MUST TO KNOW IN HEMATOLOGY

Hematology Greek:
-Haima = Blood
-Logos = Study/science

EDTA Chelates calcium

(Lavender top) Inversion: 8x
Anticoagulant of choice for hematology cell counts and cell morphology
Blood smear: prepare w/in 2 hrs
Preferred anticoagulant for platelet count:
= In some patients w/ EDTA anticoagulated blood – platelet satellitism
= Platelet satellitism: platelets adhere to neutrophils
Effect to automated platelet count Decreased
Remedy: Repeat platelet count using citrate (Rodak: Platelet count x
1.1)
EDTA = Shrinkage of cells = Hct = ESR
Not for coagulation tests:
= Inhibits fibrinogen-thrombin reaction
= Factor V is not stable in EDTA

Modified Westergren ESR 2mL EDTA + 0.5mL NSS/Citrate

(Black top tube) Ratio = 1:4 (Anticoagulant-to-Blood)
Citrate For coagulation and platelet studies
(Light blue top tube) = Preserves labile factors V and VIII
= Buffered 3.2% (0.109M) citrate
Inversion: 3-4x
Ratio = 1:9 (Anticoagulant-to-Blood)

Polycythemic patients Hct

Excess Citrate = PT, APTT
Remedy: Reduce the volume of citrate
Amount of citrate = [(100-Hct)÷(595-Hct)] x mL WB
Oxalate Double/balanced oxalate (Ratio = 2:3): Maintained cell structures
a. Potassium oxalate (Paul-Heller’s) = shrink cells
b. Ammonium oxalate (Wintrobe’s) = swell cells

Heparin Inactivation of thrombin

Anticoagulant for osmotic fragility test
Inversion: 3-4x
Not for blood film preparation:
= Distorts cells
= Produces bluish background on Romanowsky’s stain
Not for coagulation
= Inhibits thrombin and all stages of coagulation

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Order of Draw Evacuated tube:
(Henry 21st Edition) 1. Sterile blood culture tube
2. Citrate (blue)
3. Nonadditive tube (red)
4. Heparin (green)
5. EDTA (lavender)
6. Fluoride (gray)
Order of Draw 1. EDTA
(Syringe method) 2. Other anticoagulated tubes
3. Nonadditive tube

EDTA containing tubes Lavender

Pink
White
Royal blue
Tan
Skin puncture 1. Fingertips
2. Earlobe: less admixture w/ tissue juice, less pain, less free nerve
endings
3. Lateral portion of the plantar surface of the foot: <1 year old
Difference from venous specimen:
WBCs
Hgb, Hct, RBCs, platelet

Venipuncture Veins in the arms (antecubital region):

1. Median cubital = preferred, most stable
2. Cephalic (lateral)
3. Basilic (medial)
Common gauge (needle) 19, 20, 21
Routine: 20g

Common length of needle 1-1.5 inches

Color coded hub (gauge) 18 = pink
21 = green
22 = gray
23 = blue/light blue/turquoise

Angle Venipuncture: 150

BB: 450 10-200 once in the skin
Tourniquet 3-4 inches above the site (7.5-10cm)
Not exceed 1min/2mins
Prolonged application hemoconcentration

BP cuff as tourniquet 40-60 mmHg

Reassure the patient Crying = cell count

Position the patient Lying down = hemodilution ( PCV by 8%, WBC)

Lying up = hemoconcentration

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IV line Collect on the other arm
If both arms: Stop IV for 2mins
= Collect blood below the IV line
= Appropriate for all analytes except glucose and phosphorus
Hematopoiesis Cellular formation, proliferation, differentiation and maturation of blood
cells

Mesoblastic period 19th day of gestation

Yolk salk = Erythropoiesis
Embryonic hemoglobins:
a. Gower 1 = Zeta2 + Epsilon2
b. Portland = Zeta2 + Gamma2
c. Gower 2 = Alpha2 + Epsilon2
Hepatic period 3rd month of gestation
Fetal liver = Granulopoiesis, Erythropoiesis, Megakaryopoiesis
Spleen, thymus, lymph nodes
Hemoglobin production:
a. HbF = Alpha2 + Gamma2
b. HbA1 = Alpha2 + Beta2
c. HbA2 = Alpha2 + Gamma2

Myeloid period Between 5th & 6th month of gestation persist throughout life
BM = 1’ source of cell production (Hematopoiesis)
Sternum = principal source of hematopoiesis in adults
Adults HbA1 = ≥95%
HbA2 = 1.5-3%
HbF = <2%

Neonates HbF = 60-80%

HbA = 20-40%
Marrow specimens 1. Trephine (Core) Biopsy
= Trephine biopsy needle (Jamshidi needle)
2. Aspiration
= Aspiration needle (University of Illinois sterna needle)

Posterior iliac creast Safest site for BM aspirate/biopsy

M:E ratio Numeric expression comparing the relative number of granulocytic
precursors w/ the relative erythroid precursors in the BM
NV = 2:1 to 4:1 (Ave. 3:1)
Infection = 6:1
Leukemia = 25:1
Neutrophilic, Eosinophilic, Basophilic precursors = Myeloid
Erythroid precursors
Monocytic precursors = not included

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BM Cellularity Percentage of marrow space occupied by hematopoietic cells compared
w/ fat
Normocellular marrow (Adult):
Fat = 10-50%
Hematopoietic elements = 40-60% (Ave. 50%)
Yellow BM Fats

Red BM Hematopoietic cells

Marrow differential Recommended that at least 500, preferably 1000 cells be counted for a
marrow differential

Metamyelocyte/Juvenile Predominant cell (WBC) in adult BM (up to 32%)

granulocyte
Stem cells <1% cells in BM

Osteoblasts Bone forming cells

Confused w/ plasma cells
Waterbug or comet appearance
Osteoclasts Bone destroying cells
Confused w/ megakaryocytes

CD2, CD3 T cells

CD19, CD20 B cells

CD34 Stem cell marker (lymphoid and myeloid precursor)

CD16, CD56 NK cells

CD10 CALLA (Common ALL Antigen)

Erythropoietin Produced by the kidney
Primary regulator of erythropoiesis

Thrombopoietin Produced by kidney and liver

Regulator of thrombopoiesis
Erythropoiesis 1’ stimulus = Hypoxia

IL-3 Multi-CSF (Colony Stimulating Factor)

Stimulates hematopoietic cells
1. Pronormoblast Proerythroblast/rubriblast
N/C ratio = 8:1
Nucleoli = 1-2
Can produce up to 16 RBCs per 1 pronormoblast/rubriblast

2. Basophilic normoblast Prorubricyte

Intensely basophilic cytoplasm
N/C ratio = 6:1
Nucleoli usually not visible

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3. Polychromatophilic Rubricyte
normoblast Blue-gray to pink-gray cytoplasm
Last stage capable of mitosis
1st: Hgb synthesis (1st: PCPNB Reticulocyte: Last)
N/C ratio = 4:1
4. Orthochromic Metarubricyte
normoblast Small pyknotic nucleus (dark, small, nonfunctional)
N/C ratio = 1:2

5. Reticulocyte Polychromatophilic erythrocyte/Diffusely basophilic erythrocyte

Romanowsky stain = Polychromasia
Supravital stain = (+) Fine reticulum of RNA
6. Mature RBC Discocyte
6-8 µm in diameter
Life span: 120 days

3-5 days BM: Pronormoblast Reticulocyte

1-2 days PB: Reticulocyte RBC

General Cell Maturation Characteristics for Leukocytes

Immature Cells Mature Cells

Larger Smaller
(+) Nucleoli (-) Nucleoli

Chromatin: fine and delicate (most reliable) Chromatin: coarse and clumped (most reliable)
Nucleus: large and round Nucleus: round. lobulated or segmented

Cytoplasm: dark blue/basophilic (RNA) Cytoplasm: light blue (RNA)

(-) Granules (+) Granules

N:C ratio N:C ratio

Granulopoiesis Neutrophils
Eosinophils
Basophils

14 days Blast Mature granulocyte

1. Myeloblast Earliest recognizable stage in granulocytic series
N/C ratio = 4:1
Nucleoli = 2-5

2. Promyelocyte 1st: Primary granules

N/C ratio = 2:1 to 3:1
Nucleoli = 2-3

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3. Myelocyte Youngest cell in the series wherein a granulocyte can be identified
a. Neutrophil myelocyte = rose pink granules
b. Eosinophil myelocyte = orange-red granules
c. Basophil myelocyte = dark purple or blue-black granules
Last stage capable of mitosis
1st: Secondary granules
N/C ratio = 1:1
(-) Nucleoli
4. Metamyelocyte Juvenile granulocyte
Not capable of mitosis (post-mitotic pool)
Indented/kidney-shaped nucleus
Predominant WBC in BM

5. Band Stab/Staff
Youngest cell in the series present in the peripheral blood (normal)
PB = 0-6% or 0-7%
C or S shaped nucleus
Curved or Sausage shaped nucleus
Resembles Pelger-Huet cells
= PH cells: coarser chromatin than stab cells
6a. Segmented neutrophil Rose-pink granules
Nucleus: 2-5 lobes
Diurnal variation (PM)
Specific granules:
a. Lysozyme
b. Lactoferrin
c. Collagenase
d. Plasminogen activator
e. Aminopeptidase

6b. Eosinophil Reddish-orange granules

Nucleus: usually 2 lobes
Diurnal variation (ACTH)
Specific granules:
[Larger]
a. Major basic protein
b. Acid hydrolase
c. Cathepsin
d. Eosinophil cationic protein
d. Eosinophil-derived neurotoxin
e. Eosinophil protein X
f. Phospholipase
[Smaller]
a. Arylsulfatase
b. Acid phosphatase

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6c. Basophil Dark purple to blue-black granules (water-soluble)
Nucleus: generally unsegmented or bilobed (rare: 3 or 4)
Specific granules:
a. Histamine
b. Heparin
c. Eosinophilic chemotactic factor A
Monopoiesis 1. Monoblast
2. Promonocyte
3. Monocyte

Monocyte Largest cell in PB

14-20 µm in diameter
Blue-gray cytoplasm
Many azurophilic granules (ground glass appearance)
Nucleus: kidney/horse-shoe shaped, may be folded (brainlike)
Lymphopoiesis 1. Lymphoblast
2. Prolymphocyte
3. Lymphocyte

Lymphocyte Small = 8-10µm (Size = RBC)

Medium = 10-12µm
Large = 12-16µm (Rare)
Cytoplasm: bluish (Robin’s egg blue)
Nucleus: compact
T lymphocytes 60-80%
Long-lived (4-10 years)

