1

• Usually mistaken with a tart cell (monocyte with an

LEUKOCYTE MOR- Ingested lymphocyte)

PHOLOGICAL ABNOR- CLINICAL SIGNIFICANCE

• Systemic Lupus Erythematosus

MALITIES • Conective tissue disorders

Barr body (Sex Chromatin)
Lecture - Week 14 (Part 1) • Represents the 2nd X chromosome in females
GRANULOCYTE NUCLEAR • Seen in 2-3% of neutrophils
ABNORMALITIES • “DRUMSTICK” appearance of neutrophils
NUCLEAR ABNORMALITIES Pseudo-Chediak Higashi Syndrome
Hypersegmented Neutrophil Cytoplasmic inclusions that resemble the fused
• Normal neutrophils contain lysosomal granules in Chediak-Higashi
• 3-5 lobes that are separated by filaments. syndrome.
Clincal Significance:
• Hypersegmented neutrophils
Acute myeloid leukemia
• >5 lobes and are usually larger than normal
Chronic myeloid leukemia
neutrophils
Myedeloplastic syndrome
CLINICAL SIGNIFICANCE:
• Megaloblastic anemia
• Undritz anomaly → Hereditary neutrophil
hypersegmentation
• Myelodysplasia
Hyposegmented Neutrophil
• Hyposegmentation of granulocyte nucleus Hypersegmented neutrophil
• Nuclei may appear:
• Round, ovoid (Homozygous Pelger-Huet)
• Bilobed forms the characteristic spectacle-like
Hyposegmented Neutrophil
(“pince-nez”, dumbbell, peanut) morphology Pince-nez form with two rounded
with the nuclei attached by a thin filament segments connected by a filament.
(Heterozyous Pelger-Huet) Notice the dense chromatin pattern

CLINICAL SIGNIFICANCE:
• Pelger-Huet anomaly
• Pseudo or Acquired Pelger-Huet anomaly
LE Cell
• Myelodysplastic syndrome
• Acute myeloid leukemia
• Myeloproliferative neoplasms
LE Cell
• Usually a neutrophil that has ingested the Bar Body (Sex Chromatin)
antibody-coated nucleus of another neutrophil or
has engulf the homogenous, globular nuclear
mass of destroyed cell
• Presence of: ANTI-NUCLEAR ANTIBODIES, cell CYTOPLASMIC ABNORMALITIES
nuclei, phagocytes with ingested material Alder-Reilly Granules

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• Darkly staining metachromatic (purple-red) • Acute promyelocytic leukemia (APL, M3)

cytoplasmic granules in granulocytes (less often in • Acute myelomonocytic leukemia (AMML, M4)
monocytes and lymphocytes
• Abnormal large primary granules → contain Alder-Reilley Granules
degraded mucopolysaccharides due to an Note the dark granules present in
both cells. Such as granules may
enzyme defect
be seen in eosinophils & basophils.
• May resemble toxic granules (seen in infections
May-Hegglin Granules
and toxic states) Neutrophil and giant Platelet; note
CLINICAL SIGNIFICANCE the large, elongated, bluish
inclusion in the netrophil cytoplasm
• Alder-Reilly anomaly
Chediak-Higashi Granules
• Mucopolysaccharidoses (MPSs)
Neutrophil w/ large dark lysosomal
May-Hegglin Granules granules
• Gray-blue spindle shaped inclusions in the
cytoplasm Chediak-Higashi Granules
• Large Dohle body-like inclusions in neutrophils, Monocyte with large azure granules

eosinophils, basophils, and monocytes

• May-Hegglin granules: composed of precipitated
Chediak-Higashi Granules
myosin heavy chains (mRNA) Lymphocyte with one large azure
• Dohle bodies: composed lamellar rows of rough granule

endoplasmic reticulum (rRNA)

CLINICAL SIGNIFICANCE
Auer Rods
• May-Hegglin anomaly
Chediak-Higashi Granules
• Giant red, blue, to grayish round inclusions in the Auer Rods
Myeloblast; usually rod shaped but
cytoplasm may be round in appearance,
• Giant lysosomal granules in granulocytes, Single or multiple Auer rods may
be seen in malignant
monocytes, and lymphocytes promyelocytes.
• Cells in the body are affected and exhibit
abnormally large lysosomes, which contain fused
dysfunctional granules Toxic Granulations
CLINICAL SIGNIFICANCE • Appears as dark, blue-black granules in the
• Chediak-Higashi syndrome cytoplasm of neutrophils, usually in segmented
Auer Rods and band forms
• Pink or red shaped cytoplasmic granules; found in • Granulation may represent the precipitation of
myeloid and monocytic series only *not present in ribosomal protein (RNA) caused by metabolic
lymphocytes* toxicity within the cells
• Fused primary granules (Peroxidase stain • Are peroxidase positive and reflect an increase in
positive) acid mucosubstance within primary, azurophilic
• Faggot cells: granules that may enhance bactericidal activity
• Bundle of auer rods • The extent of toxic granulation is usually graded
• Seen in acute promyelocytic leukemia (APL) on a scale of 1+ to 4+, with 4+ being the most
or M3; also associated with DIC severe. Grading of the granulation is dependent
CLINICAL SIGNIFICANCE on the coarseness and amount of granulation
• Acute myelogenous leukemia (AML, M1 and M2) within the cellular cytoplasm

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• Primary granules are larger than secondary • Less encountered than toxic granules and Dohle
granules bodies
• TG CAN MIMIC GRANULATION FOUND IN • Reflect phagocytosis, either of self
ALDER-REILLY ANOMALY (autophagocytosis) or of extracellular material
• one helpful defining characteristic of toxic TYPES
granulation is that in most cases, not all • AUTOPHAGOCYTIC VACUOLES
neutrophils are equally affected • Tend to be small (approximately 2 um) and
CLINICAL SIGNIFICANCE distributed throughout the cytoplasm
• Inflammations • Induced by specimen storage in EDTA for
• Infections more than two hours, autoantibodies, acute
• Toxic states alcoholism, and exposure to high doses of
• Burns radiation
• PHAGOCYTIC VACUOLES
• Malignant disorders
Dohle Bodies/ Dohle-Amato Bodies • Tend to be large (up to 6 um) and often
accompanied by toxic granulation
• Are light/pale blue round or elongated cytoplasmic
inclusions between 1-5 um consisting of remnants • Induced by either bacteria or fungi are
of ribosomal ribonucleic acid (rRNA) arranged in suggestive of sepsis
parallel row; close to cellular membranes • When phagocytic vacuoles are seen, a
• Are typically found in band and segmented careful examination sometimes reveals
neutrophils and can appear together with toxic organisms within the vacuoles
granulations; can be seen in eosinophils, Reactive Morphologic Changes in Neutrophil
basophils, monocytes, and lymphocytes Reactive
Appearance Associated with
• Localized failure of cytoplasmic maturation Change
• PAS reaction positive Toxic Dark, blue-black Inflammation,
• A delay in preparing the blood film after collection granulation cytoplasmic Infection, G-CSF
may affect Dohle body appearance in that they granules
are more grey than blue or in some cases may not Dohle Bodies Intracytoplasmic Infection, G-CSF,
be visible pale blue round or pregnancy, burns
elongated bodies
• Confused with May-Hegglin granules
between 1 to 5um in
CLINICAL SIGNIFICANCE diameter, usually
• Infections adjacent to cellular
membrane
• Pregnancy
• Burns Cytoplasmic Small to large Bacterial
Vacuoles circular clear areas infection,
• Toxic states
in cytoplasm, rarely autophagocytosis
May-Hegglin may contain secondary to drug
Parameter Dohle Bodies organism ingestion, acute
Granules
alcoholism, or
Size Small Large excess storage of
sample before
Composition rRNA mRNA
making blood film
PAS Reaction + -
Pyknotic & Necrotic Cells
Cytoplasmic Vacuolations • Pyknotic nuclei in neutrophils generally indicate
imminent cell death

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Pyknotic nucleus Pyknotic Cells

• water has been lost and the chromatin becomes Upper cell is a neutrophil
whose nucleus is dehydrated,
dense and dark w/c makes it very dark &
• chromatin or filaments can still be seen between dense. Note that there is still
a filament between the
nuclear lobes (depending on whether the cell is a segments (pyknotic cell)
band or segmented form)
Necrotic nuclei Necrotic Cells
Neutrophil that has died. Note
• Found in dead neutrophils/necrobiotic neutrophils that the nucelus has turned
• rounded nuclear fragments with no filaments and into numerous rounded
spheres of DNA with no
no chromatin pattern
filaments (necrottic or
• Increased numbers of pyknotic or necrotic necrobiotic cell)
cells suggest that an extended amount of time
has elapsed between blood collection and blood Neutrophil anisocytosis
The neutrophil to the left is
film preparation larger than other neutrophil;
CLINICAL SIGNIFICANCE: caused by cytoplasmic
swelling
• Bacterial infection
• Drug intoxication
Cytoplpasmic Swelling
• Is a result of osmotic swelling of the cytoplasm Ehrlichia & Anaplasma
or by increased adhesion to the glass slide in • Are small, obligate, intracellular bacteria
stimulated neutrophils transmitted by ticks to humans and other
• Regardless of the cause, the result is a variation vertebrate hosts
in neutrophil size or neutrophil anisocytosis • These organisms grow as a cluster (morulae) in
• Not much of clinical significance neutrophils and monocytes
• Morulae can be mistaken for Dohle bodies in
neutrophils
• Neutrophils → Anaplasma phagocytophilum and
Reactive Morphologic Changes in Neutrophil
rarely in Ehrlichia ewingii
• Monocytes → Ehrlichia chaffeensis
Toxic granulation • Human granulocytic erlichiosis (HGE) is
transmitted by the black-legged tick (Ixodes
scapularis) and the western black-legged tick (I.
pacificus)
Histoplasma
Dohle bodies
• Histoplasma capsulatum is a fungus; this
organism lives intracellularly in cells of the
mononuclear phagocyte system (macrophage),
cells of the bone marrow, or cells from sputum or
Cytoplasmic vacuoles effusion specimens
• The fungus appears as a tiny oval body with a
clear halo surrounding a small nucleus

Abnormal Granules in Neutrophil

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Type of reactive lymphocyte that

Anaplasma phagocytophilum in a has some morphologic features
neutrophil
of plasma cells

