOLLICULAR GRANULOMATOUS PROCESSES

Cat-Scratch Disease
Cat-scratch disease frequently affects children and adolescents, although in recent, large, population-based
studies, nearly half of all patients have been over the age of 20 (e39). The adenopathic phase of the disease is
dominated by follicular hyperplasia, capsulitis, paracortical monocytoid B-cell hyperplasia, and small, neutrophil-
rich microabscesses (e195). As the lesions develop, the microabscesses coalesce, forming serpiginous and
stellate zones of eosinophilic necrosis (Figure 22-12) (e165). In the late stage, the microabscesses take on a
granulomatous appearance, with a well-formed rim of palisading histiocytes and scattered multinucleated giant
cells (Figure 22-13). Warthin-Starry staining may highlight pleomorphic and bow-shaped rods and cocci, both in
the center of abscesses and around blood vessels in the early phases of disease (e195). However, Warthin-
Starry staining is technically difficult and problematic in interpretation. PCR techniques that detect the organism
in a majority of patients with a high degree of specificity are preferred for confirmation of diagnosis (e278).
FIGURE 22-12 ▪ The abscesses in cat-scratch disease have a serpiginous or stellate contour. (Hematoxylin and
eosin stain 4×.)
FIGURE 22-13 ▪ In well-developed cases, the abscesses of cat-scratch disease have a broad, histiocyte-rich rim
with abundant neutrophils forming a so-called pyogranuloma. (Hematoxylin and eosin stain 10×.)
Histologically similar lesions may also be seen in yersinia infection, lymphogranuloma venereum, tularemia, and
infection with MAI in young children (see the following section) (66, 75, 105).
Mycobacterial Infections
The most common cause of granulomatous lymphadenitis in small children (1 to 5 years old) is infection by
nontuberculous “atypical” mycobacteria (NTM), most commonly Mycobacterium avium-intracellulare (MAI) or
Mycobacterium scrofulaceum (2,e21,e240,60). Diagnosis may be made by FNA or excisional lymph node
biopsy. Cytologically, smears show epithelioid histiocytes and granulomata with reactive lymphocytes and
plasma cells in the background as well as amorphous necrosis or necrosis associated with abundant neutrophils
(140). Histologically, the nodal architecture is partially distorted or entirely effaced by follicular hyperplasia with
well-formed granulomas composed of epithelioid histiocytes and multinucleated giant cells, which rim central
areas of caseous necrosis (eFigures 22-1 and 22-2) or necrosis containing abundant neutrophils similar to
lesions in cat-scratch disease (eFigures 22-3 and 22-4) (e88,132,e313). According to one study, well-defined
granulomas with caseous necrosis and numerous giant cells are more characteristic of Mycobacterium
tuberculosis while microabscesses are more predictive of NTM, although there is significant overlap of features
(70). Small lymphocytes are evenly distributed throughout lesional areas, and immunoblasts are
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rare or lacking. Immunocompromised patients may lack the classical granulomas and instead show looser
aggregates of histiocytes with a foamy appearance and more abundant organisms on special stains (eFigures
22-5 and 22-6) (60). Rare cases in immunocompetent patients may show mycobacterial organisms on acid-fast
stain, although greater sensitivity may be obtained using fluorescence microscopy using auramine orange (15) or
immunohistochemistry against the MPT64 mycobacterial antigen (more specific for the Mycobacterium
tuberculosis complex) (97, 109). The remainder of cases may be diagnosed via one of several polymerase chain
reaction-based techniques (7, 11, 106, 108, 135) or microbiologic culture. Most of these techniques, except for
culture, have the advantage of being applicable to fresh as well as paraffin-embedded tissue and may be
performed using cytology specimens as well as core needle biopsies or whole lymph node biopsies. Other
causes of caseating and noncaseating granulomatous lymphadenitis include infection by agents other than
mycobacteria (e147,e304,e335) and neoplastic disease, including peripheral T-cell lymphoma, nodular
lymphocyte-predominant Hodgkin lymphoma, and classical Hodgkin lymphoma (e126,e140,e246).
Chronic Granulomatous Disease

Chronic granulomatous disease of childhood, a congenital disorder caused by defective components of the
NADPH (reduced nicotinamide adenine dinucleotide phosphate) oxidase pathway (e57,e255,121), is an
extremely rare form of granulomatous lymphadenitis in which lymph nodes and other tissues are extensively
infiltrated by granulomas and neutrophil-rich abscess-like foci. Catalase-positive bacteria, specifically
Staphylococcus aureus and Gram negative bacilli, and Aspergillus are the most common agents to be recovered
in culture (e57,121) and a test for nitroblue tetrazolium reduction or other assessment of the respiratory burst by
peripheral blood leukocytes by either chemiluminescence or flow cytometry should be performed in suspected
cases (e56,128,e318). Molecular diagnostic testing may be performed to confirm the gene involved (128) (see
Chapter 5).
In 11% to 25% of cases, a careful review of clinical, radiologic, histologic, and laboratory data fails to identify a
cause of the granuloma formation (e21,e30,95), and a diagnosis of idiopathic granulomatous lymphadenitis is
rendered. In such circumstances, with all secondary causes excluded, sarcoidosis can be considered a
possibility (e180,e229,123) (Chapter 12). This disease is most common in young adults, although it is seen
occasionally in adolescents and rarely in children. Lymph node architecture is often totally effaced with no or few
residual follicles. Necrosis is rare, but when present, it is more commonly fibrinoid than caseating. Recent studies
have demonstrated an increased CD4+ FoxP3+ regulatory T-cell (Treg) population both in the peripheral blood
and the lymph nodes of patients with sarcoidosis (94, 131). Noncaseating granulomatous lymphadenitis may also
be seen in benign lymph nodes draining organs involved by tumor, and histiocytic proliferations mimicking
granulomas may be present in lymph nodes involved by lymphoma (20,e140,e239).
Interfollicular Processes with Histiocytic Proliferation

Sinus histiocytosis is a nonspecific reactive pattern that may be seen in lymph nodes draining inflammatory or
malignant processes of the skin, bowel, or lungs. In particularly striking cases, only compressed primary follicles
are seen, and germinal centers are either diminutive or absent. The subcapsular and paratrabecular sinuses are
expanded by a cellular infiltrate composed of large polygonal cells with bland nuclei and abundant pale
eosinophilic cytoplasm, some with phagocytosed debris (26). These histiocytes can be distinguished from
Langerhans cells and Rosai-Dorfman cells by their CD68+, lysozyme-positive, S100-, CD1a-, CD207- phenotype.
Sinus Histiocytosis with Massive Lymphadenopathy

Sinus histiocytosis with massive lymphadenopathy (SHML), also known as Rosai-Dorfman disease, affects
young patients (e257). Germinal centers are atrophic or lacking in most cases, and the paracortex is similarly
diminished secondary to the compressive effects of the expanded sinusoids (e258) (Figure 22-14). A
polymorphous array of lymphocytes, plasma cells, histiocytes, xanthoma cells, and “SHML” cells distend the
sinusoids, with the proportions varying from case to case. The SHML cells, which are the hallmark of this
disorder, have oval nuclei with condensed chromatin and abundant cytoplasm, which ranges from eosinophilic to
xanthomatous (e97) (Figure 22-15 and eFigure 22-7). Invariably, engulfed lymphocytes (“emperipolesis”) can be
found in the SHML cells, although this feature may be focal in some cases. Several groups have demonstrated
the utility of FNA in the diagnosis of SHML. Smears generally
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demonstrate the presence of small lymphocytes, plasma cells, few neutrophils, and SHML cells (25, 30, 72, 118).
The phenotype of the SHML cell is S100+, CD68+, but the cells lack markers for Langerhans cells (CD1a and
CD207) or dendritic cells (DRC, CD23, CNA42) (9,e75). SHML has been seen in association with neoplasms,
including mixed cellularity and lymphocyte-predominant Hodgkin lymphoma as well as non-Hodgkin lymphoma,
and in patients with immune dysregulation such as post-bone marrow transplant or in the setting of the ALPS that
should be taken into account in the evaluation of unusual cases (3,85,e97,86,119).
FIGURE 22-14 ▪ As a result of massive sinusoidal expansion by histiocytes, germinal centers are compressed in
Rosai-Dorfman disease (SHML). (Hematoxylin and eosin stain 40× magnification.)
FIGURE 22-15 ▪ The lesional cell in Rosai-Dorfman disease (SHML) has a small, cytologically bland nucleus and
abundant eosinophilic cytoplasm containing one or more lymphocytes (emperipoplesis). Although obvious in this
case, emperipolesis may be difficult to detect on routine sections. (Hematoxylin and eosin stain 200×
magnification.)
Foreign Body Sinusoidal Histiocytic Reactions

Histiocytic lymphadenopathy secondary to foreign (nonnodal) material accumulations occurs in the setting of
primary metabolic disease, in lymph nodes draining ulcerated or tumoral areas (e102,e119,e124), or adjacent to
joint prostheses (e11,e18,e137,e162,134), or after lymphangiography, although current radiographic techniques
have largely replaced lymphangiography. There is no specific manner, and the adenopathy in these patients is
rarely worrisome. In the case of accumulated contrast media, the sinusoids are dilated by foamy macrophages,
histiocytes, and multinucleated giant cells containing large lipid vacuoles (eFigure 22-8). A similar pattern may be
seen in cases of lymphadenopathy due to silicone from leaking or ruptured implants (eFigures 22-9-22-11). In the
case of joint replacement, sinuses contain pale-staining vacuolated macrophages with refractile or birefrigent
material that may be oil red O positive, depending on whether metallic particles or polyethylene particles are
present. Associated granulomas, giant cells, and fibrosis may be seen in some cases (134). Similar findings may
be noted in patients with Gaucher disease or other metabolic storage diseases (Figure 22-16). In such cases,
histiocytes resemble those seen in the bone marrow in storage diseases with either fibrillar “crumpled tissue
paper” or bubbly foamy macrophages (see Chapter 23). Little of the material is found outside the sinusoids, and
the remainder of the lymph node, although compressed or atrophic, is normal. Morbidity and mortality may be
associated with the primary process causing the accumulation of foreign material, but not with the adenopathy
itself.
FIGURE 22-16 ▪ Histiocytic proliferations caused by congenital storage disorders have a sinusoidal, often
paracortical distribution in lymph nodes. The histiocytes seen in storage disorders often have coarsely
vacuolated, (“bubbly”) or fibrillar (“crumpled tissue paper”) cytoplasm, as seen in this case of Gaucher disease.
(Hematoxylin and eosin stain 400× magnification.)
Dermatopathia
Because of the distribution of the predisposing dermatologic conditions (mycosis fungoides, psoriasis, eczema)
(e34,e326,139) dermatopathic lymphadenopathy is more frequently seen in adults. Children with dermatopathic
lymphadenopathy commonly have eczema or another chronic exanthematous disorder, and they present with
enlarged axillary or inguinal lymph nodes. At low power, involved lymph nodes show a mixed pattern of follicular
hyperplasia and sinusoidal expansion caused by an influx of histiocytes, Langerhans cells (40,e282), and
variable numbers of eosinophils (e36,e134) (Figure 22-17 and eFigure 22-12). The paracortex is expanded and
may have a pink mottled appearance because of infiltrating histiocytes and Langerhans cells (S100+, CD1a+),
some of which may contain coarsely granular brown-black melanin pigment that is positive on Fontana staining.
Occasional hemosiderin pigment is also seen. FNA of lymph nodes with dermatopathia show large clusters of
histiocytes, histiocytes with melanin pigment, few tingible body macrophages, and histiocytes with elongated or
grooved nuclei (56).
Hemophagocytic Lymphohistiocytosis
The disease profile of hemophagocytic lymphohistiocytosis may be idiopathic, familial, infection related
(e50,55,83), rheumatologic, malignancy related (e14,e91,38), related to
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antineoplastic therapy (74), or rarely preceded by Kikuchi disease (41, 79). It results from uncontrolled activation
of CD8+ T cells, macrophages, and histiocytes associated with decreased NK cell function and increased levels
of circulating proinflammatory cytokines (38, 59). Clinically significant lymphadenopathy is uncommon in patients
with hemophagocytic lymphohistiocytosis, which is diagnosed on clinical grounds when specific clinical,
laboratory, and histopathologic criteria are met, according to HLH-2004 guidelines established by the Histiocyte
Society (49). A lymph node biopsy, if performed, is generally done to exclude lymphoma. In most cases, the
follicles are small, and the germinal centers are few in number. The paracortex is depleted and has a mottled
appearance because of the presence of increased numbers of pale-staining histiocytes. The sinusoids are
distended by histiocytes, many phagocytic. The nuclear features are bland, and the cells have abundant
eosinophilic cytoplasm containing variable numbers of red blood cells and red blood cell fragments (eFigure 22-
13). Leukophagocytosis is uncommon relative to erythrophagocytosis in this condition, and, in further contrast to
SHML cells, the histiocytes in hemophagocytic lymphohistiocytosis exhibit a CD68+, S100-, CD1a-, CD207-
phenotype. Erythrophagocytosis and hemophagocytosis can be seen as a secondary phenomenon outside the
context of primary hemophagocytic lymphohistiocytosis, and all these conditions must be considered as part of
the differential diagnostic assessment. For instance, it has been reported in patients with a robust autoimmune
hemolytic anemia (e160), active systemic lupus erythematosus (e200,110,e334), X-linked lymphoproliferative
disease, ehrlichiosis (e3,31), typhoid fever (e87,126), the accelerated phase of Chediak-Higashi disease
(e134,59,e265), SHML, acute myelogenous leukemia (e267), acute lymphoblastic leukemia (101), juvenile
myelomonocytic leukemia (41, 125), and peripheral T-cell lymphoma (e51,e114,e222,122,124,e334). In some
cases, there may be a spectrum of histiocytic disorders with macrophage activation syndrome or secondary
hemophagocytic syndrome seen in patients with Langerhans cell histiocytosis (37), and patients with T-
lymphoblastic leukemia have rarely shown subsequent involvement by Langerhans cell histiocytosis or
hemophagocytic lymphohistiocytosis (116, 133).
FIGURE 22-17 ▪ The paracortical regions in dermatopathic lymphadenitis are expanded and show a pale pink
swirled appearance due to the collections of abundant histocytes and Langerhans cells admixed with small
lymphocytes. Some histiocytes contain brown melanin pigment. (Hematoxylin and eosin stain 40× magnification.)
Langerhans Cell Histiocytosis

The majority of children with Langerhans cell histiocytosis present with systemic multiorgan disease, and most
have palpable adenopathy. Common sites of involvement include bone, skin, lung, liver, spleen, bone marrow,
and pituitary (98, 120). In most cases, the disease is accurately diagnosed after biopsy of a painful bone lesion
or skin biopsy (e20,e330,120). When biopsied, lymph nodes may show a spectrum from subtle focal sinus or
paracortical involvement to total effacement of the nodal architecture involving the sinuses and/or the paracortex
(35) (eFigure 22-14). The sinuses, when involved, are expanded by an array of Langerhans cells, non-
Langerhans histiocytes, dendritic cells, lymphocytes, eosinophils, and actively phagocytic macrophages (e92)
(eFigures 22-15 and 22-16). Older lesions may have a proportional increase in non-Langerhans-type histiocytes
and xanthoma cells, and eosinophils may be scarce (e92). By immunohistochemical analysis, Langerhans cells
are reactive for CD1a (e78) and S100 (e337) but less so for CD68 (e125). A relatively new marker, CD207, or
Langerin, is a lectin associated with the Birbeck granule and is more specific for Langerhans cells than CD1a
(16, 77). Ultrastructural studies demonstrate Birbeck granules, a pathognomonic finding (e90).
MALIGNANT LYMPHADENOPATHY
The most common nodal malignancies in children are of lymphoid lineage, although mesenchymal (e201),
histiocyte/macrophage (e145,e190), and metastatic tumors (e132) may also present initially as node-based
disease. Immunophenotypic analysis is always required for an accurate diagnosis, and cytogenetic studies in
non-Hodgkin lymphomas are frequently helpful.
Like its benign counterparts, malignant adenopathy can be categorized morphologically by the architectural
changes seen in affected lymph nodes (Table 22-5). The most widely accepted current classification from the
World Health Organization (WHO) adopts a diagnostic and biologically meaningful approach based on the
lineage of the malignant cell. Within each lineage of the WHO classification, distinct diseases are defined based
on a combination of clinical, morphologic, immunophenotypic, and molecular genetic features (Table 22-6) (48).
The vast majority of pediatric cases of non-Hodgkin lymphoma fall into one of the four categories: diffuse large
B-cell lymphoma, Burkitt lymphoma, lymphoblastic lymphoma (T-cell or B-cell), and anaplastic large cell
lymphoma.
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Indolent lymphomas composed of small lymphocytes (e.g., small lymphocytic lymphoma, marginal zone
lymphoma, mantle cell lymphoma, follicle center cell lymphoma) are extremely rare in children, and they should
be diagnosed with caution.
Table 22-5 ▪ MALIGNANT CAUSES OF LYMPH NODE ENLARGEMENT CLASSIFIED

ACCORDING TO HISTOLOGIC PATTERNS
Nodular proliferations
Hodgkin lymphoma, nodular sclerosis type
Hodgkin lymphoma, lymphocyte-predominant type
Follicle center cell lymphoma
Mantle cell lymphoma (mantle zone pattern)
Diffuse and paracortical proliferations

Chloroma (granulocytic sarcoma)
Lymphoblastic lymphoma
Burkitt lymphoma
Diffuse large B-cell lymphoma
Peripheral T-cell lymphoma
Small lymphocytic lymphoma
Mantle cell lymphoma
Marginal zone lymphoma
Hodgkin lymphoma, mixed-cellularity type
Partial nodal involvement by lymphoma (any type)

Solid tumor metastasis
Posttransplant lymphoproliferative disorders
Necrotizing proliferations
Nodal infarction secondary to lymphoma or leukemia
Granulomatous or histiocyte-rich proliferations
Hodgkin lymphoma, mixed-cellularity type (some cases)
Sinusoidal proliferations
T γ/δ hepatosplenic lymphoma
T-cell anaplastic large-cell lymphoma
Solid tumor metastasis
Precursor B Lymphoblastic Lymphoma
B-cell lymphoblastic lymphoma, which represents approximately 15% of all cases of lymphoblastic lymphoma, is
most common in older children and young adults. Patients with this type of lymphoma present with rapidly
enlarging lymph nodes or soft tissue masses. In contrast to T-cell lymphoblastic lymphoma, the B-lymphoblastic
lymphoma rarely involves the mediastinum. The distinction between B-cell lymphoblastic lymphoma and
precursor B-cell acute lymphoblastic leukemia is made through examination of the bone marrow biopsy
specimen; in cases in which fewer than 25% of the marrow cells are blasts, a diagnosis of B-cell lymphoblastic
lymphoma should be made (e132). One feature typical of lymphoblastic lymphoma (B or T) is infiltration through
perinodal fat and linear infiltrates in the capsular collagen (Figure 22-18). The nodal architecture is effaced by a
diffuse proliferation of small and intermediate cells (12 to 14 mm) with fine or speckled chromatin, small or
indistinct nucleoli, and scant cytoplasm (e23,e123,e203) (Figure 22-19). The mitotic rate is frequently elevated,
and necrosis may be present. A CD45 (dim to negative), terminal deoxynucleotidyl transferase (TdT) positive,
CD19+, CD20-, sIg- phenotype sets these tumors apart from lymphomas composed of mature (nonblastoid) B-
cells, including Burkitt lymphoma (e 284) (Figure 22-19). Almost all cases are positive for CD10 (common acute
lymphocytic leukemia antigen or CALLA) (111). Important in the differential diagnosis in children are other small
blue cell tumors including
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granulocytic sarcoma/chloroma, Ewing sarcoma/primitive neuroectodermal tumor (104), embryonal
rhabdomyosarcoma that can be distinguished by phenotype, although care should be taken not to overvalue
results from any single stain (e187). For example, like Ewing sarcoma, lymphoblastic lymphomas are often CD45-
and CD99+.
Table 22-6 ▪ ABBREVIATED WHO CLASSIFICATION AS APPLIED TO PEDIATRICS
Precursor B- and T-cell neoplasms

Precursor B-lymphoblastic leukemia/lymphoma
Precursor T-lymphoblastic leukemia/lymphoma
Mature B-cell neoplasms

Marginal zone B-cell lymphoma
Follicular lymphoma
Diffuse large B-cell lymphoma
Mediastinal (thymic) large B-cell lymphoma
Burkitt lymphoma
Mature T-cell and NK-cell neoplasms

Hepatosplenic T-cell lymphomaa
Primary cutaneous CD30+ T-cell lymphoproliferative
disordersa
Primary cutaneous anaplastic large cell lymphoma
(C-ALCL)
Lymphomatoid papulosis
Angioimmunoblastic T-cell lymphoma
Peripheral T-cell lymphoma, unspecified
Anaplastic large cell lymphoma
Hodgkin lymphoma
Classical Hodgkin lymphoma
Nodular sclerosing Hodgkin lymphoma

Mixed-cellularity Hodgkin lymphoma
Lymphocyte-depleted Hodgkin lymphoma
Lymphocyte-rich classical Hodgkin lymphoma
Nodular lymphocyte-predominant Hodgkin lymphoma
aaNot discussed in this chapter.
FIGURE 22-18 ▪ Precursor B lymphoblastic lymphoma demonstrating diffuse architectural effacement of the node
in infiltration into adjacent perinodal fat and linear infiltrates of the capsular collagen. (Hematoxylin and eosin
stain 10×.)
FIGURE 22-19 ▪ The chromatin is fine and nucleoli are indistinct in lymphoblastic lymphoma. (Hematoxylin and
eosin stain 40×.)
Diffuse Large B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) is disproportionately common relative to other types of lymphoma in
patients with congenital, iatrogenic, or acquired immune deficiency states (e235). Patients with B-cell diffuse
large cell lymphoma may present with steadily enlarging peripheral lymphadenopathy or extranodal disease (soft
tissue, bone, oropharynx). Although rare, a primary mediastinal B-cell diffuse large cell lymphoma has also been
described in children (e238). DLBCL has a diffuse growth pattern and may have intermixed fibrosis (particularly
in the mediastinum). Cytoplasm is abundant and may be either amphophilic or densely eosinophilic (e320). The
cytology of the tumor cells ranges from that of reactive immunoblasts (round nucleus, thick nuclear membrane,
single large eosinophilic nucleolus, abundant cytoplasm), to polylobate and even frankly anaplastic multilobate
cells. Immunophenotypically, tumor cells mark with pan B-cell markers (CD19, CD20, CD79A, CD22) and are
surface Ig+ (except in mediastinal large B-cell lymphoma). CD10 is positive in a minority.
Burkitt Lymphoma
Burkitt lymphoma takes three epidemiologic forms (140). Endemic Burkitt lymphoma most commonly affects
children and exhibits a male predominance. It is common in equatorial Africa and New Guinea and is strongly
associated with EBV infection. Sporadic Burkitt lymphoma is less commonly related to EBV infection and affects
both children and adults, with a bimodal age distribution. Immunodeficiencyrelated Burkitt lymphoma is seen in
the setting of congenital immunodeficiency, HIV infection, and posttransplant. Burkitt lymphoma is one of the
most rapidly replicating of all human tumors, and patients frequently present with the sudden development of
large tumor masses (57). In endemic Burkitt lymphoma, the tumor shows an unexplained predilection for areas of
growth, including the sockets around deciduous teeth of young (2- to 4-year-old) children, and hormonally
responsive locations, such as the breasts of pubertal and pregnant women, ovaries, testes, and thyroid. In
sporadic and immunodeficiency-associated Burkitt lymphoma, visceral involvement, particularly of the small
bowel, is common, with initial symptoms related to obstruction or perforation.
Burkitt lymphoma diffusely effaces the nodal architecture. A monomorphic proliferation of intermediate-sized cells
is seen (nuclear size similar to that of histiocytes or endothelial cells); the round or oval nuclei have a thick
nuclear membrane and two to four nucleoli, and the cytoplasm is moderately amphophilic (65,e230). Many Burkitt
lymphomas have a somewhat cohesive appearance, and the cell borders maintain a molded contour, particularly
at the periphery. The mitotic rate is high (MIB-1/KI-67 is positive in >95% of cells), and necrosis is often present,
particularly at the periphery. Evenly distributed macrophages containing cellular debris give a mottled (“starry
sky”) appearance to Burkitt lymphoma at low power. Some classification systems make a distinction between
“Burkitt” and “non-Burkitt” morphology of small noncleaved cell lymphomas; however, the criteria are subjective,
and because of the lack of reproducibility, this histologic point is of limited clinical relevance (e331). The
immunophenotype is that of a mature surface Ig+ B-cell, and both CD19 and CD20 are expressed. CD10 is
positive. BCL-2 expression is not present. TdT expression is lacking (e8). Differential diagnostic considerations
include lymphoblastic lymphoma and rapidly replicating large cell lymphomas. Cytogenetic analyses play a key
role in confirming the diagnosis. FISH studies are very helpful in demonstrating translocations that deregulate
expression of the protooncogene c-myc (chromosome 8) paired with either the heavy-chain loci (chromosome
14) or light-chain locus (chromosome 2 and 22) (e25,e231) (Table 22-3).
Other Rare B-Cell Lymphoma in Children

Two small B-cell lymphomas are worthy of note but only rarely seen in children—follicular lymphoma and nodal
marginal zone lymphoma. Follicular lymphoma in children affects males more than females and, unlike their adult
counterparts, often presents with limited stage disease. Even though most are grade 2 or 3, many are curable.
Morphologically, they range from the typical low-power pattern of crowded monomorphic small-to-medium size
follicles to large expansive follicles and even “floral variant.” Phenotypically like adults, most cases are CD10+
and BCL-6+, but unlike adults, are often BCL-2- and most do not have underlying BCL-2/IgH (14;18)
translocation. The small minority with BCL-2 translocations appear to have a worse prognosis (84,e243).
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Pediatric nodal marginal zone lymphoma, like its adult counterpart, can be particularly difficult to diagnose in that
these lymphomas often only partially efface architecture with an interfollicular distribution. The neoplastic cells
are frequently a mix of classic monocytoid B-cells with small somewhat folded nuclei and abundant cytoplasm,
small lymphocytes with little cytoplasm, large lymphocytes, and variable numbers of plasma cells and
plasmacytoid lymphocytes. Follicular colonization may be present as well as follicles resembling those of
progressive transformation of follicular centers. Most cases have CD5-CD10-phenotype with monotypic surface
Ig. Many have monotypic cytoplasmic immunoglobulin in plasma cells on paraffin immunoperoxidase stains. Like
pediatric follicular lymphoma, these patients are predominantly males with limited stage disease (usually cervical
nodes) and have an apparently good prognosis (130).
T-Lymphoblastic Leukemia
T-cell lymphoblastic lymphoma is commonly seen in adolescents and young adults. Although a rare type of
lymphoma in the adult population, it represents approximately 30% of all pediatric non-Hodgkin lymphomas, and,
like B-cell lymphoblastic lymphoma, T-cell lymphoblastic lymphoma is distinguished from T-cell acute
lymphoblastic leukemia by the demonstration of limited bone marrow disease (<25% involvement). Because it is
frequently located in the mediastinum (e132), a rapidly growing T-cell lymphoblastic lymphoma may compress
the heart and great vessels or cause a pleural or pericardial effusion. The morphology of T-cell lymphoblastic
lymphoma is identical in every respect to B-cell lymphoblastic lymphoma, and the immunophenotype is that of an
immature T-cell with CD45 (dim-to- negative), TdT+, cytoplasmic CD3+, usually surface CD3-, CD2+, CD7+ with
variable expression of CD1a, CD4, CD5, and CD8 (e283,e324). HLA-DR is negative. CD10 is expressed in 25%
of cases. Other entities in the differential diagnosis, including B-cell lymphoblastic lymphoma, Ewing
sarcoma/primitive neuroectodermal tumor, and embryonal rhabdomyosarcoma, can be excluded with
immunophenotype studies. Tumor karyotype is less helpful in the prognosis of T-cell lymphoblastic lymphoma
than of B-cell lymphoblastic lymphoma (43). Many (although not all) translocations involve either the a and the d
T-cell receptor locus at 14q11.2, the b locus at 7q35, or the g locus at 7p14-15 (e221).
Peripheral T-Cell Lymphoma

Peripheral T-cell lymphomas including angioimmunoblastic T-cell lymphoma represent only a small fraction of
lymphomas in children (e7,e133,e235), and heterogeneity is their histologic hallmark (Table 22-7). At low power,
the lymph node architecture is either diffusely effaced or shows interfollicular expansion. Thick-walled vessels
are more prominent in peripheral T-cell lymphomas than in B-cell non-Hodgkin lymphomas, and the even mixture
of atypical small, intermediate, and large lymphoid elements is another clue to the lineage of this type of
lymphoma (e118,e168). Scattered eosinophils and plasma cells may also be seen. Irregularity of the nuclear
contour may be particularly prominent, and, in further contrast to lymphomas of B-cell lineage, these tumors in
many cases have cells with a clear cytoplasm. Detailed phenotypic studies have shown that peripheral T-cell
lymphomas are uniformly TdT-, CD45+ with most having a CD3+, CD45Ro+, CD4+, CD8- helper T-cell phenotype
(e241). Other T-cell antigens (e.g., CD2, CD5, CD7) are variably expressed, and the loss of these markers is
characteristic of peripheral T-cell lymphoma (e241). Virally mediated or drug hypersensitivity immunoblastic
reactions may mimic peripheral T-cell lymphoma, as can Hodgkin lymphoma, B-cell lymphomas rich in T-cells
(“T-cell-rich B-cell lymphomas”), and Fas mutation-related (ALPS).
Table 22-7 ▪ CATEGORIES OF PERIPHERAL T-CELL LYMPHOMA
Phenotype-specific
CD56 + (NK-like) PTCL

CD30 + PTCL (ALCL)
Organ-specific subtypes
Enteropathy-associated PTCL
Angiocentric PTCL
T γ/δ hepatosplenic PTCL
Subcutaneous panniculitic PTCL
Peripheral T-cell lymphoma, NOS
Predominantly small-cell
Mixed small- and large-cell
Predominantly large-cell
Histology-specific
Lennert PTCL
AILD type PTCL
Virally mediated
HTLV-1-related
EBV-related
NK, natural killer; PTCL, peripheral T-cell lymphoma; AILD, angioimmunoblastic lymphadenopathy with
dysproteinemia; HTLV, human T-cell leukemia/lymphoma/lymphotrophic virus; EBV, Epstein-Barr virus;
NOS, not otherwise specified.
Anaplastic Large Cell Lymphoma

T-cell anaplastic large cell lymphoma, a special type of peripheral T-cell lymphoma, affects patients of all ages,
from children to the elderly (e121,e219,e275). Cervical lymphadenopathy is particularly common in some series,
and the skin, bone, and soft tissue may be secondarily involved. Involved lymph nodes may exhibit either a
diffuse or a sinusoidal pattern (Figure 22-20) of tumor cell infiltration, and the latter may be mistaken for
metastases of a solid tumor. The tumor cells of T-cell anaplastic large cell lymphoma are often very large (>20
mm) and have bizarre, lobulated, or wreath-like nuclei (hallmark cells) with small-to-large nucleoli and abundant
eosinophilic cytoplasm (e6) (Figure 22-21). Pleomorphic (e49), sarcomatoid (e43), histiocyte-rich (e239),
neutrophil-rich (90, 127), and even monomorphic/small
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cell variants (51,e156) have been described. The common denominator in most pediatric cases being the t(2;5)
karyotype abnormality with ALK positivity (e167). Membranous staining (usually associated with Golgi positivity)
for CD30 is required for the diagnosis. Anaplastic large cell lymphoma exhibits a variable expression of pan T-
cell markers CD3, CD7, and CD5. Epithelial membrane antigen (EMA) and CD45 are usually but not always
positive (e83,65). CD4, CD2, CD43, and CD45 RO are often helpful. Most cases are positive for cytotoxic
molecules like TIA-1, granzyme B, and perforin. Only a minority are CD8+. Stains for EBV-like LMP and EBER
are consistently negative (28,e158,129). Staining for EBV, ALK, PAX-5, and a battery of T-cell and cytotoxic
granule markers is helpful in evaluating CD45-, CD30+ cases of ALCL in which the major consideration is
Hodgkin lymphoma with a syncytial growth phase.
FIGURE 22-20 ▪ Anaplastic large cell lymphoma may resemble metastatic carcinoma when it remains localized to
the sinusoids. (Hematoxylin and eosin stain 4×.)
Hodgkin Lymphoma
Hodgkin lymphoma is a primary nodal tumor of B-cell lineage (22). Recent studies in which microdissected tissue
and single-cell polymerase chain reaction methods were used have shown that the Reed-Sternberg cells in most
cases of Hodgkin lymphoma have clonal rearrangements of the immunoglobulin heavy-chain locus and exhibit
intraclonal point mutations, indicative of ongoing somatic hypermutation (e183,e216). These findings have
allowed assignment to a B-cell lineage and germinal center status to the cell of origin of Reed-Sternberg cells
(22).
FIGURE 22-21 ▪ Anaplastic large cell lymphoma with large bizarre tumor cells with multilobated nuclei with small-
to-large nucleoli and abundant cytoplasm. (Hematoxylin and eosin stain 40×.)
Hodgkin lymphoma manifests a bimodal age distribution, with peaks in young adults and older adults, and is
more common overall in males than in females (e13). The key pathological characteristic of Hodgkin lymphoma is
that the bulk of the tumor is composed of reactive leukocytes and histiocytes, with very few neoplastic cells
(46,e175). The diagnosis of Hodgkin lymphoma requires (a) the presence of neoplastic Reed-Sternberg cells of
appropriate phenotype and (b) a cytologically bland population of background inflammatory cells (e131). The
WHO classification of Hodgkin lymphoma divides these lymphomas into lymphocyte-predominant Hodgkin
lymphoma and classic Hodgkin lymphoma, which includes nodular sclerosing, mixed cellularity, lymphocyterich,
and lymphocyte-depleted subtypes (Fig 22-22).
In classic Hodgkin lymphoma, typical Reed-Sternberg cells are large (20 to 40 m), with a range of appearances.
The classic type has a bilobed or a multilobed nucleus, with a thick nuclear membrane and one or several very
large nucleoli, and abundant eosinophilic cytoplasm (47) (Figure 22-23). In the mononuclear type, the nucleus
has a single lobe and often a single central nucleolus, which may be so large that it mimics a cytomegalovirus
inclusion. The lacunar type of Reed-Sternberg cell characteristic of nodular sclerosing Hodgkin has a single-
lobed or a multilobed nucleus, usually with small nucleoli and a water clear cytoplasm that is fragmented and
retracted from the surrounding cells (Table 22-8). Phenotypically, the Reed-Sternberg cells of the different
subtypes of classic Hodgkin lymphoma share a CD45-, CD30+, CD15±, CD20± and PAX5+ (weak) phenotype. In
up to one-half of cases, Reed-Sternberg
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cells-stain for Epstein Barr virus products LMP or EBER, particularly in cases associated with immunodeficiency
(17, 42) (Table 22-9).
FIGURE 22-22 ▪ Broad bands of frequently paucicellular collagen dissect the lymph node into cellular nodules in
nodular sclerosis Hodgkin lymphoma. (Hematoxylin and eosin stain 4×.)
FIGURE 22-23 ▪ BLacunar Reed-Sternberg cells have vesicular chromatin, small nucleoli, multilobed nuclei, and
abundant cytoplasm. (Hematoxylin and eosin stain 10×.)
Nodular Sclerosing Hodgkin Lymphoma

Nodular sclerosing Hodgkin lymphoma is the most common type of Hodgkin lymphoma in children and is slightly
more common in girls than in boys. Common sites of primary disease are the cervical, mediastinal, and
supraclavicular lymph nodes. The presentation may be secondary to a mass effect (e.g., superior vena cava
syndrome, palpable adenopathy) or not directly referable to the tumor bulk (“B” symptoms). In most cases of
nodular sclerosis Hodgkin lymphoma, the low-power view is diagnostic. Broad bands of birefringent fibrosis
dissect the node into cellular nodules containing both lacunar Reed-Sternberg cells and a mixed inflammatory
infiltrate (Figures 22-22 and 22-23). The background cellularity in this subtype of Hodgkin lymphoma is quite
variable and may include a mixture of granulocytes, eosinophils, lymphocytes, and histiocytes, in which one of
these cell types may predominate (Table 22-10). Mononuclear Reed-Sternberg cell variants may be numerous,
but “classic” or “diagnostic” forms are in the minority (Table 22-8). With rare exceptions, all exhibit the typical
CD15+, CD30+, PAX5+ (weak), CD45-, and CD20± profile (e131,e242). Other entities in the differential
diagnosis, include ALCL, inflammatory myofibroblastic tumor, cat-scratch disease, and large cell lymphoma with
or without sclerosis (e48,e238), can be distinguished from nodular sclerosis Hodgkin lymphoma by light
microscopic features and immunohistochemistry.
Table 22-8 ▪ CORRELATION OF REEDSTERNBERG CELL TYPE AND SPECIFIC SUBTYPE OF

HODGKIN LYMPHOMA
NSHL MCHL LPHL LDHL
Classic + ++ -/+ ++
Mononuclear ++ ++ + ++
Lacunar ++ 0 0 0
L&H 0 0 ++ 0
L&H, lymphocyte and histiocyte; NSHL, nodular Sclerosis Hodgkin lymphoma; MCHL, mixed cellularity
Hodgkin lymphoma; LPHL, lymphocyte predominance Hodgkin lymphoma; LDHL, lymphocyte depletion
Hodgkin lymphoma; +, present; ++, numerous; 0, absent; -/+, rare to absent.
Table 22-9 ▪ IMMUNOPHENOTYPE IN HODGKIN LYMPHOMA
NSHL MCHL LPHL LDHL
Classica + + 0 +
0 0 + 0
B-cellb
aCD15+, CD30+, CD20-, CD45-.
bCD15-, CD30-, CD20+, CD45+
NSHL, nodular sclersis Hodgkin lymphoma; MCHL, mixed cellularity Hodgkin lymphoma; LPHL,
lymphocyte predominance Hodgkin lymphoma; LDHL, lymphocyte depletion Hodgkin lymphoma.
Mixed Cellularity Hodgkin Lymphoma

The other common type of classic Hodgkin lymphoma is the mixed-cellularity Hodgkin lymphoma. This is seen in
all age groups, although it is more common than nodular sclerosis Hodgkin lymphoma in older patients, and
shows a male predominance. Patients present with either isolated disease or multiple contiguous sites of
involvement centered in the neck and mediastinum. The architecture of the lymph node in mixed-cellularity
Hodgkin lymphoma is usually diffuse or interfollicular, although the cellular composition of the background
population varies from an even mixture of lymphocytes, plasma cells, eosinophils, neutrophils, and histiocytes to
a monotony of one of these elements (Table 22-10). Only rarely do the Reed-Sternberg cells in mixed-cellularity
Hodgkin lymphoma deviate from the classic phenotypic profile. Because of a close morphologic overlap with T-
cell-rich B-cell large cell lymphoma (e48,e225) and peripheral T-cell lymphoma, all cases of mixed-cellularity
Hodgkin lymphoma should be evaluated by immunohistochemistry.
Lymphocyte-Depleted Hodgkin Lymphoma

Lymphocyte-depleted Hodgkin lymphoma is exceedingly rare, if it occurs at all, in children and most often seen in
adults over 50 years of age. Involved lymph nodes are diffusely overrun by small, intermediate, and numerous
large cells, the latter representing classic and variant forms of ReedSternberg cells (e175,e208). The
proliferation is embedded within a fibrillary and fibrotic matrix (diffuse fibrosis type), or a collagenous background
and increased numbers of fibroblasts may be present (reticular type) (e122,e150). The Reed-Sternberg cells
have a classic immunophenotype profile, and the background infiltrate is composed largely of T-cells (e150)
(Table 22-9).
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Table 22-10 ▪ CORRELATION OF BACKGROUND CELLULAR AND METRIC COMPOSITION

WITH SPECIFIC TYPE OF HODGKIN LYMPHOMA
NSHL MCHL LPHL LDHL
Lymphocytes + ++ ++ +
Neutrophils ++ ++ 0 +
Eosinophils ++ ++ 0 0
Histiocytes + ++ + −/+
Bands of fibrosis ++ 0 0 0
Architecture Nodular Diffuse Nodulara Diffuse
aThe exixtence of a diffuse form of LPHL is controversial
L&H, lymphocyte and histiocyte; NSHL, nodular sclerosis Hodgkin lymphoma; MCHL, mixed cellularity
Hodgkin lymphoma; LPHL, lymphocyte predominance Hodgkin lymphoma; LDHL, lymphocyte depletion
Hodgkin lymphoma; +, present; ++, numerous; 0, absent; -/+, rare to absent.
Lymphocyte-Rich Classic Hodgkin Lymphoma

This is a rare and recently recognized subtype of Hodgkin lymphoma, which at low power has a nodular growth
pattern like nodular LPHL. Also like LPHL, the background lymphocyte population is predominantly small B-cells
(usually IgD+), unlike other subtypes of classic Hodgkin lymphoma in which CD4 small T-cells predominate.
However, the neoplastic Reed-Sternberg cells maintain a CD45-, CD30+, CD20-, CD15± phenotype unlike the
CD45+, CD20+, CD30- phenotype of LPHL (4).
Lymphocyte-Predominant Hodgkin Lymphoma

Lymphocyte-predominant Hodgkin lymphoma has a unimodal age distribution, a predilection for males, with a
peak incidence in the fourth decade. Most patients present with isolated and asymptomatic adenopathy and very
few are at an advanced stage at presentation (100, 117). The architecture is distorted by nodules of small B-
lymphocytes that are bounded by a compressed, reticulin-rich rim of blood vessels and histiocytes (117) (Figure
22-24). Scattered within the nodules are lymphocytic and histiocytic (L&H), ReedSternberg cell variants, many of
which are ringed by T-cells (e47). L&H cells are multinucleated (popcorn-like) with a thin nuclear membrane,
delicate chromatin, and inconspicuous nucleoli, and the cell has a variable quantity of cytoplasm (Figure 22-25).
Diagnostic Reed-Sternberg cells are rare (if present at all), and mononuclear variants are few in number.
Typically, the L&H cells are evenly spaced throughout the nodules, although on occasion they are present in
tighter clusters or form small aggregates. In the diffuse form of lymphocyte-predominant Hodgkin lymphoma, a
controversial entity (e244), nodule formation is minimal, and the morphologic and immunophenotypic overlap with
B-cell large cell lymphomas rich in T-cells is considerable (e130). The L&H variants exhibit a B-cell phenotype
(CD20+, CD45+) (Figure 22-25) and do not express the usual Hodgkin markers, CD15 and CD30 (e185). The
majority of the background lymphocytes are CD20+ B-cells; CD57+ T cells commonly form a collarette pattern
around the L&H cells. Transformation to DLBCL is occasionally seen. Differential diagnostic considerations are
numerous and include T-cell-rich B-cell large cell lymphoma (e46,e149,e276), lymphocyte-rich classic Hodgkin
lymphoma (e188), progressive transformation of germinal centers (e37,e48,99,e308), and florid toxoplasmosis-
related lymphadenopathy (e67).
FIGURE 22-24 ▪ In lymphocyte-predominant Hodgkin lymphoma, the nodularity is created by contrasting
populations of basophilic small mature lymphocytes centrally and eosinophilic histiocytes and compressed high
endothelial venules peripherally. (Hematoxylin and eosin stain 4×.)
Tumors of Monocyte/Macrophage Lineage

Chloroma/granulocytic sarcoma occurs more frequently in patients with simultaneous or subsequent acute
myelogenous leukemia (27), and it may also be the first sign of relapse in patients with a previous diagnosis of
acute myelogenous leukemia (e322). Lymphadenopathy or bulk disease
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in nodal groups is less common than visceral or soft tissue involvement, but when it is present, a biopsy is often
performed (e31,21,e96,e171,89,e301). The architecture of the lymph node is diffusely effaced by a proliferation
of cells of uniformly intermediate size with oval or folded nuclei, fine chromatin, and scant-to-moderate quantities
of pale or amphophilic cytoplasm. Cytoplasmic granules may be faint or absent, although a Leder stain
accentuates their presence in some cases (e207). Most cells are positive for myeloperoxidase and CD45, and
some aberrantly express macrophage markers such as CD68 or lysozyme. Rare cases may be of apparently
mixed lineage and show reactivity for polyclonal CD3 (e250,e253), CD30 (e95), or CD99 (e66), some cases may
be positive for TdT (53,e223,e250). Examinations of touch preparations of involved nodes should disclose a
blast morphology, which effectively excludes a mature B- or T-cell lymphoma. Immunohistochemistry and
correlation with clinical and laboratory parameters (92) are important in excluding nodal metastasis of Ewing
sarcoma/primitive neuroectodermal tumor or involvement by lymphoma (e328) or Langerhans cell histiocytosis.
FIGURE 22-25 ▪ The “lymphocytic and histiocytic” cell of lymphocytepredominant Hodgkin lymphoma has a thin
nuclear membrane, distinct nucleoli, and scant-to-moderate quantities of cytoplasm. (Hematoxylin and eosin stain
40×.)
SPLEEN
Embryology
The spleen develops from mesenchyme located between the two layers of the dorsal mesentery of the stomach
(the dorsal mesogastrium). As the stomach rotates during development, the dorsal mesogastrium becomes fused
to the peritoneum of the left kidney to form the lienorenal ligament, which envelops the splenic artery and vein
and the tail of the pancreas.
FIGURE 22-26 ▪ Schematic representation of the splenic red pulp (left) and white pulp (right) showing the main
compartments and structures of the human spleen. (From Van Krieken JH, Orazi A. Spleen. In Mills SE, ed.
Histology for Pathologists, 3rd ed. Philadelphia: Lippincott Williams & Wilkins, 2007:783-798).
Normal Structure and Function

In contrast to lymph nodes, which filter lymphatic fluid, the spleen is the major site of blood filtration. In the
microenvironment of the spleen, antibody-antigen binding takes place and immune complexes, such as
opsonized bacteria, are removed. The spleen is also a major site of removal of red cells with decreased flexibility
or increased osmotic fragility. The filtering function of the spleen is a dual-edged sword. Although the spleen
protects against life-threatening infections caused by encapsuled bacteria, the destruction of antibody-coated
platelets or red blood cells makes it necessary to remove the spleen in certain diseases.
The spleen is subdivided into the areas of red and white pulp (Figure 22-26). This division is useful for surgical
pathologists because most diseases primarily affect one compartment or the other. The red pulp comprises most
of the splenic volume and is the major site of removal of senescent and antibodycoated red cells and platelets
and of red cell inclusions, such as Howell-Jolly bodies (nuclear fragments) and Pappenheimer bodies (siderotic
granules). Blood enters the spleen via splenic arteries. The splenic arteries branch into progressively smaller
arteries and arterioles that ultimately empty into a network of thin-walled, endothelium-lined capillaries. These
terminate in sheathed capillaries that are lined not by endothelium but by specialized phagocytic mononuclear
cells. Blood cells traverse the sheathed capillaries and basement membrane to enter the splenic sinuses and
ultimately drain into the splenic veins. While crossing the sheathed capillaries, red blood cells with decreased
flexibility become trapped in the splenic cords and are destroyed by the phagocytic lining cells.
The white pulp of the spleen, which grossly appears as numerous gray-white spots within the red pulp, is
composed of masses of T- and B-lymphocytes. The T-lymphocytes
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surround the arterioles and become less plentiful around the more distant arteriolar branches. Within these areas
are the B-cell areas, which may contain germinal centers, particularly in the spleens of children and persons with
autoimmune disorders. Germinal centers in the spleen, as in other sites, are surrounded by a mantle zone. This
in turn is surrounded by a marginal zone of cells with relatively abundant pale cytoplasm that is usually
appreciable only in the spleen. In a retrospective review of splenic histopathology from 205 children dying
unexpectedly, there was a significant increase in reactive germinal centers in the spleen of children dying
unexpectedly in comparison to other children of the same age (e10). Germinal centers in the spleen develop in
response to immunological stimulation. The existence of a high number of germinal centers in the spleen is
evidence that an immune reaction has occurred. The histology of the normal spleen is described in greater detail
by van Krieken and te Velde (e315).
Examination of the Spleen

Many pathological processes affect the spleen of children, including congenital anomalies, benign cysts, trauma,
infection, malignancies, and other hematological disorders (50). Splenectomies in children are performed for
many reasons. The most common reasons from splenectomy include hereditary spherocytosis, hemolytic
anemias, trauma, idiopathic thrombocytopenic purpura, hypersplenism, and trauma (e45,e58,e80,136,e319).
Splenectomies to stage Hodgkin lymphoma, once the most common indication, have become rare as
chemotherapy has become the primary modality of therapy. Common causes of splenomegaly are listed in Table
22-11.
Regardless of the indication for splenectomy, all surgically removed spleens should be measured and weighed
(e204). After the capsule has been examined for color, irregularities, and tears, the spleen is sliced transversely
into thin sections. For the pathologic staging of Hodgkin lymphoma, the sections should be cut as thinly as
possible, typically about 3 mm (e154). Sections of abnormal-appearing areas should be taken (typically six to
eight sections) in addition to a section of normal-appearing spleen. We routinely order periodic acid-Schiff
staining of the section of normal spleen, as this highlights the splenic cords and makes it easier to recognize
other hematopoietic elements. If hilar lymph nodes are present, these should be dissected off and processed as
a regular fresh lymph node biopsy.
CONGENITAL ANOMALIES
Asplenia
Asplenia, congenital absence of the spleen, occurs in about 1/40,000 live births (e4). It is more common in boys
and is often associated with cardiac anomalies, such as dextrocardia, transposition of the great vessels, and
bilateral superior venae cavae, and development defects in other organs, including the liver, lungs, and
intestines (e99,e100). Primarily because of these associated defects, the prognosis of patients with congenital
asplenia is poor. In one series, nearly 80% of the patients died in infancy of cardiac failure or complications of
surgery (e259). Asplenia of any cause is associated with characteristic peripheral blood findings, including
Howell-Jolly bodies, Pappenheimer bodies, and dysmorphic and nucleated red blood cells.
Table 22-11 ▪ CAUSES OF SPLENOMEGALY IN CHILDHOOD
Nonspecific infections
Acute splenitis Hodgkin lymphoma

Autoimmune disorders Malaria
Autoimmune hemolytic anemia Myeloproliferative disorders (CML, JMML, chronic
Acute leukemias (AML, ALL) myelofibrosis)
Brucellosis Niemann-Pick disease
Cardiac failure Non-Hodgkin lymphoma
Congestion Portal or splenic vein thrombosis
Cytomegalovirus infection Rheumatoid arthritis
Echinococcosis Schistosomiasis
Gaucher disease Sickle cell anemia
Hematologic malignancies Storage diseases
Hemolytic anemias Systemic lupus erythematosus
Hepatic cirrhosis Thalassemias
Hereditary elliptocytosis Toxoplasmosis
Hereditary spherocytosis Trypanosomiasis
Histoplasmosis Tuberculosis
Typhoid fever
Other causes
Vascular tumors (hemangioma, lymphangioma,
angiosarcoma)
Myofibroblastic tumor (inflammatory
pseudotumor)
Metastatic tumors
Hamartomas
Cysts
AML, acute myelogenous leukemia; ALL, acute lymphocytic leukemia; CML, chronic myelogenous
leukemia; JMML, juvenile myelomonocytic leukemia.
Polysplenia
In contrast to asplenia, polysplenia is more common in girls (e259). The multiple small splenic masses are
located in the right upper quadrant and are often associated with dextrocardia, a right-sided aortic arch, and
pulmonary and hepatic defects (e232). The histology of the splenic tissue is normal. Although less likely to die of
cardiopulmonary defects in infancy, patients with polysplenia nonetheless have a high mortality rate. In one
series, only 25% of patients were alive at 5 years (e232).
Accessory Spleen
Accessory spleen is the most common congenital anomaly, encountered in about 16% of pediatric splenectomies
(e80). Accessory spleens are usually solitary and are most commonly located in the splenic hilum, although they
may be found in the omentum, gastrosplenic and splenocolic ligaments, or retroperitoneum
(e80,e128,e197,e214). The
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primary importance of accessory spleens is that they must be removed along with the spleen in therapeutic
splenectomies for diseases such as idiopathic thrombocytopenic purpura to prevent recurrence.
Fusion
Most cases of splenic fusion occur in white males, and most often, the spleen is fused with the left testis (e249).
Very rare cases of splenorenal or splenohepatic fusion have also been reported (e55,e113).
Hamartoma
Hamartomas of the spleen are uncommon benign tumors located primarily within the red pulp (1). They are
usually discovered incidentally after a splenectomy or at autopsy. Most are reported in the adult population, but
recent reviews suggest that 20% of reported hamartomas occur in children (1,e85). The lesions are associated
with congenital abnormalities such as tuberosis sclerosis. Often patients present with hematologic conditions
including refractory microcytic anemia, sickle cell anemia, hereditary spherocytosis, and dyserythropoietic
hemolytic anemia.
A recent report of four pediatric patients with splenic hamartomas described children ranging in age from 4 to 11
years, who presented with splenomegaly and hematological abnormalities. In each case, the spleen was
enlarged (315 to 724 g). On cut surface, single or multiple discrete bulging nodules ranging from 1.3 to 7 cm
were identified. In other studies, the nodules have been reported as large as 15 cm in diameter (e85).
Microscopically, the lesions are composed of vascular channels that resemble splenic sinusoids and lack
malpighian corpuscles. They are usually associated with histiocytic proliferations, extramedullary hematopoiesis,
lymphoplasmacytosis, fibrosis, and siderotic-calcific deposits.
Cysts
Congenital splenic cysts are rare. Hydatid or echinococcal cysts are the most common splenic cysts worldwide
but are very rare in the United States. True or primary cysts are lined by epithelium, whereas false cysts or
pseudocysts lack a cellular lining and are thought to arise after trauma (e76,e107). Splenic cysts can become
quite large (>20 cm) and are typically filled with serous fluid. Although most congenital cysts are asymptomatic,
cases of rupture associated with granulomatous peritonitis have been reported (e172).
Diseases of the Red Pulp

Diseases primarily involving the red pulp include congestion, hereditary hemolytic anemias, hemoglobinopathies,
infections, histiocytic proliferations, acute and chronic leukemias, and nonhematopoietic (particularly vascular)
tumors.
Congestion
Chronic passive congestion of the spleen is commonly the result of portal hypertension or right-sided cardiac
failure. The spleen is grossly enlarged, and microscopically the findings are nondescript. The splenic sinuses are
distended with red cells, and the capsule and splenic cords may be thickened and fibrotic. Splenic congestion is
also common in hereditary hemolytic anemias and hemoglobinopathies, which are discussed in the following
sections.
Thrombocytopenia
The spleen is the major site of both antiplatelet antibody production and platelet removal from the bloodstream.
Splenectomy is performed in patients with refractory thrombocytopenia when the disorder is refractory to steroid
or other immunosuppressive therapy (107). Refractory thrombocytopenia is defined as persistent and severe
thrombocytopenia (platelet count <20 × 109/L) and the inability of therapies to increase and sustain the platelet
count increase. Multiple factors have been identified to lead to refractoriness. These include some cases of
drug-induced thrombocytopenia. In addition, infection with HIV, hepatitis C virus, Epstein Barr virus,
cytomegalovirus, parvovirus, herpes virus 6 and 8, and Helicobacter are all chronic infections that are associated
with refractory thrombocytopenia. In China, acute thrombocytopenia in childhood is often associated with CMV
infection (107). Secondary thrombocytopenia can arise from systemic lupus as well as a number of congenital
syndromes and abnormalities seen in childhood including common variable immune deficiency, Evans syndrome,
autoimmune lymphoproliferative disorder, and paroxysmal nocturnal hemoglobinuria. Splenectomy offers a 60%
to 70% chance of cure in patients with chronic ITP.
Surgically removed spleens of patients with thrombocytopenia are usually of normal size or mildly enlarged.
Microscopically, the white pulp of spleens from patients with idiopathic thrombocytopenic purpura is usually
prominent; numerous reactive follicles are present unless the patient has recently been treated with steroids.
The red pulp shows myeloid hyperplasia and increased numbers of foamy histiocytes containing platelets and
phospholipid debris (e333).
Hereditary Hemolytic Anemias

Common inherited causes of hemolytic anemia include hereditary spherocytosis and elliptocytosis, and
hemoglobinopathies such as the thalassemias and sickle cell anemia. Many of the clinical problems of hereditary
hemolytic disorders can be controlled by splenectomy. However the use of splenectomy in a young child exposes
the patient to lifelong risk of overwhelming infections and other complication. Vasilescu et al. (136) have
proposed and demonstrated that subtotal splenectomy controls the hemolysis and maintains the splenic function.
This new model preserves the lower pole of the spleen.
Most patients with hereditary spherocytosis and elliptocytosis have splenomegaly (e163,e177). The erythrocytes
in these conditions are inflexible and become trapped as they attempt to pass through the splenic cords. Spleens
removed for these diseases typically weigh from 250 to 500 g and are firm and dark red as a result of congestion
(e329). Microscopically, the
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venous sinuses are distended with red cells and are surrounded by hemosiderin-laden macrophages and
endothelial cells. Often, the splenic white pulp is hyperplastic, with increased numbers of germinal centers.
Patients usually respond well to splenectomy. Refractory cases are most commonly the result of an
unrecognized accessory spleen (e268).
Splenomegaly is also present in the majority of young children with sickle cell anemia. In patients more than 10
years old, however, the spleen is usually decreased in size as a result of infarction and scarring (e321). Acute
rapid enlargement of the spleen may be the result of a sequestration crisis. Ultimately, the organ undergoes
“autosplenectomy,” and only a fibrotic remnant is left. Microscopically, early in the course of the disease, the
sinusoids are distended with sickled red blood cells, and the splenic cords are thickened and fibrotic; later, the
splenic parenchyma is replaced by fibrous scars containing multicolored deposits of minerals and hemosiderin
(Gamna-Gandy bodies).
Occasionally, progressive splenomegaly develops in patients with b-thalassemia who have undergone
splenectomy, and the spleen may weigh as much as 750 to 1,000 g by adolescence. The histologic findings
resemble those of the other hereditary hemolytic anemias, with extramedullary hematopoiesis a particularly
common finding.
Infection
Acute splenitis can arise as a result of many blood-borne infections. The spleen typically becomes congested,
with infiltration of the red and white pulp by neutrophils and plasma cells. Sometimes, necrotic foci develop.
Abscess formation is uncommon. Granulomatous inflammation may be seen in the spleen in disseminated fungal
or mycobacterial infections. As previously mentioned, echinococcal infections, although rare in the United States,
are the most common cause of splenic cysts worldwide.
Bartonella henselae infection usually results in selflimited lymphadenitis, cat-scratch disease, primarily a disease
of children (10,e157,e173,114). Bartonella species are small, intracellular Gram-negative rods. Bacillary infection
can also result in an unusual vascular lesion called bacillary angiomatosis. Bacillary angiomatosis usually
occurs in the skin, bone, and the brain, but a related proliferative lesion called vascular peliosis occurs in the
liver and the spleen. Bacillary angiomatosis-peliosis was first identified in HIVinfected patients with AIDS. In most
children and adolescents with intact immune systems, cat-scratch disease is confined to the lymph nodes. But
numerous examples of the systemic manifestation have been reported (10,e157,e173,114).
Splenomegaly is seen in about half of patients with infectious mononucleosis and is occasionally complicated by
fatal splenic rupture (e120,e176). Microscopically, the red pulp cords and sinuses are infiltrated by a polymorphic
population of T- and B-immunoblasts that may include large multinucleated forms resembling Reed-Sternberg
cells (e306). The clinical setting is most helpful in avoiding a misdiagnosis of Hodgkin lymphoma.
Immunohistochemically, the immunoblasts in acute EBV infection may be CD30+ but are usually CD15-) and
negative for leukocyte common antigen (e1).
DISEASES OF THE WHITE PULP