B lymphocytes 10-20%
Short-lived (3-4 days)
Can differentiate into plasma cell or memory B cells
Null lymphocytes Large granular lymphocyte
10%

Plasma cells 1. Plasmablast

differentiation 2. Proplasmacyte
3. Plasmacyte/Plasma cell
Plasma cell Large well-defined hof/perinuclear halo (light staining area in the
cytoplasm near the nucleus)
Eccentric nucleus
Deeply basophilic cytoplasm (Red/pink cytoplasm: Flame cell [Abnormal])
Chromatin: “Cart wheel pattern”

Thrombopoiesis 5 days (Megakaryoblast Platelets)

1. Megakaryoblast N/C ratio = 10:1

2. Promegakaryocyte Nucleus: may show slight lobulation (Endomitosis)

N/C ratio = 4:1 to 7:1

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3. Granular
megakaryocyte Largest cell in BM
4. Mature megakaryocyte Cytoplasm contains coarse clumps of granules aggregating into little
bundles, which bud off from the periphery to become platelets
Multiple nuclei
N/C ratio = <1:1
5. Metamegakaryocyte Disintegrated cell surrounded by platelet

6. Platelet/Thrombocyte 1-4µm in diameter

Light blue to purple
Very granular
a. Chromomere: granular and centrally located
b. Hyalomere: surrounds the chromomere, nongranular and clear to light
blue
Life span: 8-11 days
2/3 (67%) Circulating platelets

1/3 (33%) Platelets stored in the spleen

Endomitosis Nuclear division w/o cytoplasmic division

2000-4000 # of platelets a megakaryocyte can produce

1 heme molecule 1 mol O2

1 hemoglobin 4 mol O2
Mitochondria Early and late heme synthesis

141 amino acids Alpha

146 amino acids Beta, Gamma, Delta, Epsilon, Zeta

Chromosome 11 Alpha, Zeta

Chromosome 16 Beta, Gamma, Delta, Epsilon

Oxyhemoglobin Normal = Sigmoid in shape

Dissociation Curve X-axis = Hgb concentration in g/dL | Y-axis = OD
Shift to the left (ODC) CO2
Temperature
2,3-DPG
pH
Affinity of Hgb for O2
HbF

Shift to the right (ODC) CO2

Temperature
2,3-DPG
pH
Affinity of Hgb for O2

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Heme synthesis Succinyl coenzyme A + Glycine + Pyridoxal PO4
↓
(ALA synthase)
↓
D-Aminolevulinic acid
↓
(ALA dehydrase)
↓
Porphobilinogen
↓
(Uroporphyrinogen synthase)
(Uroporphyrinogen cosynthase)
↓
Uroporphyrinogen
↓
(Uroporphyrinogen decarboxylase)
↓
Coproporphyrinogen
↓
(Coproporphyrinogen oxidase)
↓
Protoporphyrinogen
↓
(Protoporphyrinogen oxidase)
↓
Protoporphyrin IX + Fe2+
↓
(Ferrocheletase)
↓
HEME
Arterial blood O2 saturation = 95%
pO2 = 95 mmHg

Venous blood O2 saturation = 70%

pO2 = 40 mmHg
P50 pO2 = 26.6 mmHg

Bohr effect pH = Hgb affinity for O2 --- (L)

pH = Hgb affinity for O2 --- (R)
Oxyhemoglobin (HbO2) Arterial blood
Blood: Bright red color

Deoxyhemoglobin (HbCO2) Venous blood

Blood: Purplish red color
Carboxyhemoglobin Blood: Cherry red color
(HbCO) Reversible
Cannot bind and carry O2
CO has 200x or 210x greater affinity for Hgb compared to O2
Smoking, gas, etc.

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Methemoglobin/ Blood: Chocolate brown color
Hemiglobin (Hi) Reversible
Fe2+ Fe3+
Methemoglobin reductase deficiency
Exposure to chemicals/drugs
Exposure to nitrite, chlorine and nitrate
HbM (Abnormal hemoglobin)
Sulfhemoglobin Blood: Mauve lavender
Irreversible
Cannot transport O2
Can combine w/ CO forming carboxysulfhemoglobin
Mixture of oxidized, partially denatured forms of hemoglobin that form
during oxidative hemolysis green hemochrome
Sulfonamides, aromatic amine drugs
Bacteremia: Clostridium perfringens
Enterogenous cyanosis

Erythrocyte membrane 50% protein:

1. Integral protein: Glycophorin A
= Contains sialic acid: (-) charge
= Zeta potential: red cells repel each other
2. Peripheral protein: Spectrin and Actin
= Responsible for biconcave shape of red cells
= Abn. Spectrin: Hereditary Spherocytosis, Hereditary Elliptocytosis
40% lipid:
1. Phospholipids
2. Cholesterol = LCAT
10% CHO
Embden-Meyerhoff Major pathway
pathway 90% glycolysis (anaerobic)
Glucose Lactic acid = 2 ATP
PK deficiency: common enzyme deficiency

Hexose monophosphate 10% glycolysis (aerobic)

shunt Provides reduced glutathione to prevent Hgb denaturation
(Pentose PO4 pathway) G6PD deficiency: most common red cell enzyme deficiency
= (+) Heinz bodies: denatured Hgb (Supravital stain: RHH)
= (+) Bite cells: due to pitting
Rapoport-Luebering Generates 2,3-DPG that regulates Hgb affinity for O2
pathway

Methemoglobin reductase Maintains Hgb iron in ferrous (Fe2+) state to be functional

pathway
Pitting Removal of inclusion by the spleen

Culling Removal of senescent/aged RBCs by the spleen

RBC lysis RBC age = Enzymes, ATP, Size, Density
1% of RBCs/day broken down by the mononuclear phagocytic system
(MPS)

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Extravascular hemolysis 90% aged red cell destruction
w/in the RES
when C’ is not/incompletely activated
Unconjugated bilirubin
Urine and fecal urobilinogen
Intravascular hemolysis 10% aged red cell destruction
w/in the blood vessels
when C’ is completely activated
Haptoglobin
Hemopexin
(+) Hemoglobinemia

Anisocytosis Variation in size

RDW Numerical expression that correlates w/ the degree of anisocytosis
NV = 11.5-14.5%

Anisochromia Variation in Hgb content (Ex. Dimorphic anemia)

MCV NV = 80-100fL (Normocytic)
<80fL = Microcytic
>100fL = Macrocytic

MCH NV = 27-32 pg
MCHC NV = 31-36% (Normochromic)
<31% = Hypochromic
>36% = Hyperchromic

Normocytic normochromic 1. Acute blood loss

anemia 2. Hemolytic anemia
3. Aplastic anemia
Microcytic hypochromic 1. Chronic blood loss = most common cause
anemia 2. Thalassemia = N-RDW
3. IDA = Iron, TIBC (Ex. Hookworm infections) | ! RDW
4. Sideroblastic anemia
5. Chronic disease

Macrocytic, normochromic 1. Megaloblastic anemia = Vitamin B12/Folate deficiency

anemia 2. Nonmegaloblastic anemia = Liver disease, alcoholism
Aplastic anemia Congenital = Fanconi’s anemia
Acquired = radiation, chemical (benzene), drugs (chloramphenicol)
Pancytopenia = WBCs, RBCs, Retics Plts

TIBC Differentiates IDA (TIBC) from other microcytic, hypochromic anemia

Degree of Hypochromia Normal = Area of palor 1/3 of the cell diameter
1+ = Area of palor 1/2 of the cell diameter
2+ = Area of palor 2/3 of the cell diameter
3+ = Area of palor 3/4 of the cell diameter
4+ = Thin rim of hemoglobin

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Megaloblastic anemia Oval macrocytes
Howell-Jolly bodies
Hypersegmented neutrophils
Ineffective erythropoiesis (pancytopenia)
Vitamin B12 (Cobalamin) w/ CNS problems
deficiency
1. Pernicious anemia
= Deficient in intrinsic factor (produced by parietal cells) for B12
absorption
2. D. latum infection
3. Vegetarian diet
4. Malabsorption syndrome
= Steatorrhea, sprue

Folic acid (Vit. B9) w/o CNS problems

deficiency 1. Pregnancy
2. Dietary deficiency
3. Steatorrhea, sprue

Polychromasia Reticulocytosis
Visible on Wright’s stain
Blue-gray coloration, pink cytoplasm
Indicates young RBCs
Erythropoietic activity
a. Hemorrhage
b. Hemolysis
Spherocytosis EOFT

Microcytic, hypochromic EOFT

Poikilocytosis Variation in shape

ESR = No rouleaux formation

Macrocytes Megalocytes: Vit. B12 or folic acid deficiency

Oval macrocytes Asynchronous development: mature cytoplasm but immature nucleus
Macroovalocytes
Acanthocyte Irregular spikes/spicules
Spur cell Abnormal L:S ratio
Thorn cell Abetalipoproteinemia
Liver diseases

Echinocyte Echinocyte: artifactual

Burr cell Burr cell: pathologic
Sea urchin cell RBCs w/ projections of equal length and distribution
Crenated RBCs ATP
Exposure to hypertonic solution
Artifact in air drying
Anemia associated w/ renal insufficiency

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Codocyte Greek: Kodon = bell
Target cell Central hemoglobinized area (bull’s eye)
Mexican hat cell Scanning EM: Bell/tall hat shaped
Surface membrane to volume ratio
Cholesterol & phospholipid
Hemoglobinopathies
Thalassemia
Liver disease, postsplenectomy, IDA

Leptocyte Thin variant of a codocyte

Spherocyte Spheroid
Ball Biconvex
Bronze cell Surface area to volume ratio
Defect of loss of membrane:
Hereditary spherocytosis = Post-splenectomy: Spherocytes
Hemolytic anemia
Burns
Banked blood stored for a long time
AIHA vs. HS AIHA = OFT, MCHC, (+) DAT
HS = OFT, MCHC, (-) DAT

Stomatocyte Greek: Stoma = mouth

Mouth, slit-like pallor area
Bowl-shaped
Permeability to Na+ (Normal: permeability to K+)
Hereditary stomatocytosis
Rhnull disease

Elliptocyte Rod/cigar shaped

Narrower than ovalocytes
Defect in cytoskeleton
Membrane protein band 4.1
Ovalocyte Egglike/oval-shaped
Wider than elliptocytes
Bipolar arrangement in Hgb
Cholesterol
Hereditary ovalocytosis
Megaloblastic BM
Myelodysplasia

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Schistocyte Fragmentation produced by damage of RBC by fibrin, altered vessel walls,
Schizocyte prosthetic heart valves
Fragmentocyte Microangiopathic hemolytic anemia
= Presence of fibrin strands in small blood vessels “clothesline-like
effect”
Burns
TTP
DIC