• fungoides)
Reed-Sternberg Cell
Ehrlichia chaffeensis in a
monocytic cell • Abnormal lymphocyte with an “owl’s eye
appearance”
• Pathognomonic sign for Hodgkin’s lymphoma
• Clinical significance: Hodgkin’s Lymphoma
Histoplasma capsulatum Rieder Cell
• Lymphocyte with a clover leaf like nucleus
• Clinical significance: Chronic lymphocytic
leukemia
Reactive Lymphocyte
LYMPHOCYTE ABNORMALITIES • stimulated when interacting with antigens in
Basket or Smudge Cell peripheral lymphoid organs
• Associated with degenerated nucleus or ruptured • Other names: variant, atypical, transformed,
cell in form or basket or smudge effector, plasmacytoid, Turk cells, Downey, and
• THUMBPRINT APPEARANCE immunoblasts
• FRAGILE LYMPHOCYTES that appear during smear • B and T lymphocyte activation results in the
preparation; ARTIFACT → appear at the end of transformation of small, resting lymphocytes into
smears proliferating larger cells
• Clinical significance: Chronic Lymphocytic • Often present as a heterogeneous population of
leukemia various shapes and sizes
Hairy Cell TYPES:
• Hair like cytoplasmic projections surrounding the • Type I: Turk’s irration plasma cytoid; lymphocyte
nucleus (fried-egg appearance) with a large block of chromatin
• Isoenzyme 5 (tartrate resistant is produced in • Type II: also known as IM cells; round mass of
abundance in hairy cell leukemia) chromatin (Ballerina skirt appearance)
• TRAP (Tartrate-resistant acid phosphatase) • Type III: Vacuolated (swiss cheese appearance)
stain positive CLINICAL SIGNIFICANCE:
• Clinical significance: Hairy cell leukemia • Infectious monucleosis
Sezary Cell • Leukemias
• Lymphocyte with a convoluted nucleus/brain-like • Viral infections
nucleus Flame Cells
• Presence indicates LEUKEMIC PHASE of • Abnormal plasma cell with red to pink cytoplasm
mycosis fungoides (Sezary syndrome) • Associated with increased Immunoglobulins
CLINICAL SIGNIFICANCE (usually IgA)
• Mycosis fungoides (cutaneous T-cell lymphoma) • Inclusion: Russel bodies (individual bodies of
Sezary syndrome (variant of mycoses Immunoglobulin)
CLINICAL SIGNIFICANCE:
Plasmacytoid lymphocyte • Multiple myeloma

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• Waldenstrom’s macroglobulinemia Abnormal Plasma Cells

Grape Cell
• Abnormal plasma cell with small colorless Flame Cell
vacuoles Associated with plasma cells
• aka Berry, Mott, Morula Cell that are producing IgA

• Inclusions: Dutcher’s bodies (intranuclear protein

inclusions) Grape Cell
Plasma cell containing
• Large protein globules giving appearance of
multiple round globules of Ig,
grapes: “Honeycomb appearance” w/c stain pink, colorless, or
CLINICAL SIGNIFICANCE: blue

• Multiple myeloma
• Reactive states
MONOCYTE OR MACROPHAGE
Basket or Smudge Cell ABNORMALITIES
Gaucher Cell
• distinctive macrophages, single or in clusters;
“CRUMPLED TISSUE APPERANCE”
• Abundant fibrillar blue-gray cytoplasm with a
Hairy Cell striated or wrinkled appearance (sometimes
described as onion skin-like)
• Positive with trichrome, aldehyde fuchsin, periodic
acid-Schiff (PAS) and acid phosphatase
Sezary Cell CLINICAL SIGNIFICANCE:
• Gaucher’s disease
Foam Cell
• Macrophages with cytoplasm packed with lipid-
filled lysosomes that appear as small vacuoles
Reed-Sternberg cells (foam) after staining
CLINICAL SIGNIFICANCE:
• Niemann-Pick’s disease
Sea-Blue Histiocytes
• Macrophages with lipofuscin, glycophospholipid,
Rieder Cells
and sphingomyelin contained in cytoplasmic
granules, 1 to 3 um in diameter, that appear blue
with Wright stain
CLINICAL SIGNIFICANCE:
Reactive Lymphocyte
Type I
• Niemann-Pick’s disease

Gaucher Cell
Reactive Lymphocyte Macrophage; note
Type II cytoplasmic striations in the
cytoplasm

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Foam Cell
Note eccentric nucleus and
bubble-like pattern of storage
deposit in the cytoplasm

Sea-Blue Histiocytes

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• HOMOZYGOUS PHA → cognitive impairment, heart

NON-MALIGNANT defects, and skeletal abnormalities may occur ; single

LEUKOCYTE DISOR- nuclei

Alder-Reilly Anomaly

DERS • Rare inherited disorder characterized by

granulocytes (monocytes and lymphocytes less
Lecture - Week 14 (Part 2) often) with large, darkly staining metachromatic
INTRODUCTION cytoplasmic granules
• Not caused by clonal or neoplastic changes in • AR anomaly was initially reported in patients with
hematopoietic precursor cells gargoylism; however, it can be seen in otherwise
• Causes can be genetic or acquired and involve healthy individuals
one or more lineages: neutrophil, lymphocyte, • Granulations also seen in mucopolysaccharidoses
monocyte, eosinophil, and basophil, affecting the (MPSs)
number of circulating cells, morphology, or both May-Hegglin Anomaly
• Many of these disorders are associated with • Rare, autosomal dominant disorder characterized
significant clinical manifestations, although some by variable thrombocytopenia, giant platelets, and
are benign in nature large May-Hegglin granules (Dohle body-like)
TYPES: • Caused by a mutation in the MYH9 gene with
• QUALITATIVE NON-MALIGNANT disordered production of myosin heavy chain type
• QUANTITATIVE NON-MALIGNANT/REACTIVE IIA, which affects megakaryocyte maturation and
STATES platelet fragmentation
Chediak-Higashi Syndrome
QUALITATIVE NON-MALIGNANT • A rare autosomal recessive disease of immune
Morphological Abnormalities Involving dysregulation
Neutrophils • Mutation in the CHS1 LYST gene
Pelger-Huet Anomally • Exhibit abnormally large lysosomes, which contain
• Autosomal dominant disorder characterized by fused dysfunctional granules
hyposegmentation • Can be present in granulocytes, monocytes, and
- ↓ nuclear segmentation and distinctive coarse lymphocytes
chromatin clumping pattern • Clinical manifestations begin in infancy with partial
• Affects all leukocytes, although morphologic albinism and severe recurrent life-threatening
changes are most obvious in mature neutrophils bacterial infections
• Mutations in the lamin B-receptor gene • Patients often have bleeding issues as a result of
• codes for lamin B receptor → inner nuclear abnormal dense granules in platelets; death
membrane protein that combines B-type lamins occurs before the age of 10 years
and heterochromatin
• plays a major role in leukocyte nuclear Defective Leukocyte Motility
shape changes that occur during normal Job’s Syndrome
maturation • Normal random movement ; abnormal
TYPES: CHEMOTACTIC/DIRECTIONAL MOTILITY
• HETEROZYGOUS PHA → normal individuals, • Patient suffer from persistent boils and recurrent
pince-nez appearance of the nucleus “cold” staphylococcal abcesses
• Associated with increased IgE

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Lazy Leukocyte Syndrome • LAD III patients experience a mild LAD I-like
• Abnormal random and chemotactic movement immunodeficiency with recurrent infections
• Cells failed to respond to inflammatory stimuli but • Othe synptoms: decreased platelet glycoprotein
have normal phagocytic and bactericidal activity IIb/IIIa, resulting in bleeding similar to that seen in
Leukocyte Adhesion Disorders (LADs) Glanzmann’s Thrombasthenia
• Are rare autosomal recessive inherited conditions Whim Syndrome
resulting in the inability of neutrophils and • WHIM → Warts, Hypogammaglobulinemia,
monocytes to move from circulation to the site of Infections, and Myelokathexis syndrome
inflammation (called extravasation) • Defect in intrinsic and innate immunity
• Consequences of these disorders are recurrent • Mutations in the CXCR4 gene
severe bacterial and fungal infections • CXCR4 protein regulates movement of white
• Hematopoietic stem cell transplant is the only blood cells between the bone marrow and
curative treatment peripheral blood
• HAS 3 TYPES (LAD I, II, III) • Neutrophils accumulate in the bone marrow
• Other: Shwachman-Bodian Diamond syndrome (myelokathexis), which results in low numbers of
circulating neutrophils
LAD I • In addition to neutropenia, lymphopenia,
• Mutation in the ITGB2 gene ; gene that encodes monocytopenia, and hypogammaglobulinemia are
CD18 (subunit of b2 integrins) present; as a result, patients experience recurrent
• b2 integrin → necessary for adhesion to endothelial bacterial infections and are highly susceptible to
cells, recognition of bacteria, and outside-in signaling human papillomavirus (HPV) infection, which
• Shortly after birth, patients suffer from recurrent leads to warts
infections, often affecting skin and mucosal
infections DEFECTIVE RESPIRATORY BURST
• Lymphadenopathy, splenomegaly, and Chronic Graulomatous Disease (CGD)
neutrophilia are common findings • A rare condition caused by the decreased ability
LAD II of neutrophils to undergo a respiratory burst after
• Mutation in the SLC35C1 gene ; leukocytes have phagocytosis of foreign organisms
normal b2 integrins • Can be X-linked recessive (60%) or autosomal
• Defective fucose transporter → no transportation of recessive (40%)
fucose (needed for selectin synthesis) → decreased • Caused by mutations in genes responsible for proteins
selectin synthesis that make up NADPH oxidase → NADPH oxidase
• Selectin - important in adhesion deficiency
• Patients have recurring infections, neutrophilia, • NADPH - important in respiratory burst
growth retardation, a coarse face, and other
physical deformities
LAD III
• Caused by mutations in Kindlin-3
• Kindlin-3 protein along with talin are required for
activation of b integrin and leukocyte rolling
• Leukocytes and platelets have normal expression
of integrins; however, there is failure in response
to external signals that normally results in
leukocyte activation

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DETECTION OF RESPIRATORY BURST

Chemiluminescense
uses dihydrorhodamine to measure
intracellular production of reactive oxygen
species
Nitroblue tetrazolium (NBT) test
NBT is a yellow, water-soluble dye
Positive: unreduced NBT (Colorless to
Yelow)
Negative: reduced NBT because of
the generation of respiratory burst (+BLUE
FORMAZAN)

• Patients experience life-threatening catalase- • MPO is low or absent in neutrophils and

positive bacterial and fungal infections monocytes but not in eosinophils
Congenital C3 Deficiency • Absence of MPO slows down bactericidal killing
• Autosomal recessive
• C3 is an opsonin LYSOSOMAL STORAGE DISEASES (LSD)
• Heterozygous: Carriers have half the normal C3 • Are a group of more than 50 inherited enzyme
activity (adequate for disease resistance) deficiencies resulting from mutations in genes that
• Homozygous: Repeated severe infections with code for the production of lysosomal enzymes
encapsulated bacteria which are poorly • These lysosomal enzyme deficiencies result is
recognized and inefficiently phagocytized because flawed degradation of phagocytized material and
of failure of opsonisation by C3 leads to buildup of undigested substrates within
G-6PD Deficiency lysosomes
• Absence affect the HEXOSE MONOPHOSPHATE • This causes cell dysfunction, cell death, and a
SHUNT range of clinical symptoms; all cells containing
• Leukocytes are unable to produce a respiratory lysosomes can be affected
burst, resulting in a defective bactericidal • LSDs are classified according to the
activity underdegraded macromolecule that accumulates
• G6PD is needed for the production of NADPH in the cell
oxidase • Examples:
Myeloperoxidae (MPO) Deficiency • Lipid Storage Disease/ Sphingollipidoses
• Autosomal recessive ; also known as ALIUS- • Mucopopolysaccharides
GRIGNASHI ANOMALY Lipid Storage Disease or Sphingolipidoses

