Case Studies

Acute Precursor B-Cell Lymphoblastic Leukemia

in a 1-Year-Old White Male: Diagnostic Evaluation
and Flow Cytometric Analysis

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Jordan M. Davis, BS, MLS(ASCP)CM,1* E. Dayan Sandler, MD 2
Lab Med Summer 2015;46:e82-e87

DOI: 10.1309/LM6CPCIWO67APBYJ

Clinical History Medical History: Treated with amoxicillin the previous week for
tonsillitis.
Patient: 1-year-old white male.
Family History: Mother has history of anemia.
Chief Complaint: Bruising, pallor, and decreased activity.
Physical Examination Results: The patient appeared pale and tired,
History of Present Illness: Bruising had first been noticed on both with dry and pale oral mucosa. Dried blood was noted on the nares
lower extremities 3 weeks before the day he arrived at the hospital bilaterally. The boy was noted to have extensive ecchymosis to the
seeking treatment. The patient experienced a fall at that time that upper and lower extremities, chest, and back; he was also noted to
resulted in a lip laceration, which resolved spontaneously without have moderate petechiae with a similar distribution. No organomegaly or
increased bleeding time. His activity level was normal until the day of lymphadenopathy was present, but mild abdominal distension was noted.
hospital admission, when he was noticed to be somewhat lethargic
and pale. He was brought to the Emergency Department after 2 Keywords: leukemia, acute lymphoblastic leukemia, flow cytometry,
episodes of epistaxis, which resolved spontaneously. Pre-B ALL, precursor, hematology

Questions
1. What are the most significant symptoms of this patient’s
illness? Do the patient’s characteristics when initially
seeking treatment suggest a preliminary diagnosis?

Abbreviations 2. Based on the results of the physical examination of this

CBC, complete blood count; RBC, red blood cell; WBC, white blood cell; patient, which tests would be most useful to narrow his
WHO, World Health Organization; ALL, acute lymphocytic leukemia; diagnosis?
AML, acute myeloid leukemia; B-ALL, B-cell acute lymphocytic
leukemia; TdT, terminal deoxynucleotidyl transferase; MLL, mixed
3. What were the most significant findings associated with
lineage leukemia; CML, chronic myeloid leukemia; FISH, fluorescence
in situ hybridization; HGB, hemoglobin; HCT, hematocrit; MCV, mean this patient’s complete blood count (CBC) and differential?
corpuscular volume; MCH, mean cell hemoglobin; MCHC, mean cell What did the bone marrow biopsy reveal?
hemoglobin concentration; RDW, red blood cell distribution width;
PLT, platelet; MPV, mean platelet volume; HLA-DR, human leukocyte 4. What is the differential diagnosis in this patient, and
antigen–D related; CALLA, common acute lymphoblastic leukemia
antigen which specialty laboratory test(s) would aid in the further
characterization of this illness?
1
Medical Laboratory Science Program University of North Florida,
Jacksonville, FL 5. Which diagnosis is derived from examination of the flow
2
Jacksonville Pathology Consultants PA, Jacksonville, FL
cytometric data?
*To whom correspondence should be addressed.
[email protected] 6. How are acute B-cell leukemias subtyped genetically?

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 Case Studies

Table 1. Peripheral Blood Laboratory Findingsa

Variable Patient Value Reference Range
WBC 101 × 10 /L9
6.00-17.50 × 109/L
RBC 1.52 × 106/μL 3.50-5.00 × 106/μL
HGB 4.1 g/dL 10.5-14.2 g/dL
HCT 13.60% 33.0-39.0%

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MCV 89.6 fL 70.0-86.0 fL
MCH 26.7 pg 23.0-31.0 pg
MCHC 29.8 g/dL 30.0-37.0 g/dL
RDW 17.30% 11.5-15.5%
PLT 8 × 109/L 150-450 × 109/L
MPV 8.6 fL 7.0-11.0 fL
WBC, white blood cell; RBC, red blood cell; HGB, hemoglobin; HCT, hematocrit;
MCV, mean corpuscular volume; MCH, mean cell hemoglobin; MCHC, mean cell
hemoglobin concentration; RDW, red blood cell distribution width; PLT, platelet;
MPV, mean platelet volume
a
Patient is a 1-year-old white male.
Image 1
Peripheral blood smear demonstrating markedly increased white
blood cell (WBC) count (Wright-Giemsa stain; original magnifica-
tion 100x).

