Hematology 1 BSMT3-1st Sem

BACHELOR OF SCIENCE IN
MEDICAL LABORATORY
A.Y 2022-2023
COLEGIO SAN AGUSTIN-

BACOLOD
ANGEL KATE DIENTE OLAGUER

THIRD-YEAR COLLEGE (1ST SEMESTER)
HEMATOLOGY
TABLE OF CONTENTS
HEMATOLOGY 1 LABORATORY ENDTERM .................................................................................................. 5

BLOOD SMEAR EXAMINATION: MAKING A BLOOD SMEAR ................................................................ 5
Performing a Manual Differential and Assessing RBC Morphology .............................................. 5
WBC Differential Count ...................................................................................................................... 9
RETICULOCYTE COUNT ......................................................................................................................... 11
LE CELLS .................................................................................................................................................. 13
ERYTHROCYTE INDICES ......................................................................................................................... 15
OSMOTIC FRAGILITY TEST ...................................................................................................................... 17
RBCs ABNORMAL MORPHOLOGY ....................................................................................................... 19
Summary of Poikilocytes and Anisocytes ....................................................................................... 22
HEMATOLOGY 1 LECTURE ENDTERM......................................................................................................... 25
MYELOPROLIFERATIVE NEOPLASM (MPNs) ......................................................................................... 25
Chronic Myelogenous Leukemia (CML) ........................................................................................ 25
Polycythemia Vera ........................................................................................................................... 29
Essential Thrombocythemia ............................................................................................................. 31
Myelofibrosis ...................................................................................................................................... 33
Other Myeloproliferative Neoplasms .............................................................................................. 35
Summary of Myeloproliferative Neoplasm .................................................................................... 38
QUALITATIVE DISORDERS OF LEUKOCYTES .......................................................................................... 41
Morphologic Abnormalities with and without Functional Defects ............................................. 41
Monocyte/Macrophage Lysosomal Storage Diseases ................................................................ 46
Genetic B and T Lymphocyte Abnormalities ................................................................................ 48
Lymphocytes ..................................................................................................................................... 50
Summary of Qualitative Disorders of Leukocytes ......................................................................... 53
LYMPHOCYTIC LEUKEMIA, LYMPHOMAS, AND PLASMA CELL DYSCRASIAS ................................... 57
Lymphocytic Leukemia .................................................................................................................... 57
Lymphomas ....................................................................................................................................... 59
Plasma Cell Dyscrasias (Paraproteinemias) .................................................................................. 60
ACUTE LYMPHOCYTIC LEUKEMIA AND ACUTE MYELOCYTIC LEUKEMIA .......................................... 63
Introduction ....................................................................................................................................... 63
Acute Lymphoblastic Leukemia (ALL) ........................................................................................... 63
Acute Myeloid Leukemia (AML) ..................................................................................................... 65
Summary ............................................................................................................................................ 73
1st Semester Endterm
HEMATOLOGY 1 LABORATORY ENDTERM
MLS 113A
BLOOD SMEAR EXAMINATION: MAKING A BLOOD SMEAR
and shape. Identifying the size and shape of

the RBC must be noted for the differentiation
and identification of anemia
• Iron Deficiency Anemia and
Thalassemia: Characterized by
microcytic and hypochromic RBCs
2. Relative hemoglobin content

3. Polychromatophilia → exhibited by
reticulocytes. These cells have a blue-
colored center.
4. Inclusions
*Neutrophil → takes up both the basic and 5. Rouleaux formation or agglutination
acid stains (methylene blue and eosin
respectively); granules are purple C. Platelets
*Eosin → takes up acid stain; granules are 1. Estimate number present
pink to orange 2. Examine for morphologic abnormalities
*Basophil → takes up basic stain; granules
are blue 2. Procedures
A. Observations Under ×10
1. Check to see if there are good counting
areas available free of ragged edges and
cell clumps.
2. Check the WBC distribution over the
smear.
3. Check that the slide is properly stained.
4. Check for the presence of large platelets,
platelet clumps, and fibrin strands.
B. Observations Under x40: WBC Estimates

1. Using the × 40 high dry with no oil.
2. Choose a portion of the peripheral smear
where there is only slight overlapping of the
RBCs.
3. Count 10 fields, take the total number of
Performing a Manual Differential and white cells, and divide by 10.
Assessing RBC Morphology 4. To do a WBC estimate by taking the
average number of white cells and
1. Principle multiplying by 2000.
A. White Blood Cells
1. Check for even distribution and estimate C. Observations Under x100: Platelet
the number present (also, look for any gross Estimates
abnormalities present on the smear). 1. Use the oil immersion lens to estimate the
2. Perform the differential count. number of platelets per field.
2. Look at 5-6 fields and take an average.
B. Red Blood Cells: Examine For: 3. Multiply the average by 20,000.
1. Size (macrocytic, microcytic, normocytic) 4. Note any macroplatelets.
Hematology 1 Laboratory MLS 113A | Blood Smear Examination: Making a Blood Smear
*Platelets per oil immersion field (OIF) 6. Normal Peripheral Blood Smear
• <8 platelets/ OIF = decreased
• 8 to 20 platelets/OIF = adequate
• >20 platelets/OIF = increased
*Platelets are remnants of megakaryocytes
3. Observing and Recording Nucleated Red

Blood Cells (NRBCs)
*If 10 or more nucleated RBCs (NRBC) are
seen, correct the White Count using this
formula:
(𝑾𝑩𝑪)(𝟏𝟎𝟎)
𝑪𝒐𝒓𝒓𝒆𝒄𝒕𝒆𝒅 𝑾𝑩𝑪 𝑪𝒐𝒖𝒏𝒕 =
𝑵𝑹𝑩𝑪 + 𝟏𝟎𝟎
*Example:
*Monocyte → big nucleus
• If WBC = 5000 and 10 NRBCs have
*Lymphocyte → small nucleus : cytoplasm
been counted
(5000)(100) ratio
• Then = 4,545.50
10+100
• The corrected white count is 4545.50. 7. Leukocytosis
*Leukocytosis, a WBC above 10,000 is usually
*NRBCs are immature RBCs due to an increase in one of the five types of
*WBC count must be corrected because you white blood cells and is given the name of
might have counted the NRBCs. NRBCs and the cell that shows the primary increase.
WBCs look the same.
Neutrophilic neutrophilia
4. Manual Differential Counts leukocytosis
*These counts are done in the same area as Lymphocytic lymphocytosis
WBC and platelet estimates with the red cells leukocytosis
barely touching. Eosinophilic eosinophilia
*This takes place under × 100 (oil) using the leukocytosis
zigzag method. Monocytic monocytosis
*Count 100 WBCs including all cell lines from leukocytosis
immature to mature. Basophilic basophilia
leukocytosis
*Reporting results
• Absolute number of cells/µl = % of cell
type in differential x white cell count
5. Observing Direction
Band Cell/ Stab
Segmented Eosinophil
Neutrophil
Neutrophil
Reactive Lymphocyte
Basophil Monocytes
*Observe one field and record the number of Lymphocyte
WBC according to the different types then

turn to another field in the snake-liked A. Stab Neutrophil/ Band neutrophil
direction *Normally found in the blood
*Avoid repeating or missing some cells *Diameter:12-16
*Cytoplasm: pink
*Granules: marrow into the bloodstream - are known as
• Primary "bands" or "stabs".
• Secondary
*Left shift:
*Nucleus: • non-segmented neutrophil > 5%
• dark purple blue • Increased bands mean acute
• dense chromatin infection, usually bacterial
• Young
B. Segmented Neutrophil • Production = Consumption
*Diameter: 12-16
*Cytoplasm: pink *Right shift:
*Granules: • hypersegmented neutrophil >3%
• primary • Increased hypersegmented
• secondary neutrophil
• Senior
*Nucleus:
• dark purple blue C. Eosinophil
• dense chromatin 2-5 lobes *Diameter: 14-16
*Cytoplasm: full of granules
*Neutrophils *Granules: large refractile, orange-red
• Neutrophils are so named because *Nucleus:
they are not well stained by either • Blue
eosin, a red acidic stain, or by • dense chromatin
methylene blue, a basic or alkaline • 2 lobes like a pair of glass
stain.
• Neutrophils are also known as "segs", *The most common reasons for an increase in
"PMNs" or "polys" the eosinophil count are
(polymorphonuclear). • Allergic reactions such as hay fever,
• They are the body's primary defense asthma, or drug hypersensitivity
against bacterial infection. • Parasitic infection
• Eosinophilic leukemia
B.1 Increased neutrophils count
(neutrophilia) *Functions in allergic reactions because it
1. Acute bacterial infection carries the major basic protein that is
2. Granulocytic leukemia responsible for inflammation
B.2 Decreased neutrophil count D. Basophil

(neutropenia) *Diameter: 14-16
1. Typhoid fever *Cytoplasm: pink
2. Brucellosis *Granules: dark blue-black obscure nucleus
3. Viral diseases, including hepatitis, *Nucleus: blue
influenza, rubella, and mumps.
*The purpose of basophils is not completely
B.3 Left-shift and Right-shift of Neutrophil understood.
*Normally, most of the neutrophils circulating *Basophil counts are used to analyzing
in the bloodstream are in a mature form, with allergic reactions.
the nucleus of the cell being divided or *An alteration in bone marrow function such
segmented. Because of the segmented as leukemia may cause an increase in
appearance of the nucleus, neutrophils are basophils.
sometimes referred to as "segs.” *The granules of the basophil contain
histamine.
*The nucleus of less mature neutrophils is not
segmented but has a band or rod-like shape. E. Lymphocyte
Less mature neutrophils - those that have *Diameter:
recently been released from the bone • small 7-9
• large 12-16
*Cytoplasm: medium blue anemia (CLL) because, in CLL, these cells are
*Granules: small agranular large a few fragile and easily smudged during smear
primary granules preparation.
*Nucleus: dark blue \round dense chromatin
*Lymphocytes are the primary components

of the body's immune system. They are the
source of serum immunoglobulins and
cellular immune response.
E.1 Two types of lymphocytes:

*B lymphocyte: Humoral immunity
*T lymphocyte: Cellular immunity
*Can also be NK cells
E.2 Lymphocytes increase (lymphocytosis) in: B. Abnormal differentials

*Many viral infections 1. 200 Cell diff:
*Tuberculosis • WBC > 15.0 (>20.0 for babies under 1
*Typhoid fever month and labor unit)
*Lymphocytic leukemia • Three or more basophils were seen.
E.3 Lymphocyte decrease 2. If more than five immature WBCs are seen
*A decreased lymphocyte (lymphopenia) (or any blasts) let someone else diff slide and
count of fewer than 500 places a patient at average results.
a very high risk of infection, particularly viral 3. Correct WBC for NRBCs if you see ten or
infections. more NRBCs/100 WBC.
4. Always indicate the number of cells
F. Monocyte counted on diff.
*Diameter: 14-20 5. If any cell type is extremely elevated (such
*Cytoplasm: grey blue as bands, monos, or eos > 20) indicate that
*Granules: dust-like lilac color granules you are aware of the abnormality by circling
*Nucleus: or checking on the card next to the results
• blue
• large irregularly shaped and folded C. Morphologic Changes Due to Area of
Smear
F.1 Disease that causes a monocytosis to
include:
*Tuberculosis
*Brucellosis
*Malaria
*Monocytic leukemia
8. Notes
A. Do not count cells that are disintegrating
*Eosinophil with no cytoplasmic membrane
and with scattered granules
*Pyknotic cell (RBC) (nucleus extremely *Thin area

condensed and degenerated, lobes • Spherocytes are really
condensed into small, round clumps with no "spheroidocytes" or flattened red
filaments interconnecting). cells. True spherocytes will be found in
• smudge cells other (Good) areas of the smear.
• Basket cells
*Thick area
*Presence of basket and smudge cells in high • Rouleaux, which is normal in such
amounts may indicate chronic lymphocytic areas. Confirm by examining thin
areas. If true rouleaux, two-three
RBCs will stick together in a "stack of
coins" fashion.
D. A well-made and well-stained smear is
essential to the accuracy of the differential
count. The knowledge and ability of the cell
morphologist are critical to high-quality
results.
E. Before reporting significant abnormalities
such as blasts, malaria, or other significant
findings on a patient’s differential, ask a more
experienced tech to review the smear for
confirmation. In clinical settings where a
pathologist or hematologist is present, the
smear is set aside for Pathologist Review. 3. Procedure
F. Never hesitate to ask questions concerning
morphology or the identification of cells. The *Using your Cell Counter
differential is one of the most difficult app, count 100
laboratory tests to learn. Learning about cells differentiated WBCs.
and their morphology is a process that
continues for as long as you perform
differentials. 4. Reporting
A. Relative Count
WBC Differential Count *Reported in percent
1. Proper Staining Reactions of Different Blood

Cells to Wright’s Stain (# 𝑜𝑓 𝑊𝐵𝐶 𝑠𝑝𝑒𝑐𝑖𝑓𝑖𝑐 𝑡𝑦𝑝𝑒)(100)
𝑹𝒆𝒍𝒂𝒕𝒊𝒗𝒆 𝑪𝒐𝒖𝒏𝒕 =
dark blue or bluish 100 𝑊𝐵𝐶 𝐶𝑜𝑢𝑛𝑡𝑒𝑑 𝑖𝑛 𝑆𝑚𝑒𝑎𝑟
Neutrophils violet nucleus; lilac

pink or reddish-lilac
fine granules *Example:
• 50 neutrophils out of 100 WBCs seen in
dark blue nucleus;
the smear
Eosinophils red to orange
*Solution:
coarse granules (50)(100)
dark blue nucleus; • = = 50% 𝑁𝑒𝑢𝑡𝑟𝑜𝑝ℎ𝑖𝑙𝑠
(100)
Basophils dark blue to black *Report:
granules • Neutrophils 60%
dark blue or deep • Lymphocytes 30%
purple-blue • Monocytes 5%
Lymphocytes nucleus; sky-blue or • Eosinophils 4%
robin-egg blue • Basophils 1%
cytoplasm • Total 100%
faint blue nucleus;
Monocytes pale blue to gray B. Absolute Count
cytoplasm
Platelets pale lilac-blue to
dark lilac 𝑨𝒃𝒔𝒐𝒍𝒖𝒕𝒆 𝑪𝒐𝒖𝒏𝒕 (#/𝒄𝒖 𝒎𝒎) =
(𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝐶𝑜𝑢𝑛𝑡)(𝑊𝐵𝐶 𝐶𝑜𝑢𝑛𝑡)
2. Blood Smear
*Example:
• relative count of neutrophil: 50%;
WBC count 10,000/cu mm
*Solution:
• = (0.50)(10,000) = 5,000/𝑐𝑢 𝑚𝑚
5. Normal Reference Range
Table. Absolute Normal Values
Neutrophil 1,600-7,260/cu mm
Eosinophils 45-440/cu mm
Basophils 45-110/cu mm
Lymphocytes 960-4,400/cu mm
Monocytes 180-880/cu mm
RETICULOCYTE COUNT
1. Reticulocyte *Reticulocyte → any nonnucleated RBC that

*Last immature stage of RBC contains two or more particles of blue-
*Contains remnant cytoplasmic ribonucleic stained granulofilamentous material after the
acid and mitochondria and ribosomes new methylene blue stain. Look for cells that
*Stains gray-blue after being stained by a still have remnants even after staining.
supravital stain
*It is a non-nucleated cell that contains RNA
remnants and organelles such as
mitochondria and ribosomes
*The reticulum is present in newly released
cells for 1-2 days before the cell reaches its
mature stage.
*They are called reticulocytes because of the
mesh-like network of ribosomal RNA that
becomes visible under a microscope once it
is stained by a supravital stain like methylene
blue.
4. Materials
*Binocular Microscope
*A Blue tip capillary tube
*Glass Slides
*New Methylene Blue (fresh and filtered) 1%
→ dissolved in sodium chloride
*Immersion Oil
*Cell counter app (online)
5. Procedure for Reticulocyte Count

*WET METHOD (New Methylene Blue Method)
1. Collect a venous EDTA sample. Finger
puncture can also be used.
2. Purpose 2. Put 5 drops of blood sample in a test
*Assess the erythropoietic activity of the tube. Label the tube.
bone marrow. 3. Dispense an equal amount of new
*It is used to determine the bone marrow's methylene blue into the tube
ability to increase RBC production in containing the sample. The ratio is 1:1.
response to anemia. It is important to use a fresh sample
since the purpose of the supravital
3. Principle stain is to stain living cells.
*Whole blood (anticoagulated with EDTA) is 4. Mix the tube. Allow standing for
stained and incubated with a supravital about 15 minutes. This incubation
stain. process will give the cells time to
*Supravital stains are those which stain living accept the stain.
material. Hence, for the detection of 5. After 15 minutes, remix the tube and
ribosomal RNA is reticulocytes, they should draw into a blue tip capillary tube the
be fixed. Blood is mixed with the stain and blood-stain mixture. Transfer a small
incubated. drop of the mixture to a glass slide.
• New Methylene Blue 6. Prepare a blood smear. Air dry for a
• Brilliant Cresyl Blue few minutes.
• Pure Azure Blue 7. Examine the smear under Oil
Immersion Objective.
*These supravital stains precipitate the RNA 8. Count 1000 RBCs (250 per field) and
and organelles found on the cell, forming a simultaneously count the reticulocyte
filamentous network of reticulum. in these areas.
9. Compute the reticulocyte count
using the formula:
Hematology 1 Laboratory MLS 113A | Reticulocyte Count
6. Computation 8. Reticulocyte Production Index for
Correction of Retics
*Perform Reticulocyte Production Index to
(# 𝑜𝑓 𝑅𝑒𝑡𝑖𝑐 𝐶𝑜𝑢𝑛𝑡𝑒𝑑)(100) correct retics because raw/uncorrected
%𝑹𝒆𝒕𝒊𝒄 =
(# 𝑜𝑓 𝑅𝐵𝐶 𝐶𝑜𝑢𝑛𝑡𝑒𝑑)(1000) retic count is misleading to some anemic
patients.
*Example: A. Step 1
• The number of reticulocytes counted
is 12 (𝑅𝑒𝑡𝑖𝑐%)(𝐻𝐶𝑇)
𝑪𝒐𝒓𝒓𝒆𝒄𝒕𝒆𝒅 𝑹𝒆𝒕𝒊𝒄 % =
*Solution: 𝑁𝑜𝑟𝑚𝑎𝑙 𝐻𝐶𝑇
(12)(100)
• %𝑹𝒆𝒕𝒊𝒄 = = 1.2%
(1000)
B. Step 2
7. Reference Range *Correct for the longer lifespan of
*ADULT: 0.5 to 1.5% (20,000 to 60,000/mm3) prematurely released retic
*INFANT: 2.5 to 6.5% → turns to adult
reference range IF the infant is 2 WEEKS of
age. 𝐶𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑅𝑒𝑡𝑖𝑐
𝑹𝑷𝑰 =
𝑀𝑎𝑡𝑢𝑟𝑎𝑡𝑖𝑜𝑛 𝐶𝑜𝑟𝑟𝑒𝑐𝑡𝑖𝑜𝑛
*↓ Reticulocyte Count → Seen in ineffective
erythropoiesis such as megaloblastic anemia
and thalassemia. RETIC SURVIVAL
HCT DAYS=MATURATION
*↑ Reticulocyte Count → Seen in blood loss CORRECTION
(E.g., Acute, and chronic blood loss) and 36-45% 1.0
hemolytic anemia. During these conditions, 26-35% 1.5
the body tries to compensate for the loss of 16-25% 2.0
blood or the destruction of RBCs. The bone 15 AND BELOW 2.5
marrow responds to the demand for
increased RBC, thereby releasing early more
immature RBCs in the blood. That is why there
is an increase in the number of reticulocytes
in the peripheral blood.
Hematology 1 Laboratory MLS 113A | Reticulocyte Count