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• Are qualitative disorders involving monocytes and • Deficiency in the enzyme acid sphingomyelinase
macrophages (ASM)
• The macrophages are particularly prone to • Associated with foam cells and sea-blue
accumulate undegraded lipid products, which histiocytes in the bone marrow
subsequently leads to an expansion of the TYPES OF NIEMANN-PICK’S DISEASE
reticuloendothelial tissue
• Acute neuronopathic form; affects mostly
• Examples: Eastern European Jews
• Gaucher’s Disease • < 5% normal sphingomyelinase activity ;
mutation in the SMPD1 gene
• Niemman-Pick’s Disease • Present in infancy and is associated with:
Gaucher’s Disease Type A • Failure to thrive
• Lymphadenopathy
• Most common of the lysosomal lipid storage • Hepatosplenomegaly
diseases; at least 1 in 17 Ashkenazi Jews are • Vision problems
• Rapid neurodegenerative decline that
carriers
results in death, usually by 4 years of age
• It is an autosomal recessive disorder caused by a
• Non-neuronopathic form ; more common in
defect or deficiency in the catabolic enzyme beta- individuals of Northern African descent
glucocerebrosidase • 10-20% normal enzyme activity ; mutation in
the SMPD1 gene
• Accumulation in sphingolipid glucocerebroside in • Presents in the first decade to adulthood with
macrophages throughout the body, including Type B a variable clinical course
osteoclasts in bone and microglia in the brain • Although there is no neurocognitive
impairment, patients experience
• Bone marrow replacement by Gaucher cells • Hepatosplenomegaly
contribute to anemia and thrombocytopenia • Heart disease
• Pulmonary insufficiency
• Pseudo-Gaucher cells can be found in bone
marrow of some patients with thalassemia, • Mutations in the NPC1 or NP2 gene
• Causes impaired cellular trafficking and
chronic myeloid leukemia, acute lymphoblastic homeostasis of cholesterol ; buildup of
leukemia, non-Hodgkin lymphoma, and plasma unesterified cholesterol in lysosomes
• Clinical presentation in type C NP is
cell neoplasms
Type C heterogeneous with regard to age of onset
Clinical Subtypes of Gaucher Disease and type and severity of neurologic and
psychiatric symptoms, as well as visceral
Type I Type II Type 3 involvement
Non- • Prognosis in type C NP is poor, with most
Acute Subacute
Neuronopathi
c
Neuronopathic Neuronopathic patients dying before the age of 25 years

Chilhood Chilhood OTHER LIPID STORAGE DISEASES

Age at presentation Infancy
Adulthood Adulthood

Hepatosplenomegal
+ → +++ + + → +++
y

Skeletal abnormality - → +++ - ++ → +++

CNS Disease - +++ + → +++

Life Span 6 to 80+ yrs < 2 yrs 2 to 60 yrs

Ashkenazi Panethnic,
Ethnicity Panethnic
Jews Swedes
Mucopolysaccharidoses
• Are a family of inherited disorders of
Niemann-Pick’s Disease
mucopolysaccharide or glycoaminoglycan (GAG)
• Characterized by accumulation of sphingomyelin
degradation
in cellular lysosomes in the liver, spleen, & lungs

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• Each MPS is caused by deficient activity of an • All the disorders within the 22q11 deletion
enzyme necessary for the degradation of syndrome (mostly associated with the TBX1 gene)
dermatan sulfate, heparan sulfate, keratan sulfate, have variable degrees of immunodeficiency
and/or chondroitin sulfate because of the absence or decreased size of the
• The partially degraded material builds up in thymus and low numbers of T lymphocytes
lysosomes and results in serious physical and • Associated with a broad range of problems such
cognitive problems and shortened survival as cardiac defects, palatal abnormalities,
distinctive facial features, developmental delays,
• Presence of Alder-Reilly bodies in neutrophils, psychiatric disorders, short stature, kidney
monocytes, and lymphocytes ; macrophages in disease, and hypocalcemia
the bone marrow can also demonstrate • Hematologic issues include thrombocytopenia and
cytoplasmic metachromatic material large platelets, autoimmune cytopenias, and
increased risk of malignancy
INHERITED DISORDERS OF LYMPHOCYTES • Death rate is high usually before 1 year of age
Bruton-Tyrosine Kinase Deficiency Examples:
• Also called as X-linked agammaglobulinemia, • Nezelof’s syndrome
Bruton’s agammaglobulinemia • DiGeorge syndrome
• Primary immunodeficiency disease characterized • Autosomal dominant Opitz GBBB
by reductions in all serum immunoglobulin • Sedlackova syndrome
isotypes and profoundly decreased or absent B • Caylor cardiofacial syndrome
cells • Shprintzen syndrome
• BTK deficiency is caused by a mutation in the • Conotruncal anomaly face syndrome
gene encoding Bruton tyrosine kinase, resulting in Severe-Combine Immune Deficiency
decreased production of BTK, which is important Gamma chain deficiency, or X-linked SCID
for B cell development, differentiation, and • most common form of SCID and is caused by
signaling
mutations in the IL2RG gene
• Without BTK, lymphocytes fail to fully mature, • IL2RG normally codes for the common gamma
leading to severe hypogammaglobulinemia and
chain in leukocyte receptors
an inability to produce specific antibodies
• Leukocyte receptor binds with interleukins 2, 4, 7,
• Infants with BTK deficiency display symptoms 9, 15 and 21
between 4 and 6 months, once maternal
- These interleukins provide growth,
antibodies have cleared.; recurring life-threatening
differentiation, and survival signals for B,
bacterial infections ensue
T, and NK cells
• Risk of fungal and viral (except enterovirus)
• Symptomatic between 3 to 6 months of age as
infection is low because of normal T cell function
protective maternal immunoglobulins are
Common Variable Hypogammaglobulinnemia
depleted, presenting without tonsils or lymph
• One or a combination of immunoglobulins is either
nodes along with severe life-threatening recurring
missing entirely or is synthesized in small
infections
quantities
• Circulating T and natural killer (NK) lymphocytes
• Inability of B-cells to mature into plasma cells
are nearly absent; B cells are adequate in number
which can be excessive production of T-
but are dysfunctional
suppressor cells
• Failure to thrive before age 2 unless treatment
22q11 Syndromes
with hematopoietic stem cell transplant is
successful

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ADA Deficiency • T cells are decreased; B cells, T cells and NK

• Autosomal recessive adenosine deaminase cells, neutrophils and monocytes are dysfunctional
deficiency represents 10% to 20% of SCID cases which leads to bacterial, viral and fungal infections
and is caused by one of many mutations in the • Risk of bleeding due to thrombocytopenia and 1
ADA gene Other SCIDs
• ADA deficiency results in an intra- and SEX-LINKED AGAMMAGLOBULINEMIA
extracellular accumulation of adenosine, which is • A defect in the helper cellular immune mechanism
lymphotoxic, leading to profound decrease in T, B, leading to agammagloulinemia
and NK cells • Defect is seen only in boys
• Patients experience a range of recurring, life- SWISS-TYPE AGAMMAGLOBULINEMIA
threatening bacterial, viral, and fungal infections • Autosomal recessive; involves loss of both T-cell
beginning early in life. In addition, there are and B-cell functions
skeletal abnormalities, neurologic deficits, and • Little or no immunoglobulin is found in the blood;
skin rashes thymus is present but lacks lymphoid elements
Wiskott-Aldrich Syndrome ATAXIA TELANGIECTASIA
• Rare X-linked disease caused by one of more • Autosomal recessive
than 400 mutations in the WAS gene, which • Ataxia: progressive loss of muscle coordination
results in decreased levels of WASp protein
• Telangiectasia: dilation of the small blood vessels
• WASp is important in cytoskeletal remodeling and
• Associated with peripheral blood lymphopenia,
nuclear transcription in hematopoietic cells
aplastic or hypoplastic thymus with minimal T-cell
(maturation)
population
• Decreased IgA and IgE, normal to increased IgG

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QUANTITATVE NON-MALIGNANT LEUKOCYTE DISORDERS

Reactive State Absolute Count Associated Disease

NEUTROPHILIA > 7 to 8 x10⁹/L Acute inflammatory

• Collagen vascular, vasculitis
Acute infectious
Most common type of • Bacterial, some viral, fungal, parasitic
leukopenia Drugs, toxins, metabolic
• Corticosteroids, growth factors, uremia, ketoacidosis
AGRANULOCYTOSIS Tissue necrosis
• Extreme neutropenia • Burns, trauma, MI, RBC hemolysis
(< 0.5) Physiologic
• Infantile: Kostmann’s • Stress, exercise, smoking, pregnancy
syndrome Neoplastic
• Carcinomas, sarcomas, myeloproliferative disorders

NEUTROPENIA < 1.75 - 1.80 x10⁹/L Drugs

• Cancer chemotherapy, chloramphenicol, sulfas/other antibiotics,
phenothiazines, benzodiazepine, antithyroids, anticonvulsants,
quinine, quinidine, indomethacin, procainamide, thiazides, radiation
Toxins
• Alcohol, benzene compounds
Intrinsic defects
• Fanconi’s, Kostmann’s, cyclic neutropenia, Chédiak-Higashi
Immune-mediated
• Collagen vascular disorders, RA, AIDS
Hematologic
• Megaloblastic anemia, myelodysplasia, marrow failure, marrow
replacement
Infectious: any overwhelming infection
Others: starvation, hypersplenism

EOSINOPHILIA > 0.7 x10⁹/L Allergic

• Urticaria, hay fever, asthma
Inflammatory
• Eosinophilic fasciitis, Churg-Strauss syndrome
Parasitic
• Trichinosis, filariasis, schistosomiasis
Nonparasitic infections
• Systemic fungal, scarlet fever, chlamydial pneumonia of infancy
Respiratory
• Pulmonary eosinophilic syndromes (Löffler’s, tropical pulmonary
eosinophilia), Churg-Strauss syndrome
Neoplastic
• CML, Hodgkin lymphoma, T cell lymphomas
Idiopathic Hypereosinophilic Syndromes
• Affects heart, liver, spleen, CNS, other organs
Others
• Certain drugs, hematologic and visceral malignancies, GI
inflammatory diseases, sarcoidosis, Wiskott-Aldrich syndrome

BASOPHILIA > 0.3 x10⁹/L Myeloproliferative disease

Allergic: food, drugs, foreign proteins
Infectious: variola, varicella
Chronic hemolytic anemia: Post splenectomy
Inflammatory:
• Collagen vascular disease, ulcerative colitis

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MONOCYTOSIS > 0.9 x10⁹/L Infectious

• Tuberculosis, subacute bacterial endocarditis, syphilis, protozoan,
rickettsia
Recovery from Neutropenia
Hematologic
• Leukemias, myeloproliferative disorders, lymphomas,
multiplemyeloma
Inflammatory
• Collagen vascular disease, chronic ulcerative colitis, sprue, myositis,
polyarteritis, temporal arteritis
Others: Solid tumor, immune thrombocytopenic purpura, sarcoidosis