Table 2. White Blood Cell Differential in

Peripheral Blooda
Variable Patient Value Reference Range
Neutrophils 0% 15%-35%
Lymphocytes 4% 41%-71%
Monocytes 0% 3%-13%
Eosinophils 0% 0-7%
Basophils 0% 0-2%
Blasts 96% 0%
Patient is a 1-year-old white male.
a

Possible Answers
Image 2
1. The most striking symptoms are the patient’s bruising,
Peripheral blood specimen from our patient, a 1-year-old white
widespread petechiae, and ecchymosis, indicative of a
male, containing L1 lymphoblasts with scant cytoplasm immature
coagulation disorder or significant thrombocytopenia. nuclear chromatin, and 1 to several nucleoli. Marked thrombocy-
Combined with his pallor and lethargy, suggestive of topenia is also evident (Wright-Giemsa stain; original magnifica-
anemia, two or more peripheral blood cytopenias are tion 1000x).

possible. Given the involvement of multiple hematological

lineages, leukemia or aplastic anemia may be implicated a complete blood count (CBC) with differential and
in the diagnosis. The patient’s characteristics on arrival for peripheral blood smear examination will be the most
examination suggest childhood leukemia most strongly;
helpful in narrowing his diagnosis. The health care
however, the boy lacks the lymphadenopathy and/or
professional should pay close attention to the results
splenomegaly sometimes associated with leukemias.
regarding number and size of platelets, as well as any
2. The patient’s characteristics on arrival for examination abnormal red blood cell (RBC) morphologic characteristics
suggest an illness of hematological origin; therefore, and/or the presence of blasts. From this information,

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Image 3 Image 4
Bone marrow aspirate smear from our patient, a 1-year-old white Bone marrow aspirate clot from our patient, a 1-year-old white
male, showing abundant blasts with scant cytoplasm (Wright- male, demonstrating predominant blasts mixed with fibrin and
Giemsa stain; original magnification 1000x). blood (Wright-Giemsa stain; original magnification 100x).

Table 3. Bone marrow Differentiala

Variable Patient Value
Neutrophils and bands 1%
Lymphocytes 0%
Erythroid precursors 2%
Granulocytic precursors 1%
Blasts 96%
a
Patient is a 1-year-old white male.

demonstrating marked leukocytosis (Table 1, Image 1).

The WBC differential revealed 96% blasts in the peripheral
blood, with the remainder of the cells being small mature
lymphocytes (Table 2, Image 2). No abnormal red blood
cell (RBC) morphologic characteristics were observed.
Image 5
Bone marrow aspirate from our patient, a 1-year-old white male, The bone marrow aspirate displays infiltration by blasts
showing abundant blasts with few, if any, residual bone marrow with scant cytoplasm (Image 3, Image 4, Image 5). This
precursors (Wright-Giemsa stain; original magnification 400x). differential reflects that observed in the peripheral blood,
with most of cells being blasts. The few remaining cells
are granulocytic and erythroid precursors, with 1 mature
a differential diagnostic list can be developed and the neutrophil noted in the differential (Table 3). The results of
patient can be assessed for need of bone marrow aspirate this examination strongly suggest leukemia.
evaluation and/or flow cytometric analysis.
4. The patient’s initial symptoms of pallor, lethargy,
3. The CBC results confirm significant anemia, with a petechiae, and bruising may be explained by a combined
hemoglobin count of 4.1 g/dL (Table 1) and a marked thrombocytopenia and anemia suggestive of leukemia or
thrombocytopenia count of 8 × 109/L. Also, the patient’s pediatric bone marrow failure syndrome (Fanconi anemia,
white blood cell (WBC) count is elevated, at 101 × 109/L, Diamond-Blackfan syndrome, etc). Also, these findings