Histiocytic Proliferations
Splenomegaly is a common feature of a variety of inborn errors of metabolism, including diseases ranging from
sphingolipidosis to glucocerebrosidosis. The accumulation of storage histiocytes engorged with storage products
within the spleen causes progressive enlargement (e164,e316).
Gaucher disease, which encompasses a spectrum of autosomal recessive disorders that result from
glucocerebrosidase deficiency, is the most common metabolic cause of splenomegaly in childhood and the most
prevalent lysosomal storage disorder (14). Of the three clinical types, type I (chronic non-neuropathic) is the most
common, particularly in individuals of European Jewish descent. Although traditionally referred to as the “adult
type,” 66% of individuals with type I Gaucher disease presents in childhood as hypersplenism develops
(e115,e124,65). In this form of Gaucher disease, macrophages containing glucosylceramideladen lysosomes
accumulate primarily in the bone marrow and spleen without involving the central nervous system.
Hepatosplenomegaly is also seen in type II disease (acute neuropathic), but involvement of the central nervous
system leads to death in early childhood. The spleen is also involved in type III disease (subacute neuropathic),
where neurologic manifestations begin in early adult life.
The basic pathology of classic, type I Gaucher disease, includes hepatosplenomegaly, bone marrow infiltration,
hematocytopenia, and bone disease. The disease is progressive and may ultimately lead to bleeding
complications, liver and lung fibrosis, pulmonary hypertension, and bone disease (44, 76, 93). Macrophage-
targeted enzyme replacement therapy has been shown to reverse or prevent many of the manifestations of
Gaucher disease type I including hepatosplenomegaly, marrow infiltration, and cytopenia.
Microscopic evaluation of the spleen shows that the sinuses are diffusely expanded by clusters and sheet large
macrophages. The storage macrophages or Gaucher cells are two to six times the size of a normal macrophage
but maintain the cytologically bland, round, oval, vesicular nuclei. There is abundant cytoplasm with a wrinkled or
a striated appearance. The cytoplasm stains with both periodic acid-Schiff and iron.
The disease is diagnosed by identifying the enzyme deficiency. The characteristic lysosomal b-
glucocerebrosidase activity is decreased or absent in leukocytes. A targeted mutational analysis has identified
four mutations (N370S, L44P, 84GG, and IVS2+1) that account for almost 90% of all disease (36). Pseudo-
Gaucher cells can be seen in a number of disorders associated with hematopoietic destruction
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such as chronic myelogenous leukemia. In these cases, the macrophage cytoplasm contains insoluble lipid
pigment that stains intensely blue (see Chapter 5).
Niemann-Pick disease is an autosomal recessive deficiency of sphingomyelinase that leads to the accumulation
of sphingomyelin within cells and tissues. Niemann-Pick disease is subclassified into two subtypes, A and B,
based on phenotypic manifestations (39, 91). Type C Niemann-Pick disease shares the eponym but is now
recognized to be a genetically distinct disorder resulting from defective intracellular trafficking of cholesterol
(e327). Type A (infantile) Niemann-Pick disease accounts for about 80% of cases, and predominantly affects
children of Jewish descent. NiemannPick disease type A presents as a severe neurodegenerative disease in
infancy. Splenomegaly develops as macrophages containing sphingomyelin accumulate in the spleen and other
organs. Niemann-Pick disease type B is panethnic and is characterized by hepatosplenomegaly,
thrombocytopenia, interstitial lung disease, and dyslipidemia. Splenomegaly is the most common presenting
manifestation. The spleen becomes significantly enlarged. Splenic volume directly correlates with liver volume
and the patient's triglyceride level and inversely correlates with the patients HDL level, their height, hemoglobin
concentration, and white cell count. Niemann-Pick cells have finely vacuolated cytoplasm that is yellow-green in
hematoxylin and eosin-stained sections and blue-green in Wright-Giemsa-stained smears. The cells are usually
positive for period acid-Schiff and lipid stains, such as Sudan black B (performed on air-dried smears or frozen
tissue), but negative for iron stains (see Chapter 5).
The GM1 and GM2 gangliosidoses are associated with splenomegaly (e65), although the most marked effects
are seen in the central nervous system (e193,e220). The GM2 gangliosidoses represent deficiencies in one of
the three enzymes needed to catabolize GM2 gangliosides. The most common of these is Tay-Sachs disease,
which is caused by deficiency of hexosaminidase A. The disease is seen relatively frequently in European Jews
and begins in infancy; death by age 3 results from progressive dementia and motor deterioration.
Microscopically, splenic macrophages are finely vacuolated and stain positively for lipids with oil red O and
Sudan black B stains.
Hepatosplenomegaly is a common feature of the mucopolysaccharidoses, particularly Hurler syndrome (MPS I
H) and Hunter syndrome (MPS II). The cells most affected in these disorders are macrophages and endothelial
and intimal smooth muscle cells, which are distended by transparent vacuoles filled with mucopolysaccharide.
These vacuoles stain positively with periodic acid-Schiff and are negative for lipids.
Chediak-Higashi Syndrome
Chediak-Higashi syndrome results in massive hepatosplenomegaly. Chediak-Higashi syndrome is an autosomal
recessive disease characterized by oculocutaneous albinisms, bleeding abnormalities, bacterial infections, and
neurologic systems. The disease results from the formation of abnormal cells containing giant, abnormal
granules due to mutation in the LYST gene. As the disease accelerates, these abnormal lymphohistiocytes are
deposited in the liver, spleen, lymph nodes, and bone marrow. The disease is treatable by bone marrow
transplant (96).
Langerhans Cell Histiocytosis

Splenic involvement is common in multifocal-multisystemic Langerhans cell histiocytosis. Typically, the spleen is
mildly enlarged, and the red pulp is infiltrated by histiocytes with bland cytologic features. The white pulp is
spared. Although they are common in other involved organs, multinucleated histiocytes and eosinophils are
infrequently seen in the spleen.
Virus-Associated Hemophagocytic Syndrome

Virus (infection)-associated hemophagocytic syndrome (VAHS) is often associated with splenomegaly, and
occasionally splenectomy is warranted to relieve the pancytopenia or reduce the risk for rupture. Microscopically,
spleens involved by VAHS show numerous erythrophagocytic histiocytes in the red pulp, so that sometimes the
possibility of malignant histiocytosis, which is extraordinarily rare, is raised. The histiocytes, however, are
cytologically bland and do not form grossly visible tumors.
Leukemia and Myeloproliferative Disorders

The spleen is frequently involved in many reactive and malignant hematopoietic disorders. The spleen is a
common site of extramedullary hematopoiesis (102). Extramedullary hematopoiesis is seen in benign reactive
conditions and hematologic and nonhematologic malignancies. It is important to distinguish the neoplastic
disorders involving the spleen from those reactive conditions discussed above. Acute lymphocytic leukemia and
acute myelogenous leukemia may result in splenomegaly. However, splenomegaly is most marked in the
myeloproliferative disorders, including chronic myelogenous leukemia and juvenile myelomonocytic leukemia
(e135,e196). O'Malley et al. (102) recently evaluated 80 splenectomy specimens to define the involvement of
benign and neoplastic disorders. The patients ranged from 2 to 84 years of age. Of the 80 splenectomy samples,
43 were involved with a neoplastic myeloid disorder and 37 with reactive extramedullary hematopoiesis. The age
of the patients in the neoplastic group was 4 to 81 years and the age of the reactive group ranged from 2 to 84
years. Only seven patients were within the pediatric age. Of the neoplastic myeloid disorders identified, 67%
were chronic myeloproliferative disorders including CML, chronic idiopathic myelofibrosis, and essential
thrombocythemia. Another 7% of patients had involvement by myelodysplasia or other myeloproliferative disorder
including one patient with juvenile myelomonocytic leukemia and two patients with chronic
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myelomonocytic leukemia. In addition, there were two other patients with myelodysplasia, nine patients with AML,
and two patients with mastocytosis.
FIGURE 22-27 ▪ Immature myeloid and megaloblastic erythroid progenitors effacing red pulp in spleen from a 2-
year-old boy with juvenile myelomonocytic leukemia. Rapid splenic enlargement prompted splenectomy.
(Hematoxylin and eosin stain, original magnification 20×.)
Although chronic myeloproliferative disorders such as idiopathic myelofibrosis are rare in children, there are
several reports in the literature. One report describes three children, two of whom were siblings that presented at
the ages of 9, 10, and 16 months of age with symptoms similar to those of idiopathic myelofibrosis (e181). All
three patients progressed rapidly with a fatal course. The occurrence in siblings suggested that this may have a
genetic cause.
Splenectomy is performed occasionally to reduce disease burden and pain, particularly in patients with chronic
myelogenous leukemia. Microscopically, spleens involved with chronic myelogenous leukemia or juvenile
myelomonocytic leukemia appear similar, with sheets of immature and maturing myeloid cells particularly
concentrated around the central arteries (Figure 22-27). Erythroid elements may also be present, but
megakaryocytes tend to be rare in chronic myelogenous leukemia (e135,e290).
Vascular Tumors
Vascular neoplasms are the most common nonhematopoietic proliferations to involve the spleen. Vascular
neoplasms are usually easily distinguished from histocytic proliferations, inflammatory myofibroblastic tumors,
and hematomas. Vascular tumors that involve the spleen include hemangiomas, lymphangiomas, littoral cell
angiomas, hemangioendotheliomas, angiosarcomas, and myoid angioendotheliomas.
Lymphangiomas and hemangiomas are closely related benign vascular tumors that may involve the spleen,
either as a solitary mass or as part of a disseminated disease (e16,e45). Splenic hemangiomas are usually
solitary and less than 2 cm in diameter. Although most are asymptomatic, larger hemangiomas are prone to
rupture or may result in a consumptive coagulopathy or thrombocytopenia (e285). Microscopically, splenic
hemangiomas are composed of masses of dilated, endothelium-lined spaces filled with erythrocytes. By
convention, tumors consisting of vessels filled with hypocellular proteinaceous fluid are considered
lymphangiomas. In some cases, it is not possible to distinguish a hemangioma from a lymphangioma definitively.
Peliosis of the spleen is usually associated with peliosis of the liver (e22,e163). In contrast to the dilated spaces
of hemangiomas, the dilated blood-filled spaces in peliosis lack an endothelial lining and are diffusely dispersed
throughout the spleen.
Littoral cell angiomas are benign vascular tumors composed of specialized tall endothelial cells that express both
endothelial and histiocytic markers. The sinusoidal spaces of littoral cell angiomas are lined by tufts and papillary
arrays of littoral cells that project into the lumen. The youngest reported patient was a 3-year-old boy. Other
vascular tumors that involve the spleen include epithelioid hemangioendothelioma (e33,e151) and epithelioid and
spindle cell hemangioendothelioma (e297).
Myoid angioendothelioma is a distinct vascular entity with features that differ from the other vascular neoplasms
(61). This is a benign tumor as originally described as a composite tumor with areas of vascular stasis intermixed
with stromal cells with myoid features. In the original description of three patients by Kraus and Dehner, two of
the patients were children, 3 and 7 years of age (70). The one remaining patient was a 43-year-old patient. A
recent publication describes a 51-year-old man with similar morphology. The reported lesions vary in size but
were otherwise histologically quite similar and all were well circumscribed. There were scattered rounded or
tubular spaces lined by cytologically bland cells throughout that documented the vascular nature of these
lesions. The predominant cell was a large, polygonal-shaped epithelioid cell with abundant eosinophilic
cytoplasm and indistinct cell borders. Nuclear configuration ranged from rounded to elongated or twisted and
hyperchromatic, and eosinophilic nucleoli were present and occasionally prominent. In many cases, the
fibroblastic-rich stroma was interspersed with chronic inflammatory cells.
Angiosarcomas of the spleen are extraordinarily rare in children and do not show the association with exposure
to vinyl chloride, arsenic, or Thorotrast established for angiosarcomas involving the liver (e84,e245). Distinction
from benign vascular tumors rests primarily on the presence of cytologic atypia and mitoses among the
endothelial cells, in addition to an infiltrative pattern of growth. The prognosis is poor (29).
Other Nonhematopoietic Tumors

A variety of mesenchymal tumors or proliferations, including Kaposi sarcoma (e211,e273), mycobacterial spindle
cell tumors (e298), and smooth muscle tumors (e74), tend to develop in patients infected with HIV.
Inflammatory myofibroblastic tumor, also known as inflammatory pseudotumor, is most common in the lungs or
mesentery but also occasionally involves the spleen (e199,e305).
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Inflammatory pseudotumors are well-circumscribed gray masses composed microscopically of spindle cells that
on immunophenotyping are of myofibroblastic origin (positive for smooth muscle and muscle-specific actin,
positive for cytokeratin) (e53). Although inflammatory pseudotumors are usually clinically benign, in rare cases,
they may undergo malignant transformation (18). Metastatic tumors to the spleen in children are uncommon. We
have seen splenic involvement by neuroblastoma or embryonal carcinoma. Direct invasion of the spleen is
occasionally seen in neuroblastoma, hepatoblastoma, or Wilms tumor.
Follicular Hyperplasia
Splenic follicular hyperplasia is common in children and especially prominent in autoimmune diseases, including
rheumatoid arthritis (e35), systemic lupus erythematosus (e333), autoimmune hemolytic anemia or idiopathic
thrombocytopenic purpura (e28,e302), and HIV infection.
Localized Lymphoid Hyperplasia

In localized lymphoid hyperplasia, proliferations of lymphocytes form solitary nodules that are suspicious for
lymphoma (e33). Two forms were originally described by Burke and Osborne (e35). In the first, the nodules are
composed of aggregates of reactive germinal centers. In the other form, the aggregates are composed of small
lymphocytes, immunoblasts, and plasma cells. The principal features distinguishing lymphoid hyperplasia from
non-Hodgkin lymphoma are the polymorphic nature of the infiltrate and the absence of cytologic atypia. In
addition, the nodules of localized lymphoid hyperplasia are usually less than 1 cm in diameter and rarely are
multiple (e33).
Non-Hodgkin Lymphoma
Splenic involvement by non-Hodgkin lymphoma, although common in adults, is rare in children. Follicular
lymphoma is so rare in childhood that the diagnosis should not be made without incontrovertible evidence of
clonality. Small noncleaved cell lymphoma involves the spleen early by colonizing germinal centers, and later by
diffusely effacing both red and white pulp. Lymphoblastic lymphoma rarely involves the spleen, usually as a
diffuse infiltrate. Large cell lymphomas, usually of B-cell origin, involve the spleen as multiple macroscopic
nodules (e86). Rare examples of hepatosplenic g/d T-cell lymphoma have been reported (e105,69).
Hodgkin Lymphoma
Splenectomies are seldom performed to stage Hodgkin lymphoma, in part because of the widespread use of
chemotherapy and the risk for postsplenectomy septicemia (e251,e263,e274). When the spleen is processed
(see section on “Examination of the Spleen”), foci of involvement should be sought carefully. These appear as
fibrotic, usually wellcircumscribed, gray-tan masses (Figure 22-28). The number of foci should be noted because
the presence of five or more can denote a worse prognosis (e69,e89). Each nodule should be examined
microscopically (a total of six to eight sections) to confirm that it represents Hodgkin lymphoma. The diagnostic
criteria for involvement in patients with known Hodgkin lymphoma are the same as in other sites: the presence of
diagnostic Reed-Sternberg cells or mononuclear variants in the appropriate cellular background. Early splenic
involvement by Hodgkin lymphoma can be subtle, with small numbers of mononuclear variants confined to the
periarteriolar lymphatic sheath or germinal center marginal zones. In more advanced involvement, the areas of
white pulp become focally confluent and eventually extend into the red pulp, often with associated fibrosis or
necrosis. Epithelioid granulomas are seen in about 9% of spleens from patients with Hodgkin lymphoma and do
not by themselves signify splenic involvement or a worse prognosis (e148,e269).
FIGURE 22-28 ▪ Focal splenic involvement by Hodgkin lymphoma. The patient was a 9-year-old boy who
underwent splenectomy for immune thrombocytopenic purpura. The spleen contains a circumscribed, whitish
nodule, 2.1 cm in greatest dimension.
Primary Immunodeficiencies
The etiology and classification of the primary immunodeficiencies are discussed in detail in the section on the
“Lymph Nodes.” In severe combined immunodeficiency, the spleen is small, with a marked decrease in the
number of lymphocytes in both the T- and the B-cell zones of the white pulp (e136,e198). In infantile-linked
agammaglobulinemia, the B-cell zones containing B-lymphocytes and plasma cells are nearly absent, and the T-
cell zones appear normal (e54,e169). In the spleens of patients with Wiskott-Aldrich syndrome, the white pulp is
depleted, with decreased numbers of both T and B lymphocytes, and the thickness of the marginal zone is
markedly reduced. These features have been proposed to be the cause of the defective response to T-cell-
independent antigens in such patients (137).
THYMUS
The thymus is a lymphoepithelial organ located in the anterior mediastinum. The important role of the thymus in
immune regulation was not appreciated until the 1960s,
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when thymus-dependent cell-mediated immunity was first described (19,e115,e116,e117). In 1965, DiGeorge
(e64) described a syndrome in which congenital absence of the thymus is associated with severely impaired cell-
mediated immunity. The very important role that the thymus plays in normal T-cell development and immunologic
responses has been extensively explored in the subsequent decades.
Embryology
The thymus originates as paired epithelial anlage derived from coordinated outpouchings of the lateral
pharyngeal pouches at invaginations of the external pharyngeal clefts (e317). The superior aspect of the third
pharyngeal pouch forms the inferior lobes of the parathyroid glands. The inferior aspects of the third pharyngeal
pouch and sometimes part of the fourth pharyngeal pouch develop into the thymus. Because the parathyroid
glands and the thymus are derived from the same pharyngeal pouch, it is not unusual for the lower lobes of the
parathyroid gland to be enmeshed within the thymus. Although they originate in the cervical region, the tubules of
the thymic primordium elongate and descend into the superior mediastinum. The thymus passes behind the
thyroid gland and the sternocleidomastoid muscle. Not uncommonly, remnants of the thymus remain in the neck
and can develop into cysts or thymic tumors (e112,e212). In humans, circulating lymphoid stem cells arising from
either the yolk sac or the liver subsequently populate the thymus by the ninth week of gestation. After this point,
thymocyte differentiation begins. The organization of the thymus into the cortex and medulla begins around the
twelfth gestational week.
Anatomy and Histology

Histologically, the thymus consists of two lobes that form a V-shaped structure extending from the thyroid gland
into the anterosuperior mediastinum. The thymic lobes are joined in the midline by connective tissue at a site
over the pericardium. The thymus weighs 10 to 35 g at birth and continues to grow to a maximum weight of 20 to
50 g at puberty (e295,e299). Normal thymic involution progresses from puberty through old age. In older adults
and the elderly, a thymic weight of 5 to 15 g is finally achieved. Thymic growth and involution vary tremendously
between persons, so that the thymic weight at any given age is variable.
Fibrous extensions of the thymic capsule divide the thymus lobes into numerous lobules; however, the lobules
are not completely separated because the medullary lymphoid region is continuous throughout the thymus
(e299). Each thymic lobule is separated into cortical and medullary regions, and both cortex and medulla are
composed of lymphocytes and epithelial cells. In addition to the subspecialized thymic epithelial cells and T-
lymphocytes, mononuclear phagocytes, myoid cells, mast cells, eosinophils, plasma cells, and a small population
of mature B-cells are present within the normal thymus.
The border between the thymic cortex and medulla is not clearly delineated (e299). The lymphocytes in the
cortex are much more closely packed than those in the medulla, so that a darker appearance of the cortex and a
lighter, spacious appearance of the medulla are characteristic. As thymic maturation proceeds, the lymphocytes
move from the outer cortex toward the medulla, where the thymocytes then enter the peripheral circulation
(e202). The cortical thymocytes express markers of immature T-cell differentiation, whereas the medullary
thymocytes express antigens representing mature, peripheral T-cells (e112,e202). Epithelial cells are located
throughout the cortex and the medulla. The epithelial cells provide much of the stroma and framework for
lymphocyte maturation. A number of distinct subtypes of thymic epithelial cells have been described. Some
investigators have separated epithelial cells by morphology, with some cells characterized by electron-dense
nucleoli and round or elongated spindle-shaped cytoplasmic processes. Others categorize the epithelial cells
according to whether or not they express MHC determinants. Although many discrepancies are found in the
literature, the epithelial cell component is essential for normal thymic development (e202). Hassall corpuscles are
concentric whorls of keratinizing epithelial cells that have undergone cystic degeneration. Their location in the
thymic medulla in addition to their function and significance are still unexplained.
Thymic Atrophy
Thymic atrophy occurs as part of normal aging and also in association with severe stress, malnutrition, and drug
use (e72) (Table 22-12). Complete thymic agenesis is associated with primary immunodeficiency in a number of
congenital defects. Similar defects in the thymus that result in immunodeficiency are seen in patients with AIDS.
Accelerated thymic involution was reported in newborns born to mothers who smoked during the prenatal period.
The thymic index and the thymic index to weight ratio of newborns from mothers who smoked greater than 1
cigarette per day were significantly lower (142).
Table 22-12 ▪ THYMIC LESIONS
Lesion Etiologic Factor
Atrophy (common finding) Stress
Cortical lymphocyte Endogenous or exogenous
Necrosis Corticosteroids
Depletion→atrophy Cyclophosphamide
Lymphoid proliferation Antigenic stimulation, phytohemagglutinin, thyroxine, hormonal agents

(rare)
Germinal center induction As in myasthenia gravis, immunostimulants, antigenic response,

hormonal
Epithelial proliferation Diethylstilbestrol (mice)
Epithelial cysts Estrogens (rats)
Adapted from Gopinath C. Pathology of toxic effects on the immune system. Inflamm Res 1996;45(Suppl
2):74-78, with permission.
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Table 22-13 ▪ IMMUNODEFICIENCY DISEASES ASSOCIATED WITH THYMIC ABNORMALITIES
T-cell Areas (Lymphocytes in Paracortex or Germinal Plasma

Germinal Plasma Periarteriolar Sheath) Centers Cells
DiGeorge syndrome A NL NL
Severe combined ↓ ↓ ↓
immunodeficiency
Autosomal A A A
recessive
X-linked ↓-A ↓-A ↓-A

Thymic ↓ ↓ ↓
hypoplasia
Ataxia ↓-A NL-↓ NL-↓

telangiectasia
During normal aging, although the overall size of the thymus does not change dramatically, the cellular
composition is markedly altered. The major change in the human thymus during aging is a replacement of the
lymphoid cellular elements with adipocytes (e295,e299). Complete lymphoid atrophy is the most evident age-
related change and represents the final state of involution. In the newborn infant, little adipose tissue surrounds
the capsule or the septa. However, during aging, the perivascular spaces and the area surrounding the capsule
and fibrous septa are gradually replaced by adipose tissue. At the same time, the number of lymphocytes
progressively decreases, as described by Steinman (e295). The number of lymphocytes gradually diminishes
until in the older person few remain. At this point, the cortical-medullary junction is poorly discriminated. A few
TdT+ lymphocytes can be identified in elderly persons.
Acquired hypoplasia results from rapid involution in young patients during severe stress, malnutrition, or
irradiation, or following the administration of cytotoxic agents (e277). The morphologic changes seen in the
thymus are similar following a number of stressors. Experimental studies in animals have demonstrated that
corticosteroid injection or the administration of radiation results in acute thymic involution immediately following
the insult (e277). The lymphocytes undergo rapid karyorrhexis, and the cortex is infiltrated with macrophages
and presents a “starry sky” appearance. The cellular destruction observed in experimental conditions also
occurs in children after a number of acute insults that cause thymic atrophy, including malnutrition and the
administration of corticotropin or radiation. Surprisingly, the Hassall corpuscles usually remain and become
multicystic large structures within the medulla.
Thymic hypoplasia is associated with primary or secondary immunodeficiency. Primary immune deficiency
disorders are a group of complex diseases characterized by abnormalities in the development and maturation of
the immune system. Significant advances in understanding the defects underlying many subtypes of primary
immunodeficiency have identified specific blocks in the normal schema of lymphoid maturation have been
identified (e260). Each block in lymphoid maturation results in a distinct immunodeficiency state. The advances
in the field have recently been reviewed by Rosen et al. (e260,e261) and Perez-Atayde and Rosen (e233). The
Rosen classification provides the diagnostic pathologist with a readily usable system for evaluating the spleen,
lymph nodes, and thymus of a child with a suspected or confirmed immunodeficiency state
(e166,e233,e260,e261). The outline encompasses immunologic defects resulting from combined
immunodeficiencies (i.e., deficiencies of both B-cells and T-cells), primary antibody (B-cell) deficiency, or primary
T-cell deficiency. Disorders resulting in a primary T-cell deficiency are caused either by a defect in primary
thymic maturation or by secondary thymic abnormalities associated with altered T-cell development. The primary
immunodeficiencies that affect thymocyte maturation include DiGeorge syndrome, reticular dysgenesis,
combined immunodeficiency disease, and ataxia-telangiectasia (Table 22-13).
DiGeorge anomaly was first identified as thymic agenesis associated with abnormalities of T-cell maturation
(e64). DiGeorge syndrome results from a failure of the normal development of the third and fourth branchial
arches, which results in abnormalities in multiple organs during the fourth to sixth weeks of embryogenesis. The
major defects include aplasia or hypoplasia of the thymus and parathyroid glands, type I truncus arteriosis, and
dysmorphic facies with micrognathia. Other associated conditions include esophageal atresia, thyroid
aplasia/hypoplasia, absence of calcitonin-containing cells of the thyroid, and endocardial cushion defects.
Although a few familial cases have been reported, the defect appears not to be hereditary but to result from an
unknown defect occurring in utero during the first trimester of pregnancy.
In cases of “complete” DiGeorge syndrome, both the thymus and the parathyroid glands are completely absent.
Most patients manifest a “partial” or “incomplete” DiGeorge syndrome, in which the thymus is hypoplastic and
otherwise histologically normal. The degree of thymocyte hypoplasia is variable, but in most instances, thymic
lobation is normal, corticomedullary differentiation is detected, and Hassall corpuscles are present. Although not
all the genetic defects are defined, many patients have either partial monosomy or a deletion of chromosome
10q11 (e59,e62,e261,e276,e332). Recent progress has been made in treating the athymia by thymus
transplantation (87). Markert et al. (87, 88) report that transplantation of cultured postnatal thymus successfully
restored many of the immune abnormalities in patients with complete DiGeorge syndrome.
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Severe combined immunodeficiency diseases are represented by several distinctive disorders with similar
clinical manifestations and distinct genetic bases. These include the lymphoid stem cell type (Swiss type) with
autosomal recessive or X-linked modes of inheritance. Infants with severe combined immunodeficiency disease
usually present by 3 months of age with thrush, monilial rashes, intractable diarrhea, and Pneumocystis jiroveci
pneumonia. In some neonates, the symptoms are similar to those of graft-versus-host disease. Death results
from overwhelming infection with herpesvirus, adenovirus, and cytomegalovirus. Hecht giant cell pneumonia,
resulting from measles infection or live measles or smallpox vaccination, is lethal to the immunocompromised
host. Laboratory evaluation of infants with severe combined immunodeficiency disease reveals a marked
lymphopenia (<1,000 lymphocytes per cubic millimeter). In the X-linked form, the number of B-cells is normal, but
the B-cells fail to mature properly. T-cells are rare and of maternal origin. One genetic defect responsible for X-
linked severe combined immunodeficiency disease is a mutation of the gene coding for the g chain of interleukin
(IL) receptor, mapped to Xq13. The g chain is a component of several IL receptors, including IL-4, IL-7, IL-11, and
IL-16. Normal lymphocyte progenitors fail to differentiate because of a lack of appropriate growth factor
stimulation. Other types of severe combined deficiency are recessive in inheritance. The most common enzyme
defects that result in immunodeficiency are of enzymes in the purine degradation pathway. The accumulation of
toxic metabolites in adenosine deaminase deficiency and purine nucleoside phosphorylase deficiency results in
lymphocyte defects (e108,e136,e138,e139,e184,e198). The symptomatology is essentially identical to that in
children with AIDS.
The difference in the lymphoid tissue among the various types of severe combined immunodeficiency disease is
minimal. The lymphocytes are generally depleted in all lymphoid tissues, including the thymus, spleen, lymph
nodes, tonsils, adenoids, and mucosa-associated lymphoid tissue. The thymus is small and dysplastic. A variable
number of T-cells at the corticomedullary junction and scattered Hassall corpuscles are found early in most
cases. Because of progressive lymphoid depletion, the thymic epithelium becomes prominent and may appear
disorganized or acquire an organoid and pseudoglandular architecture (e233). The morphology of the thymus in
other well-characterized immunodeficiencies, including ataxia-telangiectasia, Wiscott-Aldrich syndrome, and
chronic mucocutaneous candidiasis, is variable. The thymus histology can be normal or show slight lymphocytic
depletion or complete atrophy.
The Thymus in AIDS

Changes in the thymus in patients with AIDS have been controversial. In a report of 11 infants with AIDS, Joshi
(e146) described histologic changes similar to those in patients subjected to severe stress. The thymus was
located in the correct anatomic site and the lobation and blood vessels were normal, but the size, weight, and
number of lymphocytes were reduced. In some cases, more severe abnormalities were noted, including complete
involution or inflammatory changes. Animal studies suggest that transmission of the virus early in fetal
development results in more severe immune destruction. A recent study demonstrated that HIV infection results
in a high rate of spontaneous abortions, and that the thymus in spontaneously aborted fetuses demonstrates
severe abnormalities of lymphocytic differentiation and corticomedullary demarcation and an absence of Hassall
corpuscles (e146). The more severe thymic abnormalities develop earlier in gestation.
Thymic Tumors
The majority of tumors that occur in the mediastinum of children are lymphomas (41%) or tumors of neurogenic
origin. True thymic lesions including cysts, thymolipomas, thymic hyperplasia, and thymic tumors represent
approximately 2.5% of all mediastinal masses in children (13). Hyperplasia of the thymus is the most common
anterior mediastinal mass found in infants. Histologically, two types of thymic hyperplasia are recognized. True
thymic hyperplasia is characterized by increases in both the size and the depth of the gland with retention of the
normal microscopic appearance. In the second type, lymphoid hyperplasia, reactive lymphoid follicles appear
within the thymus (e293). The reactive germinal centers are identical to those seen in normal lymph nodes.
Follicular hyperplasia of the thymus can occur de novo or in association with autoimmune diseases and chronic
inflammatory states, most commonly myasthenia gravis. Approximately 70% to 80% of patients with myasthenia
gravis have follicular hyperplasia of the thymus. Although myasthenia is usually a disease of older persons,
Somnier (e293) identified a bimodal male and female age distribution. The incidence of early-onset myasthenia
gravis peaked at 21 to 30 years, but persons as young as 5 to 10 years of age were affected. The peak for early-
onset disease was approximately 10 years later in males than in females. Other autoimmune diseases, including
Graves disease, Addison disease, systemic lupus erythematosus, scleroderma, and rheumatoid arthritis, are
associated with thymic hyperplasia.
True thymic hypertrophy, enlargement of the thymus, has been reported in neonates and children up to 14 years
of age. In most cases, an enlarged thymus is an incidental finding. In other cases, the mediastinal enlargement
causes respiratory or gastrointestinal symptoms (e293). In some cases, the hypertrophy represents regeneration
following stress. The thymus in cases of hypertrophy is normal, with a normal corticalmedullary junction and
Hassall corpuscles. The diagnosis is based on the weight of the thymus at resection. Because the thymic weight
varies widely, the thymus must weigh more than approximately 100 g to be considered hypertrophic.
Neoplastic Proliferation of the Thymus

Thymic tumors account for only 1.5% of all mediastinal masses in children and include thymomas, thymic
carcinomas,
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and thymic carcinoids (13). Lymphomas are neoplastic proliferations of the lymphoid cells within the thymus and
will be discussed separately. Thymomas are neoplastic proliferations of the thymic epithelium. Although
thymomas are the most common primary neoplasms of the anterior mediastinum, they are the least frequent
mediastinal tumors in children. Fewer than 2% of all thymomas are diagnosed in the first two decades of life.
Small series of childhood cases occurring between 9 months and 15 years of life have been reported in the
literature (13, 63, 103, 113, 115). Myasthenia gravis and other autoimmune disorders occur in 30% of adults with
thymoma but are less frequent in children.
Morphologically, although thymomas are composed of neoplastic epithelial cells and lymphocytes, there is great
morphologic heterogeneity. The role of histology in prognosis has been hotly debated. The WHO in 1999
defined the histologic criteria for distinct subtypes of thymic epithelial tumors (13, 63, 103, 113, 115). Thymic
neoplasms are now subdivided into five entities: Type A, AB, B1, B2, and B3 thymomas. The classification is still
based on the extent of lymphocytic infiltration. It is important to recognize the possibility of a thymoma because
the lymphocytes express the antigens of immature thymocytes. These normal thymocytes can easily be confused
with the malignant lymphoblasts of lymphoblastic lymphoma. A variety of distinct cellular features may be seen
within the typical thymoma, including thymic cysts that may form papillary structures, germinal centers, squamous
differentiation, and keratin pearls (52). As would be expected, the typical epithelial component of a thymoma
expresses epithelium-associated antigens, including cytokeratin and EMA. The lymphocytes in a typical thymoma
express the markers of normal cortical thymocytes, medullary thymocytes, or mixtures. Immature thymocytes
express CD1, CD2, CD5, and CD7, coexpress CD4 and CD8, and express TdT.
Type C thymoma is considered thymic carcinoma. Thymic carcinomas represent 5% to 15% of all thymic
neoplasms. In contrast to benign thymoma, thymic carcinoma shows cytologically malignant epithelial cells with
nuclear prominence, increased mitotic activity, and areas of necrosis. Thymic carcinomas can present with
squamous cell, basaloid, adenosquamous, small cell, clear cell, sarcomatoid, and anaplastic large cell features.
Significant numbers of immature intraepithelial thymocytes are lacking. Type C thymoma is usually
indistinguishable from a carcinoma observed elsewhere. These malignant lesions are more likely to invade and
metastasize.
Malignant thymoma is very rare in children. In a current literature review by Yaris et al. only 14 cases of thymic
carcinoma in patients younger than 18 years old were reported. In this series of children, the median age was 13
years and there was a male predominance. Although myasthenia gravis is frequently associated with benign
thymoma, myasthenia gravis and other paraneoplastic disorders are rarely associated with thymic carcinomas
(62, 141). Dehner et al. reported that the mortality for children with thymoma is much higher than that for adults
(e62). In this series, only 3 of 11 children survived for 6 months after diagnosis.
A number of the diseases associated with thymomas are similar to those associated with thymic hyperplasia, and
they resolve following removal of the mass. Both thymic hyperplasia and thymomas are associated with
numerous autoimmune diseases: myasthenia gravis, hypogammaglobulinemia, polymyositis, systemic lupus
erythematosus, Hashimoto thyroiditis, and a variety of cytopenias, including pure red cell aplasia (e155,e293). A
recent study reports the case of a 7-year-old child with a thymoma. The child presented with facial muscle
weakness without ophthalmoplegia or ptosis. The patient had a benign thymoma. Following thymic resection, the
patient became asymptomatic (23). Of the patients reported by Dehner et al. three presented with signs of
superior vena cava syndrome (e62). Thymomas outside the thorax are seen in children when metastasis to the
lungs, bone, liver, and lymph nodes has occurred.
Malignant Lymphomas
Hodgkin and non-Hodgkin lymphomas account for approximately one third of all childhood cancers, and
lymphomas are the third most common group of cancers in children. Non-Hodgkin lymphomas represent 60% of
the lymphomas of childhood, and Hodgkin lymphoma represents 40%. The classification of non-Hodgkin
lymphomas and Hodgkin lymphoma has been discussed earlier in the section. Of all the tumors that occur in the
mediastinum, malignant lymphoma, both Hodgkin and non-Hodgkin lymphoma, represent the second most
common malignancy.
The classification system of non-Hodgkin lymphomas was developed primarily for adults. Only a relative few
subtypes of non-Hodgkin lymphomas occur in childhood. In contrast to adult cases non-Hodgkin lymphoma,
approximately 50% of childhood cases of non-Hodgkin lymphoma are of T-cell origin (e170). Low-grade and
intermediate-grade lymphomas are rare in children. The high-grade lymphomas, small noncleaved cell lymphoma
(Burkitt), and lymphoblastic lymphoma account for 70% to 80% of all non-Hodgkin lymphoma in children.
Extranodal presentation of non-Hodgkin lymphoma in sites such as the mediastinum, gastrointestinal tract, and
head and neck is much more common in children than in adults. Small noncleaved cell lymphoma in North
America commonly presents in the abdomen. Lymphoblastic lymphoma most commonly arises within thymic
remnants and presents with mediastinal involvement. Patients with mediastinal lymphomas usually present with
signs of mediastinal compression—cough, chest pain, dysphagia, dyspnea, and superior vena cava syndrome.
Hodgkin Lymphoma
Hodgkin lymphoma involves the mediastinum in approximately 50% to 70% of patients younger than 40 years.
Patients typically present with peripheral lymphadenopathy, mainly in the cervical region, and enlargement of the
mediastinal lymph nodes. The lymphadenopathy is associated
P.1004
with systemic symptoms, including fever, night sweats, and weight loss, in 25% to 30% of patients. Extreme
mediastinal involvement, which is defined as enlargement of the mediastinum to more than one third of the
diameter of the chest, is an adverse prognostic factor.
FIGURE 22-29 ▪ Lymphoblastic lymphoma results in massive enlargement of the thymus. A, B: Lymphoblastic
lymphoma effaces the normal thymic architecture with sheets of uniform malignant lymphoid small-to-medium
sized lymphocytes with very scant cytoplasm, irregular, convoluted, and inconspicuous nuclei. (Hematoxylin and
eosin stain, original magnification 40×.)
The classification scheme and the pathology of Hodgkin lymphoma have been discussed in detail in the section
on “Lymph Nodes.” In the United States, most cases of Hodgkin lymphoma in children are of the nodular
sclerosis or mixedcellularity types. The morphology of the mediastinum is that typical of nodular sclerosis
Hodgkin lymphoma, in which the lymph nodes and the thymus are replaced by dense collagen bands that divide
the tumor into discrete nodules. The typical cellular milieu of lymphocytes, plasma cells, eosinophils, classic or
diagnostic Hodgkin cells, and Reed-Sternberg cells are identified. In many patients with Hodgkin lymphoma, it is
unnecessary to obtain a biopsy specimen from the mediastinum because the disease spreads to contiguous
lymph nodes. Often, a node can be identified in the cervical or the supraclavicular area that is easier to sample.
If mediastinal specimens are obtained, the diagnosis may be difficult. In many cases, dense fibrous and
collagenous bands are infiltrated by lymphocytes. The identification of the cellular component of Hodgkin
lymphoma may be difficult, and Reed-Sternberg cells must be identified for a diagnosis to be made. In some
cases, immunohistochemistry may aid in the identification of the diagnostic Reed-Sternberg cells. In nodular
sclerosis Hodgkin lymphoma, the Hodgkin cells typically react with antibodies against CD15 and CD30, and they
fail to express CD45, the leukocyte common antigen, or CD20 or CD3, markers of B cells and T cells,
respectively.
Lymphoblastic Lymphoma
The other primary malignant lymphoma involving the mediastinum is lymphoblastic lymphoma. Lymphoblastic
lymphoma presents as a distinct clinicopathologic disorder and accounts for approximately 30% of non-Hodgkin
lymphomas in childhood (e272). Among all the non-Hodgkin lymphomas of childhood, the mediastinum is
involved in 26% of cases (Figure 22-29). The vast majority of these are lymphoblastic lymphomas. The
classification schemes for non-Hodgkin lymphoma have undergone multiple revisions during the last several
decades. The Revised European-American Classification of Lymphoma has generated a significant controversy
in addition to identifying a number of new subtypes of lymphomas (e61,e131,e262). Although the revision has
dramatically affected the subclassifications of non-Hodgkin lymphomas in adults, its effect on the diagnosis of
lymphoblastic lymphoma in children and adults has been minimal. What was formerly known as lymphoblastic
lymphoma is now categorized in the Revised European-American Classification as precursor T-lymphoblastic
lymphoma/leukemia (e131). Lymphoblastic lymphoma presents with a typical clinical picture, including a large
mediastinal mass (50% to 70% of cases) that causes symptoms associated with chest compression and cervical
or axillary lymphadenopathy. Although the largest percentage of cases of lymphoblastic lymphoma occur in
children, a bimodal age distribution has been identified, with the first peak at 16 years of age and the second at
more than 40 years of age. A marked male predisposition has been noted, with a male-to-female ratio of 2.5:1.
Histologically, lymphoblastic lymphoma demonstrates diffuse effacement of the lymph nodes or thymus. The cells
of lymphoblastic lymphoma are uniform in size and range from about 10 to 14 μm (about the size of a histiocyte
nucleus). The cells have very scant cytoplasm, so that they often give the impression of having “bare” nuclei.
The nuclei are small and inconspicuous. Mitoses are frequent. Folds or indentations in the nucleus produce
convolutions that, despite the designation of “small convoluted cell lymphoma,” are seen in only 50% of cases in
the larger series. A particular tumor may be composed predominantly of convoluted cells, a mixture of convoluted
and nonconvoluted cells, or exclusively of
P.1005
nonconvoluted cells. Nuclear convolutions are best appreciated in the smaller lymphocytes. Because studies
comparing the convoluted and nonconvoluted subtypes show no clinically significant differences (e206), we do
not attempt to distinguish between these subtypes.
As noted, about 80% to 90% of cases of lymphoblastic lymphoma are of T-cell lineage and commonly express
the pan T-cell markers CD1a, CD2, CD3 (cytoplasmic), CD7, and CD43 (e246). T-cell lymphoblastic lymphomas
most commonly express CD1a and both CD4 and CD8 or neither CD4 nor CD8, corresponding to stage II of
thymocyte maturation. Less commonly, they lack CD1 expression and express either CD4 or CD8, corresponding
to stage III of thymocyte maturation (e24,e256). In contrast, most cases of T-cell acute lymphoblastic leukemia
correspond to stage I of thymocyte maturation (negative for CD4, CD8, and CD1) (e24,e256). Importantly, almost
all cases of lymphoblastic lymphoma express TdT (e29), which can be detected immunohistochemically on air-
dried imprints, frozen tissue, or paraffin-embedded tissue (e270). TdT can also be detected on permeabilized
cells by flow cytometry (e127,e289,e300). Thus, expression of T-cell markers and TdT lends very strong support
to a diagnosis of T-cell lymphoblastic lymphoma in association with blastic histology. One important caveat is
that thymomas, which are rare in children and young adults, often contain lymphocytes that are indistinguishable
on the basis of immunophenotype from those of lymphoblastic lymphoma (e264). The less common B-lineage
lymphoblastic lymphomas typically express TdT, CD19, and CD10 without expression of surface immunoglobulin
and with or without expression of CD20, an immunophenotype similar to that of precursor B-cell acute
lymphoblastic leukemia (e237,e325).
With improved chemotherapy and aggressive management, the long-term survival of patients with lymphoblastic
lymphoma overall is reported to be from 65% to 75% (e308). The biology of lymphoblastic lymphoma overlaps
with that of acute lymphoblastic leukemia of T-cell origin. T-cell acute lymphoblastic leukemia and T-cell
lymphoblastic lymphoma often express the same antigens and present with the same clinical features. Based on
arbitrary criteria, lymphoblastic lymphoma and acute lymphoblastic leukemia are distinguished according to the
percentage of lymphoblasts in the bone marrow (e27,e132). Cases of lymphoblastic lymphoma in which
lymphoblasts comprise more than 25% of the bone marrow are subclassified as acute lymphoblastic leukemia.
Not surprisingly, acute lymphoblastic lymphoma and acute lymphoblastic leukemia demonstrate the same
molecular genetic abnormalities.
Large Cell Lymphoma

Mediastinal large cell lymphoma occurs relatively infrequently in children. Large cell lymphomas of the
mediastinum also display a typical immunophenotype and clinical-morphologic spectrum. The majority of the
patients are young women; the age range is 10 to 63 years, but the median is approximately 30 to 40 years
(e236,e238). Mediastinal large cell lymphomas, which are almost always of B-cell origin, appear to arise within
the thymus (e236,e238). The symptomatology at presentation is similar to that of a lymphoblastic lymphoma, with
evidence of a mass in the anterior mediastinum.
The histopathology of mediastinal large cell lymphoma is the same as that of diffuse large cell lymphoma. Large
cells have moderate-to-abundant amounts of pale to clear cytoplasm and vesicular nuclei with distinct to
prominent nucleoli. Occasionally, immunoblastic or anaplastic morphology is seen. In addition, mediastinal large
cell lymphoma frequently demonstrates compartmentalization, in which nodules of large cells are surrounded by
fibrous septa. Mediastinal large cell lymphomas must be distinguished from seminomas (positive for placental
alkaline phosphatase, negative for leukocyte common antigen), thymic carcinoma (positive for keratin, negative
for leukocyte common antigen), anaplastic large cell lymphoma, and Hodgkin lymphoma, in particular the
syncytial variant of nodular sclerosis Hodgkin lymphoma (positive for Reed-Sternberg cells, Ki-1, and Leu-M1;
negative for leukocyte common antigen; usually negative for other B-cell markers).
Surprisingly, although T-cells are predominantly identified in the thymus, mediastinal large cell lymphoma
typically involves mature B-cells. Large cell lymphomas within the mediastinum are classically positive for the
CD19 and CD20 antigens. Mediastinal large cell lymphoma of T-cell origin is extremely rare.
Anaplastic large cell lymphoma represents another subtype of large cell lymphoma that occurs in children. ALCL
accounts for 10% to 15% of childhood non-Hodgkin lymphomas. The diagnosis is based on proliferation of large
pleomorphic cells of a T-cell phenotype that invade into the lymph node sinuses. As elsewhere, ALCLs usually
exhibit t(2;5) translocation. Primary ALCL involves lymph nodes and extranodal sites including the skin, bone,
soft tissue, lung, and liver. Mediastinal involvement is not very common. In a recent review of 225 children
treated for anaplastic large cell lymphoma mediastinal involvement, as well as B symptoms, skin lesions, visceral
involvement, St. Jude stage 3 to 4, and Ann Arbor stage 3 to 4, elevated lactate dehydrogenase correlated with
the risk of progression and relapse (78). The overall 5-year progression-free survival rate of 81 patients with no
risk factors was 89%. Progression for survival of patients with a risk factor was 61%. Therefore, the identification
of mediastinal involvement in anaplastic large cell lymphoma is important for long-term prognosis.
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Chapter 23
The Bone Marrow
Jochen K.M. Lennerz
Anjum Hassan
Acute lymphocytic leukemia is the most common malignancy in children and classically presents with pancytopenia,
bleeding, and signs of anemia or infection. Characterized by an almost complete loss of hematopoietic elements, this
disease tragically illustrates the fragility of the otherwise harmonically orchestrated “fluid-organ,” the bone marrow.
Ultimately forming approximately 3% to 6% of the total body weight and reconstructing the peripheral blood throughout life,
this organ undergoes a fascinating embryologic development. From midfetal development on and extending throughout life,
the bone marrow is the site of origin of peripheral blood, the macrophages/dendritic cell system, mast cells, lymphocytes,
NK cells, and osteoclasts (52). At this point, we know that the potency of some of the stem cells even extends this
spectrum and that the bone marrow also contributes to solid/epithelial tissues.
DEVELOPMENT
Mesenchymal-derived primitive erythroblasts in the yolk sac are the earliest signs of hematopoiesis in the embryo at a
crown rump length of 95 mm (30). While the presence of lymphoid elements in the yolk sac is controversial, it has been
shown that the aorta [aorto-gonad-mesonephros (AGM)] (185) as well as the placenta contribute in this earliest phase to
the lymphomyeloid stem cell pool (128, 137, 186). The proposed candidates for hematopoietic stem cells (HSCs) in the
AGM express the following markers: CD34+/CD45+ and stem cell receptor c-kit (CD 117) and the transcription factor
GATA-2 (117). The cells arising in the yolk sac show myeloid restriction (184). At weeks 10 to 24, the liver is the primary
hematopoietic organ with production of red cells, granulocytes, and megakaryocytes in the primitive sinusoids. At this time,
the spleen also contributes with approximately 20% to hematopoiesis. Slowly, the production within the bone marrow takes
over, and at 4 to 5 months it will be the primary site of hematopoiesis. Typically by birth, liver and spleen show minimal
myelopoiesis. This switch is often referred to as embryo-to-fetal-to-adult-type hematopoieses (33). The development of
the bone marrow continues in a topographically organized fashion. Hematopoiesis changes from the axial and radial
skeleton (newborns) to the flat bones of the central skeleton by 12 to 16 years. Microscopically, the bone marrow is an
inhomogeneous organ, which is often illustrated by higher cellularity within deeper areas of the medullary cavity than in
subcortical zones. Due to the relatively short lifespan of peripheral blood elements, the production rates within the bone
marrow are astronomic (111). The turnaround time of neutrophils (∽2 hours) requires the production of approximately
700,000 cells per second to maintain the normal value of 5,000/μL; exponentially higher values are needed in neutrophilia
or sepsis, illustrating the dynamics of this system.
With aging, hematopoietic tissue is replaced by fat and key figures for the hematopoietic elements are: approximately 80%
until 9 years, approximately 50% until 70 years, and <30% beyond. Hematopoiesis (Table 23-1), its development and
maintenance, is an exquisitely regulated, dynamic, and highly complicated system of cell production that
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involves molecular control of cell division, differentiation commitment, and maturation carried out via the close interaction of
bone marrow microenvironmental elements with precursor cells (26, 29, 200).
Table 23-1 ▪ GENERAL FEATURES OF THE BONE MARROW AND HEMATOPOIESIS
Microenvironment with regulatory factors for stem/progenitor cells and structural support via stromal framework
and surrounding liquid matrix.
Stem/progenitor cells localize to specific niches based on complementary adhesion molecule expression
between hematopoietic cells, microenvironment and stromal cells.
Stem/progenitor cell proliferation and maturation under exquisite regulatory control; regulated “cross talk”
between stromal cells and hematopoietic cells maintains steady state.
Stimulatory and suppressive factors within microenvironmental matrix; regulatory factors consist of CSFs, ILs,
and inhibitory cytokines.
Stem cellsa are capable of self-renewal and multilineage differentiation.
Committed progenitor cellsa are destined to a specific lineage.
aNot morphologically distinct.
BONE MARROW STRUCTURE

Encased and protected by cortical bone, traversed and supported by trabecular bone, the bone marrow consists of a
highly organized thin-walled capillary network, venous sinuses, and surrounding extracellular matrix. The capillary-venous
sinus, which results from bifurcations of the nutrient or medullary arteries, is the basic structural unit of the bone marrow
(202). Within this histologic compartment, HSC and progenitor cells are exposed to the extracellular matrix that comprises
the bone marrow microenvironment (Figure 23-1). The outer adventitial reticular cells (ARCs) add connective tissue
elements and form the outer sinusoidal wall, synthesize collagen, laminin, fibronectin, and proteoglycans. All regulatory
factors, adhesion molecules, and other proteins necessary for the regulation of hematopoiesis are contained within this
matrix (52, 134). Furthermore, the ARCs are phagocytic and can become lipocytes. As outlined before, the
fat/hematopoietic ratio (“marrow cellularity”) is variable and a rough estimate can be calculated as: cellularity = 100% -age
(see below). Mitotically active cells are normally found around the supporting bone, typically paratrabecular and
perivascular from where cells mature progressively. All newly formed mature hematopoietic cells are released into the bone
marrow capillary-venous sinuses. Most cells pass through the sinus wall, but megakaryocytes reside adjacent to sinuses
and extend pseudopodia directly into the vascular space (146, 177). The capillary-venous sinuses coalesce into venules
and ultimately into veins that carry newly formed hematopoietic cells to the systemic circulation (202).
FIGURE 23-1 ▪ Bone marrow microarchitecture. A: Bone marrow biopsy from a 1-day-old boy showing hematopoietic
tissue that occupies approximately 90% of the marrow space. Only few regions of bone marrow fat are seen. The myeloid
lineage is highlighted in red (Leder stain) and the perivascular region (circle) shows lack of myeloid cells. B:
Theparatrabecular region shows myeloid and erythroid precursors. C: Perivascular distribution of precursors in a bone
marrow biopsy from an 18-year-old girl; note the delicate reticulum and extracellular matrix derived from ARCs. D: Highly
cellular (>90%) bone marrow biopsy in a preterm girl shows numerous capillaries (arrows) interspersed between the
hematopoietic cells and extracellular matrix.
STEM CELLS AND PROGENITOR CELLS

HSC can be defined by their ability to regenerate long-term multilineage hematopoiesis in myeloablated recipients.
Although not morphologically recognizable, stem cells can be detected by either functional features (the simultaneous
capability of sustained self-renewal and multilineage differentiation potential) or immunophenotype (CD34+, Thyr-1+, c-kit+,
CD38−, cytokine receptor and adhesion molecule expression) (52, 123) (Figure 23-2). HSC are estimated to constitute 1 in
104 nucleated marrow cells. In contrast, progenitor cells are progressed stem cells with lineage commitment. The process
of lineage commitment is incompletely understood; however, the resulting committed stem/progenitor cells are also
morphologically unrecognizable but immunophenotypically defined by CD34, c-kit, and CD38 expression (123, 127).
Further, maturation is characterized by the acquisition of cytologic and immunophenotypic properties of the different
morphologically recognizable hematopoietic lineages. Both proliferation and lineage maturation are regulated by the
synergistic stimulatory activities of colony-stimulating factors (CSFs) and interleukins (ILs), whereas antagonistic effects
are driven by inhibitory factors that include tumor necrosis factor (91, 112). It is known that mature hematopoietic elements
play a role in the regulation of lineage production and to maintain steady-state hematopoiesis. Even though molecular
pathways for this homeostasis are fragmentary and complicated, endocrine, paracrine, mesenchymal, and autonomic
nervous system contributions have been implicated (91, 112). The molecular regulation of bone marrow contribution and
feedback regulation during the regeneration in peripheral tissues is at this time uncharted.
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FIGURE 23-2 ▪ Selected aspects of hematopoiesis. See text for details. CLP, committed lymphoid progenitor; CMP,
committed myeloid progenitor (e.g., CFU-S: colony-forming unit—spleen); GEMM, granulo-erythro-megakaryo-monocytic;
GM, granulo-monocytic (= myelomonocytic); HPC, hematopoietic progenitor committed; HSC, hematopoietic stem cell; Im-
B, immature B-lymphocyte; PC, plasma cell; PSC, peripheral stem cell.
HEMATOPOIETIC LINEAGES
Granulopoiesis
The process of granulocytic maturation is characterized by a progressive nuclear segmentation, simultaneous decrease in
the nucleus to cytoplasmic (NIC) ratio, as well as acquisition and increase of primary and later secondary cytoplasmic
granules. The earliest morphologically recognizable cell in the granulocytic lineage is the myeloblast (20 μm; NIC ratio >
85%); the subsequent arbitrary stages of this continuous maturation process include promyelocytes, myelocytes,
metamyelocytes, band neutrophils, and segmented neutrophils (Figure 23-3). The key regulatory factors involved in
granulopoiesis are granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-
CSF), and interleukin-3 (IL-3) (167). G-CSF is an 813 amino acid membrane protein that functions by binding to its specific
cell surface receptor (G-CSFr) and activates cytoplasmic tyrosine kinases (28). Granulopoiesis is also under the control of
retinoic acid receptors (RAR), which bind to all-trans-retinoic acid and 9-cis-retinoic acid (28). The combination of four
otherwise nonmyeloid restricted transcription factors is unique to the granulocyte lineage: C/EBPa (restricted to
CD34+/CD33+ myeloid cells), PU.1 (Ets family member), CBF (AML1), and c-Myb (50, 95, 103, 197). Other transcription
factors (e.g., WT-1, Rb, and Hox) have also been implicated in granulopoiesis (197). Granulopoiesis occurs predominantly
in paratrabecular and perivascular regions within the bone marrow (134). Thus, in normal bone marrow biopsy sections,
immature granulocytic precursors selectively localize to the paratrabecular and, less conspicuously, the perivascular
regions. This distribution may be altered after cytokine treatment, chemotherapy, as well as after bone marrow
transplantation (see below). Normal localization can be highlighted by immunoperoxidase staining for myeloperoxidase.
FIGURE 23-3 ▪ Granulopoiesis. Immature granulocytic precursors (Leder positive) localize to the paratrabecular regions.
Subsequent arbitrary stages are indicated (circles) and maturation progresses to, for example, band neutrophils.
Erythropoiesis
The earliest morphologically recognizable cell in the erythroid lineage is the erythroblast (normoblast). The subsequent
maturation has been arbitrarily divided into the basophilic normoblast, polychromatophilic normoblast, orthochromic
normoblast, reticulocyte, and mature erythrocyte stages (Figure 23-4). The maturational process is characterized by
progressive nuclear condensation with ultimate extrusion of the pyknotic nucleus at the end of the orthochromic
normoblastic stage, which results in the young erythrocyte (reticulocyte). Simultaneously, the cytoplasm gradually changes
from a deeply basophilic, organelle-rich substance to one that consists almost entirely of hemoglobin. In addition to the
general growth factors (GM-CSF, IL-3, and
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IL-11), the primary growth factor responsible for red blood cell production is erythropoietin (EPO), a 30.4-kDa glycoprotein
that induces proliferation and maturation of committed erythroid progenitor cells by binding to its specific cell receptor (R-
EPO), which inhibits apoptosis and thereby regulates the rate of red cell production (65, 138). EPO does not cross the
placenta and therefore the fetus primarily controls erythropoiesis (138). Although erythroid and megakaryocytic lineages
share several transcription factors such as GATA-1 and NF-E2 (6, 118), specific growth factors act selective and allow
committed cells to differentiate and proliferate. Erythropoiesis occurs in small colonies (erythroblast islands), and even
though related to vascular structures, they appear randomly dispersed throughout the hematopoietic cavity. They are
neither paratrabecular nor perivascular in distribution (16). Erythroid architecture can be highlighted by
immunohistochemistry (Figure 23-4).
Megakaryocytopoiesis
Megakaryocytes are the largest (50 to 150 μm) nucleated cell in the bone marrow. Unlike the maturation of the other
lineages, megakaryocyte maturation from the blast to the mature cell stage is not associated with mitotic divisions.
Megakaryocyte differentiation occurs via endomitosis, resulting in increasing nuclear lobulations without cell division (194),
controlled via thrombopoietin (TPO) (92, 140, 143). The earliest megakaryocyte precursor identified in cell culture studies
is the promegakaryoblast. Subsequent maturational stages have been arbitrarily designated as megakaryoblast, basophilic
megakaryocyte, granular megakaryocyte, and plateletproducing megakaryocyte (47). The maturational sequence is
characterized by a progressive increase in the overall size, an increase in nuclear lobulations ( n = 8, 16 or 32, without
nucleoli, and the development of demarcation membranes and multiple types of (purple-red or pink) cytoplasmic granules.
Megakaryocyte production is regulated by a variety of factors, including multilineage growth factors such as GM-CSF, stem
cell factor, IL-3, IL-6, and lineage-selective factors such as IL-11 and TPO (92, 140, 143, 196). TPO binds to c-Mpl and
acts in synergy with other cytokines (see above, EPO, IFN-α, IFN-β) (129). Even though megakaryocytes appear randomly
distributed in biopsy sections, they are localized selectively to the parasinusoidal regions within the bone marrow
microanatomy. Megakaryocytes project pseudopodia into the vascular space, and proplatelets are directly released into
the blood stream by this mechanism (189).
FIGURE 23-4 ▪ A: Erythropoiesis occurs in small colonies (erythroblast islands) related to vascular structures. B:
Glycophorin A; marker of erythrocytoid differentiation. C: Subsequent stages of erythroid differentiation.
Monopoiesis and Dendritic Cell Development