Keratocyte Schistocyte w/ hornlike projections

Knizocytes Triangular cells
Pinch cells 2 palor areas

Dacryocyte Teardrop/pear-shaped
Teardrops Squeezing and fragmentation during splenic passage
MMM
Hypersplenism

Microspherocyte Severe burns

Pyropoikilocyte
Hereditary microspherocytosis
Hereditary pyropoikilocytosis
= sensitivity to temperature
= RBCs fragment at 45’C (Normal: 49’C)

Semilunar bodies Large, pale pink staining ghost of the red cell
Half-moon cell Membrane remaining after the contents have been released
Crescent cell Malaria
Burns Spherocytes
Schistocytes
Microspherocytes

Drepanocytes Crescent-shaped cell

Sickle cells Holly-leaf
Menisocytes Polymerization of deoxygenated Hgb
Sickle cell anemia
Sickle cell disease

Howell-Jolly bodies Nuclear remnants of DNA (from karyorrhexis)

Feulgen (+)
Megaloblastic anemia
Basophilic stippling Precipitation of ribosomes and RNA
Punctate basophilia 1. Fine stippling = polychromatophilia ( production of RBCs)
2. Course stippling = Lead poisoning
Pyrimidine-5-nucleotidase deficiency = Basophilic stippling

PICA In children = Lead poisoning

In adults = IDA

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Cabot rings Figure of 8
Remnant of microtubules of mitotic spindle
Megaloblastic anemia
Heinz bodies Precipitated, denatured Hgb
Multiple Heinz bodies Pitted golf ball appearance
Requires Supravital stain (RHH)
G6PD deficiency
Unstable hemoglobins (HbH)
Favism (Fava beans)
Drug-induced
Acetylphenylhydrazine/phenylhydrazine = Induce Heinz bodies formation

HbH inclusions Tetramer of beta globin chains (β4)

Requires Supravital stain (RHH)
HbCC crystals Bar of Gold
Clam shell appearance
Hexagonal w/ blunt ends and stain darkly
Solubility

HbSC crystals Washington monument shape

Dark-hued crystal of condensed Hgb distorts the RBC membrane
Solubility
Ringed Sideroblast Nucleated RBC (immature) that contains nonheme iron particles
Requires Prussian blue stain
Sideroblastic anemia (Iron overload)
MDS

Siderocyte Non-nucleated RBC (mature) containing iron granules (hemosiderin)

Sideroblastic anemia
MDS

Pappenheimer bodies Iron granules visible w/ Wright’s stain

Resembles basophilic stippling
= PB: Periphery
= BS: Homogeneous
Unused iron deposits
Sideroblastic anemia
MDS
Malaria MOT: Anopheles mosquito bite
Maturation stages: Rings > Trophozoite > Schizonts > Gametocytes
P. vivax = Worldwide
P. falciparum = Philippines
Resistant to Malaria:
= Fy(a-b-): African
= Sickle cell anemia
= G6PD deficiency

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Babesia MOT: Tick bite
“Maltese cross”
Resemble P. falciparum rings
Agglutination Cold agglutinin disease (anti-I)
Primary atypical pneumonia (anti-I)

Rouleaux RBCs in stack of coins arrangement

plasma globulin
Multiple myeloma: proliferation of Ig-producing plasma cells (ESR)
Macroglobulinemia
Drop of NSS Disperses rouleaux formation
True agglutination remains intact

Hemoglobinopathies
Hemoglobinopathies Qualitative defect in Hgb
Ex. Substitution

HbS α2β26 Glu Val

HbC α2β26 Glu Lys

HbE α2β226 Glu Lys

Sickle cell anemia 80/90-100% HbS

HbF & HbA2

Sickle cell trait 55-60% HbA1 |40-45% HbS

Thalassemia

Thalassemia Quantitative defect in Hgb

α-thalassemia Alpha-globin chain is or absent
a. Adult = β4 = HbH (deletion of 3/4 alpha genes)
b. Neonate = γ4 = Hb Bart’s (deletion of 4/4 alpha genes)

β-thalassemia Beta-globin chain is or absent

HbF and HbA2
Cooley’s anemia Thalassemia major
Homozygous β-thalassemia

Cooley’s trait Thalassemia minor

Heterozygous β-thalassemia
Cellulose acetate Hgb Alkaline pH: 8.6
electrophoresis Migration: (Cathode > Anode)
C > S > F > A1 > Barts > I > H
E D
O G
A2 Lepore
Normal: HbA1 is the fastest (most anodal)
Abnormal: HbH is the fastest (most anodal)

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Citrate agar Hgb Acid pH: 6.0-6.3
electrophoresis Migration: (Cathode > Anode)
F > A |Origin| O > S > C
E D
G
Screening test for HbS 1. Sodium metabisulfite = (+) Sickling of cells
2. Solubility test
= Sodium thiosulfite
= (+) Turbidity

HbA2 in β-thalassemia
Quantitation: Anion exchange microchromatography
HbF Alkali resistant
(+) HiCN
Tests:
1. Alkali denaturation test
= HbF resists alkali denaturation
a. Betke (NaOH)
b. Singer (KOH)
2. Acid elution test
= HbF resists acid-elution
= Cells w/ HbF = deep pink color
= Cells w/ N-HbF = ghost cells

Tests for unstable Hgb 1. Heat precipitation test: Δ50’C for 2 hrs
(HbH) 2. Isopropanol precipitation test: 17% solution
Sample Criteria for Erythrocyte Morphology Evaluation

Morphology w/in Normal 1+ 2+ 3+ 4+

Characteristics Limits (OIO) (per OIO) (per OIO) (per OIO) (per OIO)
Macrocytes (>9 µm) 0-5 5-10 10-20 20-50 >50

Microcytes (<9 µm) 0-5 5-10 10-20 20-50 >50

Hypochromia 0-2 3-10 10-50 50-75 >75

Poikilocytosis 0-2 3-10 10-20 20-50 >50

Polychromatophilia -- 1-5 6-10 >10 --

Rouleaux -- Agg. of 5-10 Numerous --

Agg. of 3-4 RBCs
RBCs aggregates

Nuclear Abnormalities

Pelger-Huet Hyposegmentation (neutrophil)

Bilobed nucleus: Dumb-bell shaped/spectacle/peanut-shaped/”Pince-
nez”
Resembles Stab cell (To differentiate: PH cell has more clumped
chromatin)
Pelger-Huet anomaly = Autosomal Dominant
Pseudo-Pelger-Huet = Acquired in myeloproliferative disorders

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Hypersegmentation ≥ 6 lobes (neutrophil)
Abnormal DNA synthesis
Undritz anomaly = hereditary hypersegmentation
Megaloblastic anemia
Cytoplasmic Abnormalities

Alder-Reilly granules Large purple-black coarse cytoplasmic granules

Accumulation of degraded mucopolysaccharides (all leukocytes)
Alder-Reilly anomaly = Autosomal Recessive
Mucopolysaccharidoses: Hurler, Hunter, Sanfilippo syndrome
Resemble toxic granules (IT)

Toxic granules Large purple to black granules resembling ALR granules

Infections
Toxic states

Toxic vacuoles Infections

Toxic states
Auer rods Pink or red rod shaped structures
Fused primary granules (peroxidase positive)
Myeloid and monocytic series only

Faggot cells w/ mass of Auer rods

M3 (APL) = associated w/ DIC
Chediak-Higashi granules Giant red, blue to grayish round inclusions (large lysosomal granules)
Seen in lymphocyte, neutrophil and monocyte
Lysosomal defects
Platelets lack dense granules
Chediak-Higashi syndrome = Autosomal Recessive (Albinism)

May-Hegglin inclusion Pale blue inclusions derived from RNA

May-Hegglin anomaly
= Autosomal Recessive
= Giant platelets
= Thrombocytopenia
Resemble Dohle bodies (IT)
Dohle bodies Single or multiple blue inclusions
Dohle-Amato bodies Aggregates of free ribosomes of rough ER
Resembles
Infections
Toxic states
IT: Infections, Toxic states Dohle bodies
Toxic granules
Toxic vacuoles

Abnormalities in Function

Page | !18
Job’s syndrome Normal random activity
Abnormal chemotactic activity
Lazy leukocyte syndrome Abnormal random and chemotactic activity

Chronic Granulomatous Inability of phagocytes to kill ingested microorganisms

Disease (CGD) Impaired NADPH oxidase
Impaired oxidative metabolism/respiratory burst
Test: NBT dye test
Cells Exhibiting Phagocytosis

LE cell Neutrophil w/ large purple homogeneous round inclusion

Believe to be a neutrophil that ingested another neutrophil
Buffy coat
Smooth and evenly stained
SLE
Tart cell Monocyte w/ ingested lymphocyte
Rough and unevenly stained

Abnormalities Involving Lymphocytes

Reactive lymphocyte a. Type I
Atypical lymphocyte = Turk’s irritation cell
Stimulated lymphocyte = Plasmacytoid lymphocyte w/ large block of chromatin
Variant lymphocyte b. Type II
Downey cell = Infectious mononucleosis: caused by EBV (target: B cells [CD21])
= Atypical lymphocyte in IM: T cells reacting to B cells infected w/ EBV
c. Type III
= Vacuolated
= Swiss cheese/moth eaten appearance

Basket cell Lymphocyte w/ thumbprint appearance

Smudge cell Due to pressure in smear preparation
Automated cell count
Remedy: Add bovine albumin
CLL

Hairy cells B cells w/ hair-like projection

Hairy cell leukemia = TRAP (+)
Sezary cells w/ cerebriform nucleus (“brain-like”)
Sezary syndrome
Mycosis fungoides

Abnormalities Involving Monocytes/Macrophages/Histiocytes

(Lipidoses/Lipid Storage Diseases)
Gaucher’s disease Accumulation of glucocerebroside
(-) glucocerebrosidase/β-glucosidase
Wrinkled/crumpled cytoplasm (Chicken scratch)

Page | !19
Niemann-Pick disease Accumulation of sphingomyelin
(-) sphingomyelinase
Foamy cytoplasm
Foam cells w/ sphingomyelin
Tay Sach’s disease Accumulation of glycolipid and ganglioside
(-) Hexosaminidase A
Vacuolated cytoplasm

Sandhoff’s disease Accumulation of glycolipid and ganglioside

(-) Hexosaminidase A & B
Vacuolated cytoplasm
Sea blue histiocytosis Unknown enzyme deficiency
Blue-green cytoplasm

Abnormalities Associated w/ Plasma Cells

Flame cell Plasma cell w/ red to pink cytoplasm
Multiple myeloma of IgA origin

Grape cell Plasma cell w/ vacuoles

Mott cell Accumulation of Russell bodies
Morula cell Multiple myeloma
Berry cell
Russell bodies Individual globules of immunoglobulin

