HEMATOLOGY: BY GERARD ANDREW RAMOS, RMT

LEUKOCYTES

Neutrophil Precursor (Maturation time: 14 days)

Stages Cell diameter
1 Myeloblast 14-20 um
2 Promyelocyte 16-25 um
3 Myelocyte 15-18 um
4 Metamyelocyte 14-16 um
5 Band / Stab 9-15 um
6 Segmented Neutrophil 9-15 um

Myeloblast: High N:C ratio (8:1 to 4:1)

Slightly basophilic cytoplasm, fine nuclear chromatin.
Two to four visible nucleoli

Promyelocyte: The nucleus is round to oval and is often eccentric.

A paranuclear halo or “hof” is usually seen
The cytoplasm is evenly basophilic and full of primary (azurophilic) granules.

Myelocyte: Last stage capable of cell division

Nucleus has more heterochromatin; cytoplasm is more lavender-pink than blue.
Start of Synthesis of Secondary Granules

Metamyelocyte: Indented nucleus (kidney bean-shaped or peanut-shaped)

The cytoplasm contains very little residual RNA (little or no basophilia)
Start of Synthesis of Tertiary Granules

Band Neutrophil: Secretory granules / secretory vesicles are formed

The nucleus is highly clumped; indentation exceeds one half the diameter of the nucleus.
Youngest stage to appear normally in peripheral circulation
“Sausage-shape nucleus”

Segmented Neutrophil: Secretory granules continue to be formed.

Presence of 3-5 nuclear lobes connected by thread-like filaments.

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Content of Neutrophil Granules
Primary Granules / Secondary Granules / Tertiary Granules Secretory granules
Azurophilic / Specific Granules
Non-specific Granules
Myeloperoxidase Lactoferrin Gelatinase (ONLY) CD11b/CD18
Acid hydrolase Lysozyme CD10, CD13, CD14, CD16
Acid phosphatase Collagenase Cytochrome b558
β-glucuronidase Aminopeptidase Complement 1q receptor
Arylsulfatase Plasminogen activator
Elastase

Neutrophils
• Once in the peripheral blood, neutrophils are divided into:
o Circulating neutrophil pool (CNP) 50%
o Marginated neutrophil pool (MNP) 50%
• H-L in the blood: 7 hrs. / Lifespan: 2-3 days
• Neutrophilia is an absolute increase in neutrophils > 7 x 109/L in adults or 8.5 x 109/L in children.
• Neutropenia is a decrease in the ANC < 2 x 109/L in adults.
• The term leukemoid reaction refers to a neutrophilic leukocytosis > 50 x 109/L with a shift to the left (ex:
Metamyelocyte; Band/Stab).

• Leukemoid Reaction is usually caused by acute / chronic infections, metabolic disease, inflammation or as part
of an inflammatory response to malignancy.
REFERENCE RANGE:
WBC
Newborns: 10-30 x 109/L
Adults: 3.6-10.6 x 109/L

DIFFERENTIAL
RELATIVE COUNT
Neutrophil: 50-70%
Lymphocytes: 18-42%
Monocytes: 2-11%
Eosinophils: 1-3%
Basophils: 0-2%

ABSOLUTE COUNT
Neutrophils: 1.7-7.5 x 109/L
Lymphocytes: 1.0-3.2 x 109/L
Monocytes: 0.1-1.3 x 109/L
Eosinophils: 0-0.3 x 109/L
Basophils: 0-0.2 x 109/L

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 Causes of Neutrophilia
• Emotional stress • Vasculitis • Leukocyte adhesion deficiency
• Strenuous exercise • Colitis • Familial cold urticaria
• Trauma/injury • Autoimmune disease • Hereditary neutrophilia
• Pregnancy: labor and delivery • Acute hemorrhage • Leukemia
• Eclampsia • Chronic blood loss • Chronic myeloproliferative neoplasm
• Surgery • Hemolysis • Steroids
• Infections: bacterial, some viral • Smoking • Lithium
• Burns • Chronic blood loss • Colony-stimulating factors (granulo-
cyte CSF)
• Myocardial infarction • Metabolic ketoacidosis
• Cortisol
• Pancreatitis • Uremia
• Epinephrine

Causes of Acquired Neutropenia

Drugs Other
• Anticancer • Levamisole (adulterated cocaine)
• Analgesics/antiinflammatories, acetaminophen, aspirin, • Immunologic
diclofenac, ibupro- fen, indomethacin, linezolid, naproxen,
sulfasalazine Radiation

Antibiotics: B-lactams, cephalosporins, chloramphenicol, • Toxins

clindamycin, gen- tamicin, penicillin, sulfonamides, • Overwhelming infections
trimethoprim-sulfamethoxazole, tetracy- cline, vancomycin
• Splenomegaly
• Antivirals: acyclovir, ganciclovir, abacavir, zidovudine
• Hemodialysis
• Antifungals, terbinafine, Amphotericin B
• Aplastic anemia
• Anticonvulsants: carbamazepine, phenytoin, valproate
• Copper deficiency
• Antihistamines: brompheniramine, chlorphenamine,
cimetidine, famotidine, ranitidine • Alcoholism
• Antimalarials: chloroquine, dapsone, quinine Tumor metastasis to bone marrow/space-occupying lesion
• Antithyroids: propylthiouracil, methimazole, carbimazole Hematologic malignancy
• Cardiovascular amiodarone, captopril, clopidogrel, • Myelodysplastic syndrome
flecainide, furosemide, methyldopa, procainamide,
propranolol, quinidine, spironolactone, thiazide diuretics, • Large granular lymphocyte syndrome
ticlopidine • Leukemia
• Antianxiety/hypnotics: benzodiazepines, meprobamate • Plasma cell neoplasm
Psychotropics: amitriptyline, amoxapine, clozapine,
doxepin, olanzapine, phenothiazines, risperidone
Hypoglycemics: chlorpropamide, tolbutamide
• Phenothiazines: chlorpromazine, phenothiazines

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Eosinophils
• Eosinophil development is similar to neutrophils.
• First maturation phase that can be identified is myelocyte stage (reddish-orange 2° granules)
• Mature eosinophils have bi-lobed nucleus
• H-L in the blood: 18 hrs. / Lifespan: 2-5 days
• Counteracts the factors released by mast cells
• Eosinophilia is associated with helminthic infections and allergic reactions.
• Specific Granules contain: 1. Major Basic Protein; “Anti-helminthic properties”
2. Eosinophil Cationic Protein
• Charcot-Leyden Crystals (STOOL/SPUTUM)
 Hexagonal bipyramidal crystals composed of Lysophospholipase
 Present in the cytoplasm of eosinophils in prolonged eosinophilic inflammation