LYMPHOCYTOSIS Adult: > 4 x10⁹/L Infections

Infants: > 9 x10⁹/L • Viral, pertussis, tuberculosis, toxoplasmosis, rickettsial
Chronic inflammatory
CMV- resembles RS • Ulcerative colitis, Crohn’s disease
cells Immune mediated
• Drug sensitivity, vasculitis, graft rejection, Graves’, Sjögren’s
Hematologic
• ALL, CLL, lymphoma
Stress: acute, transient

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DEFINITION
INTRODUCTION TO • The term leukemia is derived from the ancient
Greek words leukos (leykóç), meaning “white,”
and haima (aἷma), meaning “blood”
TABLE OF CONTENTS • Includes leukemias, lymphomas, myelomas
QUALITATIVE NON-MALIGNANT DISORDERS
(plasma cell neoplasms) and myelodysplastic
A. Morphological Abnormalities of Neutrophils
syndromes (previously called preleukemias)
Hypersegmentation
• Terms that refer to large heterogeneous groups of
Alder-Reilly Anomaly
disorders; they initiate in a hematopoietic cell as a
May-hegglin Anomaly
result of ACQUISITION OF ONE OR MORE
Chediak-Higashi Syndrome
Pelger-Huet Anomally MUTATIONS IN KEY GENES that regulate cell
Pseudo/ Acquired Pelger-Huet growth (proliferation), survival, differentiation, or
maturation
• Occur in hematopoietic cells of all lineages and at

HEMATOLOGIC NEO- various stages of their development

• Most hematologic neoplasms are not localized but

PLASMS; ACUTE rather are systemic at initiation of the malignant

process

LEUKEMIAS LEUKEMIA
Lecture - Week 15 • Originate in bone marrow, and leukemia cells
HEMATOLOGIC NEOPLASM readily pass into peripheral blood, but they can
HISTORY also infiltrate lymphoid tissues (spleen, liver,
• Hematologic neoplasms were the first human lymph nodes) as well as other organs and tissues
cancers in which a consistent genetic defect was of the body
identified • Are divided into lymphoid and myeloid lineages,
• Started during the time of Hippocrates and further into acute (precursor cell) and chronic
1839 to 1845 Virchow: 1st to recognize leukemia as a (mature cell) categories
distinct clinical disorder. He named this • ACUTE LEUKEMIAS
disorder leukemia because of the white • Onset is sudden, progression is rapid, and
appearance of the blood from patients
the outcome is fatal in weeks or months if
w/ fever, weakness, and
lymphadenopathy left untreated
• White blood cell (WBC) count is variable,
1960 Nowell and Hungerford published an
abstract that described a consistent and there is an excess accumulation of
shortened chromosome in seven precursor hematopoietic cells or blasts of a
patients with chronic myeloid or specific lineage in bone marrow and
myelogenous leukemia (CML); referred to peripheral blood because of a block in
as the Philadelphia chromosome
differentiation (maturation arrest)
1973 Rowley reported the t(9;22) translocation • CHRONIC LEUKEMIAS
in CML
• Onset is insidious and progression is slower,
1982 Taub and colleagues reported the t(8;14) with a longer survival compared with acute
translocation in Burkitt lymphoma leukemia
• The WBC count is usually elevated, and
there is a proliferation and accumulation of

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mature and maturing cells of a specific

lineage

ACUTE CHRONIC
LEUKEMIA LEUKEMIA

Predominant Precursor cell or - Mature

cell type blast

Onset Sudden Insidious

ACUTE LEUKEMIA
Symptoms at Fever (due to Variable,
• Refers to the rapid, clonal proliferation in the bone
presentation neutropenia- nonspecific; some
induced infection) asymptomatic marrow of lymphoid or myeloid progenitor cells
known as lymphoblasts and myeloblasts
Mucocutaneous • When proliferation of blasts overwhelms the bone
bleeding (due to marrow, blasts are seen in the peripheral blood
thrombocytopenia
and the patient’s symptoms reflect suppression of
)
normal hematopoiesis
Fatigue (due to • Onset is sudden, progression is rapid, and the
anemia) outcome is fatal in weeks or months if left
WBC count Variable Increased untreated
TYPES
Progression Rapid; weeks to Slower; months to
• Acute Myeloid/ Myelogenous/ Myeloblastic
w/o months years
treeatment Leukemia or Acute Non-Lymphoblastic Leukemia
(ANLL)
• Acute Lymphoblastic Anemia
LYMPHOMA CLASSIFICATION SCHEMES
• Solid tumors of lymphoid cells that usually • French - American - British (FAB) Classification
originate in the lymphatic system and proliferate in • Devised in the 1970’s
lymph nodes and other lymphoid organs and • Based on: BM morphology, cytochemical
tissues reactions, cytogenetics, T and B cell markers
MYELOMA (PLASMA CELL NEOPLASMS) (immunophenotyping)
• Cancer of the plasma cells; in myeloma, the cells • Criteria for diagnosis of Acute Leukemia:
overgrow, forming a mass or tumor that is located >30% of blasts in the blood is associated
in the bone marrow • World health Organization (WHO) Classification
• GOLD STANDARD in classifying leukemias
MYELODYSPLASTIC SYNDROMES (MDS) • Based on: BM morphology, cytochemical
• Group of acquired clonal hematologic disorders reactions, immunologic probes of cell markers,
characterized by progressive cytopenias in the clinical manifestations, cytogenetics
peripheral blood, reflecting defects in erythroid, • Criteria for diagnosis of Acute Leukemia:
myeloid, and/or megakaryocytic maturation. >20% of blasts in the blood is associated
with leukemia

ACUTE LYMPHOBLASTIC ANEMIA

• Is primarily a disease of childhood and
adolescence, accounting for 25% of childhood

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cancers and up to 75% of childhood leukemia • T-lymphoblastic leukemia/lymphoma (T-ALL)

(most common type of leukemia in children) have abnormal gene rearrangements, none of the
• Peak incidence of ALL in children is between 2 abnormalities is clearly associated with specific
and 5 years of age; although ALL is rare in biologic features
adults, risk increases with age; most adult patients • The three subtypes are differentiated based on
are older than 50 years of age morphology, including cell size, prominence of
• The subtype of ALL is an important prognostic nucleoli, and the amount and appearance of
indicator for survival; adults have a poorer cytoplasm
outlook: 80% to 90% experience complete TYPES
remission, but the cure rate is less than 40% • FAB L1 (children)
• Lymphoblasts stain PAS and TdT positive; • FAB L2 (older children and adults)
Sudan Black B (SBB) and Myeloperoxidase • AB L3 (patients with leukemia secondary to
(MPO) negative Burkitt’s lymphoma; t(8;14)

LABORATORY FINDINGS
• WBC count of >50 x 109/L ; less than 15% of FAB Siize of Nuclear
Shape Cytoplasm
patients have extreme leukocytosis of >100 Type Blasts Shape
x10⁹/L
L1 Small Indistinct Scant Invisible
• Predominance of blasts cells (>20% for WHO;
>30% for FAB) in about 50% of patients L2 Large, Indented, Large, Moderately
heterogen prominent abundant clefted
(lymphoblasts with lymphocytes and smudge ous
cells)
L3 Large Regular Prominent, Prominent
• Granulocytopenia
oval to basophilic vacuoles
• IMMUNOPHENOTYPING: round
• B-cell ALL: CD34, CD 19, CD 10, CD22, TdT
• T-cell ALL: CD2, CD3, CD4, CD5, CD7, CD8,
MORPHOLOGY OF ABNORMAL LYMPHOBLASTS
TdT
• Lymphoblasts vary in size but fall into two
CLINICAL FINDINGS
morphologic types
• Fatigue (caused by anemia), Fever (caused by
• The most common type seen is a small
neutropenia and infection), and Mucocutaneous
lymphoblast (1.0 to 2.5 times the size of a normal
bleeding (caused by thrombocytopenia)
lymphocyte) with scant blue cytoplasm and
• Lymphadenopathy, including enlargement, is
indistinct nucleoli
often a symptom
• The second type of lymphoblast is larger (two to
• Splenomegaly and hepatomegaly
three times the size of a lymphocyte) with
• Bone pain often results from intramedullary growth
prominent nucleoli and nuclear membrane
of leukemic cells
irregularities; these cells may be confused with the
• Eventual infiltration of malignant cells into the
blasts of AML
meninges, testes, or ovaries occurs frequently,
and lymphoblasts can be found in the CSF
WHO CLASSIFICATION OF ALL
• B-lymphoblastic leukemia/lymphoma (B-ALL is
subdivided into nine subtypes that are associated
w/ recurrent cytogenetic abnormalities

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CLINICAL FINDINGS:
• Infiltration of malignant cells into the gums and
IMMUNOPHENOTYPING other mucosal sites and skin
• Although morphology is the first tool used to • Splenomegaly is seen in half of AML patients, but
distinguish ALL from AML, immunophenotyping lymph node enlargement is rare
and genetic analysis are the most reliable • Hyperuricemia (caused by increased cellular
indicators of a cell’s origin turnover), hyperphosphatemia (due to cell lysis),
• Because both B and T cells are derived from and hypocalcemia (the latter two are also involved
lymphoid progenitors, both usually express CD34, in progressive bone destruction)
terminal deoxynucleotidyl transferase (TdT), and • Hypokalemia is also common at presentation
human leukocyte antigen, DR subregion (HLA- • During induction chemotherapy, especially when
DR) the WBC count is quite elevated, tumor lysis
TYPES OF ALL BASED ON IMMUNOLOGIC syndrome may occur
METHODS: LABORATORY FINDINGS:
Early B cell ALL (pro-B or pre-pre B cell ALL) • Decreased production of normal bone marrow
• 5% in children and 11% in adults elements
Intermediate (common) B cell ALL • WBC count: 5-30
• t(4;11) • Myeloblasts (>20% for WHO ; >30% for FAB) are
Pre-B cell ALL present in the peripheral blood in 90% of patients
• t(9;22) • Anemia, thrombocytopenia, and neutropenia
• Most mature B cell ALL; TdT negative, CD34 • IMMUNOPHENOTYPING: CD13, CD33, CD117,
variable CD14 or CD64
• 15% of childhood and 10% of adult B cell ALL
T cell ALL
• t(7;11)
• most often in teenaged males with a mediastinal
mass, elevated peripheral blast counts, meningeal
involvement, and infiltration of extra marrow sites.