LE CELLS
1. LE Cells
*An LE cell is a Neutrophil or Macrophage *Frequently the
that has phagocytized (engulfed) the earliest symptoms
denatured nuclear material of another cell. appear after
The denatured material is an absorbed intense exposure
hematoxylin body (basophilic particle also to sunlight.
called an LE body).
*They are a characteristic of lupus
erythematosus but are also found in similar *Leukopenia, thrombocytopenia, and an
connective tissue disorders. elevated sedimentation rate (ESR) are some
of the clinical signs of the disease.
*The LE cell was discovered in bone marrow
in 1948 by Malcolm McCallum Hargraves 3. Tart Cell
(1903–1982), a Physician and Practicing *It's a monocyte/ histocyte with engulfed
Histologist at the Mayo Clinic. viable nuclear material
*Classically, the LE cell is analyzed *It’s not to be equated with LE cell
microscopically, but it is also possible to *It has no diagnostic value
investigate this phenomenon by flow *Importance: It should not be mistaken for LE
cytometry. cell
4. LE Cell VS Tart Cell
2. SLE (Systemic Lupus Erythematosus) LE CELL TART CELL

*Persons having lupus erythematosus, one of LE cell is a neutrophil TART a mimicker of
the "collagen" diseases, have an abnormal that has engulfed a LE cell is a
plasma protein that causes swelling and homogenized monocyte
breakdown of certain blood cell nuclei in basophilic nuclear (macrophage) that
vitro. material (as in the has engulfed or
*This degenerated nuclear material attracts image) contains a nuclear
phagocytic cells, particularly segmented material with
neutrophils, which engulf this nuclear mass. regular chromatin
*The resulting phagocyte and inclusion (rather than
material is termed an "L.E." cell. homogenized).
LE Cell is seen in A TART cell can be
*Lupus erythematosus is a chronic, patients with confused with an LE
sometimes fatal, disease of unknown etiology Systemic lupus cell
or cause. erythematosus
*The peculiar skin eruption across the nose A tart cell is named
and cheeks (butterfly rash) and arthritis can after the patient Mr.
be accompanied by various visceral Tart whose marrow
manifestations. contained a lot of
*Often the rash is not present, and diagnosis Tart cells.
depends on the demonstration of the L.E.
cell.
Hematology 1 Laboratory MLS 113A | LE Cells
5. Sample *A smear is considered positive when 10 or
*Lupus erythematosus (LE) cell testing is more characteristic LE cells are seen during a
performed using any of the following: 15-minute search, associated with the
• Heparinized bone marrow presence of extracellular, amorphous,
• Heparinized venous blood nuclear masses.
• Oxalated venous blood *The presence of LE cells indicates lupus.
• Defibrinated venous blood
• Clotted venous blood A. Negative Findings
• LE factor and donor cells *Negative findings on LE cell testing exclude
(deny) a diagnosis of systemic lupus
*LE Factor: erythematosus (SLE)
• An Antibody found in the serum
found in SLE Patients. B. Positive Findings
*Positive reactions are also seen in:
6. Method of LE Cell Preparation • Rheumatoid arthritis
1. Collect 5 mL venous blood. Allow to • Chronic hepatitis(lupoid)
clot. • Scleroderma
2. Macerate clot with the use of a • Dermatomyositis
mortar and pestle. • Polyarteritis nodosa
3. Let the clot pass through a screen • Acquired hemolytic anemia
before putting into the capillary tubes • Hodgkin disease
4. Incubate the capillary tubes for an
hour at 37°C *It may also be positive in persons taking
5. Centrifuge at 3,500 rpm for 30 phenylbutazone and hydralazine
minutes.
6. Cut the capillary tubes at the area of 8. Application
the buffy coat layer. *Lupus erythematosus (LE) cell testing was
7. Make a smear and stain with Wright’s once performed to diagnose systemic lupus
stain erythematous but has been replaced for this
8. Air dry and examine under OIO purpose by antinuclear antibody testing
(ANA).
7. Interpretation *SLE is positive for ANA
*A lupus erythematosus (LE) cell test is
considered positive when approximately 2%-
30% of the cells seen on the slide in the
neutrophil count are LE cells.
Hematology 1 Laboratory MLS 113A | LE Cells

ERYTHROCYTE INDICES
1. Introduction A. Reference Range for MCH

*Generally, erythrocyte indices are closely * 27 to 33 pg (30 ±3)
related to anemia.
*Erythrocyte indices were first introduced by B. Interpretation for MCH
Wintrobe in 1929 to define the size (MCV) and *More than 33 pg = macrocyte (macrocytic
hemoglobin content (MCH, MCHC) of red anemia)
blood cells. Termed red cell indices, these *Less than 27 pg = microcyte (microcytic
values are useful in elucidating the etiology anemia)
of anemias. Red cell indices can be *Less than 22 pg = microcytic hypochromic
calculated if the values of hemoglobin, (microcytic hypochromic anemia)
hematocrit (packed cell volume), and red
blood cell count are known. 4. MCHC
*MCHC means mean corpuscular
2. MCV hemoglobin concentration.
*MCV is mean corpuscular volume. *It expresses the mean concentration of
*It is the average volume of an individual red hemoglobin in the average red blood cell.
blood cell. *Measure the proportion of each cell taken
*Indicator of red blood cell size up by hemoglobin
*Indicates the amount of hemoglobin per
unit volume.
*In contrast to MCH, MCHC correlates the
hemoglobin content with the volume of the
cell.
A. Reference Range for MCV (𝐺𝑟𝑎𝑚 𝐻𝑏)(100)

*80 to 100 fL 𝑴𝑪𝑯𝑪 = =%
𝑉𝑜𝑙𝑢𝑚𝑒% 𝐻𝐶𝑇
B. Interpretation for MCV [𝐻𝑏(𝑔/𝑑𝐿)][100]

*101 to 160 fL = macrocytes (macrocytic 𝑴𝑪𝑯𝑪 = =%
𝐻𝐶𝑇 (%)
anemia)
*72 to 79 fL = microcytes (microcytic anemia)
*50 to 71 fL = microcytes, hypochromia A. Reference Range for MCHC
(microcytic hypochromic anemia) *32 to 38% (35±3)
3. MCH *Sample: Compute the MCV, MCH, and

*MCH means mean corpuscular MCHC of the patient.
hemoglobin. • Given:
*Amount/mass of hemoglobin present in one o hemoglobin = 12 g/dL
RBC o hematocrit = 38%
*It is the ratio of hemoglobin to red cell count. o RBC = 4.1 x 1012/L
*It measures the weight of hemoglobin in the • MCV
average red cell. o 𝑀𝐶𝑉 =
[𝐻𝐶𝑇(%)][10]
12
𝑅𝐵𝐶(𝑥10 /𝐿)
(38)(10)
o = = 92.6 𝑜𝑟 93 𝑓𝐿
(4.1𝑥1012 /𝐿)
(𝐺𝑟𝑎𝑚 𝐻𝑏)(10)
𝑴𝑪𝑯 = = 𝑝𝑖𝑐𝑡𝑜𝑔𝑟𝑎𝑚(10−12 𝐿) • MCH
𝑅𝐵𝐶 𝑖𝑛 𝑚𝑖𝑙𝑙𝑖𝑜𝑛 [𝐻𝑏(𝑔/𝑑𝐿)][10]
o 𝑀𝐶𝐻 =
𝑅𝐵𝐶(𝑥1012 /𝐿)
(𝐺𝑟𝑎𝑚 𝐻𝑏)(10) (12𝑔/𝑑𝐿)(10)
𝑴𝑪𝑯 = = 𝑝𝑖𝑐𝑡𝑜𝑔𝑟𝑎𝑚 o = = 29𝑝𝑔
4.1𝑥1012 /𝐿
𝑅𝐵𝐶 𝑖𝑛 𝑚𝑖𝑙𝑙𝑖𝑜𝑛
• MCHC
[𝐻𝑏(𝑔/𝑑𝐿)][100]
[𝐻𝑏(𝑔/ 𝑑𝐿)][10] o 𝑀𝐶𝐻𝐶 =
𝑴𝑪𝑯 = = 𝑝𝑖𝑐𝑡𝑜𝑔𝑟𝑎𝑚 𝐻𝐶𝑇(%)
𝑅𝐵𝐶 (𝑥1012 /𝐿) (
12𝑔
𝑑𝐿
)(100)
o = = 31.5 𝑜𝑟 32%
38
• Patient is normocytic, normochromic
Hematology 1 Laboratory MLS 113A | Erythrocyte Indices
Hematology 1 Laboratory MLS 113A | Erythrocyte Indices

OSMOTIC FRAGILITY TEST
fragility test may be used to confirm the

presence of spherocytes.
*The test does not distinguish between
spherocytes in HS and acquired autoimmune
hemolytic anemia.
*The test only indicates that a proportion of

the red cells have decreased surface-to-
volume ratios and are more susceptible to
lysis in hypo- osmotic solutions.
*HS patients who are experiencing significant

1. Definition elevations in reticulocytes may not fall
*Osmotic fragility is a test to measure red outside of the normal range.
blood cell (RBC) resistance to hemolysis
when exposed to a series of increasingly *Cells with increased surface-to-volume
dilute saline solutions. ratios, such as occur in thalassemia and iron
*The sooner hemolysis occurs, the greater the deficiency, may show decreased osmotic
osmotic fragility of the cells. fragility.
2. Factors that affect the osmotic fragility *For patients with acute hemolysis, a normal
*Cell membrane permeability red cell osmotic fragility test result cannot
*Surface-to-volume ratio exclude an osmotic fragility abnormality
since the osmotically labile cells may be
3. Why the Test is performed? hemolyzed and not present.
*This test is performed to detect thalassemia
and hereditary spherocytosis. *Recommend testing during a state of
*Hereditary spherocytosis is a common prolonged homeostasis with stable
disorder in which red blood cells are hematocrit.
defective because of their round, ball-like
(spherical) shape. These cells are more 4. Increase and Decrease of Osmotic fragility
fragile than normal. A. Osmotic fragility decreased in:
* Thalassemia
*Spherical cells are said to have increased *Iron deficiency anemia
osmotic fragility because they are less likely *Sickle cell anemia
to expand and break open in salter water
than normal red blood cells (which are B. Osmotic fragility of red cells increased in:
indented or curved inward on both sides). *Hereditary spherocytosis
*Acquired spherocytosis
*Cells that are flatter than normal are more
likely to expand and thus have decreased 5. Osmotic Fragility Test
osmotic fragility. A. Purpose
*Thalassemia is an inherited condition that *To aid diagnosis of hereditary spherocytosis
affects the portion of blood (hemoglobin) & Thalassemia.
that carries oxygen. *To supplement a stained cell examination to
detect morphologic RBC abnormalities.
*Some red blood cells are more fragile than
normal, but a larger number are less fragile B. Materials
than normal. *Specimen: whole blood
*Collection Medium: Na Heparin tube or
*When spherocytes (HS) are suspected Lithium Heparin tube.
based on an elevated mean corpuscular *Minimum: 5 ml whole blood.
hemoglobin concentration or examination of *Rejection Criteria: Hemolyzed specimen.
a peripheral blood smear, the osmotic *Methodology: Spectrophotometer.
Hematology 1 Laboratory MLS 113A | Osmotic Fragility Test

6. Example Procedure 7. Result
1. We will do this dilution
𝐴𝑏𝑠 𝑜𝑓 𝑡𝑢𝑏𝑒
% 𝒐𝒇 𝑯𝒆𝒎𝒐𝒍𝒚𝒔𝒊𝒔 = ( ) (100)
𝐴𝑏𝑠 𝑜𝑓 𝑡𝑢𝑏𝑒 14
*Normal Range
• Hemolysis begins at 0.45% and
completes 0.35%
8. Discussion
*Interfering factors:
1. Fill the collection tube and invert it
gently several times to mix the sample
and anticoagulant thoroughly.
2. Handle the sample gently to prevent
2. Then we divide every volume into 2 accidental hemolysis.
tubes so now we get 28 tubes. 3. In some cases, RBCs don't hemolyze
3. Add 50 microns of whole blood to immediately, incubation in solution
every tube. for 141 hours improves test sensitivity.
4. let the tubes at R.T for 30 min at 2500 4. Presence of hemolytic organisms in
rpm. the sample.
5. Well-mixing by using the vortex. 5. Severe anemia or other conditions
6. Centrifuge for 5 minutes at 2500 rpm. with fewer RBCs available for testing.
7. Now we will measure the absorbance 6. Recent blood transfusion.
in the tubes by using a 7. Old sample
spectrophotometer (540 nm).
8. Calculate the % of hemolysis.
Hematology 1 Laboratory MLS 113A | Osmotic Fragility Test

RBCs ABNORMAL MORPHOLOGY
*Found in:
• Hyperfibrinogenaemia
• Hyperglobulinaemia
B. Red cell-agglutination
*Morphology:
• Irregular clumps of red cells
*Found in:
• Cold agglutinins
• Warm autoimmune hemolysis
*Terms
• Normochromic: A descriptive term
applied to a red blood cell with a
normal concentration of
hemoglobin.
• Normocytic: A descriptive term
applied to the normal size of RBC.
• Hypochromic: A descriptive term
applied to a red blood cell with a
decreased concentration of
hemoglobin.
• Macrocytic: A descriptive term
applied to a larger than a normal red
blood cell.
1. Abnormal erythrocyte morphology 3. Variation in erythrocyte size (anisocytosis)

*Abnormal erythrocyte morphology is found A. Microcytosis
in pathological states that may be: *Morphology:
• abnormalities in size (anisocytosis). • Decrease in the red cell size. Red cells
• abnormalities in shape are smaller than ± 7µm in diameter.
(poikilocytosis). The nucleus of a small lymphocyte (±
• abnormalities in hemoglobin content 8µm) is a useful guide to the size of a
or the presence of inclusion bodies in red blood cell.
erythrocytes. *Found in:
• Iron deficiency anemia
• Thalassemia
• Sideroblastic anemia
• Lead poisoning
• Anemia of chronic disease
*Comment: Most erythrocytes presented in

the picture are microcytes (compare with
the small lymphocyte). The degree of
hemoglobinization is sufficient. Normal
platelets and single ovalocytes are present.
B. Macrocytosis
*Morphology:
• Increase in the size of a red cell. Red
cells are larger than 9µm in diameter.
2. Variation in red cells Distribution
May be round or oval, but the
A. Rouleaux Formation
diagnostic significance is different.
*Morphology:
*Found in:
• Stacks of RBCs resembling a stack of
• Folate and B12 deficiencies (oval)
coins.
Hematology 1 Laboratory MLS 113A | RBCs Abnormal Morphology
• Ethanol (round) *Found in:
• Liver disease (round) • Thalassaemia major
• Reticulocytosis (round) • Hereditary ovalocytosis.
• Sickle cell anemia
4. Variation in hemoglobin content
A. Hypochromasia D. Elliptocytosis
*Morphology: *Morphology:
• Increase in the red cells' central pallor • The red cells are oval or elliptical. The
which occupies more than the long axis is twice the short axis.
normal third of the red cell diameter. *Found in:
*Found in: • Hereditary elliptocytosis
• Iron deficiency • Megaloblastic anemia
• Thalassaemia any of the conditions • Iron deficiency
leading to Microcytosis • Thalassemia - Myelofibrosis
B. Polychromasia E. Tear Drop Cells

• Red cells stain shades of blue-gray as • Red cells are shaped like a tear drop
a consequence of the uptake of both or pear.
eosin (by hemoglobin) and basic *Found in:
dyes (by residual ribosomal RNA). • Bone marrow fibrosis
Often slightly larger than normal red • Megaloblastic anemia
cells and round-round macrocytosis. • Iron deficiency
*Found in: • Thalassemia
• Any situation with reticulocytosis - for
example bleeding, hemolysis, or F. Blister cell (keratocyte)
response to heamatinic factor *Morphology:
replacement. • Have accentric hallow area.
Resemble a women's handbag
5. Variation of red cells shape (Poikilocytosis) *Found in:
A. Spherocytosis • Microangiopathic hemolytic anemia
*Morphology:
• Red cells are more spherical. Lack the G. Keratocytes (horn cell)
central area of pallor on a stained *Morphology:
blood film. • Part of the cell fuses back leaving two
*Found in: or three horn-like projections. The
• Hereditary spherocytosis keratocyte is a fragile cell and
• Immune haemolytic anemia. remains in circulation for only a few
• Zieve's syndrome hours.
• Microangiopathic haemolytic *Found in:
anemia • Uraemia
• Severe burns
B. Target Cells • EDTA artifact
*Morphology: • Liver disease
• Red cells have an area of increased
staining which appears in the area of H. Burr (crenation) cell
central pallor. *Morphology:
*Found in: • Red cells with uniformly spaced,
• Obstructive liver disease pointed projections on their surface.
• Thalassaemia (not I.D.A) *Found in:
• Haemoglobinopathies (S and C) • Hemolytic anemia
• Post splenectomy • Uremia
• Megaloblastic anemia
C. Ovalocytes
*Morphology: I. Acanthocytosis
• Oval shape red blood cell *Morphology:

• are red blood cells with irregularly B. Siderotic Granules
spaced projections, these projections *RBCs that contain no hemoglobin iron
are very in width but usually contain a granules. They appear as dense blue,
rounded end irregular granules that are unevenly
*Found in: distributed in Wright-stained RBCs.
• Liver disease Pappenheimer bodies can be increased in
• Post splenectomy hemolytic anemia, infections, and post-
• Anorexia nervosa and starvation splenectomy.
J. Schistocytosis C. Basophilic stippling

• Fragmentation of the red cells. • Considerable numbers of small
*Found in: basophilic inclusions in red cells.
• DIC *Found in:
• Micro angiopathic hemolytic anemia • Thalassemia
• Mechanical haemolytic anemia • Megaloblastic anemia
• Hemolytic anemia
K. Stomatocytosis • Liver disease
*Morphology: • Heavy metal poisoning
• Red cells with a central linear slit or
stoma. Seen as a mouth-shaped form D. Heinz Bodies
in peripheral smear. *Represent denatured hemoglobin
*Found in: (methemoglobin - Fe+++) within a cell. With
• Alcohol excess a supravital stain like crystal violet, Heinz's
• Alcoholic liver disease bodies appear as round blue precipitates.
• Hereditary Stomatocytosis *Presence of Heinz bodies indicates red cell
• Hereditary spherocytosis injury and is usually associated with G6PD
deficiency.
L. Sickle Cells
*Morphology: E. Cabot Rings
• Sickle-shaped red cells. *Reddish-blue threadlike rings in RBCs of
*Found in: severe anemias. These are remnants of the
• Hb-S disease nuclear membrane and appear as a ring or
figure 8 pattern.
M. Nucleated red blood cells *Very rare finding in patients with
*These red blood cells are released from the Megaloblastic anemia, severe anemia, lead
bone marrow early into the blood stream, poisoning, and dyserythropoiesis.
due to the need for oxygen. Normal red
blood cells do not contain a nucleus on a 7. Erythrocyte inclusion bodies
peripheral smear A. 6- Parasites of Red Cell
*Are protozoan parasites that occur in many
N. Envelope form cell species of birds and are the cause of avian
*Found in malaria.
• Thalassemia *Transmitted by mosquitoes, infection with
• Sickle cell anemia Plasmodium spp can be a cause of
hemolytic anemia
6. Erythrocyte inclusion bodies
A. Howell-Jolly Bodies 8. RBCs Abnormal morphology
*Morphology: *Depiction of red blood cell morphologies
• Small round cytoplasmic red cell that may appear on a peripheral smear,
inclusion with the same staining showing:
characteristics as nuclei A. Basophilic stippling
*Found in: B. Howell-Jolly bodies
• Post splenectomy C. Cabot's ring bodies
• Megaloblastic anemia D. Heinz's bodies