EOSINOPHILIA EOSINOPΕΝΙΑ
 Absolute eosinophil count > 0.4 x 109/L  Absolute eosinophil count 0.09 X 10/L
Causes:  Causes:
 Helminthic infections  BM hypoplasia
 allergic reactions (asthma,  autoimmune disorders
rhinitis, urticaria)  steroid therapy
 scarlet fever  stress
 primary biliary cirrhosis  sepsis
 Hepatitis  acute inflammatory states
 HIV
 autoimmune disorders
 drug reactions

Basophils
• Partially lobulated nucleus, obscured by its blue-black granules
• Closely related to Mast cells (effector cell in type 1 hypersensitivity) (involved in immediate hypersensitivity
reactions.
• Basophilia is associated with CML, allergic rhinitis, chronic infections, hypothyroidism
• Specific granules contain: 1. Histamine
2. Heparan Sulfate
3. Leukotrienes

• Basophilia is defined as an absolute basophil count > 0.15 x 109/L

 allergic rhinitis
 hypersensitivity to drugs or food
 chronic infections
 hypothyroidism
 chronic inflammatory conditions
 radiation therapy
 chronic myeloid leukemia

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MONONUCLEAR CELLS
Lymphocytes
• Subsets:
o T cells – 60-70 %
o B cells – 10-20 %
o NK cells – 10-15 %
• High N:C ratio with scanty cytoplasm
• They are not end-stage cells; they are still capable of mitosis when they are stimulated
• Lymphocytosis is usually accompanied by changes in lymphocyte morphology and are associated mainly with
viral infections such as IM, CMV infection, Hepatitis, HIV, Chickenpox, Herpes, Influenza.
• Lymphocytosis
 Children: absolute lymphocyte count > 10 x 109/L
 Adults: absolute lymphocyte count > 5 x 109/L
• Lymphocytopenia
 Children: absolute lymphocyte count < 2 x 109/L
 Adults: absolute lymphocyte count < 1 x 109/L

Surface CD2 - “E-rosette” receptor (Has the
Markers highest affinity to steep RBC)
CD3 - part of T-cell antigen receptor
CD4 - Helper T-cell / Treg cell
CD5/7 - early stages of T cells
CD8 - Cytotoxic T-cell
CD19 - part of B cell coreceptor. CD16 - receptor for IgG
CD20 - found on all stages of B cells. CD56 - no known
CD21 - receptor for C3d and EBV. function
CD22 - found mostly on mature B
cells.
CD10 - early stages of B cells/CALLA.

Stages in B cell differentiation

1. Pro-B cell Lymphocytosis Lymphocytopenia
2. Pre-B cell
Reactive Morphology Congenital Immunodeficiencies
3. Immature B-cell Infections  Severe combined
4. Mature B cell  Infectious immunodeficiency
5. Activated B cell mononucleosis disease
6. Plasma cell  Cytomegalovirus  Common variable
infection immune deficiency
Stages in T cell differentiation  Hepatitis  Ataxia-telangiectasia
1. Double negative stage  Acute HIV infection  Wiskott-Aldrich
2. Double positive stage  Adenovirus syndrome
 Chickenpox
3. Mature T cell Others
 Herpes
4. Activated T cell Infections
 Influenza
 Paramyxovirus  Acquired
Plasma cell (mumps) immunodeficiency
 Size: 8-20 um  Rubella (measles) syndrome
 Eccentric nucleus  Roseola  Severe acute respiratory
 B-Hemolytic syndrome
 Cartwheel-like chromatin
streptococci  West Nile
 Perinuclear hof  Hepatitis
 Brucellosis
 (+) CD38 and CD138  Paratyphoid fever  Influenza
 Toxoplasmosis  Herpes
 Typhoid fever  Measles
 Listeria  Tuberculosis
 Mycoplasma  Typhoid fever
 Syphilis  Pneumonia

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Monocytes
• Maturation stages: Monoblast, promonocyte, monocyte
• Appearance on stained smear:
o Nucleus is may be round, oval, kidney shaped or horse-shoe
shaped
o Has stringy or lace-like chromatin pattern
o Cytoplasm is bluish gray with fine azure granules (“ground-
glass” appearance)
o Cytoplasmic and nuclear vacuoles may also be present

• Functions include phagocytosis, antigen-presenting cell, destruction of senescent RBCs

• Monocytes remain in the circulation for approximately 3 days
• Monocytosis is associated with tuberculosis, brucellosis, leishmaniasis, SBE, Malaria, Autoimmune dis.
• Monocytosis is defined as absolute monocyte count > 1 x 109/L
• Monocytopenia is defined as absolute monocyte count in adults < 0.2 x 109/L

• Differentiates into Macrophages when they migrate to tissues:

o Liver — Kupffer Cells
o Lungs — Alveolar Macrophages / Dust Cells
o Brain / Nervous tissue — Microglial cells
o Connective tissue — Histiocytes
o Skin / Mucosa — Langerhans cells
o Spleen — Splenic macrophage
o Lymph nodes — Littoral cells
o Placenta — Hofbauer cells
o Bones — Osteoclasts
o Kidneys — Mesangial cells

MONOCYTOSIS MONOCYTΟΡΕΝΙΑ
 Tuberculosis  Aplastic anemia
 Brucellosis  Chemotherapy
 Leishmaniasis  Steroid therapy
 Subacute bacterial endocarditis  Hemodialysis
 Malaria  EBV infection
 SLE, RA  Hairy cell leukemia
 Inflammatory bowel disease
 Myositis, Sarcoidosis
 Malignancy
 Hodgkin disease
 Myelodysplastic/myeloproliferative neoplasms
 Acute myeloid leukemia

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Non-Malignant Leukocyte Disorders

1. Severe Combined Immune Deficiency

• X-linked SCID
o Gamma chain deficiency (leukocyte receptor for IL-2, 4, 7,
9, 15, 21)
o Signals from the interleukins are disrupted
• Autosomal recessive SCID
o Adenosine deaminase (ADA) deficiency
o Leads to accumulation of adenosine, which is lymphotoxic
(decreased in T, B, and NK cells)

2. Wiskott-Aldrich Syndrome
• rare X-linked disease caused by one of more than 400 mutations in the WAS gene
• T cells are decreased; B cells, T cells and NK cells, neutrophils and monocytes are dysfunctional
• There is a risk of bleeding due to thrombocytopenia and small, abnormal PLATELETS.

3. 22q11 Deletion Syndromes

• Includes Di George syndrome, Opitz GBBB, Sedlackova syndrome
• All the disorders within the 22q11 deletion syndrome have variable degrees of immunodeficiency
• There is absence or decreased size of the thymus and low numbers of T-Cells.

4. Bruton Tyrosine Kinase Deficiency / Bruton’s Agammaglobulinemia

• X-linked agammaglobulinemia
• Primary immunodeficiency disease characterized by reductions in all serum immunoglobulin isotypes and
profoundly decreased or absent B cells.