WHI CLASSIFICATION OF AML

• The 2017 WHO classification for myeloid
malignancies has categorized AMLs with recurrent
cytogenetic abnormalities into subgroups based
on the primary cytogenetic aberrations along with
a few new entities
FAB CLASSIFICATION OF AML
ACUTE MYELOID LEUKEMIA (AML) • FAB classification is synonymous with WHO
• AML is the most common type of leukemia in classification of “AML not otherwise specified”
adults, and the incidence increases with age • Based on:
• AML is less common in children • Bone marrow morphology
• The FAB classification of AML was based on • Cytochemical reactions
morphology and cytochemistry • Cytogenetics
• WHO classification relies heavily on molecular • T and B cell markers (Immunophenotyping)
characterization and cytogenetics French-American-British (FAB) Classification of AML

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Subtype Description • Presence of chloroma → green appearance of tissue

using MPO
Acute myeloid leukemia, minimally
M0 • Auer rods may be present
differentiated
ACUTE MYELOID LEUKEMIA, WITH MATURATION
M1 Acute myeloid leukemia without maturation (FAB M2)
M2 Acute myeloid leukemia with maturation • Similar to t(8;21)(q22;q22.1); RUNX1 or
RUNX1T1 mutation in WHO classification
M3 Acute promyelocytic leukemia
• A common variant that presents with <90%,
M4 Acute myelomonocytic leukemia myeloblasts and >10% maturing cells of neutrophil
Acute myelomonocytic leukemia with lineage, and <20% precursors with monocytic
M4eo
eosinophilia lineage
Acute monocytic leukemia, poorly • Auer rods are often present
M5a
differentiated • Most blasts stain with MPO, SBB, and CAE
M5b Acute monocytic leukemia, well differentiated
• Markers present: CD13, CD33, CD117, CD34
• FAB M1 and M2 account for 50% of AML cases
M6 Acute erythroleukemia
ACUTE PROMYELOCYTIC LEUKEMIA (FAB M3)
M7 Acute megakaryocytic leukemia • Comprises 5% to 10% of AML cases ; occurs in all
age groups but is seen most commonly in young
adults
• Characterized by a differentiation block at the
promyelocytic stage
• The presence of >30% abnormal promyelocytes
with bundles of auer rods (faggot cells)
• Abnormal promyelocytes can be:
FAB CLASSIFICATION OF AML • Hypergranular → associated with DIC and absence
ACUTE MYELOID LEUKEMIA, MINIMALLY of HLA
DIFFERENTIATED (FAB M0) • Hypogranular/Microgranular → aPML-M3v; 20-
• Account for less than 5% of AML, and patients are 30% of APL cases; presence of “butterfly” or coin-
generally either infants or older adults on-coin nucleus
• Auer rods typically are absent, and there is no • Promyelocytes stain positive with SBB, MPO,
clear evidence of cellular maturation and CAE
• Markers present: CD13, CD33, CD34, CD117 • Cytogenetic abnormality involves balanced
• Cells yield NEGATIVE RESULTS w/ the translocation between chromosomes 15 and 17
cytochemical stains myeloperoxidase (MPO) and t(15;17) ; fusion of PML gene and retinotic acid
Sudan black B (SBB) receptor alpha (RARa) or the PML-RARa gene
ACUTE MYELOID LEUKEMIA, WITHOUT • 95% of APL cases have the PML-RARa protein
MATURATION (FAB M1) • Treated with all-trans-retinoic acid (ATRA) and
• May comprise >90% myeloblasts, and fewer than arsenic trioxide
10% of the leukocytes show maturation to the ACUTE MYELOMONOCYTIC LEUKEMIA (FAB M4)
promyelocyte stage or beyond • Characterized by a significantly elevated WBC
• At least 3% of blasts give positive results with count and the PRESENCE OF MYELOID AND
myeloperoxidase or Sudan black B stains ; also MONOCYTOID CELLS in the peripheral blood
stain with CAE and bone marrow
• Markers present: CD13, CD33, CD117, CD34 • Characterized by >20% (WHO) or >30% (FAB)
marrow myeloblasts with >20%-80% cells of

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monocytic origin (monoblast, promonocyte, • Markers present:

monocyte); may have Auer rods • Erythroblasts: CD45, Glycophorin A (CD71)
• Also called as NAEGELI’S TYPE ; accounts for • Myeloblasts: CD13, CD15, CD33, CD117
30% of AML cases • Myeloblasts stain positive with SBB, MPO,
• FAB M4Eo → variant of FAB M4 with myeloblasts CAE ; erythroid cells are PAS-POSITIVE
and monoblasts along with abnormal eosinophils PURE ERYTHROID LEUKEMIA
• Markers present: • Remains as M6; characterized by >80% erythroid
• Myeloid: CD13, CD33, CD34, CD117 cells ; >30% proerythroblasts
• Monocytic: CD14, CD4, CD11b, CD11c, • RBC precursors have significant dysplastic
CD64, CD36, CD68, and lysozyme features ; presence of ringed sideroblasts, Howell-
• Myeloid cells stain with SBB, MPO, and CAE , Jolly bodies and other inclusions
monocytic cells with NSE ACUTE MEGAKARYOBLASTIC LEUKEMIA (FAB
ACUTE MONOCYTIC LEUKEMIA (FAB M5) M7)
• Characterized by >20% (WHO) or >30% (FAB) • Chacterized by the presence of at least >20%
marrow monoblasts ; account for 10% of AML (WHO) or >30% (FAB) blasts, of which at least
cases 50% must be of megakaryocyte origin ; Rarest
• In these leukemias, which are divided into type of AML
monoblastic (M5a) and monocytic (M5b) based on • Cells are stained with Alpha Naphthyl acetate
the degree of maturity of the monocytic cells esterase (ANAE) and PAS
present • Markers present: von Willebrand factor, CD41 (gp
SUBTYPES: IIb) , CD42b (gp Ib), CD61 (gp IIIa)
• FAB M5a/ Schilling’s type/ AML poorly
differentiated/ Acute monoblastic leukemia/ AML
without maturation
• Seen in children ; >80% monoblasts in the bone
marrow
• FAB M5b/AML well differentiated/AML with
maturation
• Seen in middle-aged adults ; <80% monoblasts
• Cytogenetic abnormality: t(8;16)(p11;p13) has
been associated with M5 (also M4)
• Markers: CD14, CD4, CD11b, CD11c, CD 64
• Cells stain positive with NSE
ACUTE ERYTHROLEUKEMIA (FAB M6)
• One of the major changes in the 2017 WHO
classification is the removal of acute
erythroleukemia (erythroid/myeloid type) – most of
these cases will now be classified as MDS with
excess blasts
ACUTE ERYTHROLEUKEMIA
• aka known as Erythemic myelosis, Di Guglielmo’s
syndrome
• Characterized by >20% (WHO) or >30% (FAB)
marrow myeloblasts and >50% dysplastic marrow
normoblasts

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• Transformation to acute leukemia

CHRONIC LEUKEMIAS
Lecture - Week 16 WHO CLASSIFICATION OF MPNs
CHRONIC LEUKEMIAS • BCR-ABL1 Mutation
• Increased mature rather than immature cells in • Chronic Myeloid Leukemia (CML)
the lymphoid or myeloid lines • JAK2V617F Mutation
• The basic defect in growth control presumably • Polycythemia vera (PV)
affects progenitor cells, but in these disorders • Essential (primary) Thrombocytopenia (ET)
most neoplastic cells differentiate • Primary Myelofibrosis (PMF)
• Progression is slower compared to acute
leukemias ; can be months up to years BCR-ABL1 MUTATION
TYPES: CHRONIC MYELOID LEUKEMIA (CML)
• MYELOPROLIFERATIVE NEOPLASMS (MPN’s) • CML is an MPN arising from a single genetic
• MATURE LYMPHOID translocation in a pluripotential HSC producing a
NEOPLASMS/LYMPHOPROLIFERATIVE clonal overproduction of the myeloid cell line
DISORDERS • CML occurs at all ages but is seen predominantly
MYELOPROLIFERATIVE NEOPLASMS in those aged 46 to 53 years; it represents about
• are clonal hematopoietic disorders caused by 20% of all cases of leukemia, is slightly more
genetic mutations in the hematopoietic stem cells common in men than in women
(HSCs) that result in expansion, excessive • CML begins with a chronic clinical phase and, if
production, and accumulation of mature untreated, progresses to an accelerated phase in
erythrocytes, granulocytes, platelets, and mast 3 to 4 years and often terminates as an acute
cells leukemia (blast crisis phase)
• Each has a chronic course that may terminate as CYTOGENETIC ABNORMALITY
acute leukemia, myelofibrosis, or a coagulopathy Presence of the BCR-ABL1 gene (Philadelphia
• Molecular analysis is now incorporated into the chromosome/Ph’)
diagnosis workup of MPNs, research molecular • Reciprocal translocation between the long arms (q
advances were capped by the discovery of the arm) of chromosomes 9 and 22  t(9;22)
JAK2, Janus kinase 2 (JAK2V617F mutation); this • This acquired somatic mutation specifically
discovery allowed for refinement of the reflects the translocation of an ABL1 proto-
classification of MPNs oncogene from band q34 of chromosome 9 to the
• Myeloproliferation largely is due to hypersensitivity breakpoint cluster region (BCR) of band q11 of
or independence of normal cytokine regulation chromosome 22 (q34;q11)
resulting from genetic mutations that reduces • The BCR-ABL protein has constitutive kinase
cytokine levels through negative feedback activity that deregulates signal transduction
systems normally induced by mature cells pathways, causing abnormal cell cycling, inhibition
• All of the MPNs involve dysregulation at the of apoptosis, and
multipotent hematopoietic stem cell (CD34), with increased proliferation of
one or more of the following shared features: cells
• Cytogenetic abnormalities • Also found in FAB M1
• Overproduction of 1 or more types of blood (3-5%) and FAB L1 and
cells w/ dominance of a transformed clone L2 (10-25%)
• Hypercellular marrow or marrow fibrosis
• Thrombotic and/or hemorrhagic bleeding
• Extramedullary hematopoiesis

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CLINICAL FINDINGS: Blast crisis 3-4 mo Generally

CHRONIC PHASE: unresponsive
• Most cases (85%) are diagnosed in this phase
• Frequent infection, anemia secondary to massive LABORATORY FINDINGS
massive pathologic accumulation of myeloid BLOOD
progenitor cells in bone marrow, peripheral blood, • Marked expansion of the granulocyte pool
and extramedullary tissues • Anemia and thrombocytopenia (variable)
• Neutrophilia eith all maturational stages present, • Left shift with few blasts in the peripheral blood
basophilia, eosinophilia • Extramedullary hematopoiesis (spleen, liver)
• Progressive fatigue and malaise, low-grade fever, BONE MARROW
anorexia, weight loss, and bone pain • Intense hypercellularity
• Night sweats and fever, associated with an • Reticulin fibers are increased in approximately
increased metabolism caused by granulocytic cell 20% of patients
turnover, may occur • Increased megakaryocyte density is associated
• Splenomegaly; splenic infarction is common with an increase in myelofibrosis
because of the abnormal overproduction and • Presence of pseudo-Gaucher cells
accumulation of granulocyte precursors in the OTHER LABORATORY FINDINGS
bone marrow, spleen, and blood • Hyperuricemia and uricosuria (from cell turnover)
• On fresh incision, extramyeloid masses appear • Detection of t(9;22) translocation (cytogenetic
green (Chloromas), presumably because of the analysis, FISH, RT-PCR)
presence of the myeloid enzyme myeloperoxidase • DECREASED LAP SCORE (LAP is used to
ACCELERATED PHASE: differentiate CML from leukemoid reaction)
• Increased splenomegaly CHRONIC MYELOID LEUKEMIA VS LEUKEMOID
• Worsening anemia and thrombocytopenia
• Gradual failure of response to treatment
• Additional cytogenetic abnormalities
• 10-19% blasts
BLAST CRISIS (ACUTE):
• involves the peripheral blood, bone marrow, and
extramedullary tissues
• Based on AL definitions, blasts constitute more
than 20% of total BM cellularity, and the
peripheral blood exhibits increased blasts
• Extramedullary growth may occur as lymphocytic
or myeloid cell proliferations; the latter are often
referred to as granulocytic sarcoma
• Clinical symptoms of blast crisis mimic those of
AL, including severe anemia, leukopenia of all
WBCs except blasts, and thrombocytopenia
Phases of Chronic Myelogenous Leukemia