Dutcher’s bodies Intranuclear protein inclusions

Platelet Abnormalities (Morphologic)

Giant platelet Bernard-Soulier syndrome

May-Hegglin anomaly
Small/ Myelodysplastic syndromes
micromegakaryocyte
Large megakaryocyte
Mononuclear
megakaryocyte
Vacuolated
megakaryocyte

Leukemia
Leukemia Abnormal, uncontrolled proliferation and accumulation of one or more of
the hematopoietic cells
Symptoms: Fever, weight loss, sweating; hepatosplenomegaly, enlarged
lymph nodes (chronic leukemia)
BMR

Acute leukemia Days to 6 months

Predominantly immature cells (blasts and “pro” stages)
Subacute leukemia 2 to 6 months

Page | !20
Chronic leukemia Variable
Minimum of 1 or 2 years
Predominantly mature cells
Leukemic leukemia WBC >15,000/µL

Subleukemic leukemia WBC <15,000/µL

(+) Abnormal and immature cells in PB
Aleukemic leukemia WBC <15,000/µL
(-) Abnormal and immature cells in PB

French-American-British Divides acute leukemias into lymphoblastic and monoblastic

(FAB) Classification of = Subdivided according to cellular morphology, cytochemical staining
Acute Leukemias results, cytogenetic studies and T & B lymphocytes marker results
Acute leukemia Normocytic, normochromic RBCs
FAB = ≥30% blasts
Henry: WHO (Now the standard for diagnosis) = ≥20%

Acute leukemia in 80% ALL

children 20% ANLL
Tests to differentiate ALL 1. MPO: Myeloperoxidase
from ANLL (AML) = (+) AML
= (-) ALL
2. SBB: Sudan Black B
= (+) AML
= (-) ALL
3. TdT: Terminal Deoxyribonucleotidyltransferase
= Marker for immature lymphocyte
= (+) ALL
= (-) ANLL

Acute Lymphoblastic Leukemias (ALL)

L1 Lymphoblasts are small and homogeneous (vary little in size)
Childhood ALL

L2 Lymphoblasts are large and heterogeneous (vary in size)

Adult ALL
L3 Burkitt-type
Rare
Lymphoblasts are large but homogeneous, and vacuolated

Acute Nonlymphocytic Leukemias (ANLL)

M1 Acute myeloblastic leukemia w/o maturation (AML w/o mat)
BM:
>30% blasts
<10% granulocytic cells

Page | !21
M2 Acute myeloblastic leukemia w/ maturation (AML w/ mat)
BM:
>30% blasts
>10% granulocytic cells
M3 Acute promyelocytic leukemia (APL)
>30% blasts
>10% granulocytic cells
>30% or >50% promyelocytes
(+) Faggot cells = Associated w/ DIC

M4 Acute myelomonocytic leukemia (AMML)

Naegeli’s leukemia
20% to <80% monocytic cells
M5a Acute monoblastic leukemia w/o maturation
Schilling’s leukemia
>80% monocytic cells (>80% monoblasts)

M5b Acute monoblastic leukemia w/ maturation

>80% monocytic cells (<80% monoblasts)
M6 Erythroleukemia
Erythremic myelosis
Di Guglielmo’s syndrome
>30% blasts
>50% erythrocytic precursors

M7 Acute megakaryocytic leukemia

>30% blasts
>30% megakaryocytic cells
Chronic Myeloproliferative Disorders

MPD Proliferation of abnormal pluripotential stem cell

Stem cell differentiates into the granulocytic (myeloid stem cell),
megakaryocytic and erythroid cell lines
1. Chronic Myelogenous (+) Philadelphia chromosome: t(9+;22-) - both long arms
Leukemia (CML) If (-) Ph’ chromosome = poor prognosis
Similar to leukomoid reaction, to differentiate:
a. Chromosome studies
b. LAP = ( in Leukomoid reaction, in CML)

2. Myelofibrosis w/ Fibrosis and granulocytic hyperplasia of BM, w/ granulocytic and

myeloid metaplasia (MMM) megakaryocytic proliferation in the liver and spleen (extramedullary)
(+) Dacryocytes
LAP
BM aspirate = impossible (dry tap)
BM biopsy = appropriate
3. Essential Thrombocytosis: 1,000 x 109/L
Thrombocythemia (ET) Functionally abnormal platelets

Page | !22
4. Polycythemia Vera (PV) BM: Panmyelosis
PB: Pancytosis/Pancythemia
RBCs, WBCs, Plts
LAP (Other polycythemia: N-LAP)
Polycythemia

1’ Absolute polycythemia Other names: Polycythemia Vera, Polycythemia Rubravera, Vaquez Osler
disease, Panmyelosis
RBC mass ( Hct)
RBCs, WBCs, Platelets
Erythropoietin (EPO)
2’ Absolute polycythemia In response to hypoxia
w/ appropriate In patients w/ pulmonary/cardiac disease
production of EPO RBCs, WBCs, Platelets
EPO

2’ Absolute polycythemia In patients w/ tumors of kidney, liver, brain, adrenal and pituitary gland
w/ inappropriate RBCs, N-WBCs, N-Platelets
production of EPO EPO
Relative polycythemia Spurious/Gaisböck polycythemia
Associated w/ stress and anxiety
N-RBC mass
Hct because of decreased plasma volume

RBC mass Differentiate absolute from relative polycythemia

RBC mass = Absolute polycythemia
N-RBC mass = Relative polycythemia
Myelodysplastic Syndrome/Dysmyelopoietic Syndrome

MDS Clonal abnormalities in hematopoietic cells

“Pre-leukemia”: can progress to ANLL if not treated
<30% blast
Differentiation % Blasts in PB % Blasts in BM Comments

Refractory anemia Mildest type

<1% <5%
(RA)
RA w/ ringed sideroblast <5% RS: Ringed sideroblast
<1%
(RARS) >15% RS

RA w/ excess blast
<5% 5-20%
(RAEB)
RAEB in transformation
>5% 20-30%
(RAEBt)

Chronic Myelomonocytic Persistent monocytosis

<5% 5-20%
Leukemia (CMML)
Lymphoproliferative disorders

LPD Proliferation of cells derived from lymphoid stem cell T/B cells
Page | !23
1. T/B cell leukemia --
2. Lymphoma Malignancy involving lymphoid tissue
a. Non-Hodgkin’s lymphoma
= proliferation of neoplastic lymphocytes
= Rappaport classification
b. Hodgkin’s lymphoma
= proliferation of cells reacting to neoplasm
= (+) Reed-Sternberg cell: large cell w/ large nucleoli (Owl’s eye)
Diagnosis: Lymph node biopsy
= Rye classification: based on histologic appearance of lymph node biopsy
= Ann-Arbor: staging based on the extent of tissue involved

3. Hairy cell leukemia Leukemic reticuloendotheliosis

Originally B cells w/ hairlike projections
(+) TRAP: Tartrate-resistant acid phosphatase

4. Mycosis fungoides and Neoplastic T cells

Sezary syndrome Sezary cells: w/ cerebriform nucleus (Brainlike)

Special Stains
Myeloperoxidase (MPO) Marker for primary granules and Auer rods
(+) AML
(-) ALL
Stain should only be done on fresh specimens

Sudan Black B (SBB) Marker for phospholipids and lipids

(+) AML
(-) ALL
Can be done on stored specimens
Parallels MPO for interpretation
Terminal DNA polymerase immunoperoxidase
deoxyribonucleotidyl Marker for immature lymphocytes
Transferase (TdT) (+) ALL
(-) AML

Periodic Acid-Schiff (PAS) Marker for glycogen, glycoproteins, mucoproteins and HMW CHO
a. Normal: all blood cells are PAS (+) except erythroblast
b. Abnormal: erythroblasts are PAS (+) = M6
(+) L1 and L2 = blocklike positivity
(-) L3
(+) M6
Naphthol AS-D Specific esterase
Chloroacetate esterase Marker for mature and immature neutrophils and mast cells
(+) AML
(-) AMoL [M5]
(-) ALL
Lasts for months

Page | !24
α-Naphthyl Acetate Nonspecific esterase
esterase Marker for monocytes, megakaryocytes and plasma cells
(+) AMoL [M5] = inhibited by fluoride
(+) AMegL [M7]
(-) AML
α-Naphthyl Butyrate Nonspecific esterase
esterase Identifies monocytes, promonocytes and monoblasts
(+) AMoL [M5] = inhibited by fluoride
(-) AML

Acid phosphatase Present in all hematopoietic cells and found in lysosomes

(+) Hairy cell leukemia = TRAP (+)
(+) T cell leukemia
(-) Non-T cell leukemia
Cannot be stored
Tartrate resistant acid Excellent marker for hairy cell leukemia
phosphatase (TRAP)

Leukocyte alkaline Neutrophil is the only leukocyte that contains this activity
phosphatase (LAP)/ Found in 3’ granules (Metamyelocytes)
Neutrophil ALP (NAP) Leukomoid reaction, MMM, PV, 3rd trimester of pregnancy
CML, CGL, PNH
Should be done on fresh specimen
Kaplow count:
= Count 100 neutrophils
= Grade 0 to 4+
NV = 30-185
LAP Score:
0 = No stain
1+ = Faint stain
2+ = Pale stain
3+ = Strong stain
4+ = Deep (Very intense) stain
Toluidine blue Binds w/ acid mucopolysaccharides in blood cells
Strongly metachromatic
Useful for recognition of mast cells and tissue basophils (metachromatic
granules)
CGL: Chronic granulocytic leukemia

Cooper & Cruickshank Stain for basophils

Reagent: Toluidine blue
Nitroblue tetrazolium test Test for CGD
(Original) NBT: Colorless to pale yellow ---(Toxic O2 molecules)---> (+) Blue formazan
Test:
Heparinized blood Centrifuge Buffy coat (WBC) + NBT (+) Formazan
NV = ≤10% formazan (+)
CGD = 0 to negligible
Infection = 70%

Page | !25
Modified NBT w/ stimulating agent
Test:
Buffy coat (WBC) + Bacterial suspension ---(NBT)---> (+) Formazan
NV = 80-100% formazan
CGD = <50% formazan
Feulgen reaction Specific reaction for DNA
Howell-Jolly bodies = Feulgen (+)

Prussian blue stain Stain for hemosiderin

(+) Sideroblast
(+) Siderocytes
Supravital stain (RHH) RHH = NCB
Reticulum = New methylene blue
Heinz bodies = Crystal violet
Hemoglobin H = Brilliant cresyl blue