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Figure 1
The results of flow cytometric analysis of specimens from our patient, a 1-year-old white male, show blasts in the R2 gate, which are
dim for CD45 expression and show low side scatter (cytoplasmic complexity). The bright CD45-positive cells are lymphocytes, with
the few remaining cells being granulocytes and their precursors. The blasts have tested positive for B lymphocyte marker CD19 and
biphenotypic for B lymphocyte marker CD20. The cells have tested positive for CD10 (common acute lymphoblastic leukemia antigen
[CALLA]) and terminal deoxynucleotidyl transferase (TdT).

might be explained by the presence of a metastatic Table 4. Immunophenotype by Flow Cytometrya

neuroblastoma, which is known to be among the most
% Cells That Have
common extracranial tumors in childhood and sometimes Variable Cells in Which is Present Tested Positive
involves the bones and bone marrow .1
CD45 (dim) Leukocytes 71
C10 Lymphoid progenitors 68
On review of the complete blood count (CBC) results, the CD19 B cells 70
marked leukocytosis rules out aplastic anemia. Further, CD20 B cells 46
the predominance of blasts in the peripheral blood leads CD22 B cells 70
to a likely diagnosis of leukemia—specifically, acute HLA-DR B cells, activated T cells, monocytes 68
CD5 T cells <1
leukemia, which requires the presence of greater than
CD7 T cells <1
20% blasts according to the classification system of the CD34 Early progenitor cells 24
World Health Organization (WHO).2 The bone marrow CD13 Myeloid cells 6
aspirate, as previously described herein, further lends CD33 Myeloid cells <1
CD14 Monocytes 6
evidence for this diagnosis.
CD36 Monocytes, granulocytes <1
TdT Immature lymphoid cells 60
The next step in characterizing this patient’s illness
HLA-DR, human leukocyte antigen–D related; TdT, terminal deoxynucleotidyl
is to identify the white blood cell (WBC) lineage transferase.
associated with his leukemia (myeloid or lymphoid). The a
Patient is a 1-year-old white male.

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Image 6
Fluorescence in situ hybridization (FISH) studies for several common translocations and fusion genes associated with leukemia, all of
which had negative results in this 1-year-old white male.

morphologic characteristics of the blasts (small, with scant

cytoplasm; Image 2 and Image 3) are suggestive of the
lymphoid lineage and, specifically, of the L1 subtype.3
However, morphologic examination is insufficient to
differentiate acute lymphocytic leukemia (ALL) from
acute myeloid leukemia (AML). Therefore, definitive
diagnosis requires further laboratory testing in the form of
immunophenotyping; this objective may be accomplished
via flow cytometric analysis.

5. The flow cytometry results reveal a population of tumor

cells consistent with a diagnosis of pre-B cell acute
lymphoblastic leukemia (Table 4, Figure 1). The cells are
positive for several lymphoid markers including CD19
and CD22, dimly positive for CD45, and biphenotypically
positive for CD20. CD10 is a common lymphoid progenitor
Image 7
marker, whereas CD19 and CD22 are markers expressed
Cytogenetic analysis shows a nonspecific genotype: 46, XY,-
specifically by B lymphocytes. Lymphoblasts in B-cell
2,?t(2;16)(q11.2;q22), der(14;17)(q10;q10) +2mar for our patient, a
acute lymphocytic leukemia (B-ALL) almost always test 1-year-old white male.
positive for CD10, CD19, and CD22, whereas CD20 and
CD34 expression is often variable, as observed in this
case.4 Terminal deoxynucleotidyl transferase (TdT), a DNA of various genetic abnormalities. Most commonly,
polymerase associated with early lymphoid cells, also this classification refers to the existence of 1 or more
tests positive in this patient. The blasts test negative for balanced chromosomal translocations resulting in fusion
myeloid and monocyte markers such as CD13, CD33, and chromosomes, although chromosome number alterations,
CD14, excluding involvement of leukemia of the myeloid aneuploidy, and even haploidy also may occur.5 After
lineage. identification of the abnormality (or abnormalities) present,
a full risk assessment can be determined, in conjunction
6. The B-cell acute lymphoblastic leukemias are with other factors such as age and white blood cell (WBC)
subtyped genetically based on the presence or absence count, to evaluate treatment options.