5. Chédiak-Higashi Syndrome
• rare autosomal recessive disease of immune dysregulation
• Leukocytes exhibit abnormally large lysosomes, containing fused dysfunctional granules
• Giant lysosomal granules are present in granulocytes, monocytes, and lymphocytes
• Patients often have bleeding issues as a result of abnormal dense granules in platelets
6. Leukocyte Adhesion Disorders
• Rare autosomal recessive inherited conditions
• Inability of neutrophils & monocytes to move from circulation to the site of inflammation (extravasation or
DIAPEDESIS).

7. Chronic Granulomatous disease (CGD)

• 60% of cases are X-linked recessive and 40% are autosomal recessive
• decreased ability of neutrophils to undergo a respiratory burst after phagocytosis of foreign organisms.

• Lab Evaluation:
1. DHR Assay — Dihydrorhodamine; Based on Flow Cytometry.
2. NBT Dye Test — Nitroblue Tetrazolium

8. WHIM Syndrome
• Warts, Hypogammaglobulinemia, Infections, and Myelokathexis syndrome (which causes Neutropenia).
• Defective movement of WBCs between the bone marrow and peripheral blood.

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Morphologic Abnormalities of Leukocytes

1. Pelger-Huet Anomaly (Hyposegmentation of Nucleus)

• autosomal dominant disorder characterized by decreased nuclear segmentation and distinctive coarse chromatin
clumping pattern.
• Pelger-Huët (PH) nuclei may appear round, ovoid, or peanut shaped. Bilobed forms - the characteristic spectacle-
like (“pince-nez”) morphology with the nuclei attached by a thin filament
• It potentially affects all leukocytes, although morphologic changes are most obvious in neutrophils
• Neutrophils are functioning normally
• N = 3-5 lobes
• PH Anomaly = 1-2 lobes
• Pseudo-Pelger Huet Anomaly
o Also known as “Acquired Pelger-Huet”
o Observed in patients with hematologic malignancy: Acute Myeloid Leukemia, MDS
o Neutrophil have abnormal function.

2. Hypersegmentation of Neutrophils
• More than 5 nuclear lobes present
• Associated with: Megaloblastic anemia, Myelodysplastic syndrome

3. Alder-Reilly Anomaly
• Autosomal recessive
• Granulocytes have dark staining granules (Reilly bodies) NOT related to infection
• Root cause: Incomplete degradation of mucopolysaccharides
• Neutrophils are functioning normally; can also be present in monocytes and lymphocytes,
• Commonly associated with Hurler syndrome, Hunter syndrome, Sanfilippo syndrome

4. Auer Rods
• Rod-like bodies representing aggregated primary granules
• Reddish-purple granules; Peroxidase (+)
• Observed in blast cells present in AML,
Especially, APL: Acute Promyelocytic Leukemia
• Faggot cells = bundles of Auer rods

5. May-Hegglin Anomaly
• Rare, autosomal dominant disorder
• Triad:
o Dohle-like inclusion bodies
= pale blue inclusion
o Thrombocytopenia
o Giant Platelets MACROTHROMBOCYTOPENIA

• There is disordered production of myosin heavy chain type IIA, which affects megakaryocyte maturation and
platelet fragmentation when shedding from megakaryocytes.
• Patients are usually asymptomatic

Reactive Morphologic Changes in Neutrophils

1. Toxic Granulation (Neutrophils)
• Dark blue-black cytoplasmic granules
• Reflects acid mucosubstance inside Primary (Azurophilic) granules

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 • Observed in severe infections, other toxic conditions, and reactive conditions
2. Dohle Bodies (Neutrophils)
• Round or elongated, pale-blue staining bodies located close to cell membrane
• Cytoplasmic remnants of RNA or free ribosomes
• Found in bacterial infections, burns, pregnancy

Lymphocyte Morphologic Abnormalities

1. Downey cell / Turk cell
• Lymphocytes that exhibit reactive morphology
• also known as Atypical, Reactive, Variant lymphocyte
• occur as lymphocytes are stimulated when interacting with antigens
in peripheral lymphoid organs
• Characteristics:
o Variation in nuclear shape and N:C ratio
o Cells have basophilic cytoplasm
o Cytoplasm may be indented by surrounding RBCs

2. Basket / Smudge cell

• caused by pressure in smear preparation; indicates nuclear fragility
• increased numbers observed in CLL: Chronic Lymphocytic Leukemia

3. Flame cell
• variant of plasma cell with irregular cytoplasmic projections (bright purple-red in Wright’s stain)
• These cells contain more immunoglobulin than normal plasma cells
• Associated with Plasma Cell Myeloma / Multiple Myeloma (Refers to the malignancy of plasma cells).

4. Grape / Mott cell / Rusell Bodies

• variant of plasma cell with round globules (Russell bodies)
• Associated with Plasma Cell Myeloma / Multiple Myeloma

5. Sezary cell
• Lymphocyte with convoluted, cerebriform nucleus (“brain-like”)
• Associated with: 1. Sezary Syndrome
2. Mycoses Fungoides

Lysosomal Lipid Storage Diseases

1. Gaucher disease
• most common of the lysosomal lipid storage diseases.
• autosomal recessive disorder caused by deficiency in the catabolic enzyme B-Glucocerebrosidase.
• there is an accumulation of unmetabolized substrate sphingolipid glucocerebroside in macrophages
• Bone marrow replacement by Gaucher cells contribute to anemia and thrombocytopenia
• Gaucher cells are distinctive macrophages, single or in clusters, exhibiting abundant fibrillar blue-gray cytoplasm
with a striated or wrinkled appearance, sometimes described as onion skin-like.

2. Niemann-Pick Disease
• characterized by an accumulation of fat in cellular lysosomes of vital organs, which impairs function
• deficiency in the enzyme Sphingomyelinase.
• Foam cells and sea-blue histiocytes can be seen in the bone marrow.
o Foam cells have cytoplasm packed with lipid-filled lysosomes (small vacuoles)

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o Sea blue histiocytes are macrophages with lipofuscin-, glycophospholipid-, and sphingomyelin
contained in -cytoplasmic granules, that appear blue with Wright stain.