BCB Stain for automated cell counter

Page | !26
 HEMOSTASIS
Primary hemostasis Involves blood vessels and platelets
Formation of platelet plug
Test: Bleeding time

Platelets Functions:
Adhesion
Activation
Release
Aggregation
Secondary hemostasis Involves the coagulation factors
Formation of stable fibrin clot
Test: Clotting time

Arteries Carry blood from the heart to the capillaries

Primary Hemostasis

Veins Return blood from the capillaries to the heart

Capillaries Injured vessel: vasoconstriction
= initiated by serotonin and thromboxane A2 derived from platelets and
endothelial cells

Maturation Stage Cytoplasmic Cytoplasmic Nuclear Thrombocytes

Granules Tags Features Visible
Megakaryoblast (-) (+) Single nucleus (-)
Fine chromatin
(+) Nucleoli

Promegakaryocytes Few (+) 2 nucleus (-)

Megakaryocytes Numerous Usually (-) 2 or more nuclei (-)

Metamegakaryocytes Aggregated (-) 4 or more nuclei (+)

Page | !27
Platelet structure 60% proteins
30% lipids
8% carbohydrate
Various minerals, water, nucleotides
1. Peripheral zone = responsible for adhesion and aggregation
a. Glycocalyx = outer surface
b. Plasma membrane = consists of 30 or more glycoprotein
c. Submembranous area
2. Sol-gel zone = platelet shape & contractile elements
a. Microfilaments: actin & myosin (actomyosin/thrombostenin)
= responsible for clot retraction
b. Microtubules = consists of tubulin: maintain the platelet shape
3. Organelle zone
= alpha & dense granules
= mitochondria, lysosomal granules
4. Membranous system
a. Dense tubular system = site of arachidonic acid metabolism
b. Open canalicular system (surface connecting system) = release of
granules
Platelet Adhesion Platelet adherence to exposed subendothelial surface (collagen)
Occurs in the presence of von Willebrand factor
In vivo: collagen
In vitro: glass

Bernard-Soulier syndrome (-) gpIb = receptor for vWF

(Giant platelet syndrome)
Von Willebrand disease (-) vWF = for platelet adhesion

Platelet Activation Morphologic and functional changes in platelets

Agonists: substance that initiate activation
Arachidonic acid ---(Cyclooxygenase)---> Thromboxane A2
TxA2 Vasoconstrictor
Stimulate platelet secretion

Aspirin Inhibits COX

bleeding time

Page | !28
Platelet Secretion Release of granules
a. Alpha granules
= Platelet factor
= Platelet derived growth factor
= Fibrinogen
= Factor V
= vWF
= β-thromboglobulin
= Thrombospondin
= Fibronectin
= Albumin
b. Dense granules (CAPAS)
= Calcium
= ADP
= Pyrophosphate
= ATP
= Serotonin
Release disorders Storage pool defects:
a. Alpha-granule deficiency
Gray platelet syndrome
Quebec platelet disorder = (-) Factor V binding protein (multimerin)
b. Dense granule deficiencies
Hermansky-Pudlak
Chediak-Higashi
Wiskott-Aldrich syndrome (α & dense granule deficiency)

Important Substances Secreted by Platelets & Their Role in Hemostasis

Promote coagulation HMWK (α)
Fibrinogen (α)
Factor V (α)
Factor VIII:vWF (α)

Promote aggregation ADP (d)

Calcium (d)
Platelet factor 4 (α)
Thrombospondin (α)
Promote vasoconstriction Serotonin (d)
Thromboxane A2 precursors (mb.PL)

Promote vascular repair Platelet-derived growth factor (α) = promotes smooth muscle growth
β-thromboglobulin (α) = chemotactic for fibroblasts
Other systems affected Plasminogen (α)
α2-antiplasmin (α)
C1 esterase inhibitor (α)

Platelet aggregation Platelet attachment to each other

Requires fibrinogen and Ca2+

Page | !29
Glanzmann’s (-) gpIIb/IIIa complex: receptor for fibrinogen
thrombasthenia
Petechiae Pinpoint hemorrhagic spots

Purpura Hemorrhage of blood into small areas of skin

Ecchymosis Blood escapes into large areas of skin

Epistaxis Nosebleed
Hemarthrosis Leakage of blood into joint cavities

Hematemesis Vomiting of blood

Hematoma Swelling or tumor in the tissues or a body cavity that contains clotted
blood

Hematuria RBC in urine

Hemoglobinuria Hgb in urine

Melena Stool containing dark red or black blood

Menorrhagia Excessive menstrual bleeding

Vascular Disorders
Hereditary hemorrhagic Most common inherited vascular disorder
telangiectasia Blood vessels are thin & lack smooth muscle
(Oslwer-Weber-Rendu
disease)

Congenital hemangiomata Tumor composed of blood vessels

(Kasabach-Merritt
syndrome)
Ehler-Danlos syndrome vascular fragility
Marfan’s syndrome

Pseudoxanthoma Elastic fibers are calcified & structurally abnormal

elasticum
Senile purpura Degradation of collagen & elastin

Scurvy (-) Vitamin C = for collagen synthesis

Defective synthesis of collagen
Henoch-Schonlein purpura Immunologic damage to endothelial cells

Quantitative Platelet Disorders

Thrombocytopenia Platelet production of BM = aplastic anemia
Survival time = platelet destruction (DIC, ITP)
Platelet sequestration by the spleen = splenomegaly
Dilution of platelet count = Thrombocytopenia α # of units transfused
Units transfused = Thrombocytopenia
Multiple transfusion: stored blood contains nonviable platelets

Page | !30
Thrombocytosis Reactive = moderate increase, asymptomatic (after hemorrhage,
splenectomy)
Autonomous = marked increase, associated w/ thrombotic/hemorrhagic
complications (Ex. ET: platelet function is abnormal)
Qualitative Platelet Disorders

Platelet adhesion 1. vWD = (-) vWF

Platelet aggregation test:
= Normal: Epinephrine, Collagen, ADP
= Abnormal: Ristocetin
2. BSS = (-) gpIb
(+) Giant platelets
Platelet aggregation test:
= Normal: Ristocetin
= Abnormal: Epinephrine, Collagen, ADP
3. Storage pool defects
= Gray platelet (α), HP, WAS, CHS (d)

Acquired Uremia: toxic metabolites

Paraproteinemias: coating of platelet membrane w/ abnormal protein
AML: abnormal megakaryocytes
MPD
Drugs: Aspirin = inhibits COX

Laboratory Tests for Primary Hemostasis

Page | !31
Platelet count 1. Direct (Neubauer counting chamber)
Plt. count/mm3 = # plt x AF x DCF x DF = # plt x 1 x 10 x 200 = # plt x
2,000
(RBC pipette = 1:200 dilution)
1 platelet uncounted = -2,000 plt/mm3
a. Reese-Ecker
- Sodium citrate
- Formaldehyde
- Brilliant cresyl blue
b. Guy and Leake
- Sodium oxalate
- Formaldehyde
- Crystal violet
c. Brecker-Cronkite
- 1% ammonium oxalate (phase contrast microscopy)
-------------------------------------------------------------------------------------------
-----------
d. Unopette
2. Indirect (smear)
Platelet count = (# of plts ÷ 1000 RBC) x RBC count
a. Dameshek
b. Fonio’s
c. Olef’s
Unopette Diluent: 1% Ammonium oxalate
Stand moist chamber for 15-20mins to allow platelets to settle
1.98mL diluent
0.02mL blood
TV = 2mL
Dilution = 1:100
Plt. count/mm3 = # plt x ACF x DCF x DF = # plt x 1 x 10 x 100 = # plt x
1,000
1 platelet uncounted = -1,000 plt/mm3

Platelet estimate 8-20 platelets/OIO

(Wedge smear) Factor: 20,000
WBC estimate (HPF) Factor: 2,000

Platelet Estimates (Smear)

Platelet Estimate of Report Platelet Estimate as

0-49,000/µL Markedly decreased

50,000-100,000/µL Moderately decreased

100,000-150,000/µL Slightly decreased

150,000-199,000/µL Low normal

200,000-400,000/µL Normal
401,000-599,000/µL Slightly increased

Page | !32
600,000-799,000/µL Moderately increased
>800,000µL Markedly increased

N-Plt count, BT Qualitative platelet abnormality

Primary vascular abnormality
vWD
Plt count, N-BT Autoimmune thrombocytopenia

Plt count, BT Simultaneous quantitative and qualitative platelet deficiency

Platelet aggregation test Aggregating agents:
a. Epinephrine
b. Collagen
c. ADP
d. Ristocetin
Sample: Citrated blood Centrifuge at 60-100g for 10mins
PRP + Agonist O.D. monitored (Aggregometer)

Platelet adhesiveness Determination of in vitro platelet adhesion

(Salzmann) Plt ct. 1 = routine collection method
Plt ct. 2 = collected through glass bead collecting system
% PA = [(PC1-PC2) ÷ PC1] x 100
NV = 26-60%
in vWD
Clot retraction time Proportional to platelet count
Clot retraction: function of thrombosthenin
a. Castor oil/Hirschboeck
(+) Dimpling/droplet like serum on the surface of blood drop
NV = 15-45mins
b. Stefanini
c. MacFarlane
CRT = (vol. serum ÷ TV) x 100
NV = 44-67%

Capillary resistance test Measures capillaries to resist pressure

Touriquet test Correlates w/ the degree of thrombocytopenia
Rumpel-Leedes test 100 mmHg for 5 mins After 15-30mins, count petechiae
(or halfway bet. systolic & diastolic pressure for 5 mins)
+1 = 0-10 petechiae (few)
+2 = 10-20 petechiae (many)
+3 = 20-50 petechiae (multiple)
+4 = >50 petechiae (confluent)
NV = 0 to occasional
Not repeated on the same arm for 7 days
Bleeding time In vivo measure of primary hemostasis
a. Duke method = fingertip/earlobe
b. Ivy method = uses blood pressure cuff (40mmHg) puncture forearm

Secondary Hemostasis

Page | !33
Coagulation factors All are produced in the liver except Factor VIII
In liver disease, all coagulation factors are except factor VIII complex
Factor VIII complex:
a. VIII: Coagulant (VIII:C)
b. vWF: produced by megakaryocytes and endothelial cells
Activated at cold temp. = VII & XI
Coagulation Factors

Numeral Preferred Name Synonyms

I Fibrinogen

II Prothrombin Prethrombin
III Tissue factor Tissue thromboplastin

IV Calcium
V Proaccelerin Labile factor
Accelerator globulin (aCg)

VII Proconvertin Stable factor

Serum prothrombin conversion
accelerator (SPCA)
VIII:C Antihemophilic factor Antihemophilic factor globulin
(AHG)
Antihemophilic factor A
Platelet cofactor 1