Phase Phase Length Treatment Status

Initial (chronic) 2-5 yrs Highly treatable

Accelerated 6-18 mo Resistance develops

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• Panmyelosis
• ↑ BM production of erythrocytes, leukocytes, and
thrombocytes
• EPO: decreased
• SECONDARY WITH APPOPRIATE EPO
PRODUCTION
JAK2V617F MUTATION • Occurs in response to hypoxia
POLYCYTHEMIA VERIA (PV) • Patients with pulmonary or cardiac disease
• Is a clonal stem cell proliferation affecting primarily • Increased BM production of erythrocytes,
the erythroid series, characterized by excessive leukocytes, and thrombocytes
proliferation of erythroid and also usually • EPO: increased
granulocytic and megakaryocytic elements in the • SECONDARY W/ INAPPROPRIATE EPO
marrow PRODUCTION
• The very slow evolution of the malignant erythroid • Tumors of kidneys, liver, brain, adrenals
clone leads to overexpansion of the red cell mass, • ↑ erythrocytes, leukocytes, and thrombocytes
hypervolemia, and splenomegalic red cell pooling • EPO: Increased
• These consequences eventually cause CLINICAL FINDINGS
generalized marrow hyperplasia with subsequent PV often presents mild symptoms occurring for
increases in the quantity of all three cell lines several years
• Associated with the JAK2 mutation, JAK2 V617F, • Increased RCM produces blood hyperviscosity,
is detected in more than 95% of patients with PV often resulting in cardiovascular disease
and is found on chromosome band 9p24 ; also • In the early stages of the disease, before
associated with JAK2 exon 12 mutation treatment, extended periods of high HCT (60%) &
JAK 2 protein hyperviscosity produce hypertension in about 50%
• Tyrosine kinase phosphorylates signal of patients with PV
transducers and activators of transcription (STAT) • Hyperviscosity and hyperproliferation and include
proteins, eventually generating transcription headache, weakness, pruritis, weight loss, and
proteins that bind promotor regions and signal fatigue
gene expression • About half of PV patients have thrombocytosis
• Controls transphosphorylation through and 1/3 experience thrombotic or hemorrhagic
conformational inhibition episodes
• Constitutive tyrosine kinase activity of the PV patients older than 60 years of age or those
mutated JAK2 protein causes continuous associated with a history of thrombosis are
activation of several signal transduction pathways considered high risk for thrombotic or hemorrhagic
that are normally activated after erythropoietin events
stimulation via the erythropoietic receptor The stable phase of PV can progress to a spent
• Mutated JAK2 is active and will phosphorylate phase in a few patients, usually within 10 years from
STAT proteins in the absence of erythropoietin or diagnosis
will overphosphorylate in its presence • Splenomegaly (palpable spleen)
TYPES OF PV • Hypersplenism
ABSOLUTE POLYCYTHEMIA VERA • BM hyperplasia
• Increased Hct: increase BM production • Cytopenias
• Types: Primary, secondary with appropriate EPO • Triad of myelofibrosis, splenomegaly, and anemia
production, inappropriate production of EPO with teardrop-shaped poikilocytes called as
• PRIMARY postpolycythemic myeloid metaplasia and

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develops in about 30% of PV patients within 20 in a variety of organ systems, including splenic or
years hepatic veins, as in Budd-Chiari syndrome
LABORATORY FINDINGS • Repeated splenic infarcts can result in splenic
• Common morphologic patterns in peripheral blood atrophy
and BM morphologic and cellular changes • Thrombosis can result in pulmonary emboli and
• BM normoblasts may collect in large clusters, neurologic complications like headache,
megakaryocytes are enlarged and exhibit paresthesis of the extremities, visual impairments,
lobulated nuclei, and BM sinuses are enlarged and tinnitus
without fibrosis • Arterial thrombi can cause myocardial infarction,
• Pseudo-Gaucher cells are rare transient ischemic attack, and cerebral vascular
• 80% of patients manifest BM panmyelosis accident
• Bleeding occurs most often from mucous
membranes in the gastrointestinal, skin, urinary,
and upper respiratory tracts.

LABORATORY FINDINGS
• Most consistent finding in the peripheral blood is
thrombocytosis that must exceed 400 x 109/L to
meet WHO criteria but often ranges from 1000 to
5000 x 109/L
• Platelets often appear normal, but giant bizarre
platelets, platelet aggregates,
micromegakaryocytes, and megakaryocyte
fragments can also be observed
• Platelets are present in clusters and tend to
accumulate near the thin edge of the blood film ;
ESSENTIAL THROMBOCYTHEMIA (ET) abnormal platelet function testing can occur
• a clonal MPN with increased megakaryopoiesis • BM has marked megakaryocytic hypercellularity,
and thrombocytosis, usually with a count greater clustering of megakaryocytes, and increased
than 600 x10⁹/L ; sometimes with a count greater megakaryocyte diameter with nuclear
than 1000 x10⁹/L hyperlobulation and density
• However, WHO criteria require a sustained
thrombocytosis with a platelet count of 400 x10⁹/L DIAGNOSIS
• AKA as primary thrombocytosis, idiopathic *WHO CRITERIA*
thrombocytosis, & hemorrhagic thrombocythaemia MAJOR CRITERIA:
• Associated with mutations in: • Megakaryocyte proliferation with large and mature
• JAK2 (64.1%) morphology, little to no granulocyte or erythroid
• MPL (4.3%) proliferation
• CALR (15.5) • Minor increase in reticulin fibers
• Must not meet any criteria for BCR-ABL1-positive
CLINICAL FINDINGS CML, PV, PMF, MDS or other myeloid neoplasms
• Vascular occlusions are often the result of • Demonstrate JAK2 V617F, CALR, or MPL
NOTE
microvascular thromboses in the digits or mutations
Based on WHO standards, a diagnosis of ET
thromboses in majo arteries and veins that occur MINOR CRITERIA
requires meeting all four major criteria or the first
three major criteria and one minor criteria

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• presence of a clonal marker or absence of • PMF occurs in patients older than age 60 and
evidence of reactive thrombocytosis equally in both genders
• The disease may be asymptomatic and usually
progresses as a slow, chronic condition
• Symptoms result from anemia, myeloproliferation,
or splenomegaly and include fatigue, weakness,
shortness of breath, palpitations, loss of appetite,
weight loss, night sweats, pruritis, pain in the
PRIMARY MYELOFIBROSIS (PMF) extremities and bones, bleeding, and discomfort
• A clonal hematopoietic stem cell MPN with or pain in the left upper quadrant associated with
splenomegaly and ineffective hematopoiesis splenomegaly
associated with areas of marrow hypercellularity • Major hemolytic episodes occur in 15% of PMF
(leukoerythroblastosis), extramedullary patients during the course of their disease and
hematopoiesis, fibrosis, and increased 10% of patients demonstrate hemosiderinuria and
megakaryocyte; it is the least common but most decreased blood haptoglobin levels suggesting
aggressive form of MPN intravascular hemolysis
• Can form de novo or as an evolutionary • Some patients develop bleeding diathesis
consequence of PV or ET resulting from a combination of thrombocytopenia,
• aka chronic idiopathic myelofibrosis, agnogenic abnormal platelet function, and hemostatic
myelofibrosis, and myelofibrosis with myeloid abnormalities suggestive of chronic disseminated
metaplasia intravascular coagulation (DIC)
• Chronic idiopathic myelofibrosis (CIM) is an
uncommon disease with an incidence one-third LABORATORY FINDINGS
that of CML • In the early stages, anemia, leukocytosis with a
• ASSOCIATED MUTATIONS: JAK2 (V617F) – left shift, and thrombocytosis are identified that are
60% ; CALR - 30% ; MPL – 5% consistent with an MPN
MYELOFIBROSIS • As fibrosis develops in the bone marrow, blood
• Fibroblasts → produce collagen to provide structural cell counts fall and pancytopenia eventually
support for HSCs develops, along with leukoerythroblastosis,
• a reactive process causes the overproduction of anisocytosis, and poikilocytosis
collagen that eventually disrupts the normal • Examination of the BM biopsy specimen provides
architecture of the BM and replaces hematopoietic most of the information for diagnosis
tissue resulting in pancytopenia DIAGNOSIS OF PMF
EXTRAMEDULLARY HEMATOPOEISIS • MAJOR CRITERIA
• recognized as hepatomegaly or splenomegaly, • Megakaryocitic proliferation with abnormal
seems to originate from the release of clonal stem morphology, usually accompanied by reticulin
cells into the circulation and/ or collagen fibrosis
• The cells accumulate in the spleen, liver, or other • Not meeting the criteria for other MPNs
organs, including the adrenals, kidneys, lymph • Evidence of JAK2V617F or other related
OTHER MYELOPROLIFERATIVE NEOPLASMS
nodes, bowel, breasts, lungs, mediastinum, mutations
CHRONIC NEUTROPHILIC LEUKEMIA
mesentery, skin, synovium, thymus, and lower • MINOR CRITERIA
CHRONIC EOSINOPHILIC LEUKEMIA
urinary tract • Leukoerythroblastosis
MYELOID/LYMPHOID NEOPLASMS WITH
• Anemia
EOSINOPHILIA AND REARRANGEMENT OF
CLINICAL FINDINGS • Increased serum lactic dehydrogenase (LDH)
PDGFRA, PDGFRB, OR FGFR1 OR WITH
levels
PCM1-JAK2
MASTOCYTOSIS
MYELOPROLIFERATIVE NEOPLASM,
Abedin, Mostaqem P. │ BSMT3 YA-1 │ HEMA311
UNCLASSIFIABLE
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• Splenomegaly least 5 x10⁹/L cells/L of

circulating B lymphocytes
for more than 3 months to
establish the diagnosis of
CLL, with confirmation of
clonality performed by flow
cytometry.