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LEUKEMIA
• refers to the rapid, clonal proliferation in the bone marrow of lymphoid or myeloid progenitor cells known as
lymphoblasts and myeloblasts.
• development of leukemia is currently believed to be a progression of mutations that give leukemic stem cells a
proliferative advantage and also hinder differentiation
ACUTE CHRONIC
Predominant cell type Precursors / blasts Mature
Onset Sudden Insidious
Symptoms Fever, fatigue, bleeding Variable; non-specific
WBC count Variable Increased
Progression w/o treatment Rapid; weeks to months Slow; months to years

Classification schemes for hematologic neoplasms:

• French-American-British (FAB) system - based on cell morphology and cytochemical staining
• World Health Organization (WHO) system - emphasizes molecular and cytogenetic abnormalities
ACUTE LEUKEMIA
1. Acute Myeloid Leukemia (AML)
• most common type of leukemia in adults, and the incidence increases with age
• WBC count ranges from 1-200 x 109/L
• Myeloblasts are present in the peripheral blood in 90% of patients.
• Common Lab findings:
o Anemia, thrombocytopenia, neutropenia
o Hypercellular BM with presence of blast cells (detected through FLOW CYTOMETRY) (>20% accdg to
WHO)
o Surface markers present: CD13, CD33, CD117
o Chemistry: Hyperuricemia, Hyperphosphatemia, Hypocalcemia

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Major types of AML (Based on WHO System) / Cytogenetics Abnormalities

AML with t (8;21) corresponds to acute myeloid leukemia with neutrophilic maturation;
also known as core binding factor leukemia

AML with inv (16) correspond to acute myelomonocytic leukemia with abnormal eosinophils; eosinophils are
increased and have large basophilic granules

APL with t (15;17) promyeloctes predominate in the BM; Azurophilic granules are abundant;
Multiple Auer Rods are usually present; DIC is common

APL: Acute Promyelocytic Leukemia

AML with t (9;11) rare leukemia (6% of AML cases) that presents with an increase in monoblasts and immature
monocytes

AML with t (6;9) associated with multilineage dysplasia and basophilia of marrow and blood; shows
variable morphology and granulocytic and/or monocytic differentiation

AML with inv (3) shows multilineage dysplasia, increased atypical megakaryocytes with monolobed or bilobed
nuclei, and normal to increased platelet counts.

AML with t (1;22) presenting as acute megakaryoblastic leukemia with hepatosplenomegaly; acute
leukemia of infants and young children, often reported in girls

AML Not otherwise specified

• These cases do not fulfill criteria for the previous categories, or cytogenetic studies were unsuccessful or could
not be performed.
• Categorization is similar to French-American-British Cooperative (FAB) Group criteria
• The FAB classification was based on the cell of origin, degree of maturity, cytochemical reactions, and limited
cytogenetic features.

M0 AML, minimally differentiated

M1 AML without maturation
M2 AML with maturation FAB Classification of AML
M3 Acute Promyelocytic Leukemia
M4 Acute Myelomonocytic Leukemia
M4Eo Acute Myelomonocytic Leukemia with eosinophilia 12
M5a Acute Monocytic Leukemia, poorly differentiated
M5b Acute Monocytic Leukemia, well differentiated
M6 Acute Erythroleukemia
M7 Acute Megakaryocytic Leukemia
M1 No clear evidence of cellular maturation; Auer rods are absent
M2 Blasts comprise 90% of nonerythroid cells in BM; closely aligned with the blasts of M0
M3 Has at least 10% maturing cells of neutrophil lineage; Auer rods are often present
M4 Monoblast and promonocytes constitute at least 20% of all marrow cells; also known as Naegelli Leukemia
M5 Monoblast, promonocytes, monocytes are present (>80% of all marrow cells); AKA: Schillings Leukemia
M6 80% or more of the bone marrow cells are erythroid; > 30% are proerythroblasts; AKA: Di Guglielmo
Syndrome
M7 Blasts comprise at least 50% of megakaryocyte origin;
M7 Identified thru immunostaining using antibodies against vWF, gpIb, gpIIb/IIIa

2. Acute Lymphoblastic Leukemia

• 25-75% of childhood leukemia (2-5 yrs of age)
• 25% of childhood ALL cases show the genetic abnormality: t(12;21) derived from a B cell progenitor
• Clinical findings: Anemia, thrombocytopenia, neutropenia, lymphadenopathy, splenomegaly
• WHO Classification: B-cell ALL and T-cell ALL
• Although morphology is the first tool used to distinguish ALL from AML, immunophenotyping and genetic analysis
are the most reliable indicators of a cell’s origin
o B-cell ALL: (+) CD34, CD19, CD10, CD22, TdT
o T-cell ALL: (+) CD2, CD3, CD4, CD5, CD7, CD9, TdT

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FAB Classification of ALL

L1 L2 L3
Cell size Small Large Large
Chromatin Homogenous Heterogenous Homogenous
Nuclear shape Regular Irregular Regular
Nucleoli Rare Present Prominent
Cytoplasm Scanty (↑ N:C ratio) Moderate Moderate & deeply basophilic
Occurrence CHILDREN ADULTS Leukemic phase of Burkitt’s lymphoma

Cytochemical Stains
AML ALL
MPO (Myeloperoxidase) + -

SBB (Sudan Black B) + -

Naphthol AS-D Chloroacetate (CAE) + -

TdT (Terminal deoxyribonucleotidyl transferase) - +

Periodic Acid Schiff (PAS) = Stains glycogen / - +

carbohydrates. Except M6

POSITIVE REACTION ON MONOCYTES CAN BE INHIBITED

Non-specific Esterases Monocytes Granulocytes
Alpha-napthyl Acetate Esterase (ANA) + -

Alpha-napthyl Butyrate Esterase (ANB) + -

(+) M4 = Acute Myelomonocytic

AML
(+) M4 = Acute Monocytic
PART #3

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Myeloproliferative Neoplasms
 Caused by genetic mutations in the stem cell that causes excessive production and accumulation of RBCs,
granulocytes and platelets in the BM, blood and tissues.

o CML = Philadelphia chromosome t (9;22) Chronic myelogenous leukemia

o PV = JAK2 gene mutation (90-95% of cases) Polycythemia vera; JAK2 = Janus 2-kinase
o ET = JAK2 gene mutation (50% of cases) Essential thrombocythemia
o PMF = JAK2 gene mutation (50% of cases) Primary myelofibrosis

1. Chronic Myelogenous Leukemia (CML)

• Arises from a single genetic translocation in a pluripotential HSC (hematopoietic stem cells) producing a clonal
overproduction of the myeloid cell line
• Associated with Philadelphia chromosome
o translocation between the long arms of chromosome 9 and 22
o results in a formation of unique chimeric gene, BCR-ABL1  causes uncontrolled production of
blood cells

• CBC findings:
o Increased Total WBC count (> 50 x 109/L)
▪ Increased in mature forms of granulocytes –
Neutrophils, Eosinophils, Basophils
▪ Myeloblasts are only 1-5%
o Normal or increased platelet count
o Normal or decreased RBCs
Acute myelogenous leukemia – blast / immature cells; >20% blasts, >30% blasts
Both leukemoid reactions & CML has high WBC count; most are mature WBCs

• Must be differentiated from Leukemoid reactions (associated with severe infections) thru NAP cytochemical
staining
o ↓ NAP score = CML
o ↑ or normal NAP score = leukemoid reactions
NAP – neutrophil alkaline phosphatase
LAP – leukocyte alkaline phosphatase

• The neutrophil alkaline phosphatase (NAP) is greatly reduced or absent in > 90% of patients with CML.
o Specimen: Blood Film
o Dye: Diazo (alkaline pH)
o Substrate: Naphthol-phosphate
o Product: insoluble blue compound
Alkaline phosphatase works on the substrate
Enzyme + substrate = product (insoluble blue compound)
o Evaluate 100 neutrophils
▪ 0 no staining
▪ 1+ faint/diffuse
▪ 2+ pale/moderate
▪ 3+ strong blue ppt
▪ 4+ deep blue ppt without visible cytoplasm
o Reference interval: 15-170
o The mean score of two examiners is reported, and they should agree within 10%

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 • BM transplantation has been successful in CML, and imatinib mesylate, a tyrosine kinase inhibitor, produces
remission in most cases.