IX Plasma thromboplastin component Christmas factor

Antihemophilic factor B
Platelet cofactor 2
X Stuart-Prower factor Stuart factor
Prower factor
Autoprothrombin III

XI Plasma thromboplastin component Antihemophilic factor C

XII Hageman factor Glass factor
Contact factor

XIII Fibrin stabilizing factor Laki-Lorand factor

Fibrinase
Plasma transglutaminase
Fibrinoligase
- Prekallikrein Fletcher factor

- High-molecular weight kininogen Fitzgerald factor

Contact activation cofactor
Williams factor
Flaujeac factor

Page | !34
Stage I: Generation of Intrinsic: XII XI IX—VIII (Cofactor) Extrinsic: III VII Common: X—
Thromboplastin V (Cofactor) ----> Common thromboplastin/Prothrombinase
(Va-Xa-Ca 2+ -PL)

Stage II: Conversion of II ---(Prothrombinase)---> Thrombin

prothrombin to thrombin

Stage III: Conversion of I ---(Thrombin)---> Fibrin clot

fibrinogen to fibrin clot XIII---(Thrombin)---> XIIIa
Fibrin clot ---(XIIIa)---> Stable fibrin clot
Fibrinogen Most concentrated

Calcium Involved in all stages of coagulation except contact phase

Contact group XII, XI, PK, HMWK
Ca2+ independent
Vit. K independent
Involved in the contact phase
XII ---(Collagen)---> XIIa (small amount)
PK ---(XIIa)--------> Kallikrein
XII ---(Kallikrein+HMWK)---> XIIa (large amount)
XI ---(XIIa)---------> XIa

Fibrinogen group I, V, VIII, XIII

Ca2+ dependent
Vit. K independent
Completely consumed during coagulation
(+) in plasma
(-) in serum
Prothrombin group II, VII, IX, X
Ca2+ and Vit. K dependent
First: VII IX X II: Last
Adsorbable factors: removed by adsorbing agents [BaSO4, Al(OH)3]
(+) in plasma
(-) in serum

Diseases BT PT APTT Stypven TT Duckert’s

Disease of 1’ hemostasis N N N N N

Fibrinogen deficiency N* N
Prothrombin deficiency N N N

Parahemophilia N N N
Factor VII deficiency N N N N N

Hemophilia A N N N N N
von Willebrand disease N N N N

Hemophilia B N N N N N

Page | !35
Factor X deficiency N N N
Hemophilia C N N N N N

Factor XII deficiency N N N N N

Factor XIII deficiency N N N N N Abn

DIC Abn
*BT may be prolonged in afibrinogenemia

Fresh Plasma Aged Plasma Adsorbed Plasma Fresh Serum Aged Serum
I + + + - -

II + + - + (<20%) -
V + - + - -

VII + + - + +
VIII + - + - -

IX + + - + +
X + + - + +

XI + + + + +
XII + + + + +

XIII + + + - -
Prothrombin:
80% is consumed during coagulation
<20% residual prothrombin

Disorders of Coagulation Causing Clotting Factor Deficiencies

Inherited Coagulopathies Acquired Coagulopathy
Factor
Inheritance Pattern Coagulopathy
I Autosomal recessive Afibrinogenemia Liver disease
Autosomal dominant Dysfibrinogenemia DIC
Fibrinolysis

II Autosomal recessive Prothrombin deficiency Liver disease

Vit. K deficiency
Anticoagulant therapy
V Autosomal recessive Owren’s disease Liver disease
Labile factor deficiency DIC
Parahemophilia Fibrinolysis

VII Autosomal recessive Factor VII deficiency Liver disease

Vit. K deficiency
Anticoagulant therapy

Page | !36
 VIII X-linked recessive Hemophilia A DIC
Autosomal dominant vWD Fibrinolysis
IX Autosomal recessive Hemophilia B Liver disease
Christmas disease Vit. K deficiency
Anticoagulant therapy

X Autosomal recessive Factor X deficiency Liver disease

Vit. K deficiency
Anticoagulant therapy
XI Autosomal recessive Hemophilia C Unclear
Rosenthal syndrome
=Common in Jewish
descent/ Ashkenazi Jews

XII Autosomal recessive Factor XII deficiency Unclear

=No bleeding tendency
XIII Autosomal recessive Factor XIII deficiency Liver disease
DIC
Fibrinolysis

PK Autosomal recessive Fletcher trait Unclear

HMWK Autosomal recessive Fitzgerald trait Unclear

Factor VIII deficiency Common inherited coagulation factor deficiency

a. Hemophilia A/Classic hemophilia/Royal disease = Queen Victoria’s son
b. vWD = most frequently inherited coagulation disorder
Major sites of coagulation Endothelium
inhibition Platelet

Protein C Degrades factor Va and VIIIa

Protein S Vit. K dependent glycoprotein
Antithrombin Major inhibitor of thrombin

Tests for Secondary Hemostasis

Clotting time Measure the period required for free form of blood to clot after it has
been removed from the body
a. Capillary blood method
= Drop or slide
= Capillary tube/Dale and Laidlaw
= Blue capillary tube
NV = 2-4mins
b. Whole blood/Lee and White
NV = 7-15mins

Page | !37
Prothrombin time Detect coagulation factor deficiencies involving extrinsic and common
pathway
Citrated blood Centrifuge at 2000g for 10mins PPP
PPP + Thromboplastin-CaCl2 rgt (+) Clot
Begin timing for clot formation on the addition of CaCl2 rgt
NV = 10-12 secs
Activated partial Detect coagulation factor deficiencies involving intrinsic and common
thromboplastin time pathway
PPP + APTT rgt:
1. Activators:
a. Micronized silica
b. Ellagic acid
c. Celite
d. Kaolin
2. Phospholipids: substitute for platelets
3. CaCl2
Begin timing for clot formation on the addition of CaCl2 rgt
NV = 25-35 secs

Stypven time/Russel viper East Indian viper venom Vipera russelli: directly activates factor X
venom time Detect coagulation factor deficiencies involving common pathway
PPP + Stypven rgt: platelin-CaCl2 rgt
NV = 6-10 secs

Thrombin time Prolonged in:

a. Fibrinogen deficiency
b. Presence of FDP or FSP
c. Presence of thrombolytic agent (Ex. streptokinase)
d. Presence of heparin
PPP + Thrombin-CaCl2 rgt
NV = 10-14 secs

Reptilase time Enzyme found in the venom of Bothrops atrox snake

Prolonged in:
a. Fibrinogen deficiency
b. Presence of FDP or FSP
c. Presence of thrombolytic agent
Not affected by heparin
PPP + Atroxin
Begin timing for clot formation upon the addition of atroxin
NV = 10-15 secs
Duckert’s test For factor XIII deficiency
5M urea solubility test Rgt: 5M Urea
Substitutes for urea:
- 1% monochloroacetic acid
- 2% acetic acid
Normal = Clot is insoluble to urea (24 hrs)
F. XIII def. = Clot is soluble to urea (24 hrs)

Circulating anticoagulants Prolonged APTT and PT not corrected

Page | !38
Lupus inhibitor Against the phospholipid portion of prothrombinase complex (Va-Xa-Ca2+-
PL)
Instrumentation for Tests of Hemostasis

Tilt tube method Visual detection of fibrin clot formation

Manual technique
End point = clot formation
Fibrometer Electromechanical detection of fibrin clot formation
Fibrin strand formation is detected using a wire loop or hook w/c has
been incorporated into a semi-automated mechanical instrument

Photo-optical detection of Detection of fibrin clot formation depends on in light scattering

clot formation associated w/ conversion of fibrinogen fibrin
Semi-automated instruments:
- Electra 750 and 750A
- Fibrintimer series
- FP 910 coagulation analyzer
Automated instruments:
- Ortho Koagulab 16S and 40A
- Coag-A-Mate X2 and XC
- MLA Electra 700 and 800

Fibrinolysis Digestion of fibrin clot

Occurs when plasminogen plasmin
Plasminogen activators 1. Intrinsic activators
- XIIa
- Kallikrein
- HMWK
2. Tissue type
- Urokinase-like PA
3. Therapeutic activators = treatment for thromboemboli
- Streptokinase
- Urokinase
- Tissue-like PA

Inhibitors of fibrinolysis α2-antiplasmin = Major inhibitor

α2-macroglobulin
Thrombospondin
PA inhibitor 1 and 2
Degradation of cross- Crosslinked fibrin (urea insoluble)
linked fibrin by plasmin ↓
(Plasmin)
↓
Complex DD/E | Complex YD/DY | Complex YY/DXD
(D-dimer)

Page | !39
Degradation of non- Fibrinogen or
crosslinked fibrin and Noncrosslinked fibrin (urea soluble)
fibrinogen by plasmin ↓
(Plasmin)
↓
Fragment X
↓
(Plasmin)
↓
Fragment Y | Fragment D
↓
(Plasmin)
↓
Fragment D | Fragment D
Primary fibrinolysis No fibrin monomer
No fibrin polymer
No D-dimer
Plasminogen activators from damaged/malignant cells
Converts plasminogen plasmin in the absence of fibrin formation
Prostatic carcinoma

Secondary fibrinolysis DIC = uncontrolled, inappropriate formation of fibrin w/in the blood
vessels
w/ fibrin monomer
w/ fibrin polymer
w/ D-dimer = most specific for DIC
Infection: N. meningitidis
Neoplasm
Snake bite
HTR

Laboratory Evaluation of Fibrinolysis

Whole blood clot lysis Clot should remain intact for approximately 48 hrs at 37’C
time (WBCLT) Clot lysis prior to 48 hrs indicates excessive systemic fibrinolysis

Euglobulin lysis time Euglobulins: proteins that ppt. when plasma is diluted w/ H2O & acidified
- Plasminogen
- Plasmin
- Fibrinogen
- Plasminogen activators
Plasma + Acetic acid Ppt. euglobulin
Euglobulin + thrombin Clot euglobulin
Euglobulin Dissolved in buffer
Normal = No clot lysis (2 hrs)
Fibrinolysis = Clot lysis <2 hrs
Protamine sulfate test Detects the presence of fibrin monomers (2’-DIC) in the plasma
Plasma + Protamine sulfate + ETOH (+) gel-like clot (paracoagulation)

Page | !40
Ethanol gelation test Detects the presence of fibrin monomers (2’-DIC) in the plasma
Plasma + NaOH (pH) + ETOH (+) pptn/gel
Latex D-dimer assay Most specific test for DIC