Monocytes, at 12 to 20 μm the largest leukocyte, are derived from the same precursor cells that give rise to neutrophils,
and M-CSF is instrumental in influencing the progenitor cells to differentiate into monocyte-macrophages (78). Gradual
nuclear folding and the acquisition of cytoplasmic granules characterize the stages of maturation, designated as
monoblast, promonocyte, and mature monocyte. Although characteristically monocytes have fewer and smaller granules
than neutrophils, neither monoblasts nor promonocytes are generally recognizable in normal bone marrow. Monocytes
circulate in the blood and subsequently migrate to solid tissues to become macrophages or various types of immune
accessory cells. This accessory role and evidence that these cells play an integrated, multifaceted role in humoral and
cellular immunity beyond simple phagocytosis, the former designation mononuclear phagocyte system (100) has been
replaced. Foucar and Foucar (63) proposed the alternative name mononuclear phagocyte and immunoregulatory effector
(M-PIRE) system as a more accurate descriptor. The M-PIRE system includes monocytes, macrophages, multiple dendritic
cells (e.g., Langerhans and dendritic reticulum cells), and their bone marrow precursors. Some evidence suggests a
common cell of origin (73). Because the constituent cells show unique immunophenotypic and functional properties and
are therefore viewed as distinct cell lineages,
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the M-PIRE designation remains controversial. Regardless of the name, both macrophages (histiocytes) and dendritic cells
are inconspicuous normal constituents of virtually all organ systems, and mature cells of monocyte-macrophage lineage
also remain as a major constituent of the bone marrow microenvironment.
Lymphopoiesis
T- and B-lymphocytes are derived from the same stem cells that give rise to all hematopoietic elements. Factors that are
known to influence B-cell proliferation, differentiation, and functional activities include IL-1, IL-2, IL-4, IL-10, adhesion
molecules, and IFN-γ; analogous T-cell factors include IL-1 through IL-9 (163). The bone marrow microenvironment serves
as the “bursal equivalent” in humans and is the primary site of postnatal B-cell development, whereas T-cell precursors
migrate from the marrow to the thymus for maturation and differentiation. Antigenetically mature T- and B-cells can
proliferate in response to a variety of cytokines.
The stages of maturation of both B- and T-lymphocytes are generally defined by the surface antigen profile rather than by
morphologic features (Figure 23-2). The earliest immunologically recognizable B-cells express nuclear terminal
deoxynucleotidyl transferase (TdT), surface CD34 (progenitor cell antigen), CD79a, and HLA-DR; CD 10 expression is
variable but common (59, 108). Further maturation is characterized by the acquisition of cytoplasmic mu heavy chain, and
later, surface immunoglobulin. B-cell precursors are generally infrequent in normal bone marrow, although these immature
cells are much more prominent in specimens from infants and young children. When they are abundant, the term
hematogones has been applied to immature lymphocytes (see below).
T-cell maturation is characterized by the presence of cytoplasmic and, later, surface CD3 together with the expression of
many other antigens associated with T-cells (132). Terminal maturation is defined by the development of either a helper
(CD4+) or a suppressor surface antigen profile (CD8+).
Although the terms lymphoblast and prolymphocyte have been applied to developing lymphoid cells and are utilized in
leukemia classification, the distinction is not easy in normal bone marrow specimens. Lymphocytes migrate from blood to
specific tissue sites throughout the body, selectively homing to B- or T-cell regions of lymph node, spleen, and thymus, and
to widespread extranodal regions. T-lymphocytes are characteristically long lived and periodically recirculate.
Development of Natural Killer Cells

Natural killer (NK) cells are unique among mature cells in that they were initially defined by a functional activity, nonmajor
histocompatibility complex-restricted cytotoxicity, before either morphologic or immunophenotypic characteristics were
delineated (107). These cells were subsequently found to perform many other functions (132, 171, 192). Evidence
suggests a common T/NK progenitor cell, and the thymus may be an additional site of NK-cell maturation.
On immunophenotype analysis, NK cells are defined by the expression of such adhesion molecules as CD56, CD57, and
CD16. However, the expression of these adhesion molecules is not restricted to NK cells. The fact that true NK cells lack
CD3 and CD8 expression facilitates their distinction from cytotoxic/suppressor T-cells, which share other features with true
NK cells, including large granular lymphocyte morphology and adhesion molecule expression (CD57, CD16).
Cells with NK activity (both cytotoxic/suppressor T-cells and true NK cells) are concentrated within the large granular
lymphocyte population of peripheral blood mononuclear cells. The mature cells have round nuclei, condensed chromatin,
inconspicuous nucleoli, and moderate amount of pale blue cytoplasm that contains a small number of prominent, coarse,
azurophilic granules. The granules contain cytolytic perforin and associated granule proteases (e.g., granzyme) essential
for their cytolytic activity.
NORMAL HEMATOPOIETIC PARAMETERS

The peripheral blood and bone marrow profile are characterized by prominent age-related physiologic variations (Tables
23-2 and 23-3). As previously outlined, bone marrow cellularity decreases with age (115) and is classically best evaluated
on biopsy sections or imprints. Particle sections are next best choice and aspirate smears may be difficult to evaluate;
however, section imprints and aspiration smears are all reported as equally reliable (130). While earlier references
specified 100% cellularity at birth, more recent studies show that bone marrow cellularity is somewhat lower than
previously estimated (66); therefore, the percentage should be taken as a representative figure. The distribution of
erythroid and lymphoid elements also varies by age, whereas the proportion of bone marrow devoted to granulopoiesis is
generally stable. A dramatic decline in erythroid elements parallels the drop in EPO levels that occurs after birth in normal-
term infants (139). Erythropoiesis returns to normal steady-state levels following resolution of this so-called physiologic
anemia of infancy. Likewise, dramatic age-related variations occur in the proportion of bone marrow lymphoid cells, with up
to 40% lymphocytes in bone marrow specimens from very young children and infants (18). The proportion of lymphocytes
decreases in bone marrow specimens and B-cell production in general declines with age (2).
Age-related variations in peripheral blood values are well delineated (Table 23-2) and the most dramatic changes are
found in erythrocyte, neutrophil, and lymphocyte parameters (17).
HEMATOLOGIC PROFILE OF THE NEONATE

The first month of life is characterized by remarkable physiologic changes in erythrocyte and white blood cell parameters
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(Table 23-3), and these parameters vary between term and preterm neonates (17). In the normal-term neonate, the
hematocrit, mean corpuscular volume, red cell count, and white cell count are higher than normal at any age. The neonatal
period is also the only time when circulating erythroid precursors are physiologic. The nucleated red blood cells are
cleared rapidly from the blood and do not normally persist beyond the first 3 to 4 days of life (62). In healthy neonates, the
relative hypoxia in utero is reversed at birth, so that a marked, transient, abrupt decline in erythropoiesis (so-called
physiologic anemia of infancy) occurs. These physiologic changes are exaggerated in preterm infants (139).
Table 23-2 ▪ NORMAL VALUES FOR BONE MARROW AND DIFFERENTIAL CELL COUNTS
Parameter (Unit) Cord Week Week 1 Child Adult

Blood 1 4 year
Hemoglobin (g/dL) 16.5 17 14 12 13.5 M-16 F-14
Hematocrit (%) 53 54 43 37 40 M-47 F-41
RBC (×106/μL) 5.3 5 4 4.6 4.6 M-5.2 F-4.6
MCV(μm3) 115 100 98 80 84 90
MCHC (g/dL) 32 33 33 34 34 34
Reticulocytes (% of RBC) 3-7 0-1 0 0-1 0-1 0-1

Nucleated RBC (per 100WBC) 500 0 0 0 0 0
WBC (×109/L) 20 12 10 10 7 6
Absolute neutrophil count 13 5 4 4 3 3

(×109/L)
Absolute lymphocyte count 5 5 6 6-8 4 3

(×109/L)
Platelet count (×109/L) 290 250 250 250 250 250
Cell Type Normal Range (%) Cell Type Normal Range

(%)
Myeloblasts 0-3 Basophils and precursors 0-1
Promyelocytes 2-8 Monocytes 0-1
Myelocytes 10-13 Erythroblasts 0-2
Metamyelocytes 10-15 Other erythroid elements 10-25
Band/neutrophils 25-40 Lymphocytes 10-35
Eosinophils and precursors 1-3 Plasma cells 0-1
Table 23-3 ▪ HEMATOLOGIC PROFILE DURINGTHE FIRST MONTH OF LIFE AND IN YOUNG INFANTS
I. Term infants to 1 month
Hgb and Hct drop from 16.5 g/dL and 53% at birth to an average of 14 g/dL and 43% at 1 month of age
MCV declines from 115 μm3 at birth to about 98μm3 at 1 month
Reticulocyte count drops from 5% to 7% at birth to ∽0% at 1 month
Nucleated red blood cells are present at birth but disappear in first week of life
Marked leukocytosis with neutrophilia is normal at birth and lymphocytes predominate by 1 month
II. Preterm infants
Lower Hgb and Hct levels at birth than term neonates

Higher MCV, more nucleated red blood cells and higher reticulocyte counts compared with term neonates
More rapid and pronounced physiologic nadir
Lower leukocyte counts than term neonates III. Young infants
Neonatal assessment complex because of dramatic physiologic variations in conjunction with potential
maternal, familial, obstetric, and other fetal and neonatal factors
Maternal factors: infections, medications, obstetrical complications, and underlying illnesses
For example, maternal and paternal incompatibility for red blood cell antigens can result in hemolysis (hemolytic
disease of the newborn)
Numerous constitutional hereditary disorders of hematopoietic cell production and survival including:
Diamond Blackfan anemia (red cell aplasia)
Thalassemias (hemoglobinopathy)
Congenital neutropenia (granulocyte aplasia) ± thrombocytopenia with absent radii (megakaryocyte aplasia)
Constitutional disorders can manifest at birth or in early infancy
Fetomaternal hemorrhage or internal hemorrhage can produce neonatal anemia
Other causes include various congenital malformations and congenital neoplasms
Hct, hematocrit; Hgb, hemoglobin; MCV, mean corpuscular volume.
The neonate assessment for a hematologic disorder is uniquely challenging because of the complex interplay between
possible maternal, familial, and obstetric factors in conjunction with the pronounced physiologic variations (62), all of which
must be included in the workup of any hematologic aberration.
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Table 23-4 ▪ INDICATIONS FOR BONE MARROW EXAMINATION IN CHILDREN
Peripheral blood abnormality (undetermined after regular workup)

Evaluation of possible constitutional hematopoietic disorder
Evaluation for leukemia, myelodysplasia, myeloproliferative/myelodysplastic disorders, and chronic
myeloproliferative disorders
Evaluation for fever of unknown origin, storage diseases, and unexplained splenomegaly
Staging and management of patients with certain types of neoplasms (e.g., Hodgkin and non-Hodgkin
lymphoma, various other solid tumors)
Evaluation of patient with atypical but nondiagnostic lymphoreticular process in other sites
Evaluation of patient who does not follow predicted course of initial diagnosis (e.g., patient with presumed
idiopathic thrombocytopenic purpura who does not respond to therapy)
Ongoing monitoring of response to therapy in patients with a variety of hematologic and lymphoreticular
disorders
Bone marrow assessment prior to autologous bone marrow transplantation
EXAMINATION OF THE BONE MARROW IN CHILDREN

Indications for bone marrow examination in children are listed in Table 23-4. The decision to examine the bone marrow is
made on an individual basis by correlating laboratory and hematologic findings with the clinical history. While the posterior
iliac crest is the preferred site for the evaluation in older children, aspirates and even biopsy specimens can be obtained
from the tibia in young infants (178). Before performing a bone marrow examination, careful consideration must be given to
what types of specimens are necessary for optimal evaluation of the most likely differential diagnosis (Table 23-5). Except
for cultures, as a general rule, all specialized studies should be delayed until the bone marrow aspirate smears have been
reviewed. When verified as adequate, the appropriate specialized tests can be ordered. Flow cytometry is one of the
routine techniques for immunophenotyping and aids in determining the lineage and stage of “maturation” of neoplastic
bone marrow infiltrates. Cytogenetic evaluation provides essential diagnostic and prognostic information, not only in acute
lymphoid and myeloid leukemias but also in other myeloid disorders. Other, special techniques are also useful to assess
for minimal residual disease in patients with leukemias/lymphomas, and to evaluate metastatic processes (Table 23-5).
Table 23-5 ▪ SPECIALIZEDTECHNIQUES IN BONE MARROW EXAMINATIONS
Technique Specimen Indications

Required
Culture Aspirate, Workup for infection

sterile
Cytochemical stains Air-dried Lineage identification for immature cells

aspirate
smears
Immunohistochemical Paraffin- Numerous antibodies available to assess for lymphoid, myeloid,

stains embedded erythroid, and megakaryocytic antigens as well as to determine lineage
tissues of metastatic processes
Selected antibodies to assess immaturity (e.g., CD34) also available
Immunophenotyping Aspirate, Useful in determining immunophenotypic profile of wide variety of

(by flow cytometry) sterile neoplastic disorders (e.g., leukemias and lymphomas) as well as
benign infiltrates (e.g., hematogones)
Cytogenetics Aspirate, Yield prognostic and diagnostic information in acute leukemias,

sterile myeloid neoplasms, and lymphoma
Essential in the evaluation of acute leukemias
Fluorescence in situ Air-dried Assess for specific cytogenetic abnormality if probe available
hybridization smears, cell Useful in minimal residual disease assessment
culture
smears
Molecular analysis Paraffin- Useful in determining B- and T-cell clonality, as well as gene
imbedded rearrangements and other genetic aberrations
tissues
(PCR)
Aspirate, Useful in detecting gene amplifications in metastatic neuroblastoma.

sterile (other
methods)
Constitutional Hematopoietic Disorders

Bone marrow biopsies for constitutional hematologic disorders are only rarely encountered in clinical practice. The different
entities represent a heterogeneous group of diseases and involve individual lineage defects of, for example, erythroid,
megakaryocytic, and histiocytic elements (Table 23-6). Many of these constitutional disorders (e.g., thrombocytopenia with
absent radii, see below) are evident at birth or shortly thereafter, whereas the multilineage abnormalities that characterize
the constitutional aplastic anemias usually develop more gradually, sometimes not until adulthood (40, 41, 125). Another
interesting pattern is that constitutional hematologic disorders are frequently associated with a variety of abnormalities in
other organ systems (Table 23-6), while the bone marrow picture is largely one of individual aplasia or multilineage failure
without distinctive morphologic aberrations (41). Exceptions include marked dyserythropoiesis in congenital
dyserythropoietic anemia, and erythroid hyperplasia in various constitutional erythrocyte survival disorders (81, 180).
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Table 23-6 ▪ CONSTITUTIONAL HEMATOLOGIC DISORDERS
Fanconi anemia
DNA repair defect (autosomal/X-linked recessive) with increased incidence of AML
Aplastic anemia in >90%, prominent neonatal cytopenia; pancytopenia by midchildhood
Gradual development of single and multilineage aplasia
Associated congenital anomalies of bone, skin, kidney; mental retardation
13 genes (A-N) identified—most FANCA (16q24.3; exon 43) ∽60%

common: FANCC (9q22.3; exon 14) ∽10%
FANCG (9p13; exon 14) ∽10%
Dyskeratosis congenita
DNA repair defect (uncharacterized genetic subtype in 50%)
Gradual development of pancytopenia and aplastic anemia ∽80%)
Initial hypercellularity common
Associated with congenital anomalies of skin, nails, mucosa; frequent mental retardation
Four genes identified: X-linked recessive (∽30%) dyskerin (Xq28; exon 15)
Autosomal dominant (10%) TERC (3q26; exon 1)
TERT (5p15; exon16)
Autosomal recessive (∽1 %) NOP10 (15q14; exon 2)
TERT(5p15;exon 16)
Diamond-Blackfan anemiaa
70% uncharacterized genetic subtypes (autosomal dominant and recessive described)
Likely intrinsic progenitor cell defect
Constitutional red cell aplasia (rare erythroblasts present)
Some patients develop marrow failure
Associated with congenital anomalies, especially skeletal (30%-40%)

Three genes identified: Autosomal dominant (∽30%) RPS19 (19q13.2; exon 6)
RPS24 (10q22-23; exon 7)
RPS17 (15q25.2; exon 5)
Congenital dyserythropoietic/idiopathic aplastic anemiaa
Erythroid hyperplasia/aplasia
Associated with distinctive bone marrow abnormalities including multinucleation, nuclear bridging, and
megaloblastic changes/bone marrow failure
Chromosomal instability and increased incidence of malignancy (repair defect)
Heterozygous mutations in TERC and TERT are risk factors for some cases
Schwachman-Diamond syndromeb
Constitutional neutropenia with frequent development of aplasia (∽20%)
Associated with congenital anomalies including exocrine pancreas insufficiency
One gene identified: Autosomal recessive (∽90%) SBDS (7q11; exon 5)
Thrombocytopenia with absent radiic (TAR)
Constitutional thrombocytopenic disorder with reduced megakaryocytes and bone anomalies
Probable autosomal recessive disorder MPL (1q34; exon12)
Congenital amegakaryocytic thrombocytopeniac
Decreased megakaryocytes
Different inheritance pattern than TAR, some cases X-linked
High incidence of dysplasia
Lysosomal enzyme defects/storage disorders (multiple types):
Over 40 genetic disorders (∽1 in 7,000 live births) with mostly secondary hematologic manifestations
Accumulation of substrate protein within histiocytes/macrophages
Increased bone marrow histiocytes with distinctive morphology
Classification into six groups: Lipid storage disorders (Gaucher, Niemann-Pick)

Gangliosidosis (Tay-Sachs disease)
Leukodystrophies (ADL, MLD, Krabbe, Refsum, Pelizaeus-

Merzbacher)
Mucopolysaccharidosis (Hunter syndrome, Hurler disease)
Glycoprotein storage disorders (mucolipidosis, pseudo-Hurler)
Mucolipidoses (ML type I-IV; sialidosis)
aConsidered a constitutional erythrocyte disorder; this group also includes hemoglobinopathies, membrane
defects and enzyme defects; For example, thalassemias, sickle cell disorders, hereditary spherocytosis, and
pyruvate kinase deficiency (not discussed here)
bConsidered a constitutionalgranulocyte disorder; this group also includes Kostmann agranulocytosis syndrome,
cyclic neutropenia, and Chédiak-Higashi syndrome (see Table 23-7).
cConsidered a constitutional megakaryocytic disorder.
ALD, adrenal leukodystrophy; MLD, metachromatic leukodystrophy.
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The related group of storage diseases typically occurs as a consequence of lysosomal enzyme defects, affecting mainly
histiocytes. Tissues throughout the body are affected and cells exhibit distinctive morphologic abnormalities caused by the
accumulation of substrate proteins. Although not a primary hematologic disorder, the accumulation of abnormal histiocytes
in the bone marrow produces secondary hematologic effects (41) (see Chapter 5).
Aplastic Anemia in Children

Aplastic anemia in children can be separated into constitutional/inherited versus acquired (173). This heterogeneous group
of disorders, characterized by bone marrow failure with/without somatic abnormalities, typically presents with bone marrow
failure in childhood. Eventually, severe trilineage hypoplasia develops; however, despite the name (aplastic), initial
presentation is often trilineage hyperplasia, megaloblastic changes, or single lineage aplasia. It is noteworthy that some
cases may not present until adulthood, highlighting the importance not only for pediatric pathologists. Since cloning of the
first aplastic-anemia-related gene in 1992 [Fanconi anemia ( FA)-gene], considerable advances in the syndromic entities
have been made (41). It is clear that approximately 20% of bone marrow failure syndromes in children are inherited and
approximately 10% represent secondary causes. The latter includes radiation, drugs (typically busulfan, chloramphenicol,
nonsteroids), viruses (e.g., hepatitis), and immunological causes (e.g., systemic lupus erythematosus). The classic
diepoxy-butane/mitomycin C-induced chromosome fragility testing in cytogenetics has been complemented by targeted
molecular approaches (183). The former test assayed the underlying constitutional DNA repair defect in FA, which
represents the most common genetic aplastic anemia (Table 23-6). Our current understanding of the molecular pathology
underlying this group of diseases is convergence in the DNA repair-FBRCA pathway (41). The diseases affect telomere
maintenance in dyskeratosis congenita-related genes (e.g., dyskerin, TERC, TERT, NOP10), ribosome biogenesis in
Shwachman-Diamond syndrome and Diamond-Blackfan anemia genes (SBDS and RPS19/24/17, respectively) (67), or
congenital amegakaryocytic thrombocytopenia-associated genes (e.g., MPL encoding for TPO) (38, 69, 174, 183, 188).
Despite the availability of mutational information and mode of inheritance (Table 23-6), the majority (∽70%) of “classical”
bone marrow failures is “idiopathic” or uncharacterized and therefore the main/primary pathogenesis remains unknown (41,
93). The peak incidence for secondary and idiopathic aplastic anemia in children is 3 to 5 years of age, and the
morphologic features in the bone marrow are generally absent or severely reduced hematopoiesis.
Benign Erythroid Disorders in Children

Non-neoplastic erythroid disorders (also known as pure red-cell aplasia) consist primarily of congenital and acquired
anemias (Table 23-6) (145). The congenital form is induced by intrauterine damage to early erythroid precursors (180).
Although the uncommon familial or tumor-associated polycythemia/erythrocytosis can be seen in the neonatal period, the
most common neonatal polycythemia is the physiological subtype, resulting from intrauterine hypoxia. The prevalence of
specific types of anemia varies by patient age and ethnicity. In neonates, anemias secondary to blood loss predominate,
followed by immune and nonimmune hemolytic processes. Anemias secondary to either maturation or proliferation defects
are uncommon in infants and include constitutional red cell aplasia and congenital dyserythropoietic anemias (180).
Depending on the ethnic features in a given practice area, constitutional erythrocyte survival disorders, including
hemoglobinopathies and erythrocyte membrane disorders (36), are relatively common causes of anemia in infants.
However, bone marrow examination is generally not required for diagnosis.
A relatively common diagnostic challenge in bone marrow biopsies in children is the classification of red cell aplasia. The
three primary causes of red cell aplasia in young children are Diamond-Blackfan anemia, transient erythroblastopenia of
childhood, and red cell aplasia secondary to parvovirus infection (71). The latter (also-called acquired pure red-cell
aplasia) is typically transient and self-limited. If a variety of clinical, laboratory, hematologic, and bone marrow morphologic
findings are integrated, the types of red cell aplasia in young children can generally be distinguished. In all types of
constitutional and acquired red cell aplasia, the bone marrow is characterized by a profound decrease in maturing erythroid
elements, although usually a variable number of erythroblasts are apparent. In addition, distinctive intranuclear inclusions
within the residual enlarged erythroblasts are the hallmark of parvovirus infection, but these may not be readily apparent in
all cases and immunohistochemistry can be helpful (Figure 23-5). Consequently, acute parvovirus infection should always
be excluded by serologic or molecular studies in cases of red cell aplasia, even when the morphologic features of
parvovirus infection are lacking. Another distinctive bone marrow finding, increased hematogones (88, 96), may
accompany any type of red cell aplasia in children, especially in very young patients.
In older infants and children, the most common cause of anemia is iron deficiency; other causes of anemia in this age
group include chronic disease, such as HIV-1 infection, and red cell aplasia. The frequency of constitutional hemolytic
anemias varies by ethnicity. Other nutritional anemias, including vitamin B12 and folate deficiency, occur in both
constitutional and acquired forms in children, but the incidence is low. For many of these types of anemia, the diagnosis
can be established by integrating clinical and laboratory parameters, and bone marrow examination may not be required;
however,
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bone marrow evaluation is necessary for diagnosis in most patients with red cell aplasia, pancytopenia, and suspected
congenital dyserythropoietic anemia (55, 203).
FIGURE 23-5 ▪ Parvovirus. A,B: Intranuclear viral inclusions in a patient with Parvo B19 induced red cell aplasia (arrows)
and corresponding Parvovirus immunohistochemistry. C: Morphology of intranuclear inclusion on smear (Wright-Giemsa
stain).
Finally, drugs and chemicals are associated with the development of pure red-cell aplasia (166). Common examples
include ampicillin, azathioprine, carbamazepine, cephalothin, chlormadinone, cotrimoxazole, D-penicillamine, erythromycin,
estrogens, furosemide, gold, indomethacin, rifampicin, and valproic acid (49).
Inherited and Congenital Hematopoietic Syndromes Affecting Granulocytes

Disorders affecting granulocytes result from known genetic patterns of inheritance, from known mutations and/or from
mechanisms that are still poorly understood. Conventionally, if present at birth, these are designated as congenital and if
diagnosed later in infancy, these are termed inherited. An important note of caution in the diagnosis of these disorders is
the exclusion of much more common, reactive causes such as inflammatory conditions, infections, nutritional deficiencies,
and/or therapy effect. These are much more common causes of marked changes in the peripheral blood counts, such as
leukocytosis, monocytosis, and neutropenia.
Table 23-7 ▪ SELECTED CONSTITUTIONAL HEMATOLOGIC DISORDERS INVOLVING

GRANULOCYTES
Immunodeficiency Mode of Inheritance and Predominant

Disorder Morphologic Findings Clinical Observations
Phagocytic/motility/adhesion defects in granulocytes
Chronic X-linked or autosomal recessive. Abscess and Defective microbial killing by

granulomatous granulomas common; normal leukocyte phagocytic cells; any site can be
disease (e23) morphology involved, most documented are GI
lesions and lungs; incomplete
response to infections
Leukocyte adhesion Autosomal recessive (mutations in chromosome Delayed wound healing, delayed
defect (e1,e13) 18). Distinct lack of neutrophils at sites of attachment of umbilical cord,
infection despite peripheral neutrophilia and recurrent infections
myeloid hyperplasia in bone marrow; normal
morphology
Chediak-Higashi Autosomal recessive; functionally defective Recurrent pyogenic infections, partial

syndrome (e2,e3) neutrophils with giant cytoplasmic granules oculocutaneous albinism, progressive
neuropathy
Cyclic neutropenia Autosomal dominant or sporadic; absence of Cyclic hematopoiesis with periods of
(e10,e19) granulocytic precursors in neutropenic phase; neutropenia lasting from 9 to 21 days
normal morphology followed by neutrophila. Increased
infections correspond to neutropenic
cycle
Kostman syndrome Autosomal recessive or sporadic; severe Recurrent bacterial infections;

(e4,e5,e25) neutropenia with sustained myeloid aplasia in myelodysplasia and acute myeloid
bone marrow leukemia may be seen in those
treated with G-CSF therapy.
In general, the congenital disorders of granulocytes are typically associated with isolated neutropenia and further
subclassified into three categories: (a) severe congenital neutropenia, (b) cyclic neutropenia, and (c) chronic benign
neutropenia. The inherited disorders, on the other hand, cause morphologic and/or functional changes in the leukocytes
with or without cytopenias. Among the numerous disorders in this group, the more common ones are Pelger-Huet anomaly,
May-Hegglin anomaly, and Chediak-Higashi syndrome (Table 23-7).
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FIGURE 23-6 ▪ Hematogones, in a bone marrow core biopsy; cells exhibit condensed nuclear chromatin (inset).
INHERITED IMMUNODEFICIENCY DISORDERS

General Considerations
In the peripheral blood and bone marrow of children, lymphocytes are a prominent component, constituting up to 40% of
nucleated elements. Typically, these are evenly dispersed without formation of aggregates. If aggregation is encountered,
autoimmune or inflammatory disorders should be considered.
Hematogones are a type of benign lymphocyte that can be found in very large numbers in the bone marrow of young
children (106, 195). These cells characteristically have round-toirregular nuclei with homogeneously condensed chromatin,
inconspicuous nucleoli, and a very high nuclear-to-cytoplasmic ratio (Figure 23-6). Bone marrow hematogones can be
substantially increased in a variety of constitutional and acquired hematologic disorders, in children undergoing
nonhematologic tumor staging, and in children recovering from bone marrow suppression (106, 114).
Immunophenotypically, hematogones are B-lymphocytes that express an antigenic spectrum ranging from immature B- to
mature (polyclonal) B-cells (157) (Figure 23-7). This maturational spectrum is best evaluated by flow cytometry and useful
in distinguishing hematogonerich lymphocytosis from the chief differential diagnostic consideration, acute lymphoblastic
leukemia (ALL) (157). In addition, although CD34+, TdT+ lymphocytes are evident on clot/biopsy sections, hematogones
are generally dispersed rather than clustered, another feature useful in the distinction from ALL (157, 158).
FIGURE 23-7 ▪ A-D: Immunohistochemistry of hemato gones (same case as Figure 24-6), showing variable CD20 ( A) and
CD79a ( B) expression with no Tdt ( C) and strong CD10 ( D) expression.
Specific Inherited Immunodeficiency Disorders

This group of disorders can be classified into deficiencies of B-cells, T- cells, or combined deficiencies. Some authors also
include defects of the phagocytic and complement systems in this category (199). While several of the disorders in this
group have been defined genetically (e.g., severe combined immunodeficiency, X-linked agammaglobulinemia adenosine
deaminase deficiency), the classification and genetic basis of many other disorders remains to be determined (Table 23-8).
Platelet and Megakaryocytic Disorders

Transient thrombocytopenia of newborns affects up to 4% of new births and is most commonly seen in distressed
neonates (43, 135). In most cases, no cause is identified and remission is spontaneous. Known causes of neonatal
thrombocytopenia include maternal illnesses, maternal drug therapy, maternal alloimmunization against fetal platelets, fetal
or neonatal infections, and chromosomal abnormalities. If a bone marrow biopsy is deemed necessary, megakaryocytes
are usually slightly increased in number and morphologically normal. Decreased number of bone marrow megakaryocytes
is usually
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seen in constitutional disorders such as thrombocytopenia with absent radii or X-linked amegakaryocytic thrombocytopenia
(Table 23-6). Down syndrome (DS) is associated with multiple platelet and megakaryocytic abnormalities including giant
platelets, circulating megakaryocytes, and thrombocytopenia in some cases (4, 10, 22).
Table 23-8 ▪ SELECTED CONSTITUTIONAL HEMATOLOGIC DISORDERS INVOLVING B-CELLS

ANDT-CELLS
Antibody deficiency disorders
Combined variable Heterogeneous group of disorders with Recurrent sinupulmonary

immunodeficiency intrinsic B-lymphocyte defect; T-lymphocyte infections, malabsorption;
(e7,e9,e12) defects described in some; complications like chronic lung
lymphadenopathy with hyperplastic germinal disease, chronic
centers, no plasma cells. gastroenteritis, or liver failure.
X-linked Mutations in B-lymphocyte-specific tyrosine Sinopulmonary, Gl, skin and

(Bruton)Agammaglobulinemia kinase gene; hypoplastic lymphoid organs joint infections caused by
(e12,e16,e27) with atritic germinal centers; decreased B- pyogenic bacteria and
lymphocytes, absent plasma cells enteroviruses
Selective IgA deficiency Most common and mildest; varying modes of Heterogeneous clinical
(e8,e24) inheritance; nonspecific findings of villous presentation; mostly no
blunting and follicular hyperplasia in Gl significant illness; recurrent
biopsies sinopulmonary infections; food
allergy; celiac disease.
Hyper IgM syndromes (e21) Mostly X-linked; inapparent germinal centers; Similar clinical findings to other
B- lymphocytes are present with abundant antibody deficiency disorders
plasma cells
Predominantly T-cell deficiency disorders
Severe combined X-linked; defects in all stages of T-cell Several subtypes with varied
immunodeficiency (e8,e9) development; B-lymphocytes affected in presentations; severe and
some types; involuted thymus; decreased recurrent systemic infections
lymphocytes
Ataxia telangiectasia A single genetic defect localized to Sinopulmonary infections,

(e6,e17) chromosome 11 q22-23; loss of cerebellar telangiectasia; progressive
Purkinje cells and granular layer; ataxia, and hypersensitivity to
pneumonia; chronic hepatitis; hypoplastic ionizing radiation
lymph nodes
Wiscott-Aldrich syndrome Deletions on Xp11.22-23; varying degrees of Thrombocytopenia with

(e18,e26) lymphoid depletion in lymphoid organs; petechiae or bleeding;
poorly formed or absent germinal centers recurrent infections, eczema;
immunodeficiency
NEOPLASTIC DISORDERS IN BONE MARROW

Myeloproliferative Disorders in Down Syndrome (DS)
Transient myeloproliferative disorder (TMD) and acute mekaryoblastic leukemia (AMKL) are the two most common myeloid
malignancies encountered in DS. Mutations in GATA-1 have been classically detected in DS-TMD and DS-AMKL;
however, more recently the same mutations were found in blasts of non-DS TMD and AMKL, suggesting an etiologic and a
clonal link between the two disorders (83, 85, 204).
Transient Myeloproliferative Disorder

TMD is the most frequently encountered myeloproliferative disorder in neonates. TMD generally occurs in the neonatal
period but has been documented in utero (64). In newborns with DS or trisomy 21 mosaicism, it affects up to 10% of
newborns (70, 206). Although it resembles congenital acute leukemia (99) and can show up to 50% blasts and a clonal
chromosome × inactivation in all lineages, it resolves spontaneously in 1 to 2 months (99, 159). The white blood cell count
is characteristically markedly elevated, exceeding 50,000/mm3 and shows normal appearing neutrophils with an otherwise
variable hemogram. The hallmark of TMD is the striking number of circulating heterogeneous blasts. Morphological,
cytochemical, and immunophenotypic studies show a predominance of erythroblasts and megakaryoblasts (9, 105) (Figure
23-8). Despite spontaneous resolution, a substantial portion of patients have been reported to develop acute myeloid
leukemia, usually, acute megakaryoblastic leukemia (9, 42, 105, 121).
Acute Leukemia
The incidence of overt acute leukemia is markedly increased in children with DS irrespective of an antecedent TMD (9, 32,
187, 205). The affected children are generally older and present with evidence of severe bone marrow failure and
hepatosplenomegaly. The type of acute leukemia seen in children with DS is age dependent. In children less than 3 years
of age, acute megakaryoblastic leukemia generally develops with an admixed erythrocytic component, whereas ALL
predominates in older children (32, 85). In an overt acute leukemia, the bone marrow is replaced by blasts,
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which are generally cytochemically, morphologically, and phenotypically homogeneous. Additional clonal chromosomal
abnormalities along with trisomy 21 are more common in acute leukemias than in TMD (32).
FIGURE 23-8 ▪ AB: Bone marrow in a newborn with TMD associated with DS, showing myeloid left shift (A) and
prominence of immature cells including blasts (B).
Congenital Acute Leukemias

Congenital leukemias are by definition acute leukemias presenting at birth until 1 month of age. Likely to have originated in
utero, these are extremely rare and described with rates of one in 5 million births (151). Congenital leukemias are
predominantly myeloid; however, lymphoid types have been described in biologic subsets associated with translocations
involving 1 1q23 (MLL gene) (31, 74, 122, 153, 164). The myeloid leukemias typically demonstrate a prominent
monoblastic component, marked leukocytosis, and extramedullary disease; hepatosplenomegaly and skin lesions are
especially prominent (153, 164). Distinctive features of 11q23-associated congenital ALL include central nervous system
disease and a CD 10−, CD15+ B-cell precursor phenotype with frequent myeloid antigen coexpression (31, 153). The
second biologic subset of congenital acute leukemias are associated with t(1;22)(p13;q13). This subtype is an acute
megakaryoblastic leukemia that occurs in infants less than 1 year of age (198), and the clinical picture resembles a solid
tumor. Both bone marrow and extramedullary infiltrates are extremely fibrotic and tumor cells often appear as isolated nests
(25, 104). On the hemogram t(1;22) associated leukemia presents typically with severe pancytopenia, while in contrast the
11q23 leukemia is characterized by marked leukocytosis. Although the prognosis is poor in all subtypes of congenital
leukemia, those cases that present within the first month have the worst prognosis. Lineage switch in congenital acute
leukemias is characterized by MLL gene rearrangements with t(4;11), t(9;11), and other translocation partners (98, 175).
Acute Lymphoblastic Leukemia

ALL is a clonal B- or T-cell neoplasm characterized by a loss of normal hematopoietic elements and the predominance of
immature B- or T-cells capable of minimal, if any, maturation. ALL represents the most prevalent of pediatric leukemias with
an incidence of approximately 3/100,000 children annually. ALL predominates in children between 2 and 9 years of age
and 75% of all ALL patients are younger than 15 years (1, 97). Boys are affected more often, and a substantially increased
incidence of ALL has been documented in patients with genetic disorders such as DS and 11q23 abnormalities (85).
Patients typically present with fever, bleeding, splenomegaly, or hepatosplenomegaly. In infants, remarkable
organomegaly, high white cell count, and central nervous system involvement predominate. In older children, the typical
presentation is a mediastinal mass with or without leukocytosis. Infection and bleeding are the consequence of cytopenias,
and organomegaly is secondary to the infiltration of solid tissues by leukemic blasts (1). The white blood cell count is
highly variable and ranges from less than 4,000/mm3 to more than 1,000,000/mm3. In cases with a low white blood cell
count, careful blood smear and bone marrow examinations are necessary for diagnosis.
The most widely accepted classification system for ALL was initially proposed by the French-American-British (FAB)
Working Group in 1976 and revised in 1981 to improve concordance (11, 12) (Table 23-9). Although the
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FAB criteria are valid for identification and distinction of most myeloid from lymphoid processes, the lineage-based
classification does not allow distinguishing biologic subsets of acute leukemia. In 2003, the World Health Organization
(WHO) sponsored the development of a new classification system that focused on defining distinct clinicopathologic
entities based on morphologic, immunophenotypic, genetic, and clinical features (77).
Table 23-9 ▪ CLASSIFICATION OF ALL
French-American British
L1a High nuclear/cytoplasmic ratio

Nuclei have inconspicuous nucleoli
Blasts variable in size but often small
L2a Moderate-to-abundant amounts of cytoplasm

Nuclear have one or more distinct, prominent nucleoli
Blasts, medium to large
L3b Scant-to-abundant amounts of deeply basophilic cytoplasm
Abundant cytoplasmic (distinct) vacuoles
Nuclear chromatin homogeneous
One or more, generally indistinct nucleoli
Blasts, medium to large
FIGURE 23-9 ▪ A-C: The three types of lymphoblasts (FAB classification) include FAB-L1 blasts with high nuclear
cytoplasm ratio (A), FAB-L2 blasts with moderate-to-abundant cytoplasm (B) and FAB-L3 blasts with deeply basophilic and
highly vacuolated cytoplasm (C).
Morphologic Basis of ALL Classification
In ALL, the bone marrow blasts characteristically account for more than 90% of all nucleated cells. Whether B- or T-
precursor derived, cases of prototypic ALL are morphologically indistinguishable (94). Lymphoblasts, termed L1 and L2
(FAB-criteria), consist of intermediate-to-large cells with variable amounts of cytoplasm and nuclei exhibiting finely
dispersed chromatin and variably prominent nucleoli (11, 12) (Figure 23-9A,B). The nuclei of prototypic lymphoblasts
usually exhibit subtle irregularities and convolutions that are best appreciated on high power.
Although very rare (<1%), the abnormal ALL population can be indistinguishable from tissue infiltrates of Burkitt lymphoma.
Burkitt cells are defined by both nuclear and cytoplasmic features, including deeply basophilic cytoplasm with distinct lipid
vacuoles and moderately sized round nuclei with several indistinct nucleoli (19, 35) (Figure 23-9C). Although Burkitt
leukemia is, by convention, included in the FAB-classification of ALL, the morphologic and immunophenotypic profile is
consistent with a mature B-cell lymphoma rather than the prototypic ALL. On bone marrow biopsy sections, cells of Burkitt
leukemia/lymphoma are more homogeneous and have round nuclear contours with one to three small basophilic nucleoli
(35). Mitotic activity is very brisk, and the abundance of tingible body macrophages may sometimes impart a “starry sky”
appearance to bone marrow sections, similar to that seen in other solid tissue sites of disease (Figure 23-10). Cytoplasmic
vacuolation is best appreciated in aspirate smears.
Granular ALL, comprising up to 7% of pediatric ALL cases, is characterized by coarse cytoplasmic granulation in at least
5% of the blasts. It can be mistaken for myeloid leukemia; however, the granules are myeloperoxidase negative. This
morphologic subtype confers a poor prognosis (27).
Phenotypic Basis of ALL Classification

Most cases of ALL are B-lineage leukemias while T-lineage ALL accounts for only 10% to 25% of cases. In comparison
with normal B- and T-cell maturation profiles, most cases of ALL display aberrant or asynchronous antigen expression,
including adhesion molecule expression (68, 75). The optimal flow cytometric panel for ALL should contain a variety of B-,
T-, and myeloid monoclonal antibodies along with Tdt and CD34, which are assessed on the weak CD45+ blast population
(15, 34) (Figure 23-11).
Biologic Basis of ALL Classification

Combining cytogenetic and molecular techniques, chromosomal abnormalities are detected in 80% to 90% of ALL cases
(53, 119, 161). The most frequent numeric chromosomal abnormality in ALL is hyperdiploidy (−50% of B-cell precursor
ALL), with 15% having 47 to 50 chromosomes (119). Hyperdiploidy can be determined with
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standard cytogenetic techniques or by flow cytometric measurement of DNA content. Hyperdiploidy, with a chromosome
number of 50 or greater, or a DNA content index (ratio of patient DNA content to normal control DNA content) of 1.16 or
greater is an established good prognostic indicator in ALL. This prognostic advantage has been attributed to higher
response rates to antimetabolite-based therapy. However, cases with extreme hyperdiploidy (i.e., near triploidy, tetraploidy,
or >65 chromosomes) exhibit a poor outcome (149, 152).
FIGURE 23-10 ▪ A, B: A “starry sky” pattern is evident in this bone marrow showing extensive involvement by Burkitt
lymphoma; bone marrow aspirate shows cytoplasmic vacuoles in malignant cells.
FIGURE 23-11 ▪ A-D: Composite flow-cytometry histograms showing immunophenotypic profile of a precursor B-acute
lymphoblastic leukemia. Note that the cells are dim CD45+ (A), TdT+ (B), CD34+ (C) and coexpressing CD10 and CD19
(D).
Approximately 40% of pediatric ALL cases demonstrate chromosomal translocations by standard cytogenetic analysis
(161). The most common translocation, t(12;21), can be detected by molecular methods in 20% to 25% but remains cryptic
on routine cytogenetics (<0.5% detection). The prognosis of t(12;21) is similarly favorable to those of hyperdiploid ALL. In
contrast, t(1;19) presents with high-risk disease and carries a worse prognosis. Blasts show a pre-B phenotype with
negative CD34, cytoplasmic immunoglobulin positivity, and partial CD20 expression. ALL with t(5;14) and eosinophilia
tends to occur in older children and is characterized by striking tissue eosinophilia with consequent organomegaly and
usually an aggressive clinical course. The eosinophils exhibit striking dysplasia. (124, 161). As illustrated above, a
significant proportion of cases may be missed by classic cytogenetics, and PCR- or fluorescence in situ hybridization
(FISH)-based analysis is strongly indicated for therapeutic stratification. Philadelphia chromosome (Ph)-positive ALL is
another biologic subtype of ALL that results from the classic t(9;22)(q34;q11) translocation (Figure 23-12A,B). In most
pediatric ALL cases, the chimeric bcr/abl transcript encodes for the p190 protein. Although Ph-positive ALL cases show no
difference in terms of clinical presentation, these patients usually have poor responses to chemotherapy, necessitating
alternative therapies including bone marrow transplant (156, 168). With few exceptions, the prognosis in ALL can be
determined by integrating age, white blood cell count, sex, genotype, and other parameters (Table 23-10). The clinical,
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immunophenotypic, and prognostic significance of a variety of these chromosomal translocations is summarized in Table
23-11.
FIGURE 23-12 ▪ A, B: Precursor B-acute lymphoblastic leukemia with translocation t (9;22) as demonstrated by dual color
fusion FISH probes.
Table 23-10 ▪ CLINICAL AND LABORATORY FEATURES IN PEDIATRIC ALL ASSOCIATED WITH
POOR PROGNOSIS
Clinical features
Age <1 year old or >10 years
Mediastinal massa
Marked hepatosplenomegalya
Non-Caucasian racea
CNS disease at the time of presentation
Suboptimal response to induction chemotherapy
Laboratory features
High white blood cell count (>50 × 109/L
Blast immunophenotype (T-cell, CD10-, L3 morphology)
Biologic factors (see Table 23-12)
aNo longer considered statistically significant prognostic variables (e22).
Acute Myelogenous Leukemia

Acute myelogenous leukemia (AML) is a clonal hematopoietic disorder characterized by a predominance of immature cells
capable of minimal, if any, maturation. AML can be derived from progenitors of any lineage and hence multilineage
differentiation can be noted. AML occurs in patients of all ages but is more prevalent in adults. Nevertheless, most
congenital leukemias are myeloid in origin. The incidence of AML is low throughout childhood and early adulthood, but the
proportion of cases of acute leukemia that are myeloid steadily increases during these years. Factors linked to an
increased incidence of AML include constitutional genetic disorders, acquired bone marrow diseases, smoking,
occupational/environmental exposures, and therapeutic agents (13, 85, 141, 179). Numerous studies have documented an
increased incidence of AML in patients receiving chemotherapy, especially alkylating agents or topoisomerase II
inactivators (13, 144, 151). Other environmental exposures associated with AML include radiation, benzene, and other
chemicals (13, 141, 179).
Table 23-11 ▪ INCIDENCE AND CLINICAL FEATURES ASSOCIATED WITH GENOTYPIC

ABNORMALITIES IN PEDIATRIC ALL
Numerical Incidence Comments/Prognosis

Abnormalities
Hypodiploid (1) 2%-8%; Older than 10, poor risk by NCI criteria, Poor prognosis
Hyperdiploid 25%-40% Most frequently seen genetic abnormality; low risk by NCI criteria,
favorable response to antimetabolite therapy; trisomies 4 and 10 likely
linked to improved outcome; outcome worse in cases of additional,
worse outcome translocations (see below)
Structural
abnormalities
Cryptic 20%-40% TEL/AML 1; most commonly seen genetic translocation; good

t(12;21) prognosis
(p13;q22)
t(9;22) 3%-5% BCR/ABL; may be cryptic; may be associated with additional

(q34;q11) abnormalities; poor response to therapy
t(1;19) ≤5% by routine E2A/PBX 1; mostly in neonates and infants; high-risk disease at
(q23;p13) cytogenetics; 20%- presentation; usually pre-B with cy immunoglobulin; poor outcome
25% if molecular
techniques are
employed
t(v;11)(v;q23) 2%-11% High-risk presentation; inferior treatment outcome; predominates in

(2, 3) therapy-related and congenital leukemias
t(8;14) <5% MYC dysregulation; B-ALL with L3 (Burkitt) morphology; mature B-cell
(q24;q32) also phenotype
t(2;8)
andt(8;22)
t(11;14) 30% of T-ALL by TAL1 dysregulation on chromosome 1p32 or TCR gene dysregulation;
(p15;q11), molecular usually older patients; prominent extramedullary disease
t(1;14) techniques
(p32;q11), and
t(1;7)
(p32;q35)
t(5;14) Rare Older patients; aggressive disease course; neural and cardiovascular
(q31;q32) complication due to striking eosinophilia secondary to IL-3 related
stimulation
Modified from Foucar KM. The bone marrow. In: Pediatric Pathology, 2nd ed. Stocker JT, Dehner LP, eds.
Philadelphia: Lippincott Williams & Wilkins. 2001;1135-1162. v = variable.
Similar to ALL, the most widely accepted classification system for AML is the FAB-system (Table 23-12) (12).
Subsequently, new classification strategies have been devised to include differing clinical features of de novo AML and
therapy or myelodysplasia-related AML (79, 87, 151). The most significant change is the blast percentage (20%) required
for diagnosis of AML (FAB: 30%). Several other features, however, both clinical and genetic, play a role in establishing the
final diagnosis (51).
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Table 23-12 ▪ ORIGINAL FAB CLASSIFICATION OF AMLa
AML-MO ≥30% blasts
<3% Sudan black B/myeloperoxidase positivity
Myeloid antigen expression by immunophenotyping or myeloperoxidase expression by electron

microscopy
AML-M1 ≥30% blasts
≥3% Sudan black B/myeloperoxidase positivity in blasts
<10% cells exhibiting maturation beyond blast stage
AML-M2 ≥30% blasts
≥3% Sudan black B/myeloperoxidase positivity in blasts
>10% granulocytic cells exhibiting maturation beyond blast stage
<20% monocytic cells
AML-M3 >30% blasts + hypergranular promyelocytesb
Intense myeloperoxidase/Sudan black B reaction in virtually all cells
AML- Same criteria, except that granules within most promyelocytes very inconspicuous and nuclei highly
M3m grooved and reniform
AML-M4 Monocytosis (≥5 × 109/L); increased lysozyme
≥30% myeloblasts + monoblasts + promonocytes
>20% Sudan black B/myeloperoxidase-positive cells
>20% NSE positive cells
AML-M4 Same criteria as AML-M4 + abnormal eosinophils in bone marrow

eos
AML-M5 ≥30% myeloblasts + monoblasts + promonocytes

a,b
<20% Sudan black B/myeloperoxidase-positive cells
>80% NSE positive cells
Monoblasts predominate in M5a
Promonocytes predominate in M5b
AML-M6 ≥30% of nonerythroid cells are myeloblasts

a,b
>50% erythroid elements
Erythroblasts predominate (suggested AML-M6b category)

AML-M7 ≥30% blasts (myeloblasts + megakaryoblasts)
>30% megakaryocytic elements defined by immunophenotyping or ultrastructural electron

microscopy
aCurrently the required blast percentage for morphologic AML diagnosis is 20% or more.
Morphologic and Immunophenotypic Basis of AML Classification

The morphologic diagnosis of AML depends on the identification of a variety of types of blasts and other immature cells
that define the subtype of AML. Accordingly, cytochemical stains are valuable in delineating specific types of immature
myeloid cells and greatly enhance accuracy in the diagnosis of AML (Table 23-13). Flow cytometry is useful in determining
the lineage and stage of maturation in many cases of AML; immunophenotyping is also critical in the successful
identification of both undifferentiated myeloid leukemias and acute megakaryoblastic leukemias (198). Although marker
selection is limited, paraffin immunoperoxidase techniques can be used to assess for immaturity (CD34, TdT),
myeloid/monocytic maturation (myeloperoxidase, CD43, lysozyme, CD68, CD15), erythroid maturation (hemoglobin A), and
megakaryocyte maturation (CD61, factor VIII) (126, 160, 191) (Figure 23-13A,B).
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Table 23-13 ▪ MORPHOLOGIC FEATURES OF BLASTS AND OTHER IMMATURE CELLS
Type of Cell Key Morphologic Features Cytochemistry Immunophenotypic

Features
Myeloblast Large nucleus with finely dispersed chromatin SBB+, MPO+ HLA-DR, CD33,
and variably prominent nucleoli. Relatively high CD13, anti-MPO,
nuclear/cytoplasmic ratio CD34
Variable number of cytoplasmic granules, may
be concentrated in limited portion of cytoplasm
Promyelocyte Nuclear chromatin slightly condensed; nucleoli SBB+, MPO+ CD33, CD13, anti-
variably prominent; nucleus often eccentric and MPO
Golgi zone may be apparent
Numerous cytoplasmic granules that may be
more dispersed throughout cytoplasm
In APL intense cytoplasmic granularity usually
present, and nuclear configuration variable, but
nuclear folding and lobulation characteristic of
microgranular variant of APL
Monoblast Moderate-to-low nuclear to cytoplasmic ratio, NSE+ HLA-DR, CD33,

nuclear chromatin finely dispersed with variably CD13, vCD14, CD4
prominent nucleoli; nuclei round to folded
Abundant, slightly basophilic cytoplasm
containing fine granulation and occasional
vacuoles
Promonocyte Slightly condensed nuclear chromatin; variably NSE+ HLA-DR, CD33,

prominent nucleoli CD13, CD14, CD4
Abundant finely granular blue/gray cytoplasm
that may be vacuolated
Very monocytic appearance with nuclear
immaturity
Erythroblast Relatively high nuclear/cytoplasmic ratio PAS+ Glycophorin A, Hgb

Nucleus round with slightly condensed A
chromatin; nucleoli variably prominent
Moderate amounts of deeply basophilic
cytoplasm that may be vacuolated
Megakaryoblast Highly variable morphologic features PAS+ CD41, CD61, HLA-

Often not recognizable without special studies DR, v Factor VIII
May be lymphoid-appearing with high nuclear to
cytoplasmic ratio
Nuclear chromatin fine to variably condensed
Cytoplasm may be scant to moderate, is usually
agranular or contains a few granules; blebbing
or budding of cytoplasm may be evident
Blasts may form cohesive clumps
SBB, Sudan black B; MPO, myeloperoxidase; APL, acute promyelocytic leukemia; NSE, neuron specific esterase;
PAS, periodic acid-Schiff; Hgb, hemoglobin.
FIGURE 23-13 ▪ A: Acute myeloid leukemia with maturation. B: Flow cytometry histograms showadim CD45 population,
coexpressing CD34, CD33, CD13, and CD117.
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Table 23-14 ▪ COMMON GENOTYPIC ABNORMALITIES INAML

Numerical Abnormalities Comments
-5,-7,-X,-Y,+8,del(5q), del(7q),+21 Found in 30%-40% of AML in children
Linked to adverse outcome
Structural Abnormalities
t(8;21)(q22;q22) AML 1/ETO; 10%-15% of pediatric AML
t(15;17)(q22;q11-21) PML/RARα;8%-15% of pediatric AML
Inv16(p13q22) CBFβ/MYH11; 5%-12% of pediatric AML
11q23 MLL (multiple partner genes); 5%-20% of pediatric AML
Biologic Basis of AML Classification

Because of the implications for treatment and prognosis, cytogenetic analysis is now a standard of care for patients with
AML. Clonal cytogenetic abnormalities are identified less frequently than in ALL, approximating 50% to 80% of cases (161)
(Table 23-14). Cytogenetic abnormalities, specific for a FAB subtype of AML, are rarely encountered. The strongest
association between FAB-classification and karyotype is in AML-M3 with most cases demonstrating t(15;17). Less clear
associations include t(8;21) with AML-M2, and inv(16) with AML-M4 and eosinophilia. Abnormalities of 11q23 have been
correlated with AML of monocytic differentiation. In the WHO classification of AML, the traditional lineage-based
classification is retained and distinct biologic subtypes based on genotype are integrated (Table 23-15). Cases of AML
characterized by reciprocal translocations occur more commonly in children than in adults (119, 161). The four most
frequent types of translocation-induced AML represent distinct clinicopathologic entities with characteristic morphologic
and clinical parameters.
Table 23-15 ▪ SUMMARY OFTHE WHO CLASSIFICATION OF ACUTE MYELOID LEUKEMIAS
A. Acute myeloid leukemia with recurrent genetic abnormalities
Disease Clinical Morphology Immunophenotype Prognosis
Acute myeloid Often presents Blasts with long slender Auer CD13+, CD33+, Favorable
leukemia with with rods, abnormal granulation
MPO+, CD19+,
t(8;21)(q22;q22); extramedullary
(AML1/ETO) disease CD34+, CD56+
Acute myeloid Occasionally Abnormal eosinophils with CD13+, CD33+, Favorable

leukemia with presents with large basophilic granules,
MPO+; frequently
abnormal bone extramedullary decreased lobation
marrow disease CD4+, CD14+,
eosinophils CD11b+, CD11c+,
inv(16)(p13q22) or CD64+, CD36+,
t(16;16)(p13;q22) lysozyme+
(CBFb/MYH11)
Acute Coagulopathy, Abnormal promyelocytes with CD13+ Favorable
promyelocytic normal/low multiple Auer rods (heterogeneous),
leukemia (AML WBC predominate CD33+ (bright),
with t(15;17) (hypergranular
HLA-DR-, CD34−
(q22;q12); variant); high
(PML/RARa and WBC
variants) (hypogranular
variant)
Acute myeloid Frequently Monocytic blasts predominate Variable CD13 and Intermediate
leukemia with occurs in CD33+, CD4+, survival
11q23 (MLL) children
CD14+, CD11b+,
abnormalities
CD11c+, CD64+,
CD36+, lysozyme+
B. Acute myeloid leukemia with multilineage dysplasia
Following a myelodysplastic syndrome or myelodysplastic syndrome/myeloproliferative disorder

Without antecedent myelodysplastic syndrome
C. Acute myeloid leukemia and myelodysplastic syndromes, therapy related
Alkylating agent related

Topoisomerase type II inhibitor-related (some may be lymphoid)
Other types
D. Acute myeloid leukemia not otherwise categorized
Disease Clinical Morphology/Cytochemistry Immunophenotype Prognosis
Acute myeloid Usually <3% of blasts MPO+, <3% of Often CD13+, Unfavorable
leukemia presents in
blasts NBE+ CD33+, CD117+,
minimally adulthood,
differentiated cytopenias CD34+, CD38+,
HLA-DR+
Acute myeloid Usually Blasts comprise ≥90% of Often CD13+, Unfavorable

leukemia without presents in nonerythroid cells; ≥3% of
CD33+, CD34+,
maturation adulthood, blasts MPO+, ≥3% of blasts
cytopenias, CD117+, MPO+
NBE+
occasionally
with markedly
increased WBC
Acute myeloid Variable age ≥3% of blasts MPO+, ≤3% of Usually CD13+, Variable
leukemia with range and
blasts NBE+ CD33+, CD15+;
maturation symptomatology
variable CD117+,
CD34+, HLA-DR+
Acute Anemia, fever, >20% blasts; ≥monocytes Usually CD13+, Variable

myelomonocytic fatigue; WBC and precursors; ≥neutrophils
leukemia usually elevated and precursors; ≥3% of blasts CD33+; Often CD4+,
MPO+, ≥3% of blasts usually CD14+, CD11b+,
NBE+* CD11c+, CD64+,
CD36+, lysozyme+
Acute monoblastic Most common >80% monocytic cells, of Variable CD13+, Unfavorable
leukemia in children, which >80% are monoblasts;
CD33+, CD117+;
often presents <20% neutrophils and
with precursors; <3% of blasts Often CD14+, CD4+,
extramedullary MPO+, >3% of blasts NBE+ CD11b+, CD11c+,
disease, CD64+, CD68+,
bleeding CD36+, lysozyme+
disorders
Acute monocytic Most common ≥80% monocytic cells, of Variable CD13+, Unfavorable
leukemia in adults, often which the majority are
CD33+, CD117+;
presents with promonocytes; <20%
extramedullary neutrophils and precursors; Often CD14+, CD4+,
disease, <3% of blasts MPO+, ≥3% of CD11b+, CD11c+,
bleeding blasts NBE+ CD64+, CD68+,
disorders CD36+, lysozyme+
Acute erythroid Adults; anemia ≥50% of entire nucleated Erythroblasts are Unfavorable
leukemia population is erythroid and glycophorin A+ and
(erythroid/myeloid) ≥20% myeloblasts in hemoglobin A+;
nonerythroid population; >3% myeloblasts are
of blasts may be MPO+ CD13+, CD33+,
CD117+, and MPO+
Pure erythroid Extremely rare >80% of cells are immature Blasts are Unfavorable
leukemia erythroid cells; no significant sometimes
myeloblast component; <3% glycophorin A+ and
of blasts MPO+, ≥3% of hemoglobin A+
blasts NBE+
Acute Cytopenias Dysplastic megakaryocytes, Usually CD41+, Poor

megakaryoblastic Blasts often have cytoplasmic
CD61+; occasionally
leukemia pseudopods. Abnormal
platelets and megakaryocyte CD13+, CD33+;
fragments in peripheral blood; CD34-, CD45-, HLA-
DR-
usually <3% of blasts MPO+
and <3% of blasts NBE+
Acute basophilic Very rare Blasts are toluidine blue+; Usually CD13+, Difficult to
leukemia usually <3% of blasts MPO+, predict due
CD33+, CD34+,
<3% of blasts NBE+ to low
HLA-DR+, CD9+
number of
reported
cases,
probably
poor
Acute Very rare, Pan-hyperplasia, dysplastic CD13+, CD33+, Poor
panmyelosis with adults, megakaryocytes; increased
CD117+, MPO+;
myelofibrosis pancytopenia reticulin fibrosis
some cases express
with no/minimal
erythroid or
splenomegaly
megakaryocytic
antigens
MPO, myeloperoxidase; NBE, naphthyl butyrate esterase; WBC, white blood count (e14).
AML with t(8;21)