NAP STAINING
Evaluate 100 neutrophils
0 x 93 = 0
1+ x 5 = 5
2+ x 2 = 4
3+ x 0 = 0
4+ x 0 = 0
9 NAP score = ↓

Ref range: 15-170

 ↑ Leukemoid reaction, PV, PMF, third trimester of pregnancy
 ↓ CML, PNH

2. Polycythemia Vera
• characterized by excessive proliferation of erythroid, granulocytic and megakaryocytic elements in the bone
marrow (panmyelosis). Splenomegaly is usually present
• ↑ RBC, granulocytes, megakaryocytes, except for lymphocytes

• WHO Criteria for diagnosis:

o ↑ hemoglobin, hematocrit, or red cell mass = Absolute polycythemia
▪ Men: Hgb > 16.5 g/dL and Hct > 49%
▪ Women: Hgb > 16 g/dL and Hct > 48%
o Hypercellular BM with trilineage growth (panmyelosis)
o Identification of the JAK2 V617F mutation or the JAK2 exon 12 mutation
o Low EPO levels

• The increased red cell mass produces blood hyperviscosity, often resulting in cardiovascular disease
• Treatment includes:
o therapeutic phlebotomy (during early stages)
o alkylating agent hydroxyurea
o JAK inhibitors (e.g. ruxolitinib)
Polycythemia
1. Absolute – Hct & Red cell mass
a. Primary - ↑ RBC ct, ↓ EPO levels (ex: PV)
b. Secondary - ↑ RBC ct, ↑ EPO levels (ex: Renal tumors, hypoxic conditions:
smoking, ↑ altitude areas)
2. Relative – reduction in plasma volume
- ↑ Hct, Normal red cell mass (ex: Dehydration, burns, diuretics)

3. Essential Thrombocythemia
• clonal MPN with increased megakaryopoiesis and thrombocytosis
• Clinical manifestations
o vascular occlusions (thromboses in major arteries and veins)
o splenic infarcts resulting to atrophy
o pulmonary emboli and neurologic complications
o bleeding in mucous membranes
Normal platelet count: 150-400 x 109/L

• Lab findings:

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 o Thrombocytosis (>400 x 109/L)
o Platelets often appear normal, but there could be presence of giant bizarre platelets, platelet aggregates,
micromegakaryocytes, and megakaryocyte fragments
o Leukocytosis is usually present and ranges from 22 to 40 x 109/L.
o Segmented neutrophils may be increased
• Treatment includes:
o Plateletpheresis
o Hydroxyurea therapy
o Low-dose aspirin
o JAK2 inhibitors

4. Primary Myelofibrosis
• It is the least common but most aggressive form of MPN
• Chronic, progressive clonal panmyelosis characterized by megakaryocytic and often granulocytic hyperplasia
with varying degrees of reactive fibrosis of the marrow & extramedullary hematopoiesis  production of
blood cells outside the bone marrow
• Splenomegaly is present in 90% of patients with PMF, and 50% have hepatomegaly

• Bone marrow findings:

o Panmyelosis (megakaryocytic and often granulocytic hyperplasia: neutron, eo, and baso)
o Varying degrees of reactive fibrosis
o Collagen overproduction disrupting the normal architecture of the BM
o It is usually impossible to aspirate marrow  “DRY TAP”, and biopsy is necessary for
examination

• CBC findings:
o Normocytic anemia, anisocytosis, and poikilocytosis including Dacryocytes
o Pancytopenia resulting from fibrosis of BM

• Treatment includes a variety of approaches, including transfusions, hydroxyurea, interferon-γ, busulfan,

androgens, erythropoietin, and JAK2 inhibitors

Myelodysplastic syndrome (MDS)

• Historically referred to as refractory anemia, smoldering leukemia, oligoblastic leukemia, preleukemia.
• The median age of diagnosis is 76 years old.
• Group of acquired clonal hematologic disorder characterized by:
o Progressive CYTOPENIA  ↓
Blood cell ct & morphology is
abnormal
o Dyserythropoiesis (abnormal
morphology)
▪ Oval macrocytes
▪ Hypochromic microcytes
▪ RBC precursors with abnormal nuclear shapes
▪ Ring sideroblasts (young RBCs with iron deposits)
▪ Howell-Jolly bodies, basophilic stippling
o Dysmyelopoiesis (abnormal morphology)
▪ Persistent basophilia of mature WBCs
▪ Abnormal granulation of WBCs
▪ Abnormal nuclear shape

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 o Dysmegakaryopoiesis (abnormal morphology)
▪ Giant platelets
▪ Abnormal granulation of platelets
▪ Abnormal nuclear shapes in the megakaryocytes
▪ Circulating micromegakaryocyte
• FAB Classification of MDS:
1. Refractory anemia
2. Refractory anemia with ring sideroblasts (RARS)
3. Refractory anemia with excess blasts (RAEB)
4. Chronic myelomonocytic leukemia
5. Refractory anemia with excess blasts in transformation (RAEB-t)
Lymphoproliferative disorders
• Includes the Lymphoid leukemia and Lymphoma
• classified by WHO thru their biological features and not the anatomical distribution

Mature B cell Neoplasms

1. Chronic Lymphocytic Leukemia (CLL) (common in males than females)
Children – ALL; Adults - CLL
• most common leukemia in adults in Western countries
• median age of diagnosis is approximately 72 years, with a slight male preponderance
• accumulation of small lymphoid cells in the BM, peripheral blood and lymphoid organs
• Lab detection:
o Small lymphocytes (“soccer ball”) appearance, scant cytoplasm, absent nucleoli.
o Prolymphocytes are present up to 55%
o Numerous smudge cells/basket cells on blood smear. (indicating ↑ nuclear fragility of
lymphocytes)
o Surface markers: CD19, CD20, CD23

2. Hairy Cell Leukemia

• median age of diagnosis is approximately 50 years, with a slight male preponderance
• Small lymphocytes with abundant cytoplasm and hairy cytoplasmic projections
• Leukemic cells are found primarily in BM and spleen.
• Common clinical findings: Splenomegaly and Pancytopenia (↓ blood cell ct)
• Lab detection:
o TRAP (Tartrate-resistant acid phosphatase) cytochemical stain (+) - hairy cells show red granular
cytoplasmic staining.
o Annexin A = protein expression that can be used as specific marker to HCL.
o Surface markers:CD19, CD20, CD22.