CYTOGENIC ABNORMALITIES
• 13q14.3 del → DLEU2/MIR15A/MIR16A
• 6q21 del
• Trisomy 12
• ATM gene → 11q23 del
MATURE LYMPHOID NEOPLASMS • TP53 gene → 17p13 del
• Are a diverse collection of disease entities with
varying clinical presentations and natural histories; • Typical CLL presents with >85% of the
however, common to all is identification with a lymphocytes appearing small and mature w/ scant
particular lineage cytoplasm & a dense nucleus w/ a condensed
• Disease states in large part can be identified with chromatin pattern without a defined nucleolus
a normal counterpart in the blood, bone marrow or • Characteristic chromatin pattern has been labeled
lymph nodes. Neoplastic transformation of these as “cobblestone” or likened to a soccer ball
cells results in abnormal changes in growth and ATYPICAL CELL
differentiation patterns, resulting in disease • Seen in 20% of patients
• Lymphoproliferative disorders in which the primary • Prolymphocytes or atypical lymphoid cells
site of disease is the blood or bone marrow are represent less than 10% or less than 15% of
classified as leukemias; disorders in which the circulating lymphocytes
localization of disease is in the lymph nodes and Associated wit
spleen are considered lymphomas Leukemia Type Solid Tumor Counterpart
RELATIONSHIP OF LEUKEMIAS & LYMPHOMAS
Stem cell leukemia Lymphoma, undifferentiated

Lymphoma, poorly leukemia,

CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) Acute lymphoblastic
differentiated; lymphocytic
• CLL is the most common leukemia in adults in
Western countries Lymphoma, well leukemia
Chronic lymphocytic
differentiated; lymphocytic
• Clonal proliferation of small B lymphocytes
involving BM, blood, and lymph nodes (LYMPH Monocytic leukemia Reticulum cell sarcoma
NODE INVOLVEMENT IS CALLED SMALL Acute myelogenous Chloroma granulocytic leukemia
LYMPHOCYTIC LYMPHOMA)
Plasma cell leukemia Myeloma
• CLL is generally a disease of the older adult; the
median age of diagnosis is approximately 72 • h biologic markers indicating more aggressive
years which is common in males disease
• Most patients are asymptomatic on presentation, • Subtypes:
and the disease is often detected by an • CLL/PLL →  >10% but <55% of total
abnormality in a routine CBC lymphocytes (>55% is associated with
• The International Workshop on Chronic Lymphoid Prolymphocytic leukemia)
Leukemia (IWCLL) requires the presence of at

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• Mixed-type/atypical CLL → >15% large atypical • Staging systems are important prognostic
lymphoid cells determinants for CLL and are used to guide
• SMUDGED LYMPHOCYTES are a common clinical management
finding on peripheral blood film review in CLL • The Rai and Binet staging systems have been
CLINICAL FINDINGS: used for this purpose
• The typical patient with CLL is asymptomatic, and • Both systems divide patients into risk categories
the disease is usually discovered at the time of a reflecting the degree of organomegaly and
routine physical examination lymphadenopathy or compromise of bone marrow
• Common symptoms include malaise, low-grade function
fever, and night sweats • PROGNOSTIC INDICATORS: presence indicates
• Weakness, fatigue, anorexia, and weight loss worse prognosis
• Physical examination usually reveals cervical and • del(17q)(TP53)
supraclavicular adenopathy • IGHv mutational status
• Hepatosplenomegaly • Testing for CD38 and ZAP (zeta-associated
IMMUNOPHENOTYPING protein of 70 kDa)
MARKERS
• POSITIVE:
• CD19, CD20, CD23, CD5, CD23, CD79a
• dim surface Ig (single light chain with IgM or
IgM and IgD)
• NEGATIVE:
• CD10, BCL-6, and cyclin D1
• This pattern is very useful in distinguishing
CLL/SLL from mantle cell, follicular, and marginal
zone lymphomas
• Antigen expression is sometimes variable; not all
cases fit into a classic CLL immunophenotypic
profile, to account foR this variability and
differentiate CLL from related B cell neoplasms, a
scoring system has been developed
• Each criterion is given 1 point, and the total score
is cumulative:
• 4 or more: Chronic lymphocytic leukemia
• 1 or 2: Non-Hodgkin’s lymphoma
• 3 (Intermediate): remains problematic, and
these cases may require a biopsy of a node
or molecular data to establish the diagnosis

STAGING AND PROGNOSIS

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PROLYMPHOCYTIC LEUKEMIA (PLL)

• Originally described as a variant of CLL
• Is characterized by a very marked lymphocytosis MYCOSIS FUNGOIDES
(usually greater than 100 × 103/μL), massive & SEZARY SYNDROME
splenomegaly, moderate hepatomegaly, and • Is the most familiar form
inconspicuous lymphadenopathy of cutaneous T cell
• The malignant lymphoid cells have large vesicular lymphoma
nucleolus, condensed nuclear chromatin, and a • MF is the more common
moderate amount of cytoplasm disorder, making up
• > 55% of blood leukemic cells are prolymphocytes approximately 60% to 70% of the cutaneous T cell
IMMUNOPHENOTYPING: lymphoma cases
• Positive: CD19, CD20, FMC-7, bright surface Ig • MF is largely confined to the skin, although in the
• Negative: CD5, CD23 later stages of the disease, dissemination to
lymph nodes, organs, and blood can be seen
HAIRY CELL LEUKEMIA • MF is generally an indolent disease with a slow
• Hairy cell leukemia (HCL) is an indolent disease of progressive course; SS is defined as a systemic
B cell lineage most commonly found in middle age disorder with peripheral blood involvement
(median age, 50 years); more common in males (leukemic phase of MF) and a worse prognosis
• The major focus of the disease is on the spleen, than MF
blood, and bone marrow SEZARY CELLS:
HAIRY CELLS: • Abnormal appearance with scant cytoplasm and a
• Have round to ovoid nuclei, lack nucleoli, and cerebriform, folded nucleus, variably condensed
have relatively abundant cytoplasm with ragged chromatin, and inconspicuous nucleoli
projections that extend circumferentially around IMMUNOPHENOTYPING:
the entire cell • CD2, CD3, CD4, and CD5
CYTOGENETIC ABNORMALITY: LABORATORY FINDINGS:
• BRAF (V600e) mutation • Biopsies of the skin reveal bandlike lymphocytic
IMMUNOPHENOTYPING: infiltrates in the dermis often with admixed
• CD 20, CD22, CD79a, CD103, CD25, CD11c histiocytes and occasional eosinophils
• Annexin A is specific to HCL and helps to • T cells with irregular nuclei show single-cell
differentiate HCL from related B cell disorders infiltration of the epidermis and often form clusters
CLINICAL FINDINGS: known as Pautrier’s abscesses
• Fatigue • Presence of Sezary cells (Sezary syndrome)
• Anemia, leukocytopenia, thrombocytopenia,
splenomegaly (most predominant finding), and CLINICAL FINDINGS
marrow fibrosis (bone marrow aspirates frequently • MYCOSIS FUNGIODES
are unsuccessful) • Psoriatic-like skin lesions require a repeated
• Bleeding and infection can be present biopsy before a definitive diagnosis is made.
• • Skin biopsy can show a characteristic
localization of atypical cells in Pautrier

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microabscesses in the epidermis, but this • Malignant bone marrow–based, plasma cell
occurs in only a minority of cases neoplasm associated with abnormal protein
• SEZARY SYNDROME production
• Characterized by erythroderma, generalized • Plasma cell leukemia is an increased number of
lymphadenopathy, and the presence of plasma cells in the peripheral blood and should be
clonal T cells in the skin, lymph nodes, & considered a form of multiple myeloma and not a
peripheral blood separate entity
• An absolute Sézary cell count greater than 1 IMMUNOPHENOTYPING
x10⁹/L is required to make a diagnosis of SS • CD38, CD138 (Kappa or lambda clonal excess)
CLINICAL FINDINGS
PLASMA CELL NEOPLASMS/MYELOMAS • Include bone pain (typically in the back or chest)
• Also known as Plasma cell dyscrasias • Weight loss and night sweats are not prominent
• Malignant disorders of terminally differentiated B until the disease is advanced
cells (plasma cells) • Abnormal bleeding may be a prominent feature
• Although they constitute approximately 1% to 2% • In some patients, the major symptoms result
of cancers, PCNs are the second most common from acute infection, renal insufficiency,
hematologic malignancy hypercalcemia, or amyloidosis
• Plasma cells secrete monoclonal immunoglobulin • Approximately 90% of patients suffer from
(paraproteins) is an essential characteristic of the broadly disseminated destruction of the
PCNs skeleton ; bone marrow failure
• Associated with rouleaux formation and elevated • Major cause of death are infection and renal
ESR insufficiency
• TYPES: LABORATORY FINDINGS
• Monoclonal Gammopathy of Undetermined • Bone marrow plasma cells: >30%
Significance (MGUS) • Increased plasma volume caused by monoclonal
• Multiple (Plasma cell) myeloma protein commonly produces hypervolemia
• Waldenstrom’s Macroglobulinemia • Rouleaux formation and elevated ESR
• Serum electrophoresis:
MONOCLONAL GAMMOPATHY OF UNCERTAIN • Overproduction of IgM; “M-spike”
SIGNIFICANCE (MGUS) • Presence of monoclonal serum protein (mostly
• Benign monoclonal proliferation of plasma cells is IgG, can be IgA or IgD)
usually considered with the plasma cell • Presence Bence Jones proteins (free light chains
neoplasms because it represents a precursor —kappa or lambda) found in the urine
state for myeloma
• Presence of circulating monoclonal protein WALDENSTROM’S MACROGLOBULINEMIA
WITHOUT ASSOCIATED PLASMA CELL • aka LYMPHOPLASMACYTIC LYMPHOMA
DYSCASIA or other neoplasms • B-cell neoplasm characterized by
• Detections rely on detection of paraproteins in lymphoplasmoproliferative disorder with infiltration
serum or urine of the bone excessive IgM (macroglobulin) and
decreased production of the other
immunoglobulins
• High levels of IgM can result in a hyperviscosity
MULTIPLE (PLASMA) CELL MYELOMA syndrome
• CYTOGENETIC ABNORMALITY: mutation in the
Myeloid differentiation factor 88 (MYD88) gene

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CLINICAL FINDINGS: • Presence of Reed-Sternberg cells (a usual

• Symptoms of WM are due to the extent of tumor pathognomonic sign for Classical Hodgkin’s
infiltration and to elevated Lymphoma)
• Weakness, fatigue attributable to anemia, and • IMMUNOPHENOTYPING: CD15, CD30, CD45
bleeding • SUBTYPES: Nodular sclerosis, Mixed cellularity,
• Bone pain is virtually nonexistent; Lymphocyte depleted, Lymphocyte-rich
HYPERVISCOCITY SYNDROME
• Lymphadenopathy and Hepatosplenomegaly REDD-STEINBERG CELLS VARIANTS
LABORATORY FINDINGS: HODGKIN CELLS
• Pleomorphic B-lineage cells at different stages of • Large mononuclear lymphoid cells with an oval
maturation, such as small lymphocytes, nucleus, thick nuclear membrane, distinct
lymphoplasmacytoid cells (abundant basophilic eosinophilic nucleolus, and abundant cytoplasm
cytoplasm but lymphocyte-like nuclei), and plasma MUMMIFIED CELLS
cells Degenerated or apoptotic cells with a pyknotic
• IMMUNOPHENOTYPING: CD154 nucleus and condensed cytoplasm
• Absolute lymphocytosis LACUNAR CELLS
• IgM levels in the blood circulation (IgM >3 g/dL); • Lobated nucleus and artifactual retraction of
“M-spike”serum electrophoresis cytoplasm secondary to formalin fixation
• Rouleaux and elevated ESR • Occurs predominantly in the nodular sclerosis
variant of Classical Hodgkin’s Lymphoma
HODGKIN’S LYMPHOMA • Appear to be situated in a clear space
• Hodgkin lymphoma (HL) is primarily a lymph (“LACUNA”)
node-based disease, distinct from NHL that
involves the lymph nodes
• The hallmark of classical HL is the Reed sternberg
cell, which is a large binucleated, multinucleated,
or mononuclear (Hodgkin) cell with each nucleus
bearing a very large inclusion-like nucleolus
• DIAGNOSIS involves tissue biopsy,
Immunophenotyping, cytogenetics, DNA analysis