The fusion gene AML1-ETO produced by t(8;21)(q22;q22) accounts for 10% to 15% of childhood AML cases and usually
shows myeloid maturation (119, 161). Patients may present with extramedullary myeloid tumors. The bone marrow is
characteristically effaced by myeloblasts and maturing myeloid elements, which may exhibit an odd, salmon-colored
cytoplasm with a peripheral basophilic rim. Dysplastic findings may lead to a mistaken diagnosis of myelodysplasia,
especially if the blast count is less than 20%. Auer rods with tapered ends are typically readily apparent (136).
Coexpression of CD19 and CD56 has been noted in cases of t(8;21) AML (86, 148). AML t(8;21) has a favorable prognosis
in adults, however, additional KIT activating mutations confer poorer prognosis.
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AML with t(15;17)
Acute promyelocytic leukemia (APL) is a distinct clinicopathologic entity with t(15;17)(q22;q11-12) resulting in a PML-RARa
fusion gene (119, 161) (Figure 23-14). Older children and young adults are most commonly affected and present with
pancytopenia, profound thrombocytopenia, and marked coagulopathy (12). In the common, hypergranular subtype,
promyelocytes are inconspicuous in blood, whereas the bone marrow is effaced by intensely granulated promyelocytes
(Figure 23-15). Auer rods are numerous and often stacked in bundles (Figure 23-15, inset). A microgranular variant
accounts for about one-fourth of cases and is characterized by leukocytosis and hypogranular promyelocytes exhibiting
marked nuclear folding. Intense staining with Sudan black B and myeloperoxidase characterizes both the common and the
microgranular subtypes. Immunophenotypic studies show a CD34 and HLA-DR negative phenotype that indicates maturity,
but is not diagnostic of APL. The clinical outcome is good if coagulopathy is arrested by all-trans retinoic acid therapy;
conventional chemotherapy is also required (56, 182).
FIGURE 23-14 ▪ The translocation (15;17) is exclusively observed in APL; arrows indicate breakpoints.
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FIGURE 23-15 ▪ Several intensely granular promyelocytes are evident in the aspirate smear showing APL; Auer rods can
be seen (inset).
Acute Myeloid Leukemia with inv (16)

Acute myelomonocytic leukemia (AMML) with bone marrow eosinophilia is often associated with inv16(p13q22) and CBFb-
MYH11 fusion gene formation (Figure 23-16). Another translocation that is less commonly identified is t(16;16). Together,
these are seen in approximately 10% of pediatric patients. This subtype may be associated with extramedullary myeloid
cell tumors (myeloid sarcoma). The bone marrow eosinophils often exhibit mixed eosinophilbasophil granules (8) (Figure
23-17). However, not all cases with this cytogenetic abnormality exhibit eosinophilia or AMML morphology. Prognosis is
good with high likelihood of cure by chemotherapy and prolonged disease free course (102, 116).
AML with 11q23 Abnormalities

11q23 is the defining translocation in this AML with multiple partner genes. In children, the MLL gene rearrangement is
most frequently noted in congenital myeloid leukemias, often monoblastic, especially in t(4;11) (31, 74, 164). The MLL
gene rearrangement also typifies the therapy-related monocytic/monoblastic leukemias that occur following topoisomerase
II inhibitor therapy (Figure 23-18). These occur 1 to 3 years after epipodophyllotoxin or related therapies and are
characterized by the abrupt onset of cytopenias and bone marrow effacement by promonocytes/monoblasts. Although
initial remissions are generally achieved, the overall outcome is poor in therapy-related AML with 11q23 translocations
(150, 153).
FIGURE 23-16 ▪ Florescent in situ hybridization (FISH) analysis of the AMML cells demonstrates inversion of chromosome
16 utilizing breakapart FISH probes (two green, two red signals).
FIGURE 23-17 ▪ AMML demonstrates myeloid blasts and immature monocytic cells; scattered cells with eo-baso- granules
were evident (inset).
AML with Monosomy 7

This abnormality is much more commonly seen in pediatric myelodysplasias; however, approximately 5% to 7% of pediatric
de novo AML cases are associated with monosomy 7. The vast majority occurs in patients with a preceding history of
myelodysplasia, monosomy 7, or chronic myelomonocytic leukemia. (89, 110, 142). The prognosis is uniformly poor.
FIGURE 23-18 ▪ 11q23-associated acute monocytic leukemia evolving in post-therapy setting; the monocytic nature is
confirmed by NSE staining (inset).
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FIGURE 23-19 ▪ Acute myeloid leukemia evolved from myelodysplastic syndrome. Note dyserythropoiesis (arrowheads)
and abnormally granular myelocytes with nuclear cytoplasmic asynchrony (arrows).
Myelodysplastic and Chronic Myeloproliferative Disorders in Children

In young children, a chronic myelodysplastic/myeloproliferative disorder is characterized by leukocytosis with both
neutrophilia and monocytosis, a left shift to blasts, multilineage dyspoiesis, anemia, and thrombocytopenia (Figure 23-19;
Table 23-16). Typically, hepatosplenomegaly and skin lesions also develop (48, 109, 120). In some children, a
constitutional monosomy 7 has been identified, and the term infantile monosomy 7 syndrome has been utilized to describe
such cases. In other infants, a very similar blood, bone marrow, and clinical picture develops in conjunction with increased
levels of hemoglobin F and various RAS or NF1 gene mutations (48, 57, 176). The latter disease, originally termed juvenile
chronic myelogenous leukemia (CML) and later called juvenile myelomonocytic leukemia, is differentiated from CML by
the Philadelphia chromosome-breakpoint cluster region-abl gene rearrangement (58, 60, 61). Despite the disparate
terminology, so-called infantile monosomy 7 syndrome and juvenile myelomonocytic leukemias are currently thought to
comprise a spectrum of infantile myelodysplastic/myeloproliferative processes that are frequently associated with
monosomy 7/del(7q) (109, 120). The WHO Working Group placed these disorders into a hybrid
“myelodysplastic/myeloproliferative disease” category to distinguish them from both conventional chronic myeloproliferative
disorders and “adult-type” myelodysplasia (77).
Table 23-16 ▪ MYELODYSPLASTIC DISORDERS IN CHILDHOOD
I. Patients ≤5 years
Hybrid myelodysplastic/myeloproliferative picture

Leukocytosis with monocytosis, left shift, dysplasia
Frequent extramedullary disease with organomegaly, lymphadenopathy
Association with constitutional genetic/immune disorders
Striking incidence of monosomy 7
Male predominance
Terms infantile monosomy 7 syndrome, juvenile CML (JCML), CMML, and JMML used
Variable disease course, overall poor prognosis
II. Patients >5 years
Typical features of “adult” type MDS

Includes therapy-related cases
More high-grade forms of MDS predominate
Monosomy 7 common finding but often have other cytogenetic
FIGURE 23-20 ▪ Severe dyserythropoiesis and myeloid left shift in a young patient with leukocytosis, anemia, and
thrombocytopenia. Note megakaryocyte with separate lobes (center field). Cytogenetic studies showed complex
cytogenetics including del(7).
Conventional chronic myeloproliferative disorders, including CML (Ph+), also occur in children. CML accounts for
approximately 5% of childhood leukemias and usually affects adolescents; cases in earlier childhood have been reported
(58, 165). Clinical manifestations and course of Ph+ CML are similar to adult cases. In addition, rare reports describe
familial/constitutional myeloproliferative disorders, including essential thrombocythemia and chronic idiopathic myelofibrosis
(44, 90, 170). “Adult-type” myelodysplastic syndromes, characterized by cytopenias and multilineage dysplasia, also affect
children (Figure 23-20). The children are typically older and, in some cases, the condition is therapy related.
NEOPLASTIC HISTIOCYTIC DISORDERS IN BONE MARROW

Neoplastic Histiocytoses
Clonal disorders characterized by increased numbers of bone marrow monocytes/histiocytes include acute and chronic
monocytic/myelomonocytic leukemias (described earlier), Langerhans cell histiocytosis, and malignant
histiocytosis/histiocytic sarcoma. Minimal-to-extensive bone marrow involvement is evident in at least 20% of patients with
multisystem Langerhans cell histiocytosis (7), and
P.1033
patients with extensive bone marrow replacement often exhibit pancytopenia and extensive extramedullary disease,
associated with a poor prognosis. On smear preparations, the nuclear chromatin of Langerhans cells is delicately clumped,
and the cytoplasm is abundant and pale and may contain vacuoles or granules. More characteristic Langerhans cell
morphology is appreciated on clot or biopsy sections in the form of small, inconspicuous clusters (169) or discrete
collections of cells with admixed neutrophils, eosinophils, and multinucleated giant cells. Other nonspecific abnormalities,
such as eosinophilia and mild reticulin fibrosis, may be present. S100 and CD1a are useful immunophenotypic markers for
Langerhans cell histiocytosis, and both stains can be performed on paraffin sections (169).
FIGURE 23-21 ▪ Histiocytic sarcoma extensively involves spleen in a patient with disseminated malignant histiocytosis.
Anaplastic large cell lymphoma must be ruled out.
The bone marrow infiltrates in cases of so-called malignant histiocytosis (proposed WHO designation of disseminated
histiocytic sarcoma), unlike those in acute and chronic leukemias, are often inconspicuous, accounting for only a minority
of the total nucleated cells (72). Malignant histiocytosis/histiocytic sarcoma is characterized by a diffuse admixture of
hematopoietic elements, very large cells, and marked nuclear atypia (Figure 23-21). If present, phagocytosis is minimal. On
biopsy sections, the cells are either individually dispersed or in small clusters and easily overlooked. The
immunophenotypic features of malignant histiocytosis are somewhat variable, but most cells express myeloid and
monocytic antigens, whereas others appear more akin to immune accessory cells, demonstrating S100, CD21, and/or
CD35 reactivity (181). Although often weak, nonspecific esterase (NSE) activity is a useful marker for these cells.
The morphologic and clinical features of several disorders substantially overlap with those of malignant histiocytosis,
including hemophagocytic lymphohistiocytosis, anaplastic large cell lymphoma expressing Ki-1 antigen, acute monoblastic
leukemias, and rare NK-cell and B-cell lymphomas (46, 131, 133). The distinction between malignant histiocytosis and
anaplastic large cell lymphoma is based on immunophenotypic, cytogenetic, and molecular studies. Unlike malignant
histiocytosis/histiocytic sarcoma, anaplastic large cell lymphomas are usually T-cell neoplasms with a unique chromosomal
abnormality, t(2;5) (p23;q35), if ALK positive (14). Because T-cell neoplasms, and more rarely NK-cell or B-cell neoplasms,
can closely mimic histiocytic sarcoma, immunophenotyping and molecular techniques should be used to exclude more
common T-, NK-, or B-cell neoplasm in cases of possible histiocytic sarcoma (20, 46, 133).
The distinction between malignant histiocytosis/histiocytic sarcoma and acute monoblastic leukemia is occasionally
problematic and somewhat semantic, as both neoplasms are derived from the same hematopoietic lineage. Correct
diagnosis of malignant histiocytosis reportedly occurs with a frequency of 0.5 to 1/1,000 compared to other malignant
lymphomas (101). Some authors recommend using the percentage of neoplastic cells in bone marrow to distinguish
between monocytic leukemia (>25% blasts) and malignant histiocytosis/histiocytic sarcoma (<25% malignant cells in bone
marrow) (20). Presence of a mass lesion, cytologic dysplasia, and immunophenotypic studies in addition to the clinical
presentation are helpful in establishing the correct diagnosis. Historically, an incorrect diagnosis of malignant histiocytoses
has been rendered in the setting of a histiocyterich lymphoma with a relatively small lymphoid component, or in the setting
of anaplastic large cell lymphoma (histiocytic variant) or in the setting of hemophagocytic syndrome which causes life-
threatening illness and raises concern of a hematopoietic malignancy (21, 113).
Histiocytoses of Varied Biologic Behavior

This group of disorders is characterized by an unpredictable biologic behavior and includes dendritic cell-related disorders
for example xanthogranuloma disseminatum (within the family of juvenile xanthogranuloma “JXG”; systemic JXG) and
macrophage-related disorders ranging from benign hemophagocytic histiocytoses to solitary histiocytomas to name a few
(24, 54).
Metastatic Disorders in Bone Marrow

The incidence of metastasis of childhood solid tumors to bone marrow is quite variable. A significant contributing factor is
the adequacy of the bone marrow biopsy sample for optimal staging. Marrow metastases are frequently seen in
neuroblastomas, peripheral neuroectodermal tumor, rhabdomyosarcoma, synovial sarcoma, and lymphomas. The
metastatic rates are extremely low for Wilms tumor, hepatoblastomas, and retinoblastomas (37, 154, 155). In children,
metastatic small blue cell tumors, such as Ewing sarcoma, rhabdomyosarcoma, retinoblastoma, and neuroblastoma, may
exhibit blast-like nuclear features and can be seen individually dispersed on aspirate smears, closely mimicking acute
leukemia in bone marrow (3, 37, 45, 76, 80, 147) (Figure 23-22). In fact, neuroblastoma ranks as the second most frequent
cause of bone marrow infiltration in children;
P.1034
the first being ALL. Confusion with acute leukemia is especially likely when the primary lesion is occult and when the bone
marrow is extensively replaced by metastatic tumor. Although some evidence of tumor cell cohesion is usually apparent on
aspirate smears or biopsy sections, a variety of immunologic techniques can be used to distinguish these metastatic
lesions conclusively from a primary hematologic neoplasm. Immunoperoxidase staining for various solid tumor-associated
antigens can be employed and bone marrow biopsies are considered more effective in diagnosis of metastatic disease as
the aspirates are often “aspicular” due to fibrosis (201).
FIGURE 23-22 ▪ A,B: Bone marrow biopsy shows extensive effacement by metastatic neuroblastoma. The malignant cells
tend to form cohesive clusters in the smear; a mimic of leukemia.
Bone Marrow Transplantation

Bone marrow transplantation is currently available as a therapeutic option for a variety of neoplastic and non-neoplastic
disorders (5, 82). It is particularly considered as an option in refractory acute leukemias and lymphomas and untreatable
solid tumors (84). Two kinds of bone marrow transplants are usually available: autologous and allogeneic (HLA-matched
donor). The concept behind bone marrow transplantation is reconstitution of normal bone marrow elements after cure of
the pathologic marrow. In diseases involving clonal stem cell abnormalities, an allogeneic transplant is the best chance for
curing the disease. This follows intense chemotherapy with or without total body irradiation to eradicate the bone marrow
and immune cells as well as the neoplastic cells. In other instances, collections of bone marrow or peripheral blood stem
cells from the patient may be used for an autologous bone marrow transplantation (172). This does not require marrow
ablation and can be performed if the bone marrow is free of neoplastic process. Autologous transplantation does not carry
the risk of serious complications associated with allogeneic transplants such as graft versus host disease, post-transplant
lymphoproliferative disorders, myelodysplasia, secondary hematologic malignancies, solid tumors, and graft rejection
(190).
Morphologic Expectations in Post-Transplant Bone Marrow Samples

In the immediate post-transplant period (1 to 7 days), findings resemble chemoablation, that is, bone marrow fibrosis,
serous atrophy, and nonspecific edema with a predominance of stromal cells (162). Bone marrow engraftment starts as
early as 7 days and is characterized by small colonies of red blood cells (which usually appear first), followed by white
blood cells and megakaryocytes (39). Peripheral blood counts recover in the same general order (193). Failure of bone
marrow cellularity to normalize (50% of normal cellularity by day 21) and persistent peripheral cytopenia is a significant
cause for concern of graft failure (failure of transplanted cells to engraft by day 28). Significant increase in peripheral blood
counts may, on the other hand, herald recurrence of primary disease or cytokine effect (23). Once graft failure has been
excluded as a potential cause, engraftment status and recurrence of disease is monitored.
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Chapter 24
Soft Tissue
Louis P. Dehner
The pediatric surgical pathologist who is presented with a “soft-tissue tumor” in a child may be confronted with a
wide spectrum of pathology ranging from an enlarged lymph node, fibroinflammatory process in the superficial
soft tissues, maldevelopment of vessels or lymphatics, or a true neoplasm (Table 24-1). Some of the soft-tissue
tumors (STTs) arise in the skin (infantile myofibroma) with involvement of the subcutis, subcutis (infantile
fibromatosis or lipofibromatosis), or at the level of the fascia and the deep soft tissues. Soft-tissue sarcomas
(STSs) in children may be organ based as in the case of embryonal rhabdomyosarcoma (ERMS), whereas
others [such as Ewing sarcoma-primitive neuroectodermal tumor (EWS-PNET), alveolar rhabdomyosarcoma
(ARMS), and synovial sarcoma (SS)] have the more familiar pattern of presenting in the peripheral soft tissues of
the extremities. Immunohistochemistry (IHC) and molecular diagnostic studies have facilitated the diagnostic
evaluation of STSs in children, but the entire exercise is initiated with a differential diagnosis.
One of the more common clinical diagnoses accompanying a pediatric surgical specimen is “rule out soft-tissue
tumor.” If this is the case, the neoplasm is benign in the majority of cases and more often than not is a vascular
tumor of one type or another. Of course, there is the dilemma in some vascular tumors of a true neoplasm or
malformation, which may or may not bear upon the surgical management. Vascular, neurogenic, fibrous-
myofibroblastic, and myogenic tumors account for the majority (70% to 85%) of all soft-tissue neoplasms in
children. Most of these tumors are benign (60% to 70% of cases) where there is a predilection for the trunk,
extremities, and head and neck region in descending order of frequency; these tumors as a group generally
come to clinical attention at or before 10 years of age. Some of the more aggressive STS are diagnosed in the
second decade such as EWS-PNET, ARMS, and SS; however, like all generalizations, there are exceptions that
each one of these neoplasms is recognized in the first 2 to 3 years of life (142). Arguably one of the most
malignant and treatment-resistant soft-tissue neoplasms of childhood is the malignant rhabdoid tumor (MRT) with
its many primary organ-based primary sites (kidney, liver, and central nervous system), which is also seen in a
variety of nonorgans soft-tissue locations including the head and neck and mediastinum. Congenital STTs are
mainly restricted to vasoformative proliferations, teratomas arising in the sacrococcygeal soft tissues,
retroperitoneum, and head and neck without a specific localization to an organ. Other less common STTs
presenting at birth or in the first month of life are congenital infantile fibrosarcoma (CIFS),
myofibromamyofibromatosis, granular cell tumor (GCT) (oral cavity), and embryonal or rarely ARMS. However,
one should be prepared for the unanticipated when the clinical impression is a STT in a child.
Table 24-1▪SOME EXAMPLES OF “SOFT TISSUE TUMORS” IN CHILDREN
Developmental cyst (e.g., dermoid cyst)
Inflammatory process (e.g., granulomatous lymphadenitis, fibroinflammatory process in soft tissues,

deep granuloma annulare)
Vascular malformation or neoplasm
Neoplasms (e.g., pilomatrixoma, myofibroma)

The classification of STTs in children is accommodated for the most part by the World Health Organization
(WHO) classification (75). Traditionally, STTs are classified on the basis of tissue differentiation and phenotype
as determined by IHC. Another ancillary technique, molecular genetics, has come to occupy an increasingly
important role in the diagnosis of STSs in children (74, 170, 187) (Table 24-2). One of the first STS specific,
nonrandom chromosomal abnormalities, t(11;22) (q24;q12) translocation, was identified in EWS-PNET, the
second most common STS of childhood (92). Emerging from this initial observation is the appreciation that there
is a family of STSs with a predilection for children, adolescents, and young adults in which the EWS gene on
chromosome 22q is the fusion partner in a number of non-random translocations; these chromosomal
perturbations can be detected utilizing fluorescent in situ hybridization (FISH) on nuclear preparations obtained
from formalin-fixed, paraffin-embedded tissue.
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Table 24-2 ▪ SOFT TISSUE NEOPLASMS IN CHILDREN AND THEIR RECURRING

CYTOGENETIC ABNORMALITIES
Tumor Type Cytogenetic Abnormality LOCI
EWS-PNETa t(11;22)(q24;q12) EWS-FLI1 (85%)

t(21;22)(q22;q12) EWS-ERG (10%)
t(7;22)(p22;q12) ETV1-EWS
t(17;22)(q12;q12) E1AF-EWS
t(2;22)(q33;q12) FEV-EWS
DSRCTa t(11;22)(q13;q12) EWS-WT1
CCS of soft tissue t(12;22)(q13;q12) EWS-ATF1

(melanoma of soft
parts)a
EMCa t(9;17)(q22;q11.2) EWS-CHN/TEC

t(9;15)(q22;q21) RBP56-CHN/TEC
t(12;22)(q13;q12) TCF12-CHN/TEC
AFHa t(9;22)(q22;q12) EWSR1-ATF1

t(12;16)(q13;q11) FUS-ATF1
Myxoid LPSa t(12;16)(q13;p11) FUS-CHOP

t(12;22)(q13;p12) EWSR1-CHOP
ERMS LOHat 11p15, gains 2+, 7+, 8+, 11+, 12+, 20+,
21+, 13q 21+,
20+; Losses 1p35-36-, 3-, 7-, 6-, 9q22-, 14q 21-
32-, 17-
ARMS t(2;13)(q35;q14) PAX 3-FKHR
t(1;13)(p36;q14) PAX7-FKHR
CIFS t(12;15)(p13;q25) ETV6-NTRK3

SS t(X;18)(p11;q11) SYT-SSX1(biphasic)
SYT-
SSX2(monophasic)
SYT-SSX4
ASPS der(17)t(X;17)(p11.2;q25) ASPL-TFE3

MRT deletion and mutation in 22q11 SMAR CB1/INI1
Lipoblastoma 8q11-13 (hSNF5/INI1)
IMT Translocations involving 2p23 (ALK) t(2;17) PLAG1
(p23;q23) ALK-CLTC
ALK-TPM3
ALK-TPM4
ALK-CAR5
ALK-RANBP2
ALK-TMP4
ALK-SEC31L
LGFS t(7;16)(q32-34;p11) FUS-CREB3L2

t(11;16)(p11;p11) FUS-CREB3L1
aExtended EWS family of tumor.
The approach to the pathologic diagnosis of a STT from a child has the same starting point as one in an adult, a
careful gross examination which is followed by the selection of tissue blocks from representative areas of the
tumor based on the macroscopic features (if one has digital photographic capability, a gross illustration can often
substitute for a long narrative description). The decision about the number of tissue blocks is guided by the size
of the specimen and the variability of gross features from viable areas to those with a necrotic or hemorrhagic
appearance. Many blocks may be required to identify any residual tumor in those cases with preoperative
adjuvant therapy. If the specimen is submitted as a gross resection, the peripheral margins and any attached
organs or bony structures should be identified and the margins tattooed with India ink or other dyes that will
survive processing in order to evaluate the adequacy of the surgical margins. Margins of surgical resection are
generally reported as “free of tumor” or not. Determining the distance between the tumor and the tumor-free
margin by gross and microscopic examination is difficult in many cases. Some margins are limited by the
constraints of the anatomy as it relates to neurovascular bundles or bony structures and can be even more
challenging in an infant or small child.
If the specimen is a small biopsy and submitted for intraoperative frozen section consultation, very little tissue
may remain for permanent sections; thus, another tissue sample should be obtained, if at all possible, as a
contingency. Once the biopsy has been examined, it should be marked with an appropriate dye and placed in a
small tea bag before routine tissue processing. If facilities are available and the tissue sample is judged to be
more than adequate for histological examination, cytogenetics and tissue banking should be considered as well.
The world of STTs in children can be divided into three morphologic spheres: vascular structures of varying
morphology, spindle cells, and small or not so small round cells (Table 24-3). Various spindle cell tumors are
listed in this table, and many of them are familiar to the pediatric
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pathologist. Some of the diagnoses are made with relative ease through one’s own experience with the
appreciation of certain histologic features, which separate that particular round cell or spindle cell proliferation
from all of the other similar appearing tumors. As one peruses Table 24-3, it becomes apparent that some of
these STTs occur almost exclusively in the first two decades of life whereas others are seen more often in
adolescence or early adulthood (65).
Table 24-3 ▪ MORPHOLOGIC THEMES IN SSTS IN CHILDREN SOME DIFFERENTIAL

DIAGNOSTIC CONSIDERATIONS
Morphology Tumor Types
Round and not so “rounded” RMS (both ERMS and ARMS)

small cells EWS-PNET
Mononuclear histiocytic-like cells
LCH and JXG
Non-histiocytic hematolymphoid neoplasms (lymphoma,
granulocytic sarcoma)
MRT
ASPS
PEComa
Epithelioid vascular tumor
ES
Spindle cells Fibrous tissue reaction

NF
Fibrous tumors—fibromatosis
CIFS
SS
IMT
DFSP
Schwannoma
KHE
LGFS
MPNST
Spindle cell RMS
Blood vessels and lymphatics Hemangiomas of diverse subtypes

Vascular malformations
Lymphangioma
Cystic hygroma
Table 24-4 ▪ MALIGNANT ROUND CELL TUMORS AND THEIR IMMUNOPHENOTYPE

Tumor VIM CK DES MYOD1- CD99 CD43 WT1 CHR BAF47
MYOG
RMS + − + + − − − − −
NB ± − − − − − − + −
EWS/PNET + ± − − + − − − −
WT-BL + − ± − − − + − −
DSRCT + ± + − ± − + ± −
SS-PD + + − − ± − − − −
MRT + + − − ± − − − +
UDS + ± − − ± − − − −
HPN + − − − ± + − − −
RMS, rhabdomyosarcoma; NB, neuroblastoma; EWS/PNET, Ewing sarcoma—primitive

neuroectodermal tumor; WT-BL, Wilms tumor—blastemal predominant; DSRCT, desmoplastic small
round cell tumors; SS—PD, synovial sarcoma—poorly differentiated; MRT, malignant rhabdoid tumor;
UDS, undifferentiated sarcoma; HPN, hematopoietic neoplasm to include lymphoid and non-lymphoid
tumors; VIM, vimentin; CK, cytokeratin; DES, desmin; MyoD1-Myog, MyoDI—myogenin; WT1, Wilms
tumor; CHR, chromogranin.
Ancillary IHC studies have had a profound, determinant effect upon the practice of surgical pathology over the
past 25 years, but especially so in the diagnosis of soft tissue and hematopoietic neoplasms; the same can be
said about the role of cytogenetics and molecular genetics for these two phenotypic categories. In the case of
malignancies in children, several of the more common neoplasms, soft tissue or otherwise, are morphologically
similar from the perspective of their more or less uniform composition of small or large malignant round cells
(Table 24-4). Of course, there are other accompanying features, which should be incorporated into the
differential diagnosis without the need to utilize every commercially available antibody in one’s IHC laboratory.
However, it is acknowledged that there are those cases in which successive waves of newly ordered
immunostains may be necessary in order to arrive at a final diagnosis or the realization for the need to send the
case to an outside consultant. In the meantime, the titer of anxiety is on the rise for all concerned. A difficult case
is a difficult case for no other reason than that it is a difficult case as an existential reality. Attention to the clinical
aspects including clinical laboratory studies can be helpful in crafting the differential diagnosis and guiding the
selection of stains. In the course of this chapter, there will be frequent references to Table 24-4, which should be
familiar to most pathologists with some level of experience with the malignant round cell tumors of childhood,
which are not necessarily all “small blue cells.” Each of these tumor types, less the undifferentiated round cell
sarcoma, has one or more molecular aberrations with diagnostic and prognostic implications in some cases.
There are other “round cell” neoplasms presenting in childhood, though also in adults, which are not included in
Table 24-4 but are cited in Table 24-3. These are neoplasms with the added characterization as “epithelioid”
whose cytomorphologic features are polygonal contours, a central nucleus, and abundant eosinophilic to clear
cytoplasm. Alveolar soft part sarcoma (ASPS), perivascular epithelioid cell tumor (PEComa), epithelioid
hemangioma and hemangioendothelioma (HE), and epithelioid sarcoma (ES) are among the principal tumor
types in this category.
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STSs in adults are commonly graded pathologically on the basis of mitotic activity [mitoses per 10 or 50 high
power fields (×400)], nuclear pleomorphism, and necrosis. Pleomorphic sarcoma (formerly many of these
sarcomas were interpreted as “malignant fibrous histiocytoma”) and leiomyosarcomas are the two most common
STSs in adults, which lend themselves to this traditional grading scheme. Pathologic grading of
nonrhabdomyosarcomas (RMSs) in the Children Oncology Group (COG) system relies on a combination of
specific histologic types of sarcomas in the grade 1 and grade 3 categories together with an assessment of
mitotic activity [<5 mitoses per 10 high-power fields (40× objective) and <15% of surface area necrosis, grade 2].
Those sarcomas not included in the grade 3 category but with an excess of 5 mitoses and/or greater than 15%
surface area necrosis are grade 3 sarcomas. Needless to say, differences in pathologic grading may arise
between observers in a particular soft-tissue sarcoma. CIFS has a low risk for metastasis and is a COG grade 1
sarcoma but can have considerable mitotic activity and extensive necrosis. Only a malignant peripheral nerve
sheath tumor (MPNST) with rhabdomyoblastic elements is a grade 3 sarcoma, but other MPNSTs are
presumably graded on mitotic activity and/or necrosis. The COG system of grading STS is less than satisfactory
in our opinion. Pathologic staging of STSs in children differs in several respects from its adult counterpart and
especially so in the case of RMSs in children where the primary site may have a significant impact upon the
stage and prognosis. Whether metastatic disease is detected or not in the unfavorable histology STS in children,
like ARMS and EWS-PNET, the assumption is made that micrometastases already exist and their presumed
presence serves as the rationale for systemic chemotherapy whether the tumor is localized at the time of clinical
presentation or not. A final point in these introductory comments about STSs in children is whether there is a
difference in the clinical behavior of the pathologically identical neoplasms, based upon age at diagnosis (65, 93,
191). There are some data to support that argument in the case of SS.
VASCULAR TUMORS
Vascular tumors of one type or another are among the most common STTs in children and account for 20% to
30% of all cases (40). As many as one third of all vascular tumors in childhood are diagnosed in the first year of
life and are one of the most frequently recognized tumors at or shortly after birth (39). Cutaneous and even deep
organ vascular tumors may present with multifocal sites of involvement in infancy. One such example is the infant
with a HE of the liver with multiple extrahepatic hemangiomas, often in the skin and less often in the spleen.
Traditionally, vascular lesions (or tumors) have been divided on the basis of their resemblance or appearance to
blood vessels or lymphatics. In some cases, this distinction between the two types of vessels may require IHC. A
pathologic distinction is also made between a vascular neoplasm and malformation, which has been incorporated
into a classification of “vascular anomalies” (33, 45) (Table 24-5). This classification is probably more widely
utilized by clinicians whereas the WHO classification of vascular tumors is more familiar to most pathologists (75)
(Table 24-5). The latter classification does not include vascular malformations or a separate category for
lymphatic or lymphangiomatous lesions. The rationale for the latter is the stated difficulty in reliably differentiating
vascular from lymphatic endothelium on the basis of histology alone; however, the monoclonal antibody D2-40 is
directed against a specific epitope on lymphatic endothelium (Fig 24-1). In regard to vascular anomalies and
malformations, a number of mutations have been identified in genes, which are important in vasculogenesis.
Benign tumors. Hemangioma or HE with any number of qualifying prefixes was found to be the most common
pathologic diagnosis of a STT seen in the first two decades of life during a 20-year period with an excess of
1,500 cases (40). The most common site is the skin in 25% of all vascular tumors. There is a preference for the
head and neck region in the case of the skin and soft tissue. Other sites include the deep soft tissues, bone,
orbit, parotid gland, skeletal muscle, and upper air passages including the nasal cavity and larynx. The
overwhelming majority (70% to 90%) of hemangiomas, and HEs for that matter, are initially recognized in the first
6 months (38).
Lobular capillary hemangioma (LCH) and hemangioma with the designation of “infantile,” “juvenile,” or
“capillary” are the two most commonly diagnosed vascular lesions in the skin and subcutaneous and/or deeper
soft tissues in children, respectively (153, 154). Both types of vascular tumors are characterized by a lobular
growth pattern, but hemangiomas in the subdermal soft tissues may have a more infiltrative pattern, often with
extension into the overlying dermis. The LCH or so-called pyogenic granuloma of the skin is a raised,
erythematous nodule with or without epidermal ulceration. Lobules of diminutive, hypercellular vascular spaces
with mitotic figures to the formation of patent capillaries with a fibrous stroma reflect the proliferative and
involutional stages in the evolution of LCH (Figure 24-1). Within the vascular lobules, this process can be
appreciated with the formation of patent capillaries. Arcades of feeding vessels are found at the base of the LCH.
The polypoid configuration of the LCH makes it prone to trauma with ulceration and inflammation to the point that
the underlying pathology may be obscured.
Hemangiomas of the soft tissues are found in the subcutis with or without a dermal component and may extend
into the fascia or less often into skeletal muscle. Though these tumors may appear well circumscribed clinically,
microscopic examination frequently demonstrates a more diffuse pattern. One of the characteristic features is the
superimposition of the vascular growth on existing structures such as lobules of subcutaneous fat or lymph
nodes. In addition to the circumscribed lobular foci, a more diffuse pattern of small and even larger vessels is
often present at the margins of excision. Like the LCH, the cellularity of these vascular
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lobules and the presence of patent vascular spaces are a manifestation of the proliferative phase of growth. It is
in the involutional or regressive phase that there is the formation of thrombi in varying stages of organization and
subsequent dystrophic calcifications and fibrosis. The latter process is well documented in the infantile HEs of
the liver. Organization of the clot may be associated with papillary endothelial hyperplasia (PEH, vegetant
hemangioma of Masson), which is also seen in hematomas. Though referred to as a hemangioma, most
examples of PEH are simply an exuberant organization of a thrombohematoma and should not be mistaken for
an angiosarcoma. Lack of nuclear pleomorphism and marked atypism in the PEH should give pause to the
diagnosis of angiosarcoma.
Table 24-5 ▪ CLASSIFICATION OF VASCULAR ANOMALIES
ISSVA WHO
Tumors
Hemangioma Hemangiomas (benign)
Infantile Subcutaneous—deep
Tufted angioma Capillary
Epithelioid Cavernous
Spindle cell Arteriovenous
Capillary Venous
Lobular capillary (pyogenic Intramuscular
granuloma)
HE Synovial
Kaposiform Epithelioid
Papillary intralymphatic (Dabska Angiomatosis
tumor) Lymphangioma
Retiform
Spindle cell HE (intermediate)
Epithelioid Kaposiform HEA
Angiosarcoma Retiform HEA

Papillary intralymphatic
angioendothelioma
Composite HEA
KS
Malignant
Epithelioid HEA
Angiosarcoma
Malformations
Simple (slow flow)
Capillary (portwine,
angiokeratoma)
Lymphatic (lymphangioma)
Venous (cavernous
hemangiomas)
Simple (fast flow)
Arterial (arteriovenous
hemangiomas)
Combined
AVM
Capillary—venous
Capillary—lymphatic venous
Lymphatic—venous
Capillary AVM
Other morphologic variants of hemangioma include the epithelioid, arteriovenous, and tufted types. The
cavernous and arteriovenous hemangiomas are considered examples of malformations in the International
Society for the Study of Vascular Anomalies (ISSVA) classification (Figure 24-2) (Table 24-5). Epithelioid
hemangioma is recognized in the skin (angiolymphoid hyperplasia with eosinophilia), in blood vessel in soft
tissues (vasocentric) and bone. The histologic hallmark is a prominent polygonal endothelial cell resembling an
epithelial cell. The endothelial cells may have cytoplasmic vacuoles that form small, capillary-sized vascular
spaces, which may resemble small glands (Figure 24-3). Lymphocytes and/or eosinophils accompany the
vascular proliferation and their presence is a useful clue to the diagnosis. However, epithelioid endothelial cells
have some similarity to Langerhans cells as well so that one may wish to include CD1a for Langerhans cells in
addition to CD31, CD34, and factor VIII-related antigen (endothelial markers) in the panel of IHC stains. Pericytes
are present at least focally around these small vessels, which can be demonstrated by smooth muscle actin
(SMA) positivity.
Hemangiomas in children are known to occur in specific sites in the soft tissues such as the synovium and
skeletal muscle. A network of capillary-sized vascular spaces occupies the supporting stroma of the synovium
and surrounding periarticular soft tissues, thus explaining hemarthrosis as the clinical presentation. Prominent
hemosiderin deposition and synovial hyperplasia are also features
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of hemophiliac synoviopathy-arthropathy and pigmented villonodular synovitis, which should be considered in the
differential diagnosis. Skeletal muscle hemangioma presents in the muscles of the head and neck region and
extremities, usually in children older than 10 years of age. All or a portion of the skeletal muscle is occupied by
an infiltrative process consisting of capillary-sized vascular spaces with or without a component of larger blood
vessels and adipose tissue, which has resulted in the alternative designation of infiltrating angiolipoma (Figure
24-4). The vessels occupy the interstitial tissues and occur within the muscle itself as small vessels infiltrating
between skeletal muscle fibers. An entire muscle can be involved by this diffusely infiltrative process. The
distinction from an angiomatosis is not always clear; however, the latter tends to involve multiple tissue layers
from the skin to bone. When an extremity is the site of involvement, as it is in cases of angiomatosis, the
differentiation between the skeletal muscle hemangioma and the latter may be one of degree or diagnostic
preference. The pathogenesis of both is more in the realm of a malformation than a neoplasm. Venous
malformations of skeletal muscle also have a predilection for the head and extremities and most cases (70% to
80%) are noted at birth (95). Often some smooth muscle accompanies the vessels as appropriate for veins. In
contrast to vascular neoplasms, glucose transporter-1 is not expressed by the endothelial cells of vascular
malformations (147).
FIGURE 24-1▪LCH presented on the shoulder of this young female. A: A polypoid mass is composed of lobules
of capillary-sized vascular spaces. B: In the more cellular or proliferating areas, vascular spaces are difficult to
appreciate and mitotic figures are found with ease. C: The involuting areas are characterized by well formed,
patent capillaries. Some congenital hemangiomas do not involute.
FIGURE 24-2▪Arteriovenous hemangioma presented in a 3-year-old female with an enlarging mass on the heel
of the left foot. Vascular nodules are composed of venous and arteriole-like structures. This vascular lesion is
probably a malformation.
FIGURE 24-3▪Epithelioid hemangioma presented as a cutaneous mass on the arm of a 3-year-old male.
Compact small vessels lined by epithelioid or histiocytoid endothelial cells, some with vacuolated cytoplasm and
an accompanying lymphocytic infiltrate characterize this lesion.
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FIGURE 24-4▪Skeletal muscle hemangioma presented as a soft tissue mass on the back of a 2-year-old female.
A: Vascular spaces and adipose tissue are present in the skeletal muscle. B: Other areas consist of vascular
spaces within the skeletal muscle.
Intermediate tumors. This category of vascular neoplasms includes several entities, which are designated as
HEs whose clinical behavior is characterized as locally aggressive or rarely metastatic in the WHO classification
(75) (Table 24-5). Some HEs have complex mixed pattern features as in the case of the so-called composite HE
(80).
Kaposiform hemangioendothelioma (KHE) occurs almost exclusively in children with a mean age at
diagnosis between 2 and 4 years old, but as early as antenatally, with nonimmune hydrops or pericardial effusion
(133). The extremities and head and neck are the anatomic sites of predilection. However, KHE has been
reported in the retroperitoneum, mediastinum, intestinal tract, middle ear, or as diffuse multifocal sites. As many
as 50% of cases are complicated by the Kasabach-Merritt syndrome and also have evidence of
lymphangiomatosis. The tumor may be confined to the skin or present in the deeper soft tissues as multiple
nodules with a diffusely infiltrating pattern. Though a mass may be palpable, KHE usually does not form a well-
circumscribed, solitary mass, which is also generally true for most “vascular anomalies.” Microscopically, the
nodules have a distinctive spindle cell appearance (thus the designation of Kaposi sarcoma [KS]-like) (Figure 24-
5). The tumor cells have uniformly bland features in the absence of nuclear hyperchromatism and mitotic figures.
Erythrocytes may or may not be present among the spindle cells, but the small eosinophilic globules of KS are
usually not seen. Nodules may also have the morphology of a more conventional capillary hemangioma. A
lymphangiomatous component may be present as well. Glomeruloid nodules have a resemblance to the
formations of a tufted angioma, which is usually confined to the dermis with the formation of so-called
cannonball-like lesions. It has been reported that D2-40 expression may assist in the differentiation of KHE from
tufted angioma (4). Unlike KS, KHE does not harbor HHV-8.
Spindle cell and retiform HEs occur throughout life without a particular predilection to children. Papillary
intralymphatic angioendothelioma (PILA, Dabska tumor) presents in not only older children and adolescents, but
also in young adults (60). A dermal nodule is a poorly circumscribed lesion whose size varies from a few
centimeters to more than 30 cm. Papillary or glomeruloid structures with the phenotype of lymphatics are found in
enlarged thin-walled vascular
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structures. Though initially considered a malignant vascular tumor, PILA rarely metastasizes and infrequently
recurs.
FIGURE 24-5▪KHE presented as multiple masses in the intestinal tract and retroperitoneum in a 7-year-old
female. A: Many of the nodules are composed of compact spindle cells with interposed erythrocytes resembling
KS. B: Other nodules had a more lobulated and tufted appearance. C: Factor VIII-related antigen
immunostaining labeled the spindle cells.
Malignant vascular tumors. Epithelioid hemangioendothelioma (EHE) is a low-grade vascular neoplasm

with microscopic features, which are similar to the benign counterpart, but with more cytologic atypia and an
aggressive infiltrating pattern. The one example of EHE, which we have seen most often in the first two decades
of life has presented in the liver. Small groups and individual tumor cells with a cytoplasmic vacuole are
surrounded by a pale hyaline to almost chondroid appearing stroma unlike the more compact pattern of vascular
spaces in the epithelioid hemangioma. Overall, EHE has a metastatic rate of 15% to 20%. The lung is the
favored site of metastasis of the hepatic EHE where the pulmonary lesions were once referred to as
intravascular bronchoalveolar tumor of Leibow.
Conventional high-grade angiosarcoma is uncommon in adults and rarer yet in children. Though reported in the
peripheral soft tissues, the few angiosarcomas in children that we have seen presented in the liver. These
tumors, usually in children 10 years old or less, had the characteristic sinusoidal growth pattern by atypical
endothelial cells with hyperchromatic nuclei and the occasional mitotic figure. We acknowledge that there are
examples of “infantile hemangioendothelioma” of the liver with the so-called type II pattern whose microscopic
features are borderline to a degree of uncertainty about the potential for malignant behavior. However, we have
not encountered yet as infantile HE with an exclusive, small vessel type I pattern, which has pursued a malignant
course. However, the type II endovascular papillary pattern is less reliably reassuring in terms of ultimate
outcome.
Kaposi sarcoma (KS) is known to occur in childhood as either lymph node-based disease or in a variety of
extranodal sites in an immunocompromised child with AIDS or an organ transplant recipient. There is the same
association with HHV-8 as in adults. The lesions of KS occur in mucosal sites of the oral cavity, gastrointestinal
tract, and lung. Cutaneous lesions or inguinal lymphadenopathy are other presentations. Hepatosplenic KS is
also known to occur in children.
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The histopathologic features are those of a relatively lowgrade spindle cell proliferation and entrapped
erythrocytes. These spindle cells have membrane immunoreactivity for CD31 and CD34, and the nuclei are
positive for HHV-8.
FIGURE 24-6▪Mixed venous and lymphatic malformation presented as a mass on the chest wall of a 17-year-old
female. A: A mixture of muscle associated venous structures are intermixed with thin-walled lymphaticlike
spaces. B: Some of these spaces stain positively for D2-40, a lymphatic marker. C: Many of the vascular
channels are associated with SMA positivity
Lymphatic Tumors
Cystic lymphangioma (hygroma) and the other variants of lymphangioma are classified simple, low flow
malformations in the ISSVA classification (Table 24-5). A compressible soft-tissue mass in the neck or more
extensive involvement of surrounding anatomic structures such as the orbit, parotid gland, bone or into the
mediastinum, and/or axilla in the clinical presentation. Solitary or more generalized cystic lesions may be present
in the retroperitoneum and/or mesentery. Bone involvement may result in so-called disappearing bone disease
(Gorham-Stout syndrome). Variably sized lymphatic spaces lined by inconspicuous endothelium and a watery
eosinophilic coagulum in some lumina are the basic microscopic features. These spaces are irregularly
distributed in the soft tissues with extension along septal planes and into the interstitium between lobules of
salivary gland in the neck or the thymus when there is involvement of the mediastinum. Some of these
malformations may include capillary or venous elements (Figure 24-6). In the latter case, smooth muscle may
accompany some of the larger vascular spaces. Another complication in the pathology of a lymphangioma is in
the recurrence whose vascular pattern has been altered by a reactive fibrous stroma with some resemblance to
a fibromatosis.
The ISSVA classification reflects the fact that some vascular malformations are a collage of vessels of different
types from capillaries to arteries (21, 22, 153) (Table 24-5). The classic arteriovenous malformation (AVM),
usually encountered in the central nervous system or extremities, is a racemose of arteries and veins often
accompanied by secondary features such as thrombi in various stages of organization, fibrosis, hemosiderin
deposition, and dystrophic calcifications. There are several hereditary disorders whose predominant feature is
the formation of AVMs, which have been reviewed by Tille and Pepper (201). Mutations in several genes, which
are involved in angiogenesis have been
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detected in hereditary hemorrhagic telangiectasia (three subtypes, each with a different mutation), Klippel-
Trenaunay syndrome (AGGF1 mutation) mutation, and other disorders with AVMs (37, 45, 103, 209). Rarely,
AVMs are complicated by the development of angiosarcoma.
FIGURE 24-7▪Glomangioma (glomuvenular malformation) in a 20-yearold female presented as paraspinal and

retroperitoneal masses. The vascular spaces are accompanied by a circumferential population of small,
basophilic appearing cells beneath the endothelium.
Glomus tumor (glomuvenous malformation) is variably regarded as a neoplasm or malformation of the

neuromyoarterial body. Solitary glomus tumors (90% of cases) have a predilection for subungual sites on the
hand. Autosomal dominant multiple glomus tumors or glomangiomas (10% of cases) are characterized by a
germ-line mutation in GLMN gene on 1p22. It is also of interest that there may be an increase in glomus tumors
in the setting of neurofibromatosis type 1 (NF1). Approximately 10% to 15% of solitary glomus tumors present
before the age of 20 years, usually in the second decade. The glomus cell is a modified smooth muscle cell.
Cuffs of small uniform, basophilic cells are present beneath an intact, inconspicuous endothelium (Figure 24-7).
FIBROUS, MYOFIBROBLASTIC, AND PERICYTIC TUMORS