8. RBCs Abnormal morphology Continuation
Summary of Poikilocytes and Anisocytes
Cell Description Causes

Anisocytes Abnormal variation in RBC *Hemolytic, megaloblastic,
volume or diameter Iron deficiency anemia
*Megaloblastic anemia
Large RBC (> 8um in *Myelodysplastic syndrome
Macrocytes diameter), MCV >100 fL *Chronic liver disease
*Bone marrow failure
*Reticulocytosis
Macro-ovalocytes Large oval RBC *Megaloblastic anemia
*Iron deficiency anemia
*Anemia of chronic
Microcytes Small RBC (<6 um in inflammation
diameter), MCV <80 fL *Sideroblastic anemia
*Thalassemia
*HbE disease and trait
Abnormal variation in RBC *Severe anemia
Poikilocytes shape *Certain shapes are helpful
diagnostically
*Hereditary spherocytosis
Spherocytes Small, round, dense RBC with *Immune hemolytic anemia
no central pallor *Extensive burns (along
schistocytes)
*Hereditary elliptocytosis,
Elliptical (cigar-shaped), oval Hereditary ovalocytosis, Iron
Elliptocytes (egg-shaped) deficiency anemia
Thalassemia major,
Myelophthisic anemia
*Hereditary stomatocytosis,
Stomatocytes RBC with the slit-like area of Rh deficiency syndrome,
central pallor Acquired stomatocytosis (liver
disease, alcoholism), Artifact
*Microangiopathic hemolytic
Fragmented RBC due to anemia (along with
Schistocytes rupture in the peripheral microspherocytes)
circulation *Traumatic cardiac hemolysis
*Extensive burns (along with
microspherocytes
Keratocytes RBC fragment in the shape of *Same as schistocyte
a helmet
Sickled cells RBC with membrane folded *Hb SC disease
over *Hb C disease

Small, dense RBC with few *Severe liver disease (spur cell
irregularly spaced projections anemia)
Acanthocytes of varying length *Neuroacanthocytosis
(abetalipoproteinemia,
McLeod syndrome)
RBCs with blunt or pointed,
short projections that are
usually evenly spaced over *Uremia
Burr cells the surface of the cell; *Pyruvate kinase deficiency
present in all fields of blood
film but in variable numbers
per field
RBC with a single pointed *Primary myelofibrosis
Dacryocytes extension resembling a *Myelophthisic anemia
teardrop or pear *Thalassemia
*Megaloblastic anemia


HEMATOLOGY 1 LECTURE ENDTERM
MLS 113A
MYELOPROLIFERATIVE NEOPLASM (MPNs)
*Are clonal hematopoietic disorders caused by genetic mutations in the hematopoietic stem cells
that result in expansion, excessive production, and accumulation of erythrocytes, granulocytes,
and platelets. The problem is the hematopoietic stem cell, not the mature cell.
*Myeloproliferation is due to hypersensitivity or independence of normal cytokine regulation
(proliferation even in the absence of EPO, G-CSF, etc.) that reduces cytokine levels through
negative feedback systems normally induced by mature cells.
*When cells reach a certain level, the fas L and the fas receptor trigger the apoptosis of these
cells. However, in the case of MPN, there is no such thing, hence increasing the proliferation of
these cells.
*Expansion occurs in varying combinations in the bone marrow, peripheral blood, and tissues.
*MPNs are predominantly chronic with accelerated, subacute, or acute phases.
Chronic Myelogenous Leukemia (CML) • Anemia → due to the small number of

RBCs produced since the bone
marrow is busy producing WBCs.
• Bleeding → lots of platelets produced
BUT are nonfunctional.
• Splenomegaly → there is
extramedullary hematopoiesis since
the bone marrow is too crowded.
*All secondary to massive pathologic

accumulation of myeloid progenitor cells in
bone marrow, peripheral blood, and
extramedullary tissues.
*Peripheral blood shows:

• Neutrophilia with all maturational
*Is an MPN arising from a single genetic stages present → even the young
translocation in a pluripotential WBCs that are supposed to be in the
hematopoietic stem cell producing a clonal bone marrow.
overproduction of the myeloid cell line, • Basophilia → supposedly, this is not
resulting in a preponderance of immature common in peripheral blood
cells in the neutrophilic line. because our blood only comprises
about <1% of basophilia.
*CML begins with a chronic clinical phase • Eosinophilia
and, if untreated, progresses to an • Thrombocytosis → increase in platelet
accelerated phase in 3 to 4 years and often count.
terminates as acute leukemia.
1. Incidence of Chronic Myelogenous
*The clinical features are: Leukemia
• frequent infection → WBCs released *Occurs at all ages but is seen predominantly
are immature, hence cannot fight in those aged 46 to 53 years.
infection *20% of all cases of leukemia start from CML
Hematology 1 Lecture MLS 113A | Myeloproliferative Neoplasm (MPNs)

*Slightly more common in males than in *BCR-ABL is a mutation that is formed by the
females. Symptoms associated with clinical combination of two genes, known as BCR
onset are usually of minimal intensity and and ABL. It's sometimes called a fusion gene.
include fatigue, decreased tolerance of *The BCR gene is normally on chromosome
exertion, anorexia, abdominal discomfort, number 22.
weight loss, and symptomatic effects from *The ABL gene is normally on chromosome
splenic enlargement. number 9.
*The BCR-ABL mutation happens when
2. Cytogenetics of the Philadelphia pieces of BCR and ABL genes break off and
chromosome switch places.
*Most common cause of CML *The mutation shows up on chromosome 22,
*A unique chromosome, the Philadelphia where the piece of chromosome 9 has
chromosome, is present in proliferating attached itself.
hematopoietic stem cells and their progeny *The mutated chromosome 22 is called the
in CML and must be identified to confirm the Philadelphia chromosome because that's
diagnosis. the city where researchers first discovered it.
*The Philadelphia chromosome was first *The BCR-ABL gene is not the type of
identified as a short chromosome 22 in mutation that is inherited from your parents. It
Philadelphia. is a type of somatic mutation, which means
*The Philadelphia chromosome is a you are not born with it. You get it later in life.
reciprocal translocation between the long
arms of chromosomes 9 and 22. 3. Peripheral blood and bone marrow
*A dramatic left shift is noted that extends
down to the promyelocyte stage and
occasionally even produces a few blasts in
the peripheral blood.
*The platelet count is often elevated.
*Extramedullary granulopoiesis may involve
sinusoids and medullary cords in the spleen
and sinusoids, portal tract zones, and solid
areas of the liver.

5. Leukocyte Alkaline Phosphatase
Figure. Peripheral blood films in the chronic

phase of chronic myelogenous leukemia. A,
Leukocytosis is evident at scanning power *Initial testing of the cells for leukocyte
(×100). B, Bimodal population of segmented alkaline phosphatase (LAP) enzyme activity
neutrophils and myelocytes (×500). C, may be useful in some settings for preliminary
Increased basophils, and immature differentiation of CML from a leukemoid
neutrophils (×1000). reaction due to severe infections.
4. Other Laboratory Findings of Chronic *Phases of CML

Myelogenous Leukemia • The Progression of Ph + CML that
*Hyperuricemia and uricosuria from occurs when the condition is left
increased cell turnover may be associated untreated is described in three
with secondary gout, urinary uric acid stones, phases:
and uric acid nephropathy. o Chronic Phase CML
o Accelerated CML
*Approximately 15% of patients exhibit total o Blast Crisis CML
white blood cell (WBC) counts greater than
300 × 109 /L. (Normal WBC Count: 5 to10 x 109/
L)
• Symptoms in these patients are
secondary to vascular stasis and
possible intravascular consumption of
oxygen by the leukocytes.
*Fluorescence in situ hybridization *LAP (Leukocyte Alkaline Phosphatase)

• The diagnosis of CML is confirmed by • LAP is an enzyme found in the
demonstrating the presence of the t membranes of secondary granules of
(9; 22) translocation by cytogenetic neutrophils.
analysis, detection of the BCR/ABL1 • In the procedure, a blood film is
fusion gene using fluorescence in situ incubated with a naphthol-
hybridization, and/or detecting the phosphate substrate and diazo dye
BCR/ABL1 fusion transcript by at an alkaline pH.
qualitative reverse transcriptase • The LAP enzyme hydrolyzes the
polymerase chain reaction. substrate, and the liberated naphthol

reacts with the dye producing a 7. Progression of Chronic Myelogenous
colored precipitate on the granules. Leukemia
The slide is examined microscopically, *In the pre-imatinib era, most cases of this
and 100 segmented neutrophils and disease would eventually transform into
bands are counted and rated from 0 acute leukemia.
to 4+ based on the intensity of the *Additional chromosome abnormalities
staining. reflect the evolution of the malignant clone
and may appear, associated with enhanced
*The LAP score is calculated by multiplying dyshematopoietic cell maturation patterns,
each score by the number of cells and and increases in morphologic and functional
adding the products. abnormalities in blood cells.
*There is often an increasing degree of
*For example: anemia and, in the peripheral blood, fewer
• 5 cells with 4+ mature leukocytes, more basophils, and
• 5 cells with 3+ fewer platelets, with a greater proportion of
• 25 cells with 2+ abnormal platelets, micromegakaryocytes,
• 45 cells with 1+ and megakaryocytic fragments.
• 20 cells with 0 stainings calculate a *Blast crisis involves the peripheral blood,
LAP score of 130. bone marrow, and extramedullary tissues.
*Because scoring is subjective, the mean 8. Related diseases of Chronic Myelogenous

score of two examiners is reported, and they Leukemia
should agree within 10%. *Several diseases exist that are clinically like
• A sample reference interval for the CML but do not exhibit the Philadelphia
LAP score is 15 to 170, but every chromosome and express only a few
laboratory establishes its own. pseudo-Gaucher cells.
• The LAP score is decreased in 1. Chronic neutrophilic leukemia
untreated CML, and normal or 2. Juvenile myelomonocytic leukemia
increased in leukemoid reactions. 3. Adult chronic myelomonocytic
• Individuals with polycythemia vera or leukemia
those in the third trimester of
pregnancy also have higher LAP *The peripheral blood of adults with chronic
scores. myelomonocytic leukemia may have
characteristics like those seen in refractory
6. Treatment for Chronic Myelogenous anemias, such as oval macrocytes and
Leukemia reticulocytopenia. The peripheral WBC
concentration may reach 100 × 109 /L.
According to WHO criteria, absolute
monocytosis (more than 1 × 109
monocytes/L) must be present to make the
diagnosis.
9. Goals of the Therapy for CML

*Goals of therapy include complete
hematologic, cytogenetic, and molecular
remission indicated by a normalized CBC
and differential, absence of Ph1 by
karyotype analysis, and absence of
measurable BCR/ABL transcripts,
respectively.
*Busulphan → to decrease cell growth in the • Imatinib is a type of cancer growth
bone marrow blocker called a tyrosine kinase
*Hydroxyurea → antimetabolite, slows the inhibitor (TKI). Tyrosine kinases are
growth of cells. proteins that cells use to signal to
each other to grow. They act as
chemical messengers. There are

several different tyrosine kinases and the EPO receptor-signaling pathway without
blocking them stops the cancer cells EPO.
from growing. *PV progenitor cells do not divide more
rapidly but accumulate because they do not
*Although imatinib has proven to be a die normally (There is no apoptosis).
successful form of therapy, a major limitation
is the development of imatinib resistance *Because approximately 5% of PV patients
resulting in relapse. Approximately 25% to do not possess the JAK2 V617F mutation and
30% of patients with newly diagnosed CML because PV has a familial predisposition, it is
will discontinue imatinib therapy within 5 thought that other mutations must be
years due to lack of remission, resistance, or involved in the pathogenesis of PV, and
toxicity. some must precede and possibly predispose
the JAK2 V617F mutation.
Polycythemia Vera
2. Diagnosis of Polycythemia Vera
*Polycythemia vera (PV) is a neoplastic *Based on the WHO standards, the diagnosis
clonal myeloproliferative disorder that of PV requires that two major criteria and one
commonly manifests with panmyelosis in the minor criterion be met or that the first major
bone marrow and increases in erythrocytes criterion listed, and two minor criteria be met.
(significant increase), granulocytes, and • 2 major + 1 minor
platelets in the peripheral blood. • 1 major + 2 minor
Splenomegaly is common.
*The disease arises in a hematopoietic stem A. The two major criteria are:
cell. 1. An elevated hemoglobin (Hb) level
• Men: > 18.5 g/dL
• Women: > 16.5 g/dL
2. Identification of the JAK2 V617F mutation,

the JAK2 exon 12 mutation, or a similar JAK2
mutation.
B. The three minor criteria are:

1. Pathogenic Mechanism of Polycythemia 1. Panmyelosis in the bone marrow
Vera 2. Low serum erythropoietin levels
*In PV, neoplastic clonal stem cells are 3. Autonomous, in vitro erythroid colony
hypersensitive to, or function independently formation.
of, erythropoietin for cell growth.
*Understanding of the pathologic C. Additional diagnostic features of PV
mechanism explaining this phenomenon in include:
PV was significantly advanced in 2005 with *An increased RBC mass of 36 mL/kg or
the discovery of a consistent mutation in the greater in males and 32 mL/kg or greater in
JAK2 gene. The specific JAK2 mutation, females
JAK2V617F, is detected in 90% to 97% of *An arterial oxygen saturation of 92%
patients with PV. (normal) or greater.
*Splenomegaly
*Example:
• Erythroid progenitor cells have *Other features of PV are:
receptors for EPO to receive the EPO. • thrombocytosis of greater than 400 ×
However, in polycythemia vera, even 109platelets/L
in the absence of EPO, the receptor is • leukocytosis of greater than 12 × 109
ACTIVATED and STIMULATED, hence cells/L without fever or infection
the proliferation of RBCs. • increases in leukocyte alkaline
phosphatase (LAP)
*Experiments performed with murine cell lines • serum vitamin B12, or unbound vitamin
showed that the mutated JAK2V617F was B12 binding capacity.
constitutively active, and able to activate
3. Peripheral Blood and Bone Marrow panmyelosis (hematoxylin and eosin stain,
*Not only are quantitative changes seen, but ×400).
bone marrow normoblasts may collect in
large clusters 4. Clinical Presentation of Polycythemia Vera
*Megakaryocytes are enlarged and exhibit *PV is always associated with increased RBC
lobulated nuclei mass. This is the stable phase of PV, which
*Bone marrow sinuses are enlarged without progresses to a spent phase in a few patients.
fibrosis
*Approximately 80% of patients manifest *In the spent phase, patients experience
bone marrow panmyelosis, and 100% of progressive splenomegaly (palpable spleen)
bone marrow volume may exhibit or hypersplenism (large spleen with bone
hematopoietic cellularity. marrow hyperplasia and peripheral blood
*Although the bone marrow pattern may cytopenias) and pancytopenia.
mimic that of other MPNs, the peripheral
blood cells appear normal, with normocytic, *They may also exhibit the triad of bone
normochromic erythrocytes; mature marrow fibrosis, splenomegaly, and anemia
granulocytes; and normal-sized, granulated with teardrop-shaped poikilocytes. The latter
platelets. The other 20% of patients exhibit pattern is called postpolycythemic myeloid
lesser degrees of cellularity in the bone metaplasia, and its morphologic features are
marrow and peripheral blood. like those of PMF.
*Splenomegaly, hepatomegaly, generalized
vascular engorgement, and circulatory *Peripheral WBC and RBC counts vary, and
disturbances increase the risk of nucleated erythrocytes, immature
hemorrhage, tissue infarction, and granulocytes, and large platelets are
thrombosis. present.
*Usually, splenomegaly is secondary to

extramedullary hematopoiesis.
*Myelofibrosis occurs within the bone

marrow and may come to occupy a
significant proportion of bone marrow
volume, with subsequent ineffective
hematopoiesis.
*One MPN can become another MPN

Figure. Peripheral blood film in stable phase
• Polycythemia vera can become
polycythemia vera with essentially
Essential Thrombocythemia. Both can
normocytic, normochromic erythrocytes
also become Primary Myelofibrosis.
(×500).
Figure. Bone marrow biopsy specimen in

stable phase polycythemia vera showing

*Majority of cases occur in individuals
between the ages of 50 and 60 years, but a
second peak occurs primarily in women in
the childbearing years, approximately 30
years of age.
2. Pathogenic mechanism of Essential

Thrombocythemia
*Most of the mutations described in PV also
occur in ET but usually at a lower frequency.
1. The JAK2 V617F occurs in
approximately 55% of patients with ET.
2. MPL exon 10 mutations (MPL
W515L/K) are observed in 3% of ET
patients
3. TET2 (4.4% to 5%)
5. Treatment and Prognosis for Polycythemia 4. ASXL1 (5.6%)
Vera 5. LNK (3% to 6%)
A. Treatment 6. IDH1/2 (0.8%).
*The treatment of choice for PV is therapeutic
phlebotomy at a frequency necessary to *The way these mutations alter normal
maintain the hematocrit at less than 45%. cellular functions is like PV.
*Low-dose aspirin has been shown
efficacious to minimize thrombosis in all risk 3. Clinical Presentation of Essential
categories. Thrombocythemia
*Vascular occlusions are often the result of
*The alkylating agent hydroxyuria is microvascular thromboses in the digits or
recommended in high-risk patients with PV thromboses in major arteries and veins that
and can be substituted for INF-γ in younger occur in a variety of organ systems, including
patients and busulfan in older patients who splenic or hepatic veins, as in Budd-Chiari
develop intolerance or resistance to syndrome.
hydroxyuria.
*Bleeding occurs most frequently from
B. Prognosis mucous membranes in the gastrointestinal
*Prognosis for patients with PV is good, with a and upper respiratory tracts.
median survival exceeding 15 to 20 years.
However, the disease progresses to acute *Splenomegaly is observed at presentation in
leukemia in 15% of patients. 50% of patients.
*Treatment with modern JAK inhibitors
Essential Thrombocythemia
*Essential thrombocythemia (ET) is a clonal

MPN with increased megakaryopoiesis and
thrombocytosis, usually with a count greater
than 600 × 109 /L and sometimes with a count
greater than 1000 × 109 /L. However, WHO
criteria require a sustained thrombocytosis
with a platelet count of 450 × 109 /L or
greater. Over the years, ET has been known
as primary thrombocytosis, idiopathic Figure. Peripheral blood film in stable phase
thrombocytosis, and hemorrhagic essential thrombocythemia shows increased
thrombocythemia. numbers of platelets and mature neutrophils
(×500).
1. Incidence of Essential Thrombocythemia
*Incidence is estimated to be between 0.6
and 2.5 cases per 100,000 persons per year
were refractory or intolerant to hydroxyuria.
In 15 of the 39 patients who completed 18
weeks of treatment, 83% (15/18) achieved
some degree of spleen size reduction, 20%
(3/15) had a 15% decrease in JAK2 allele
burden, and 15% developed thrombosis and
had frequent gastrointestinal events
*Patients with ET experience relatively long

survival provided they remain free of serious
thromboembolic or hemorrhagic
complications.
5. Clinical Symptoms of Essential