CATEGORIES CLINICAL FINDINGS

NODULAR LYMPHOCYTE-PREDOMINANT • Painless enlargement of the lymph node (usually
HODGKIN’S LYMPHOMA in the neck)
• Is a B-cell neoplasm composed relatively rare • Mediastinal mass
neoplastic cells called “popcorn cells” or • Enlarged cervical nodes
lymphocytic histiocytic cell (L&H cell) • Fever (over 100.4oF)
• Popcorn cells are large lymphoid cells with • Night sweats
abundant cytoplasm and vesicular multi-lobated • Weight loss (>10%)
nuclei (popcorn nuclei) LABORATORY FINDINGS
• IMMUNOPHENOTYPING: CD 45, CD20, BCL-6
CLASSICAL HODGKIN’S LYMPHOMA
• Comprises a heterogenous group of lymphoid
neoplasm derived from the germinal center; can
be associated with EBV infections

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MYELODYSPLASTIC SYNDROMES (MDS)

• Characterized by a decreased in one or more
types of peripheral blood cells due to abnormal
maturation in the bone marrow
• Clonal stem cell disorders are characterized by:
• Maturation defects
• Ineffective erythropoiesis
NON-HODGKIN’S LYMPHOMA • Peripheral pancytopenia is spite of marrow
• The most frequent type of NHL is diffuse large B- cellularity
cell lymphoma, which accounts for approximately • FAB classification of myelodysplastic syndromes
40% of new cases of lymphoma based on the presence of dysmyelopoiesis and
• More than half of patients with diffuse large B-cell the quantification of myeloblasts (Type I and Type
lymphoma are older than 60 years of age II) or erythroblasts:
• Three different types of diffuse large B-cell • REFRACTORY ANEMIA (RA)
lymphomas can be defined based on gene • REFRACTORY ANEMIA W/ RINGED
expression subgroups: SIDEROBLASTS (RARS)
• Germinal center, B-cell–like lymphoma that • REFRACTORY ANEMIA W/ EXCESS
expresses high levels of genes characteristic BLASTS (RAEB)
of germinal center, B- cell-like lymal • CHRONIC MYELOMONOCYTIC LEUKEMIA
germinal center B cells (CMML)
• Activated B-cell–like lymphoma, w/c • REFRACTORY ANEMIA W/ EXCESS
expresses genes characteristic of BLASTS IN TRANSFORMATION (RAEB-T)
mitogenically activated blood B cells
• New subgroup, type 3 diffuse large B-cell
lymphoma, which has a heterogeneous
gene expression that suggests it includes
more than one subtype of lymphoma

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• POSITIVE RESULT: Peroxidase activity produces

CYTOCHEMISTRY RED BROWN GRANULES in cytoplasm of
WEEL 17 granulocytes and monocytes
CYTOCHEMISTRY
• Techniques such as flow cytometry, cytogenetic
analysis, and molecular testing are now commonly
used in the diagnosis of acute and chronic
leukemias; However, older techniques, such as
cytochemical stains, still retain their importance
• Cytochemistry involves the use of cytochemical
stains
• The GENERAL PRINCIPLE of most cytochemical
stains is to incubate cells on the blood smear with
the substrate that reacts with the intracellular
marker
• CLASSIFICATION OF CYTOCHEMICAL STAINS:
• Enzymatic
• Non-enzymatic
• Immunocytochemical
SUDAN BLACK B (SBB)
• is a fat-soluble dye; stains
phospholipids neutral fats
& sterols;
• Can be done on stored
specimens
MYELOPEROXIDASE • For differentiation of AML
• MPO is present in the primary granules of most (+) from ALL (-);
granulocytic cells, beginning at the promyelocyte PARALLELS MPO FOR INTERPRETATION
stage and continuing throughout maturation; • POSITIVE RESULT: DARK PURPLE-BLACK
leukemic myeloblasts are usually positive for MPO GRANULES in neutrophil precursors
• LYMHPOCYTES DO NOT EXHIBIT MPO
ACTIVITY
• Stain should be DONE ONLY ON FRESH
SPECIMENS because it deteriorates
• Used to differentiate acute myelogenous leukemia
(POSITIVE) from acute lymphoblastic leukemia
(NEGATIVE)
• In many cases of the AMLs (without maturation,
with maturation, and promyelocytic leukemia), it ESTERASES
has been found that more than 80% of the blasts • used to differentiate myeloblasts and neutrophilic
show MPO activity; Auer rods found in leukemic granulocytes from cells of monocytic origin
blasts and promyelocytes test strongly MPO • Nine isoenzymes are present in leukocytes
positive • Esterase with low substrate specificity is known as
• Lymphoblasts in ALL and lymphoid cells are MPO non-specific esterase (Alpha-Naphthyl acetate
negative and butyrate) ; called non-specific because these

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 34

stains may also produce a positive result with

other cells
• Esterase with high specificity to the substrate is NON-SPECIFIC ESTERASE

known as specific esterase (Naphthol AS-D

Chloroacetate Esterase); called specific because
granulocytic cells show staining
• Esterase activity is inhibited by varying degrees of
mercury, acid solutions, heat, and iodine (false PHOSPHATASES
negative results) • widely distributed in mammalian tissue
POSITIVE RESULT: • The two major classifications, based on pH, are
• SPECIFIC ESTERASE (Chloroacetate esterase): alkaline and acid phosphatase
• BRIGHT RED GRANULES in cytoplasm of • Microscopic determinations of these enzymes are
neutrophils, neutrophil precursors and mast cells based on methods that produce a visible
• NON-SPECIFIC ESTERASE (acetate and precipitate of the organic phosphates hydrolyzed
butyrate): by these enzymes
• DARK BROWN GRANULATION in the cytoplasm ACID PHOSPHATASE (ACP)
of Monocytes, Megakaryocytes, erythroblasts, • Almost all blood cells contain the acid
plasma cells phosphatase enzyme and show positivity with acid
• IMPORTANT NOTE: alpha-naphthyl esterase is phosphatase stain; is one of many acid
inhibited by sodium fluoride hydrolases that have been demonstrated in
lysosomes
• Acid phosphatase hydrolyzes the naphthol AS-B1
phosphoric acid in the incubation mixture
releasing insoluble naphthol, which then couples
with pararosanilin, giving a red-colored precipitate
• Most leukocytes exhibit a positive reaction in
varying degrees to this test; however, monocytes
demonstrate a more intense positive reaction than
do neutrophils
• Although lymphocytes display little activity, T cells
SPECIFIC ESTERASE
do exhibit intense positivity in the Golgi region,
whereas B cells may be positive or negative. For
this reason, the procedure is useful in
differentiating subgroups of ALL
NON-SPECIFIC ESTERASE TARTRATE RESISTANT ACID PHOSPHATASE
(TRAP STAIN)
• In the presence of L(+) tartaric acid (Tartrate
resistant acid phosphatase), the activity of most
acid phosphatase is inhibited, the exception is
NON-SPECIFIC ESTERASE ACP Isoenzyme 5 (present in Hairy cell leukemia),
which is resistant to L(+) tartaric acid
• POSITIVE RESULT: RED COLOR PRECIPITATE

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TRAP STAIN
PERIODIC ACID-SHIFF (PAS)
• The PAS reaction is
important in
carbohydrate
histochemistry (PA is
LEUKOCYTE ALKALINE PHOSPHATASE (LAP)
used as an oxidizing
• Alkaline phosphatase is present in the cytoplasm
agent and Schiff is
of neutrophils in varying degrees and referred to
used as the stain)
as leukocyte alkaline phosphatase (LAP) or
• Positive staining
Neutrophil alkaline phosphatase (NAP)
reactions indicate the
• Eosinophils are rarely positive and B lymphocytes;
presence of glycogen, a polymer of glucose, and
all other leukocytes are negative
other 1,2-glycol–containing carbohydrates
• Leukocyte alkaline phosphatase (LAP) activity can
• Lymphoblasts, malignant erythroblasts, and
be increased, normal, or decreased in a variety of
megakaryocytic cells STAIN POSITIVE with this
conditions
stain; myeloblasts and normal erythrocytic cells
• This procedure is frequently used to distinguish
are NEGATIVE with this stain
between leukemoid reactions (INCREASED) and
• Useful in the diagnosis of erythroleukemia (FAB
chronic myelogenous leukemia (DECREASED)
M6) and acute lymphoblastic leukemia (ALL)
• IMPORTANT NOTE: Capillary blood is preferred
• POSITIVE RESULT: BRIGHT FUCHSIA PINK,
to anticoagulated whole blood; if anticoagulated
MAGENTA TO PURPLE pattern of staining which
whole blood must be used, heparin is preferred
varies with each cell type
and EDTA must be avoided
• POSITIVE RESULT: BLUE (Naphthol AS-MX +
fast blue RR salt) or VIOLET (Naphthol AS-MX +
fast violet B salt) pigment
LAP SCORE/ GRADING
• Also called as KAPLOW’S COUNT
• The deposits of blue or violet pigment viewed
microscopically reflect the sites of granulocytic
alkaline phosphatase activity, The granulocyte
population is rated on the basis of the number of
PER’S PRUSSIAN BLUE
cells stained and the intensity of the pigment
• Free iron precipitates into small blue/green
deposits
granules in mature erythrocytes; cells are called
• A 100 cell count (band and segmented
siderocytes
neutrophils) is done and is rated on a scale of 0 to
• Iron inclusions are called siderotic granules
4+ based on the quantity and intensity of the
(Prussian blue) or Pappenheimer bodies (Wright’s
precipitated dye within the cytoplasm of cells
stain)

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• Sideroblasts are nucleated RBCs in bone marrow • Cooper-cruickshank stain – stain for basophils
that contain iron granules (normal)
• Ringed sideroblasts contain iron that encircles the
nucleus (abnormal) CYTOCHEMICAL REACTIONS IN
• An increased percentage of siderocytes is seen in
SELECTED LEUKEMIAS
severe hemolytic anemias (e.g., beta-thalassemia
major), iron overload, sideroblastic anemia, and
post-splenectomy; ringed sideroblasts are seen in
bone marrow of myelodysplastic syndrome
(refractory anemia with ringed sideroblasts) and
sideroblastic anemias

Test AML M4 AML M1-M3 AML M5

RINGED SIDEROBLAST SBB/ MPO + + -

SE + + -

NSO + - +

TERMINAL

DEOXYRIBONUCLEOTIDYL TRANSFERASE
(TdT)
• TdT is an intranuclear enzyme that catalyzes the
irreversible addition of deoxynucleotides to the 3-
prime region on the end of DNA
• Present in almost 90% of ALL cases ; MARKER
FOR LYMPHOID PRECUSORS
• Used to differentiate ALL (POSITIVE) from AML
(NEGATIVE)

TOLUIDINE BLUE
• Binds w/ acid
mucopolysaccharides in
blood cells
• It is used for recognition
of mast cells and
basophils (purplish red)

Abedin, Mostaqem P. │ BSMT3 YA-1 │ HEMA311