This section is concerned with a category of non-neoplastic and neoplastic entities which have in common a
spindle cell with the morphologic and phenotypic features of a fibroblast, and/or a transitional type mesenchymal
cell with the composite attributes of a fibroblast and smooth muscle cell, the myofibroblast, a cell with a smooth
muscle phenotype and vascular perithelial localization (75). In the setting of one of the unique fibrous tumors of
childhood, infantile myofibromatosis-myofibroma, the proliferating subintimal myofibroblasts have the capacity to
differentiate into a cell with the morphology and immunophenotype of pericytes with contractile attributes of
smooth muscle.
FIGURE 24-8▪Keloid presented as a mass in the posterior auricular region of a 5-year-old female. Dense
acellular bundles of collagen are separated by fibroblasts.
Scars, keloids, and fasciitis. The scar, a reactive fibrous and myofibroblastic proliferation, occurs in all tissue
types and organs (brain excepted with its reactive gliosis) and is a programmed process of repair.
Morphologically, the myofibroblasts and fibroblasts can acquire a degree of atypia, especially in a field of
radiation, which can be a source of concern about its benign or malignant nature. A resected sarcoma after
radiation therapy is one circumstance when a highly atypical fibroblastic reaction can be mistaken for persistent
tumor.
Keloids and hypertrophic scars are similar in many respects with the formation of nodules of reactive fibroblasts
in the dermis, whose presence has obliterated or replaced the normal microanatomy. Keloids are additionally
characterized by groups of thickened intensely eosinophilic bundles of collagen (Figure 24-8). Similar bundles of
collagen may be seen in desmoids fibromatosis in the mesentery or nodular fasciitis (NF). The formation of
keloids and hypertrophic scars is regarded as an abnormality in the normal woundhealing process; the frequency
of both processes is increased in some families and is more common in individuals of African descent (12, 185).
There has been considerable interest in attempting to understand the pathogenesis of these presumably related
processes (118).
Nodular fasciitis (NF) and other pseudosarcomatous myofibroblastic lesions remain important because of their
potential for misdiagnosis as a sarcoma despite the many admonitions in the literature over the last 50 to 60
years (180). It remains underappreciated for the most part that NF occurs in children including those in the first
few years of life (47). One of the more dramatic examples of a fasciitis in infancy is cranial fasciitis presenting as
a large mass with compression of the underlying brain in some cases (171). In our experience, approximately
40% of all cases of NF present in the first two decades, particularly in the first
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decade of life, as a mass with a predilection for the head and neck region in 35% to 40% of our cases. Often NF
arises as a rapidly developing mass, generally measuring less than 3 cm, in the orbital and periorbital,
periparotid, premaxillary and intramaxillary, auditory canal, and intraoral soft tissues. There is often clinical
concern about ERMS, which may not abate even after a biopsy due to its pseudosarcomatous features. The
subcutis is the tissue level of origin for most NFs, followed by the fascia, lower dermis, muscle, and rarely the
joint space. Grossly, a circumscribed, nonencapsulated nodule with a glistening mucoid appearance is reflected
in the histologic features. Several histologic patterns coexist in the nodule with dense, spindle cell areas forming
short fascicles adjacent to less cellular foci with separation of the spindle cells by mucoid-myxoid extracellular
material (so-called tissue culture pattern) and transitional areas with both patterns (Figure 24-9). Mitotic figures
are readily identified with some nuclear atypia (absent atypical mitotic figures and anaplasia). Microcysts with
mucin, a variable number of histiocytes, foci of interstitial hemorrhage, and scattered inflammatory cells in the
background are the constellation of diagnostic features. Scattered multinucleated cells and a storiform-like
pattern may suggest a fibrohistiocytic proliferation; the compact spindle cell proliferation with mitotic figures
serves to raise concern for fibrosarcoma (FS) or leiomyosarcoma; and immature fibroblasts in the tissue culture-
like foci are the features to suggest the possibility of ERMS. The myofibroblasts of NF express vimentin and SMA
but desmin, myoD1, and myogenin are all nonreactive by IHC. In general, NF is regarded as a nonrecurring
process and if there is a recurrence, one should consider the likelihood of a misdiagnosis. The difficulty in the
differentiation of cranial fasciitis from a fibromatosis is presented in a study where β-catenin was expressed in
the nuclei of a putative recurring cranial fasciitis. Similarly, this may explain the local recurrence of 20% in a
series of NF in children, which are in reality examples of desmoid type fibromatosis. However, β-catenin nuclear
positivity may be seen infrequently in NF. Proliferative fasciitis and myositis are regarded as related entities to
the more common NF.
FIGURE 24-9▪NF in a 10-year-old female presented as a 2 cm mass in the posterior triangle of the neck. A: The
abrupt interface exists between the spindle cells and the adjacent nonlesional collagen. Note the presence of
interstitial hemorrhage. B: Intersecting fascicles and nodules of loosely arranged spindle cells and interstitial
mucin are some of the characteristic features. The nuclei failed to immunostain for β-catenin. C: The presence of
multinucleated cells may cause confusion with a fibrohistiocytic lesion.
Myositis ossificians (MO), either the solitary, sporadically occurring soft-tissue mass or the multifocal
fibrodysplasia ossificans progressiva (FOP), has its own potential
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for diagnostic miscues. Most cases of MO are sporadic, solitary, and may or may not be accompanied by a
history of trauma to possibly explain the male predominance. It is seen uncommonly in the first decade of life and
more often in later childhood or adolescence in which case there may be a history of incidental or organized
(sports) blunt trauma. The classic presentation is a circumscribed intramuscular mass, or alternatively the
formation of a parosteal, calcified mass, or a mass attached to the surface of the bone by a pedicle. The sites of
predilection are the thigh, buttock, and abdominal wall (50). In terms of size, MO can measure in excess of 10 to
15 cm. The inner portion of the 3-zone mass is composed of plump spindle and polygonal myofibroblasts, blood
vessels and histiocytes, which are surrounded by a zone of immature osteoid and an outer shell of mature bone
(Figure 24-10). The diagnostic trap is set if a biopsy is obtained from the central, proliferating zone (180).
Despite the initial impression of marked cellularity and some degree of atypia, the realization is that there are few
mitotic figures and certainly no atypical mitoses. Nuclear anaplasia is absent.
FIGURE 24-10▪Myositis ossificans presenting as a soft tissue mass in the posterior neck of a 10-year-old male.
A: The center of this mass is composed of compact spindle cells with some nuclear atypia but in the absence of
atypical mitotic figures. B: The transition zone is between the central spindle cells and osteoid formation. C: The
peripheral zone is represented by the active new bone formation.
Heterotopic ossification with fibro-osseous features has been reported in the auditory canal of young individuals.
Cutaneous osteoma occurs sporadically or may be a manifestation of Albright hereditary osteodystrophy (AHO)
or pseudohypoparathyroidism type Ia with or without the AHO phenotype. There are inactivating mutations of the
GNAS gene (20q13).
FOP is an autosomal dominant disorder, which is characterized by the progressive transformation of soft tissues
and skeletal muscle to heterotopic bone (112). The mutation has been mapped to chromosome 2q23-24, the site
of activin A type I receptor/activin-like kinase 2 (ACVR1/ALK2), a bone morphogenetic protein type I receptor. In
addition to the characteristic great toe malformations, there is the development of soft-tissue swelling or masses
on the back, which are described as “spreading” through the subcutaneous tissues and deeper. Biopsy reveals
a spindle cell and myxoid transformation of the subcutis with a resemblance to infantile subcutaneous
fibromatosis or lipofibromatosis, more so than NF. A biopsy site may enlarge due to metaplastic ossification,
which appears to accelerate in foci of trauma.
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Other forms of so-called pseudosarcomatous proliferations of the soft tissues and periosteum are florid
reactive periostitis, fibrous pseudotumor of the digit, a form of localized MO, and bizarre parosteal
osteochondromatous proliferation (Nora lesion) (180). These various lesions are problematic if the specimen is a
biopsy without adequate clinical information and characterization of the imaging features. Once again, the
atypical histology is not accompanied by overtly malignant features, as discussed in the previous sections on NF
and myositis ossificans, in particular as it relates to the absence of atypical mitoses and anaplasia.
Fibroblastic-myofibroblastic tumors in the WHO classification include both NF and myositis ossificans.
However, this section focuses upon a group of neoplasms or presumed neoplasms, some of which occur
predominantly in children, and others that are seen in adults as well (41, 75, 211).
Most of the unique fibroblastic-myofibroblastic tumors of childhood with some exceptions are recognized in the
first 5 years of life and many at or before 2 years of age. These fibrous tumors of childhood include the following:
fibromatosis colli, myofibroma-myofibromatosis, fibrous hamartoma of infancy (FHI), inclusion body fibromatosis
(infantile digital fibroma), infantile fibromatosis (lipofibromatosis), Gardner-nuchal fibroma, juvenile aponeurotic
fibroma, nasopharyngeal angiofibroma, congenital-infantile FS. Palmar and plantar fibromatoses (superficial
fibromatosis) and desmoid-type fibromatosis are seen in all age groups. Desmoid-type fibromatosis, Gardner
fibroma, and nasopharyngeal angiofibroma are known manifestations of familial adenomatous polyposis (FAP)
including Gardner syndrome (43). Infantile myofibromatosis has an autosomal dominant pattern of inheritance in
a minority of cases whereas juvenile hyaline fibromatosis (JHF) with its allelic syndrome, systemic hyalinosis, is
an autosomal recessive disorder (2).
FIGURE 24-11▪Infantile myofibroma (myofibromatosis) presented as deep mass in the posterior neck of a 3-
month-old female. A: The sharply demarcated mass measuring 3.0 cm is composed of uniform spindle cells in a
pale eosinophilic stroma. B: Sweeping arrays of spindle cells are present in a fibrohyaline stroma with a small
vascular space which has been compressed by spindle cells.
Infantile myofibromatosis (myofibroma), the most common of various fibrous tumors of childhood, accounts for
20% to 25% of all cases. A solitary cutaneous or subcutaneous nodule (90% of cases) measures less than 3 cm
in most cases, presents in the first 5 years of life, and may be noted at birth and occurs in the head and neck
region (40% to 60% of cases) followed by the trunk and extremities (192). However, the bone and various
organs including the brain, dura, liver, intestinal tract, lung, and testicle are some of the other less common sites.
Multifocal lesions, usually restricted to the skin-subcutis and/or bone, are seen in 5% to 8% of cases, and in 1%
to 2% of cases, there are more widespread skin, soft tissue, and visceral lesions, which are recognized in an
infant less than 6 months old. The clinical outcome is poor in these infants because of pulmonary venous
occlusion by the formation of intravascular myofibromas. On the other hand, solitary lesions are known to
undergo spontaneous regression.
A firm nodular non-encapsulated mass measuring 1 to 3 cm in greatest dimension may also be accompanied by
calcifications, cysts, and central hemorrhage with a microscopic pattern of a hemangiopericytoma (HPC) with
hemorrhage and coagulative type necrosis. The cellularity is most apparent toward the periphery where the
compact spindle cells are arranged in short fascicles or within hyaline-myxoid, almost chondroid-appearing
stroma, which separates or largely replaces the spindle cells (Figure 24-11). If the nodule is located in the
dermis, there are often multiple discrete nodules with normal intervening cutaneous structures and dermal
collagen and if in the subcutis, there is overgrowth and entrapment of fat. A more infiltrative pattern may be seen
in the dermis, but the small, SMA positive nodules are best seen in the superficial dermis. However, the
myofibroma does not have the infiltrative growth of a desmoid-type fibromatosis. At the periphery
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of some nodules, a compressed vessel is a hallmark feature of the myofibroma and can be identified in many
cases (Figure 24-12). The associated HPC-like pattern can be dominant in some tumors with only peripheral,
nodular myofibromatous foci (Figure 24-13). Central degeneration without overt necrosis is yet another feature.
The myofibromatous pattern is immunopositive for SMA whereas the HPC-like foci are positive for CD34. In
addition to the HPC-like areas, dense spindle cell foci can simulate CIFS but without the potential implications
nor cytogenetics of the latter tumor (Figure 24-14) (see Table 24-2). Local recurrence is seen in less than 10% of
cases. It should be noted that the myofibroma is seen in older children and even adults.
FIGURE 24-12▪Infantile myofibroma (myofibromatosis) presented as a soft tissue mass in the neck of a β-
monthold boy. A: The smaller nodules of spindle cells are associated with a compressed vessel at the periphery
which is useful in the recognition of a myofibroma. B: Other fields are composed of fascicles and nodules with a
fibromyxoid appearance.
The myofibroblast and pericyte also coexist in skin tumor and STT, the myopericytoma (MPC), with its
resemblance to infantile myofibromatosis (59). This tumor occurs in older children and adolescents though
mainly in adults. The nodular pattern of MPC consists of small vessels surrounded by concentric collarettes of
spindle cells with a resemblance to the metanephric stromal tumor of the kidney. On the theme of pericytes, there
is the solitary fibrous tumor (SFT), initially described in the pleura, but now recognized in many extrapleural sites
and the classic HPC (64). These two neoplasms are regarded as a single spectrum entity, and both tumors are
distinct from the fibroblastic-myofibroblastic tumors (71).
Infantile fibromatosis includes three pathologic patterns based in part on the level of tissue involvement:
subcutis with the alternative designation of lipofibromatosis; skeletal muscle with diffuse infiltration of the muscle
by immature appearing spindle cells and desmoid-type fibromatosis without any specific microscopic features to
differentiate it from any other desmoid tumor without respect to age (211). Infantile subcutaneous fibromatosis
(lipofibromatosis) accounts for approximately 5% to 10% of fibrous tumors of childhood with a predilection for the
distal extremities, though it may occur on the trunk and head and neck region (70). The growth pattern of
variably dense spindle cells with a collagenous background extends along and around the interlobular septa of
the subcutaneous fat and is not well circumscribed either clinically or pathologically (Figure 24-15). There is
partial overgrowth of the fat by the spindle cells with a remote resemblance to dermatofibrosarcoma protuberans
(DFSP). In fact, a fibrous-appearing DFSP should be considered in the differential diagnosis even in a young
child. CD34 expression may be present focally in the infantile subcutaneous fibromatosis (usually diffuse in
DFSP), but the spindle cells of infantile fibromatosis are variably positive for SMA. These tumors are known to
recur which is not surprising, given their diffuse growth pattern. Infiltration into the deep soft tissues is
uncommon. The diffuse pattern of infantile fibromatosis is recognized as an infiltrating tumor involving a skeletal
muscle in the head and neck and often the tongue. Immature spindle cells sweep through the interstitum of the
muscle with retention of some architectural landmarks of the separated bundles of muscle (Figure 24-16).
Because of the relatively immature appearance of the tumor cells, a fetal rhabdomyoma (FRM) or RMS may be
considered in the differential diagnosis. Appropriate immunohistochemical stains for myoD1 and/or myogenin
should resolve the dilemma since these are only expressed in rhabdomyoblasts. Complete surgical resection is
complicated by the morbidity of tumor location and its diffuse pattern. The desmoid-type fibromatosis rarely
occurs in infants but its fibrous, spindle cell pattern with irregular infiltration and replacement of skeletal muscle
along the invasive borders are identical to those of desmoid tumors in older children and adolescents. Any one of
the infantile fibromatoses can involve the lower dermis, whereas
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involvement of the deeper soft tissues including the skeletal muscle is confined to the diffuse and desmoid types.
FIGURE 24-13▪Infantile myofibroma (myofibromatosis) presented as a mass in the groin of a 4-month-old male.
A: This tumor has a mixed pattern of myofibroma and HPC which in this field has the former features. B: The
HPC areas are usually present centrally with more ovoid cells surrounding small, clefted vascular spaces. C:
Immunohistochemical staining for SMA highlights the myofibromatous pattern without reactivity in the contiguous
HPC-like foci. D: A contrasting pattern of immunoreactivity for CD34 is seen in the CD34-positive HPClike foci
and absence of staining in myofibromatous focus.
FIGURE 24-14▪Infantile myofibroma (myofibromatosis) presented in a 7-day-old female as a mass on the back.
A: One of the two patterns in this tumor includes uniform spindle cells in a pale, eosinophilic background. B:
Other foci are more hypercellular and mitotically active with a resemblance to CIFS. Despite the similarities, a
t(12;15) is not found in these worrisome foci.
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FIGURE 24-15▪Infantile subcutaneous fibromatosis (lipofibromatosis) in this 3-day-old boy in the head and neck
region shows a proliferation of immature appearing spindle cells within the subcutaneous fat. The entrapment
rather than overgrowth distinguishes this fibromatosis from DFSPGCF which is also seen in infancy.
Fibrous hamartoma of infancy (FHI), another unique fibrous tumor of childhood, accounts for no more than
5% of all such neoplasms (41). This tumor occurs almost exclusively in the 2 to 3 years of life, often in the first
few months where the trunk, axilla, inguinal region, and extremities are the sites of predilection in descending
order (28, 55). The subcutaneous, poorly circumscribed fibrofatty tumor generally measures less than 5 cm. It
shares many of the same gross and microscopic features with the infantile subcutaneous fibromatosis except for
the small nodules of immature, spindled mesenchymal cells in a pale basophilic background (Figure 24-17).
These may be found as isolated structures in the fat or along the periphery or within bundles of more mature
appearing spindle cells. Focal extension may be found in the overlying dermis in which case the predominantly
subcutis nature of the tumor is not readily apparent. The local recurrence rate is only 10% to 15%, which is low
in light of the fact that FHI is incompletely resected in most cases.
FIGURE 24-16▪Infantile fibromatosis of the diffuse type presented on the upper back of a 4-month-old boy. The
loosely arrayed immature spindle cells are infiltrating through the skeletal muscle rather than its destructive
overgrowth as in desmoid fibromatosis.
FIGURE 24-17▪FHI presented in the axillary region of a 4-monfh-old boy. A: The pattern of subcutaneous
infiltration by bland appearing spindle cells resembles infantile subcutaneous fibromatosis. B: The presence of
small bundles of immature spindle cells at the periphery or within the midst of the more mature fibroblasts is the
diagnostic feature.
Inclusion body fibromatosis (infantile digital fibroma) presents on the lateral and/or dorsal aspect of finger
and/or toe with usual sparing of the thumb and great toe as a firm nodule(s) in an infant or child 5 years of age or
less at diagnosis (124). More than one digit is involved in 25% to 30% of cases. This tumor measures 1 to 2 cm
in most cases and has a uniform
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white, fibrous appearance similar to a desmoid-type fibromatosis. The dermis is commonly effaced by a uniform
spindle cell proliferation, forming short fascicles, and with a collagenous background with isolated hair follicles or
sweat glands (Figure 24-18). Confluent, contiguous extension into the subcutis is associated with overgrowth of
fat. There is a microscopic resemblance to the desmoid-type fibromatosis, except for the presence of
eosinophilic, paranuclear inclusions in variable numbers; these inclusions are usually more readily identified
inatrichrome stain (Figure 24-18) (15). The infiltrative growth around and through neurovascular structures in the
digit limits complete resection in most cases, which accounts for a local recurrence rate in excess of 50%. It is
important to take note of the fact that more than one digit may be involved.
FIGURE 24-18▪Inclusion body fibromatosis (infantile digital fibroma) presented on the fifth toe of a 7-monthold
female. A: The dense, relatively hypocellular spindle cell proliferation has effaced the dermis. B: Trichrome stain
demonstrates uniform pattern of collagen deposition. C: Paranuclear eosinophilic bodies are best seen at higher
magnification. D: These filamentous bodies of actin are demonstrated to better advantage in the trichrome stain.
A small subset of fibroblastic-myofibroblastic tumors, typically presenting in the first 2 years of life, are seemingly
composed of more than one histologic pattern (composite fibrous tumor). The most common example is the
infantile myofibromatosis—HPC with concurrent patterns of both. Other combinations are the infantile
fibromatosis with CIFSlike foci and infantile myofibroma. These tumors demonstrate the morphologic plasticity of
the fibroblast-myofibroblast and its capacity to simultaneously express itself with several microscopic patterns
and in a sense reflect the relationship of these separate fibrous tumors of childhood to each other. We have
seen examples of composite fibrous tumor behave in the fashion of multifocal or generalized infantile
myofibromatoses.
Desmoid-type fibromatosis (desmoid tumor, musculoaponeurotic fibromatosis) is the most common fibrous
neoplasm presenting in the first two decades of life (60% to 70% of all fibrous tumors) with cases presenting
throughout
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childhood and adolescents with a bimodal age distribution in the first 2 years and later in older children (41). The
extremities (including brachial plexus) and trunk are the sites of predilection in older children, but these tumors
are also seen in the head and neck (orbit, paranasal sinus, mandible), intrathoracic, and abdominal (mesentery
and pelvis) sites. Desmoid tumors arising in the shoulder-axilla or gluteal-thigh region have a local recurrence
rate of 30% or greater (23). Most tumors occur sporadically (90% to 97% of cases), but there is a strong
association with Gardner syndrome—FAP in less than 5% of cases.
FIGURE 24-19▪Desmoid fibromatosis (desmoid tumor) presented as a deep soft tissue mass in the posterior
thigh of a 15-year-old female. The cut surface of this 12 cm circumscribed mass has a tan-white trabecular
appearance. Note the pushing growth into the skeletal muscle at the periphery.
A small incisional or needle biopsy can be challenging since other non-neoplastic and neoplastic fibrous
proliferations arise in the differential diagnosis. An operative resection yields a gray-white mass with a uniform
mucoid to trabeculated appearance whose dimensions range from a few centimeters to >10 cm (Figure 24-19).
When skeletal muscle is present at the periphery of the resection, irregular infiltration by the usually bland
spindle cell proliferation into the muscle can be appreciated; this same feature is seen to a more limited extent in
some cases of NF arising in a muscle. The periphery of the mass should be tattooed with India ink (or other
appropriate dye) since the status of surgical margins correlates with a local recurrence rate of 35% to 70%.
Fascicles of spindle cells or a loosely organized pattern of spindle cells are accompanied by a variably pale,
myxoid to edematous or more collagenized background. The spindle cells may have the features of mature
fibroblasts or display variation in the size and configuration of the stromal cells to reflect their less mature, more
myofibroblastic attributes, which is manifested by immunopositivity for SMA (Figure 24-20). Mitotic figures can be
identified among the myofibroblasts. A myxoid background, proliferating myofibroblasts, and some interstitial
hemorrhage and edema portray a more NF-like appearance. The infiltrative margins rather than peripheral
nodularity characterize the desmoid tumor in contrast to NF in most cases. Scattered lymphoid nodules at the
interface with the surrounding normal soft tissues also usefully distinguish a desmoid tumor from other fibrous
proliferations. There is little to differentiate a recurrent desmoid from the newly diagnosed tumor except for the
findings of earlier surgery including scarring and foreign body giant cells and a more circumscribed margin in the
primary tumor.
The relationship of the desmoid tumor to FAP has provided the opportunity to understand some of the molecular
pathology of this neoplasm. Sporadic desmoid tumors have somatic mutations in the β-catenin gene (CTNNB1
on 3p21), which regulates the Wnt signaling pathway whereas the FAP-associated desmoids have an APC gene
(5q22) mutation. Nuclear expression of a β-catenin is a useful marker to differentiate the sporadic and familial
desmoid tumor from other fibrous tumors including NF in most cases (Figure 24-20) (13, 26). However, some of
the other fibrous tumors may have nuclear positivity for β-catenin so that it is not absolutely specific for desmoid
tumors (200).
Gardner-nuchal fibroma is a distinctive paucicellular, densely collagenized mass presenting in the first decade
of life with a predilection for the posterior truncal-paraspinal region (43). Other sites of involvement include the
head and neck and extremities (144). Approximately 70% of affected individuals have a family history of FAP or
represent a new mutation (43). The tumor is poorly circumscribed with a plaque-like growth in the subcutis or
deeper soft tissues. It can be difficult to judge the peripheral margins of a fibroma from the normal fibrous
connective tissues. A desmoid tumor may accompany a fibroma or evolve from a recurrent fibroma. Like the
desmoid tumor, there is nuclear positivity for β-catenin in 60% to 70% of cases. Cyclin-D1 is expressed in the
nuclei in virtually all cases. Nuchal and Gardner fibromas have virtually identical pathologic features.
Palmar-plantar fibromatosis is seen in children but more commonly in adults. These are poorly circumscribed
fibrous tumors with a pattern of spindle cell foci separated by bland hypocellular collagenized stroma (69).
JHF is an autosomal recessive disorder, which is allellically related to infantile systemic hyalinosis (ISH) with
a loss of function mutation in the capillary morphogenesis gene-2 (CMG2 on 4q21) (2, 128). Large, painful
nodules in the head and neck region (including marked gingival hypertrophy) and around joints evolve from small
cutaneous papules, which are first noted in infancy and accelerate in growth throughout childhood. Osteolytic
bone lesions develop, as do joint contractures. Firm, white nodules in the soft tissues and dense fibrous
effacement of the dermis, resembling to some extent morphea-scleroderma, are some of the pathologic features
(Figure 24-21). The nodules are circumscribed and consist of homogeneously dense hyaline collagen with focal
paucicellular and more cellular foci, consisting of ovoid stromal cells residing in apparent lacunae with a
chondrocyte-like appearance (Figure 24-22). Unlike JHF, ISH has
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visceral involvement in addition to papulonodular lesions of the skin and soft tissues. The heart, intestinal tract,
spleen, and skeletal muscle are infiltrated by the fibrohyaline tissue with a resemblance to amyloid. Protein-
losing enteropathy is a complication of small intestinal hyalinosis. Only infantile myofibromatosis among the other
fibrous proliferations has visceral involvement by a more cellular, vasocentric nodular proliferation than the
diffuse interstitial hyalinosis of ISH.
FIGURE 24-20▪Desmoid fibromatosis (desmoid tumor) presented in the posterior thigh of a 15-year-old female.
A: A bland proliferation of fibroblasts is seen in a non-homogeneous collagenous background. B: The fibroblasts
maintain their myofibroblastic phenotype with immunostaining for SMA. C: Most desmoids express nuclear β-
catenin by IHC.
FIGURE 24-21▪JHF presented as a firm mass around the knee of a 17-year-old female who had several other
similar masses excised previous to this one. This well-circumscribed mass had a glistening, slightly nodular
appearance on cut surface. The consistency of the mass was described as firm with a chondroid-like quality.
Calcifying aponeurotic fibromatosis (juvenile aponeurotic fibroma) is one of the least common of the fibrous
tumors of childhood (1% to 2% of all cases) with a predilection for the distal extremities (75). Usually, older
children and adolescents present with a mass in the ankle or wrist in the deep subcutis, fascia, or tendon. A
poorly circumscribed mass measuring less than 5 cm has a firm, gritty, gray-white appearance of cut surface. An
infiltrative process of spindle cells (fibroblasts) is accompanied by less cellular, hyalinized areas in which foci of
granular calcifications are found. Without the calcifications, there is a resemblance to infantile subcutaneous
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fibromatosis. Recurrences are reported in 50% or more of cases. Because of the distal, periarticular localization,
monophasic SS may be briefly considered in the differential diagnosis.
FIGURE 24-22▪JHF presented as multiple masses in this 17-year-old female. A: The dense, hypocellular
nodules of collagen are characteristic of this tumor. B: Rounded stromal cells within lacunar spaces resembling
chondrocytes is another histologic feature. No other fibrous lesion in childhood approaches this degree of dense,
uniform hyalinization with the possible exception of a Gardner fibroma which lacks nodularity.
Juvenile nasopharyngeal angiofibroma (JNA) is a tumor whose ambiguous histogenesis has resulted in its
uncertain classification in the past as a fibroma or vascular tumor. Most cases present in older male children or
adolescents with epistaxsis. These tumors are seen in the setting of FAP. Like desmoid-type fibromatosis, the
nuclei of JNA express β-catenin. A firm lobulated or pedunculated mass measuring 5 to 10 cm is the gross
appearance. A uniform population of spindled to stellate fibroblasts lacks a fasciculated pattern and is interrupted
by evenly distributed thin-walled vascular spaces. Mast cells are commonly distributed within the background.
These tumors have pushing rather than the infiltrating borders of a desmoid-type fibromatosis.
Fibromatosis colli is infrequently seen as a surgical specimen though it is one of the more common fibrous
tumors of childhood since spontaneous regression occurs in more than 90% of cases (39). A firm, white
lobulated fibrous mass measuring 1 to 3 cm typically arises in the lower one-third of the sternocleidomastoid
muscle where it has infiltrative borders like the desmoid-type fibromatosis though fibromatosis colli is usually
more cellular with a less collagenized stroma. There is some similarity to NF, which only rarely arises in skeletal
muscle.
Inflammatory myofibroblastic tumor (IMT) is a distinctive clinicopathologic entity, which has emerged from a
somewhat poorly defined group of idiopathic fibroinflammatory processes collectivity known as inflammatory
pseudotumors (88). In the WHO Classification, the IMT is regarded as an “intermediate, rarely metastasizing”
neoplasm, which principally occurs in the first three decades with cases seen as early as the first year of life into
early adulthood with a mean age at diagnosis between 10 and 15 years without the inclusion of older adults (42,
75). The lung, gastrointestinal tract, mesentery, liver, and bladder are the principal primary sites, which in
aggregate account for 70% to 75% of all cases in children (Table 24-6). This tumor is also ubiquitous in terms of
its other less common sites of presentation including the dura, orbit, kidney, uterus, and upper respiratory tract.
Peripheral soft tissues and bones are rarely affected. In a small proportion of cases, multiple lesions may be
detected at presentation or develop over a prolonged clinical course. It is not always clear whether multiple IMTs
are metastatic lesions or independently developing multifocal tumors (149). Constitutional or B-symptoms with
fever, failure to thrive, and weight loss together with microcytic hypochromic anemia and polyclonal gammopathy
are present in 5% to 15% of cases; these children may be a diagnostic dilemma for weeks to months. There is
IL-6 production in association with IMTs, which often falls to normal levels after surgical resection.
The tumors range in size from less than 1 to 15 cm in greatest dimension, with the larger IMTs arising in the
abdomen. In the lung, IMT measures 4 to 6 cm, but in the mesentery or retroperitoneum, IMT is generally in
excess of 10 cm. A well-circumscribed, non-encapsulated tumor has a glistening tan-white to gray-tan
homogeneous and nodular appearance, with minimal hemorrhage and absence of necrosis in most cases.
Calcifications are seen more often in the pulmonary IMTs (where it is the most common primary neoplasm of the
lung in childhood) but occur in extrapulmonary sites as well. Microscopically, three basic patterns are recognized;
they are not necessarily in equal proportions nor is each represented in every case. The first of these is
characterized by a dense spindle cell proliferation with some fascicular formation in association with a variably
prominent population of lymphocytes and mature plasma cells in the background.
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Adjacent foci may be composed of loosely arranged spindled to plump stromal cells in a myxoedematous
background resembling NF to yet a third pattern of paucicellular dense fibrosis with some inflammatory cells in
the background (Figure 24-23A). Dystrophic calcifications, osseous metaplasia, and collections of histiocytes are
other features. Mitotic figures are found in the spindle cell foci; however, atypical mitotic figures and anaplasia
should suggest a high-grade pleomorphic sarcoma. Necrosis is present in those IMTs, which have undergone
sarcomatous changes and may be accompanied by overt nuclear pleomorphism and hyperchromatism. Most
IMTs are immunoreactive for vimentin, SMA (Figure 24-23B), and cytokeratin in a minority of cases in children.
Approximately 50% to 60% of IMTs are ALK-1 positive with a membrane or cytoplasmic pattern to reflect a
specific ALK-1 translocation (Figure 24-23C) (53, 168, 215) (Table 24-2). Coffin and associates found that ALK-
1-positive IMTs pursue a less aggressive course than those which are ALK-1-negative (42). Surgical resection is
the treatment of choice with a recurrence-free survival of 80% or greater.
Table 24-6 ▪ SITE OF IMT IN THE FIRSTTWO DECADES OF LIFE
Sites No. Mean Age and Range Sex Sex Total (%)
(M/F)
Abdomen 61 (50)
Small Intestine 36 9 years (3 months-17 years) 13/23
Omentum-mesentery
Liver 11 4 years (2 months-9 years) 6/5
Stomach 5 9 years (6 years-14 years) 2/3
Retroperitoneum 5 7 years (4 months-14 years) 3/2
Pancreas 2 12 years, 7 1/1
Spleen 2 16 years, 9 years 2/0
Genitourinary Tract 16 (13)

Bladder 13 11 years (4 years-17 years) 6/7
Scrotum 2 7 years, 15 years 2/0
Prostate 1 17 years 1/0
Thorax 32 (26)
Lung 17 10 years (2 years-20 years) 13/4
Larynx-Trachea 6 7 years (12 days-12 years) 3/3
Heart 9 2 1/2 years (1 month-12 6/3

years)
Superficial and Deep Soft 12(10)

Tissues
Extremities-trunk 5 6 years (1 month-13 years) 3/2
Head and neck 3 2 years(1 year-3 years) 2/1
Perirectal-pelvic 4 8 years (3 years-13 years) 1/3
Brain 2 (2)
2 1 year, 15 years 0/2 123 (-

100)
From the files of the Lauren V. Ackerman Laboratory of Surgical Pathology, St. Louis Children’s
Hospital, Washington University Medical Center, St. Louis, MO.
The differential diagnosis includes low-grade myofibroblastic sarcoma, NF, inflammatory leiomyosarcoma,
myxofibrosarcoma and calcifying fibrous pseudotumor, desmoid-type fibromatosis, and gastrointestinal stromal
tumor (GIST). The latter two neoplasms, when arising in the mesentery or intestine, display β-catenin (nuclear)
and CD117 immunopositivity, respectively. Calcifying fibrous tumor is thought to be a distinctively different entity
from IMT; this tumor is recognized more commonly in adults than IMT but also occurs in the mesentery-omentum
and intestinal tract like the latter. Irregular dystrophic and/or psammomatous calcifications are found in a uniform
fibrous background. These tumors are CD34-positive, show sparse reactivity for SMA, and are uniformly ALK-1-
negative.
Myxoinflammatory fibroblastic sarcoma and myxofibrosarcoma are tumors of the peripheral soft tissues typically
seen in adults (71). Inflammatory leiomyosarcoma is likewise a sarcoma of adults, which is desmin- and SMA-
positive. NF shares a histologic pattern with IMT but is small (2 cm or less), and superficial in most cases. SMA is
positive in both, but ALK-1 is not expressed in NF. Low-grade myofibroblastic sarcoma is a rare sarcoma with
many overlapping morphologic and immunohistochemical features in common with IMT except for the fact that it
generally does not occur in children and adolescents and is seen in the head and neck region and extremities
rather than the lung and abdomen as in the case of IMT. Finally, we acknowledge that it is not always clear in
some cases when the diagnosis of inflammatory pseudotumor should be applied to a fibroinflammatory mass.
However, there are other entities in addition to the IMT, which were designated as inflammatory pseudotumors in
the past such as the dendritic cell (DC) tumor. Most deep circumscribed mass lesions in children composed of
myofibroblasts and inflammatory cells, which are either ALK-1-positive or -negative are probably examples of
IMT.
Fibrosarcoma (FS) includes several specific pathologic entities: congenital infantile FS, low-grade fibromyxoid
sarcoma (LGFS), and sclerosing epithelioid FS, all of which are seen in children and one, congenital infantile FS,
which occurs almost exclusively in the first few years of life and is
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distinguished pathologically from the adult-type FS (75). In other diagnostic settings, FS or spindle cell sarcoma
is the differential diagnosis for monophasic SS, MPNST, leiomyosarcoma, spindle cell RMS, and the disputed
infantile rhabdomyofibrosarcoma when the latter five neoplasms have been excluded after a through
immunohistochemical and molecular-cytogenetic evaluation. In other words, adult-type FS is in a sense a
pathologic diagnosis of exclusion. However, there is a CD34-positive variant of adult-type FS, which occurs in
children and adults (68, 72). Adult FSs in most cases in children are grade 2 neoplasms based upon a mitotic
count of five mitoses or fewer per 10 high-power fields (Figure 24-24). These tumors tend to be less than 6 cm in
diameter, are well circumscribed, and lack necrosis (Figure 24-25).
FIGURE 24-23▪IMT presented as an abdominal mass in a 7-year-old male. A: The cellular areas are composed
of spindle cells arranged in fascicles and accompanied by a variably prominent population of lymphocytes,
plasma cells and finely vacuolated histiocytes. B: The spindle cells are immunopositive for SMA in most cases.
C: ALK-1 immunopositivity with a membraneous and cytoplasmic pattern is present in over 50% of cases in
children.
Congenital infantile fibrosarcoma (CIFS) generally presents in the first year or two of life as a large mass
occupying a substantial portion of the involved site (hand, foot or entire extremity, trunk) or an obstructing mass
in the small or
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large intestine of an infant. These tumors can be quite hemorrhagic and may be mistaken for a vascular tumor
clinically. In the past, this tumor was managed by extensive surgery including amputation, but today CIFS is
treated by low dosage adjuvant chemotherapy with often impressive reduction in its size. A circumscribed, but
non-encapsulated mass measuring 6 to 15 cm in greatest dimension has either uniform, glistening tan-white cut
surface or a cystic, hemorrhagic, and friable character whose features may suggest something other than CIFS.
Fascicles of uniform spindle cells with or without the so-called herringbone pattern are the classical features, but
we have been impressed by the histologic diversity of these tumors that have a poorly organized pattern of
immature and even primitive mesenchymal cells; the more primitive appearing CIFSs can be more aggressive in
behavior than their typical spindle cell counterpart (Figure 24-26). A primitive RMS or an undifferentiated
sarcoma (US) may be considered in the differential diagnosis before IHC and/or molecular genetic studies are
applied to sort out the diagnosis. Though the histologic pattern may be problematic, CIFS has the ETV6-NTRK3
fusion transcript, t(12;15)(p13;q25), in most cases (136) (Table 24-2). Other cytogenetic abnormalities include
trisomy 11, t(12;13), gains in chromosomes 8, 11, 17, and 20; and deletion of 17q. Foci resembling CIFS may be
found in other fibrous tumors of childhood including infantile myofibromatosis, but the characteristic translocation
is not present in these cases. The other neoplasm with the t(12;15) translocation is the cellular mesoblastic
nephroma or infantile FS of the kidney. After chemotherapy, the resected specimen may have minimal residual
tumor with only fibrosis, histiocytes, and hemosiderin deposition. Metastasis occurs in less than 5% of cases. In
the lung, the infantile peribronchial myofibroblastic tumor has a histologic resemblance to CIFS but lacks the
signature translocation of the latter tumor.
FIGURE 24-24▪FS (adult type) presented in the region of the right ankle in this 7-year-old male. The cut surface
of this 4.5 cm mass has a faintly multinodular tan-white glistening appearance.
FIGURE 24-25▪FS (adult type) presented in the ankle of a 7-year-old male. A: The pattern is that of densely
apposed spindle cells with fusiform nuclei with prominent nucleoli. Mitotic activity is brisk. B: The tumor cells are
uniformly positive for vimentin. C: There is diffuse membrane positivity for CD34 and negative for all other
markers and did not have the t(x; 18) translocation of SS.
Low-grade fibromyxoid sarcoma (LGFS) (Evans tumor) is a generally slow-growing soft-tissue neoplasm with
a preference for the lower extremity and trunk (78, 156). Approximately 20% of cases are discovered before the
age of 20 years and have been seen as early as 4 years old (89). A well-circumscribed, nonencapsulated
fibrous-appearing tumor has a distinctive microscopic appearance of bland spindle cells with an alternating pale
myxoid background to a more collagenous stroma (Figure 24-27). Foci of epithelioid cells are found in those
tumors with a hyalinized stroma and in some of these cases, hyalinizing giant rosettes are present to establish
the linkage between LGFS and the hyalinizing spindle cell tumor with giant rosettes (205) (Figure 24-27C). A
shared translocation, t(7; 16)(q34;p11), has been identified in 90% of cases as well as a second less common
translocation, t(11;16) (p11;q11). Immunohistochemically, LGFS is diffusely positive for vimentin and focally for
epithelial membrane antigen (EMA) in greater than 75% of cases.
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FIGURE 24-26▪CIFS presented as a mass in the jejunum of a 3-month-old female. A: A uniform proliferation of
spindle cells with and without a fascicular growth pattern is the characteristic appearance of this tumor. B: The
spindle cells are consistently immunopositive for vimentin, but little else. This tumor was translocation positive.
FIGURE 24-27▪LGFS presented as a soft tissue mass on the forearm of a 12-year-old male. A: The margins are
well circumscribed, usually in the absence of a well-formed pseudocapsule. B: The alternating pattern of more
cellular and the less cellular myxoid foci is a characteristic feature. C: Hyalinizing rosettes are also another
typical, but inconsistent finding.
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Sclerosing epithelioid fibrosarcoma (SEFS), an uncommon subtype of FS, is seen on occasion in
adolescents but more often in the latter decades of life (75, 162). The lower extremity and pelvis are the
preferred sites of presentation and reside in the deep soft tissue where bone involvement may be present. A
hyalinized matrix like stroma contains the small aggregates and individual epithelioid cells, which are only
immunoreactive for vimentin. The SEFS is an aggressive neoplasm, which metastasizes to the lungs in 50% to
70% of cases. A rearrangement of chromosome 10p11 is reported. Sclerotic areas in LGFS have a resemblance
to SEFS.
Solitary fibrous tumor (SFT) and hemangiopericytoma (HPC) have been wedded as a pathologic continuum
and classified with the fibroblastic-myofibroblastic tumors in the same WHO category of intermediate, rarely
metastasizing neoplasms (75). The relationship of SFT and HPC to each other has been reviewed by Gengles
and Guillou (85). Most cases of SFT arise from the pleura in adults, but any number of nonpleural sites of origin
have been documented in both children and adolescents so that it is important to consider this diagnosis when
presented with a bland appearing spindle cell neoplasm with a collagenous stroma (172). The cellular foci may
alternate with less cellular fibrous areas. Some SFTs may be more uniformly cellular with a variety of patterns
associated with FS (fascicular or herringbone), nerve sheath neoplasm especially in the presence of a myxoid
background or LGFS (palisading) or storiform (fibrous histiocytoma and dermatofibrosarcoma). Both SFT and
HPC are immunoreactive for vimentin and CD34; this immunophenotype is shared with DFSP, but in most cases,
a distinction is made by the clinical presentation of a dermal-subcutaneous-based neoplasm in the case of DFSP
rather than a deep soft tissue or serosal-based mass in the case of a SFT. In terms of HPC, a differentiation is
made between infantile myofibromatosis-associated HPC and HPC presenting in the soft tissues in older
children, adolescents, and adults. The HPC-like pattern may be encountered in other soft-tissue neoplasms
including monophasic SS, congenital infantile FS, mesenchymal chondrosarcoma (MCS) and MPNST
Dermatofibrosarcoma protuberans (DFSP) and the related giant cell fibroblastoma (GCF) have been
regarded variously as fibrohistiocytic or fibroblastic neoplasms. Given the fibrosarcomatous progression in some
DFSPs, the tumor may have declared itself in a histogenetic sense. Approximately 8% to 10% of DFSPs are
diagnosed in the first two decades of life, but some tumors, which are finally diagnosed in adults have been
present clinically since childhood (35, 36). The early suggestion that GCF is the juvenile variant of DFSP has
been validated by the demonstration of a shared translocation, t(17;22)(q22;q13) as well as concurrent histologic
patterns of DFSP and GCF in the same tumor (107) (Table 24-2). The earliest clinical presentation is a tumor
noted shortly after birth (10% of cases) (86). A nodule or hypertrophic or atrophic plaques on the trunk or
proximal extremity are two of the more common presentations. Other less common sites include an acral or
inguinal-perineal localization. Except for the more frequent pattern of GCF in children (75% or so of cases <20
years old), DFSP is a tumor that occupies the mid-to-lower dermis with contiguous extension into the subcutis
with overgrowth of fat and extension along fibrous septa and into the deep fascia.
Three basic histologic patterns account for the microscopic variation and the diagnostic challenge offered by
DFSP: uniform low-grade compact spindle cell proliferation with or without storiform profiles, spindle cells with
fibroblastic features, and a collagenous stroma resembling a fibrous tumor and a pale myxoid background with
separation of spindle cells (Figure 24-28). GCF has a similar pattern of infiltration as the classic DFSP.
Pigmented cells are found in the socalled Bednar tumor or pigmented DFSP (176). The floretlike giant cells of
GCF appear to reside in tissue clefts and spaces. The mesenchymal cells can display substantial cytologic
variability and have a somewhat primitive appearance in a fibromyxoid background. A more fibrous appearance
may suggest a fibromatosis. The diagnosis is eased if there are areas of classic storiform DFSP. Perivascular
lymphocytes in GCF are useful in the diagnosis. Vimentin and CD34 are expressed by the tumor cells (Figure 24-
28C) (197). It has been reported that DFSP and GCFs can be immunopositive for CD99 (in addition to SS,
angiomatoid fibrous histiocytoma (AFH) and other EWS family of tumors, MRT, SFT, HPC, and MCS).
The differential diagnosis of DFSP includes fibrohistiocytic tumors of the skin, and in some cases, even after
thorough immunohistochemical evaluation, there may remain some uncertainty about the final diagnosis. Factor
XIIIa immunoreactivity is often useful to establish the identity of a fibrohistiocytic tumor whereas CD34 is negative
in most cases. Juvenile xanthogranuloma (JXG) with a predominant spindle cell pattern can be mistaken for
DFSP when Touton giant cells are not present. The medallion-like dermal dendrocyte hamartoma must be
considered in any rounded, atrophic lesion on the upper trunk, which has a congenital clinical presentation. A
spindle to oval cell proliferation replaces the dermis with concentric proliferation around small vessels and nerve.
There is extension into the subcutis like the DFSP; these tumors are immunopositive for factor XIIIa and CD34
but lack the t(17;22) translocation of DFSP.
FIBROHISTIOCYTIC TUMORS
WHO classification of fibrohistiocytic tumors conveys a certain element of skepticism in referring to them as the
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so-called fibrohistiocytic tumors (75).” The pathway to this state of affairs was the result of the decline and fall of
the malignant fibrous histiocytoma (MFH) as it reemerged as the “undifferentiated pleomorphic sarcoma,” which
was premised on the argument that MFH was nothing more than the final common morphologic and biologic
pathway for several specific types of STSs mainly in adults (54). Pleomorphic sarcomas are uncommonly
encountered in children and some of these have been second malignant neoplasms in a survivor of a first
childhood malignancy. In a review of STSs in children exclusive of RMS, Hayes-Jordan and associates reported
that 11% of cases were diagnosed as MFHs in addition to the more common SS (24% of cases) and MPNST
(15% of cases) (93).
FIGURE 24-28▪DFSP presented as a soft tissue mass in the breast of a 2-year-old female. A: Uniform spindle
cells with pale staining nuclei are arranged in broad fascicles. B: Among the spindle cells, there are scattered
giant cells similar to those in the GCF. C: The tumor cells are diffusely immunoreactive for CD34.
Fibrous histiocytoma in some respects appears as often as a histogenetic concept as a specific diagnosis in a
child or adult. Dermatofibroma (DF) of the skin (benign cutaneous fibrous histiocytoma) is the most common
“conceptual” representative of fibrohistiocytic tumors in children and adults in our experience. Even the latter
statement is the subject of disagreement by Zegler and associates who refer to DF as “fibrosing dermatitis”
rather than a true neoplasm (218). Another viewpoint is that the DF is a neoplasm of dermal dendrocytes, which
explains some of its overlapping microscopic and immunohistochemical features to those of JXG (52). In addition
to a pure spindle cell proliferation in the dermis with its characteristic collagen trapping at its CD34-
immunopositive lateral margins, DF may contain hemosiderin-laden macrophages (hemosiderotic DF) with focal
hemorrhage, prominent erythrocyte-filled lakes (aneurysmal DF), multinucleated giant cells, or epithelioid
histiocytes (Figure 24-29). The histiocytic component among the spindle cells may have finely xanthomatized or
foamy cytoplasm. Touton-like giant cells may raise the possibility of JXG which cannot be resolved with IHC
since the latter and DF both express factor XIIIa. However, JXG in the skin has a “pushing” rather than infiltrating
margins into the dermis of a DF. DF can extend into the deep dermis and subcutis to cause concern about
DFSP, but infiltration into the subcutaneous fat is not a feature of DF (14).
A problematic fibrohistiocytic lesion is the so-called benign fibrous histiocytoma (BFH) of the subcutis and
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deep soft tissues with a recurrence rate of 15% (compared to the DF with a local recurrence rate of 5% or less)
and a rarely expressed potential for metastasis (87). Microscopically, BFH is a hypercellular spindle cell
neoplasm, more so than a DF, whose cells tend to have more cytoplasm but can resemble the more common DF.
Multinucleated giant cells and mitotic figures may be present in BFH. Atypical mitoses and anaplasia are present
in the rare atypical fibroxanthoma of skin in a child. If metastasis develops, it is more often than not after multiple
local recurrences of a BFH.
FIGURE 24-29▪Hemosiderotic DF in the lower extremity of a 16 year old male. A: The tumor is composed of
plump spindle cells with interstitial hemorrhage. B: In this field there are collections of hemosiderin laden
macrophages.
The differential diagnosis of a suspected fibrous histiocytoma is determined to some degree on the presenting
site. In the skin, DFSP and JXG are the principal diagnostic considerations whereas in the subcutis or deeper
soft tissues, NF with a storiform pattern with or without multinucleated giant cells has a resemblance to a fibrous
histiocytoma. In the bone, fibrous histiocytoma and nonossifying fibroma are often a microscopic distinction
without a difference. Fibrous histiocytoma in the airway or lung has some features in common with the IMT. JXG
also rarely presents as a solitary mass in the upper airway.
Giant cell tumor (GCT) of tendon sheath has two patterns: the more common nodular (nodular tenosynovitis)
and the less common diffuse (extra-articular pigmented villonodular tenosynovitis) types (51, 183). The diffuse
GCT may be composed almost exclusively of mononuclear cells despite its appellation and is located in the deep
soft tissues, often in or around a large joint, but extra-articular in location (188). The localized nodular GCT
presents in the finger or wrist as a firm nodule, measuring 2 cm or less and well circumscribed by a fibrous
capsule. The nodule is composed of bland appearing mononuclear cells with a variable number of multinucleated
cells. Spindle cells, xanthomatized histiocytes, and hemosiderin are other features.
Pigmented villonodular synovitis is reported in children, typically over the age of 10 years, presenting in the
knee joint with a chronic joint effusion (129). Papillary-appearing hemorrhagic tissues are characterized by
synovial cell hyperplasia with a hypercellular stroma and hemosiderin-laden mononuclear cells. Chronic
hemarthropathy of hemophilia and synovial hemangiomatosis are other considerations in the differential
diagnosis.
Two fibrohistiocytic tumors, AFH and plexiform fibrohistiocytic tumor (PFHT), occur predominantly in children and
are both regarded as intermediate or low malignant potential neoplasms (75).
Angiomatoid fibrous histiocytoma (AFH) is a slowly enlarging tumor of the extremities or trunk in a child older
than 10 years or in a young adult (61). The tumor may present in the vicinity of a lymph node so that an apparent
lymphoid-based neoplasm may be the initial microscopic impression in the presence of nodular collections of
lymphocytes around the periphery of the mass but in the absence of a fibrous capsule and subcapsular
sinusoids. Constitutional manifestations like those of the IMT have been observed in a small minority of cases. A
sharply demarcated, but nonencapsulated mass measures from 1 to 8 cm in diameter. Cystic areas of
hemorrhage are commonly seen on cut surface but may be absent either grossly or microscopically with the
seeming contradiction of a nonangiomatoid AFH (Figure 24-30). An incomplete fibrous pseudocapsule contains
prominent collections of small lymphocytes and plasma cells. The tumor cells are ovoid to spindle shape and are
arranged in densely cellular nodular cells with faint storiform configuration. There is minimal nuclear atypia and
mitotic figures in most cases, but some tumors can display individual nuclear pleomorphism and considerable
mitotic activity, even atypical mitotic figures. Occasionally, giant cells are present. Immunohistochemically, these
tumors are reactive for
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vimentin (100% of cases), desmin (40% to 50% of cases), CD68, EMA (5% to 10% of cases), and CD99 (diffuse
membrano-cytoplasmic staining similar to EWS-PNET in 50% or more of cases) (Figure 24-30D). The CD99
positivity is interesting in light of the molecular genetics of EWS gene fusion in the EWSR1-ATF1 translocation
(186, 199) (Table 24-2). There is a local recurrence rate of 10% to 15% and distant metastasis in 5% or less of
cases. The rare case may have metastatic involvement of a regional lymph node upon initial clinical
presentation.
FIGURE 24-30▪AFH in a 14-year-old male presented in the upper arm. A: A lymphocytic infiltrate is present at
the periphery of the mass in addition to lymphoid follicles can be mistaken for a lymph node-based neoplasm. B:
The angiomatoid characterization of this tumor is based upon the presence of red cell filled spaces. These
spaces are seen in most but not all cases which can lead to diagnostic difficulties. C: The tumor cells have ovoid
to polygonal-shaped nuclei and eosinophilic cytoplasm. The cell borders are poorly defined. Scattered mitotic
figures are present and in some cases, atypical mitotic figures may be seen. D: Immunohistochemical staining for
CD99 shows diffuse membrane positivity. This tumor had an EWS breakapart by FISH.
Plexiform fibrohistiocytic tumor (PFHT) is a distinctive neoplasm of the dermis and/or subcutis whose
morphologic variability contributes to some of its difficulties in pathologic diagnosis (148). A firm nodule on the
forearm, lower extremity, or trunk in a child over 10 years of age and into early adulthood is the clinical
presentation. The multinodular growth pattern at low magnification is characteristic; these nodules may be
composed of fibroblast-like cells and/or mononuclear cells with osteoclast-like giant cells whose numbers can
vary from inapparent to several in the midst of the mononuclear cells. In those PFHTs with a predominant
fibroblastic pattern, an inflammatory component is often present (Figure 24-31). Infiltration of the subcutaneous
fat has some similarities to infantile subcutaneous fibromatosis. Adding to the challenge is that the fibroblasts are
often immunoreactive for SMA. Overall, PFHT has a locally nonaggressive appearance in contrast to a
fibromatosis and has minimal mitotic activity in most cases. A background of hyalinized collagen can accentuate
the nested character of the tumor and can have some resemblance to the clear cell sarcoma (CCS) of tendon
sheath. The mononuclear cells of
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PFHT are immunoreactive for CD68 whereas the tumor cells in the CCS express S100 proteins and HMB-45
(196). Other considerations in the differential diagnosis when the dermis is involved by multiple nodules of
histiocytic cells is a melanocytic proliferation (Spitz or cellular blue nevus) and neurothekeoma especially in
those PFHTs with myxoid features. One histogenetic perspective is that PFHT and cellular neurothekeoma may
be related neoplasms. Like other fibrohistiocytic neoplasms, PFHT has a reported local recurrence rate of 15%
to 40%, but a metastatic rate of less than 2% of cases.
FIGURE 24-31▪PFHT in a 5-year-old female presented on the lower extremity. A: A mass measuring 2 cm in
greatest dimension involved the lower dermis and underlying subcutis and composed of nodules of pale staining
histiocyte-like cells with lymphocytes. B: Some or many of the nodules have one or several multinucleated giant
cells.
Dendritic cell (DC) neoplasms are composed of cells whose normal function is antigen presentation
represented by four distinctive types: follicular DC, interdigitating DC (IDC), Langerhans cell, and histiocytic-
fibroblastic cell. The latter cell may serve as the neoplastic progenitor for the DF and JXG. The Langerhans cell
and Langerhans cell histiocytosis (LCH) are familiar topics in pediatric pathology. JXG is likewise well known as
a cutaneous lesion in a young child, but approximately 5% of cases of JXG in children present as a mass in the
subcutis or within the skeletal muscle (52, 106). These children are less than 1-year-old at diagnosis and the
mass may even be present at birth. The head and neck and trunk are the sites of predilection for a nodule
measuring 3 cm or less. A well-circumscribed, nonencapsulated proliferation may be composed predominantly of
mononuclear cells, a combination of mononuclear and spindle cells or infrequently only of bland appearing
spindle cells (Figure 24-32). The presence of xanthomatized mononuclear cells should alert to the possibility of
JXG since classic Touton giant cells are often not present in the extracutaneous lesions of JXG. Eosinophils can
be prominent and their presence may lead to concern about LCH but the cells of JXG do not express CD1a;
however, like most DC proliferations, there is often S100 protein reactivity. Pseudorheumatoid nodule or deep
granuloma annulare, a nonneoplastic lesion of the soft tissues of the head and lower extremities in young
children, can be mistaken for a histiocytic proliferation (Figure 24-33).
Follicular DCs are found in the germinal centers where they are characterized by CD21, CD35, CD138, and
clusterin positivity and less often for S100 protein and nonreactive for CD1a. These spindle cell tumors with
concentric whorls of cells can be mistaken for a fibrohistiocytic neoplasm of unspecified type or IMT. Though a
rare neoplasm, follicular DC tumor is recognized in children.
IDC tumors are seemingly less common than the follicular DC tumors and have been documented in children.
There is a report of a histiocytic sarcoma with IDC differentiation in a 3-month-old boy (121). These tumors may
be composed of highly pleomorphic large polygonal cells whose features have some resemblance to the cells of
the MRT as well as a spindle cell component, which is not a feature of MRT. Multinucleated giant cells have
been observed as well. The tumor cells are reactive for vimentin, CD68, and S-100 protein but not for CD30
(positive in anaplastic large cell lymphoma), ALK-1, CD 1a, or CD21.
ADIPOCYTIC (LIPOMATOUS) TUMORS

Just as there is a “gray zone” between what constitutes a vascular neoplasm versus a malformation, an
analogous dilemma is encountered in some examples of adipocytic or lipomatous tumors. Since lipomatous
lesions in children are resected for a number of reasons other than the presence of a mass, it may be the case
that the pathologist is not always provided with a complete clinical profile on the patient. However, there are
several important and well-characterized syndromes in which the specimen is not just another “lipoma” (Table
24-7). Each of these syndromes is associated with multiple lipomatous tumors with the less than
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settled question whether these fatty masses are hamartomas or neoplasms. The lipomatosis in
encephalocraniocutaneous lipomatosis (ECCL) is diffuse overgrowth of subcutaneous fat of the scalp and
eyelids, and ipsilateral to unilateral porencephalic cysts (193). It has been questioned whether another
syndrome, oculocerebral syndrome, may not represent a milder variant of ECCL. There is also the possible
relationship of congenital infiltrating lipomatosis to multiple familial lipomatosis.
FIGURE 24-32▪JXG presented as a soft tissue mass in the posterior thigh of a 10-week-old male. A:
Mononuclear, pale staining histiocyte-like cells and Touton giant cells are the classic microscopic features. B:
Other foci are composed of xanthoma-like cells. C: A spindle cell component is infiltrating into the skeletal
muscle. D: Factor XIIIa and CD68 (shown) are expressed by the tumor cells which are immunonegative for
CD1a.
FIGURE 24-33▪Deep granuloma annulare presented in the ankle region of a 4-year-old male who also had a
second similar mass over the tibia. A central focus of necrobiotic collagen is surrounded by a densely cellular
mantle of histiocytes. Numerous such lesions comprise the mass effect. This diagnosis may be elusive when the
central necrobiosis is relatively inconspicuous in which case a histiocytic proliferation or vascular lesion is often
considered in an involuting lesion.
Phosphatase, tensin homologue, and deleted on chromosome TEN (PTEN) hamartoma tumor
syndromes comprise four entities, all of which are characterized to a greater or less degree by a germ-line
mutation in the tumor suppressor gene PTEN on 10q23.3; these syndromes, all phenotypically distinctive, are
associated with hamartomatous overgrowths to include the development of lipomas. Lipomatous lesions develop
in over 90% of those with Proteus syndrome. Some of these tumors have features of well-circumscribed
mature lipomas whereas others are more diffuse with overgrowth of mature fat with accompanying fibrous septa
resembling the microanatomy of the subcutis
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with associated nerves and blood vessels. Infiltration of skeletal muscle by mature adipose tissue is another
feature. Vascular lesions with hemangioma and lymphangioma-like features occur as well.
Table 24-7 ▪ SYNDROMIC-ASSOCIATED LIPOMA-LIPOMATOSIS
Congenital infiltrating lipomatosis (Slavin-Cols)
ECCL (Haberland)
Congenital lipomatous overgrowth, vascular malformations and epidermal nevi (Sapp)
PTEN (10q23.3) hamartoma tumor syndromes
Cowden syndrome
Bannayan-Kiley-Ruvaleaba syndrome
Proteus and proteus-like syndrome
Bannayan-Zonana syndrome
Multiple familial lipomatosis
Macrodystrophia lipomatosa
Macrodystrophia lipomatosa and macrodactyly are characterized by an overgrowth of an entire extremity or

a digit. A substantial component of the overgrowth phenomenon is mature adipose tissue in all tissue layers
below the dermis. In addition to the fatty overgrowth, fibrous, vascular, and neural components either in excess
or resembling other lesions such as fibromatosis, hemangioma, and neuromatous proliferations can lead to
uncertainty about a neoplastic or hamartomatous process. We have elected to refer to these as “soft-tissue
dysplasia-overgrowth.” Lipofibromatous hamartoma of the median nerve or other nerves in the upper extremity is
found in association with 25% to 30% of cases of macrodactyly.
Congenital intraspinal lipoma is seen with some frequency in those institutions with an active neural tube
defect-spinal dysraphia program (158, 214). One-third to one-half of all lipomas in children in our own experience
are diagnosed in the latter setting. A mass in the subcutis is detected in the midline of the lower back. A
circumscribed, multinodular mass of pale tan to yellow tissue is composed predominantly of lobules of mature
adipose tissue with neural elements and a variety of other tissues indigenous to this site including bone and
cartilage. Microscopic foci of immature nephrogenic tissue and enteric- and/or respiratory-lined cysts offer the
possibility of a teratoma as an alternative interpretation, but the context of a spinal defect and the predominance
of adipose tissue should be kept in mind before a diagnosis of a sacral teratoma is made. One potential problem
in the differential diagnosis is the Currarino syndrome (point mutations in the HLXB9 homeobox gene, 7q36) with
sacral anomalies, tethered spinal cord or lipoma, various anorectal malformations and presacral teratoma. The
spinal lipoma is distinct from the mature teratoma. In some cases, the presacral cyst can have some limited
features of an enteric duplication.
Lipomas are neoplasms and perhaps more hyperplasias or hamartomas in some cases which in either instance
are circumscribed masses of lobules of mature adipocytes. In adults, lipomas comprise 50% or more of all STTs
whereas in children lipomas constitute only 5% of all STTs compared to vascular tumors at 30% in this age
group. Other than the sacral lipomas in association with spinal dysraphia, lipomas in children have a preference
for the superficial soft tissues of the head and neck and trunk. Deep and intramuscular lipomas are uncommon in
children. The differential diagnosis of the intramuscular lipoma includes the skeletal muscle hemangioma, which
can have a substantial component of adipose tissue. Angiolipoma presents in the subcutis of the extremities
(forearm predilection) or trunk, usually in adolescents and young adults. Multiple angiolipomas, often tender, may
have an autosomal dominant or recessive pattern of inheritance. Deep angiolipomas, as an apparent
hamartoma, is seen in Proteus syndrome. Angiolipoma, like the common lipoma, is composed of one or more
lobules of mature adipose tissue but with small peripheral capillaries, which are congested or contain fibrin
thrombi. Although rarely performed as an ancillary study, cytogenetic analysis of a true lipoma demonstrates
supernumerary rings and giant rod chromosomes reflecting amplification of 12q14-15, the site of the MDM2
oncogene (91). Myxoid foci at the periphery of a fatty lobule(s) in an otherwise mature lipoma in a child may
represent the residual immature fat in a lipoma-like lipoblastoma or alternatively myxoid degeneration (Figure 24-
34). There are a number of morphologic subtypes of lipomas, which are largely seen in adults.
Lipoblastoma is the distinctive lipomatous neoplasm of childhood, presenting between early infancy to 10 years
of age (one-third of cases at or before 1 year old) with a predilection for the extremities (60% to 65% of cases)
(Table 24-8). Individual cases have been detected in utero by fetal ultrasonography. There are two growth
patterns, localized, and diffuse (so-called lipoblastomatosis), but most lipoblastomas are a well-circumscribed
mass in the subcutis rather than diffusely infiltrating into the deeper soft tissues (56, 96). A well-circumscribed,
lobulated yellow-tan to grayish mucoid mass measures from 1 to 10 cm in greatest dimension (Figure 24-35).
The lobules are composed of immature lipocytes with or without central mature lipocytes in a background of
delicate, branching capillaries whose vascular pattern has a resemblance to myxoid liposarcoma (LPS) (Figure
24-36). Pools of acellular, basophilic mucoid matrix are another feature, which is also seen in myxoid LPSs. IHC
generally does not provide any assistance in the diagnosis, which may be vexed by a positive desmin stain in a
population of immature mesenchymal cells. The local recurrence rate is reportedly as high as 50% but is closer
to 20% to 25% in our experience. We have had to resort to molecular diagnostic studies in some cases to
confirm that the immature-appearing lipomatous neoplasm, especially in the older child, is not a myxoid LPS.
Lipoblastoma is characterized by a chromosomal rearrangement of 8q11-13 region (8q12), which harbors the
developmentally regulated zinc finger gene, PLAG1 (11, 18). Lipoblastoma-like hamartoma
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is seen in soft tissues of the extremity in young child in a focus of lipoatrophy. A final note is that children with a
lipoblastoma may have neurodevelopmental problems as well as other systemic manifestations (Coffin, personal
communication, 2009).
FIGURE 24-34▪Lipoma with myxofibrous features presented as a soft tissue mass on the foot of an 8-year-old
male. A: Lobules of mature lipocytes are separated by prominent fibrous septa. B: Some of the lobules of fat
have a myxomatous appearance. In the presence of immature lipocytes, it may support the interpretation of a
lipomalike lipoblastoma.
Liposarcoma (LPS) is a rare soft-tissue sarcoma of childhood, which accounts for approximately 3% of all STSs
in children; less than 5% of all LPSs are diagnosed in the first two decades of life; LPS in children typically
presents in the second decade (average age of 15 to 17 years), has a female predilection (2F:1M), and has a
preference for the lower extremity (60% to 70% of cases) (1). Myxoid LPS accounts for 80% to 90% of cases and
most of these tumors are conventional myxoid-round cell types (Figure 24-37). Variation in the morphology
includes spindle cell foci and pleomorphic features. Lipoma-like or well-differentiated and pleomorphic LPSs that
together account for 65% to 70% of LPSs in adults represent 10% or less of cases in children. The prognosis of
LPS in children is similar to the experience in adults. Cytogenetics is helpful in those cases of myxoid LPS with
the differential diagnosis of lipoblastoma with the demonstration of the t(12;16) (q13;p11) (FUS-CHOP fusion
transcript) or EWSR1-CHOP rearrangement (141). The latter translocation would seem to qualify some myxoid
LPSs as a distant cousin in the extended EWS family (Table 24-2). In the COG grading scheme of STSs in
children, myxoid LPS is a grade I neoplasm.
Table 24-8 ▪ SITES AND AGE AT PRESENTATION OF LIPOBLASTOMAS FROMTHE FILES OF

LAURENV ACKERMAN LABORATORY OF SURGICAL PATHOLOGY (1989 TO 2009)
Site No. (%) Age (Range and Mean) Sex (Male:Female)
Lower extremitya 16(32) 1-10 years (3.5 years) 8M/8F
Trunk 10(20) 3 months-9 years (2.7 years) 6M/4F
Neckb 6(12) 1-5 years (3 years) 2M/4F
Axilla—upper extremity 6(12) 6 months-4 years (2 years) 2M/4F
Retroperitoneum—omentum 3(6) 1-2 years (1.6 years) 1M/2F
Scrotum 3(6) 2 years-6 years (5 years) 1M/2F
Mediastinum 2 (4) 8 months, 6 years 1M/1F
Orbit 2 (4) 1 year, 1 year 2M
Scalp 1 (2) 1 year 1M
Vulva 1 (2) 1 year 1F
50 (100) Mean (2.8 years) 26M/24F
aGluteal region, thigh, foot.
bParotid (1 case).
From the files of the Lauren V. Ackerman Laboratory of Surgical Pathology, St. Louis Children’ Hospital,
Washington University Medical Center, St. Louis, MO.
Another distinctive lipomatous tumor is the hibernoma, which infrequently presents before 20 years of age
(<10% of cases). The extremities and head and neck region are the sites of predilection. A lobulated, yellowish-
brown mass is composed of lipocytes with eosinophilic, finely
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vacuolated cytoplasm resembling immature adipocytes in the retroperitoneum of infants. Not surprisingly,
lipoblastoma may have hibernoma-like foci.
FIGURE 24-35▪Lipoblastoma in the neck of a 5-year-old female has a well circumscribed, yellowish-white, has a
faintly lobulated appearance on cut surface and measures 8 × 4 × 1.5 cm. This tumor recurred several months
after the initial excision.
A lipomatous lesion whose pathologic features are characterized by fatty infiltration and replacement of the right
ventricle of the heart by mature adipose tissue is arrythmogenic right ventricular cardiomyopathy
(dysplasia). There is progressive replacement of the entire thickness of the apical, inferior, and infundibular wall
of the right ventricle by mature adipose tissue, which begins from the epicardium or from the midmyocardium as a
seeming metaplasia rather than fatty infiltration (198).
FIGURE 24-36▪Lipoblastoma in the neck of a 5-year-old female presented some difficulty in the differential
diagnosis from myxoid LPS. A: The background is composed of immature mesenchymal cells with pale staining
features. These cells may stain positively for desmin. The interspersed lipocytes show varying stages of
maturation. B: A pale myxoid background with a delicate arborizing network of capillaries separate both immature
and more mature lipocytes.
PERIPHERAL NERVE SHEATH TUMORS