3. Burkitt Lymphoma
• aggressive cancer of mature B cells associated with a fulminant clinical presentation
• has three subtypes: endemic, sporadic, and HIV associated
• morphologic findings:
o medium-sized lymphoid cells which have deeply basophilic cytoplasm (strong blue); highly vacuolated
o Bone marrow and lymph node biopsies may show a classic “starry sky” appearance on Low power field

4. Mantle cell Lymphoma

• median age at diagnosis is 68 years, and males are affected more than females
• patients present with extensive lymphadenopathy, and GI tract is also affected (extranodal disease)
• accurate diagnosis requires demonstration of either t(11;14) or overexpression of cyclin D1

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 5.
Diffuse Large B-cell Lymphoma
• Most common form (25-30% of cases) of Non-Hodgkin’s Lymphoma
• Clinical presentation is a rapidly expanding, painless lymphadenopathy in one or more sites
• Lab findings:
o Large lymphoid cells with diffuse histologic growth pattern.
o Surface markers: CD5, CD10, CD30

Mature T cell Lymphoma

• Mycosis Fungoides
o most common type of cutaneous lymphoma (60-70% of cases)
o Small lymphoid cells that have predilection to epidermis and dermis (tumor-like lesions)  caused by
malignant T cells affecting the skin
o In later stages, disseminates to lymph nodes, organs, and blood
o Malignant T cells (Sézary cell) have cerebriform, folded nucleus, variably condensed chromatin

• Sezary syndrome
o Aggressive lymphoma with leukemic presentation (peripheral blood
involvement).
o characterized by erythroderma and generalized lymphadenopathy.
o An absolute Sézary cell count greater than 1 x 109/L is required to make a
diagnosis.

Plasma cell Neoplasms

 malignant disorders of terminally differentiated B cells
 essential characteristic is the ability of the plasma cells to secrete monoclonal immunoglobulin (Ig)
 Types:

o Monoclonal Gammopathy of Undetermined Significance (MGUS)

 benign monoclonal proliferation of plasma cells; represents precursor state for myeloma
 absence of end organ damage

o Plasma cell Myeloma (MM) – old name: Multiple myeloma

 characterized by monoclonal proliferation of plasma cells in the BM
 clinical findings: bone lesions and hypercalcemia
 Lab findings:
• Monoclonal gammopathy (SPE – Serum protein electrophoresis) / M protein /
paraproteins – monoclonal peak in the gamma region
• Bence-Jones proteins in urine – composed of homologous light chains
excreted in the urine, not detected in reagent strip (negative); sensitive only
for albumin; SSA (positive) – precipitates between 40-60C, dissolves at 100C
• Presence of flame cells, mott cell / grape cells in peripheral smear
• malignant plasma cells express high levels of CD38 and CD138

o Waldenström’s Macroglobulinemia (WM) – excessive synthesis of IgM

 low-grade lymphoplasmacytic lymphoma associated with aberrant secretion of IgM.
 high levels of IgM can result in a hyperviscosity syndrome
 involves a somatic mutation in the myeloid differentiation factor 88 (MYD88) gene

Hodgkin’s Lymphoma
• primarily a lymph node-based disease

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 • has two distinct forms which differ pathologically and clinically:

o Classic HL
▪ makes up 95% of cases
▪ characterized by the presence of REED-STERNBERG CELLS
 “owl’s eye” appearance
 has abundant cytoplasm & bilobed nucleus with prominent eosinophilic nucleoli
 B-cell origin and almost never found in peripheral blood (lymph node biopsy)
 Surface markers: CD15 and CD30

o Lymphocyte-predominant HL
▪ malignant cell is a lymphocytic histiocytic cell
 L&H cells are large with scant cytoplasm and a folded single nucleus;
 they are labeled “popcorn” cells because of their distinct morphology.
 50% express epithelial membrane antigen (EMA)

LAB PROCEDURES IN HEMATOLOGY

Specimen collection
• K2 EDTA less shrinkage of RBC; is the most common anticoagulant for routine hematology testing (CBC)
• Blood for CBC should be analyzed within 6 hrs (if stored at room temp) or within 24 hrs (4°C)
o Consequences: old specimen – RBC swelling, ↑MCV = presence of vacuoles on WBCs,
disintegration of platelets
• Peripheral blood films should be prepared within 3-4 hrs of collection

Manual cell count

• Uses a hemacytometer, most common is the Levy chamber with improved Neubauer ruling
• Hematocytometer - Composed of two grids, separated by H-shaped moat
• Each grid has nine 1 mm x 1 mm squares, with a total area of 9 mm2 (9 large squares)

o Each of the four corner Large Squares is subdivided into 16 small squares
o Center Large Square is subdivided into 25 small squares
o Distance between each counting surface and the coverslip is 0.1 mm
Manual Cell Count
 WBCs are counted in the four large corner squares
 RBCs are counted in the central square but, 5 small square are used; 5 out of 25 small squares
are used for RBC count
 Platelets are counted in whole area of central large square; 25 out of 25 small squares
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 • General Formula
Cells counted x Dilution Factor
Area counted x 0.1mm

AREA
1 WBC square = 1 mm2
1 RBC square = 0.04 mm2
Platelet - Central large square = 1 mm2

Blood diluting pipette (Unopette or Thoma pipet)

RBC pipette WBC pipette

Units 0.5, 1.0, 101 0.5, 1.0, 11
Dilutions 1:100, 1:200 1:10, 1:20

RBC pipette – larger bulb size; has red bead

WBC pipette – smaller bulb size; has white bead

Most common dilutions

Diluting fluids Dilution Objective lens Area counted
WBC count 1% ammonium oxalate; 1:20 10x 4 mm2
3% acetic acid; 1% HCl 1:100 10x 9 mm2
Acid – lyses RBCs  LPO
RBC count NSS 1:100 40x 0.2 mm2
 HPO
PLT count 1% ammonium oxalate 1:100 40x 1 mm2

1 WBC square – 1mm2 x 4 = 4 mm2

1 RBC square – 0.04 mm2 x 5 = 0.2 mm2

Sample computations:
1. WBC count
145 WBCs are counted on four WBC large squares.
Dilution used by the technologist is 1:20
Formula: Cells counted x Dilution Factor
Area counted x 0.1mm  constant (depth)