Test Primary Fibrinolysis Secondary Fibrinolysis

WBCLT < 48 hrs < 48 hrs

Euglobulin clot lysis < 2 hrs < 2 hrs

Ethanol gelation - +

Protamine sulfate - +
D-dimer - +

Heparin Injected
Action: inhibits thrombin
Monitoring: APTT = sensitive, method of choice (CAP)
Neutralize w/ protamine sulfate
Warfarin Oral
Coumarin WARF: Wisconsin Alumni Research Foundation
Coumadin Action: Vit. K antagonist, inhibits II, VII, IX, X
Monitoring: PT (reported in INR)
Neutralize w/ Vitamin K, FFP

INR International normalized ratio

INR = (Patient PT ÷ Normal PT)ISI
INR = 2-3
- Prevents MI, embolism & thrombosis
INR = 2.5-3.5
- For patients w/ mechanical heart valves

ISI International Sensitivity Index (PT rgt)

PT rgt is calibrated w/ Manchester rgt = from human brain thromboplastin

Page | !41
 HEMATOLOGY PROCEDURES
Brightfield microscopy Examine blood films

Oil immersion microscopy Erythrocyte morphology & leukocyte differential

Phase-contrast microscopy For manual platelet counts

Fluorescence microscopy ANA and T/B cell studies

Electron microscopy Observation of fine ultrastructures of cells (100,000x magnification)

Basic component of Diopter rings: adjust for focusing differences between eyes
Standard light microscope Rubber eyeguard: adjust for comfort
Eyepiece tube clamp screw: loosen to rotate head
Reverse facing nosepiece: for ease in specimen manipulation
Revolving nosepiece: use to rotate objectives
Objectives: lenses w/c form primary image of specimen
Field diaphragm: aperture diaphragm w/c restricts area of illumination
Field diaphragm control ring: adjust size opening of field diaphragm
Coarse focus knob: brings slide into view
Fine focus knob: sharpens image
Lamp socket: holds light source
Interpupillary distance scale: indicates distance between eyes
Eyepiece: magnify image formed by objective lens
Stage: holds specimen
Slide holder: holds slide in place
Condenser control ring: adjusts size opening of condenser
Condenser: aperture diaphragm that controls light
Condenser centering screws: center the field of view
Condenser focus know: focuses light onto slide
X/Y travel knobs: moves slides on stage
Brightness control dial: turns microscope on/off, adjusts light intensity

Total magnification TM = Ocular x Objective

(10)(LPO: 10) = 100x
(10)(HPO: 40) = 400x
(10)(OIO: 100) = 1,000x

Hemoglobin Determination
Hemoglobin !AM, "PM
! Strenuous muscular activity
! Altitude = ! Hgb
" Lying down

Acid hematin Rgt: 0.1N HCl

Comparing the brownish yellow color of solution to standard comparator
block
Alkali hematin Rgt: 0.1N NaOH
Not for newborns (HbF: alkali resistant)

Gasometric (Van Slyke) 1g Hgb = 1.34mL O2

Page | !42
Chemical 1g Hgb = 3.47mg Fe2+
(Kennedy’s, Wong’s)
SG method CuSO4 method (See BB notes)

Cyanmethemoglobin/ Manual and automated

Hemiglobincyanide Dilution = 0.02mL blood: 5mL rgt (1:251)
method Reagents:
Drabkin’s reagent (Brown container)
a. Potassium ferricyanide = ferrous ferric
b. Potassium cyanide = Hi HiCN
c. Nonionic detergent = lysis of red cells, decreases turbidity
d. Sodium bicarbonate (Orig. Drabkin’s) = result is read after 15mins
e. Dihydrogen potassium phosphate (Mod. Drabkin’s) = result is read after
3 mins
*Color intensity is measured at 540nm
*All forms of Hgb are measured except SulfHb
*Overanticoagulation does not affect result
♫ Turbidity = False !
a. High WBC count: to correct, centrifuge read supernatant
b. HbS & HbC: to correct, dilute 1:1 w/ H2O result x 2
c. Lipemic blood: prepare patient’s blank (pt. plasma + HiCN rgt)

Rule of three 3 x RBC = Hgb

3 x Hgb = Hct
Apply only to normocytic, normochromic red cells
Hematocrit Determination

Macromethods Large volume of blood

1. Wintrobe & Landsberg = Double oxalate
2. Van Allen = Sodium oxalate
3. Haden = Sodium oxalate
4. Sanford-Magath = Sodium oxalate
5. Bray = Heparin
Centrifuge: 2000-2300g for 30mins
Layers (Spun Hct):
1st: Fatty layer = barely visible unless the patient is lipemic
2nd: Plasma
3rd: Buffy coat (1mm = 10,000 WBCs/mm3)
Bottom: Packed cells
Wintrobe tube Length = 11.5cm
Bore = 3.0mm
Calibration = 0-100
a. Left: 0-100 (ESR)
b. Right: 100-0 (Hct)

Page | !43
Micromethod (Adam) Capillary tube:
Length = 7.0-7.5cm (70-75mm)
Bore = 1mm
Anticoagulant: Red (Heparin)
No anticoagulant: Blue
Centrifuge: 10,000-15,000g for 5mins
Trapped plasma: plasma that remains in RBC portion after spinning
= ! in poikilocytosis
♫ Advantages:
a. Better packing of cells
b. Less blood
c. Less time consumed
♫ Layers:
Top: Plasma
2nd: Platelets
3rd: Leukocytes
4th: Retics & nRBCs
5th: Mature RBCs
Bottom: Clayseal (4-6mm)
Automated methods Hct = only computed
Hct = MCV x RBC count

RBC Count

RBCs !AM, "PM

RBC diluting fluids Isotonic solutions
1.) NSS
2.) 3.8% Sodium citrate
3.) Dacies or formol citrate
4.) Hayem’s
5.) Toisson’s
6.) Bethell’s
7.) Gower’s
Dilution (RBC pipette) = 0.5:100 (Blood: Diluent) = 1:200

RBC count RBC/mm3 = # RBC x AF x DCF x DF = # RBC x 5 x 10 x 200 = # RBC x 10,000

WBC Count

WBCs "AM, !PM

WBC Diluting fluids Hypotonic solutions: lyse non-nucleated RBCs
1.) 1-3% acetic acid
2.) 1% HCl
3.) Turk’s diluting fluid: Gentian violet + glacial acetic acid (solid at 17’C)
+ H2 O
Mix = 3 mins (To allow lysis of RBCs)
Dilution (WBC pipette) = 0.5:10 (Blood: Diluent) = 1:20
Leukocytosis = Use RBC pipette (1:100 or 1:200)

Page | !44
WBC count WBC/mm3 = # WBC x AF x DCF x DF
Counting Chamber

Fuch’s Rosenthal 2 counting areas

Each CA w/ 16 1mm2 squares
Depth = 0.2mm
Depth factor = 5
Volume = Area x Depth x # CA = 16mm2 x 0.2mm x 2 = 6.4mm3
Volume/counting chamber = 3.2mm3
Speir’s Levy 4 counting areas
Each CA w/ 10 1mm2 squares
Depth = 0.2mm
Depth factor = 5
Volume = Area x Depth x # CA = 10mm2 x 0.2mm x 4 = 8mm3
Volume/counting chamber = 2mm3

Improved Neubauer 2 primary squares

Each 1’ square w/ 9 1mm2 2’ squares
Depth = 0.1mm
Depth factor = 10
Volume = Area x Depth x # CA = 9mm2 x 0.1mm x 2 = 1.8mm3
Volume/counting chamber = 0.9mm3
RBC count Center square:
w/ 25 3’ square
Each 3’ square w/ 16 small squares
25 x 16 = 400
5 (counted) x 16 = 80 small squares

WBC count 4 corners:

Each 2’ square w/ 16 3’ squares
4 x 16 = 64 3’ squares
Nucleated RBCs Not lysed by WBC diluents
Falsely counted as WBCs

NV:
Adult = ≥5 nRBC/100 WBC differential
Newborn = ≥10 nRBC/100 WBC differential

Formula for WBC Corrected WBC = uncorrected WBCs x 100

correction 100 + NRBCs
Preparation and Staining Procedures for the Blood Smear

Techniques 1. Cover glass smear (Ehrlich)

2. Cover glass and slide (Beacom)
3. Wedge smear/Push/Spreader slide technique
4. Spun smear/Spun/Spinner’s technique = Automated: Hemaspinner
250 (30-400) Angle between 2 slides

22 x 22mm Square coverslip

Page | !45
2-3mm Drop of blood
0.25 inch (1cm) Distance of blood drop from the frosted edge of the slide

0.5 inch Smear terminates near the end of the slide (automated spreader)
Buffy coat smear For patients w/ WBC count of <1 x 109/L
Demonstration of LE cell

Thick blood smear For blood parasites

Methanol Fixative for blood and BM smears
Toxic, causes permanent blindness

Romanowsky’s stains Wright’s

Giemsa = preferred stain for blood parasites
Modified Wright’s-Giemsa
Leishman
Jenner
May-Grunwald
-------------------------------------------------------------------------------------------
-----------
Contains:
= Methylene blue (or Azure B - oxidized): basic
= Eosin: acidic
w/in 2-3 hours of Time blood smears should be stained
specimen collection

pH 6.8 Blood and bone marrow staining

pH 7.2 Malarial parasite staining

Excessively blue stain Thick films

Prolonged staining time
Inadequate washing
Too high alkalinity of stain
Diluents tends to cause excessive basophilia
Excessively pink stain Insufficient staining
Prolonged washing time
Mounting coverslips before they are dry
Too high acidity of the stain
Buffer may cause excessive acidophilia

Cross-sectional or Blood film is moved from side to side

crenellation method
Longitudinal method WBCs are counted from the tail toward the head of the smear

Battlement method Near the tail on a horizontal edge: count 3 consecutive horizontal edge
fields, count 2 fields towards the center of the smear, count 2 fields
horizontally, count 2 fields vertically to the edge

Page | !46
WBC Counting 100 cells = routine
50 cells = if patient WBC count <1.0 x 109/L
200 cells:
= >10% eosinophils
= >2% basophils
= >11% monocytes
= lymphocytes > neutrophils (except in children)
PV patients Thinner smear:
- smaller blood drop
- slow spread
- low angle

Anemic patients Thicker smear:

- larger blood drop
- fast spread
- increase angle
Neutrophils Relative = 47-77%
Absolute = 1.8-7.8 x 109/L

Lymphocytes Relative = 20-40%

Absolute = 1.0-4.8 x 109/L
Monocytes Relative = 2-10%
Absolute = 0.01-0.8 x 109/L