This category of tumor accounts for as many as 15% of all soft-tissue neoplasms in childhood with neurofibroma
(NF) and schwannoma as the two most common types. There are several morphologic subtypes in the latter two
categories reflecting variability in the histologic features from the growth pattern (localized, diffuse, and plexiform
in the case of NF) to cellularlity and regressive atypia (in the case of the schwannoma). Both NF and
schwannoma have pigmented variants; the psammomatous melanotic schwannoma arises in spinal nerve roots,
bone, skin, and upper intestinal tract and may be a manifestation of the Carney complex. Also included among
the peripheral nerve sheath tumors (PNSTs) are the perineurioma, GCT, and neurothekeoma or nerve sheath
myxoma (75). Although Weiss and Goldblum have listed CCS of tendon and aponeuroses as a malignant PNST,
it is discussed with the extended EWS family of neoplasms in this chapter (211). MPNST occurs as a sporadic
tumor or in the setting of NF1 in children and adults alike (8, 82). In children, MPNST is the second or third most
common non-rhabdomyosarcomatous STS, accounting for 15% of all sarcomas compared to SS, representing
25% of cases in the pediatric age group (63, 92, 93). There is also a category of tumefactions of a reparative-
reactive type, which includes the traumatic neuroma, postamputation nerve hypertrophy, and presumed
hamartomatous lesions such as the neuromuscular hamartoma and the neural lipofibroma involving the median
nerve (Figure 24-38).
Neurofibroma (NF) is the most common PNST in children, accounting for almost 70% of cases. Localized NFs
are restricted to the dermis whereas the diffuse NF with involvement of the dermis and subcutis as well as the
deeper
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plexiform NF are invariably a manifestation of NF1 with the development of multiple cutaneous, subcutaneous,
and deep soft-tissue masses (62, 181). Plexiform NFs in NF1 are detected in 40% to 50% of children by 5 years
of age with a anatomic preference for the trunk and extremities, although the infiltrative plexiform NF may involve
deep anatomic structures in the head and neck region (203, 204, 207) (Table 24-9). NFs are less common in
NF2, where the characteristic tumor type is the schwannoma, which can have plexiform features to be
differentiated from the plexiform NF. Diffuse NF is composed of uniform, bland appearing spindle cells in a pale
staining eosinophilic background. There is often overgrowth of the dermis with contiguous growth into the
subcutis. Plexiform NF is composed of rounded to more serpentine nodules of spindle cells in a pale staining
myxoid background. These nodules are found in the skin and/or subcutis with or without an accompanying
pattern of diffuse NF (Figure 24-39). The nodules can be found in and around salivary glands in the head and
neck region or grossly as thickened and tortuous peripheral nerves. The smaller plexiform NFs are less readily
traceable to a peripheral nerve. The presence of enlarged, pleomorphic nuclei and even a few mitotic figures
should be viewed with concern about sarcomatous transformation of a plexiform NF. These pleomorphic nuclei
often display p53 staining whose presence should be correlated with any other features to suggest malignant
transformation (127).
FIGURE 24-37▪Myxoid LPS presented on the anterior abdominal wall of a 13-year-old male. A: Mucinous filled
cysts are separated by moderately cellular foci with a myxoid and vascularized background. B: The individual
tumor cells have enlarged nuclei which are moderately hyperchromatic. The delicate network of capillaries is
present in the background.
FIGURE 24-38▪Traumatic neuroma of the peroneal nerve occurred in a 17-year-old female who had sustained
deep soft tissue injury to the lower extremity. Several nerve fascicles are present in the soft tissues as individual
structures separated by collagen. Smaller nerve bundles are adjacent to one of the larger nerve bundles.
Schwannoma is a neoplasm with morphologic and immunophenotypic features of Schwann cells forming the
nerve sheath (122). There is a predilection for the head and neck region and upper extremity in the case of
sporadic schwannomas in children (114, 115). It is unusual for a schwannoma to present before the age of 10
years (182). In children with NF2, nodular or plaque lesions in the skin or subcutis are well circumscribed,
encapsulated spindle cell neoplasms with or without plexiform features; these tumors show strong diffuse S100
protein positivity unlike the less uniform pattern of S100 protein staining of the NF (182). Grossly, the
schwannoma varies in size from 1 to 10 cm in greatest dimension, is encapsulated, and has a glistening, mucoid,
and a pale tannish to yellowish-tan appearance (Figure 24-40). Hemorrhage, cystic degeneration, and fibrosis
are uncommon secondary features in schwannomas in children in contrast to schwannomas in adults. The
challenge in the pathologic diagnosis is the variability in the
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histologic patterns, but the two basic ones are the spindle cell pattern with or without Verocay body formation
(Antoni A) and the alternating less cellular myxoid foci (Antoni B) (Figure 24-41). Foci resembling a NF may be
seen in some cases, but keep in mind the presence of a capsule in the schwannoma. Lymphocytes and foamy
histiocytes may be more or less apparent in a particular schwannoma. Mast cells are present in variable
numbers. Nuclear enlargement and hyperchromatism are present in some cases and should not be viewed with
concern about potential malignancy. Sporadically schwannomas can have a hypercellular spindle cell pattern,
and mitotic figures can be seen in the cellular variant. In the setting of NF2, caution is advisable with a diagnosis
of MPNST when the schwannoma is both cellular and mitotically active. Rather than a diffuse pattern, a
multinodular pattern is also documented in children and these tumors are mistaken for a NF (213). Bilateral
schwannoma of the VIII cranial nerve is a diagnostic manifestation of NF2 (138, 166) (Table 24-9). Finally without
the presence of ganglion cells, a ganglioneuroma with its predominant neuromatous-stromal component has a
convincing resemblance to a schwannoma or NF (Figure 24-42).
Table 24-9 ▪ GENETICS AND MANIFESTATIONS OF NFTOSESTYPE 1 AND 2 AND

SCHWANNOMATOSIS
NF Type 1 NF Type 2 Schwannomatosis
Incidence 1:3,000 1:30-40,000 1:1.7 million

(Finnish)
Gene NF1: 17q11.2 NF2: 22q12.2 Possible 22q 11

which harbors
SMARCB1/INI1
Neurofibromin, negative regulator of Merlin, inhibits cell

RAS-MAPK proliferation in
response to cellular
adhesion
Phenotype Café au lait macules (infancy) Schwannomas Two or more

Diffuse and plexiform NFs Bilateral VIII nerve schwannomas
Optic nerve glioma (pilocytic schwannomas without VIII nerve
astrocytoma)Ependymoma schwannomas
MPNST (lifetime risk 8%-13%)
Pseudarthrosis
Vascular dysplasias
Compiled fromYohay, K. Neurologist 2006;12:86-93; McClatchey AI. Neurofibromatosis. Annu Rev

Pathol 2007;2:191-216; MacCollin M, Chiocca EA, Evans DG, et al. Diagnostic criteria for
schwannomatosis. Neurology 2005;64(11):1838-1845; Hadfield KD, Newman WG, Bowers NL, et al.
Molecular characterisation of SMARCB1 and NF2 in familial and sporadic schwannomatosis. J Med
Genet 2008;45(6):332-339; Brems H, Beert E, de Ravel T, et al. Mechanisms in the pathogenesis of
malignant tumours in neurofibromatosis type 1. Lancet Oncol 2009;10(5):508-515
FIGURE 24-39▪Plexiform NF arose in soft tissues of the lower back in a 14-year-old female with a history of NF1.
A: This field discloses both the plexiform and diffuse pattern of growth, both characteristic of NF1. B: Plexiform
transformation occurs in peripheral nerves at all levels of the nerve. C: Diffuse pattern is often found in
association with plexiform tumors. D: The plexiform nodules are usually hypocellular with or without coarse
eosinophilic bundles. Increased cellularity and mitotic figures should be viewed with concern about malignant
progression.
FIGURE 24-40▪Schwannoma presented as a paraspinal mass in a 17-yearold male. This encapsulated tumor
measured 10 cm in greatest dimension and had a uniform, glistening yellowish-tan cut surface.
FIGURE 24-41▪Schwannoma presented in the paraspinal region of a 17-year-old male. The encapsulated tumor
had a predominant Antoni B pattern of loosely arrayed spindle cells in an edematous to myxoid background.
FIGURE 24-42▪Ganglioneuroma presented in the lumbosacral region of a 12-year-old female. A: The tumor is
well circumscribed with a capsule or pseudocapsule and is composed of bundles of fusiform spindle cells with a
resemblance to a schwannoma. B: Other microscopic fields contain individual or small groups of mature ganglion
cells.
Schwannomatosis, like NF2, is characterized clinically by the presence of two or more schwannomas, but
unlike NF2, vestibular nerve involvement is not a feature (135) (Table 24-9). Some 15% of affected individuals
develop schwannomas in the first two decades of life. The suspected genetic mutation is near the NF2 gene
locus at 22q11 where the INI1 gene resides as an important oncogenic site (90).
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FIGURE 24-43▪MPNST presented in a 5-year-old male with NF1 with an intracranial presentation of a large
mass arising in the region of the frontal lobe. A: A high grade spindle cell sarcoma consists of uniform cells with
enlarged, elongated hyperchromatic nuclei. B: Nodules of hypercellular cartilage are present focally. C: The
tumor cells are uniformly reactive for vimentin, but only focally for S100 protein. D: The CD57 immunostaining is
diffuse throughout all microscopic fields.
Malignant peripheral nerve sheath tumor (MPNST) presents as a sporadic neoplasm or as a complication of
NF1. The other STS in the setting of NF1 is ERMS, which presents earlier in life than does the MPNST in most
cases. It is rare for a MPNST in NF1 to develop before 5 years of age, but the incidence increases in
subsequent decades with a lifetime risk of 10% to 15% (20, 63). Most MPNSTs generally measure in excess of 5
cm and have a gelatinous grayish-tan to white surface with or without necrosis and hemorrhage. When the
MPNST arises as a plexiform NF, as it often does in NF1, there is often widespread sarcomatous involvement of
the nerve with the formation of one or more masses. The mass(es) is usually not sharply demarcated from the
plexiform component. The basic histologic pattern of a MPNST is a spindle cell sarcoma with fascicular profiles
of interweaving cells. At low magnification, the fascicles may have an alternating “light cell-dark cell” quality due
to more cellular and less cellular foci with lucency between the cells, often with a pale mucoid to myxoid
appearance. Fusiform to more ovoid nuclei display varying degrees of hyperchromatism and mitotic activity.
Anaplasia is uncommon. Residual foci of plexiform NF are often present in NF1-associated MPNSTs; overgrowth
or infiltration by the sarcoma is appreciated in these transitional zones with residual NF. It is for this reason that a
biopsy may yield equivocal findings for MPNST other than scattered atypical spindle cells intermixed with a
plexiform NF. Though the biopsy may not be satisfactory for an unequivocal diagnosis of MPNST, it should
prompt a re-biopsy especially in the presence of an enlarging, previously stable deep soft-tissue mass. In
addition to the features of a spindle cell sarcoma, other findings include individual and small collections of
rhabdomyoblasts (Triton tumor), gland-like structures, a multinodular pattern with overgrowth of a plexiform NF,
an epithelioid pattern with tumor cells resembling those of a MRT, nodules of cartilage or a small cell, rosette-like
pattern with a resemblance to EWS-PNET, or neuroblastoma (Figure 24-43).
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Formations resembling tactile bodies are found in both NFs and MPNSTs. Sporadically occurring MPNSTs are
more difficult to diagnose with certainty when major nerve involvement is not obvious. IHC is not always helpful
but is useful in the differentiation of MPNST from monophasic SS or adult-type FS. In addition to vimentin,
MPNST is immunoreactive for S100, CD57, and collagen type IV in some but not all cases (Figure 24-43).
Another issue in the prognostic assessment of MPNST is the histologic grade and outcome; however, there is
the opinion that all MPNSTs should be viewed as high-grade sarcomas regardless of their individual pathologic
features even in the presence of “low-grade” histology. More relevant is the adequacy of the surgical resection,
which can be problematic when a major nerve is the primary site.
Other types of PNSTs of the soft tissues include the perineurioma, nerve sheath myxoma, and neurothekeoma.
Perineurioma is an uncommon neoplasm, which is seen in children and adults alike (134). Extraneural and
intraneural variants are recognized (17). A well-circumscribed subcutaneous mass measuring less than 7 cm is
composed of spindle cells or more epithelioid appearing cells in a fibrous and emptier appearing background.
Tight whorls of spindle cells and storiform profiles have some resemblance to DFSP. These tumors may be
immunoreactive for CD34 like DFSP but are also positive for EMA and vimentin (both markers are positive in
meningiomas) as well as collagen type IV. Unlike schwannomas, these tumors do not express S100 protein.
Intraneural perineurioma is even less common than extraneural or soft-tissue variant. Over 50% of cases are
diagnosed by 20 years of age as a soft-tissue mass arising in a major nerve or the brachial plexus. There is
some resemblance to a plexiform NF in terms of gross involvement. The spindle cell areas like those in the
extraneural perinerioma are EMA-positive and S100 protein-negative. Nerve sheath myxoma is a predominant
myxoid neoplasm of the dermis and subcutis (68). It is composed of spindled and epithelioid cells arranged in
cords and nests. These Schwann-like cells are immunoreactive for S100 protein, glial fibrillary acidic protein, and
CD57 with some EMA-positive, presumed perineural cells. Neurothekeoma is a neoplasm of young individuals
with 60% of cases presenting before the age of 20 years, and also a tumor whose features overlap with the
nerve sheath myxoma (67). These tumors have been histologically subtyped as cellular, myxoid, and mixed
(102). Multiple, small nodules of spindled to epithelioid cells with or without a prominent myxoid matrix are
accompanied by osteoclast-like giant cells. The immunophenotype of these tumors includes expression of NK1-
C3, neuron specific enolase, CD10, and CD68 whereas they are nonreactive for S100 protein. Quite frankly,
some of these cases are difficult to distinguish from the plexiform fibrohisticytic tumor (104).
Granular cell tumor (GCT) is one of the ubiquitous neoplasms in terms of its anatomic distribution whose
phenotype characterizes it as either neural (S100 protein positive) or nonneural (S100 negative) in type. Two
examples of the latter are the so-called congenital epulis or GCT presenting on the anterior alveolar border of
the maxilla or mandible of a neonate and the so-called primitive polypoid GCT of the dermis (Figure 24-44) (19,
34). The skin, oral cavity, and upper respiratory tract are among the more frequent primary sites in children
(Table 24-10). Multifocal GCTs occur in the presence of a positive family history or in the setting of Noonan
syndrome. A firm, poorly demarcated yellowish-tan to white mass measuring less than 2 cm in most cases is the
usual gross appearance. Nests of granular cells of varying sizes and shapes are composed of polygonal to more
ovoid cells with prominent eosinophilic cytoplasm and a central nucleus (Figure 24-45). The cytoplasm contains
eosinophilic or granular bodies representing lysosomes; these bodies account for the CD68-granular positivity
(Figure 24-44). Nuclear pleomorphism is present in some cases without any implications about outcome, but
mitotic figures should be viewed with concern. Perineural involvement with plexiform features is seen in a small
minority of cases (Figure 24-45). With the noted exceptions, most GCTs are diffusely immunoreactive for S100
protein as well as inhibin-a (125). Malignancy in GCT is rare, especially in children. Mitotic activity, nuclear
pleomorphism, spindle cell morphology, and deep invasion of soft tissues should alert to the possibility of
malignancy. We have seen malignant GCT in the vulva of a 4-year-old female and in the thigh of a 17-year-old
female.
Table 24-10 ▪ GCTS IN CHILDREN AND ADOLESCENTS
Site No. (%) Age Range (Mean) M:F
Solitary skin 21 (34) 2-16 years (10 years) 7/14
Softtissuea 9 (15) 11-17 years (12.5 years) 4/5
Oral cavity (congenital)b 9 (15) 1 day-1 month 0/9

(10 days)
Breast 5 (8) 14-18 years (17 years) 0/5
Orbit 4 (7) 15-17 years (16 years) 1/3
Lip 4 (7) 9-14 years (12 years) 2/2
Multiple skin 4 (7) 5-16 years (13 years) 2/2
Larynx 3 (5) 4-18 years (11 years) 1/2
Tongue 1 (2) 10 years 0/1
Esophagus 1 (2) 16 years 1/0
61 (∽100) 18:43
aOne case in the thigh of 13M with regional lymph node metastasis
bAnterior maxilla (5 cases), anterior mandible (2 cases), hard palate (1 case), frenulum of tongue (1
case).
Hospital, Washington University Medica Center, St. Louis, MO.
Glioneuronal heterotopia, usually presenting in the head and neck region as a polypoid or mass, is usually
seen in early childhood. One example is the so-called nasal glioma. Islands of heterotopic glial tissue are
accompanied by a fibrous stroma in the nasal glioma. Neurons are identifiable in some cases. Glial heterotopias
have been described on the trunk as a soft-tissue mass in children. Extramedullary soft tissue ependymomas
and myxopapillary ependymomas are forms of a heterotopic neoplastic process.
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FIGURE 24-44▪Congenital GCT (congenital epulis) presented as an intraoral mass attached to maxillary gingival
ridge in a neonatal female. A: The tumor cells are compactly arranged against the overlying squamous mucosa.
B: The individual tumor cells are characterized by the pale eosinophilic granular cytoplasm. C: The tumor cells
are immunoreactive for CD68 with a granular pattern of positivity. D: This tumor is regarded as a “non-neural”
type of GCT since the S100 protein is nonreactive in the tumor cells. Note the positivity of the Langerhans and
DCs in the mucosa.
FIGURE 24-45▪GCT presented on the arm of a 7-year-old female. A: Small collections of granular cells are seen
in the superficial dermis. B: This tumor also had a plexiform growth pattern with its intraneural and perineural
involvement (see J Cutan Pathol 2009;36:1174-1176).
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FIGURE 24-46▪PEComa presented as a mass in the abdominal mid-line and was associated with the umbilical
vein-ligamentum teres in a 4-year-old female. The tumor on cut surface has a well circumscribed appearance
with a slightly nodular tannish-brown appearance. It measured 3.5 cm in greatest dimension.
PERIVASCULAR EPITHELIOID CELL NEOPLASM (PECOMA)

This category of neoplasms is a histogenetic concept in a sense since there is no known normal counterpart cell
at the present (101). What has emerged from the concept is a group of neoplasms that includes the
angiomyolipoma of the kidney and the so-called sugar tumor of the lung. Although these tumors lack morphologic
homogeneity, they share a unique immunophenotype of smooth muscle (SMA, calponin) and melanocytes (HMB-
45, melan-A, and microphthalmic transcription factor) (79). The S100 protein is often nonreactive.
The PEComa arises in soft tissues and various visceral structures and organs including the falciform ligament
(socalled myomelanocytic tumor), uterus, vagina, and liver where the epithelioid angiomyolipoma can be
mistaken for a hepatic adenoma (Figure 24-46) (159). The tumor cells of the PEComa can have a predominantly
clear cell appearance or large epithelioid cells with abundant eosinophilic cytoplasm (Figure 24-47). The
differential diagnosis includes malignant melanoma, ASPS, and CCS of tendon sheath and aponeuroses. There
is the conundrum of the histogenetic relationship of the PEComa to the Xp11.2 translocation renal neoplasm with
its TFE3 gene fusion, ASPS (der TFE3 fusion), and melanoma. The PEComa, though having some
immunophenotypic overlap with the CCS of tendon sheath, is not one of the extended EWS family neoplasms.
GASTROINTESTINAL STROMAL TUMOR

The GIST is a well-established clinicopathologic entity with its signature activating pathway mutations of KIT
(type III tyrosine receptor kinase) or PDGFRA (platelet-derived growth factor receptor a) on 4q12 with resulting
immunohistochemical positivity for CD117 (c-kit) and CD34 (145, 167). These tumors are uncommon in the first
two decades of life accounting for only 1% of all cases. However, GISTs in younger individuals have some
distinctive features in contrast to the adult counterpart. The general experience with sporadic GISTs in all age
groups is that the stomach (50% to 60% of cases) and small intestine (20% to 30%) are the two most common
primary sites. In individuals 21 years of age or less, the stomach is the preferred site of presentation (>80% of
cases). A small subset of GISTs in children (usually over the age of 10 years at diagnosis) is associated with the
Carney triad (pulmonary chondroma, paraganglioma, and GISTs in addition to adrenal adenomas in young
females) and NF1 (58, 137, 212). Most GISTs have an activating mutation in KIT (85% to 90% of cases) or
PDGFRA (10% or less of cases), but in children and those with NF1, the wild type KIT without mutations is the
rule rather than the exception and the gastric tumors tend to be multifocal rather than solitary as in adults (105,
146). Histopathologically, GISTs have a predominant spindle or epithelioid or mixed morphology. An epithelioid
pattern has been observed with greater frequency in GISTs in the children. Spread into the peritoneal cavity and
regional lymph node, metastases are reportedly more common in children, but paradoxically the clinical course is
more indolent despite this aggressive behavior by the tumor. In some respects, the risk assessment based upon
the size of the tumor and mitotic rate loses some of its predictive value in GISTs in children. Though not likely to
be confused with a GIST, CCS of the kidney may be immunopositive for CD117.
SKELETAL AND SMOOTH MUSCLE NEOPLASMS

Not surprisingly, the subject of myogenic tumors in children is dominated by one neoplasm, RMS, which
accounts for 40% to 60% of all STSs in the first two decades of life (92). Benign myogenic tumors represented
by the adult, fetal and cardiac rhabdomyomas, and leiomyoma are uncommon by comparison with RMS in
children.
Rhabdomyomas in children present either in the heart or as a mass in the subcutis of the head and neck region
or chest wall in children in the first 2 years of life. Over 50% of cardiac tumors in children are rhabdomyomas and
50% to 80% of those cases are associated with the tuberous sclerosis complex (25). These tumors are
diagnosed in utero in 0.1% to 0.2% of prenatal ultrasounds. Fetal hydrops and findings to suggest hypoplastic
left heart syndrome are among some of the clinical presentations. Well-circumscribed solitary mass or multifocal
pale tan masses in the left or right ventricle are the two most common sites of involvement but not restricted to
the ventricle. Histologically, the enlarged myocytes have clear to vacuolated to eosinophilic cytoplasm with
scattered cells having strands of cytoplasm from the nuclear to cell membrane producing the so-called spider cell
(Figuer 24-48).
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FIGURE 24-47▪PEComa has characteristic microscopic and immunophenotypic features. A: A uniformly nested
neoplasm is composed of rounded to ovoid tumor cells with uniform clear cytoplasm. B: The
immunohistochemical profile included positivity for vimentin. C: The tumor cells are immunoreactive for SMA. D:
A similar diffuse pattern of positivity is seen for HMB-45.
FIGURE 24-48▪Cardiac rhabdomyoma in a 3-week-old female presented as an obstructing mass in the left
ventricle. A: Large tumor cells with abundant clear cytoplasm are accompanied by interspersed cells with
strands of cytoplasm producing the features of the so-called spider cells. B: The tumor cells are strongly
immunopositive for desmin.
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FIGURE 24-49▪FRM presented in a 4-month-old female with a 2.7cm mass on the upper chest wall. A: The
pattern consists of fetal appearing myotubes separated by uniform immature mesenchymal cells without
appreciable atypia, rhabdomyoblastic differentiation nor mitotic figures. B: Desmin immunostaining enhances the
pattern of positively staining myotubes and non reactive immature mesenchymal cells.
Fetal rhabdomyoma (FRM) is a rare, sporadic tumor (also known to occur in the basal cell carcinoma
syndrome) with two histologic patterns: myxoid and cellular (111). Unlike RMS with its presentation in deep soft
tissue or visceralbased tumors, FRM presents in the deep dermis and/or subcutis (208). A vague multinodular
pattern is composed of an orderly, almost layered, arrangement of small immature cells with interposed immature
myotubes (Figure 24-49). The nuclei of both cell types are uniform and are neither enlarged nor hyperchromatic.
The presence of any mitotic figures warrants reconsideration of a diagnosis of FRM to a well-differentiated
ERMS. Adult rhabdomyoma is a rare tumor overall, but even more so in children (119). Over 90% of all cases
present in the head and neck region are known to form multifocal masses occurring almost exclusively in males.
These tumors have some resemblance to the cardiac rhabdomyoma. The differential diagnosis includes a
nonneoplastic disorder of skeletal muscle, focal myositis, presenting as a mass or masses in the deep soft
tissues of the extremities (9, 83). This inflammatory process forms a well-circumscribed mass in the skeletal
muscle with a combination of inflammatory and myopathic changes (Figure 24-50). The adjuvant muscle often
has accompanying injury with degenerative and regenerative features.
FIGURE 24-50▪Focal myositis in an 11-year-old female presented with multiple soft tissue masses in the lower
extremity. A: The biopsy shows a circumscribed multinodular pattern of skeletal muscle with atrophy and
myopathic changes. B: Multifocal lymphocytic infiltrates are found among muscle fibers with degenerating
features.
Smooth muscle tumors in children include the rare smooth muscle hamartoma arising on the lower trunk
usually in infants. Leiomyoma of the soft tissues is a rare soft-tissue
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neoplasm regardless of age (210). Multifocal smooth muscle tumors of undetermined malignant potential and
leiomyosarcoma have been reported in the immunosuppressed child and in some cases; these tumors are
another example of Epstein-Barr virus-associated neoplasm. A variant of Alport syndrome is associated with
diffuse smooth muscle masses in the esophagus and perineal region (84).
Rhabdomyosarcoma (RMS), together with neuroblastoma and Wilms tumor, is one of the most familiar and
wellstudied malignant neoplasms of childhood. Its two distinctive subtypes, ERMS (65% to 75% of cases) and
ARMS (20% to 25%), constitute approximately 45% to 55% of all STSs in children and 6% to 8% of all
malignancies in the first two decades of life (139, 164). In the United States, approximately 900 newly diagnosed
cases are seen per year compared to 60 cases per year in the United Kingdom. In common with several other
solid malignancies of childhood, the majority of cases (65% to 79%) are diagnosed before the age of 10 years
with an initial age peak between 1 and 4 years and a later smaller age peak between 15 and 19 years to reflect
the more numerous ERMS in the younger children and ARMS in older children and adolescents (194). However,
it should be kept in mind that ARMS can present in infancy and early childhood just as an ERMS occurs in
adolescence as in a case of paratesticular embryonal RMS. The anatomic distribution of RMS is well established
in the following sites and organ systems: genitourinary tract (25% to 30% of all cases) to include the bladder,
prostate, vagina, cervix and pelvic soft tissues, head and neck (30% to 35% of all cases) to include oral cavity,
oropharynx, nasal cavity and nasopharynx, pterygopalatine fossa, middle ear and orbit (Figure 24-51),
extremities (15% to 20% of all cases), and miscellaneous other sites (15% to 30% of all cases). Some of the
miscellaneous sites are ones in which there is minimal clinical suspicion about RMS such as in the skin, common
bile duct, chest wall, retroperitoneum, and perianal-perineum. In a small minority of cases, there is widespread
disease with involvement of bone and lymph node upon initial clinical presentation as in the case of ARMS but
rarely in embryonal RMS. Both ERMS and ARMS spread to regional lymph nodes and beyond to the lungs and
bone marrow. Approximately 15% to 20% of children who present with RMS have evidence of regional or distant
metastatic disease. In the specific instance of ARMS, a higher proportion of children will have lymph node and/or
bone marrow involvement at diagnosis (30% to 35% of cases). Regardless of the pathologic stage of RMS at
presentation, the decision for chemotherapy is based on the premise that there is at least micrometastatic
disease at the time without specific pathologic documentation.
FIGURE 24-51▪ERMS arising in the soft tissues of the orbit. Though rarely necessary today, this specimen from
an orbital exenteration shows a soft, glistening, whitish neoplasm infiltrating around the globe.
Most children with RMS do not have any predisposing genetic conditions or risk factors (95% to 98% of cases)
except in the minority of children with Li-Fraumeni syndrome, NF1, Beckman-Wiedemann syndrome, Costello
syndrome, Noonan syndrome, and the familial pleuropulmonary blastoma tumor predisposition syndrome (216).
Other neoplasms of childhood may have a malignant rhabdomyoblastic component, yet are not regarded as
RMSs per se: triton tumor (MPNST with RMS component), malignant ectomesenchymoma (PNET with RMS
component) or gangliorhabdomyosarcoma (Figure 24-52), pleuropulmonary blastoma in its three pathologic
subtypes (Figure 24-53), RMS arising in a germ cell neoplasm, Sertoli-Leydig cell tumor of the ovary with
heterologous elements in the form of RMS and congenital melanocytic nevus with RMS elements (76).
The staging of RMS, as with any other solid malignancy, is a combined clinical and pathologic endeavor, which
incorporates the extent of disease beyond the primary site, but it also includes the specific primary site since the
latter influences outcome in addition to the presence or absence of regional lymph node metastasis and distant
metastasis to the bone marrow and/or lungs as well as to other sites. Those tumors arising in the head and neck
region may be parameningeal (nasopharynx, middle ear, or pterygopalatine fossa) or not (orbit). Parameningeal
RMS is more likely to spread to the meninges and brain (174).
Though the pathologic subtype of RMS is not formally incorporated into the staging of the disease, ARMS is
known to have advanced stage disease at presentation more often than ERMS, as a manifestation of the more
aggressive behavior of ARMS. RMS is divisible into three pathologic-prognostic categories: favorable,
intermediate, and unfavorable (169) (Table 24-11). In addition to favorable, intermediate, and unfavorable
pathologic subtypes of RMS, these tumors have also been subclassified on the basis of genotype, which
correlates with the histologic subtype (48) (Table 24-2).
The overall 5-year disease-free survival for RMS in children is 70% to 75% today compared to 15% or less 30
years ago. As Dr. Jesse Ternberg, chief of pediatric surgery at St. Louis Children’s Hospital from 1972 to 1990,
summarized the outcome for these children in the early years, “If I could not cut it out entirely, the child was a
goner.”
Embryonal RMS constitutes slightly over 80% of all RMSs in our experience with the head and neck region and
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genitourinary tract together accounting for 80% of all cases (143). The nasal cavity and nasopharynx were the
most common primary sites in the head and neck region (23 of 76 cases, 30%) and the paratesticular soft tissues
(17 of 59 cases, 29%) and vagina (15 of 59 cases, 25%) together were the most common sites in the
genitourinary tract (Table 24-12).
FIGURE 24-52▪Gangliorhabdomyosarcoma (malignant ectomesenchymoma) presented as an extratesticular

scrotal mass in a 2-year-old boy. A: Focal areas of the tumor are composed in part of mature ganglion cells in a
background with a neuromatous appearance. B: Other foci show an ERMS with differentiated rhabdomyoblasts
accompanied by less mature appearing malignant cells. C: Desmin immunostaining is shown in the
rhabdomyosarcomatous areas. D: The neuromatous stroma is strongly positive for S100 protein.
FIGURE 24-53▪Pleuropulmonary blastoma, cystic or type I presented as a cystic lung lesion in a 2-day-old male.
Beneath the epithelial lining of the cysts, there is a cambium layer-like population of primitive round cells, some
of which have the bright eosinophilic cytoplasm of rhabdomyoblasts. Previously these neoplasms were
considered as an example of embryonal RMSs arising in a congenital lung cyst.
Grossly, the size of ERMS correlates with the primary site with the largest tumors presenting in the
abdomenpelvis and extremities where the mass commonly exceeds 6 to 8 cm in greatest dimension. The typical
macroscopic appearance of an untreated ERMS is a soft gelatinous mass with a glistening, mucoid grayish-white
cut surface with or without hemorrhage and necrosis. Most tumors are well circumscribed in the absence of a
well-formed capsule. Since a primary resection is generally unusual today, most RMSs are unlikely to have the
latter features but rather reflect the effects of preoperative chemotherapy which can reduce the tumor to no more
than a small yellowish-white scar composed of histiocytes and fibrosis with or without any remnants of
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the tumor other than differentiated rhabdomyoblasts (7, 44). When residual tumor is identified, a fibrous capsule
may surround a cystic and/or solid focus with focal hemorrhage and fibrosis.
Table 24-11 ▪ PATHOLOGIC-PROGNOSTIC CATEGORIES OF CHILDHOOD RMS
Incidence
Favorable
Embryonal RMS, sarcoma botryoides 4%-6%
Embryonal RMS, spindle cell 2%-3%
Intermediate
Embryonal RMS, patterns other than sarcoma botryoides and spindle cell types 45%-50%
Unfavorable
Alveolar RMS 25%-30%
US 3%-5%
Pleomorphic RMS 1%-2%
The usual initial encounter with a RMS by the pathologist is a needle or open biopsy, which can establish the
terms of the diagnostic challenge. In some respects, ERMS is one of the most histologically diverse of the solid
neoplasms of childhood, which reflects the broad range in the differentiation of the rhabdomyoblasts from small
primitive cells displaying considerable heterogeneity in nuclear size and shape with minimal evidence as to the
exact nature of the tumor (Figure 24-54). In fact, the cytologic diversity of individual tumor cells, which may be
accompanied by a pale, mucoid to myxoid background is an important clue to the diagnosis. The nuclei are
densely hyperchromatic, and mitotic figures are variably prominent. Scattered among the smaller tumor cells,
larger individual cells with eosinophilic cytoplasm may be present; these latter cells are the ones most likely to
demonstrate positivity for desmin, muscle specific actin, myoD1, and myogenin whereas the small primitive tumor
cells may stain diffusely for vimentin, only focally for desmin if at all and may have limited nuclear reactivity for
myoD1 and/or myogenin (31, 184). Other microscopic patterns include condensation of small primitive cells
beneath an epithelial-lined surface in the sarcoma botryoides or solid sheets of tumor cells interspersed by
nested collections of primitive tumor cells resembling the blastemal pattern of Wilms tumor; the blastema-like
pattern is composed predominantly of polygonal-shaped tumor cells with scattered rhabdomyoblasts with clear to
eosinophilic cytoplasm. A spindled population can be seen in association with the blastemal-like pattern, but one
and possibly a second type of ERMS is composed almost exclusively of spindle cells with a differential diagnosis
inclusive of CIFS and leiomyosarcoma. However, most spindled ERMS have scattered immature
rhabdomyoblasts within the background or other minor foci of more primitive appearing small tumor cells. In the
uterine cervix, ERMS is often seen in association with heterologous cartilage (Figure 24-55). When the primary
site is the paratesticular region, ERMS of the spindle cell type is the tumor to be excluded on the basis of IHC.
The other presumed type of RMS with an exclusive spindle cell pattern is the infantile rhabdomyofibrosarcoma
with its usual bland microscopic features unlike the spindle cell embryonal RMS. These tumors can have a
resemblance to CIFS, but unlike the latter tumor, there is immunopositivity for myoD1 and myogenin and they do
not have the ETV6-NTRK3 translocation.
Table 24-12▪EXPERIENCE WITH RMS IN CHILDREN AND ADOLESCENTS FROM 1989 TO

2009
Anatomic Sites ERMS ARMS Total (%)

Head and neck 68 8 76 (34)
Genitourinary tract 58 1 59 (26)
Chest wall-trunk 11 4 15 (7)
Extremity 11 18 29 (13)
Abdomen-pelvis 19 6 25 (11)
Perianal region 5 5 10 (4)
Retroperitoneum 6 — 6 (3)
Skin 3 — 3 (1)
181 (81) 42 (19) 223
Anaplasia may be seen on occasion and if the suspected RMS is a tumor in or near the chest or lung, it is likely
that the neoplasm is a pleuropulmonary blastoma especially in the presence of a collage of high-grade
sarcomatous patterns including nodules of malignant-appearing cartilage. Nodules of immature cartilage or other
teratoid elements are present with some frequency in sarcoma botryoides of the uterine cervix. Another
uncommon feature of ERMS is the presence of a hyalinized or sclerotic stroma. Virtually all ERMSs are
immunoreactive for vimentin, but the number of tumor cells, which express desmin and muscle specific actin is
quite variable from one tumor to another as a manifestation of the spectrum of myogenic differentiation. Likewise,
the number of tumor cells with nuclear positivity for myoD1 and myogenin varies from case to case. One ERMS
demonstrates diffuse nuclear positivity, whereas another, especially the more immature or primitive ones, may
have only a few labeled nuclei. Myogenin and myoD1 have a high degree of sensitivity exceeding 95% and a
specificity of virtually 100%.
The molecular genetics of ERMS is different from those of alveolar RMS in that there is no signature or non-
random translocations. Rather there is loss of heterozygosity on 11p15.5 in the region of IGF-2. Gains and
losses of chromosomes or chromosomal regions have also been identified (Table 24-2).
ARMS is the less common of the two subtypes and in our series accounted for 19% of cases with the soft tissues
of the extremities, lower greater than upper, as the preferred primary site of 43% of our cases (Table 24-12). In
the perianal region, ARMS accounted for 50% of cases.
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FIGURE 24-54▪ERMS presented in the paratesticular soft tissues of a 7-year-old male. A: One pattern consists
of small, polymorphic appearing cells including spindle cells with embryonal features. B: Other foci are
composed of more monotonous pleomorphic round cells arranged in loosely cohesive groups with an alveolar-
like appearance. C: Rhabdomyoblasts are staining for desmin. D: The areas concerning for ARMS show only
scattered nuclear positivity for myogenin unlike ARMS with its diffuse nuclear positivity. FISH studies failed to
demonstrate a FKHR breakapart.
FIGURE 24-55▪ERMS presented as a polypoid mass arising from the uterine cervix of a 15-year-old female. A:
Primitive appearing rhabdomyoblasts are present with population of enlarged, more pleomorphic malignant cells.
B: Unique among ERMS is the presence of nodules of cartilage when this tumor presents in the cervix. There is
the question of the relationship of this tumor to the adenosarcoma of the uterus. This tumor may be found in
association with the pleuropulmonary blastoma complex with DICER1 mutation.
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FIGURE 24-56▪ARMS present in the foot of a 3-month-old female. The tumor demonstrates the three histologic
features of this neoplasm. A: Most areas had the septal growth of uniform malignant round cells attached to
fibrovascular stroma and the remaining individual tumor cells seemingly suspended in space. B: Other foci
display the individual tumor cells in loose sheets with the so-called nascent alveolar pattern. C: Large,
multinucleated tumor cells are seen in a background of monotonous round cells. FISH studies identified a FKHR
breakapart.
This tumor more so than ERMS accommodates to the characterization of a malignant round cell neoplasm in that
the cells are uniformly polygonal with high-grade rounded nuclei and variably prominent cytoplasm. Mitotic
figures and nuclear debris are more prominent than in ERMS. The tumor cells may form solid sheets of
nonoverlapping cells with foci in which the tumor cells tend to fall away from each other, producing the so-called
nascent alveolar pattern (Figure 24-56). When the biopsy is a more generous one with stroma in the
background, the tumor is more likely to have a nested-septal pattern in which the alveolar pattern of central
disaggregated individual tumor cells is surrounded by individual tumor cells attached to the septal stroma. A
similar alveolar pattern is seen in some cases of EWS-PNET (Figure 24-57). The presence of larger
multinucleated tumor cells with prominent eosinophilic cytoplasm among the mononuclear tumor cells in these
solid or more obvious septal-alveolar foci is virtually diagnosis of ARMS. In a minority of cases, one may
encounter foci of ARMS with a pattern resembling ERMS; these cases are very uncommon in our experience and
molecular genetic studies are very helpful in terms of diagnosis, but one complication is the emergence of the
fusion-negative ARMS, now accounting for over 40% of currently diagnosed ARMS, unlike the historic figure of
20% of all ARMS as fusion-negative tumors.
Most ARMSs are consistently immunopositive for vimentin and desmin as well as myoD1 and myogenin (150,
184). The latter three markers are diffusely positive in most cases of ARMS and less consistently so in ERMS.
Diffuse nuclear staining for myogenin in ARMS has been described as a useful discriminating reaction from the
more limited nuclear positivity in ERMS, and the diffuse nuclear staining is correlated with an unfavorable
outcome (94).
There are two well-documented translocations in ARMS involving PAX3-FKHR (FOX01) and PAX7-FKHR
(FOX01) gene fusions, t(2;13) (q35;q14) and t(1;13) (p36;q14) in 60% and 20% of fusion-positive cases,
respectively (48, 165). PAX3-FKHR-positive ARMS is associated with a poorer outcome than the PAX7-FKHR-
positive and fusion-negative ARMS. Amplification of 12q13-q14 has an adverse effect upon prognosis. The
t(1;13) ARMS is seen more often in younger children (190).
What is clear about ARMS is that it is prognostically unfavorable. Because ARMS can present with a lymph node
metastasis or as disseminated disease in bones and bone marrow, the pathologic diagnosis can be challenging if
ARMS is not considered in the differential diagnosis.
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A nonlymphoid hematopoietic neoplasm, MRT, EWS-PNET, and neuroblastoma are other childhood
malignancies, which are candidate neoplasms with some qualified clinical and pathologic overlap with ARMS.
Pleomorphic RMS is recognized in childhood as a rarely occurring tumor (81). A sclerosing variant of RMS has
been reported but is probably not a specific pathologic subtype with prognostic implications.
FIGURE 24-57▪EWS-PNET can have microscopic features with rosette formation to suggest a classic
neuroblastoma. A: This tumor displays a septal pattern resembling ARMS. B: Diffuse CD99 membrane positivity
and the EWS breakapart by FISH corroborated the diagnosis.
SARCOMAS OF UNCERTAIN HISTOGENESIS

Undifferentiated sarcoma (US) with various descriptors such as “round cell,” “small blue cell,” “anaplastic,” or
“spindle cell” existed for generations in the past and still does in a more limited sense for those malignant
neoplasms in children whose morphological and immunohistochemical features as well as cytogenetic and
molecular diagnostic studies are not diagnostic for a known tumor entity (175). At one time, as many as 25% of
all STSs in children were assigned to the US or a similar category of “sarcoma of undetermined differentiation or
histogenesis.” This latter category was only exceeded by RMS, which accounted for 50% to 60% of STSs in
children. Over the past 20 to 25 years, the diagnosis of US has steadily declined with the application of IHC and
cytogenetics to establish the identity of such tumors as primitive embryonal RMS, myeloid-monocytic neoplasms,
the various representative tumor types in the extended EWS family and MRT. The pathologic classification of
RMS in children has a category of “unfavorable histology undifferentiated sarcoma.” Somers and associates
were among the first to report their experience with 13 cases of US in children between the ages of 1 month and
16 years at diagnosis; these tumors were described as “diffuse sheets of tumor cells with high cellularity” with
round or spindle cell features (189). The diagnostic “full court press” on these tumors failed to establish a
specific tumor type. In our own experience, the diagnosis of US is made in the presence of a malignant round cell
neoplasm with high-grade nuclear features, often more atypical and pleomorphic than those of EWS-PNET and
more like the MRT (Figure 24-58). Some of these tumors closely resemble EWS-PNET with periodic acid-Schiff
(PAS)-positive granular cytoplasm, which is diastase sensitivity, but without the EWS breakapart by FISH.
Possibly, some of these US are examples of a primitive round cell sarcoma with translocation t(4;19) (q35;q13.1).
We are aware of another round cell “sarcoma” with the t(15;19) translocation of the undifferentiated carcinoma of
the upper aerodigestive tract in children (Figure 24-59). Other examples of USs in children include the entities
undifferentiated embryonal sarcoma of the liver and anaplastic sarcoma of the kidney (152, 173, 206). The
specific tumor types with primitive round cell features are the primitive ERMS (with only rare cells positive for
desmin and myoD1 or myogenin), MRT (usually <5 years old and few, if any, rhabdoid cells by microscopic
examination but microscopically are more obvious in the vimentin and/or cytokeratin immunostain and lack
BAF47 nuclear positivity to indicate an INI1 deletion) or a nonlymphoid hematopoietic neoplasm (vimentin and
CD43 positivity). One would question whether some of the USs in the study of Orbach and associates were
possibly MRTs (160).
Ewing sarcoma-primitive neuroectodermal tumor (EWS-PNET) and the extended EWS family of tumors
have evolved as a result of the recognition that EWS and several other neoplasms are chacterized by an EWS
breakapart to form a number of unique non-random translocations. Soon after the initial reports of an EWS-
equivalent neoplasm in the soft tissues and a malignant small cell tumor of the chest wall and paraspinal region
(Askin tumor), cytogenetic studies established that these two presumably separate entities
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had the same t(11;22) (q24;q12) translocation (24, 131, 177). Eventually, it became apparent that there was no
longer any reason to maintain the dichotomy between EWS and the peripheral PNET from a pathologic,
therapeutic, or prognostic perspective so that today these cases are diagnosed as “EWS-PNET.” The past
category of “sarcoma of uncertain histogenesis,” the second most common STS of childhood, is now largely
occupied by EWS-PNET (92).
FIGURE 24-58▪Undifferentiated round cell sarcoma presented as a mass in the inguinal region of an 11-year-old
male. A: These neoplasms are most commonly round cell neoplasms whose features resemble EWS-PNET
though with somewhat more polymorphic and pleomorphic features. B: The nuclei tend to be more irregular than
EWSPNET but are accompanied by a clear to vacuolated cytoplasm. C: A PAS stain demonstrates the granular
positivity of glycogen. D: Vimentin immunopositivity is a consistent finding, but CD99, if reactive, does not display
the diffuse positivity of EWS-PNET. An EWS breakapart was not identified in this case by FISH studies.
EWS-PNET of soft tissues, the archetype of the extended EWS family of tumors, is a neoplasm whose origin is
probably a mesenchymal stem cell whose oncogenesis is triggered with the formation of unique fusion genes
consisting of the transactivation domain of EWS and the DNA finding domain of one of five ETS family
transcription factors (178). EWS was originally described in the bone of young individuals in the 1920s and
almost 50 years later was recognized in the soft tissues with a predilection for the chest wall, extremities, and
paraspinal region (179) (Table 24-13). It is now recognized that the EWS-PNET can present in the kidney, lung,
salivary gland, skin, and any number of other sites including the vulva, dura, and brain, which requires its
inclusion in the differential diagnosis of any malignant round cell neoplasm in a young individual. Most cases of
EWS-PNET present in the second decade with an average age at diagnosis of 12 years. However, almost 40%
of tumors presented at or before 10 years of age with the two youngest cases, 1-year-old males with paraspinal
and floor of the mouth tumors, respectively (Table 24-13). When EWS-PNET presents in the retroperitoneum
and paravertebral locations, the tumor must be differentiated from undifferentiated and poorly differentiated
neuroblastoma, especially in a child less than 6 years old.
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FIGURE 24-59▪Malignant round cell neoplasm presented in the abdomen of a 2-year-old male. A biopsy consists
of crowded, uniform malignant round cells infiltrating through mesenteric fat. MRT, nonlymphoid hematopoietic
neoplasm (granulocytic sarcoma) and undifferentiated neuroblastoma were considered in the differential
diagnosis. The tumor cells only expressed vimentin and cytogenetic studies revealed a t(15;19) translocation
(BRD4-NUT fusion) of the type associated with the childhood upper airway carcinoma. (Contributed by Bahig M.
Shehata, M.D., Atlanta, Georgia).
The pathologic evaluation of a suspected EWS-PNET of soft tissue and bone has been thoroughly outlined by
Carpentier and associates who have appropriately stated that the “first priority should always be given to
formalin-fixed tissues for morphologic evaluation” (27). The biopsy specimen is more often than not “small” by
most measures plus the fact that there may be as much hemorrhage and necrosis as viable, well-preserved
neoplastic tissue (Figure 24-60). The other reality is that the biopsy may be the last opportunity to document the
pathologic features since preoperative chemotherapy often results in total or near-total ablation of tumor.
Table 24-13 ▪ EWS-PNET OF SOFTTISSUE AND BONE IN CHILDREN AND ADOLESCENTS
Soft Tissuea Boneb
Site No. (%) Site No. (%)
Chest wall 14 (39) Pelvic bones 14 (40)

Extremity 7 (19) Humerus 5 (14)
Paraspinal 4 (11) Tibia 4 (11)
Scalp, face, floor of mouth, submandibular gland 4 (11) Femur 4 (11)
Neck 3 (8) Rib 3 (8)
Retroperitoneum 2 (5) Radius 2 (6)
Nasopharynx 1 (3) Fibula 2 (6)
Trachea 1 (3) Mandible 1 (3)
36 (100) 35 (100)
2Average age at diagnosis, 12 years, (age range 1 to 20 years), 14 (39%) children 10 years or less at
diagnosis; 22 males and 14 females
bAverage age at diagnosis, 14 years (age range 2 to 20 years), 6 (17%) children 10 years old at
diagnosis, 17 males and 18 females. From the files of the Lauren V. Ackerman Laboratory of Surgica
Pathology, St. Louis Children’s Hospital, Washington University Medical Center, St Louis, MO.
FIGURE 24-60▪EWS-PNET presented in the paraspinal region of a 15-year-old male. This 10 cm mass with
cystic and hemorrhagic features has soft, gray-white viable tumor at the periphery. Hemorrhage and necrosis are
common gross features.
A well-preserved and fixed biopsy demonstrates a diffuse and/or nested-lobular pattern of a monotonous,
monolayer of rounded or polygonal tumor cells. Where there is an apparent nested or lobular pattern, there is an
accompanying fibrous stroma. Other features can include focal necrosis, pools, or lakes of erythrocytes with a
pelioid appearance and collections of tumor cells with apparent loss of cohesion with an alveolar-like quality
reminiscent of ARMS. As a monolayer of nonoverlapping tumor cells, the central nuclei are similar with a
uniformly dispersed or subtly clumped chromatin with one or more micronucleoli (Figure 24-61). The cytoplasm is
clear to finely vacuolated and often contains abundant PAS-positive, diastase-digestible glycogen granules
(Figure 24-62A,B). Any compression of the tissue results in the loss of the latter cytologic features with more
hyperchromatic nuclei with fusiform contours and inapparent cytoplasm. Mitotic figures are usually modest in
number, and anaplasia is absent. Some tumors may display the presence of pyknotic, shrunken tumor cells
scattered in the background of better preserved tumor cells. On occasion, diminutive extracellular pools of a
mucoid to an almost chondrohyaline stroma material are noted. The characteristic immunophenotype includes
diffuse vimentin and CD99 positivity with a dotlike and/or perinuclear reactivity and a uniform
cytoplasmicmembranous pattern, respectively (Figure 24-62C,D). A similar pattern of cytoplasmic positivity for
cytokeratin is present in 20% to 25% of cases; many fewer cells are reactive compared to vimentin. It is well to
keep in mind that these two markers alone will not differentiate EWS-PNET from lymphoblastic lymphoma,
desmoplastic small round cell tumor (DSRCT), or MRT (Table 24-2). It is unusual for a EWS-PNET not to
express CD99; however, we have seen several examples, which subsequently were shown
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to have an EWS breakapart to emphasize that FISH has an important role in the evaluation of any suspected
EWS fusion-associated neoplasm.
FIGURE 24-61▪EWS-PNET presented as a deep soft tissue mass in the paraspinal retroperitoneum. A:
Substantial hemorrhage and necrosis can accompany these tumors in which some microscopic fields may
contain few tumor cells. B: These tumors like ARMS and hematolymphoid neoplasms qualify as pure round cell
neoplasms. Wellpreserved areas of tumor show cells with uniform central nuclei surrounded by clear to
vacuolated cytoplasm.
Desmoplastic small round cell tumor (DSRCT) is the second neoplasm to have been recognized as a
member of the extended EWS family with an EWS fusion partner, but with WT1 rather than FLI1. Originally
described as a multifocal, multinodular neoplasm arising from the peritoneum, DSRCT is known to occur in the
posterior fossa, pleura, scrotum, ovary, and kidney (15). Because of its unique immunophenotype of vimentin,
cytokeratin, desmin, and WT1 positivity, it was suggested initially that the DSRCT might be a primitive
mesothelial neoplasm since this phenotype is shared with mesothelial cells (32, 161). The most common
presenting site is the abdomen, which coincides with our own experience in 19 cases, of which 16 (84%)
occurred in the abdominal cavity as multiple peritoneal nodules. The remaining three cases presented on the
pleura and in the pancreas and parotid gland. The patients ranged in age from 3 to 18 years (mean age 10
years) with 11 tumors presenting in the second decade, but this tumor is well documented into the third decade
and beyond. The male-to-female ratio in our experience was 9 males and 10 females. Virtually all of our cases
were seen as biopsies since the clinical presentation does not lend itself to primary surgical resection. The basic
microscopic features are summarized in the name of the tumor with a dense
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fibrous background containing numerous, variably sized nests of undifferentiated small cells, which are not
necessarily all rounded or polygonal (Figure 24-63). Gland-like structures, solid squamoid nests, and even
rhabdoid cells can be seen. However, a desmoplastic fibrous stroma is also a feature of metastatic Wilms tumor
and RMS, which can create a microscopic and immunohistochemical dilemma with DRSCT, which may be
resolved by MyoD1 or myogenin staining in the case of RMS and the lack of an EWS breakapart in the case of
Wilms tumor.
FIGURE 24-62▪ EWS-PNET can be characterized histochemically and immunohistochemically. A: The clear
cytoplasm of the tumor cells reflects the abundant glycogen as seen in this PAS stain. B: Following diastase
digestion of the PAS stain, the tumor cells reacquire the clear cytoplasm.
FIGURE 24-62*▪(continued) C: Virtually all tumors are immunopositive for vimentin with a perinuclear
cytoplasmic or dot-like pattern of reactivity. D: These tumors are uniformly positive for CD99 with a diffuse
pattern of reactivity.
Clear cell sarcoma (CCS) of soft tissues (malignant melanoma of soft parts), another member of the extended
EWS family, is characterized by an EWS translocation, t(12;22) (q13;q12), which is found in 75% or more of
cases; this same translocation is found in AFH (98) (Table 24-2). Though the CCS is phenotypically a melanoma
as defined by the presence of melanosomes and the expression of S100 protein, HMB-45, melan-A, and
micropthalmic transcription factor, it does not have the activating mutations of BRAF kinase, which are the basic
molecular events of cutaneous melanoma. A slowly enlarging soft-tissue mass in the distal extremities (lower
more often than upper) and rarely in bone, small intestine, and kidney in adolescents or young adults is the
clinical presentation (57, 109). This tumor is infrequent in children 5 years old or less. A mass in the region of a
tendon or aponeurosis, measuring 5 cm or less, is firm and well circumscribed with a grayish-tan surface. Like
the cutaneous melanoma, the spindled or more epithelioid cells are arranged in cohesive
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groups with a delicate fibrous stromal network in the background or with a more prominent hyalinized stroma and
can be highly infiltrative (140). Other patterns have a resemblance to the disaggregated cells of an ARMS or
EWS when the tumor cells are more polygonal in appearance. Multinucleated giant cells are seen with some
frequency. Clear to eosinophilic cytoplasm can add to the EWS-like appearance. The rounded to ovoid nuclei
are modestly hyperchromatic and vesicular with amphophilic nucleoli (Figure 24-64). Pseudoinclusions are more
or less prominent. Mitotic figures are not especially numerous. Other immunophenotypic attributes include the
expression of CD99 and neuron-specific enolase. Tumors larger than 5 cm and those with necrosis are regarded
as having unfavorable prognosis. Regional lymph node metastasis is a common mode of spread. Overall survival
is approximately 50%. The differential diagnosis includes SS (common sites of presentation), PEComa
(similarities in immunophenotype, but without EWS breakapart), paraganglioma (similar immunophenotype, but
absence of HMB-45 or melan-A expression) and cutaneous melanoma (similar immunophenotype, but absence
of EWS breakapart).
FIGURE 24-63▪DSRCT in a 17-year-old male presented with abdominal pain. Multiple masses were identified by
imaging studies. A: A biopsy shows the presence of discrete and interconnecting nests of crowded malignant
small basophilic tumor cells surrounded by a fibrous stroma. B: The tumor shows strong diffuse positivity for
vimentin.
FIGURE 24-63▪(continued) C: Scattered tumor cells show dot-like and perinuclear pattern of cytokeratin
positivity. D: Desmin expression may be strong and diffuse as in this case or may be more limited. These tumors
also show strong nuclear positivity for WT1 (not illustrated). The EWS breakapart was identified by FISH.
FIGURE 24-64▪CCS of tendon sheath (melanoma of soft part) presented as a mass over the clavicle in a 10-
year-old male. A: A nodular tan-white mass measuring 2.5 cm consisted of ill-defined nests of rounded to
spindled-shaped tumor cells in a background of fibrous stroma. B: Immunohistochemical staining showed strong
positivity for HMB-45.
Extraskeletal myxoid chondrosarcoma (EMC) is seemingly the least common representative of the extended
EWS family tumors and the least frequently of these neoplasms in children and adolescents. A large combined
institutional experience revealed no cases in the first decade and only 2
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(2%) of 86 cases in individuals between 11 and 20 years old, though the individual case has been seen in
younger children. Three translocations have been identified to date and one of these is an EWS fusion partner
(Table 24-2). The proximal lower extremity is the most common site of presentation in all age groups (3). These
tumors arise in the subcutis or deeper soft tissues as a well-circumscribed, multinodular mass with a complete or
incomplete fibrous capsule, usually less than 10 cm in diameter and have a gelatinous whitishtan to tannish
appearance. Cartilage is generally not identified grossly or microscopically leading to question whether EMC
should be regarded as chondrosarcoma since it is also inconsistently immunoreactive for S100 protein. Fibrous
septa separate the tumor into lobules, which are composed of delicate lacelike strands, more solid appearing
nests, spindle cells, and high-grade round cells. The background has a variable myxoid or mucoid appearance.
In some cases, it is the multilobulated architecture at low magnification, which provides the subtle clue to the
diagnosis while other SSTs are under consideration in the differentiated diagnosis like poorly differentiated or
monophasic SS, MRT (rhabdoid cells in EMC), neurothekeoma, myoepithelial tumor of soft tissues, chordoma,
parachordoma (if it exists as a distinct entity), and EWS-PNET (117). Immunohistochemically, EMC is reactive for
vimentin (75% to 80% of cases), neuronspecific enolase (50% to 95%), EMA (10% to 15%), S100 protein (15%
to 20%), synaptophysin (40% to 50%), and glial fibrillary acidic protein (2% to 5%) (97). These tumors are
generally nonreactive for CD99, c-MET, and CAM 5.2 (the two latter markers are also positive in chordomas)
and have normal expression of BAF47 (INI1).
FIGURE 24-64▪(continued) C: The tumor cells are also positive for S100 protein. An EWS breakapart was
demonstrated by FISH.
Mesenchymal chondrosarcoma (MCS), like myxoid chondrosarcoma and EWS-PNET, has a primary soft
tissue and skeletal presentation (30, 46). Its relationship to the other extended EWS family of tumors is not
entirely clear, though a t(11;22) translocation has been reported in addition to other karyotypic abnormalities
(trisomy 8, 20-, 12+) (151). Most MCSs present in children beyond the age of 10 years and into the third decade
(30). Very rarely, MCS is seen in the neonate (49). Approximately 60% to 70% of MCSs arise in the soft tissues
or nonosseous sites like the dura, orbit, pelvis, sinonasal tract, peripheral soft tissues, and kidney. The tumor is
characterized microscopically by nodules or islands of atypical hyaline cartilage and an accompanying population
of primitive round cells resembling EWS or a more spindle cell stroma with clefted vascular spaces resembling
HPC. We have seen cases of MCS in which the two patterns appeared to segregate from each other. These
tumors are immunopositive for vimentin, CD99, and reportedly desmin and myogenin (155). The 5-year survival
is 40% to 50%, and MCSs arising in the soft tissues and with the HPC-like pattern fared worse than tumors
arising in the bone and with EWS-like features (30).
Myoepithelial tumor of soft tissue is a neoplasm presenting predominantly in the extremities but not to the
exclusion of other regional sites. The largest series to date reported that approximately 20% of tumors are
diagnosed before the age of 20 years (100). The multilobulated and reticulated growth pattern of epithelioid and
spindle cells has some overlapping features with EMC, proximal type epithelioid sarcoma (ES), and
parachordoma. These tumors are immunoreactive for cytokeratin (AE1/AE3) and/or EMA, S100 protein, calponin,
p63, and glial fibrillary acidic protein. Pathologic grade and resectability are the two principal correlates of
outcome.
Synovial sarcoma (SS) is one of the most common non-RMSs in the first two decades of life together with the
MPNST. There is some variance from one series to another, but 15% to 30% of SSs are diagnosed at or before
20 years of age (66, 74, 191). Less than 15% of all SSs in children and adolescents are diagnosed before the
age of 10 years. In our own experience with SSs, there were 57 cases, which were diagnosed between 4 and 20
years of age (mean age, 14 years) with 12 (20%) of the cases seen in children 10 years old or less.
Approximately 60% of cases presented in the extremities, which is in accord with other larger series, which have
reported up to 70% of tumors in the extremities (Table 24-14). Various sites in the head and neck region
accounted for 10% of our cases. With molecular genetic testing for the t(X;18) translocation, SS has been
documented in a number of previously unrecognized sites such as the lung-pleura, heart, and intestinal tract as
some examples. Several cases of pleuropulmonary SS were originally submitted for review as possible examples
of type I or cystic pleuropulmonary blastomas (PPB), but four of the patients were adolescents between 13 and
15 years of age whereas the median age for type I PPB is 9 months. A firm, well-circumscribed fibrous appearing
mass may be accompanied by focal hemorrhagic cysts and dystrophic calcifications or metaplastic bone (Figure
24-65). In those tumors with minimal fibrous stroma, the consistency is soft and the cut surface has a glistening,
mucoid appearance resembling an ERMS or CIFS. Attention to the dimensions of the tumor
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is important since there is a consistent correlation between size (> or <5 cm) and outcome; those tumors in
excess of 5 cm have a significantly poorer prognosis (non-RMSs in children, regardless of pathologic type, have
a poor outcome if>5 cm in greatest dimension) (126).
Table 24-14 ▪ SS IN CHILDREN AND ADOLESCENTS
Anatomic Site No. (%)
Lower extremity 22 (39)
Proximal 14
Distal 8
Upper extremity 11 (19)
Proximal 6
Distal 5
Chest wall 7 (12)
Lung-Pleura 5 (9)
Abdominal wall 3 (5)
Retroperitoneum 2 (3)
Neck 2 (3)
Paranasal sinus—pharynx 2 (3)
Scalp 1 (2)
Orbit 1 (2)
Posterior mediastinum 1 (2)
57 (100)
37 males, 20 females (mean age, 14 years).
From the files of the Lauren V. Ackerman Laboratory of Surgica