Area of 1 WBC square – 1 mm2

Area of 1 RBC square – 0.04 mm2

145 x 20 = 2,900
4 mm2 x 0.1 mm2 0.4 mm2

= 7,250/mm2 = ML = x 10 6/L

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= 7.25 x 10 9/L
Si unit = x 10 9/L

2. Platelet count

3. RBC count

Procedure for WBC count

1. Prepare dilution. Mix thoroughly.
2. Allow dilution to sit for ________ (complete lysis of RBCs)
3. Mix again and charge both sides of hemacytometer
4. Place hemacytometer in a moist chamber for ______ (allows cells to settle)
5. Count cells using LPO in both grids (difference should be less than 10%). Get the average count
6. Edge rule: Cells touching the left and top lines are counted. Cells touching bottom and right are ignored
7. Any NRBCs present in the specimen are not lysed by the diluting fluid. The NRBCs are counted as WBCs
because they are indistinguishable when seen on the hemacytometer.
8. If five or more NRBCs per 100 WBCs are observed on the differential count on a stained peripheral blood film, the
WBC count must be corrected.
o Formula for Corrected WBC count:

Hemoglobin Determination
1. Visual Colorimetry
• Acid Hematin (Sahli-Hellige method)
o Procedure: 20 uL blood + 0.1N HCl = Acid hematin
o Allow the mixture to stand for _________ to develop color
o Mixture is then diluted using distilled water until its color matches the brown glass standard
o Hb F, Hi, HbCO and SHb cannot be converted into acid hematin

• Alkali Hematin
o Procedure: 20 uL of blood + 0.1N NaOH = Alkali hematin
o Hb F is resistant to akali denaturation

2. Photoelectric Colorimetry
• Cyanmethemoglobin method (HiCN)
o Recommended because it measures all forms of Hb except Sulfhemoglobin
o Specimen: 20 uL blood
o Reagent: 5 mL Drabkin’s solution
 Potassium ferricyanide
 Potassium cyanide
 Dihydrogen potassium phosphate

o Mix and allow to stand for ________ (conversion of Hb to HiCN)

o Use cyanmethemoglbin reagent as blank
o Product: HiCN measured at 540 nm

• Sources of error:
o Cyanmethemoglobin reagent is sensitive to light. It should be stored in brown bottle / dark area.
o Turbidity of sample can cause false-increase values

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 High WBC count (>20 x 109/L) High
PLT count (>700 x 109/L)
Lipemia
Hb S and Hb C
Centrifuge reagent-sample mixture and measure the supernatant.
Mix 0.01 mL of patient’s plasma + 5 mL of cyanmethemoglobin reagent,
and use it as reagent blank
Prepare 1:2 dilution with distilled water and multiply the result by 2

o Abnormal globulins associated with MM or WM may precipitate in the reagent.

o Reagent contains cyanide which is highly toxic; automated analyzers use cyanide-
free SLS.

3. Indirect method
• Chemical (Principle: 1 gHb contains 3.47 g Fe)
• Gasometric (Principle: 1 g Hb contains 1.34 mL O2)

4. Gravimetric
• Specific gravity (CuSO4 method; sp. Gr. Of 1.053)

23
Microhematocrit determination
• Ratio of volume of RBCs to that of whole blood expressed as percentage or in ratios
• Unreliable as an estimate of anemia

1. Macrohematocrit Method of Wintrobe

• Used only in the past; large amount of blood needed
• Centrifugation at 2,000-2,300 g for 30 mins
• Large error due to trapped plasma

2. Microhematocrit Method of Adams

• Most accurate manual method
• Centrifugation at ______________________
• Blood filled ___________
• Clay Seal ____________
• False results:
o Falsely elevated
▪ Inadequate centrifugation
▪ Including buffy coat in the measurement
▪ Trapped plasma (causes 1-3% higher than value obtained in automated analyzers)
• macrocytic anemia
• hypochromic anemia
• sickle cell anemia
• thalassemia
• spherocytosis
o Falsely low
▪ incomplete sealing
▪ overanticoagulation
▪ IV fluid contamination
▪ interstitial fluid from a skin puncture

• Rule of Three
o Can be used to quickly check the results of Hgb and Hct
o Applies only to specimens that have normocytic normochromic RBCs
o Hgb x 3 = Hct (± 3)

Reticulocyte count
• used to assess the erythropoietic activity of the bone marrow
• A sample from EDTA-whole blood is stained with a supravital stain, such as new methylene blue
• Procedure:
o Mix equal amounts of blood and NMB stain (2 to 3 drops, or approximately 50 uL each)
o Allow to incubate at room temp for 3 to 10 minutes. Remix and prepare two wedge films.
o Observe under OIO and calculate % reticulocytes
o Formula:

o To improve accuracy, another tech counts the other film; counts should agree within 20%
o Inserting a Miller disc into the eyepiece of microscope can facilitate counting

24
  RBCs are counted in the smaller square, and retics are counted in the larger square
 A minimum of 112 RBCs should be counted in the small square

o Formula:

Erythrocyte Sedimentation Rate

• Measures the degree of settling of RBCs in plasma in anticoagulated whole blood
• ESR result is the distance in millimeters that the RBCs fall in 1 hour
• Non-specific measurement used to detect and monitor INFLAMMATORY responses
• Most important factor affecting ESR _______________________
• Clinical Significance:
o Increased in
 Acute/Chronic infections
 Multiple myeloma / Waldenstrom’s
 Hyperfibrinogenemia
 Anemia, Macrocytosis
 AMI, ESRD, RA, DM, Gout
 Neoplastic disease
 Pregnancy, menstruation

o Decreased in:
 Polycythemia / Pancytosis
 Poikilocytes (Sickle cells, Spherocytes, Acanthocytes)
 Microcytosis
 Hypofibrinogenemia
 Hypogammaglobulinema
 Newborn
• Mechanical / Technical factors

o Falsely-High ESR = High temperature, tilted ESR tube, vibrations \

o Falsely-Low ESR = Low temperature, old sample, bubbles in ESR tube

• Temperature: 20-25°C
• Should be performed within 4 hours after blood collection if kept at room temp
• Anticoagulant: _____________________
• ESR columns:
o Westergren Method
 Length = 300 mm
 Ext bore diameter = 5.5 mm
 Int bore diameter = 2.5 mm
 Graduation = 0-200 mm
o Wintrobe Method
 Length = 115 mm
 Int bore diameter = 3 mm
 Graduation = 0-100 mm

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Peripheral Blood Films
• Should be made within 3-4 hrs after blood is collected using EDTA o Possible problems that can
be encountered in certain conditions:
▪ EDTA-inducted platelet satellitosis
▪ EDTA-induced platelet clumping
• Types of blood films:
o Wedge method
▪ most convenient and most commonly used technique
▪ requires at least two glass slides (dimensions: 75 x 25 mm or 3 inch x 1 inch)

▪ Size of drop of blood: __________

▪ Distance of blood from edge of slide __________
▪ Angle of pusher slide __________
▪ Length: __________
▪ Drying of smear: __________

▪ Features of well-made film:

• Finger-shaped blood film
• Slightly rounded feathery edge
• Lateral edges of film are visible
• Smooth without irregularities

o Automated slide making and staining:

▪ Example: Sysmex SP-10
▪ Based on the hematocrit reading, the system adjusts the size of the drop of blood used and the
angle and speed of the spreader slide
▪ Patient information, such as name, number, and date is printed on the slide.