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 Case Studies

Some common translocation-based abnormalities include 2. Harris N, Jaffe E, Diebold J, et al. World Health Organization
classification of neoplastic diseases of the hematopoietic and
the mixed lineage leukemia (MLL) rearrangement, a lymphoid tissues: report of the Clinical Advisory Committee
translocation at band 11q23 with any of several potential meeting—Airlie House, Virginia, November 1997. J Clin Oncol.
1999;17(12):3835-3849.
fusion partners.5 Blasts typically test positive for CD19 but
3. Penchansky L. Acute lymphoblastic leukemia and lymphoma. In:
test negative for CD10 and CD24; patients usually exhibit Penchansky L. Pediatric Bone Marrow. Heidelberg, Germany:
high WBC counts of greater than 100 × 109/L. Prognosis Springer-Verlag; 2004:215-245.

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generally is considered poor for patients who exhibit this 4. Borowitz MJ, Chan JKC. B lymphoblastic leukaemia/lymphoma,
not otherwise specified. In: Swerdlow SH, Campo E, Harris NL,
translocation. Another well-characterized translocation et al, eds. WHO Classification of Tumours of Haematopoietic and
is ETV6-RUNX1, t(12;21)(p13;q22). This abnormality is Lymphoid Tissues. 4th ed. Lyon, France: IARC Press; 2008:168-170.

common in children, accounting for approximately 25% 5. Borowitz MJ, Chan JKC. B lymphoblastic leukaemia/lymphoma with
recurrent genetic abnormalities. In: Swerdlow SH, Campo E, Harris
of all B-ALL cases.5 Blasts show a phenotype of CD19 NL, et al., eds. WHO Classification of Tumours of Haematopoietic
and CD10 positivity, but show negativity for markers and Lymphoid Tissues. 4th ed. Lyon, France: IARC Press; 2008:171-
175.
CD9 and CD20; most test CD34 positive. Unlike the MLL
6. Hu Y. Acute lymphoblastic leukemia: genetic events and molecular
rearrangement, the ETV6-RUNX1 translocation has a signatures. Am J Biomed Sci. 2014;6(4):238-253.
favorable prognosis.6 More rare, but still significant, is
the BCR-ABL1 translocation, t(9;22)(q34;q11.2), which
forms the Philadelphia chromosome normally associated
with chronic myeloid leukemia(CML). Blasts typically test
positive for CD10, CD19, and terminal deoxynucleotidyl
transferase (TdT), with accompanying increased
expression of myeloid markers CD13 and CD33 without
involvement of the myeloid lineage. Although uncommon
in childhood B-ALL, the prognosis for t(9;22) patients is
considered to be poor.6

The presence or absence of these genetic abnormalities

is assessed using fluorescence in situ hybridization
(FISH), with probes specific for the DNA sequences of the
genes that may become altered (Image 6). This patient
exhibits a nonspecific genotype, which tests negative for
the translocations previously discussed herein (Image 7)
although still demonstrating an abnormal karyotype.

Acknowledgements
We thank the faculty of the University of North Florida
Medical Laboratory Science program for their commitment
and knowledge, the laboratory staff of Baptist Medical
Center Jacksonville for guidance, support, and friendship,
and Jeffrey Goldstein, MD, for providing high quality
images for this case study.

References
1. Brodeur GM, Seeger RC, Barrett A, et al. International criteria for
diagnosis, staging, and response to treatment in patients with
neuroblastoma. J Clin Oncol. 1998;6(12):1874-1881.

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