Pathology, St. Louis Children’s Hospital, Washington University
Medical Center, St. Louis, MO.
There are two basic histologic patterns of SS: monophasic spindle cell proliferation and biphasic spindle-
glandular type (Figure 24-66). A third poorly differentiated pattern, constituting 5% or less of cases, has some
similarities to EWS-PNET including a similar immunophenotype of vimentin, cytokeratin, and CD99 positivity but
in the absence of a EWS breakapart but with a SYT-SSX breakapart. The fourth purely glandular pattern
resembling well-differentiated adenocarcinoma is very rare. Necrosis and rhabdoid cells are other features of
poorly differentiated SS. Uniform spindle cells with either ovoid or fusiform contours are arranged in dense
sheets or short fascicles (Figure 24-67). The nuclei have a pale, finely granular chromatin, and the cytoplasm is
inconspicuous. Mitotic figures are not prominent. Like the MPNST, which the monophasic spindle cell SS
resembles, there may be alternating “light cell-dark cell” areas reflecting variable cellular density. A HPC-like
pattern is another useful clue to the diagnosis. In addition, myxoid foci convey an impression of MPNST or
DFSP. Mast cells are nonspecific, but their presence should evoke concern about SS or MPNST in the
appropriate setting. The glandular profiles are either subtle, residing as small tubular structures interspersed in a
predominant spindle cell background or are overly obvious large glands or cysts. Immunohistochemically, SS is
diffusely positive for vimentin in the spindle cell component whereas the epithelial component expresses
cytokeratin AE1/AE3, cytokeratin 7, and EMA; the cytokeratins may be limited to individual tumor cells or entirely
nonreactive (Figure 24-68). The EMA is more likely to stain small groups of tumors cells with a membranous
pattern. Both CD99 and bcl2 are consistently expressed in SS. An antibody to TLE1 has shown some diagnostic
value in cases of SS. SS is also known to be immunopositive for S100 protein. Though most SSs have the
t(X;18) translocation, whether it is the fusion transcript, SYT-SSX1 (present more often in biphasic SS) or SYT-
SSX2 (more often monophasic SS) does not seem to have prognostic significance though there is some
evidence to suggest that the latter fusion transcript is correlated with a less favorable outcome. All SSs are at
least grade 2 sarcomas but increased mitotic activity and necrosis are grade 3 features. However, COG regards
all SSs as grade 3 based upon the tumor type. A spindle cell sarcoma with marked nuclear pleomorphism,
anaplasia, and extensive necrosis is unlikely to represent a SS. The overall survival of children and adolescents
is approximately 80%, whereas it is lower in adults (60%) (195).
FIGURE 24-65▪This SS presented in the ankle of an 8-year-old male. Some tumors are associated with
dystrophic calcifications.
FIGURE 24-66▪SS presented in the left foot of this 18-year-old male. This neoplasm had a classic biphasic
pattern of gland formation in a spindle cell background.
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FIGURE 24-67▪SS presented in the wrist of an 11-year-old male. A: A monotonous population of uniform spindle
cells characterizes this monophasic neoplasm. B: Some areas had a more myxoid appearance with the
separation of tumor cells.
FIGURE 24-68▪Most SSs have a characteristic immunophenotype. A: The vimentin immunostain is typically
diffusely positive. B: These tumors are also immunopositive for cytokeratin 7 and the pattern of positivity is one
of individual tumor cell reactivity. These tumors are often more diffusely positive for EMA. C: Another useful
marker is CD99 which may be diffusely or focally positive. With this immunophenotype one can then move on to
FISH studies to evaluate for the t(X;18) breakapart.
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Table 24-15 ▪ EXTRARENAL AND RENAL MRTs IN CHILDREN (1989 TO 2009)
No. (%)
Soft tissue (1 week-16 years, mean 3.9 years) 47 (48)
Neck (16)
Paraspinal region (11)
Axilla (5)
Abdominal wall (3)
Arm (3)
Back (2)
Other (7)
Central nervous system (1 year-18 years, mean 5 years) 15 (15)
Kidney (3 days-5 years, mean 1.3 years) 12 (12)
Liver (3 months-8 years, mean 1.6 years) 11 (11)
Abdomen (2 months-2 years, mean 1 year) 4 (4)
Mediastinum (7 months-3 years, mean 1.2 years) 4 (4)
Dessiminated (3 days-3 weeks, mean 1.5 weeks) 3 (3)
Bladder (5 years, 10 years) 2 (2)
98 (100)
53 males and 45 females.
From the files of the Lauren V. Ackerman Laboratoryears of Surgical Pathology, St. Louis Children’s
Malignant rhabdoid tumor (MRT) and its counterpart in the brain and spinal cord, atypical teratoid/rhabdoid
tumor (ATRT), have evolved into a single pathologic and molecular genetic entity, which is characterized by
biallelic inactivation of the hSNF5/INI1/SMARCB1 on 22q11.2 through deletion or mutation (EWS gene is located
on 22q12) (110, 157). Though the MRT was initially described in the kidney, our experience revealed that almost
50% of cases presented in a variety of extrarenal sites in the soft tissues with a preference for the neck and
paraspinal region (Table 24-15). Three neonates had disseminated disease including the soft tissues of the
head and neck and multiple organs. Like congenital neuroblastoma, the placenta may contain micrometastases
in the chorionic villi. The ATRTs presented as a supratentorial (nine cases), posterior fossa (four cases), and
spinal cord (two cases) mass. The MRT, regardless of the primary site, is a neoplasm of early childhood and 53
(54%) of our cases presented in children 1-year-old or less with the youngest, a 3-day-old female with a renal
MRT and another 3-day-old infant with disseminated MRT with disfiguring facial masses and widespread
metastases including the placenta. Approximately 80% of our cases were diagnosed at or before 5 years of age.
FIGURE 24-69▪MRT as in this case of a 2-year-old male proved to be an elusive diagnosis when it presented as
a anterior and middle mediastinal mass which involved the thymus. A: Small nests of uniform polygonal
malignant cells are separated by a dense fibromyxoid stroma. Though filamentous cytoplasmic inclusions were
not identified histologically, prominent nucleoli are seen. B: Dense vimentin-positive inclusions are noted by IHC
and they correspond to the rhabdoid inclusions by routine light microscopy.
FIGURE 24-69▪(continued) C: Cytokeratin staining is sparing but the tumor cells are strongly immunopositive for
EMA. D: BAF47 IHC demonstrates nuclear positivity in stromal and inflammatory cells in the background, but the
nuclei of the tumor cells are nonreactive. INI1 deletion was confirmed by FISH.
The gross features of a MRT are optimally demonstrated in a resected mass from the kidney or liver; these
tumors are soft and have a bosselated, glistening tan-white surface. In the soft tissues, fibrous stroma can
accompany these tumors so that there is a nesting pattern or a fibromyxoid alteration with remote resemblance to
chondroid matrix. The number and prominence of rhabdoid cells with intensely eosinophilic filamentous inclusion
in the cytoplasm can vary considerably from one MRT to another and from one microscopic field to another in the
same tumor. Small biopsies can be especially problematic without appropriate immunohistochemical stains
and/or molecular studies (Figure 24-69). Another important morphologic attribute is the large, eccentric vesicular
nucleus with its prominent nucleolus. Rhabdoid cells may be inconspicuous in a background of more epithelioid
appearing cells without inclusions to populations of smaller, lymphoid-like cells, which are seen with some
frequency in ATRTs, which have led to a diagnosis of a central PNET or medulloblastoma in the past. It is
advisable to consider MRT in the differential diagnosis of a malignant round cell neoplasm in a child before
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settling on an interpretation of “undifferentiated round cell sarcoma.” IHC for vimentin and cytokeratin
demonstrates intense cytoplasmic positivity in the configuration of a spherical inclusion that totally occupies the
cytoplasm (Figure 24-68). In general, vimentin staining produces the more diffuse pattern of positivity, but the
cytokeratin staining of the rhabdoid inclusions often stands out in a background of nonreactive tumor cells.
Membrane-cytoplasmic positivity for CD99 does not produce the diffuse pattern of a EWS-PNET. SMA positivity
is consistently present in the ATRT in contrast to nonneural MRTs. The nuclei fail to stain for BAF47 to reflect
the inactivated INI1 gene, but the interpretation can be complicated by the presence of numerous inflammatory
cells or fibroblasts with their functioning INI1 gene with intense nuclear staining as the internal control, but these
cells may also obscure small groups of nonreactive tumor cells in the background (99) (Figure 24-68). Originally
reported as INI1-negative small cell hepatoblastoma, these tumors are now considered primary MRTs of the
liver. Other round cell neoplasms of infancy and young children with INI1 loss but without the dire clinical
outcome of MRT have been reported (16, 120, 202). It remains to be determined how these relate to MRT.
Epithelioid sarcoma (ES) is a rare STS regardless of age at diagnosis. It accounts for 1% to 2% of all STSs
overall, but because it has a predilection for adolescents and young adults, it may represent as many as 5% to
8% of non-RMStous STSs in the first two decades of life. There are two types of ES: the classic or the distal
type presenting as slowgrowing nodule or nodules in the hand or forearm in almost 50% of cases and the
proximal or axial presenting type (6, 73). The latter type of ES is a neoplasm whose pathologic,
immunophenotype, and molecular genetic features are very similar to the MRT with an abbreviated aggressive
clinical course like the MRT and unlike the classic ES (116, 130). The classic ES is a more superficial neoplasm
in the dermis or subcutis in contrast to the proximal ES in the deep soft tissues. Classic ES has a multinodular or
diffuse pattern of uniform epithelioid cells with abundant eosinophilic or clear, but vacuolated cytoplasm with or
without a spindle cell component. The nodules are composed of a mantle of epithelioid cells with central necrosis
or hyalinization. In the presence of nodules with central necrosis, necrobiotic granulomas of the granuloma
annulare type may arise in the differential diagnosis, both clinically and pathologically. IHC can settle the
diagnostic dilemma in that the histiocytes of granuloma annulare are CD68-positive whereas the cells of ES
coexpress vimentin and pancytokeratin as well as EMA (123). In at least one series, 93% of all ESs (both classic
and proximal types) had loss of INI1 staining blurring the distinction from MRT (219). Approximately 50% of ES
are CD34-positive, which can be problematic if the differential diagnosis includes an epithelioid vascular tumor
since there is a variant of the latter, which can mimic ES.
Alveolar soft part sarcoma (ASPS) is a neoplasm, which occurs in a similar age group as ES and also
represents only 1% of all STSs in the first two decades of life. Unlike ES, ASPS may present in early childhood
as a mass in the base of the tongue (113, 217). The head and neck region are preferred sites for the
presentation of ASPS in children. The deep soft tissue of the proximal lower extremity is the single most common
primary site (29). There are a number of diverse sites in which ASPS has been documented including heart,
lung, bone, and uterus as a sampling of some of the non-soft tissue primary sites (132). There is a female
predilection in contrast to most other STSs in childhood. Although the histogenesis of ASPS remains uncertain, it
has a nonrandom unbalanced translocation, der(17)t(x;17)(p11;q25),
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which produces an ASPL-TFE3 fusion gene; the balanced fusion translocation involving the same fusion
partners is present in the Xp11.2 renal cell carcinoma (nonmelanotic and melanotic types) of childhood (5, 10,
77, 163).
FIGURE 24-70▪ASPS presented as a soft tissue mass in the lower leg of a 9-year-old male. A: The
circumscribed 7 cm tumor is composed of uniform polygonal cells with prominent nucleoli surrounded by delicate
stromal envelopes. B: Microvascular invasion is found at the periphery of the tumor. C: Pale granular
cytoplasmic staining was present in the PAS stain. D: Only isolated tumor cells are immunoreactive for vimentin.
Note the prominent staining of the stromal envelopes.
Grossly, a circumscribed mass with a grayish to yellowish to hemorrhagic cut surface is the rather nonspecific
appearance. The alveolar characterization refers to distinct collections of uniform polygonal cells with
eosinophilic to granular cytoplasm surrounding a central nucleus with a variably sized nucleolus, which is
surrounded by delicate vascular envelopes (Figure 24-70). Larger groups of tumor cells may be separated by
dense, hyaline stroma or the overall pattern may be one of sheets of tumor cells without the alveolar architecture
(Figure 24-71). The latter appearance has been seen with some frequency in ASPSs in children in our
experience and can be the source of some diagnostic perplexity. Other variations include the presence of more
gigantiform rounded or elongated tumor cells in a background of smaller but more typical appearing cells
identifiable by routine histology or PAS staining after diastase digestion.
P.1099
Necrosis, hemorrhage, and an inflammatory infiltrate are other secondary findings. In the presence of a classic
alveolar pattern, the diagnosis is reasonably straightforward together with corroborating IHC, which includes
reactivity for vimentin, muscle specific actin, and desmin. On the other hand, the only immunoreactivity is for
vimentin, which outlines the delicate vascular envelope around groups of tumor cells and tumor cells may or may
not be positive. The most specific marker is TFE3 nuclear positivity. The differential diagnosis of ASPS includes
ARMS, but in most cases, the distinction is made without difficulty given the absence of true organized alveolar
pattern, the presence of marked nuclear hyperchromatism, and mitotic activity in the ARMS; these features
contrast with those of ASPS. Desmin is diffusely positive in ARMS and only focally so, if at all, in ASPS. If the
cells of ASPS have vacuolated cytoplasm and a less than obvious alveolar pattern, diffuse GCT of tendon
sheath is worthy of consideration. The emergence of the PEComa is another neoplasm in the differential
diagnosis of ASPS. The short-term survival is relatively favorable, but ASPS is known for its delayed metastatic
behavior to the brain and/or lung as long as 5 to 10 years after the original diagnosis. This tumor is an example
of a grade 3 sarcoma in the COG grading scheme based on its pathologic type rather than individual pathologic
features, which are commonly low grade.
FIGURE 24-71▪ASPS presented as a mass in the right atrium of the heart of an 11-year-old female. The tumor
has a diffuse pattern of tumor cells more like the nascent alveolar pattern of ARMS. This diffuse pattern can be
confusing initially but is not uncommon in those tumors presenting in childhood.
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Chapter 25
The Skin
Vijaya B. Reddy
Aliya N. Husain
Although any adult dermatologic disease can be seen in children, there are several conditions that are of clinical
significance that occur with greater frequency, or at times exclusively, in children and neonates. Table 25-1 lists
the pediatric dermatologic diseases seen by the general pathologist at two tertiary care medical centers over a
period of 19 years. Although the vast majority of the specimens were those of benign pigmented lesions and
plastic repair and debridement, a variety of benign and malignant neoplasms as well as life-threatening
inflammatory dermatoses can occur in children and require accurate diagnosis and timely management. This
chapter, while covering the spectrum of dermatologic diseases, focuses specifically on the clinicopathologic
features of the diseases encountered in children. Although a majority of the congenital diseases involving the
skin are diagnosed clinically and rarely need biopsy, a significant number of diseases can only be diagnosed
with specificity on histopathologic grounds. A specific diagnosis can be rendered by using a simple algorithmic
approach based on low-power pattern analysis (Table 25-2). As in other areas of pathology, clinicopathologic
correlation is an integral part of the diagnostic process.
Table 25-1 ▪ DISEASES SEEN IN CONSECUTIVE PEDIATRIC SKIN SPECIMENS AT LOYOLA

UNIVERSITY MEDICAL CENTER OVER 10YEARS (1986-96) AND AT RUSH UNIVERSITY
MEDICAL CENTER OVER 9YEARS (1996-2004)
Loyola (%) Rush (%) Total (%)
Benign pigmented lesions 742 (51.5) 554 (43.1) 1296 (47)
Scars, keloids, debridement, plastic repair 421 (29.1) 431 (33.5) 852 (31)
Viral infections 82 (5.7) 51 (4) 131 (4.8)
Inflammatory conditions 68 (4.7) 96 (7.5) 164 (6)
Vascular lesions 47 (3.25) 26 (2) 73 (2.7)
Cysts 42 (2.9) 47 (3.7) 89 (3.3)
Benign neoplasms 38 (2.6) 58 (4.5) 96 (3.5)
Malignant melanoma 2 13 (1) 15 (0.5)
Urticaria pigmentosa 2 0 2
Langerhans cell histiocytosis 1 1 2

Dermatofibrosarcoma protruberans 1 3 4
Infantile fibrosarcoma - 2 2
Metastases - 2 2
Basal cell carcinoma - 1 1
Total 1446 1285 2731
EMBRYOLOGY
The epithelial structures of the skin, namely epidermis, folliculo-sebaceous units, and apocrine and eccrine
sweat glands, are derived from the ectoderm. The dermis and its mesenchymal constituents, namely vessels,
smooth muscle, and nerve bundles, originate from the mesoderm.
There is a third component of skin that is composed of the migratory cells that originate at different sites and
populate the skin. The melanocytes, Merkel cells, and Langerhans histiocytes form an integral part of the
epidermis, while the mast cells and dendritic cells are present in the dermis. Melanocytes, Merkel cells, and
perineural cells are neural-crest derivatives. Mast cells and Langerhans cells are derived from mesenchymal
precursors of bone marrow.
Skin development starts as a single layer of cells or periderm, which can be recognized in a 3-week-old
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embryo. There is progressive stratification of the epidermis, and by the end of the first trimester, several layers of
glycogen-rich cells can be seen in the epidermis. Cornification of the epidermis is completed during the 6th
month of gestation (Figure 25-1). Defects in cornification (ichthyoses) can be diagnosed through fetal skin
biopsies at this stage. At about 12 weeks, folliculo-sebaceous units and sweat glands begin as buds of basal
cells that protrude into the mesenchyme of the dermis. Ectodermal dysplasias, which are characterized by
absence of follicles and sweat glands, can be detected through fetal skin biopsies after the second trimester.
Table 25-2A ▪ ALGORITHMIC APPROACH TO SPECIFIC DIAGNOSIS: INFLAMMATORY

DERMATOSES
1. Superficial perivascular dermatitis
• Consider: Urticaria
Telangiectasia macularis eruptiva perstans
Vitiligo
Spongiotic dermatoses:
Contact dermatitis
Nummular dermatitis
Atopic dermatitis
Drug-hypersensitivity reaction (especially with eosinophils)
Viral exanthem
Dermatophytosis
2. Superficial and deep perivascular dermatitis
• Consider: Mucha-Habermann disease — pityriasis lichenoides et varioliformis acuta (interface

dermatitis)
Lymphomatoid papulosis
Insect bite reactions (with eosinophils)
3. Vasculitis
Small vessels vasculitis with neutrophils-leukocytoclastic vasculitis

Medium vessel vasculitis with neutrophils-polyarteritis nodosa
Large vessel vasculitis-nodular vasculitis (erythema induratum):
4. Granulomatous dermatitis
Palisading granulomas
Granuloma annulare
Necrobiosis lipoidica
Rheumatoid nodule
Caseating and noncaseating granulomas
Infectious
Sarcoidosis
5. Vesiculobullous dermatoses
Subcorneal:
Subcorneal pustular dermatosis of childhood
Impetigo
Intraepidermal:
Staphylococcal scalded skin syndrome
Pemphigus vulgaris
Herpesvirus infection
Darier disease
Subepidermal:
Dermatitis herpetiformis/Linear IgA dermatosis
Bullous cutaneous lupus erythematosus
6. Folliculitis and perifolliculitis
• Consider: Dermatophytosis
Eosinophilic pustular dermatosis

Acne vulgaris
Alopecia areata
Lupus erythematosus
Trichotillomania
7. Fibrosing dermatitis
Scar
Dermatofibroma
Scleroderma/morphea
8. Panniculitis
Septal panniculitis
Erythema nodosum
Scleroderma
Eosinophilic fasciitis
Lobular panniculitis
Sclerema neonatorum
Subcutaneous fat necrosis of newborn
α-1-antitrypsin deficiency
Weber-Christian disease
Cytophagic panniculitis
Table 25-2B ▪ ALGORITHMIC APPROACH TO SPECIFIC DIAGNOSIS: NEOPLASTIC

CONDITIONS
1. Epithelial
2. Melanocytic
3. Mesenchymal
4. Hematopoietic
Benign Malignant
Small Large
Well circumscribed Poorly circumscribed
Symmetric Asymmetric
Smooth margins Infiltrating margins
May be ulcerated
By the end of the first trimester, the dermoepidermal junction can be recognized, and at about 6 months, the
dermal papillae become recognizable. The dermis, which begins as loosely arranged mesenchymal cells in a
myxoid background, continues to be modified throughout the third trimester and beyond.
Because fetal skin biopsies are becoming increasingly useful in the diagnosis of genodermatoses, an
understanding of the embryology of skin is critical in not only selecting the time of biopsy but also in
interpretation of the biopsy findings.
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FIGURE 25-1▪ A: Skin of an early, second-trimester fetus showing stratification of epidermis, beginning of the
follicular germs, and immature dermis. B: Skin from a 28-week fetus with stratum corneum and adnexal
structures in a collagenized dermis. (Hematoxylin and eosin stain, original magnifications ×200.)
NORMAL HISTOLOGY
Fetal skin is characterized by a virtually absent stratum corneum and an epidermis that is only a few cell layers
thick. Depending on the gestational age, the dermis is relatively hypocellular and myxoid. Rudiments of adnexa
including hair follicles and sweat glands can be identified starting from 20 to 24 weeks of gestation. In a
premature baby, subcutaneous fat is virtually absent.
The dermis is thin in children compared with that in adults, with proportionately larger amount of subcutaneous
fat. With increasing age, the epidermis and the stratum corneum increase in thickness and the dermis becomes
more compact and thick. Anatomic variations exist within the normal spectrum, and awareness of these features
may prove helpful in localizing the site of biopsy when clinical information is lacking. These include numerous
terminal hair follicles in the scalp, many vellus hair follicles and sebaceous lobules in facial skin, apocrine glands
in the axilla and genitalia, and eccrine glands in acral skin. A prominent stratum granulosum and stratum
corneum characteristic of chronic trauma are present in biopsies from the palm and sole.
BIOPSY TECHNIQUES
Skin samples can be obtained using various biopsy techniques, including punch, shave, excision, and curettage.
Selection of the appropriate biopsy technique depends on the clinical impression and the kind of information
anticipated by the clinician.
Punch biopsy is generally the choice of technique in evaluation of inflammatory dermatoses. This technique
allows the histologic examination of the full thickness of the skin including the subcutaneous fat. A 4-mm punch
biopsy provides an adequate sample. In small children and cosmetically important areas, a 3-mm punch may be
substituted. The area selected for biopsy should be a well-developed lesion and representative of the pathologic
process.
Shave biopsy is the technique used in the evaluation of lesions that appear to be confined to the epidermis and
superficial dermis, and is best for the clinical diagnosis of keratoses and other benign neoplastic lesions. It may,
on occasion, be used for diagnostic confirmation of basal cell or squamous cell carcinoma.
Excisional biopsy is the technique of choice for suspected malignancies or atypical pigmented lesions. Excisional
biopsies generally allow for the evaluation of surgical margins, and as such, the lateral and deep margins should
be inked before sectioning. Excisional biopsy or an incisional biopsy can also be used when panniculitis is
clinically suspected.
Curettage is the technique some clinicians employ in examining clinically benign lesions. From a pathologist’s
point of view, this is not a preferred method because the fragments of tissue so obtained are often small and
superficial, precluding accurate analysis. Furthermore, if a clinically benign lesion turns out to be malignant on
histologic examination, vital information such as invasion or thickness cannot be obtained. Curette fragments are
difficult to orient.
Scrape preparation is useful in evaluation of viral vesicles, when cells are scraped off a vesicular or pustular
lesion and analyzed after a quick stain.
Fine-needle aspiration biopsy is a popular method of biopsy in the evaluation of subcutaneous bumps and
lumps. However, it requires an experienced cytopathologist for proper handling and interpretation of the material
obtained.
SPECIMEN HANDLING
Routine Processing
Biopsy specimens should be placed immediately in a fixative. The fixative of choice for the majority of the
specimens is 10% buffered formalin. Punch and shave biopsies larger
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than 3 mm in diameter should be bisected for optimal fixation as well as for appropriate plane of sectioning
through the lesion, which is usually located in the center of the specimen. Excisional biopsies should be inked
and sectioned at 2- to 3-mm intervals. Sections cut at 3 to 5 μm are routinely stained with hemotoxin and eosin.
Special Processing
Specimens for direct immunofluorescence (IF) testing of bullous diseases are ideally obtained by biopsy of
perilesional skin. A well-established lesion is best for suspected cases of lupus, whereas an early lesion is ideal
for suspected case of vasculitis. Michel fixative is a good transport medium because IF testing can be performed
for approximately 7 days. Alternatively, the specimen can be placed in normal saline and transported immediately
to the laboratory. Frozen sections are incubated with fluorescein-labeled antibodies typically against IgG, IgA,
IgM, C3, C1q, and fibrinogen and evaluated with IF microscope.
Electron microscopy may be of use in the diagnosis of undifferentiated neoplasms and can be invaluable in
establishing the diagnosis of various types of epidermolysis bullosa and also in metabolic disorders like Fabry
disease. Specimens for electron microscopy should be fixed immediately in 2% glutaraldehyde or
paraformaldehyde.
Skin and subcutaneous tissue may be used for cytogenetic analysis. Sterile specimens should be placed in a
transport medium such as RPMI.
ALGORITHMIC APPROACH TO SPECIFIC DIAGNOSES

A systematic approach in analysis of biopsy sections allows for a smooth and accurate diagnosis. The pattern
analysis method, which was introduced by Wallace H. Clark Jr, popularized by A. Bernard Ackerman and
followed widely by most dermatopathologists today, involves the evaluation of sections at a scanning
magnification. Examination at scanning magnification can be highly informative and usually helps in classifying a
disease process either as neoplastic or inflammatory. Table 25-2 is rather simplistic but representative of the
more common dermatologic conditions that can be diagnosed with specificity in the pediatric patient. The
following is a description of diseases grouped according to etiology and pathogenesis.
CONGENITAL DISEASES (GENODERMATOSES)

Genodermatoses are a large and diverse group of disorders presenting with cutaneous involvement and an
underlying genetic defect. Only those genodermatoses that can be diagnosed in the fetus, neonate, or child will
be discussed. It must be emphasized that some of these will be rarely seen by the pathologist, while others are
not uncommon and are frequently included in the differential diagnosis.
Aplasia Cutis Congenita

Aplasia cutis congenita, or localized absence of skin, presents as a single or multiple skin defects typically
involving the scalp. It presents at birth with a deep ulcer-like lesion, in which the subcutaneous fat is exposed. If
the lesion occurs in utero, it manifests as a healed scar at birth. A sporadic lesion of aplasia cutis has no
significant clinical consequences. More often, aplasia cutis may be a part of a variety of inheritable or
noninheritable syndromes including trisomy 13, amniotic bands, and cardiac anomalies (29, 159).
A histologic section of aplasia cutis shows a full-thickness skin defect with healing at the edges. A fully healed
lesion shows scar with absence of adnexal structures. Formerly considered as synonymous, congenital absence
of skin, characterized by absence of epidermis only, is now regarded as part of the epidermolysis bullosa group
of disorders.
Ichthyosis
Ichthyoses are a heterogeneous group of disorders of epidermal cornification that are characterized by dryness
and scaling of the skin. Ichthyoses are generally inherited, although acquired forms are described, especially in
association with hematopoietic malignancies. The hereditary forms are divided into (a) the primary forms, which
include ichthyosis vulgaris, recessive X-linked ichthyosis, epidermolytic hyperkeratosis (bullous congenital
ichthyosiform erythroderma), classical lamellar ichthyosis, and nonbullous congenital ichthyosiform erythroderma;
(b) ichthyosiform disorders such as Harlequin ichthyosis, erythrokeratoderma variabilis, and CHILD (congenital
hemidysplasia with ichthyosiform erythrodermal and limb defects) syndrome; and (c) other related disorders of
differentiation such as Darier disease, HaileyHailey disease, and porokeratosis (143).
Ichthyosis vulgaris is a common disorder, inherited in an autosomally dominant pattern that presents with fine
white to larger scales involving large areas of the body but most prominent on the extensor surfaces of the
extremities with relative sparing of the flexural areas. Histologically, there is moderate hyperkeratosis with a
decreased or an absent granular layer and follicular plugging (Figure 25-2).
FIGURE 25-2 ▪ Ichthyosis vulgaris showing hyperkeratosis and a prominent stratum corneum with a diminished
granular cell layer. (Hematoxylin and eosin stain; original magnification ×200.)
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FIGURE 25-3▪ Congenital bullous ichthyosiform erythroderma showing marked hyperkeratosis and epidermolytic
hyperkeratosis with vacuolar degeneration of the stratum spinosum and granulosum, which is responsible for the
formation of bullae. (Hematoxylin and eosin stain; original magnification ×200.)
X-linked ichthyosis inherited as a recessive disease presents early in infancy with large brown scales involving
the entire body with accentuation on the neck and behind ears and relative sparing of the flexural areas.
Histologically, there is hyperkeratosis with normal or thickened granular layer.
Bullous congenital ichthyosiform erythroderma or epidermolytic hyperkeratosis has an autosomal dominant
pattern of inheritance and presents with generalized erythema and blistering at birth. Microscopic features
include hyperkeratosis, a characteristic vacuolization of the cells in spinous and granular layers and prominent
keratohyaline granules (Figure 25-3).
Lamellar ichthyosis is inherited as an autosomal recessive disorder and is characterized by large plate-like
scales involving face, trunk, and extremities with a predilection for flexor areas. Microscopic changes are
nonspecific and include hyperkeratosis with or without foci of parakeratosis and mild epidermal hyperplasia
(Figure 25-4). Lamellar ichthyosis can present as a collodion baby, in which the infant is encased in a keratinous
membrane and superficially resembles a harlequin fetus. However, the membrane is usually shed in 10 to 14
days, following which the clinical features of lamellar ichthyosis become apparent.
FIGURE 25-4▪Lamellar ichthyosis showing hyperkeratosis and mild psoriasiform changes. (Hematoxylin and
eosin stain; original magnification ×200.)
Nonbullous congenital ichthyosiform erythroderma is inherited as autosomal recessive disorder, is milder than
lamellar ichthyosis, and has a more prominent erythrodermic component.
Fetal harlequin ichthyosis is an autosomal recessive disorder that can be fatal. Fetal skin biopsy can be
diagnostic and shows massive hyperkeratosis. In utero, the massive hyperkeratosis interferes with normal
development. At birth, the child is encased in a thick, fissured, scaly cast, associated with ectropion.
Darier Disease
Darier disease, also known as keratosis follicularis, is a relatively uncommon disease that is inherited in an
autosomal dominant pattern. Darier disease gene has been localized to chromosome 12 (201). It typically
presents in children aged 5 to 15 years as keratotic papules distributed in the seborrheic areas such as face,
neck, and upper trunk (28). Oral mucosa and nails can also be involved (78, 208). The histopathologic findings
are characterized by suprabasal acantholysis covered by dyskeratotic cells (corps ronds) and parakeratosis
(corps grains), in addition to papillomatous epidermal hyperplasia and hyperkeratosis (Figure 25-5).
Occasionally, these lesions are centered around the hair follicles.
Most cases of Darier disease have a benign but protracted course with exacerbations during summer.
Hailey-Hailey Disease
Hailey-Hailey disease is an autosomal dominant genodermatosis that initially manifests typically only after
puberty (late
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teens or early 20s). It is characterized by recurrent vesicles and erosions on the neck, axillae, and groin. Mucosal
involvement is uncommon. Histologic features include suprabasal acantholysis resulting in a dilapidated brick
walllike appearance (Figure 25-6). Most cases of Hailey-Hailey disease have a fairly stable course. The
cutaneous lesions are exacerbated by heat, humidity, and bacterial and candidal infections.
FIGURE 25-5▪Darier disease with focal intraepidermal acantholysis, dyskeratosis, and hyperkeratosis.
FIGURE 25-6▪Hailey-Hailey disease showing intraepidermal acantholysis with a dilapidated brick wall-like
appearance. There is no significant dyskeratosis which helps in the differential diagnosis from Darier disease.
Porokeratosis
Porokeratosis is inherited as an autosomal dominant disorder that manifests in childhood and infancy as
asymptomatic keratotic papules that enlarge progressively to form plaques with peripheral keratotic ridges. Four
variants of porokeratosis can be seen in the pediatric population and include the classic plaque type of Mibelli,
linear porokeratosis, porokeratosis palmaris, plantaris et disseminata, and punctate porokeratosis that is limited
to palms and soles. A fifth type, disseminated superficial actinic porokeratosis, is a disease of adulthood (36, 91,
141, 168). Histopathologic features common to all types of porokeratoses include a cornoid lamella, which is a
column of parakeratosis that corresponds to the peripheral keratotic ridges seen clinically (Figure 25-7). The
cornoid lamella overlies an area of epidermal invagination where there is a diminished granular zone and
vacuolated and dyskeratotic keratinocytes that correspond to an abnormal clone of keratinocytes. In
porokeratosis of Mibelli, the epidermal invagination is more pronounced and deep compared with other types of
porokeratosis.
In addition to the inherited form, porokeratosis has been described in various immunosuppressive states
including Crohn disease, renal transplantation, and HIV infection. Squamous cell carcinoma and Bowen disease
have been reported to develop in lesions of porokeratosis.
FIGURE 25-7▪Porokeratosis showing a column of parakeratosis that is inclined toward the center (cornoid
lamella). Dyskeratotic keratinocytes may be seen at the base of the column. (Hematoxylin and eosin stain,
Restrictive Dermopathy
Restrictive dermopathy is an uncommon autosomal recessive disorder that presents with prematurity; rigid and
tense skin with erosions, denudations, and multiple joint contractions; fixed facial expression; and perineal
anomalies (Figure 25-8). Histologic features include a thickened epidermis with flattening of rete ridges and
hyperkeratosis. The dermis is thin with absent elastic fibers, collagen bundles oriented parallel to the surface,
and poorly developed adnexal structures (Figure 25-9). The disease is fatal, with most infants dying within weeks
after birth. Abnormalities in collagen and abnormal synthesis of keratin have been proposed as the underlying
defects.
Ectodermal Dysplasia
Ectodermal dysplasias form a large and heterogeneous group of congenital disorders that share the involvement
of structures of ectodermal origin and may include trichodysplasia, odontodysplasia, onychodysplasia, and
disorders of sweating. More than 100 syndromes encompassing all forms of Mendelian inheritance have been
described clinically, two forms of ectodermal dysplasia are recognized: hidrotic and anhidrotic or hypohidrotic.
The hidrotic form, with an autosomal dominant pattern of inheritance, is primarily a disorder of keratinization. It is
characterized by hypotrichosis, dystrophic nails, and palmoplantar hyperkeratosis. The hypohidrotic form is an X-
linked recessive disorder localized to the q1 1-q21.1 region of X-chromosome with full expression in men, who
show the tetrad of anhidrosis or hypohidrosis, hypotrichosis, dental hypoplasia, characteristic facies, and
frequently dystrophy of nails. In addition to aplasia and hypoplasia of sweat glands, the submucosal glands of
the trachea and bronchus may be affected, leading to frequent respiratory infections. Histologically, both forms
show hypoplasia of hair and sebaceous glands. In addition, the anhidrotic form shows aplasia or hypoplasia of
eccrine glands and occasionally of apocrine glands.
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FIGURE 25-8▪A: Restrictive dermopathy showing severe contractures in the absence of overt bony
abnormalities. B,C: The tight, shiny skin and characteristic fixation of the mouth and perineum.
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FIGURE 25-9▪Restrictive dermopathy without a stratum corneum, but the granular layer is prominent, indicating
that the stratum corneum may have been lost in the postmortem interval. The rete ridges are flattened and most
of the adnexal structures are atrophic. (Hematoxylin and eosin stain; original magnification ×250.)
Focal Dermal Hypoplasia

Focal dermal hypoplasia was originally described by Libermann (119) as part of ectodermal and mesodermal
dysplasia in association with osseous defects. The cutaneous aspects were first detailed by Goltz et al. (76).
Because a majority of the patients are women, it has been assumed to have X-linked dominant mode of
inheritance. Focal dermal hypoplasia is a multisystem condition in which developmental defects of skin are
associated with ocular, dental, and skeletal system abnormalities. The clinical course is dictated by the extent of
systemic involvement. The skin findings, which are present from birth, consist of widely distributed asymmetric
linear streaks of atrophy or hypoplasia of the skin, often with associated telangiectasia that follow Blaschko lines.
Some lesions may present as soft yellow nodular outpouchings caused by herniation of fat through an atrophic
dermis in linear array. Various mutations in X-linked PORCN, a putative regulator of Wnt signaling, have been
identified in focal dermal hypoplasia (83, 202).
Histological features of focal dermal hypoplasia include a marked decrease in the thickness of the dermis, with
collagen distributed as thin fibrils rather than bundles, which may be interrupted by presence of adipocytes. The
latter corresponds to the clinically apparent soft yellow nodules. The frequent presence of adipocytes high up in
the dermis raises the possibility of nevus lipomatosus in the differential diagnosis. Nevus lipomatosus, however,
lacks the collagen abnormalities and the frequent X-linked chromosomal abnormalities of focal dermal
hypoplasia.
Epidermolysis Bullosa
Epidermolysis bullosa is a heterogeneous group of inherited disorders with variable modes of transmission,
characterized by bullous lesions that develop spontaneously or secondary to minor trauma and includes
approximately 20 subtypes (65). Based on the presence or absence of scarring, mode of inheritance, cleavage
plane of the blister, and the presence or absence of structural elements of skin, epidermolysis bullosa is
traditionally divided into three major forms: simplex, junctional, and dystrophic (19, 196).
FIGURE 25-10▪Junctional epidermolysis bullosa in a 6-week old infant showing extensive blistering and
sloughing of the skin. (Courtesy of Sarah Stein, M.D., Department of Medicine, University of Chicago Medical
Center.)
Epidermolysis bullosa simplex, including Cockayne, and Dowling-Meara forms, is typically transmitted in an
autosomal dominant pattern and generally associated with good prognosis because the blisters heal without scar
formation. Histologic sections show intraepidermal separation, generally within the basal cell layer. A periodic
acid-Schiff (PAS) stain is helpful in localizing the level of cleavage above the basement membrane zone. Gene
defects of keratins 5,14 are implicated and may be identified with IF mapping.
Junctional epidermolysis bullosa is inherited as an autosomal recessive disorder and includes the fatal Herlitz
type, in which blistering begins at birth and death occurs within the first 2 years (Figure 25-10), and the non-
Herlitz type, which manifests similar to the Herlitz type but with a generally better overall prognosis. The
cleavage plane occurs in the lamina lucida of the basement membrane at the dermoepidermal junction. Similar
changes may involve the gastrointestinal, respiratory, and urinary tracts. Gene defects involving laminin 5 chain
and collagen are identified.
Dystrophic epidermolysis bullosa includes the dominant form, which has a good prognosis, and the recessive
form, which has a poor prognosis due to extensive erosions and ulcerations that heal with scarring. The level of
cleavage is in the papillary dermis below the basement membrane (Figure 25-11). The principal gene defect
involves collagen VII.
Immunomapping studies are useful in localizing the cleavage plane and determining the presence, increase, or
absence of the structural protein for which the gene is mutated in epidermolysis bullosa. These studies are
essential for accurate classification of the type of epidermolysis bullosa, which in conjunction with clinical
presentation forms the basis of prognostic information and genetic counseling. Furthermore,
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fetal skin biopsies during the third trimester can be diagnostic in the most severe forms of epidermolysis bullosa.
FIGURE 25-11▪Epidermolysis bullosadystrophicahas a subepidermal blister with mild dermal inflammation.

Incontinentia Pigmenti
Incontinentia pigmenti (Bloch-Sulzberger syndrome) is an X-linked dominant dermatosis that affects mostly
women (55, 149). Affected hemizygous male fetuses are generally thought to die in utero although recent
literature suggests that some male individuals may show cutaneous and extracutaneous features of incontinentia
pigmenti in a limited distribution that allow survival (142). The characteristic cutaneous manifestations evolve
from crops of vesicles and bullae on the extremities arranged in linear or whorled pattern at birth or shortly
thereafter that heal with hyperkeratotic verrucous lesions. As the verrucous lesions subside, characteristic
streaks and whorls of hyperpigmentation develop, being most pronounced on the trunk. Faint hypochromic or
atrophic lesions in a linear pattern may be seen on the lower extremities in some women and rarely in children
(20).
Histologically, the vesicular stage is characterized by eosinophilic spongiosis and intraepidermal vesicle
formation and eosinophil-rich dermal inflammatory cell infiltrate (Figure 25-12). The verrucous stage is
characterized by hyperkeratosis and papillomatous epidermal hyperplasia with focal dyskeratosis. The
hyperpigmented stage corresponds to numerous melanophages in the dermis as in any other postinflammatory
pigmentary change.
In approximately 80% of patients, systemic involvement, particularly of the central nervous system and the eye,
and teeth abnormalities may be present. Although the skin manifestations are self-limiting, the clinical course is
guided by the extent of systemic involvement.
Acrodermatitis Enteropathica
Acrodermatitis enteropathica is an autosomal recessive disorder characterized by defective intestinal absorption
of zinc presenting with the triad of dermatitis, diarrhea, and alopecia in infancy at the time of weaning (128).
Acquired acrodermatitis enteropathica-like syndromes can occur in exclusively breast-fed preterm infants, infants
who are fed on breast milk low in zinc, infants with organic acid urea, and any other acquired zinc deficiency
states including human immunodeficiency virus infection. Cutaneous manifestations are characterized by
vesiculobullous lesions with acral and periorificial distribution. The histopathologic findings include intraepidermal
bullae with epidermal necrosis or spongiosis and superficial perivascular mixed inflammatory cell infiltrate. A well-
established lesion shows parakeratosis, marked pallor, ballooning of keratinocytes, and a markedly diminished
granular zone. The histologic changes can be identical to those in glucagonoma syndrome and pellagra,
conditions associated with nutritional deficiencies of factors essential for normal maturation and metabolism of
epidermal keratinocytes.
FIGURE 25-12▪Incontinentia pigmenti showing the initial skin changes with an intense eosinophilic infiltration
within mildly spongiotic epidermis and the dermis. (Hematoxylin and eosin stain, original magnification ×200.)
NONINFFECTIOUS ACQUIRED VESICULOBULLOUS DISEASES
Linear IgA Bullous Dermatosis
Linear IgA bullous dermatosis, also known as chronic bullous dermatosis of childhood, presents with large tense
bullae in prepubertal children often younger than 5 years of age. The lesions are widespread in distribution and
vesicles and bullae, sometimes arranged like a string of pearls, occur at the periphery of a healing lesion. Areas
of predilection include the lower part of the trunk, including the groin and genitalia and perioral areas. Rare
cases have been described in neonates. Microscopic features are essentially indistinguishable from dermatitis
herpetiformis and consist of neutrophilic microabscesses at the tips of dermal papillae in early lesions and
subepidermal bulla filled with neutrophils or eosinophils in well-established lesions
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(Figure 25-13). Direct IF testing shows a distinct linear pattern of staining at the basement membrane zone with
IgA, in sharp contrast to the granular IgA deposits seen in dermatitis herpetiformis. Direct IF testing is crucial in
differentiating lupus erythematosus and chronic bullous dermatosis of childhood from other childhood bullous
diseases like bullous pemphigoid and lichen planus. Chronic bullous disease of childhood has generally a benign
course with a spontaneous remission before puberty. Rare cases heal with scarring when the disease process
seems to overlap with childhood cicatricial pemphigoid, which some consider to be another morphologic
expression of linear IgA dermatosis of childhood and adults. Linear IgA dermatosis of both children and adults is
also similar, both IgA1-mediated diseases (204) with some cases of chronic bullous dermatosis of childhood
relapsing into adulthood. Distinction of linear IgA bullous disease from dermatitis herpetiformis is important
because linear IgA bullous disease is not typically associated with gluten-sensitive enteropathy.
FIGURE 25-13▪Linear IgA dermatosis with a subepidermal blister with neutrophils.

FIGURE 25-14▪Dermatitis herpetiformis: A,B: subepidermal separation with neutrophilic microabscesses at the
tips of dermal papillae. Differentiation from linear IgA dermatosis is possible only on IF studies. [Hematoxylin and
eosin stains, original magnification *200(A),X400(B).]
Dermatitis Herpetiformis
Dermatitis herpetiformis presents as an intensely pruritic papulovesicular eruption that is typically distributed on
the scalp, the extensor aspects of extremities, and the back. The lesions may be grouped in herpetiform fashion
and symmetrical in distribution. They are characterized by small papules and tense vesicles that rupture easily
(165). Although dermatitis herpetiformis generally manifests as a skin disease, approximately 75% to 90% of the
children with this disorder have an associated gluten-sensitive enteropathy and a high frequency of HLA
antigens, including HLA B8, DR3, and DqW2 (105, 107). Histologic sections of a papular lesion show the
characteristic neutrophilic microabscesses at the tips of the dermal papillae (Figure 25-14). Sections of a
clinically apparent vesicle show a subepidermal bulla filled with neutrophils and a varying mixture of eosinophils
and fibrin. Microabscesses are present at the edge of the blister. Direct IF testing is positive for granular deposits
of IgA at the tips of dermal papillae in almost all patients (6). A gluten-free diet is effective in controlling the
intestinal and cutaneous manifestations in most children.
Herpes Gestationis
Pemphigoid gestationis (herpes gestationis) is a rare acquired autoimmune bullous disease that affects pregnant
women most commonly during the second trimester (32) and, in a small percentage of cases, can be transmitted
to the neonates born to these women. The affected neonate may present with macules, or papulovesicular or
bullous lesions at birth or shortly thereafter. In neonates, the condition is transient, with complete resolution of
the lesions occurring within a month, and it is attributed to the transplacental transfer of maternal antibodies (9).
Studies have failed to show significant association between pemphigoid gestationis and increased incidence
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and risk of fetal morbidity or mortality. Histopathologic and IF findings may be identical to those seen in bullous
pemphigoid and consist of subepidermal bulla with eosinophils and linear deposits of C3 and IgG at the
basement membrane zone. Additionally, sera from patients with pemphigoid gestationis like those with bullous
pemphigoid are positive for antibodies against a 180-kD epidermal antigen (46).
Epidermolysis Bullosa Acquisita

Epidermolysis bullosa acquisita is an acquired form of epidermolysis bullosa that is seen more commonly in
adults but can sometimes be seen in children (30, 195). Clinically, it may resemble the autosomal dominant
dystrophic type of epidermolysis bullosa acquisita and manifests with tense blisters on the extensor aspects that
heal with scarring and milia formation. However, epidermolysis bullosa acquisita is an autoimmune disease
characterized by IgG antibodies to type VII collagen of the basement membrane (129, 205).
Light microscopic and IF findings are indistinguishable from bullous pemphigoid, which can also be seen in
children (54). Both conditions are characterized by subepidermal bullae with linear deposits of IgG and C3 at the
basement membrane zone. Indirect IF studies localize the IgG deposits to the roof of salt-split skin, in which the
cleavage runs through the lamina lucida of the basement membrane in pemphigoid and the floor (beneath the
lamina lucida) in epidermolysis bullosa acquisita.
Erythema Multiforme, Stevens-Johnson Syndrome, and Toxic Epidermal Necrolysis

Erythema multiforme, Stevens-Johnson (S-J) syndrome, and toxic epidermal necrolysis (TEN) (Lyell syndrome)
form the clinical and histopathologic spectrum of a potentially lifethreatening group of disorders characterized by
epidermal necrosis with formation of bullae, which can involve a large part of the skin surface and mucosa. The
high mortality rate in patients with TEN is directly related to the resultant fluid loss and sepsis. S-J syndrome is
more common in childhood than erythema multiforme or TEN.
Erythema multiforme is distinguished clinically by the characteristic iris or targetoid lesions that can occur on any
part of the body but most commonly on the palms and soles. Erythematous and purpuric macules that progress
to flaccid bullae and detach from the underlying dermis are characteristic of S-J syndrome and TEN. The
detachment is extensive in TEN while mucosal involvement is more prominent in S-J syndrome.
The majority of cases of erythema multiforme in children is etiologically related to herpes simplex virus infection;
other viral infections including Epstein-Barr virus and orf and mycoplasma infections have also been implicated
(115). Drugs such as sulfonamides and penicillins play an important role, especially in the more severe S-J
syndrome and TEN (66, 111). No cause can be identified in a significant number of cases.
FIGURE 25-15▪Erythema multiforme with basket-weave orthokeratosis, vacuolar alteration of the basal cell layer,
necrotic keratinocytes, and mild superficial perivascular inflammation. (Hematoxylin and eosin stain, original
Histopathologic features include interface dermatitis with vacuolar alteration of the basal cell layer and mild
perivascular infiltrate of lymphocytes, which are also present along the dermoepidermal junction. The histologic
hallmark of this group of diseases is the necrotic keratinocyte, which may be few in milder forms and numerous
with confluent areas of necrosis in more established lesions (Figure 25-15). In TEN, full-thickness epidermal
necrosis leads to subepidermal separation and loss of epidermal surface with the eroded clinical appearance of
skin originally described by Lyell (Figure 25-16). An unaltered stratum corneum in skin biopsies attests to the
acute nature of the assault on the skin. Immune complex mediated reactions of type III and IV and helper T-cell-
mediated immunoreactions are believed to play a role in the pathogenesis of erythema multiforme/TEN (198).
Erythema multiforme/S-J syndrome/TEN are potentially life-threatening disorders that require hospitalization,
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withdrawal of recent drugs, and supportive care. Potential infectious causes should be sought and treated. The
benefits of specific treatment including corticosteroids and intravenous administration of immunoglobulin are still
under debate and further investigation (70, 133, 173, 189).
FIGURE 25-16▪Toxic epidermal necrolysis with full-thickness epidermal necrosis with separation at the
dermoepidermal junction and sparse inflammatory cell infiltrate. An unaltered cornified layer attests to the
acuteness of the event. (Hematoxylin and eosin stain, original magnification ×400.)
MISCELLANEOUS NONINFECTIOUS VESICULOPUSTULAR DISEASES

A variety of benign vesiculopustular diseases are commonly seen in neonates and infants. It is important to
differentiate these conditions from the more serious vesiculopustular diseases that can affect children (200).
Erythema Toxicum Neonatorum

Erythema toxicum neonatorum (toxic erythema of newborn) is an asymptomatic, transient, self-limiting, common
eruption that occurs in the first 24 to 48 hours of life of full-term newborns. The lesions are characterized by
macules, papules, and tiny pustules that can affect any part of the body but favor the trunk and proximal
extremities. Classical clinical presentation rarely requires a skin biopsy that would reveal eosinophils in the
pilosebaceous units and differentiate erythema toxicum neonatorum from other neonatal pustular dermatoses
including incontinentia pigmenti (131, 138).
TRANSIENT NEONATAL PUSTULAR MELANOSIS

Transient neonatal pustular melanosis is a benign, self-limiting condition that predominantly affects black infants.
The skin eruption begins as superficial sterile pustules that rupture easily and typically heal with hyperpigmented
macules with collarettes of fine scale. Similarities between transient neonatal pustular melanosis and erythema
toxicum neonatorum are emphasized by some authors who proposed “sterile transient neonatal pustulosis” as a
unifying term (63). However, the pustules in transient neonatal pustular melanosis show abundance of
neutrophils.
Acropustulosis of Infancy
Acropustulosis of infancy presents as recurrent crops of pruritic vesicles and pustules on distal extremities with
predilection for palms and soles, primarily in black infants during the 1st year of life. Most cases show
spontaneous resolution by the age of 2 years (50).
Smears from the pustule or histologic sections of the subcorneal pustules will show abundant neutrophils.
ECZEMATOUS DERMATITIS
“Eczema” is the term often used to describe erythematous, scaling vesicular lesions with serum crust.
Eczematous dermatitis is characterized histologically by epidermal spongiosis and, therefore, is often referred to
interchangeably as spongiotic dermatitis. A specific diagnosis is based on clinical history, morphologic
appearance, and distribution of lesions. This group of disorders includes nummular dermatitis, contact dermatitis,
dyshidrotic dermatitis, and atopic dermatitis.
Nummular Dermatitis
Nummular dermatitis is characterized by coin-shaped, pruritic, erythematous, scaly crusted plaques on the
extensor aspect of the extremities. It is believed to be a manifestation of xerosis and is more commonly seen in
older patients.
Atopic Dermatitis
Atopic dermatitis is an inherited chronic pruritic skin disease and is the most common skin disease seen in
children, with an estimated incidence of as high as 20% (147). About onethird of the cases are diagnosed before
the age of 1 year and before 5 years of age in vast majority of patients (85). Sites of predilection are the face in
young infants, extensor surfaces of extremities in children younger than 1 year of age, and the popliteal and
antecubital fossae, face, and neck in older children and adolescents. The major abnormality in this disease
appears to be the overproduction of allergen-specific IgE, and some authors suggest that demonstration of such
antibodies be a requisite for the diagnosis of atopic dermatitis (184). Cytokines, T-cells, and antigen-presenting
cells in addition to abnormalities of skin barrier appear to play a role in the pathogenesis (34).
Contact Dermatitis
Contact dermatitis includes primary irritant dermatitis and allergic contact dermatitis. Primary irritant dermatitis is
frequently seen in children on the cheeks caused by saliva, extremities in response to harsh soaps or
detergents, and the diaper area from toiletries (171). Allergic contact dermatitis presents with pruritic, edematous
papules, plaques, and occasionally vesicles 12 to 24 hours after exposure to an allergen such as poison ivy,
fragrances, nickel, and rubber compounds (16). Allergic contact dermatitis occurs more frequently in children with
atopic tendencies (93).
Dyshidrotic Dermatitis
Dyshidrotic dermatitis (pompholyx) typically presents with numerous, pinpoint, recurrent, pruritic vesicles along
the sides of the fingers and toes and on palms and soles that usually last a few weeks and frequently relapse.
Histopathology of Spongiotic Dermatitis

Irrespective of the specific type of disease, spongiotic dermatitis shows a similar spectrum of changes. In the
acute phase, there is epidermal spongiosis, sometimes marked,
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with vesiculation (Figure 25-17). In the subacute phase, the spongiosis is milder, but associated parakeratosis
with plasma cells, neutrophils, eosinophils and epidermal hyperplasia may be present. In the chronic phase, the
spongiosis is mild to absent, but changes of chronicity are reflected in a hyperkeratotic cornified layer, marked
epidermal hyperplasia, and fibrotic papillary dermis. Superficial perivascular lymphohistiocytic infiltrate is present
to varying degrees in all the phases of spongiotic dermatitis.
FIGURE 25-17▪Eczematous dermatitis showing marked epidermal spongiosis with formation of intraepidermal
vesicles and moderate perivascular mixed inflammation. (Hematoxylin and eosin stain, original magnification
×400.)
NONINFECTIOUS PAPULOSQUAMOUS DERMATOSES
This includes a group of diverse disorders characterized by papular and scaling lesions and associated
epidermal proliferation. Approximately 10% of the patients seen in a pediatric dermatology clinic present with
papulosquamous skin disorders (170). The following is a brief discussion of the more common dermatoses
traditionally regarded as the papulosquamous dermatoses.
Psoriasis Vulgaris
Psoriasis vulgaris accounts for 4% of all dermatoses encountered in children younger than the age of 16 years
(176) and in about 30% of the patients, psoriasis manifests in the first or the second decade of life (18). Of the
various forms of psoriasis, namely, plaque type, guttate, pustular and erythrodermic psoriasis, plaque type is the
most common one seen in children followed by guttate psoriasis (61). Pustular psoriasis and psoriatic
arthropathy are less common in children (161). The clinical presentation is characterized by asymptomatic scaly
erythematous plaques in the plaque type and by slightly pruritic small red droplike scaly lesions in guttate
psoriasis. Silvery scales that, on scraping, leave pinpoint areas of bleeding (Auspitz sign) are typical of psoriasis.
Lesions are distributed in a bilaterally symmetrical pattern with predilection for scalp and extensor aspects of
extremities. Involvement of face is more common in children than in adults and needs to be distinguished from
atopic dermatitis. Similarly, psoriasis may involve the diaper area in up to 13% of patients where it must be
differentiated from infantile seborrheic dermatitis and other causes of diaper dermatitis (24). Classic histologic
features of psoriasis include confluent parakeratosis with neutrophils (Munro microabscesses), regular
elongation of epidermal rete with thin suprapapillary plates, dilated vessels in dermal papillae, and mild
superficial perivascular inflammation (Figure 25-18). Dermatophytes can produce a psoriasiform dermatitis.
FIGURE 25-18▪Psoriasis showing confluent parakeratosis and a regular epidermal hyperplasia in which the rete
ridges are of equal length. (Hematoxylin and eosin stain, original magnification ×200.)
Psoriasis, a multifactorial disorder with a genetic basis, typically runs a chronic course with remissions and flare-
ups.
Seborrheic Dermatitis
A chronic dermatosis of unknown cause, seborrheic dermatitis is quite common in infants aged 2 to 10 weeks
and in adolescents. In infants, seborrheic dermatitis begins as an erythematous scaly rash typically involving the
scalp, face, and diaper area. In adolescents, it appears as a dry fine exfoliation of the scalp (dandruff) and
expands to the face with the clinical features sometimes overlapping with those of psoriasis.
Histopathologic features overlap with psoriasis and spongiotic dermatitis and consist of epidermal hyperplasia
and spongiosis with exocytosis and patchy parakeratosis, which is often present at the openings of the follicular
infundibula. A mild superficial perivascular lymphohistiocytic inflammation is present in the dermis.
Infantile seborrheic dermatitis may clinically mimic Langerhans cell histiocytosis, which is a potentially serious
disorder.
Lichen Planus
More commonly a disease of adulthood, lichen planus, generally a self-limiting pruritic eruption, is generally
considered uncommon in children (120). However, children of
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South Asian subcontinent appear to be more susceptible to developing lichen planus (14). The clinical
appearance of the eruption is distinctive and consists of flat-topped violaceous papules involving flexor aspects
of the extremities and lower back. Lichen planus can also involve hair, nails, and mucous membranes in a
significant number of cases. The histologic features are distinctive and consist of hyperkeratosis,
hypergranulosis, irregular epidermal hyperplasia, and a bandlike lymphohistiocytic infiltrate that obscures the
dermoepidermal junction (lichenoid dermatitis) where there are vacuolar alterations and colloid bodies (Figure
25-19). Melanophages are seen in the infiltrate in older lesions.
FIGURE 25-19▪Lichen planus showing hyperkeratosis, hypergranulosis, irregular epidermal hyperplasia, and a
bandlike lymphohistiocytic infiltrate that obscures the dermoepidermal junction. (Hematoxylin and eosin stain,
The etiology of lichen planus is unknown in most cases, whereas in others, various drugs have been implicated.
Pityriasis Rosea
Pityriasis rosea is an acute, self-limiting papulosquamous eruption appearing in children, especially adolescents,
up to 45% of the time (40). It typically presents with a single large scaly plaque, the herald patch on the trunk that
is followed within a week by more disseminated smaller oval scaly pink papules along the lines of skin cleavage.
In addition to the trunk, the neck and proximal extremities may be involved. Histologic sections show focal
parakeratosis, focal spongiosis, and a mild superficial perivascular lymphohistiocytic infiltrate. Extravasated red
blood cells are often present in the papillary dermis and may extend into the epidermis (Figure 25-20). Biopsy of
the herald patch also shows epidermal hyperplasia and denser infiltrate of inflammatory cells. A viral etiology has
been suspected for a long time, and in recent years, viruses such as human herpes virus 7 and parvovirus have
been implicated in the etiology (26). Most cases of pityriasis rosea resolve within 6 to 12 weeks with no specific
treatment.
FIGURE 25-20▪Pityriasis rosea showing patchy parakeratosis (mounding parakeratosis) and focal spongiosis
and extravasated red cells and inflammatory cells in the superficial dermis. (Hematoxylin and eosin stain, original
Pityriasis Rubra Pilaris