Staining of Blood Films

• Widely used stains: Polychrome stains
o Methylene blue – basic dye stains acidic cellular components (e.g. nucleic acids)
o Eosin – acidic dye and stains basic cellular components (e.g. hemoglobin)
• Fixative: Methanol
• Wright’s stain (Romanowsky stain)
o Methyl alcoholic solution of: Eosin, Methylene blue (50-75%), Azure B
o Buffer solution (K+ phosphate / Na phosphate, pH _____ )
• Errors on staining:
o Causes of excessive blue stain:
 too alkaline buffer
 prolonged staining time
 inadequate washing
 thick films
o Causes of excessive pink stain:
 too acidic buffer
 insufficient staining time
 prolonged washing
 mounting coverslips before they are dry

Blood film examination

• LPO (10x)
26
 o overall film quality, color, and distribution of cells can be assessed
o can be scanned to check for the presence of fibrin strands, rouleaux
formation, agglutination, abnormal cells such as blasts, reactive
lymphocytes and parasites

• HPO (40x)
o use to select the correct area of the film in which to begin the differential count
o WBC estimation can be performed (Factor used: ___________)

• OIO (100x)
o WBC differential count is performed
 “Battlement” track pattern is preferred to minimize distribution
errors.
 100 WBCs are counted and classified through the use of push-
down counters.
 To increase accuracy, it is advisable to count at least 200 cells
when WBC > 40 x 109/L.
o Evaluation of RBC, WBC, and platelet morphology.
o Platelet estimation can be performed (Factor used: ___________)

REFERENCE RANGES

Hemoglobin Newborns: 16.5-21.5 g/dL MCV 80-100 fL

Adult male: 13.5-18 g/dL MCH 26-32 pg
Adult female: 12-15 g/dL MCHC 32-36% or g/dL
RDW 11.5-14.5%

Hematocrit Newborns: 0.48-0.68 Platelet ct 150-400 x 109/L

Adult male: 0.40-0.54 MPV 6.5-12 fL
Adult female: 0.35-0.49

RBC Newborns: 4.1-6.1 x 1012/L Retic ct 0.5-2.5%

Adult male: 4.2-6.0 x 1012/L
Adult female: 3.8-5.2 x 1012/L ARC 20-115 x 109/L

WBC Newborns: 10-30 x 109/L

Adults: 3.6-10.6 x 109/L ESR Male: 0-15 mm/hr
Westergren Female: 0-20 mm/hr

Differential RELATIVE COUNT ABSOLUTE COUNT

Neutrophil: 50-70% Neutrophils: 1.7-7.5 x 109/L
Lymphocytes: 18-42% Lymphocytes: 1.0-3.2 x 109/L
Monocytes: 2-11% Monocytes: 0.1-1.3 x 109/L
Eosinophils: 1-3% Eosinophils: 0-0.3 x 109/L
Basophils: 0-2% Basophils: 0-0.2 x 109/L

27
BONE MARROW EXAMINATION
• Indications: Neoplasia diagnosis, BM failure, infections, monitoring of treatment •
• Before BM collection (< 24 hrs), blood is collected for CBC with blood film examination
• Site of collection:
o Preferred: __________________ of the pelvis.
o Others sites: Sternum, anterior medial surface of the tibia
• Types of needles that are used for collection:
o Jamshidi biopsy needle
o Westerman-Jensen biopsy needle
o University of Illinois aspiration needle
o Snarecoil biopsy needle

• Preparation of BM samples.
o Direct aspirate smears
o Anticoagulated aspirate smears
o Crush smears of aspirate
o Buffy coat smears
o Core Biopsy Imprints / Touch Preparations
o Histologic Sections / Cell Block of the Core Biopsy

BM aspirate BM core biopsy

Cytologic assessment BM architecture

Uses Cell Morphology observation BM cellularity
Differential count Detection of focal lesions

Stains for evaluation

Bone Marrow Cellularity

• Normocellular
o For children: 80% cellularity

o For ages 30-70 yrs old: 50% cellularity

o For ages > 70 yrs old: (100% - Patient age) ±10%

• Hypocellular / Hypoplastic: ↓ cells and ↑ fat

• Hypercellular / Hyperplastic: ↑ cells and ↓ fat

Myeloid to Erythroid Ratio

• Normal: 1.5 to 3.3 : 1
o Increased in: Infections, leukemia
o Decreased in: Depression of leukopoiesis

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Other Definitive Tests on BM samples
• Cytochemical studies
• Cytogenetic studies
• Molecular studies
• Flow cytometry
• Fluorescence in situ hybridization

AUTOMATION

• Electronic impedance
o based on the detection and measurement of changes in electrical resistance produced by cells as
they traverse a small aperture.
o electrical resistance between the two electrodes, or impedance in the current, occurs as the cells
pass through the sensing aperture, causing voltage pulses that are measurable.
 The number of voltage pulses is proportional to ______________________.
 The height of the voltage pulse is directly proportional to _________________.
o Blood sample is distributed into two channels: RBC/PLT and WBC/HGB.
o The data are plotted on a frequency distribution graph, or volume distribution histogram.
o The histogram produced depicts the volume distribution of the cells counted.

• Radiofrequency
o usually used in conjunction with electronic impedance
o the cell interior density is proportional to pulse height or change in the RF signal.
o Conductivity is determined using a high frequency electromagnetic probe that provides
information on the cell internal constituents.

• Optical Light Scatter

o As cells pass through the sensing zone & interrupt the beam, light is scattered in all directions.
o The detection of scattered rays and their conversion into electrical signals is accomplished by
photodetectors at specific angles.
 _______________ (0 degrees) correlates with cell volume.
 _______________ (90 degrees) correlates with degree of internal complexity.
o Scatter properties at different angles may be plotted against each other to generate twodimensional
cytograms or scatterplots

29
CAUSES OF ERRONEOUS RESULTS WITH AUTOMATION

Condition Parameters affected

Cold agglutinins

Lipemia, icteric sample

Lyse-resistant RBCs with abnormal Hb

Microcytes or schistocytes

Nucleated RBCs, megakaryocyte fragments

Platelet clumps

WBC > 100,000 / uL

Old specimen

30