Thrombocythemia
Figure. Source: Bone marrow biopsy *Clinical symptoms associated with
specimen in essential thrombocythemia thromboembolic vaso-occlusive events
showing marked megakaryocytic include the syndrome of:
hypercellularity (hematoxylin and eosin stain, • erythromelalgia (throbbing and
×400). burning pain in the hands and feet,
accompanied by mottled redness of
4. Treatment and Prognosis for Essential areas)
Thrombocythemia • transient ischemic attacks
*Treatment involves prevention or early • seizures
alleviation of hemorrhagic or vasoocclusive • cerebral or myocardial infarction.
complications that occur as the platelet
count increases. The production of platelets
must be reduced by suppressing marrow Aquagenic pruritus
megakaryocyte production with an
alkylating agent like hydroxyuria. Present in Essential Thrombocythemia and
Polycythemia vera. It is characterized by
*JAK2 inhibitors are being investigated in ET strong itching, stinging, tingling, or burning
patients who are refractory or intolerant to sensations following contact with water
hydroxyuria or are otherwise high risk. without visible changes in the skin
*INCB018424 (ruxolitinib) was studied in 39

patients with ET at a dose of 25 mg b.i.d. In all *Other symptoms include:
39 patients, the median platelet count • headache, dizziness, visual
reduced from 884 to 558 × 109 /L, and the 11 disturbances, and dysesthesias
patients who had leukocytosis achieved a (decreased sensations). Hemorrhagic
normal WBC count after 6 months of complications include bleeding from
treatment. Four patients who demonstrated oral and nasal mucous membranes
splenomegaly showed spleen size reduction; or gastrointestinal mucosa and the
40% to 75% of patients had a 50% or greater appearance of cutaneous
improvement in one or more of the following: ecchymoses.
pruritus, bone pain, night sweats, and
peripheral tingling/numbness. Only 13% (5 *The median survival for patients with ET is 20
patients), achieved complete remission. years, including cases in which the process
However, a follow-up report at 10.4 months arises in younger patients. However, some
of treatment showed that 92% were still patients may develop post-ET myelofibrosis,
participating in the study, no grade 3 or 4 which reduces survival.
hematologic complications were noted, and
although cytopenias were observed in 10% *Patients whose cells manifest chromosome
to 20%, they were grade 2 (mild). abnormalities may have a poorer prognosis.
*CEP701 (Lestaurtinib) was studied in 27

patients with PV and 12 patients with ET who
Myelofibrosis *A. Leukemia (ANLL-M7, ALL)
*Hairy Cell Leukemia
1. Primary Myelofibrosis *Metastatic Carcinoma
*Primary myelofibrosis (PMF), previously *Infection: E.g., Tbc, Fungal infection
known as: *SLE, Systematic sclerosis
• chronic idiopathic myelofibrosis *Systematic Mastocytosis
• agnogenic myelofibrosis *Hypovitamin D, renal osteodystrophy
• myelofibrosis with myeloid metaplasia *Hypoparathyroidism, Hyperparathyroidism,
etc.
*Is a clonal MPN in which there is
splenomegaly and ineffective 3. Myelofibrosis
hematopoiesis associated with areas of *The myelofibrosis in this disease consists of
marrow hypercellularity, fibrosis, and three of the five types of collagens: I, III, and
increased megakaryocytes. IV.
*Megakaryocytes are enlarged with *In approximately 30% of patients, biopsy
pleomorphic nuclei, coarse segmentation, specimens show no fibrosis.
and areas of hypochromia. *Increases in these collagens are not a part
of the clonal proliferative process but are
considered secondary to an increased
release of fibroblastic growth factors.
*Marrow fibrosis causes expansion of marrow
sinuses and vascular volume, with an
increased rate of blood flow.
*Increased production of megakaryocytes
4. Hematopoiesis and Extramedullary

Hematopoiesis
*Since there’s overcrowding and overuse of
the bone marrow, the body will find other
sites for hematopoiesis.
Figure. The peripheral blood film exhibits *Extramedullary hematopoiesis, clinically
immature granulocytes and normoblasts, recognized as hepatomegaly or
dacryocytes (teardrop-shaped RBCs), and splenomegaly, seems to originate from the
other bizarre RBC shapes. release of clonal stem cells into circulation.
The cells accumulate in the spleen, liver, or
2. Causes of Myelofibrosis other organs, including adrenals, kidneys,
lymph nodes, bowel, breasts, lungs,
mediastinum, mesentery, skin, synovium,
thymus, and lower urinary tract.
*The disease is associated with an increase in

circulating hematopoietic cells, but
fibroblasts are a secondary abnormality and
not clonal.
*B and T cells may be involved.
*There is an increase in circulating unilineage
and multilineage hematopoietic progenitor
cells, and the number of CD34+ cells maybe
300 times normal.
*Body cavity effusions containing

hematopoietic cells (extramedullary
hematopoiesis in the cranium, the intraspinal
*Idiopathic Myelofibrosis and PRV, ET, CML, epidural space, or the serosal surfaces of
MDS pleura, pericardium, and peritoneum).
*Lymphoma, Multiple Myeloma

4. Hematopoiesis and Extramedullary *Platelets show impaired aggregation in
Hematopoiesis Continuation response to epinephrine, decreased
A. Complications adenosine diphosphate concentration in
*Portal hypertension, with its attendant dense granules, and decreased activity of
consequences of ascites, esophageal and platelet lipoxygenase.
gastric varices, gastrointestinal hemorrhage
*Hepatic encephalopathy arises from the C. Immune Response
combination of a massive increase in *Humoral immune responses are altered in
splenoportal blood flow and a decrease in approximately 50% of patients and include
hepatic vascular compliance. the appearance of autoantibodies to
erythrocyte antigens, nuclear proteins,
B. Incidence and clinical presentation gamma globulins, phospholipids, and organ-
*The disease occurs in patients older than specific antigens.
age 60 and may be asymptomatic. *Circulating immune complexes, increased
*PMF generally presents with: proportions of marrow-reactive lymphocytes,
• Fatigue and the development of amyloidosis are
• Weakness evidence for active immune processes.
• Shortness of breath *Collagen disorders coexist with PMF, which
• Palpitations suggests that immunologic processes may
• Weight loss stimulate marrow fibroblast activity.
• Discomfort or pain in the left upper
quadrant (associated with 5. Diagnosis of Myelofibrosis
splenomegaly).
*Abnormalities in
erythrocytes noted on
peripheral blood films
include the presence of
dacryocytes, other
bizarre shapes,
nucleated RBCs, and
polychromatophilia.
*Granulocytes are increased, normal, or
decreased in number and may include
immature granulocytes, blasts, and cells with 6. Treatment and Prognosis of Myelofibrosis
nuclear or cytoplasmic anomalies. *Treatment has been targeted at the
*Platelets may be normal, increased, or amelioration of anemia,
decreased in number, with a mixture of hepatosplenomegaly, and constitutional
normal and abnormal morphologic features. symptoms.
Micromegakaryocytes may be observed.
*Bone marrow biopsy specimens exhibit *Severe anemia has been treated with
intense fibrosis, granulocytic and androgen therapy, prednisone, danazol,
megakaryocytic hypercellularity, thalidomide, or lenalidomide, and hemolytic
dysmegakaryopoiesis, dysgranulopoiesis, anemia with glucocorticosteroids.
and numerous dilated sinuses containing • The most common constitutional
luminal hematopoiesis. symptoms encountered by patients
with PMF include fatigue (84%), bone
*Neutrophils may exhibit pain (47%), night sweats (56%),
impairment of pruritus (50%), and fever (18%).
physiologic functions
such as phagocytosis, *The development and testing of JAK
oxygen consumption, inhibitors were directed at PMF because the
hydrogen peroxide symptoms and outcomes are worse
generation, and decreased compared to those of PV and ET.
myeloperoxidase and glutathione reductase *Reduction of myelofibrosis and marrow and
activities. tissue hypercellularity has been

accomplished with busulfan hydroxyurea dominate, but the increase in bands,
and, in a few patients, interferon-α and metamyelocytes, myelocytes, and
interferon-γ. promyelocytes in combination usually
*Chemotherapy is partially successful in comprise fewer than 5% of WBCs but can be
reducing the number of CD34+ cells and as many as 10%.
immature hematopoietic cells, marrow *Neutrophils do not appear dysplastic, but
fibrosis, and splenomegaly. they often contain toxic granules.
• Busulfan
• 6-thioguanine *RBC and platelet morphology are normal in
• Chlorambucil the peripheral blood.
*The bone marrow reflects the peripheral
*The most successful treatment to date for blood in that it is hypercellular with
patients younger than age 60 is allogeneic predominantly a proliferation of neutrophils,
stem cell transplantation. including myelocytes, metamyelocytes,
bands, and segmented neutrophils.
Other Myeloproliferative Neoplasms
*The myeloid-to-erythroid ratio is at least 20:1.
1. Chronic Neutrophilic Leukemia RBCs and platelets are normal in number,
*Chronic neutrophilic leukemia (CNL) is a and no cell line exhibits significant dysplastic
clonal disorder in which a hyperproliferation morphology.
of neutrophilic cells in the bone marrow
produces sustained neutrophilia in the 2. Chronic Eosinophilic Leukemia
peripheral blood and hepatosplenomegaly. *Chronic eosinophilic leukemia (CEL) is a
*CNL must be differentiated from CML, clonal proliferation of eosinophils from
based on the absence of the Philadelphia eosinophil precursors that dominate in the
chromosome and the BCR/ABL fusion gene, bone marrow and peripheral blood.
as well as from both a reactive neutrophilic *Infiltrating eosinophils degranulate to
process and other MPNs. release cytokines, enzymes, and other
granular proteins that damage the
A. Clinical Presentation of CNL surrounding tissue, which results in organ
*Hepatosplenomegaly is the most common dysfunction.
finding, but 25% to 30% of patients report
bleeding from mucocutaneous sites like the A. Clinical Presentation of CEL
gastrointestinal tract. *Although some patients may be
*Other symptoms include gout from WBC asymptomatic when found to have
turnover and pruritus that may be associated eosinophilia, most have signs and symptoms
with neutrophil infiltration of tissues and of fever, fatigue, cough, angioedema,
organs. muscle pain, and pruritus.
B. Peripheral Blood and Bone Marrow of CNL *A more severe sequela of CEL involves the
heart.
• Fibrosis can form in the heart
(endomyocardial fibrosis), which can
evolve into cardiomegaly. Within the
heart, scar tissue may form in the
mitral and tricuspid valves, affecting
valve function and predisposing to
thrombi formation.
*Other serious complications:

• peripheral neuropathy
• central nervous system dysfunction
• pulmonary symptoms from
eosinophilic infiltrate
• rheumatologic problems
*Patients have a WBC count of more than 25
× 109 /L with a slight left shift. Neutrophils
2. Chronic Eosinophilic Leukemia *Systemic mastocytosis
Continuation • indolent systemic mastocytosis
B. Peripheral Blood and Bone Marrow of CEL • smoldering mastocytosis
*Peripheral eosinophilia must be observed, • aggressive systemic mastocytosis
with most eosinophils appearing normal. • systemic mastocytosis with
*The bone marrow is hypercellular owing to associated hematological neoplasia
eosinophilic proliferation and can (SM-AHN)
demonstrate Charcot-Leyden crystals. • mast cell leukemia
*Myeloblast numbers are elevated but below *Mast cell sarcoma (isolated focal
the 20% threshold necessary to classify the involvement of an extracutaneous organ or
disorder as acute leukemia. tissue)
*Erythrocytes and megakaryocytes are B. Incidence of Mastocytosis

normal in number but sometimes * Mastocytosis can occur at any age.
demonstrate dysplastic morphologic • Approximately 80% of patients with
features. mastocytosis show skin involvement
*Bone marrow fibrosis occurs due to the regardless of the type of mastocytosis
release of eosinophilic basic protein and diagnosed.
eosinophilic cationic proteins from the
eosinophil granules. Bone marrow fibrosis *Cutaneous mastocytosis occurs in the skin
contributes to the premature release of (mostly in children)
eosinophils into circulation, and they deposit *Systemic mastocytosis usually involves the
in a variety of tissues. bone marrow and other organ systems like
the spleen, lymph nodes, liver, and
gastrointestinal tract. (Occurs after the
second decade of life)
*Mast cell leukemia is characterized by mast
cells in the peripheral blood.
C. Clinical Presentation of Mastocytosis
C. Diagnosis of CEL
*The diagnosis of CEL requires eosinophilia
with a count of more than 1.5 × 109 cells/L
and the presence of malignant features, and
the elimination of reactive eosinophilia and
other malignancies that have concomitant
eosinophilia.
3. Mastocytosis
*Mastocytosis is a broad term referring to a
clonal neoplastic proliferation of mast cells,
which accumulate in one or more organ
systems, but it can present differently and
manifest in a range of severities.
*The WHO group has classified mastocytosis
into seven subcategories: cutaneous
mastocytosis, indolent systemic mastocytosis, *Patients present with urticarial lesions (wheel
systemic mastocytosis with associated clonal and flare) that may become activated when
hematologic non–mast-cell-lineage disease, stroked upon physical examination. Skin
aggressive systemic mastocytosis, mast cell lesions also tend to have melanin
leukemia, mast cell sarcoma, and pigmentation.
extracutaneous mastocytoma. *Four categories of symptom severity have
been described in mastocytosis:
A. WHO Revised Classification 2016 • constitutional systems like fatigue and
*Cutaneous mastocytosis weight loss; skin manifestations;
mediator-related systemic events mastocytosis occurs in three forms: (all of
such as abdominal pain, which occur predominantly in children
gastrointestinal distress, headache • urticaria pigmentosa
• respiratory symptoms • diffuse cutaneous mastocytosis
• musculoskeletal complaints like bone • mastocytosis of the skin
pain, arthralgias, and myalgias. Extracutaneous mastocytoma also
exhibits a unifocal mast cell tumor,
*Hematologic findings include: but it is of low-grade pathology.
• Anemia
• Leukocytosis E. Prognosis of Mastocytosis
• Eosinophilia *Cutaneous mastocytosis in children has a
• Neutropenia favorable prognosis and may regress
• Thrombocytopenia spontaneously around puberty. Milder
versions like cutaneous mastocytosis and
*In patients with systemic mastocytosis with indolent cutaneous mastocytosis follow a
associated clonal hematologic non–mast- benign course and are associated with a
cell-lineage disease the most common normal lifespan
associated hematologic finding is chronic
myelomonocytic leukemia, but any myeloid 4. Myeloproliferative neoplasm,
or lymphoid malignancy can occur, unclassifiable
although myeloid versions predominate. *The category myeloproliferative neoplasm,
unclassifiable (MPN-U) is designed to capture
D. Diagnosis of Mastocytosis disorders that clearly express
myeloproliferative features but either fail to
meet the criteria of a specific condition or
have features that overlap two or more
specific conditions.
*Most patients with MPN-U fall into one of

three groups:
• Patients with an early stage of PV, ET,
or PMF in which the criteria that
define the disorders are not yet fully
developed.
• Patients presenting with features
indicative of advanced disease
resulting from clonal evolution that
masks the potential underlying
condition.
• Patients who have clear evidence of
an MPN but who have a concomitant
condition like a second neoplasm or
an inflammatory condition that alters
*The typical skin lesion is the first diagnostic the MPN features.
clue to mastocytosis. Cutaneous

Summary of Myeloproliferative Neoplasm
1. Chronic Myeloid Leukemia
2. Polycythemia Vera

3. Essential Thrombocythemia 4. Primary Myelofibrosis
PARAMETER CML PV ET PMF

Normal or Normal or slightly Normal,
WBC Increased decreased increased increased or
decreased
RBC Normal or Increased Normal or slightly Normal or
decreased increased decreased
Normal or Normal or Normal,
Platelets decreased increased Increased increased or
decreased
Molecular JAK2 V617F or
abnormalities BCR-ABL1 other JAK2 ±JAK2 ±JAK2
mutation
*CML, Chronic myelogenous leukemia; ET, Essential thrombocythemia; PMF, Primary myelofibrosis;
PV, Polycythemia vera; RBC, Red blood cells; WBC, White blood cells.
MYELOPROLIFERATIVE NEOPLASM (MPNS) KEY POINTS
*Genetic mutation in hematopoietic stem cells = expansion, excessive production, and

accumulation of RBCs, WBCs, and platelets.
*There is no apoptosis = continuous cell proliferation
1. Chronic Myelogenous Leukemia (CML)

*Continuous release of immature WBCs leading to infection
*Lesser RBCs leading to anemia
*Many BUT non-functional platelets lead to bleeding
*Splenomegaly
* Translocation between the long arms of chromosomes 9 and 22.
*BCR-ABL mutation
*Philadelphia chromosome → mutated chromosome 22
*It is a somatic mutation, not inherited
*Diagnosis of CML: the presence of t (9;22) translocation or the detection of BCR/ ABL1 fusion gene
*Treatment: Busulphan and Hydroxyurea
*Can progress to acute leukemia
2. Polycythemia Vera
*Panmyelosis
*↑ RBCs, granulocytes, and platelets

*Splenomegaly
*Consistent mutation/ continually active JAK2 gene (JAK2V617F)
*The receptors are still activated even in the absence of G-CSF
*Accumulation of PV progenitor cells
*Increased RBC mass
*Spent phase → patient experiences progressive splenomegaly, hypersplenism, and
pancytopenia
*Treatment:
• Therapeutic phlebotomy
• Hydroxyuria
• If intolerance or resistance to hydroxyuria:
o INF-γ for infants
o Busulfan for adults
3. Essential Thrombocythemia
*Increased megakaryopoiesis and thrombocytosis
*Mutations described in PV also occur in ET BUT usually at the lower frequency
*Clinical Presentations:
• Vascular occlusion
• Bleeding
• Splenomegaly
*Treatment: Production of platelets must be reduced by suppressing marrow megakaryocyte
production using an alkylating agent like hydroxyuria

QUALITATIVE DISORDERS OF LEUKOCYTES
Morphologic Abnormalities with and without 2. Pseudo- or Acquired Pelger-Huët Anomaly

Functional Defects *Neutrophils with PHA morphology can be
observed in patients with hematologic
MORPHOLOGIC ABNORMALITIES WITHOUT malignancies such as:
FUNCTIONAL DEFECTS • Myelodysplastic Syndromes (MDS)
• Acute Myeloid Leukemia
1. Pelger-Huët Anomaly (PHA) • Chronic Myeloproliferative
*Pelger-Huët anomaly (PHA), also known as Neoplasms → E.g., polycythemia
true or congenital PHA, is an autosomal vera, CML, essential
dominant disorder characterized by thrombocythemia. The bone marrow
decreased nuclear segmentation (bilobed, keeps on producing cells that could
unilobed). There is nuclear lead to cancer.
hyposegmentation.
*Pseudo-PHA neutrophils can also be seen in
*Characteristic coarse chromatin clumping infections:
pattern potentially affecting all leukocytes. • HIV Infection
• Tuberculosis
• Mycoplasma Pneumoniae
• Severe Bacterial Infections
*Drugs are known to induce pseudo-PHA to

include:
• Mycophenolate mofetil
• Valproate
• Sulfisoxazole
• Ganciclovir
*A result of a mutation in the lamin β-receptor • Ibuprofen
gene. • Chemotherapies such as paclitaxel
• The lamin β receptor is an inner and docetaxel.
nuclear membrane protein that
combines β-type lamins and
heterochromatin and plays a major
role in leukocyte nuclear shape.
*The nuclei may appear round, ovoid, or

peanut-shaped. Bilobed forms—the
characteristic spectacle-like (“pince-nez”)
morphology with the nuclei attached by a
Figure. Wright stain showing a Pseudo-Pelger-
thin filament—can also be seen.
Huet neutrophil and a band on the
*Neutrophils in Pelger-Huët anomaly appear
peripheral blood of a patient with a Chronic
to function normally.
Myeloproliferative Disorder, Unclassified.
Differentiating Between True PHA and

Pseudo-PHA
True PHA Pseudo-PHA
Number of cells Number of cells
present with PHA present with PHA
morphology: The morphology: The
number of affected number of affected
cells is much higher cells is much lower
*63% to 93% *< 38%
Figure. Pelger-Huët cell. Pince-nez form with all WBC lineages the phenomenon is
two rounded segments connected by a are potentially usually seen only in
filament.
Hematology 1 Lecture MLS 113A | Qualitative Disorders of Leukocytes
affected in terms of neutrophils, except *Myelokathexis refers to a rare hereditary
nuclear shape and for some cases of condition characterized by normal
chromatin structure. MDS where granulocyte production; however, there is
monocytes, impaired release into the circulation that
eosinophils, and leads to neutropenia.
basophils may *Occurs because of mutations that increase
exhibit PHA the function of the chemokine receptor
morphology. CXCR4.
In true PHA, Hypogranular • The CXCR4 receptor upon binding its
neutrophils exhibit neutrophils are a ligands triggers multiple signaling
normal granulation. common finding in pathways that orchestrate cell
MDS-related migration, hematopoiesis and cell
pseudo-PHA. homing, and retention in the bone
Peripheral blood marrow.
smears of family *Neutrophils appear hypermature. There
members may may be hypersegmentation, hyper
reveal similar condensed chromatin, and pyknotic
findings in true PHA. changes. Cytoplasmic vacuoles may also be
observed.
*Myelokathexis is a component of an
extremely rare inherited disorder, WHIM
Syndrome, a syndrome in which warts,
neutropenia, hypogammaglobulinemia,
infections, and myelokathexis are common
findings.
Figure. PHA Eosinophil. This cell is supposed to

be segmented but it does not show any
segmentation. It looks like a metamyelocyte
BUT with a smaller nucleus. Metamyelocytes
have a bigger nucleus.
Figure. True PHA
Figure. PPHA in MDS. Bilobed nucleus but it’s

hypogranular.
3. Myelokathexis
*(from the Greek, meaning ‘retained in the
bone marrow)