Pityriasis rubra pilaris is a chronic follicular-based erythematous papular eruption of unknown etiology that can
manifest in children (5). Although most cases are acquired, a familial form is also recognized. Keratoderma of the
palms and soles develops in a majority of the affected children and about 40% show cephalic involvement.
Histologic findings include epidermal hyperplasia, alternating hyperkeratosis and parakeratosis oriented in both
vertical and horizontal directions, and a mild superficial perivascular lymphocytic infiltrate. Follicular plugging is
present in biopsies of the follicular papules (Figure 25-21).
Spontaneous resolution of the cutaneous rash is expected in a majority of the cases within 2 to 3 years, whereas
recurrences and a protracted course may occur in others.
Pityriasis Lichenoides
Pityriasis lichenoides is a self-limiting cutaneous eruption of unknown cause that can occur in pediatric patients,
commonly
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during the first decade of life (59). The cutaneous eruption may be delineated along a spectrum including an
acute, more severe form, pityriasis lichenoides et varioliformis acuta (PLEVA, Mucha-Habermann disease), and a
chronic milder form, pityriasis lichenoides chronica. Transitional forms in between the two extremes are
recognized in children. In addition, a more severe but rare variant, the acute febrile ulceronecrotic form, which is
more common in children, has also been described (109). PLEVA is characterized by an extensive papular,
papulonecrotic, and occasionally, vesiculopustular eruption on the trunk and proximal extremities that resolves
within a few weeks. As the older lesions resolve, crops of newer lesions continue to appear, and the overall
course may be protracted to several months. The ulceronecrotic form is characterized by large coalescing
ulceronecrotic nodules and plaques associated with high fever.
FIGURE 25-21▪Pityriasis rubra pilaris showing mild epidermal hyperplasia with alternating layers of
hyperkeratosis and parakeratosis and follicular plugging. (Hematoxylin and eosin stain, original magnification
x200.)
The chronic form of pityriasis lichenoides chronica is characterized by recurrent crops of reddish-brown papules
with an adherent scale that typically resolve within 3 to 6 weeks without scarring. Transient postinflammatory
pigmentary changes may occur.
Histopathologic findings in pityriasis lichenoides include interface dermatitis with parakeratosis, epidermal
spongiosis, necrotic keratinocytes, and a perivascular lymphocytic infiltrate. Papillary dermal edema and
extravasated red cells may be present. In PLEVA, the inflammatory cell infiltrate is dense and deep, and
spongiosis and epidermal necrosis are more marked with eventual erosion or ulceration of the epidermis with
overlying parakeratotic scale crust containing neutrophils (Figure 25-22).
Clinical and histopathologic findings may show some overlap with lymphomatoid papulosis, a benign, recurrent
self-healing dermatosis that falls within the spectrum of CD30-positive cutaneous lymphoproliferative disorders.
Studies have shown T-cell clonality in pityriasis lichenoides, especially in the acute form (122, 203), suggesting
that host immune reaction prevents further progression to lymphoma. Although evolution to cutaneous T-cell
lymphoma has been reported (144), in a longterm follow-up study of 89 children with pityriasis lichenoides, the
clinical course was essentially benign, with no evolution into lymphomatoid papulosis or lymphoma (73).
FIGURE 25-22▪Pityriasis lichenoides et varioliformis acuta with mounds of parakeratosis containing neutrophils
and interface dermatitis with vacuolar alteration of the basal cell layer and necrotic keratinocytes. (Hematoxylin
and eosin stain, original magnification ×200.)
Papular Acrodermatitis of Childhood

Papular acrodermatitis of childhood, or Gianotti-Crosti syndrome, is a self-limiting papular and papulovesicular
eruption involving the face, extremities, and buttocks of children aged 2 to 6 years with an underlying viral
infection. Since the original description of the cases in association with hepatitis B infection, a variety of other
viruses including Coxsackie, Epstein-Barr, parainfluenza, pox, parvovirus B19, and HIV, and, certain bacteria as
well as immunizations have been shown to be associated with similar cutaneous eruptions. In recent years, there
has been a striking shift from HBV to EBV as the most common cause, although the exact mechanism of how the
infectious agents cause the cutaneous eruption continues to reside in the realm of the unknown (25).
The histologic features are not specific and include focal parakeratosis, varying degrees of epidermal spongiosis
with exocytosis, and a mild perivascular lymphohistiocytic infiltrate. Spongiotic vesicles, when present, contain
lymphocytes and Langerhans cells.
The cutaneous eruption generally lasts for about 3 to 4 weeks, and relapses are not reported.
Lichen Sclerosus
Lichen sclerosus is generally a skin disease of adults of unknown etiology that can be seen in children (151).
The majority of the affected children have involvement of the anogenital area by ivory-colored flattened papules
and plaques. Human papillomavirus (HPV) has been shown to be present in some pediatric cases of lichen
sclerosus (51), although the exact significance of this finding and the risk of squamous cell carcinoma in pediatric
onset lichen sclerosus are undefined (150). The clinical and histopathologic findings are essentially similar to
those seen in adults. The histologic features include hyperkeratosis, epidermal atrophy, and a zone of papillary
dermal sclerosis, beneath which there may be a band of lymphocytes (Figure 25-23). Lichen sclerosus in
childhood generally has a better prognosis, with spontaneous resolution occurring in up to 60% of the affected
girls before puberty. There is some morphologic overlap with morphea.
INFECTIOUS DISEASES
Bacterial Infections
Bacterial infections of skin are a common cause for pediatric outpatient visits. Skin infection may be primary or a
complication of an underlying skin disease. Occasionally, skin involvement may be a manifestation of a systemic
infection.
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Only the more common bacterial infections that often affect children are discussed.
FIGURE 25-23▪Lichen sclerosus showing atrophy of the epidermis with zone of sclerosis underneath, which is
aband of inflammatory cells. (Hematoxylin and eosin stain, original magnification ×200.)
Impetigo
Impetigo is the most common bacterial infection of the skin seen in children. Two clinical forms are recognized:
nonbullous and bullous forms.
Nonbullous Impetigo (Impetigo Contagiosa)
Nonbullous impetigo or the crusted form of impetigo accounts for the majority of cases. It was historically often
caused by group A β-hemolytic streptococci but now appears to be more commonly caused by Staphylococcus
aureus. It is characterized by highly infectious 1 to 2 mm vesiculopustular lesions that quickly rupture to be
covered by heavy yellow crusts. Lesions may involve any part of the body but occur most frequently on the
exposed parts of the body such as face, neck, and extremities.
Histologic sections from a vesiculopustule show a subcorneal pustule, which may contain Gram-positive cocci
(Figure 25-24). Sections of the crusted lesions show a neutrophilic scale crust covering the epidermis. Impetigo
contagiosa may be superimposed on pre-existing skin diseases such as atopic dermatitis (4). Complete
resolution of the lesions, either spontaneously or with treatment with antibiotics, occurs in most cases. Acute
glomerulonephritis, a wellrecognized sequela in a small percentage of patients, appears to be decreasing in
incidence partly due to changing patterns in the infecting agents.
Bullous Impetigo
Bullous impetigo, caused almost always by S. aureus, generally affects newborn infants and children and can be
thought of as a localized form of staphylococcal scalded skin syndrome (SSSS), caused by the same exfoliative
toxins. It presents with small vesicles that may progress to flaccid bullae of more than 1 cm, with no associated
erythema. The bullae are filled with clear fluid.
FIGURE 25-24▪Impetigo with its subcorneal pustule. Gram stain may show Gram-positive cocci. (Hematoxylin
and eosin stain, original magnification ×200.)
Histologic sections of the bullae show a cleavage plane in the uppermost part of the epidermis at or below the
level of the granular layer, similar to the findings in SSSS. The underlying dermis shows a perivascular
neutrophilic infiltrate that may also involve the epidermis. Unlike that in impetigo contagiosa, the bullous cavity
contains few or no inflammatory cells.
When impetigo appears to be rapidly spreading, prompt treatment with systemic antibiotics avoids the risk of
worsening infection or hospitalization (87). Although skin infections due to methicillin-resistant S. aureus (MRSA)
are still relatively uncommon in children, given the evolving epidemiology, skin swabs should be cultured and
sensitivity tests performed (21, 114).
Staphylococcal Scalded Skin Syndrome

SSSS is a generalized blistering disease seen most often in neonates and children younger than 5 years of age.
Like bullous impetigo, this disease is caused usually by epidermolytic toxin-producing S. aureus. The pathogen
cannot be isolated from the lesions of SSSS; instead, a distant source of staphylococcal infection in the form of
purulent pharyngitis, conjunctivitis, rhinitis, or umbilical infection may be present. Exfoliative toxins, ETA and
ETB, produced by S. aureus target desmoglein 1, a cell-to cell-adhesion molecule found in the desmosomes of
superficial epidermis and cause the cleavage in the superficial granular layer of the epidermis, typical of SSSS
(7). The clinical manifestations appear to depend on serotypes of the exfoliative toxins with ETA associated with
bullous impetigo and ETB with generalized SSSS (206).
SSSS is characterized by an abrupt onset of fever and diffuse erythema that evolves into large flaccid sterile
bullae
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filled with clear fluid. Within a short time, the bullae rupture and large sheets of epidermis peel off, giving the
typical scalded appearance. The scaly desquamation resolves within 3 to 5 days without scarring.
FIGURE 25-25▪Staphylococcus scalded skin syndrome showing intraepidermal clefting at the level of granular
zone with minimal to absent inflammation is characteristic because the lesion is caused by toxin. [Hematoxylin
and eosin stain, original magnification ×200(A), ×400(B).]
Histologic findings are identical to those seen in bullous impetigo, with the cleavage plane at or below the
granular layer. However, in contrast to bullous impetigo, the superficial dermis in SSSS is usually free of
inflammatory cells (Figure 25-25). Despite the clinical similarities, SSSS can be easily distinguished from TEN, a
potentially fatal skin loss disorder, based on the histologic finding of full-thickness epidermal necrosis in the
latter. In addition, mucosal involvement, often seen in TEN, is lacking in SSSS. Treatment is directed at
eradicating the nidus of staphylococcus infection and management of fluids and electrolytes with complete
recovery within 2 weeks expected in most pediatric patients. Fatalities are generally related to sepsis from the
primary source.
Toxic Shock Syndrome

Toxic shock syndrome, although classically described in menstrual women, can occur in nonmenstrual form that
is much more common now and occurs in a variety of clinical settings (98) It is an acute life-threatening
multisystem disease characterized by fever, hypotension, a generalized rash, and involvement of three or more
organ systems, caused by TSS toxin-1 and enterotoxins produced by a strain of S. aureus. In children, the most
common sources of S. aureus infection are upper airway infections such as sinusitis and tracheitis, burns, and
minor skin infections. Communityassociated MRS A may be isolated from some patients (37). The cutaneous
eruption is a diffuse macular erythroderma resembling scarlet fever or sunburn. The histologic findings are
nonspecific and may include a spongiotic dermatitis with necrotic keratinocytes and exocytosis of neutrophils,
papillary dermal edema, and perivascular and interstitial infiltrate of neutrophils and eosinophils (97). Toxic shock
syndrome, similar to that caused by S. aureus, is occasionally caused by group A β-hemolytic streptococcus and
may be associated with localized infection such as necrotizing fasciitis (127). TSS is managed with supportive
therapy, identification, and aggressive treatment of source of infection.
Ecthyma
Ecthyma is an ulcerative pyoderma caused by group A β-hemolytic streptococci commonly affecting children.
Like impetigo, it begins as a superficial vesicle that evolves into a vesiculopustule. This lesion enlarges and
becomes crusted. Unlike impetigo, in ecthyma, the organism infects not only the epidermis but also the dermis,
and consequently, the lesions heal with a scar. A history of antecedent trauma is present in most cases.
Histologic features are those of ulcerative dermatitis with dense neutrophilic infiltrate. Gram-positive cocci may
be identified.
Ecthyma Gangrenosum
Ecthyma gangrenosum is an ulcerative cutaneous lesion caused by Pseudomonas aeruginosa generally in
association with pseudomonas sepsis (35). Underlying predisposing conditions such as immunodeficiency,
cancer, chemotherapy, burns, and treatment with multiple antibiotics may be present. Rarely, ecthyma
gangrenosum can occur in previously healthy children (212). The cutaneous lesions start as hemorrhagic bullae
that rupture and form punched-out ulcers with a necrotic base. Nonulcerating nodules may be simultaneously
present, which demonstrate cellulitis caused by the bacilli. Histologic sections of the ulcerated lesion
demonstrate a necrotizing vasculitis at the base of the ulcer, with only a scant neutrophilic infiltrate. It is believed
that the pseudomonas bacilli invade the walls of the deep subcutaneous vessels and spread along the
periadventitial tissues to the dermal vessels, with resultant vascular necrosis and ulcer formation (49). The
presence of Gram-negative bacilli can be demonstrated in and around the ulcer. Ecthyma gangrenosum in the
absence of underlying bacteremia has a better prognosis. However, the presence of underlying pseudomonas
sepsis can be rapidly fatal and requires early diagnosis, treatment with appropriate antibiotics, and surgical
excision of progressive lesions to prevent mortality (110).
Erysipelas
Erysipelas is a form of superficial cellulitis of the skin caused most commonly by group A β-hemolytic
streptococcus and rarely by non-group A streptococci, S. pneumoniae and other organisms (58). Factors that
predispose pediatric patients to erysipelas included very young age, diabetes, immunocompromised states, and
nephritic syndrome (33). The characteristic lesion is a well-demarcated, slightly indurated, dusky red area with
an advancing border, typically on the face and recently more commonly seen on legs, especially in association
with chronic lymphatic obstruction (82). Histologic sections show marked dermal edema with diffuse infiltrate of
predominantly neutrophils. Dilated lymphatics and capillaries
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are present. Gram stain is positive for Gram-positive cocci. Septicemia, abscess formation, and rarely,
necrotizing fasciitis may complicate some cases of erysipelas.
Viral Infections
Human Papillomavirus
HPV, a member of the Papovaviridae family, is a group of DNA viruses. With advances in molecular biology
techniques, more than 67 types of HPV have been identified, some with specific cellular tropism. Transmission of
HPV is by direct contact. Clinical patterns of HPV infection include verruca vulgaris or common wart, verruca
plantaris or palmaris, verruca plana, and condyloma acuminatum. Certain HPV types manifest with characteristic
type of lesions such as HPV types 2, 4, and 7 in verruca vulgaris; HPV type 3 in verruca plana; HPV types 1,2,
and 4 in palmoplantar warts; and HPV types 6 and 11 in condyloma acuminatum in children (140). However,
more than one type can share the same cellular tropism.
The characteristic histologic changes of HPV infection, irrespective of the clinical pattern, are epithelial
hyperplasia, which can be papillomatous, hyperkeratotic and parakeratotic, especially at the tips of the papillary
projections. The cytopathic effect of HPV is manifested as an irregular and hyperchromatic nucleus surrounded
by a halo of clear cytoplasm or koilocyte (Figure 25-26).
In children, verruca vulgaris is the most common pattern of HPV infection seen. In most immunocompetent hosts,
spontaneous regression is the expected course. In immunocompromised patients, including patients with
epidermodysplasia verruciformis (both autosomally inherited and acquired forms), widespread infection with HPV
and progression to squamous cell carcinoma can occur. Oncogenetic types of HPV, such as HPV type 16, can
be identified by DNA hybridization in these lesions. Sexual abuse can be a source of condyloma acuminatum in
children and requires careful evaluation of the clinical findings and history (125, 182). However, most cases of
anogenital warts in children are likely to be the result of nonsexual transmission, that is prenatal mode and
maternal history of warts may be obtained in a significant number (102).
FIGURE 25-26▪Verruca vulgaris. A: Hyper- and parakeratosis and papillomatous epidermal hyperplasia. B:
Hypergranulosis and koilocytosis typical of papilloma virus infection. As warts involute, koilocytes and
papillomatosis become less apparent. (Hematoxylin and eosin stain, original magnification ×100.)
Molluscum Contagiosum
Molluscum contagiosum is a common pediatric cutaneous infection caused by a DNA poxvirus that spreads
through person-to-person contact or autoinoculation. It most commonly presents in children younger than 5 years
of age with discrete, dome-shaped umbilicated waxy papules varying in size from 1 to 5 mm, involving the face,
neck, axilla, abdomen, and thighs.
The histologic findings are classic and consist of epidermal hyperplasia with surface invaginations. Within the
epidermal cells, there are large intracytoplasmic inclusion bodies—called molluscum bodies—that compress the
nuclei to a thin crescent at the periphery of the cell (Figure 25-27). The molluscum bodies increase in size as the
infected cells move toward the surface. Basophilic molluscum bodies are found along with the cornified layer
within the invaginations. Occasionally, molluscum contagiosum can rupture into the dermis and induce an
inflammatory response.
In most immunocompetent hosts, spontaneous regression of the lesions is seen without treatment. In the context
of immunosuppressed states, especially HIV infection, hundreds of lesions of molluscum contagiosum may be
seen with no tendency toward resolution. Hundreds of lesion in a child is cause for concern about
immunodeficiency.
Herpes Virus Infection

Herpes viruses are a family of large DNA viruses that include herpes simplex virus, varicella zoster virus,
cytomegalovirus, Epstein-Barr virus, and human herpesviruses 6-8.
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FIGURE 25-27▪Molluscum contagiosum with characteristic eosinophilic round intranuclear and intracytoplasmic
inclusions are seen within the hyperplastic epithelium of the follicular infundibula. (Hematoxylin and eosin stain,
Herpes Simplex
Two forms of herpes simplex virus infections are recognized—orofacial type caused by herpes simplex virus type
1 and genital type caused by herpes simplex type 2—and both can present as primary or recurrent infections.
Primary infection with HSV-I is largely a childhood disease that can manifest as gingivostomatitis and rarely as
Kaposi varicelliform eruption and keratoconjunctivitis. HSV-2 is primarily acquired through sexual contact and
can rarely be seen in infants owing to in utero infection or direct contact in the birth canal. Most primary HSV
infections are asymptomatic. Recurrent HSV infection occurs in people with previous infections and is
characterized by repeated episodes of lesions at the same site.
FIGURE 25-28▪Herpetics lesions regardless of the specific virus have similar histologic features. A: An
intraepidermal vesicle surrounded by multinucleated keratinocytes. B: Characteristic intranuclear inclusions are
present at the margins of the vesicle. [Hematoxylin and eosin stain, original magnification ×200(A), ×400(B).]
Varicella and Herpes Zoster

Herpes zoster virus commonly manifests in children as chicken pox due to primary infection with varicella zoster
infection. Chicken pox is a highly contagious generalized vesiculopustular eruption that spreads centrifugally,
with lesions in different stages of development. Herpes zoster is caused by reactivation of latent varicella-zoster
virus that resides in a dorsal root ganglion and presents as grouped vesicles in a dermatomal distribution.
Herpes zoster can develop any time after a primary infection and is often triggered by immunocompromised
state. Because varicella vaccine is a live attenuated virus, herpes zoster can develop in a vaccine recipient. In
young children, herpes zoster has a predilection for areas supplied by the cervical and sacral dermatomes (118).
The histologic findings are identical in herpes simplex and varicella-zoster infections. Intraepidermal vesicles with
acantholysis are the characteristic feature. Balloon degeneration and multinucleated keratinocytes with
eosinophilic intranuclear inclusions are seen. Epidermal necrosis with neutrophilic scale crust characterizes older
lesions (Figure 25-28). Leukocytoclastic vasculitis may develop in some cases of herpes simplex.
Human Immunodeficiency Virus

In the acute stage, HIV can present with a transient viral exanthem not unlike other viral exanthems. More
commonly, the cutaneous manifestations in HIV are related to immunocompromise and opportunistic infections.
Mucocutaneous candidiasis, severe seborrheic dermatitis, eosinophilic pustular folliculitis, and lichenoid
dermatitis of AIDS are some of the manifestations (152). In addition, a range of persistent infections including
fungal infections, prolonged varicella, severe cases of herpes zoster, herpes gingivostomatitis, verrucae or
condyloma acuminatum, and molluscum contagiosum can be seen in HIV-infected patients (79).
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Fungal Infections
Fungal infections of the skin can be classified as superficial and deep forms.
Superficial fungal infections of the skin include dermatophytosis (Tinea) typically caused by three genera,
namely, Trichophyton, Microsporum, and Epidermophyton. In addition, Pityrosporum and Candida can also
cause superficial fungal infections of the skin.
Tinea capitis is a fungal infection of the scalp and hair that is common in prepubertal children and is most often
caused by Tinea tonsurans in the United States (57). Tinea capitis presents as one or more scaly patches of
alopecia (167). With some species, such as T. verrucosum or Microsporum canis, large boggy swellings called
kerion can develop in the infected areas. Tinea corporis is also common in children and characteristically
presents with annular scaly lesions with an active inflammatory border (ringworm). The lesions can be seen
anywhere on the body.
Tinea versicolor caused by Pityrosporum ovale involves upper trunk with areas of brownish discoloration that
later appear hypopigmented.
Primary cutaneous infection with Candida is often seen in the diaper area of infants and characteristically
presents as an eczematous dermatitis. Oral candidiasis (thrush) is not uncommon in infants, especially those
born to HIV-positive mothers. The diagnosis of superficial fungal infections is best accomplished by
demonstration of the organism by culture. KOH preparation offers a rapid method of diagnosis if the organism
can be demonstrated. A biopsy of the lesion and demonstration of the organism is another reliable method of
establishing diagnosis (1).
Histologically, dermatophytoses generally show mild nonspecific superficial perivascular inflammation and
occasionally subcorneal neutrophilic pustules. Fungal hyphae, best seen with PAS stain, are present in the
cornified layer in tinea corporis, and within the cornified layer as well as the follicle and hair shaft in tinea capitis
(Figure 25-29). In kerion celsi, a marked, mixed inflammatory response with formation of dermal abscesses is
seen. A granulomatous response to disrupted hair shafts may also be present.
FIGURE 25-29▪A,B: Tinea capitis showing involvement of a hair follicle and shaft by numerous spores.
Histologic sections from a biopsy of pityriasis versicolor show minimal inflammatory reaction. However, the short
nonbranching hyphae and spores of Malassezia are easily identified within the cornified layer, even on
hematoxylin and eosin-stained sections.
Deep mycosis can be primarily a cutaneous fungal infection with a propensity to involve deeper tissues or be
part of systemic infections such as those involving the respiratory system or reticuloendothelial system. Primary
subcutaneous mycoses often caused by saprophytic organisms include sporotrichosis, chromoblastomycosis,
histoplasmosis, coccidioidomycosis, blastomycosis, and cryptococcosis. Most of these infections manifest with
suppurative and granulomatous inflammation of the dermis and subcutis, with a frequent pseudoepitheliomatous
epidermal hyperplasia. PAS and silver stains often reveal the characteristic morphology of the fungal organism
(Figure 25-30). Deep mycosis may be part of a systemic infection, especially in immunocompromised children.
Necrotizing skin lesions with vasculitis and granulomas can be seen with disseminated aspergillosis,
mucormycosis, and fusarial infection. A deep necrotizing process in the subcutis should alert to a deep
angioinvasive infection.
Infestations
Scabies is a highly contagious pruritic papular vesicular and pustular eruption caused by Sarcoptes scabiei (88).
Children are often affected with rapid spread through person-to-person contact. The adult female mite lays eggs
within burrows in the superficial epidermis, most commonly involving the soles, wrists, interdigital spaces, thenar
eminences, and genitalia. Erythematous papules and pustules with intense pruritus and multiple excoriations
characterize the clinical presentation.
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A positive diagnosis can be made by scraping a burrow and examining the scrapings under a drop of mineral oil.
A more aggressive approach is to biopsy a suspected lesion. Histologic sections show a superficial and deep
perivascular mixed inflammatory cell infiltrate with frequent eosinophils suggestive of a hypersensitivity reaction.
A definite diagnosis can be made only when the mite or eggs of S. scabiei are identified within the parakeratotic
cornified layer.
FIGURE 25-30▪Blastomycosis. A: Pseudoepitheliomatous hyperplasia with suppurative and granulomatous

inflammation. Broad-based budding yeast forms of blastomycosis can be seen within the cytoplasm of
multinucleated giant cells or within the microabscesses, or both. (Hematoxylin and eosin stain, original
magnification ×400.) B: GMS stain highlights the characteristic broad based budding yeast form.
NONINFECTIOUS INFLAMMATORY DERMATOSES

Acute Febrile Neutrophilic Dermatosis
Acute febrile neutrophilic dermatosis (Sweet syndrome) is generally a disease of adults characterized by fever,
leukocytosis, violaceous plaquelike lesions on the face, trunk, and extremities, and a diffuse dermal neutrophilic
infiltrate without vasculitis (42). Although the condition is rare, several reports of Sweet syndrome in children
have been documented (89, 96), and as in adults, many of these cases are associated with underlying
malignancies or inflammatory diseases, which most often dictate the overall clinical prognosis.
Eosinophilic Cellulitis
Eosinophilic cellulitis, or Well syndrome, originally described in adults, is a rare, recurrent inflammatory
dermatosis of uncertain pathogenesis. Cases of eosinophilic cellulitis have been reported in children (8) in
association with various precipitating events such as viral infections and insect bites. A possible genetic factor is
also suggested. Histologic features include a dense diffuse dermal infiltrate of eosinophils. Foci of collagen
degeneration deposited with eosinophilic granules, referred to as flame figures, may be present.
Neutrophilic Eccrine Hidradenitis and Idiopathic Palmoplantar Hidradenitis

Neutrophilic eccrine hidradenitis is a generally self-limiting inflammatory dermatosis often seen in association
with chemotherapy for various malignancies. Neutrophilic eccrine hidradenitis has been reported in children in
association with chemotherapy for non-Hodgkin lymphoma and acute myelogenous leukemia (106). The
condition is characterized by the appearance of numerous erythematous papules and plaques on the trunk and
extremities within several weeks of beginning chemotherapy. Histologic sections show a dense neutrophilic
infiltrate within and around the coils of eccrine glands and ducts.
Idiopathic palmoplantar hidradenitis was first described by Stahr et al. (186) primarily in otherwise healthy
children and young people. It is characterized by the abrupt onset of tender erythematous papules, plaques, and
nodules on the soles and, less often, palms of young patients. The ages of the patients range from 1.5 to 15
years, with increased prevalence in autumn and spring (163, 179). Skin biopsy shows a dense neutrophilic
infiltrate with abscess formation in and around the eccrine coil. However, special stains and microbiologic
cultures are generally negative for organisms. The presence of neutrophilic abscesses and absence of
squamous syringometaplasia in idiopathic palmoplantar hidradenitis aid in differentiation from neutrophilic
eccrine hidradenitis. There is complete resolution of the lesions within 2 to 3 weeks with supportive therapy
alone. Approximately 50% of the patients may experience a relapse that resolves spontaneously (181).
Pyoderma Gangrenosum
Pyoderma gangrenosum is an uncommon idiopathic ulceronecrotic skin disease that can present in children 4%
to 5% of the time (22). A systemic illness, most often inflammatory
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bowel disease or hematologic disorder, is present in 50% to 74% of the patients (130). As in adults, the lower
extremities are often involved. In addition, head and neck and anogenital areas appear to be more commonly
involved in infants and children. A typical lesion of pyoderma gangrenosum consists of ulceration with a necrotic
center, mucopurulent exudate, a violaceous undermined border, and an erythematous periphery. Histologic
findings are nonspecific and vary depending on the area biopsied from a typical ulcer with necrosis and
neutrophilic abscesses in the center of the lesion to endothelial swelling, fibrinoid necrosis, thrombosis, and
extravasated red cells and a lymphocytic infiltrate at the erythematous periphery. Biopsy of the undermined edge
shows mixed inflammatory cell infiltrate and early neutrophilic abscesses. A lymphocytic or a leukocytoclastic
vasculitis was observed at the border of the lesion by some authors (199). Pyoderma gangrenosum is one in the
family of neutrophilic dermatoses.
NONINFECTIOUS GRANULOMATOUS DERMATOSES

Granulomatous reaction may be seen in response to a variety of agents including infections, foreign body, and
degenerative changes of collagen. In some cases, as in sarcoidosis, the inciting agent is not apparent. An
infectious process should be appropriately excluded in all cases. Some granulomatous reactions such as those
seen in response to ruptured cyst contents, degenerating collagen (necrobiotic granulomas), and sarcoidosis are
so characteristic that a specific diagnosis can usually be rendered.
Granuloma Annulare
Granuloma annulare is a benign disorder of unknown etiology associated with degenerated collagen and is often
seen in children. It is characterized by a single or multiple asymptomatic ringed papules most commonly on the
dorsa of hands and feet and often mistaken for tinea.
Histologic findings are diagnostic of granuloma annulare. They consist of zones of degeneration of collagen
within the upper half of the dermis, sometimes with mucinous deposits. These zones are surrounded by
histiocytes arranged in a palisade (Figure 25-31). Perivascular lymphocytic infiltrates may also be present. A
subcutaneous form of granuloma annulare, also known as pseudorheumatoid nodule, is more commonly seen in
children than adults. This form presents commonly on the pretibial area or lower legs and head and neck as
asymptomatic deep dermal or subcutaneous nodules. Histologic sections show large foci of myxoid degeneration
of collagen surrounded by palisades of histiocytes within the deep dermis and subcutaneous tissue (187).
Although mucinous degeneration rather than fibrinoid degeneration of the collagen and the absence of arthritis
help differentiate subcutaneous granuloma annulare from rheumatoid nodule, this distinction is not always
possible. However, the majority of patients with granuloma annulare show no serologic evidence of IgM
rheumatoid factor. Deep granuloma annulare in a child may present as a soft tissue tumor.
FIGURE 25-31▪Granuloma annulare as an upper dermal granuloma showing central myxoid degeneration of the
collagen surrounded by a palisade of histiocytes. (Hematoxylin and eosin stain, original magnification ×200.)
Although an association of granuloma annulare with systemic diseases such as diabetes, lymphoma, and other
malignancies and sarcoidosis has been suggested in adult patients, most of the children are otherwise healthy
and progression to systemic disease of any kind is not common (62). Rare cases of underlying immune defects,
such as IgA-IgG2 deficiency, have been reported in children (113). The clinical course of granuloma annulare is
spontaneous regression with occasional recurrences.
Necrobiosis Lipoidica
Necrobiosis lipoidica is a degenerative disease of the dermal collagen often seen in association with diabetes. It
is a disease of young adults and is rarely reported in children (148, 197). Clinically, it is characterized by oval
plaques, most commonly on the shins. The center of the plaque may later become atrophic with a distinctive
yellow waxy hue. Histologic sections show a palisading granulomatous inflammation surrounding zones of
degenerated collagen. The process may involve the entire dermis and extend up to the subcutaneous fat.
Plasma cells are a frequent component of the inflammatory cell infiltrate. Late lesions show marked sclerosis and
deposits of fat in the epidermis.
Rheumatoid Nodule
Juvenile rheumatoid arthritis is a chronic debilitating disease of childhood. Classic rheumatoid nodules are,
however, uncommon in this form of rheumatoid arthritis. Rheumatoid nodules occur as subcutaneous nodules
over the extensor surfaces. Histologically, the lesions are characterized by palisading granulomas surrounding
large zones of fibrinoid degeneration of collagen. These lesions occur in patients with rheumatoid arthritis and
elevated rheumatoid factors. Similar lesions occurring in the absence of rheumatoid
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arthritis and more commonly seen in children and referred to as pseudorheumatoid nodule most likely represent
subcutaneous granuloma annulare (60).
Sarcoidosis
Sarcoidosis is a multisystem disorder characterized by noncaseating granulomas (Chapters 12 and 22). Although
rare, sarcoidosis can be seen in children younger than the age of 15 years (132). Skin involvement is seen in
approximately a 25% of the patients with sarcoidosis and up to 50% of patients have eye involvement. A
subgroup of childhood sarcoidosis manifests in preschool children younger than 6 years of age with skin, joint,
and eye involvement without any pulmonary lesions, which may be confused with juvenile rheumatoid arthritis
(178). The cutaneous lesions of sarcoidosis are red to yellow or violaceous papules and plaques that, on
histologic examination, show typical noncaseating epithelioid granulomas with little or no necrosis, similar to
lesions seen in other organs. Sarcoidosis must be differentiated from infectious conditions particularly
mycobacterial and deep fungal infections (124).
PANNICULITIS
Inflammation of the fat may predominantly involve either the lobules of the fat, that is, lobular panniculitis, or the
fibrous septae, that is, septal panniculitis. It is important to recognize that, on histologic examination,
considerable overlap may exist. Panniculitis may be a manifestation of underlying systemic disease, most notably
connective tissue diseases such as lupus, dermatomyositis, polyarteritis nodosa, and juvenile rheumatoid
arthritis. The histologic changes are typical and diagnostic in some entities, like erythema nodosum, whereas in
others, they are nonspecific and require extensive clinical, microbiologic, and often serologic support.
Erythema Nodosum
Patients with erythema nodosum present with sudden onset of symmetric, tender, erythematous subcutaneous
nodules on the extensor aspects of lower legs. A prodrome of sore throat and respiratory symptoms may be seen
in some children. Histologic sections that contain subcutaneous fat show a predominantly septal pattern of
inflammation with acute and chronic inflammation and thickening of the septae with some involvement of the
periphery of lobules. In older lesions, granulomatous inflammation with multinucleated giant cells may be present
(Figure 25-32). Necrosis within the granulomatous foci should prompt a search for microorganisms. Overt fat
necrosis and vasculitis are uncommon findings.
Erythema nodosum-like reaction patterns can be seen in a variety of infections including tuberculosis,
streptococcal infection, histoplasmosis, coccidioidomycosis, and occasionally mumps. Another well-recognized
association is with inflammatory bowel disease.
FIGURE 25-32▪Erythema nodosum with a septal pattern of panniculitis and marked fibrous thickening of the
septa and granulomatous inflammation. (Hematoxylin and eosin stain, original magnification ×200.)
In children, erythema nodosum is a self-limiting disease, with resolution of the lesions occurring within a few
weeks. Elimination of the precipitating factor and treatment of infection, if identified, is generally sufficient (137).
Subcutaneous Fat Necrosis of the Newborn

Subcutaneous fat necrosis of the newborn is a relatively uncommon, painless, self-limiting disease that affects
fullterm and post-term infants (68). It manifests at 1 to 6 weeks of age as asymptomatic, firm nodules on cheeks,
shoulder, back, buttocks, and thighs.
Histologic sections show a predominantly lobular involvement with foci of fat necrosis and infiltration by
macrophages and multinucleated giant cells. Within the cytoplasm of the macrophages and giant cells, lipid is
present as needleshaped crystals arranged in a radial array (Figure 25-33). Deposits of calcium may be seen.
The etiology of subcutaneous fat necrosis is largely unknown. Maternal factors and obstetric trauma are
implicated in some cases. Spontaneous resolution of the lesions occurs within the first few months of life.
Hypercalcemia is a rare complication, which if present, should be actively treated (123).
Sclerema Neonatorum
Sclerema neonatorum is a rare, rapidly spreading, diffuse hardening of the subcutaneous tissue of back,
shoulders, and buttocks usually affecting premature, ill newborns. Histologic features include diffuse involvement
of fat lobules by fat cells containing radially arranged crystals of lipid. Inflammation is minimal or absent, a feature
that histologically distinguishes sclerema neonatorum from subcutaneous fat necrosis of newborn (209). The
prognosis is generally poor, with a fatal outcome. In a case control study of neonates with sepsis and sclerema,
exchange transfusion has been shown to improve survival (164).
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FIGURE 25-33▪Subcutaneous fat necrosis. A: Lobular pattern of panniculitis with lymphohistiocytic infiltrate. B:
Multinucleated histiocytes contain characteristic needle-shaped crystals of lipid. Histologic differential diagnosis
includes sclerema neonatorum. [Hematoxylin and eosin stain, original magnification ×200(A), ×400(B).]
VASCULITIS
Cutaneous vasculitis may be a primary disorder, but more commonly, it is a manifestation of an underlying
systemic disease such as collagen vascular disease. A simple classification of vasculitis considers the type of
blood vessel involved, namely, capillary, venule, or artery, and the type of inflammatory cell infiltrate involved,
namely, lymphocytes and neutrophils. Although there is some debate regarding the actual classification
schemes, it is generally agreed that the minimum criteria for the diagnosis of vasculitis include demonstration of
actual damage to the vessel wall in the form of fibrinoid necrosis, a perivascular inflammatory cell infiltrate and
red cell extravasation.
Leukocytoclastic Vasculitis
Henoch-Schönlein Purpura
Henoch-Schönlien purpura, a form of leukocytoclastic vasculitis, is the most common type of vasculitis seen in
children (75, 169) following streptococcal upper respiratory infection, with a peak incidence between 4 and 8
years of age. In addition to palpable purpura on buttocks and lower extremities, affected children often have
arthralgias and arthritis, abdominal pain, and hematuria. A skin biopsy is of great value in the diagnostic workup
of these patients. Histologic features typical of leukocytoclastic vasculitis are usually present and include
superficial perivascular infiltrates of neutrophils, neutrophilic nuclear dust (leukocytoclasia), and extravasated red
blood cells (Figure 25-34). The vessels show endothelial swelling and deposits of fibrin within the walls. IF
studies are of help in differentiating other causes of leukocytoclastic vasculitis from Henoch-Schönlein purpura.
Deposits of IgA in association with C3 and fibrinogen are present within the vessel walls (207).
Henoch-Schönlein purpura is a self-limiting immune complex disorder, with complete resolution occurring within 6
to 16 weeks (23).
Leukocytoclastic vasculitis may be seen secondary to infections due to either direct invasion of vessels or
immunemediated mechanisms. Meningococcal infection is a frequent cause of infectious leukocytoclastic
vasculitis in children (53), in whom meningococci can be found within the endothelial cells and neutrophils.
Leukocytoclastic vasculitis can also be seen in association with autoimmune diseases and secondary to use of
certain drugs. An unusual variant of leukocytoclastic vasculitis, acute hemorrhagic edema of childhood
(Finkelstein disease) generally affects children younger than of 3 years of age (77) and has many similarities to
Henoch-Schönlein purpura (44). However, the lesions are larger and not associated with systemic symptoms.
IgA may not be present by IF studies. Leukocytoclastic vasculitis in children has a relatively benign course,
especially in those cases associated with infection and drugs.
Lymphocytic Vasculitis
A histologic diagnosis of lymphocytic vasculitis with authentic vascular damage and infiltration of the vessel walls
with lymphocytes is only rarely documented. A lymphocytic vasculitis may be seen in insect bite reactions,
PLEVA, lymphomatoid papulosis, and collagen vascular diseases. Lichen aureus and Schamberg-Majocchi
purpura represent benign pigmented purpuras characterized by chronic petechiae in legs and elsewhere.
Histologically, there is a superficial perivascular lymphocytic infiltrate and extravasated red cells. In older lesions,
hemosiderin-laden macrophages may be seen that give the characteristic pigmented appearance. The lesions
are asymptomatic and may last for months to years.
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FIGURE 25-34▪Henoch-Schönlein purpura. A: Superficial perivascular and interstitial infiltrate of neutrophils and
extravasated red cells. B: Neutrophils, extravasated red blood cells, and neutrophilic dust are present. Fibrinoid
necrosis of the vessel wall is seen only in later lesions. [Hematoxylin and eosin stain, original magnification
×200(A), ×400(B).]
Other rare causes of childhood vasculitis in children include polyarteritis nodosa, Wegener granulomatosis, and
Churg-Strauss syndrome, which can rarely present with cutaneous symptoms (47, 177). Some cases are a
manifestation of a drug reaction.
FOLLICULITIS AND PERIFOLLICULITIS

Acne
Acne is a common cause of visits to the physician’s office, especially among adolescents. Acne vulgaris is most
common on the face and anterior and posterior trunk, where there are abundant sebaceous glands that produce
more sebum in response to androgens. The primary lesion is intrafollicular hyperkeratosis, together with
collection of desquamating cells and sebum with subsequent obstruction of the follicular infundibula leading to
the formation of a comedone. Open comedones (blackheads) appear as large pores with central black-brown
cores. Closed comedones (whiteheads) are often associated with inflammation and rupture to produce a pustule
or nodule when the inflammation is deep. Nodulocystic acne and acne conglobata are severe expressions of
acne vulgaris, whereas neonatal acne is a transient eruption in newborns secondary to maternal and infant
androgen response (17). Persistent acne in an infant should raise suspicion of a possible androgen-producing
neoplasm (100).
Eosinophilic Pustular Folliculitis

Eosinophilic pustular folliculitis, or Ofuji disease, originally described in healthy Japanese adults, can present in
infancy with white-yellow pustules on the scalp and upper forehead (52). However, it is more likely that this is
simply a histologic pattern that can be seen in association with a variety of diseases including scabies and insect
bite reactions rather than a specific entity (211).
Histologic findings include eosinophilic spongiosis, subcorneal pustule with eosinophils, and a dense
perifollicular inflammation with frequent eosinophils. Microbiologic cultures are generally negative for organisms.
The eruption resolves without scarring. Eosinophilic pustular folliculitis may occur in association with HIV
infection and other immunocompromised states.
SYSTEMIC DISEASES WITH CUTANEOUS MANIFESTATIONS

Collagen Vascular Diseases
Lupus erythematosus is the most common collagen vascular disease to present in childhood, followed by
juvenile rheumatoid arthritis and dermatomyositis (175). Polyarteritis nodosa, scleroderma, and other collagen
vascular diseases are less common.
Lupus Erythematosus
Although all forms of lupus can affect children, systemic lupus erythematosus is the most common form.
Childhood systemic lupus erythematosus peaks in early adolescence, with about 60% of cases occurring
between the ages of 11 and 15 years. Cutaneous manifestations are the second most frequent finding (77%)
next to renal involvement (84%) in pediatric patients with systemic lupus erythematosus. Discoid lupus
erythematosus without clinical serologic evidence of systemic disease can occur rarely in children (166).
However, discoid lupus erythematosus may be a part of systemic lupus erythematosus syndrome. Cutaneous
changes of lupus erythematosus include
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malar rash, oral ulcerations, photosensitivity, alopecia, and discoid lupus erythematosus (92).
Neonatal lupus erythematosus is seen in newborn infants born to anti-Ro (SS-A) antibody-positive mothers
(117), with the development of skin lesions and or heart block at birth to 2 months of age (134). The skin lesions
consist of erythematous, nonscaling, sharply demarcated lesions with a predilection for involvement around the
eyes and sometimes annular polycyclic type of lesions commonly seen in subacute cutaneous lupus
erythematosus.
Sections from early lesions of systemic lupus erythematosus corresponding to the erythematous malar rash show
only nonspecific changes. The histologic changes seen in well-established systemic lupus erythematosus,
subacute cutaneous lupus erythematosus, neonatal lupus erythematosus, and discoid lupus erythematosus are
essentially similar, varying only in degree. The characteristic changes are those of interface dermatitis with
marked vacuolar alteration of the basal cell layer, where there is also a lymphocytic infiltrate that obscures the
dermoepidermal junction. Additional findings include hyperkeratosis with epidermal atrophy and follicular
plugging, most prominent in discoid lesions, and perivascular and periadnexal lymphocytic infiltrate. A thickened
basement membrane, best seen with PAS stain, and separation at the dermoepidermal junction are seen in older
lesions (Figure 25-35). Interstitial dermal mucin is also seen. Direct IF reveals a continuous granular deposit of
C3, IgG, and occasionally, IgM along the dermoepidermal junction in involved and uninvolved skin in systemic
lupus erythematosus and only in involved skin in discoid lupus erythematosus.
Neonatal lupus erythematosus is a transient disorder, and prognosis is generally good in the absence of heart
block (158). Some of these infants may develop systemic lupus erythematosus as young adults. The prognosis
in childhood systemic lupus erythematosus, like that in adults, has improved with aggressive therapy. Renal
complications generally dictate the survival (see Chapter 17).
FIGURE 25-35▪Lupus erythematosus: mild hyperkeratosis, atrophy of the epidermis with vacuolar alteration of
the basal cell layer, smudging of the basement membrane and interface dermatitis extending around the hair
follicle. (Hematoxylin and eosin stain, original magnification ×400.)
Scleroderma or Progressive Systemic Sclerosus

Scleroderma or progressive systemic sclerosus in children shows a significantly less frequent involvement of all
organs, a higher prevalence of arthritis and myositis, and a better outcome than in adults (213). The localized
form of scleroderma or morphea is a disease of children and young adults. It can present as plaque, linear,
guttate, or generalized forms. Histologic findings vary according to the duration of the lesion. In active lesions,
there is a superficial and deep perivascular and interstitial lymphocytic infiltrate that extends into the
subcutaneous tissue associated with thickened collagen bundles. In older lesions, the inflammatory component is
mild or absent, and hyalinized collagen bundles replace the entire dermis and extend into the septa of the
subcutaneous fat (Figure 25-36). The prognosis of morphea is generally good, with the lesions healing with
atrophy and eventual cessation of new lesions occurring. Sclerodermoid skin changes may occur in chronic
graft-versus host disease.
GRAFT-VERSUS-HOST DISEASE
Graft-versus-host disease is a response seen in immunocompromised hosts to immunocompetent donor cells. In
children, this is most often seen as a complication of hematopoietic stem cell transplantation in the treatment of
acute leukemia or following a nonirradiated blood transfusion in an immunocompromised infant (99). The
cutaneous findings of acute graft-versus-host disease include a pruritic maculopapular
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eruption, which can become exfoliative. The histologic findings in acute graft-versus-host disease closely
resemble those of erythema multiforme and TEN, and consist of vacuolar alteration of the basal cell layer with
necrotic keratinocytes, some of which are surrounded by lymphocytes, the so-called satellite necrosis (Figure 25-
37). In severe cases, there is marked epidermal necrosis with formation of subepidermal bullae. A subacute
lesion of graft-versus-host disease resembles lichen planus, with a dense bandlike lymphocytic infiltrate that
obscures the dermoepidermal junction. In the chronic form, the histologic changes closely resemble those of
scleroderma, with hyalinization of collagen bundles. Vacuolar alteration at the basal cell layer and satellite
necrosis, if present, may help in the differential diagnosis in all stages.
FIGURE 25-36▪Scleroderma showing the characteristically rectangular biopsy with dense dermal sclerosis and
thickening of the septae in the subcutaneous fat. (Hematoxylin and eosin stain, original magnification ×200.)
FIGURE 25-37▪Acute graft-versus-host disease showing vacuolar alteration of the basal cell layer with scattered
“apoptotic” keratinocytes surrounded by few lymphocytes. The changes are those of an interface dermatitis.
The prognosis for graft-versus-host disease is generally good when the disease is localized to skin alone. Early
treatment may be of help in preventing joint contractures and disability associated with chronic graft-versus-host
disease. In recent reports, a significantly higher mortality was observed in children with sclerodermatous graft
versus host disease (194). The differential diagnosis includes host lymphocyte recovery and drug reaction in the
first 30 days.
METABOLIC DISORDERS
Calcinosis Cutis
Cutaneous calcifications may be of the localized dystrophic type or systemic metastatic type. One type of
localized calcinosis is subepidermal calcific nodules seen on the heels of infants following repeated heel sticks
(Figure 25-38). Idiopathic subepidermal calcific nodules can be seen at birth (84). Calcinosis may be a
manifestation of systemic disease such as dermatomyositis and, rarely, scleroderma and renal failure (193).
Tumoral calcinosis seen around joint areas is mainly a disease of children which presents as a soft tissue tumor.
Other metabolic diseases like amyloidosis, porphyrias, and mucinoses can involve the skin but are not common
in pediatric age groups.
FIGURE 25-38▪Subepidermal calcified nodule at the site of a heel stick. (Hematoxylin and eosin stain, original
Mucopolysaccharidoses are lysosomal enzyme deficiency disorders that manifest with abnormal accumulations
of mucopolysaccharides in many organs including the skin. In all types of mucopolysaccharidoses, the skin may
appear thickened and inelastic. Biopsy sections stained with Giemsa stain show metachromatic granules within
fibroblasts. By electron microscopy, membrane-bound finely granular deposits can be seen in the cytoplasm of
fibroblasts (Figure 25-39) (see Chapter 5).
CYSTS, NEOPLASMS, AND HAMARTOMAS

Epidermal Nevi
Epidermal nevi are proliferations of epidermal keratinocytes that present as rough-surfaced lesions at birth or
shortly thereafter. Clinically, they may be localized to palms and soles, widespread or segmental in distribution.
Clinical patterns of expression include linear, zosteriform, and whorled. Histologic sections usually show a single
pattern throughout the lesion. Various histologic patterns that are seen in epidermal nevi include epidermolytic
hyperkeratosis, focal acantholytic dyskeratosis, verrucous hyperkeratosis, inflammatory linear verrucous
epidermal nevus, seborrheic keratosis-like (Figure 25-40), and veruciform xanthomatosis (190). Abnormally
formed pilosebaceous units, often in excess numbers, are the features of nevus sebaceus of Jadassohn.
Epidermal nevi are generally stable benign lesions, and transformation to benign or malignant neoplasms is rare
but well described in adults. Some epidermal nevi are associated with extracutaneous manifestations, notably of
the central nervous, skeletal, and renal systems, and are called epidermal nevus syndromes that are likely due
to specific genetic defects (162, 191).
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FIGURE 25-39▪A: Hurler syndrome showing a skin biopsy specimen with questionable increase in dermal
metachromasia. (Toluidine blue stain, original magnification ×400.) B: Electron microscopy of fibroblasts,
endothelial cells, and macrophages disclosed numerous membrane-bound vacuoles, some with granular and
lamellar electron-dense contents. (Uranyl acetate and lead citrate stain, original magnification ×40,000.)
Non-neoplastic epithelial cysts are among the most common tumorous lesions seen in children.
Epidermal Inclusion Cyst

Epidermal inclusion cyst or keratinous cyst presents as a single firm dermal nodule that shows a keratinizing
stratified squamous epithelial-lined cyst filled with laminated keratin or pilar keratin in the case of a tricholemmal
cyst. Multiple epidermal inclusion cysts are seen in Gardner syndrome (71). Milia that can be seen occasionally
on the face of a neonate are small epidermal inclusion cysts. Milial cysts are not associated with a formed hair
follicle.
Dermoid Cyst
Dermoid cysts are developmental in origin and arise along lines of embryonic suture closures. Common sites of
involvement are the periorbital region, midline of nose, scalp, and anterior neck (154). Dermoid cysts are lined by
keratinizing squamous epithelium. In contrast to epidermal inclusion cysts, the lining also contains
folliculosebaceous and apocrine units, and sebum and hair are seen in addition to laminated keratin in the cyst
contents. There is a resemblance to a steatocystoma. Simple excision is the treatment of choice. Midline dermoid
cysts may be accompanied by a sinus tract and should be evaluated radiologically before surgery.
FIGURE 25-40▪Epidermal nevus showing hyperkeratosis and papillomatous epidermal hyperplasia. Note that the
adnexal structures are normal. (Hematoxylin and eosin stain, original magnification ×200.)
Eruptive Vellus Hair Cyst

Eruptive vellus hair cysts occur as multiple soft asymptomatic follicular papules of sudden onset in children and
young adults. The sites of predilection are anterior chest, extremities, face, neck, and posterior trunk. Histologic
sections show a squamous epithelial-lined cyst filled with laminated keratin and numerous hairs cut transversely
and obliquely. An autosomal dominant developmental abnormality of vellus hair follicles is believed to be the
underlying etiology.
Steatocystoma Multiplex
Steatocystoma multiplex is an autosomal dominant disorder seen as multiple small cystic lesions most commonly
in the axillae, sternal region, and on the arms. The cysts are lined by stratified squamous epithelium, with only
two to three cell layers and covered with a thick homogeneous eosinophilic cuticle (Figure 25-41). Flattened
sebaceous lobules can be
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seen in the vicinity. It is generally believed that eruptive vellus hair cysts and steatocystoma multiplex are
variable expressions of the same disorder with overlapping clinical and histologic features (39, 146).
FIGURE 25-41▪Steatocystoma: cyst lined by stratified squamous epithelium with only two to three cell layers and
a thick homogeneous eosinophilic cuticle. (Hematoxylin and eosin stain, original magnification ×200.)
ADNEXAL TUMORS
Adnexal tumors occur in children less commonly than in adults. Of the adnexal tumors, tumors with follicular
differentiation account for the majority. Pilomatrixoma, also known as calcifying epithelioma of Malherbe, is
perhaps the most common adnexal neoplasm seen in the pediatric age group (41). Pilomatrixomas present with
increased frequency in the first and the sixth decades, with the head and neck area being the most common site.
Clinically, they present as a hard dermal or a subcutaneous nodule (103). Familial occurrences and multiple
lesions are documented (12, 155).
Histologic changes follow a distinct chronologic sequence. Early lesions begin as cystic structures lined by
matrical and supramatrical cells similar to those in the bulb of normal hair follicles. As the cells mature, the nuclei
disappear and leave ghosts of completely cornified cells, or the “shadow cells.” Fully developed lesions show
irregularly shaped and sized lobules of matrical and supramatrical cells. Each lobule shows maturation toward
the center in the form of masses of “shadow cells” (Figure 25-42). With time, the lesion shows signs of
regression in the form of less apparent or even absent peripheral epithelial elements and consists mostly of the
shadow cells, which may be surrounded by granulation tissue and granulomatous inflammation. Late lesions
show no epithelial component and consist only of masses of cornified cells with extensive calcification and
occasionally ossification (3).
At all times, the benign nature of the neoplasm is apparent from the sharp circumscription seen at the periphery.
In early lesions, mitotic figures may be frequent in keeping with the proliferative phase of the neoplasm and do
not imply malignancy.
FIGURE 25-42▪Pilomatrixoma is a well-circumscribed cystlike lesion with proliferation of basaloid cells that
cornify in a peculiar pattern resulting in formation of shadow or ghost cells. (Hematoxylin and eosin stain, original
Trichoepithelioma often presents as solitary, flesh-colored papules occurring on the face. Less commonly, it
presents as multiple lesions, transmitted as an autosomal dominant disorder.
Histologically, the silhouette is that of a benign neoplasm composed of germinative cells embedded in a cellular
fibrocytic stroma. The germinative cells can be arranged as nodules or cribriform and retiform patterns, and are
usually encircled by mesenchymal cells like those of the embryonic perifollicular sheath. Infundibulocystic
structures filled with cornified cells may be prominent trichoblastoma, a less differentiated follicular neoplasm
composed of germinative cells, is another expression of trichoepithelioma.
Eccrine Neoplasms
Syringoma is a relatively common adnexal neoplasm that differentiates toward the acrosyringium of the eccrine
duct. It is seen in children with greater frequency in association with trisomy 21 syndrome (172). It can present as
a sudden onset eruption of small papules, usually on the face and sometimes on the vulva (72, 183).
Histologically, the lesions are characterized by multiple, small epithelial structures that may be solid or tubular.
The tubular structures may contain granular material within the lumina. Some of the epithelial nests may have
elongated or tadpole-like shapes. An important histologic feature is the confinement of the neoplasm to the upper
half of the dermis, a feature helpful in distinguishing syringoma from microcystic adnexal carcinoma, especially in
adults.
Other eccrine neoplasms such as eccrine poroma and eccrine acrospiroma occur infrequently in children.
Sebaceous and apocrine neoplasms: True sebaceous and apocrine neoplasms are uncommon in children.
Nevus sebaceus of Jadassohn is a hamartoma that contains most elements of normal skin and subcutaneous fat
and is best designated as an organoid nevus. Nevus sebaceus commonly occurs as a yellowish round-to-oval
hairless plaque on the scalp, forehead, and lateral portions of the face. The clinical and histologic appearances
vary considerably and follow a chronologic sequence. The yellowish pebbly appearance of these lesions at birth
corresponds to prominent sebaceous lobules, a result of the effects of maternal hormones.
After infancy, the appearance and development of the sebaceous lobules in the lesions follow the growth of
sebaceous units elsewhere. They are small and the epidermis is flat until puberty, when sebaceou

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EDITORS

Richard M. Conran MD, PhD, JD

Michael K. Fritsch MD, PhD

Jason A. Jarzembowski MD, PhD

Raj P. Kapur MD, PhD

Jochen K. M. Lennerz MD, PhD

Rish K. Pai MD, PhD

Joseph R. Siebert PhD

Mariko Suchi MD, PhD

Christopher R. Weber MD, PhD

THE STANDARD PEDIATRIC AUTOPSY

Table 1A-1 ▪ INSTRUMENTS USED IN PERFORMING THE PEDIATRIC AUTOPSY

b. Medium sized, straight or curved for opening bowel.

c. Large or heavier ones for opening calvarium and vertebral column.

a. Small and medium sized

b. Straight and curved

a. No. 10 size curved for most routine work

b. No. 1 size with pointed tip for delicate cutting

Balances for weighing

a. Standard hanging balance for weighing neonates or small children

b. Electronic balance (accurate to 0.1 g) for weighing organs

FACE, EYES, EARS, MOUTH (EXTERNAL AND INTERNAL)

FIGURE 1A-2 ▪ Fullbody view showing crown-hell measurement.

ARMS, HANDS, FINGERS

CHEST: FRONT AND BACK

ABDOMEN: FRONT AND BACK INCLUDING ANUS/VAGINA, URETHRA

LEGS, FEET, TOES

Special Techniques and Studies

TISSUE FOR CYTOGENETICS

REMOVING THE ORGANS

EXAMINATION OF THE BODY CAVITY

VERTEBRAL COLUMN/SPINAL CORD

SEPARATION AND EXAMINATION OF HEART/LUNG

EXAMINATION OF THE RESPIRATORY SYSTEM

EXAMINATION OF THE ABDOMINAL ORGANS

Central Nervous System

Preparing the Remains for Disposition

Table 1B-1 ▪ ADVANTAGES OF FNA

No general anesthesia necessary

Minimal trauma with little morbidity

Can be used in conjunction with other diagnostic modalities

(e.g., immunocytochemistry, electron microscopy, microbiologic culture, flow cytometry)

Allows pathologist to have direct patient interaction

Table 1B-2 ▪ INDICATIONS FOR FNA

Mass lesion of unknown cause Any tumor >0.5 cm

Alternative to surgery High-risk surgical candidate

Documentation of a nonresectable Large neuroblastoma-tissue diagnosis necessary before

Confirmation of a metastasis Pulmonary lesion in a child with previous Wilms tumor

Support of a clinical diagnosis Persistent lymphadenopathy

Preoperative planning Salivary gland lesion

Table 1B-3 ▪ EQUIPMENT NEEDED FOR FNA

Needles (22- to 25-gauge)

Aspiration gun (optional)

Nonbacteriostatic normal saline solution

Flow cytometry medium with heparin

Specific Molecular Techniques

Table 1C-1 ▪ TYPICALTISSUE REQUIREMENTS FOR COMMONLY USED MOLECULAR DIAGNOSTICTECHNIQUES

Methodology Tissue Format Tissue Amount

Fresh Frozen FFPE

Flow cytometry <1 cm3 ✓