*Also, in some patients with Alder-Reilly
anomaly, the granules are found in
LYMPHOCYTES and MONOCYTES, ruling out
toxic granulation, which is exclusive to
NEUTROPHILS.
Figure. Two neutrophils from a patient with

Alder-Reilly anomaly. Note the dark granules
present in both cells. Such granules may also
be seen in eosinophils and basophils. Source:
(Courtesy Dennis R. O’Malley, MD, US Labs,
Irvine, CA.)
A. Toxic Granulation
*Toxic granulation can be associated with
infection and inflammation. Increased
granulation of neutrophils may also be
present in some genetic disorders, following
treatment with myeloid growth factors (G-
CSF or GM-CSF), in a marrow responding to
myelosuppressive therapy, with pregnancy,
and in uremia.
Figure. Hypersegmented neutrophils
4. Alder-Reilly Anomaly
*Alder-Reilly anomaly is transmitted as a
recessive trait and is characterized by
granulocytes with large, darkly staining
metachromatic cytoplasmic granules
composed primarily of partially digested
mucopolysaccharides. The problem is not
anymore in the nucleus BUT in the granules.
*The granules are referred to as Alder-Reilly
bodies or Reilly bodies. The morphology may
resemble heavy toxic granulation.
*The following are usually associated with

TOXIC GRANULATION and are NOT SEEN in
ALDER-REILLY ANOMALY.
• Neutrophilia
• Döhle bodies → remnant of RER
• left shift

A. Toxic Granulation Continuation granules result in leukocyte dysfunction and
recurrent pyogenic infections.
Figure. Three cells from a patient with

Figure. The presence of increased numbers
Chédiak-Higashi syndrome. A, Neutrophil
of circulating non-segmented neutrophils.
with large dark lysosomal granules. B,
Monocyte with large azure granules. C,
4. Alder-Reilly Anomaly Continuation
Lymphocyte with one large azure granule.
*The basic defect is the incomplete
degradation of mucopolysaccharides.
A. Chédiak-Higashi Syndrome (CHS)
Affecting the Platelets
*Reilly bodies are most associated with:
*Patients often have bleeding issues due to
• Hurler syndrome
abnormal dense granules in platelets.
• Hunter syndrome
• Maroteaux-Lamy polydystrophic
*Chédiak-Higashi syndrome is associated
dwarfism
with a mutation in the CHS1/ LYST Lysosomal
trafficking regulator gene on a chromosome
*LEUKOCYTE FUNCTION IS NOT AFFECTED in
that encodes for a protein involved in vesicle
Alder-Reilly anomaly
fusion or fission.
*Fusion of different lysosomal granules.
Morphologic Abnormalities with and without
Functional Defects Continuation
*Platelets have 3 types of granules:
• Alpha granules
MORPHOLOGIC ABNORMALITIES WITH
• Dense granules → important
FUNCTIONAL DEFECTS
because it contains several
substances that are necessary for
1. Chédiak-Higashi Syndrome (CHS)
platelet aggregation. E.g., ADP
*Chédiak-Higashi syndrome is a rare, fatal,
(Adenosine diphosphate) → allows
autosomal recessive disease.
the platelet to bind to another
platelet to form aggregation.
*The disease is characterized by abnormal
Abnormality in the platelet (dense
FUSION OF GRANULES making them BIG/
granules) leads to bleeding tissues
GIANT in most cells that contain granules
because there is no platelet
throughout the body. This does not only
aggregation.
affect WBCs but also affects all the cells that
• Lysosomes
contain granules such as platelets.
*The fused granules are large and mostly
DYSFUNCTIONAL.
*Hematopoietic cells are affected, but

disease manifestations can be found in hair,
skin, adrenal and pituitary glands, and
nerves.
*Hematologic findings in include giant

lysosomal granules in granulocytes,
monocytes, and lymphocytes. These fused

variable thrombocytopenia, GIANT
PLATELETS (as big as the RBC), and large
Döhle body–like inclusions in neutrophils,
eosinophils, basophils, and monocytes.
*There is disordered production of myosin
heavy chain type IIA (non-muscle myosin)
which affects megakaryocyte maturation
and platelet fragmentation. The basophilic
Döhle body–like leukocyte inclusions in the
May-Hegglin anomaly is composed of
precipitated myosin-heavy chains. (Looks
like a Döhle body BUT it is not) Döhle bodies
are composed of lamellar rows of the rough
endoplasmic reticulum.
*Clinically, most individuals with May-Hegglin
anomaly are asymptomatic, but a few have
B. Manifestations of Chédiak-Higashi mild bleeding tendencies that are related to
Syndrome (CHS) the degree of thrombocytopenia.
*Staph/ Strep infections
*Partial albinism Figure. A neutrophil and
*Neurologic symptoms a giant platelet from a
*Accelerated phase → lymphoma patient with May-
*Early bone marrow transplantation Hegglin anomaly. Note
the large, elongated,
2. May-hegglin anomaly bluish inclusion in the
*May-Hegglin anomaly is a rare, autosomal neutrophil cytoplasm.
dominant platelet disorder characterized by

Monocyte/Macrophage Lysosomal Storage *The MPSs have been subdivided according

Diseases to which enzyme is defective, which GAG is
being stored, and whether the symptoms are
*Monocyte/macrophage lysosomal storage severe or attenuated.
diseases can be subdivided into: *The peripheral blood of a patient with MPSs
1. Mucopolysaccharide (or may appear relatively normal; however,
glycosaminoglycan [GAG]) storage metachromatic Reilly bodies may be seen in
diseases neutrophils, monocytes, and lymphocytes.
2. Lipid storage diseases *Bone marrow may reveal macrophages
with large amounts of metachromatic
*As a group, they represent inherited enzyme material.
deficiencies or defects that result in the *Diagnosis relies on assays for the specific
flawed degradation of phagocytized enzymes involved. Treatment has consisted
material and the buildup of the partially of enzyme replacement therapy or
digested material within the phagocyte. All hematopoietic stem cell transplantation.
cells containing lysosomes can be affected,
including T lymphocytes. Figure. Lymphocyte on
the blood film for a
1. Mucopolysaccharidoses (MPSs) patient with a
*The mucopolysaccharidoses (MPSs) are a mucopolysaccharide
family of inherited disorders of (GAG) storage disorder known
glycosaminoglycans degradation. as Hurler disease. Notice
the dark cytoplasmic
*Each MPS is caused by deficient activity of granules
an enzyme necessary for the degradation of:
• dermatan sulfate 2. Lipid Storage Diseases
• heparan sulfate A. Gaucher Disease
• keratan sulfate *The most common of the lysosomal lipid
• and/or chondroitin sulfate storage diseases.
*It is an autosomal recessive disorder caused
*The partially degraded GAG builds up in the by a defect or deficiency in the catabolic
lysosomes and eventually results in physical enzyme β-glucocerebrosidase, which is
abnormality and sometimes mental necessary for glycolipid metabolism.
retardation.
*There is an accumulation of unmetabolized *In these diseases, pseudo-Gaucher cells
substrate sphingolipid glucocerebroside in form as a result of excessive cell turnover and
macrophages throughout the body, overwhelming the glucocerebrosidase
including osteoclasts in bone and microglia enzyme rather than a true decrease in the
in the brain. enzyme.
*Electron microscopy can distinguish
A.1 The clinical triad used in diagnosis is: between the two cells because pseudo-
• Hepatomegaly Gaucher cells do not contain the tubular
• Gaucher cells in the bone marrow inclusions described in Gaucher cells.
• Increase in serum phosphatase
*Gaucher disease has been subdivided into

three types based on clinical signs and
symptoms. Neurologic symptoms play a key
role in differentiating between the three
subtypes.
*The phenotype is quite heterogeneous, with
some patients being completely
asymptomatic (seen in Type I), while others
experience a multitude of clinical problems.
Clinical findings are mostly related to the
patient’s age and the degree of enzyme
deficiency.
Figure.
‘Onion Skin’
Characteristic
macrophages with
cytoplasmic striations
found in the bone *62-year-old female with a history of ethnic
marrow of a patient with leukopenia, who developed CML and
Gaucher disease. became severely leukopenic while on
Source: (From Carr JH, Rodak BF: Clinical treatment with imatinib.
hematology atlas, ed 4, St. Louis, 2013, *Repeat marrow revealed infiltration with
Saunders.) Pseudo-Gaucher cells (secondary to CML).
Pseudo-Gaucher cells are histiocytes with
Figure. The rounded, blue, lamellar cytoplasm
delicate resembling "onion skin" that can be found in
cytoplasm of up to 40% of the bone marrow of patients
Gaucher cells in a with CML. These are like glucocerebroside-
bone marrow stuffed histiocytes seen in Gaucher disease.
smear resembles
crinkled tissue 2. Lipid Storage Disease Continuation
paper. B. Niemann-Pick Disease
*Types of Niemann-Pick Disease
A.2 Treatment of Gaucher disease • Types A and B → almost similar
*Use of enzyme replacement therapy with because of the absence of
recombinant glucocerebrosidase sphingomyelinase. Type A is the
*Stem cell transplantation absence of sphingomyelinase. Type B
*Glucocerebroside inhibitors is only a deficiency in
sphingomyelinase.
A.3 Pseudo-Gaucher Cells • Type C
*A secondary effect of a specific disease
*In the bone marrow in some patients with: B.1 Types A and B Niemann-Pick Disease
• Thalassemia *Types A and B are caused by a missing or
• Chronic Myelogenous Leukemia malfunctioning enzyme called
• Acute Lymphoblastic Leukemia sphingomyelinase. This affects the body's
ability to metabolize fat (cholesterol and
lipids), resulting in a buildup of fat in cells. This *The prognosis in type C is poor. Most patients
causes cell dysfunction and, over time, cell die before the age of 25 years.
death.
• Type A occurs mainly in infants, who Figure. Niemann-Pick cell
show severe, progressive brain with an eccentric
disease. There is no cure, so most nucleus and
children do not live beyond their first bubble/foam-like
few years. pattern of storage
• Type B usually occurs later in deposit in the cytoplasm.
childhood and is not associated with Source: (From Carr JH,
primary brain disease. Most people Rodak BF: Clinical hematology atlas, ed 4, St.
affected with type B survive into Louis, 2013, Saunders.)
adulthood.
Genetic B and T Lymphocyte Abnormalities
*Functional B and T lymphocyte

abnormalities are genetic disorders that
B.2 Type C Niemann-Pick Disease
generally result in the decreased production
of B cells, T cells, or both.
*They are all associated with an increased risk
of infection and secondary malignancy.
1. T Lymphocyte Abnormality (DiGeorge

Syndrome)
*The problem IS NOT in the ENZYME. The

problem is in the GENE.
*Niemann-Pick type C is a rare inherited
disease. There is a decrease in cholesterol
effluxing from the intracellular
*Characterized by the absence or
endosome/lysosome to the cytosol. Genetic
underdevelopment of the thymus and thus
mutations of this type cause cholesterol and
markedly decreased numbers of T
other fats to accumulate in the liver, spleen,
LYMPHOCYTES.
or lungs. The brain is eventually affected too.
*It is associated with a microdeletion in *BTK is important for B cell development,
chromosome band 22q11.2 differentiation, and signaling. Without BTK, B
lymphocytes fail to reach maturity and will
*Defective parathyroid glands, cardiac die prematurely. Results in nonproduction of
abnormalities, abnormal facial antibodies.
development, neurologic disorders, and
hypocalcemia. A. Symptoms of Sex-linked
Agammaglobulinemia
*Less than 1% of patients with this deletion are *First few months of life are relatively normal
athymic, a condition sometimes referred to (maternal Ig)
as complete DiGeorge syndrome. *Tonsils are small, lymph nodes are barely
*Many of these patients are treated with palpable
thymus transplantation. *Recurrent infection of sinuses and the
middle ear. Pneumonia
*Pyogenic bacteria → permanent tissue
damage caused by enzyme release from
bacteria and phagocytes bronchiectasis,
chronic lung disease.
• Haemophilus influenzae,
Streptococcus pneumoniae,
Staphylococcus aureus
*Do not give vaccines such as the Oral polio
vaccine disseminates and causes
poliomyelitis because the baby does not
have antibodies.
*T-cell responses to intracellular bacteria is
normal (Mycobacteria).
2. B Lymphocyte Abnormality [Sex-linked *Lack of mature b cells plasma cells in the
Agammaglobulinemia (XLA)/ Bruton’s periphery.
Agammaglobulinemia]
3. Combined B and T Lymphocyte
Abnormalities
*SCID can be divided into two types: Both
result in depletions of T, B, and natural killer
(NK) lymphocytes.
A. Severe Combined Immunodeficiency

(SCID)
A.1 Adenosine Deaminase Deficiency (ADA)
*Is a B CELL DISEASE that is caused most

frequently by a mutation in the gene
encoding Bruton tyrosine kinase (BTK).

A.1 Adenosine Deaminase Deficiency (ADA) 2. Combined immunodeficiency
Continuation (Humoral and cellular)
*Results in excess amounts of its natural 3. Eczema
substrates (adenosine and 2′- 4. Thrombocytopenia with small
deoxyadenosine), which cause lymphocyte platelets (↑ Production; ↑ Destruction)
depletion through a variety of mechanisms. 5. Increased risk of autoimmune
*Autosomal recessive disorders and cancers
*#1-4 are Triad (only in 1/3rd)
A.2 X-linked SCID
*Is the more common of the two and is Lymphocytes
caused by a mutation in the gene encoding
the IL-2 receptor γ chain (responsible for the 1. Lymphocytosis
maturation and migration of immune cells), *The definition of lymphocytosis varies with
which is shared by several interleukins. the age of the individual.
*The mutation results in T cell lymphopenia, *Children older than 2 weeks and younger
dysfunctional B cells, and a lack of NK cells. than 8 to 10 years normally have higher
absolute lymphocyte counts than adults.
3. Combined B and T Lymphocyte *Lymphocytosis in young children -absolute
Abnormalities Continuation lymphocyte count greater than 10.0 × 109 /L
B. Wiskott-Aldrich Syndrome (WASp) *Lymphocytosis in adults - greater than 4.5 ×
*Is also X-linked and is caused by a mutation 109 /L.
in a gene that encodes a protein called
WASp. 2. Lymphocytosis (Reactive)
*The mutation results in low levels or absence A. Infection
of the WAS protein, and affected individuals *Infectious mononucleosis
have immunodeficiency, eczema, and *Cytomegalovirus Infection
thrombocytopenia. (TIE) *Hepatitis
*Acute HIV infection
*Adenovirus
*Chickenpox
*Herpes
*Influenza
*Paramyxovirus (mumps)
*Rubella (measles)
*Roseola
*Mumps
*ß-Hemolytic streptococci
*Brucellosis
*Absence of WASp affects migration, *Paratyphoid fever
adhesion, and activation of a variety of *Toxoplasmosis
leukocytes, including T cells, B cells, and NK *Typhoid fever
cells. *Listeria
*Mycoplasma
*Symptoms in infancy *Syphilis
• Recurrent, severe infections
• Eczema B. Miscellaneous
• Thrombocytopenia (petechiae) *Idiosyncratic drug reactions
*Postvaccination
*Low levels of IgM *Sudden onset of stress from myocardial
*Increased risk of hematologic malignancy infarction
*Treatment: Manage bleeding/ infections, *Allergic reaction
BMT *Hyperthyroidism
*Malnutrition
*Clinical Features:
1. Affects 1 in 10 of every 1 million male C. Nonreactive Morphology
newborns *Bordetella pertussis (whooping cough)

*Acute infectious lymphocytosis *Nutritional/dietary
*Polyclonal B-lymphocytosis • Ethanol abuse
• Zinc deficiency
3. Lymphocytopenia
*The definition of lymphocytopenia is age
dependent. LYMPHOCYTES
*Lymphocytopenia in young children-
absolute lymphocyte count below 2.0 × 10 9 *Over the years, reactive morphologic
/L changes in lymphocytes have been
*Lymphocytopenia in adults → count below described using various terms, including:
1.0 × 109 /L. • Variant Lymphocytes
• Reactive Lymphocytes
4. Lymphocytopenia (Inherited) • Effector Lymphocytes
A. Congenital immunodeficiency diseases • Transformed Lymphocytes
*Severe combined immunodeficiency • Turk cells
disease • Downey cells
*Common variable immune deficiency • Immunoblasts
*Ataxia-telangiectasia
*Wiskott-Aldrich syndrome *Atypical Lymphocytes Atypical is commonly
*Others used, but it is probably the least suitable of all
because it implies that the cells are abnormal
B. Acquired when in fact the lymphocytes are reacting to
*Aplastic anemia antigens in a normal manner.
*Infections
• Acquired immunodeficiency *Occur as lymphocytes are stimulated when
syndrome interacting with antigens in peripheral
• Severe acute respiratory syndrome lymphoid organs.
• West Nile *B and T lymphocyte activation results in the
• Hepatitis transformation of small, resting lymphocytes
• Influenza into proliferating larger cells.
• Herpes *Often present as a heterogeneous
• Measles population of various shapes and sizes.
• Tuberculosis • There is variation in the
• Typhoid fever nuclear/cytoplasmic ratio, nuclear
• Pneumonia shape, and chromatin pattern, which
• Rickettsiosis is generally clumped, but some cells
• Ehrichiosis may demonstrate chromatin patterns
• Sepsis that are less mature (less clumped).
• Malaria Nucleoli may be visible.
*Iatrogenic *Most obvious in reactive

• Immunosuppressive agents lymphocytes is an
• Stevens-Johnson syndrome increase in basophilic
• Chemotherapy cytoplasm that may vary
• Radiation in intensity within and
• Platelet or stem cell apheresis between cells.
collection
• Major surgery *Reactive (variant)
lymphocytes from a
*Systemic disease patient with infectious
• Autoimmune diseases mononucleosis.
• Hodgkin lymphoma
• Carcinoma
• Primary myelofibrosis *A plasmacytoid lymphocyte is a type of
• Protein-losing enteropathy reactive lymphocyte that has some of the
• Renal failure morphologic features of plasmacytes.

*However, because reactive lymphocytes
may be activated by T or B cells, it is
important to understand that plasmacytoid is
a morphologic term and does not imply
lineage.
Figure. Reactive
(variant) lymphocyte
(plasmacytoid)
1. Common Clinical Manifestations of IM
EPSTEIN-BARR VIRUS (EBV)–RELATED

INFECTIONS: Infectious mononucleosis (IM)
*Sore throat
*Dysphagia
*Fever
*Chills
*When primary infection with Epstein-Barr *Cervical lymphadenopathy
virus occurs in childhood, it often goes *Fatigue
unnoticed *Headache
*The incubation period of infectious
mononucleosis is about 3 to 7 weeks, and 2. Hematologic Findings of IM
during this time the virus preferentially infects *WBC count is usually elevated to a range of
B lymphocytes through the attachment of 10—30 × 109 /L or more with an absolute
viral envelope glycoprotein 350/220 to CD21 lymphocytosis.
(C3d complement receptors). *There is a wide variation in lymphocyte
morphology, with up to 50% or higher
*The oropharynx epithelial cells are also exhibiting reactive features.
infected, but the mechanism is unclear
because these cells do not express CD21. 3. Complications of IM
*The cellular response in IM is important in the *Hepatosplenomegaly (elevated
control of the infection and is characterized transaminases)
by the proliferation and activation of natural *Hemolytic Anemia
killer (NK) lymphocytes, CD4+ T cells, and CD8 *Moderate Thrombocytopenia
+ memory cytotoxic T cells (EBV-CTLs) in *Aplastic Anemia
response to B cell infection. *Disseminated Intravascular Coagulation
*Most of the circulating reactive *Thrombotic Thrombocytopenic Purpura
lymphocytes seen in circulation represent *Hemolytic Uremic Syndrome
activated T cells. *Guillain-Barré syndrome

*Other neurologic complications *Definitive testing for EBV infection includes a
panel of antigen and antibody tests for VCA,
4. Laboratory Examinations of IM EBNA, and IgG/IgM antibodies against VCA
*Rapid screening tests for the detection of and EBNA.
heterophile antibodies, antibodies *Cytomegalovirus can cause a
stimulated by the EBV that cross-react with mononucleosis syndrome with a similar
antigens found on sheep and horse red cells. clinical feature.
Summary of Qualitative Disorders of Leukocytes
MORPHOLOGIC ABNORMALITIES WITHOUT • Heavy toxic granulation: exclusive to

FUNCTIONAL DEFECTS NEUTROPHILS ONLY
• Alder-reilly anomaly: granules are
1. Pelger-Huet Anomaly (PHA) found in LYMPHOCYTES and
*Aka true or congenital PHA MONOCYTES
*Hyposegmentation (bilobed, unilobed)
• Bilobed appears in pince-nez form *Partially digested/ incomplete degradation
*All leukocytes have coarse chromatin of mucopolysaccharides due to lack of
clumping lysosomal enzymes
*Lamin B-receptor gene mutation → major *Problem is in the GRANULES
role in leukocyte nuclear shape *Granules aka alder-reilly bodies/ reilly bodies
*Neutrophils function normally. *Leukocytes function normally
*Present in patients with hematologic
malignancies MORPHOLOGIC ABNORMALITIES WITH
FUNCTIONAL DEFECTS
*Differences:
• Affected cells are higher 1. Chédiak-Higashi Syndrome (CHS)
• Affects all WBC lineage *Characterized by abnormal fusion of
• Normal granulation of neutrophils granules = GIANT granules = DYSFUNCTIONAL
= Recurrent pyogenic infections
2. Pseudo or Acquired Pelger-Huet Anomaly *Affects ALL cells that contain GRANULES
*Pseudo-PHA neutrophils can be seen in (E.g., Platelets = Dysfunctional = results in
infections and drugs. bleeding issues)
*Mutation in CHS1/LYST Lysosomal trafficking
*Differences: regulator gene.
• Affected cells are lower
• Usually affects neutrophils 2. May-Hegglin Anomaly
• Hypogranular neutrophils (MDS- *Autosomal dominant PLATELET DISORDER
related pseudo-PHA) *GIANT platelets + Large Döhle body-like
inclusions
3. Myelokathexis *Disordered production of myosin heavy
*Neutrophil retention in the bone marrow chain type IIA = affects megakaryocyte
*Normal granulocyte production BUT maturation and platelet fragmentation
impaired release = NEUTROPENIA *Patients are asymptomatic, but some have
*Mutation in chemokine receptor CXCR4 = mild bleeding tendencies
trigger hematopoiesis, cell migration, and *MYH9 mutation
cell homing
*Neutrophils are hypermature MONOCYTE/MACROPHAGE LYSOSOMAL
*Is a component of WHIM syndrome STORAGE DISEASES
4. Alder-Reilly Anomaly *Types: Mucopolysaccharide (GAG) and

*Appearance of granulocyte: large, darkly lipid storage diseases
staining metachromatic cytoplasmic *Is an inherited enzyme deficiency or defect
granules = flawed degradation and build-up of
*RESEMBLES Heavy toxic granulation material within the phagocyte.
*All cells containing lysosomes are affected.

1. Mucopolysaccharides (MPSs) NPC2 proteins = Impair intracellular
*Deficient activity of an enzyme to degrade: transport = Abnormal storage of lipids
• Dermatan sulfate and cholesterol
• Heparan sulfate o Prognosis is poor
• Keratan sulfate o Bubble/ foam-like pattern of
• Chondroitin sulfate storage deposit in the
*Partially degraded GAG builds up in the cytoplasm
lysosomes
*Peripheral blood of patient: may present GENETIC B AND T LYMPHOCYTE
metachromatic reilly bodies in neutrophils, ABNORMALITIES
monocyte, lymphocytes
*Treatment: 1. T Lymphocyte Abnormality (DiGeorge
• Enzyme replacement therapy Syndrome)
• Hematopoietic stem cell *Symptoms: C.A.T.C.H. 22
transplantation *Absence/ underdeveloped thymus = ↓ T
Lymphocytes
2. Lipid Storage Diseases *Microdeletion in chromosome band 22q11.2
A. Gaucher Disease *1% of patient with this disease is athymic aka
*DEFECT/ DEFICIENCY in enzyme B- complete DiGeorge Syndrome
glucocerebrosidase = Accumulation of *Treatment: Thymus transplantation
sphingolipid glucocerebrosidase
*Onion skin appearance of macrophages 2. B Lymphocyte Abnormality [Sex-linked
*Delicate Gaucher cells resemble crinkled Agammaglobulinemia (XLA) or Bruton’s
tissue paper Agammaglobulinemia]
*HAS tubular inclusions *A mutation in a gene encoding Bruton
*Treatment: tyrosine kinase (BTK) = B cell maturation stops
• Enzyme replacement therapy with at pre-B-cell stage = B cell dies prematurely =
recombinant glucocerebrosidase Nonproduction of antibodies.
• Stem cell transplantation *Symptoms:
• Glucocerebrosidase inhibitors • The first few months of life are normal
due to maternal Ig
*Pseudo-Gaucher cells • Recurrent infection
• Seen in patients with thalassemia, • The pyogenic bacterial infection
CML and ALL as a secondary disease leads to bronchiectasis and chronic
• There is NO DECREASE in the enzyme lung disease
• B-glucocerebrosidase enzyme is *Do not give vaccines like the oral polio
overwhelmed = pseudo-gaucher vaccine because babies lack antibodies.
cells This will cause poliomyelitis in babies.
• NO tubular inclusions
3. Combined B and T Lymphocyte
B. Niemann-Pick Disease Abnormalities
*Types: A. Severe Combined Immunodeficiency
• Type A → SMPD1 gene mutation = (SCID)
Absence of sphingomyelinase = *Adenosine deaminase deficiency (ADA)
accumulation of sphingomyelins • NORMAL ADA converts
o Occurs in infants deoxyadenosine (which is toxic) to a
o Severe progressive brain non-toxic substance
disease and no cure = death • ABNORMAL ADA cannot bind to
• Type B → SMPD1 gene mutation = deoxyadenosine = deoxyadenosine
Deficiency in sphingomyelinase = levels rise = kills B and T cells =
decrease in ceramides infection by bacteria and viruses
o Occurs in childhood
o NO primary brain disease = *X-linked SCID
live until adulthood • Mutation in gene encoding IL-2
• Type C → NPC1 or NPC2 gene receptor γ chain (responsible for
mutation = Dysfunction in NPC1 or

maturation and migration of immune 3. EPSTEIN-BARR VIRUS (EBV) – RELATED
cells) INFECTIONS: Infectious Mononucleosis (IM)
• Mutation results in: *Infection is not noticeable among children
o T cell lymphopenia *This virus preferentially infects B
o Dysfunctional B cells LYMPHOCYTES through the attachment of
o Lack of NK cells viral envelope glycoprotein 350/220 to CD21
(C3d complement receptors)
B. Wiskott-Aldrich Syndrome (WASp)
*Mutation in a gene that encodes for protein *Oropharynx epithelial cells are infected BUT
WASp = leading to immunodeficiency, the mechanism is unknown since these cells
eczema, and thrombocytopenia do not express CD21
*Absence of Wasp = affects migration,
adhesion, and activation of T, B, and NK cells *Symptoms:
*↓ IgM • Sore throat
• Swollen lymph nodes, liver, tonsils,
LYMPHOCYTES and spleen
• Body aches, fatigue, fever
*Reactive lymphocytes may be activated by *Treatment:
T and B cells; it is important to understand that • Fluids, rest, and avoidance of
plasmacytoid is a morphologic term and contact sports
does not imply lineage. • Acetaminophen or ibuprofen for pain
and fever
1. Lymphocytosis *Hematologic Findings in IM
*ADULT: > 4.5 x 109/L • Elevated WBC Count: 10 to 30 x 109/L
*CHILDREN: > 10.0 x 109/L • Variation in Lymphocyte morphology
(50% have reactive features)
2. Lymphocytopenia *Laboratory Findings of IM
*ADULT: <1.0 x 109/L • Rapid screening test for detection of
*CHILDREN: < 2.0 x 109/L heterophile antibodies


LYMPHOCYTIC LEUKEMIA, LYMPHOMAS, AND PLASMA CELL DYSCRASIAS
Lymphocytic Leukemia *Anemia is a result of the sequestration of red

cells in lymphoid tissues.
CHRONIC LYMPHOCYTIC LEUKEMIA *Lymphocytes contain more glycogen than
usual giving a positive PAS stain. (Periodic-
*Proliferation and accumulation of Acid Schiff)
lymphocytes (B cells) that are relatively *Plasma immunoglobulins may be reduced
unresponsive to antigenic stimuli. Bone marrow aspiration is not necessary.
*The antigen stimulates the B cells to
differentiate into plasma cells which will
produce the antibodies. BUT in CLL, even in
the presence of antigen, the B cells are not
stimulated and unresponsive.
*Antibodies are produced by B cells
(specialized white blood cells). When an
antigen encounters a B cell, it causes the B
cell to divide and clone. These cloned B cells
— or plasma cells — release millions of
antibodies into your bloodstream and lymph
system.
1. Clinical Presentation of CLL

*Disease primarily of the elderly with 90% of
patients being over 50 years of age.
5. Three Modes of Therapy in CLL
*Chemotherapy involves the use of alkylating
2. Symptoms of CLL
agents such as cyclophosphamide or
*Fatigue
chlorambucil either singly or in a
*Exercise intolerance
combination with vincristine (plant alkaloid)
*Bruising
and prednisone (corticosteroid).
*Pallor
*Radiation is used primarily to tear enlarged
*Jaundice associated with anemia
lymph nodes and splenomegaly that have
*Fever
proved resistant to chemotherapy.
*Infection
*Leukapheresis is especially useful in
*Bone tenderness
reducing the number of lymphocytes in the
*Weight loss
peripheral blood when the patient has
*Edema from lymph node obstruction
symptoms of blood hyperviscosity.
3. Physical Findings of CLL
PROLYMPHOCYTIC LEUKEMIA
*Enlarged lymph nodes
*Splenomegaly
1. Pathophysiology of PL
*Accumulation of abnormal lymphoid cells in
4. Laboratory Findings of CLL
the spleen, bone marrow, and to a lesser
*Lymphocytosis
extent, the liver.
*Lymphocytes are somewhat larger than
*Immunologic deficiencies such as low
normal, have nuclei with clumped or
gamma globulins and generally low
condensed chromatin, and may have
immunoglobulins. Lacks B cells because
prominent nucleoli.
these are the proteins that are produced by
*Cytoplasm may be abundant, nongranular,
B cells.
and moderately basophilic, or it may be
*T lymphocytes are below normal.
relatively scanty.
*Smudge cells → are lymphocytes broken
2. Clinical Presentation of PL
when the film is made.
*Fatigue, weakness, weight loss, sweats, and
*Neutrophil decreased, granulocytopenia,
fever.
anemia, and thrombocytopenia.
Hematology 1 Lecture MLS 113A | Lymphocytic Leukemia, Lymphomas, and Plasma Cell
Dyscrasias
*Prognosis is poorer than for either CLL or HCL. • The presence of mononuclear cells
Survival is reported to be less than 1 year. with numerous cytoplasmic
projections (that is why it is hairy).
3. Physical Findings of PL
*Splenomegaly and less often, 1. Pathophysiology HCL
hepatomegaly. *The growth and accumulation of hairy cells
*Lymphadenopathy is less common in the spleen, blood, and bone marrow
account for the complications, which fall
4. Laboratory Findings of PL essentially into two groups:
*Leucocyte count is high from 25 x109/L to • Those related to cytopenias and
1000 x109/L. splenomegaly, such as anemia,
*Prolymphocytes is a relatively large bleeding, infection, and
mononuclear lymphoid cells with oval to paraneoplastic complications,
round nucleus coarse-appearing chromatin including autoimmune syndrome and
strands and one or two large vesicular less often, paraproteinemia.
nucleoli with perinuclear condensations of
chromatin. The cytoplasm is agranular and *Mean survival has been said to be 5 years.
basophilic.
2. Clinical Presentation of HCL
*Absolute neutrophil counts can range from *Bleeding, weakness, fatigue, infection, and
low to high. abdominal discomfort.
*There may be absolute monocytosis.
3. Physical Findings of HCL
*Bone marrow examination reveals almost *Splenomegaly
total replacement of the marrow by
prolymphocytic infiltrations. 4. Laboratory Findings of HCL
*Hairy cells (Lymphocytes with cytoplasmic
projections) are found in the peripheral
blood in greater than 90% of patients.
*Hairy cells have scanted to abundant,
agranular, light grayish-blue cytoplasm. The
plasma membrane appears irregular with
hairlike projections. These cells have round to
oval nuclei, the nucleus is folded or bilobed.
The chromatin is loose and lacy, and one or
two nucleoli are seen.
*Pancytopenia results from infiltration of the
marrow with malignant cells.
*Bone marrow biopsy shows lymphoid
infiltration with greater space between cell
5. Treatment for PL nuclei.
*The goal is to reduce the lymphocyte mass *Tartrate-resistant acid phosphatase (TRAP) is
in the blood, marrow, and tissues. positive in hairy cells but negative in other
*PLL responds poorly to the modes of therapy lymphoid cells.
successful in CLL which is why the prognosis is
bad.
HAIRY CELL LEUKEMIA
*Described as leukemic
reticuloendotheliosis.
*Two characteristics have commonly seen:
• Large spleen 5. Treatment for HCL
*If the patient is asymptomatic, no therapy is
needed.
Dyscrasias
*Alkylating agents may reduce tumor *Bone Marrow:
burden very little, and their toxicity will make • seldom involved except in the
cytopenias worse. advanced stage.
*Corticosteroids have rarely helped in
treating HCL because of the association of *Other Lab Findings
their use with infections. • Reduced serum iron and total iron
*Splenectomy is the treatment for patients binding capacity (TIBC)
with cytopenias, due to splenic sequestration • A rarely direct antiglobulin test (DAT)
of cells. is a positive signifying hemolytic
component to anemia.
Lymphomas • Elevations of erythrocyte
sedimentation rate, fibrinogen,
HODGKIN’S DISEASE haptoglobin, serum globulins,
ceruloplasmin, and copper.
1. Epidemiology of Hodgkin’s Disease • Elevated leukocyte alkaline
*HD has unique bimodal incidence curve. phosphatase (LAP), lysozyme, lactic
*Begins to rise after the age of 10 and peaks acid dehydrogenase, and
in the 20s then declines to age 45 after which transaminase.
incidence rises steadily with advancing age. • Hyperuricemia- leads to acute renal
failure
2. Clinical Features of Hodgkin’s Disease
*Painless enlarging lymph node, usually in the
neck.
*Mediastinal mass
*Fever, night sweats, weight loss, or a
combination thereof which are referred to as
the B symptoms.
3. Laboratory Findings in Hodgkin’s Disease

*The only definitive test is lymph node biopsy.
Figure. Rye Classification
*Peripheral Blood:
• EARLY → absolute increase in
*Good prognosis
monocytes and eosinophils.
• no anemia and platelets are normal
4. Treatment for Hodgkin’s Disease
in number.
*Chemotherapy
• large, abnormal-appearing
*Combination Chemotherapy
lymphocytes with very little cytoplasm
*Radiation alone or radiation plus
and irregular nuclear chromatin.
chemotherapy
• As the disease progress, there is
leucocytosis, granulocytosis which
NON-HODGKIN LYMPHOMAS
mimics leukemia (leukemoid
reaction)
1. Epidemiology of Non-Hodgkin Lymphomas
• ADVANCE STAGE → granulocytes
*Incidence rises steadily with age starting
with toxic granulation.
around age 40.
• Plasma cells are known as Reed-
*More common in men than women
Sternberg Cells “Owl Eyes”
2. Physical Findings of Non-Hodgkin
Lymphomas
*Painless lymph node enlargement. Cervical
nodes are most often involved.
3. Symptoms of Non-Hodgkin Lymphomas

*Fever
*Weight loss
Dyscrasias
*Night sweats Plasma Cell Dyscrasias (Paraproteinemias)
PLASMA CELLS
*Capable of synthesizing heterogeneous

proteins - immunoglobulins (Ig) with the
capacity to bind the particular antigens that
stimulated their production.
*Plasma cells are the one that produces the
antibodies.
*Types of Immunoglobulins
• IgG → exists as a monomer,
responsible for secondary immune
response, precipitating antibodies,
virus-neutralizing antibodies,
Figure. Classifications of Non-Hodgkin
hemagglutinins, hemolysins, and
Lymphomas
activators of the classical
complement pathway.
4. Treatment for Non-Hodgkin Lymphomas
• IgA → often exists as a dimer, the
*Radiation and chemotherapy → have the
second most abundant
same effects on bone marrow and
immunoglobulin. Provides the first line
peripheral blood.
of defense on mucosal surfaces.
• IgM → the largest Ig that has a
MYCOSIS FUNGOIDES AND SEZARY
pentameric arrangement. It is the first
SYNDROME
antibody to respond to antigenic
challenges.
• IgD → monomer, surface Ig on blood
lymphocytes and may have
lymphocyte activation and
suppression activity.
• IgE → monomer, reaginic antibody
found in the respiratory and
gastrointestinal tracts. Respond to
allergic reactions. Attach to mast
cells and basophils and mediates
allergic reaction and play role in the
parasitic response.
MULTIPLE MYELOMA
*Most common malignant disease of plasma

cells and generally affects older individuals
(50-70 years).
1. Pathophysiology of Multiple Myeloma

*Clonal proliferation of malignant plasma
*Mycosis is a fungal infection cells begins in the bone marrow, and multiple
*Rare lymphomas caused by neoplastic T tumors appear as patchy infiltrates in skeletal
lymphocyte that migrates to the skin. structures, producing osteoporosis and lytic
*Laboratory abnormalities like non-Hodgkin bone disease.
lymphomas. *More than 50% of the myeloma produces
*Classic cell lymphocyte that is larger than proteins of the IgG class. The second most
normal with scant cytoplasm and frequent monoclonal proteins are IgA.
cerebriform nucleus with or without nucleoli
and with fine chromatin.
Dyscrasias
*The lower molecular weight Light chains
(Bence Jones Proteins) are readily filtered by
the renal glomeruli.
2. Clinical Presentation of Multiple Myeloma

*Chief complaint is skeletal pain. Initial back
pain associated with centrally located bones
progresses to severe pain of the spontaneous
(pathological) bone fracture. Accompanied
by anemia and renal insufficiency. *Dutcher Bodies
• Immunoglobulin-filled cytoplasm
3. Laboratory Findings in Multiple Myeloma invaginating into the nucleus creates
*Normochromic, normocytic anemia with Hb the appearance of an intracellular
levels between 7 and 12 g/dL. inclusion.
The Erythrocyte sedimentation rate is • PAS + ve
elevated because of serum globulins. • Identified by Dutcher and Fahey
*Rouleaux formation and neutropenia
PLASMA CELL LEUKEMIA
*Myeloma cells → may be large with
immature-appearing chromatin or small with *Circulating plasma cells exceed 2x109/L.
clumped chromatin and may be pale or
dark depending on the amount of 1. Clinical Presentation of Plasma Cell
cytoplasmic RNA. Leukemia
*Less bone pain and less osteolysis.
*Cellular inclusions such as:
• intranuclear (Dutcher) bodies 2. Laboratory Findings in Plasma Cell
(nuclear inclusion), the crystalline Leukemia
structure of abnormal Ig, or *Greater than 2x109/L, pancytopenia with
• rounded accumulation of Ig in the erythroblastic findings, and an elevated ESR.
cisternae (Russell bodies) *Abnormal plasma cells are small with little
(cytoplasmic inclusion). cytoplasm and with pronounced nuclear:
cytoplasmic asynchronism.
*Bone marrow infiltration is diffuse, and the
proliferating cells exceed 45% of the
population.
Figure. Dutcher Bodies (Nuclear Inclusion) WALDENSTROM MACROGLOBULINEMIA
*Characterized by a large concentration of

clonal IgM
1. Clinical Presentation of WM
*Gradual onset of symptoms as IgM
concentration mounts.
Figure. Russel Bodies (Cytoplasmic Inclusion) *Patients are predominantly men and older
than 50 years of age.
Dyscrasias
*Fatigue, weight loss, blurred vision, and *Interferon treatments
bleeding episodes, especially epistaxis (nose
bleeding). HEAVY CHAIN DISEASES
*Hepatosplenomegaly, lymphadenopathy,
and retinal abnormalities preceding retinal *Immunoproliferative disorders are
hemorrhage. characterized by abnormal synthesis of the
Fc portion of a particular Heavy chain.
2. Laboratory Findings in WM
*Normocytic, normochromic anemia.
*Reticulocyte count is usually normal or
decreased.
*Increased ESR secondary to the rouleaux
formation
*Bone marrow biopsy reveals a plasmacytoid
lymphoma.
*Mast cells may be increased.
*Differential diagnostic data are obtained
from serum protein electrophoresis and
immunoelectrophoresis.
*Other serum studies show increased plasma AMYLOIDOSIS
viscosity. Significantly, monoclonal IgM may
exhibit cryoglobulin activity demonstrated by *Amyloidosis results from the accumulation of
precipitation or gel formation during pathogenic amyloids—most of which are
refrigeration at 4˚C. aggregates of misfolded proteins—in a
variety of tissues.
3. Treatment for WM
*Plasmapheresis to reduce viscosity *Light chain and heavy chain disease can
*Alkylating agents possible with prednisone cause amyloidosis.
Dyscrasias
ACUTE LYMPHOCYTIC LEUKEMIA AND ACUTE MYELOCYTIC LEUKEMIA
Introduction Figure. A, LYMPHOBLASTS (bone marrow,

Wright stain, ×500). Cells have a diameter of
*The broad term leukemia is derived from the two to three times the normal lymphocyte
ancient Greek words leukos (λευκóç), diameter, scant blue cytoplasm, coarse
meaning “white,” and haima (αἷμα), chromatin, deeper staining than myeloblasts,
meaning “blood.” As defined today, acute and inconspicuous nucleoli. B, MYELOBLASTS
leukemia refers to the rapid, clonal (bone marrow, Wright stain, ×500). Cells have
proliferation in the bone marrow of lymphoid a diameter three to five times the
or myeloid progenitor cells known as lymphocyte diameter, moderate gray
lymphoblasts and myeloblasts, respectively. cytoplasm, uniform fine chromatin, two or
When the proliferation of blasts overwhelms more prominent nucleoli, and possibly Auer
the bone marrow, blasts are seen in the rods.
peripheral blood and the patient’s symptoms
reflect suppression of normal hematopoiesis. Acute Lymphoblastic Leukemia (ALL)
*Acute lymphoblastic leukemia (ALL) is

primarily a disease of childhood and
adolescence, accounting for 25% of
childhood cancers and up to 75% of
childhood leukemia.
*The peak incidence of ALL in children is
between 2 and 5 years of age.
Hematology 1 Lecture MLS 113A | Acute Lymphocytic Leukemia and Acute Myelocytic
Leukemia
*Patients with B CELL ALL typically present 2. Morphology of Lymphoblast
with: *Lymphoblasts vary in size but fall into two
• fatigue (caused by anemia) morphologic types.
• fever (caused by neutropenia and • The most common type seen is a
infection) small lymphoblast (1.0 to 2.5 times the
• mucocutaneous bleeding (caused size of a normal lymphocyte) with
by thrombocytopenia). scant blue cytoplasm and indistinct
• Lymphadenopathy, including nucleoli
enlargement, is often a symptom. • The second type of lymphoblast is
(Lymphadenopathy is cancerous if it larger (two to three times the size of a
is painful due to infection. lymphocyte) with prominent nucleoli
Lymphadenopathy is due to and nuclear membrane
leukemia if it is painless) irregularities. These cells may be
• Enlargement of the spleen confused with the blasts of acute
(splenomegaly) and the liver myeloid leukemia (AML).
(hepatomegaly) may be seen.
• Bone pain often results from the Acute lymphoblastic
intramedullary growth of leukemic leukemia. Large
cells. Eventual infiltration of malignant lymphoblast with
cells into the meninges, testes, or prominent nucleoli and
ovaries occurs frequently, and membrane irregularities
lymphoblasts can be found in the (peripheral blood,
cerebrospinal fluid. ×1000). Source: (From
Carr JH, Rodak BF: Clinical hematology
*In T CELL ALL, there may be: atlas, ed 4, St. Louis, 2013, Saunders.)
• a large mass in the mediastinum
leading to compromise of regional 3. Prognosis for ALL
anatomic structures. *The prognosis for ALL depends on the age at
• Like B-ALL, T-ALL may present with the time of diagnosis, lymphoblast load
anemia, thrombocytopenia, (tumor burden), immunophenotype, and
organomegaly, and bone pain, genetic abnormalities. Children rather than
although the degree of leukopenia is infants or teens do the best. Chromosomal
often less severe. translocations are the strongest predictor of
adverse treatment outcomes for children
1. B Lymphoblastic Leukemia/Lymphoma and adults.
with Recurrent Genetic Abnormalities (2008
World Health Organization Classification) *Peripheral blood lymphoblast counts
• B lymphoblastic leukemia/lymphoma greater than 20 to 30 × 109/L,
with t(9; 22)(q34; q11.2); BCR-ABL1 hepatosplenomegaly, and
• B lymphoblastic leukemia/lymphoma lymphadenopathy all are associated with
with t(v; 11q23); MLL rearranged worse outcomes.
• B lymphoblastic leukemia/lymphoma
with t(12; 21)(p13; q22); TEL-AML1 *Immunophenotyping
(ETV6-RUNX1) • Although morphology is the first tool
• B lymphoblastic leukemia/lymphoma used to distinguish ALL from AML,
with hyperdiploidy immunophenotyping and genetic
• B lymphoblastic leukemia/lymphoma analysis are the most reliable
with hypodiploidy indicators of a cell’s origin. Because
• B lymphoblastic leukemia/lymphoma both B and T cells are derived from
with t(5; 14)(q31; q32); IL3-IGH lymphoid progenitors, both usually
• B lymphoblastic leukemia/lymphoma express CD34, terminal
with t(1; 19)(q23; p13.3); E2A- deoxynucleotidyl transferase (TdT) ←
PBX1(TCF3-PBX1) DNA polymerase, and HLA-DR. Four
*Lymphoma → produces a mass types of ALL have been identified by
*Leukemia → no mass immunologic methods: early B-ALL
Leukemia
(pro-B, or pre-pre-B), intermediate *Infiltration of malignant cells into the gums
(common) B-ALL, pre-B-ALL, and T- and other mucosal sites and skin also can be
ALL. seen.
• ALL → positive for TdT *Splenomegaly is seen in half of AML patients,
• AML → negative for TdT but lymph node enlargement is rare.
Cerebrospinal fluid involvement in AML is rare
4. World Health Organisation (WHO) system and does not seem to be as ominous a sign
*Doctors mostly use the World Health as in ALL. Patients with AML tend to have few
Organisation (WHO) system. It's based on the symptoms related to the central nervous
type of lymphocyte (white blood cell) that system, even when it is infiltrated by blasts.
has become cancerous and the
characteristics the cell has. This system helps 2. Laboratory Findings of AML
your doctors to plan treatment and predict *Common abnormalities in laboratory test
how well the treatment will work. results include:
• hyperuricemia (caused by increased
*There are 3 different subtypes: cellular turnover)
• pre (precursor) B cell ALL, this is the • hyperphosphatemia (due to cell lysis)
most common type in adults • hypocalcemia (the latter two are
• pre (precursor) T cell ALL, this is more also involved in progressive bone
likely to affect young adults and is destruction).
more common in men • Hypokalemia is also common at
• mature B cell ALL, this type is identified presentation.
by particular genetic changes
*During induction chemotherapy, especially
Acute Myeloid Leukemia (AML) when the WBC is quite elevated, tumor lysis
syndrome may occur. Consolidation
*AML is the most common type of leukemia chemotherapy is a treatment after signs of
in ADULTS, and the incidence increases with leukemia are no longer present. This is done
age. AML is less common in children. to prevent the recurrence of leukemia.
• The French-American-British (FAB)
classification of AML was based on *Tumor lysis syndrome
morphology and cytochemistry • is a group of metabolic
• The WHO classification relies heavily complications that can occur in
on cytogenetics and molecular patients with malignancy, most
characterization. notably lymphomas and leukemias,
with and without treatment of the
1. Clinical presentation of AML malignancy. These complications are
*The clinical presentation of AML is caused by the breakdown products
nonspecific but reflects decreased of dying cancer cells, which in turn
production of normal bone marrow cause acute uric acid nephropathy
elements. and renal failure. Tumor lysis
*Most patients with AML have a total WBC syndrome is characterized by
count between 5 and 30 × 109/L, although hyperkalemia, hyperphosphatemia,
the WBC count may range from 1 to 200 × hyperuricemia and hyperuricosuria,
109/L. and hypocalcemia. Hyperkalemia
alone can be life-threatening.
*Myeloblasts are present in the peripheral
blood in 90% of patients. 3. Subtypes of acute myeloid leukemia and
*Anemia, thrombocytopenia, and related precursor neoplasms
neutropenia give rise to the clinical findings *Laboratory diagnosis of AML begins with a
of pallor, fatigue, fever, bruising, and complete blood count, peripheral blood film
bleeding. In addition, disseminated examination, and bone marrow aspirate and
intravascular coagulation and other biopsy specimen examination.
bleeding abnormalities can be significant.
Leukemia
3. Subtypes of acute myeloid leukemia and *The t(8; 21)(q22; q22); RUNX1/
related precursor neoplasms Continuation RUNX1T1 mutation is found in about 5% of
*The total WBC count may be normal, AML cases. Seen predominantly in children
increased, or decreased; anemia is usually and young adults, AML with this translocation
present, along with significant has myeloblasts with dysplastic granular
thrombocytopenia. cytoplasm, Auer rods, and some maturation,
like the FAB M2 classification. Various
*The bone marrow is usually hypercellular, anomalies, such as pseudo–Pelger-Huët cells
and greater than 20% of cells typically are and hypogranulation, can be seen.
marrow blasts, although if certain genetic Eosinophilia is possible. Prognosis is generally
abnormalities are present, the 20% blast favorable but may be negatively impacted
threshold is not necessary for the diagnosis of if unfavorable additional abnormalities, such
AML. as monosomy 7, occur. The diagnosis of this
subtype is based on genetic abnormality,
*Acute Myeloid Leukemia and Related regardless of blast count.
Precursor Neoplasms (2008 World Health
Organization Classification) Acute myeloid leukemia
• Acute myeloid leukemia with with t(8; 21). Myeloblasts
recurrent genetic abnormalities with granular cytoplasm
• Acute myeloid leukemia with and some maturation
myelodysplasia-related changes (bone marrow,
• Therapy-related myeloid neoplasms ×500). Source: (From Carr
• Acute myeloid leukemia, not JH, Rodak BF: Clinical
otherwise specified hematology atlas, ed 4, St. Louis, 2013,
• Myeloid sarcoma Saunders.)
• Myeloid proliferations related to
Down syndrome A.2 Acute myeloid leukemia with
• Blastic plasmacytoid dendritic cell inv(16)(p13.1q22) or t(16; 16)(p13.1; q22);
neoplasm CBFB-MYH11.
*Accounting for approximately 5% to 8% of all
*Acute Myeloid Leukemia with Recurrent AML cases, core-binding factor (CBF) AML
Genetic Abnormalities (2008 World Health occurs at all ages, but it is found
Organization Classification) predominantly in younger patients. The
• AML with t(8; 21)(q22; q22); RUNX1- genetic aberration is sufficient for diagnosis
RUNX1T1 regardless of blast count.
• AML with inv(16)(p13.1q22) or t(16; *Myeloblasts, monoblasts, and
16)(p13.1; q22); CBFB-MYH11 promyelocytes are seen in the peripheral
• APL with t(15; 17)(q22; q12); PML- blood and bone marrow. In the bone
RARA marrow, there may be eosinophilia with
• AML with t(9; 11)(p22; q23); MLLT3- dysplastic changes.
MLL *The incidence of the extramedullary disease
• AML with t(6; 9)(p23; q34); DEK- is higher than in most types of AML, and the
NUP214 central nervous system is a common site for
• AML with inv(3)(q21q26.2) or t(3; relapse. The remission rate is good, but only
3)(q21; q26.2); RPN1-EVI1 one-half of patients are cured.
• AML with t(1; 22)(p13; q13) RBM15-
MKL1 Acute myeloid leukemia
• Provisional entity: AML with with inv(16). There is an
mutated NPM1 increase in myeloid and
• Provisional entity: AML with monocytic lines.
mutated CEBPA Eosinophilia may also be
present (peripheral
A. AML with recurrent genetic abnormalities blood, ×1000). Source:
A.1 Acute myeloid leukemia with t(8; 21)(q22; (From Carr JH, Rodak
q22); RUNX1/RUNX1T1.
Leukemia
BF: Clinical hematology atlas, ed 4, St. Louis, Acute myeloid leukemia
2013, Saunders.) with t(15; 17), or
promyelocytic
A.3 Acute myeloid leukemia with t(15; leukemia. A, Low-power
17)(q22; q12); PML-RARA. view of the more
*Also known as acute promyelocytic common hypergranular
leukemia (APL), AML with the t(15; 17)(q22; variant (peripheral
q12); PML-RARA (Retinoic acid receptor blood, ×500). B, Oil
alpha ) mutation comprises 5% to 10% of AML immersion view of the
cases. It occurs in all age groups but is seen microgranular variant showing bilobed
most commonly in young adults. nuclear features (peripheral blood,
×1000). Source: (B from Carr JH, Rodak
*This disorder is characterized by a BF: Clinical hematology atlas, ed 4, St. Louis,
differentiation block at the promyelocytic 2013, Saunders.)
stage. The abnormal promyelocytes are
considered to be comparable to blasts for A.4 Acute myeloid leukemia with t(9; 11)(p22;
the purpose of diagnosis. Detection of the 15; q23); MLLT3-MLL.
17 translocation is sufficient for diagnosis *SAML (Secondary AML) with t(9; 11)(p22;
regardless of blast count. q23); MLLT3-MLL represents a specific
subgroup of the previous classification of
*Characteristics of this presentation are the AML with 11q23 abnormalities, and AMLs with
abnormal hypergranular promyelocytes, other MLL abnormalities should not be
some with Auer rods. When promyelocytes placed in this group.
release primary granule contents, their
procoagulant activity initiates disseminated *AML with t(9; 11) is rare leukemia (6% of AML
intravascular coagulation; however, cases) that presents with an increase in
thromboembolic events may occur at monoblasts and immature monocytes. The
presentation and during treatment. blasts are large with abundant cytoplasm
and fine nuclear chromatin. The cells may
*In one variant of APL, the granules are so have motility, with pseudopodia seen
small that because of the limits of light frequently.
microscopy, the cells give the appearance
of having no granules. This microgranular *Granules and vacuoles can be observed in
variant, accounting for 30% to 40% of APL the blasts. Typically, this disease occurs in
cases, may be confused with other children and may be associated with
presentations of AML, but the presence of gingival and skin involvement and/or
occasional Auer rods, the “butterfly” or disseminated intravascular coagulation. The
“coin-on-coin” nucleus, and the clinical prognosis is intermediate to poor.28
presentation are clues.
Acute myeloid leukemia
*The treatment of APL is significantly different with t(9; 11)
from all other types of acute myeloid abnormalities. Both
leukemia, and it is therefore important to monoblasts and
arrive at an accurate diagnosis. Treatment immature monocytes are
includes all-trans-retinoic acid (ATRA) and increased (bone marrow,
arsenic trioxide. ×500).
*Immature monocytes have granules
*ATRA is a vitamin A analogue and induces *Mature monocytes do not have granules
differentiation of the malignant
promyelocytes. In adults who achieve B. Acute myeloid leukemia with
complete remission, the prognosis is better myelodysplasia-related changes
than for any other type of AML. *AML with myelodysplasia affects primarily
older adults and has a poor prognosis.
Leukemia
3. Subtypes of acute myeloid leukemia and D.1 Acute myeloid leukemia with minimal
related precursor neoplasms Continuation differentiation. (M0)
B. Acute myeloid leukemia with *The blasts in AML with minimal differentiation
myelodysplasia-related changes are CD13+, CD33+, CD34+, and CD117+. Auer
Continuation rods typically are absent, and there is no
*This subcategory of AML with clear evidence of cellular maturation. The
myelodysplasia-related changes cells yield negative results with the
incorporates leukemias with at least 20% cytochemical stains, myeloperoxidase, and
blasts, multilineage dysplasia, a history of Sudan black B. These cases account for less
MDS or MDS/myeloproliferative neoplasm than 5% of AML, and patients are generally
(MPN), or a specific MDS-associated either infants or older adults.
cytogenetic abnormality and the absence
of AML with recurrent genetic abnormalities. Acute myeloid leukemia,
*MPN can lead to AML minimally differentiated
*Significant dysplastic morphology includes (French-American-British
pancytopenia with neutrophil classification M0). Blasts
hypogranulation or hypergranulation, lack myeloid
pseudo–Pelger-Huët cells, and unusually morphologic features
segmented nuclei. Erythrocyte precursors and yield negative
have vacuoles, karyorrhexis, megaloblastoid results with
features, and ringed sideroblasts. There may myeloperoxidase and Sudan black B
be dysplastic micromegakaryocytes and staining. Auer rods are not seen. CD34 is
dysplastic megakaryocytes. frequently present (bone marrow,
×500). Source: (From Carr JH, Rodak
C. Therapy-related myeloid neoplasms BF: Clinical hematology atlas, ed 4, St. Louis,
*Treatment with alkylating agents, radiation, 2013, Saunders.)
or topoisomerase II inhibitors has been
associated with the development of a D.2 Acute myeloid leukemia without
secondary AML, MDS, or MDS/MPN. These maturation. (M1)
therapy-related neoplasms account for 10% *Closely aligned with the blasts in minimally
to 20% of AMLs, MDSs, and MDSs/MPNs. differentiated AML, the blasts in AML without
maturation are also CD13+, CD33+, and
*Generally, these disorders occur following CD117+, and CD34 is present in about 70% of
treatment for a prior malignancy, but they cases. Blasts may comprise 90% of
have also been associated with intensive nonerythroid cells in the bone marrow, and
treatment of patients with nonmalignant fewer than 10% of the leukocytes show
disorders requiring cytotoxic therapy. maturation to the promyelocyte stage or
beyond. Blasts have Auer rods and usually
*Therapy-related myeloid neoplasms are give positive results with myeloperoxidase or
similar in morphology to AML with Sudan black B stains.
myelodysplasia, monocytic/monoblastic
leukemia, or AML with maturation, and the Acute myeloid leukemia
prognosis is generally poor. without maturation
(French-American-British
D. Acute myeloid leukemia, not otherwise classification M1). Blasts
specified constitute 90% of the
*Because the leukemias in the “not otherwise nonerythroid cells; there
specified” category do not fit easily into the is less than 10%
WHO subtypes described earlier, they are maturation of the granulocytic series beyond
grouped according to morphology, flow the promyelocyte stage (bone marrow,
cytometric phenotyping, and limited ×500).
cytochemical reactions, as in the FAB
classification.
Leukemia
*Monocytic cells (monoblasts and
promonocytes) constitute at least 20% of all
marrow cells. The monoblasts are large with
abundant cytoplasm containing small
granules and pseudopodia. The nucleus is
large and immature and may contain
multiple nucleoli. Promonocytes also are
present and may have contorted nuclei. The
cells are positive for the myeloid antigens
CD13 and CD33 and the monocytic antigens
CD14, CD4, CD11b, CD11c, CD64, and
CD36. Nonspecific cytogenetic changes are
found in most cases.
D.3 Acute myeloid leukemia with maturation.

(M2)
*AML with maturation is a common variant
that presents with greater than 20% blasts, at
least 10% maturing cells of neutrophil lineage
(), and fewer than 20% precursors with
monocytic lineage. Auer rods and other
aspects of dysplasia are present.
Acute myeloid leukemia

with maturation. Blasts Acute myelomonocytic
constitute 20% or more of leukemia. Both myeloid
the nucleated cells of and monocytic cells are
the bone marrow, and present. Monocytic cells
there is maturation comprise at least 20% of
beyond the all marrow cells, with
promyelocyte stage in more than 10% of the monoblasts and
nonerythroid cells (bone marrow, ×1000). promonocytes present (peripheral blood,
×1000). Source: (From Carr JH, Rodak BF:
D.4 Acute myelomonocytic leukemia. (M4) Clinical hematology atlas, ed 4, St. Louis,
*Acute myelomonocytic leukemia is 2013, Saunders.)
characterized by a significantly elevated
WBC count and the presence of myeloid and D.5 Acute monoblastic and monocytic
monocytoid cells in the peripheral blood and leukemias. (M5)
bone marrow. *In these leukemias, which are divided into
monoblastic and monocytic based on the
Leukemia
degree of maturity of the monocytic cells myeloid), in which 50% or more of nucleated
present in the marrow and peripheral blood, bone marrow cells are normoblasts and
more than 80% of the marrow cells are of greater than 20% are myeloblasts. In the FAB
monocytic origin. These cells are CD14+, classification, this subtype was known as M6.
CD4+, CD11b+, CD11c+, CD36+, CD64+, and
CD68+. *The second type is PURE ERYTHROID
LEUKEMIA. In this type, 50% or more
*Blasts are large with abundant, often nucleated cells are pronormoblasts and 30%
agranular cytoplasm and large prominent or more are basophilic normoblasts.
nucleoli. When some evidence of maturation Together, these two erythroid components
is present, the cells are comprise more than 80% of the bone
called promonocytes. Promonocytes in marrow. The myeloblast component is not
monocytic leukemias with differentiation is significant. Complex rearrangements and
considered to be blast equivalents. hypodiploid chromosome numbers are
common. Chromosomes 5 and 7 are
*Nonspecific esterase testing usually yields frequently affected.
positive results.
*Acute monoblastic/monocytic leukemia *The red blood cell (RBC) precursors have
comprises fewer than 5% of cases of AML and significant dysplastic features, such as
is most common in younger individuals. multinucleation, megaloblastoid
*Extramedullary involvement, including asynchrony, and vacuolization. The
cutaneous and gingival infiltration, and nucleated RBCs in the peripheral blood may
bleeding disorders are common. Nonspecific account for more than 50% of the total
cytogenetic abnormalities are seen in most number of nucleated cells. Ringed
cases. sideroblasts, Howell-Jolly bodies, and other
inclusions may be present. Abnormal
megakaryocytes may be seen. Both types of
erythroid leukemia have an aggressive and
Gingival enlargement in rapid clinical course.
monoblastic and
monocytic leukemia. Acute erythroid
leukemia. Erythroid
precursors show
dysplastic features,
including multinucleation
A, Acute monoblastic and megaloblastic
leukemia. More than asynchrony (bone
80% of the bone marrow marrow, ×500). Source: (From Carr JH, Rodak
cells are of monocytic BF: Clinical hematology atlas, ed 4, St. Louis,
origin (bone marrow, 2013, Saunders.)
×500).
B, Acute monocytic
leukemia with
promonocytes.
Promonocytes are considered blast
equivalents. Source: (B from Carr JH, Rodak
BF: Clinical hematology atlas, ed 4, St. Louis,
2013, Saunders.)
D.6 Acute erythroid leukemia (M6) D.7 Acute megakaryoblastic leukemia (M7)
*According to the WHO classification, there *Patients with acute megakaryoblastic
are two subtypes of acute erythroid leukemia usually have cytopenias, although
leukemia, based on the presence of a some may have thrombocytosis. Dysplastic
significant component of myeloblasts. The features are often present in all cell lines.
first is ACUTE ERYTHROLEUKEMIA (erythroid/
Leukemia
*Diagnosis requires the presence of at least syndrome. Somatic mutations of
20% blasts, of which at least 50% must be of the GATA1 gene have also been detected
megakaryocyte origin. and are linked to both leukemogenesis and
*Megakaryoblast diameters vary from that of high cure rates. Approximately 10% of
a small lymphocyte to three times their size. newborns with Down syndrome present with
*Chromatin is delicate with prominent transient abnormal myelopoiesis, which is
nucleoli. morphologically indistinguishable from AML.
*Immature megakaryocytes may have light *Spontaneous remission generally occurs

blue cytoplasmic blebs. within a few months.
*Megakaryoblasts are identified by
immunostaining, employing antibodies 6. Blastic plasmacytoid dendritic cell
specific for cytoplasmic von Willebrand neoplasm
factor or platelet membrane antigens CD41 *Blastic plasmacytoid cell neoplasm is a rare
(glycoprotein IIb), CD42b (glycoprotein Ib), or clinically aggressive tumor derived from
CD61 (glycoprotein IIIa). precursors of plasmacytoid dendritic cells. It
presents with skin lesions and may ultimately
progress to involve peripheral blood and
bone marrow.
7. Acute leukemias of ambiguous lineage

(ALAL)
*Acute leukemias of ambiguous lineage
(ALALs) include leukemia in which there is no
clear evidence of differentiation along a
single cell line and are commonly referred to
Figure. Acute megakaryocytic
as acute undifferentiated leukemias (AULs).
leukemia. A, Note heterogeneity of blasts,
one small with scant cytoplasm, two with
*Other cases of ALAL that demonstrate a
cytoplasmic blebbing, and one quite large
multiplicity of antigens where it is not possible
(peripheral blood ×1000). B, Positive reaction
to determine a specific lineage are
for CD42b (bone marrow, ×1000). Source:
called mixed phenotype acute
(A from Carr JH, Rodak BF: Clinical
leukemias (MPALs).
hematology atlas, ed 4, St. Louis, 2013,
Saunders.)
*Classification of Acute Leukemia of
Ambiguous Lineage (ALAL) (2008 World
Health Organization Classification)
• Acute undifferentiated leukemia
(AUL)—synonyms: ALAL without
differentiation, primitive acute
leukemia, stem cell leukemia
• Mixed phenotype acute leukemia
(MPAL)—synonyms: biphenotypic
4. Myeloid sarcoma acute leukemia, bilineal leukemia,
*Myeloid sarcoma refers to the mixed lineage acute leukemia, dual
extramedullary proliferation of blasts of one lineage acute leukemia, hybrid
or more myeloid lineages that disrupts tissue acute leukemia:
architecture. Tissue architecture must be • MPAL with t(9; 22)(q34; q11.2); BCR-
effaced for the neoplasm to qualify for this ABL1
diagnosis. • MPAL with t(v;
11q23); MLL rearranged
5. Myeloid proliferations related to down • MPAL B/myeloid, not otherwise
syndrome specified
*Unique patterns of malignancy occur in • MPAL T/myeloid, not otherwise
persons with trisomy 21 resulting in Down specified
Leukemia
8. Cytochemical stains and interpretations for *In contrast, lymphoblasts in ALL and
AML lymphoid cells are MPO-negative. The
*Techniques such as flow cytometry, reaction only in the blast cells must be used
cytogenetic analysis, and molecular testing as the determining factor for the
are now commonly used in the diagnosis of differentiation of acute leukemias. This is true
acute leukemias. However, older techniques for MPO and for the other cytochemical
such as cytochemical stains still retain their stains used in determining cell lineage that is
importance. An advantage of cytochemical mentioned in this chapter. The fact that
stains is that they are relatively cheap and maturing granulocytes are MPO-positive is
can be performed by laboratories normal and has little or no diagnostic
throughout the world, including in areas significance.
where resources and access to advanced
techniques are limited. B. Sudan black B
*SBB staining is another useful technique for
A. Myeloperoxidase (MPO) the differentiation of AML from ALL. SBB stains
*Myeloperoxidase (MPO) is an enzyme found cellular lipids. The staining pattern is quite like
in the primary granules of granulocytic cells that of MPO; SBB staining is possibly a little
(neutrophils, eosinophils, and, to a certain more sensitive for the early myeloid cells.
extent, monocytes). Lymphocytes do not
exhibit MPO activity. This stain is useful for Sudan black B reaction.
differentiating the blasts of acute myeloid The positivity increases
leukemia (AML) (Positive) from those of acute with the maturity of the
lymphoblastic leukemia (ALL). (Negative) granulocytic cell (bone
marrow, ×1000).
Positive reaction to
myeloperoxidase stain in *Sudan Black B Interpretation
early myeloid cells. Note • Granulocytes (neutrophils) show a
Auer rod at arrow (bone positive reaction to SBB from the
marrow, ×1000). myeloblast through the maturation
series. The staining becomes more
intense as the cell matures as a result
Strong positive reaction of the increase in the number of
to myeloperoxidase stain primary and secondary granules.
in leukemic Monocytic cells can demonstrate
promyelocytes from a negative to weakly positive staining
patient with acute due to various changes that occur
promyelocytic leukemia during differentiation. Lymphoid cells
(bone marrow, ×1000). generally do not stain. In ALL, fewer
than 3% of the blast cells show a
*MPO Interpretation positive reaction.
• MPO is present in the primary granules
of most granulocytic cells, beginning C. Esterases
at the promyelocyte stage and *Esterase reactions are used to differentiate
continuing throughout maturation. myeloblasts and neutrophilic granulocytes
Leukemic myeloblasts are usually from cells of monocytic origin. Nine
positive for MPO. In many cases of isoenzymes of esterases are present in
AMLs (without maturation, with leukocytes. Two substrate esters commonly
maturation, and with promyelocytic used are α-naphthyl acetate and α-naphthyl
leukemia), it has been found that butyrate (both nonspecific). Naphthol AS-D
more than 80% of the blasts show chloroacetate (specific) also may be used.
MPO activity. Auer rods found in “Specific” refers to the fact that only
leukemic blasts and promyelocytes granulocytic cells show staining, whereas
test strongly MPO positive. nonspecific stains may produce positive
results in other cells as well.
Leukemia
*Esterases Interpretation reaction in the monocytes is inhibited (bone
• Esterase stains can be used to marrow, ×1000).
distinguish acute leukemias that are
granulocytic from leukemias that are A diffuse positive α-
primarily of monocytic origin. When naphthyl butyrate
naphthol AS-D chloroacetate is used esterase reaction is seen
as a substrate, the reaction is positive in monocytes. α-
in the granulocytic cells and negative Naphthyl butyrate is less
to weak in the monocytic cells. sensitive than α-naphthyl
Chloroacetate esterase is present in acetate, but it is more
the primary granules of neutrophils. specific. Granulocytes
and lymphoid cells generally show a
Positive reaction to AS-D negative reaction, although a small positive
chloroacetate esterase dot may be seen in lymphocytes. In
stain in two granulocytic myelomonocytic leukemia, positive AS-D
cells (bone marrow, chloroacetate activity and positive α-
×1000). naphthyl butyrate or α-naphthyl acetate
activity should be seen because myeloid
and monocytic cells are present. In
Positive reaction to α- myelomonocytic leukemia, at least 20% of
naphthyl acetate the cells must show monocytic differentiation
esterase stain in that is nonspecific esterase positive and is
monocytes (bone inhibited by sodium fluoride. In pure
marrow, ×1000). B, Same monocytic leukemias, 80% or more of the
specimen with the blasts are nonspecific esterase positive and
addition of sodium fluoride. The esterase specific esterase negative.
Summary
*The development of leukemia is currently believed to be a stepwise progression of mutations, or

“multiple-hits,” involving mutations that give leukemic stem cells a proliferative advantage and
also hinder differentiation.
*For most acute leukemias, causes directly related to the development of the malignancy are
unknown, but a few exceptions exist. Some known causes include environmental toxins, certain
viruses, previous chemotherapy, and familial predisposition.
*There are several classification schemes for leukocyte neoplasia, including the FAB system, based
primarily on morphology and cytochemical staining, and the WHO system, which retains some
elements of the FAB scheme but emphasizes molecular and cytogenetic changes.
1. ALL
*Only half of the patients with ALL have leukocytosis, and many do not have circulating
lymphoblasts, but neutropenia, thrombocytopenia, and anemia are usually present.
*In children ALL is a disease in which the “good prognosis” subtypes are associated with a 95%
rate of complete remission, but adults with ALL have a poorer outlook.
*Infiltration of malignant cells into the meninges can occur, with lymphoblasts found in the
cerebrospinal fluid, testes, and ovaries.
*Prognosis in ALL depends primarily on age at the time of diagnosis, lymphoblast load (tumor
burden), and immunophenotype. Chromosomal translocations seem to be the strongest predictor
of adverse treatment outcomes for children and adults.
*The t(12; 21) marker is found in a significant number of patients with childhood ALL.
*There are two main subtypes of ALL according to the WHO classification system: B lymphoblastic
leukemia/lymphoma and T lymphoblastic leukemia/lymphoma.
Leukemia
*Tumor lysis syndrome is an increasingly common complication of treatment, especially in patients
with a high tumor burden.
2. AML
*Although morphology is the first tool in distinguishing ALL from AML, immunophenotyping is often
the only reliable indicator of a cell’s origin.
*The incidence of AML in adults increases with age.
*The clinical presentation of a patient with AML is nonspecific and reflects the decreased
production of normal bone marrow elements, an elevated WBC count, and the presence of
myeloblasts. Anemia, thrombocytopenia, and neutropenia give rise to the clinical findings of
pallor, fatigue, bruising and bleeding, and fever with infections.
*The classification of AML is complicated by the presence or absence of multiple cell lines defined
as “myeloid” in origin, specific cells within these cell lines, and specific karyotype abnormalities.
*Leukemias with ambiguous lineage include leukemias in which there is no clear evidence of
differentiation along a single cell line.
*FAB Classification
• M0- Acute Myeloid Leukemia with minimal differentiation
• M1- Acute Myeloid Leukemia without Maturation
• M2- Acute Myeloid leukemia with maturation
• M3- Acute Promyelocytic Leukemia
• M4- Acute Myelomonocytic Leukemia
• M5- Acute Monocytic Leukemia
• M6- Acute Erythroleukemia
• M7- Acute Megakaryocytic Leukemia
*Cytochemical techniques are often used in conjunction with morphologic analysis,

immunohistochemical methods, flow cytometry, cytogenetic analysis, and molecular biologic
techniques in establishing a diagnosis.
*Cytochemical reactions may be enzymatic or nonenzymatic. Fresh smears must be used to
detect enzymatic activity, whereas nonenzymatic procedures may be performed on specimens
that have been stored at room temperature.
*MPO stains primary granules and is useful in differentiating granulocytic from lymphoid cells.
*SBB stains lipids and results parallel those with the MPO stain.
*Esterases help differentiate granulocytes and their precursors from cells of monocytic origin.
Butyrate esterase testing gives positive results in monocytes but not in granulocyte precursors,
whereas naphthol AS-D chloroacetate esterase stains granulocyte precursors.
Leukemia
Leukemia
Leukemia

Hematology 1 BSMT3-1st Sem

Uploaded by

Copyright:

Available Formats

Hematology 1 BSMT3-1st Sem

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Hematology 1 BSMT3-1st Sem

Uploaded by

Copyright:

Available Formats

BACHELOR OF SCIENCE IN

COLEGIO SAN AGUSTIN-

ANGEL KATE DIENTE OLAGUER

HEMATOLOGY 1 LABORATORY ENDTERM .................................................................................................. 5

BLOOD SMEAR EXAMINATION: MAKING A BLOOD SMEAR

and shape. Identifying the size and shape of

2. Relative hemoglobin content

B. Observations Under x40: WBC Estimates

*Platelets are remnants of megakaryocytes

3. Observing and Recording Nucleated Red

WBC according to the different types then

B.2 Decreased neutrophil count D. Basophil

*Lymphocytes are the primary components

E.1 Two types of lymphocytes:

E.2 Lymphocytes increase (lymphocytosis) in: B. Abnormal differentials

*Pyknotic cell (RBC) (nucleus extremely *Thin area

1. Proper Staining Reactions of Different Blood

Neutrophils violet nucleus; lilac

1. Reticulocyte *Reticulocyte → any nonnucleated RBC that

5. Procedure for Reticulocyte Count

Hematology 1 Laboratory MLS 113A | Reticulocyte Count

4. LE Cell VS Tart Cell

2. SLE (Systemic Lupus Erythematosus) LE CELL TART CELL

Hematology 1 Laboratory MLS 113A | LE Cells

1. Introduction A. Reference Range for MCH

A. Reference Range for MCV (𝐺𝑟𝑎𝑚 𝐻𝑏)(100)

B. Interpretation for MCV [𝐻𝑏(𝑔/𝑑𝐿)][100]

3. MCH *Sample: Compute the MCV, MCH, and

Hematology 1 Laboratory MLS 113A | Erythrocyte Indices

fragility test may be used to confirm the

*The test only indicates that a proportion of

*HS patients who are experiencing significant

Hematology 1 Laboratory MLS 113A | Osmotic Fragility Test

Hematology 1 Laboratory MLS 113A | Osmotic Fragility Test

1. Abnormal erythrocyte morphology 3. Variation in erythrocyte size (anisocytosis)

*Comment: Most erythrocytes presented in

B. Polychromasia E. Tear Drop Cells

Hematology 1 Laboratory MLS 113A | RBCs Abnormal Morphology

J. Schistocytosis C. Basophilic stippling

Hematology 1 Laboratory MLS 113A | RBCs Abnormal Morphology

Summary of Poikilocytes and Anisocytes

Cell Description Causes

Hematology 1 Laboratory MLS 113A | RBCs Abnormal Morphology

Hematology 1 Laboratory MLS 113A | RBCs Abnormal Morphology

Hematology 1 Laboratory MLS 113A | RBCs Abnormal Morphology

MYELOPROLIFERATIVE NEOPLASM (MPNs)

Chronic Myelogenous Leukemia (CML) • Anemia → due to the small number of

*All secondary to massive pathologic

*Peripheral blood shows:

Hematology 1 Lecture MLS 113A | Myeloproliferative Neoplasm (MPNs)

Hematology 1 Lecture MLS 113A | Myeloproliferative Neoplasm (MPNs)

5. Leukocyte Alkaline Phosphatase

Figure. Peripheral blood films in the chronic

4. Other Laboratory Findings of Chronic *Phases of CML

*Fluorescence in situ hybridization *LAP (Leukocyte Alkaline Phosphatase)

Hematology 1 Lecture MLS 113A | Myeloproliferative Neoplasm (MPNs)

*Because scoring is subjective, the mean 8. Related diseases of Chronic Myelogenous

9. Goals of the Therapy for CML

Hematology 1 Lecture MLS 113A | Myeloproliferative Neoplasm (MPNs)

2. Identification of the JAK2 V617F mutation,

Pyknotic cell (RBC) (nucleus extremely Thin area

Fluorescence in situ hybridization LAP (Leukocyte Alkaline Phosphatase)

Iatrogenic Most obvious in reactive