Eosinophils Relative = 0-6%

Absolute = 0-0.6 x 109/L
Basophils Relative = 0-1%
Absolute = 0-0.2 x 109/L

Neutrophilia Appendicitis
Myelogenous leukemia
Bacterial infections
Eosinophilia Allergies
Scarlet fever
Parasitic infections (T. spiralis)
Eosinophilic leukemia

Lymphocytosis Viral infections

Whooping cough
IM
Lymphocytic leukemia
Lymphoma

Page | !47
Monocytosis Brucellosis
Tuberculosis
Monocytic leukemia
SBE
Typhoid
Rickettsial infections
Collagen disease
Hodgkin’s disease
Gaucher’s disease
Shift to the left (+) immature granulocytic cells
Leukemia
Bacterial infections

Shift to the right Hypersegmented neutrophils (≥6 lobes)

Reticulocyte count NV:
Adult = 0.5-1.5% (Ave: 1.0%)
Newborn = 2-6%

% Retics [# Retics ÷ # RBC (1000)] x 100

Absolute reticulocyte ARC = (% Retics ÷ 100) x RBC count (1012/L) x 1,000

count NV = 25-75 x 109/L
Corrected reticulocyte CRC = % Retics x (Patient Hct ÷ Normal Hct [0.45L/L])
count NV = 1

Reticulocyte production General indicator of the rate of erythrocyte production increase above
index/Shift correction normal in anemias
Indicates BM response to anemia
RPI = CRC ÷ Maturation time of retics in the blood
NV = 1 (Hct: 45%)
Maturation time of retics 1.0 day = Hct: 45 ± 5%
in the blood 1.5 days = Hct: 35 ± 5%
2.0 days = Hct: 25 ± 5%
2.5 days = Hct: 15 ± 5%

RPI > 3 Adequate response of BM to anemia

- Chronic hemolysis
- Recent hemorrhage
- Response to therapy
RPI < 2 Inadequate response of BM to anemia
- Aplastic anemia
- Ineffective erythropoiesis (megaloblastic anemia)

Miller disk % Retics = Retics (A) ÷ [RBC (B) x 9] x 100

Eosinophil count NV = 50-350 x 106/L

Page | !48
Eosinophilia Allergic reactions
Parasitic infections
Brucellosis
Leukemias
Eosinopenia Hyperadrenalism (Cushing’s disease)
Shock
Administration of ACTH

Eosinophil diluting fluids Composition:

a. Phloxine/eosin/neutral red iodide = stains eosinophils
b. Propylene glycol = lyses RBCs
c. Na2CO3 = lyses WBCs except eosinophils, intensifies staining of granules
d. Heparin = prevents clumping
Diluting fluids:
1. Pilot’s = phloxine
2. Manner’s = phloxine
3. Randolph’s = phloxine
4. Hinkleman = eosin
5. Tannen’s = neutral red iodide
Thorn’s test Assess adrenocortical function
Sample 1: fasting specimen
Sample 2: 4 hrs after administration of ACTH ("eo. count)
Normal: Eo. count: 1 > 2 (lower by 50%)
Abnormal: Eo. count: 1 = 2 (hypoadrenalism)

Erythrocyte Indices (Wintrobe Indices)

RBC indices Classify anemia according to RBC morphology

MCV MCV = (Hct ÷ RBC) x 10

NV = 80-100 fL (old: µm3)
MCH MCH = (Hgb ÷ RBC) x 10
NV = 27-32 pg (old: µµg)
Rarely used

MCHC MCHC = (Hgb ÷ Hct) x 100

NV = 31-36% (31-36 g/dL)
Defective centrifuge Values affected:
= Hct
= MCV
= MCHC

MCHC >38% does not Incorrect calculation

occur (+) cold agglutinins
MCHC will not fall <22% Lipemia
(+) HbS & HbC

Erythrocyte Sedimentation Rate (ESR)

Page | !49
ESR Nonspecific measurement used to detect & monitor an inflammatory
response to tissue injury
! ESR Erythrocytes:
*Macrocytes
*Anemia
Plasma composition: most important determinant
*Fibrinogen
*α1-globulin
*α2-globulin
*β-globulin
*γ-globulin
*Cholesterol
Technical factor:
*Tilting = !30 angle = 30% error
*!Temp.
" ESR Erythrocytes:
*Microcytes
*Poikilocytes
*Polycythemia
*Anisocytes
Plasma factor: most important determinant
*Albumin
*Lecithin
Technical factor:
*Overanticoagulation = !EDTA = shrinkage of RBC = "Hct, "ESR

Stages of ESR 10 mins = 1. Initial rouleaux

40 mins = 2. Rapid settling of RBCs
10 mins = 3. Final sedimentation of RBCs
60 mins = Total
Wintrobe & Landsberg Requires smaller amount of blood
Involves no dilution
Length: 11.5cm (115mm)
Internal bore: 3.0mm
Anticoagulant: Double oxalate

Standard/Original Most sensitive

Westergren Requires more blood
Length: 300mm
Internal bore: 2.65 ± 0.15mm
Anticoagulant: Citrate (black)
Anticoagulant-to-Blood ratio = 1:4
Modified Westergren Anticoagulant: 2mL EDTA + 0.5mL NSS/Citrate

Zeta Sedimentation Ratio Not affected by anemia

(ZSR) Major disadvantage: requires special capillary tubes and Zetafuge
ZSR = (%Hct ÷ %Zetacrit) x 100

Page | !50
Erythrocyte Osmotic Anticoagulant: Heparin
Fragility test % NaCl = # drops NaCl x 0.02
(Griffin and Sanford Add RBCs, stand for 2hrs at room temp
method) Check for hemolysis (pink/red supernatant)
NV:
- Initial hemolysis = tube 21 or 22 (0.42-0.44%)
- Complete hemolysis = tube 16 or 17 (0.32-0.34%)
Ascorbate cyanide Detects deficiencies in the pentose phosphate pathway:
screening - G6PD
- glutathione peroxidase
- glutathione reductase
Rgts:
- Na ascorbate
- Na cyanide
Normal = red
(-) Enzyme = brown

G6PD fluorescent G6P + NADP ---(RBC: G6PD)---> 6-phosphogluconate + NADPH

screening (fluorescence)
Normal: Max fluorescence at 10mins
G6PD def: Little or no fluorescence
Paroxysmal nocturnal Acquired disorder in w/c red cells are abnormally sensitive to
hemoglobinuria (PNH) complement
(-) DAF

Sucrose hemolysis test Screening test for PNH

Patient RBCs + ABO compatible serum + sucrose solution
Normal = (-) Hemolysis
PNH = (+) Hemolysis
Ham’s acidified serum Confirmatory test for PNH
test Tube 1: Patient RBCs + normal serum + weak acid (0.2N HCl)
Tube 2: Patient RBCs + patient serum + weak acid (0.2N HCl)
Tube 3: Patient RBCs + normal inactivated serum + weak acid (0.2N HCl)
Normal = (-) Hemolysis on all tubes
PNH = (+) Hemolysis except on Tube 3 (inactivated serum)

Patient has received Patient w/ PNH + blood transfusion ---(Ham’s test)---> " Hemolysis
normal RBCs
PCH IgG autoanti-P = biphasic hemolysin
- Cold = attaches to RBCs
- Warm = RBC lysis

Donath-Landsteiner test Test for PCH

Ctrl: Patient WB incubate at 37’C for 30mins incubate at 37’C for 30mins
Test: Patient WB incubate at 4’C for 30mins incubate at 37’C for 30mins
Normal = (-) hemolysis on test and control
PCH = (-) hemolysis on control but (+) hemolysis on test sample

Page | !51
Autohemolysis test Blood alone ---(48 hrs)---> Hemolysis: >0.2 to 2%
Blood + glucose ---> Hemolysis: 0-0.8%
Blood + ADP ---> Hemolysis: 0-0.9%
-------------------------------------------------------------------------------------------
-----------
G6PD deficiency (PPP) = corrects w/ glucose only
PK deficiency (EMP) = corrects w/ ADP only
H. Spherocytosis = corrects w/ ADP and glucose
Potential Causes of Erroneous Results with Automated Cell Counters

Parameter Causes of Spurious Increase Causes of Spurious Decrease

WBC count Cryoglobulin Clotting
Cryofibrinogen Smudge cells
Heparin Uremia plus immunosuppressants
Monoclonal proteins
Nucleated RBCs
Platelet clumping
Unlysed RBCs

Platelet count Cryoglobulin Clotting

Cryofibrinogen Giant platelets
Hemolysis Heparin
Microcytic RBCs Platelet clumping
RBC inclusions Platelet satellitism
WBC fragments
RBC count Cryoglobulin Autoagglutination
Cryofibrinogen Clotting
Giant platelets Hemolysis
WBC >50,000/µL Microcytic RBCs

Hemoglobin HbCO >10% Clotting

Cryoglobulin Sulfhemoglobin
Cryofibrinogen
Hemolysis
Heparin
WBC >50,000/µL
Hyperbilirubinemia
Lipemia
Monoclonal proteins
Hematocrit (automated) Cryoglobulin Autoagglutination
Cryofibrinogen Clotting
Giant platelets Hemolysis
WBC >50,000/µL Microcytic RBCs
Hyperglycemia >600mg/dL

Hematocrit (microhct) Hyponatremia Excess EDTA

Plasma trapping Hemolysis
Hypernatremia

Page | !52
MCV Autoagglutination Cryoglobulin
WBC >50,000/µL Cryofibrinogen
Hyperglycemia Giant platelets
Reduced red cell deformability Hemolysis
Microcytic RBCs
Swollen RBCs
MCHC Autoagglutination WBC >50,000/µL
Clotting Spuriously low Hgb
Hemolysis Spuriously high Hct
Spuriously high Hgb
Spuriously low Hct

Cryoglobulin Increased: WBC count, RBC count, Platelet count, Hgb, Hct
Cryofibrinogen Decreased: MCV
Heparin Increased: WBC count, Hgb
Decreased: Platelet count

Clotting Increased: MCHC

Decreased: WBC count, RBC count, Platelet count, Hgb, Hct
Hemolysis Increased: Hgb, MCHC, Platelet count
Decreased: RBC count, Hct, MCV

Autoagglutination Increased: MCV, MCHC

Decreased: RBC count, Hct
Defibrinated blood Blood Glass Beads/clips
Tests: “OAA”
- OFT
- Autohemolysis test
- Acidified serum test

Automated Cell Counter

Optical light scattering Blood cells when subject to light will create forward & side light scatters
w/c are detected by photodetector
Forward LS = cell size
Side LS/900/right angle scatter = cell granularity
Ex. Technicon autoanalyzer

Electrical impedance Blood cells are nonconductors of electricity. they create impedance or
resistance of current when passed in a solution that conduct electricity
Ex. Sysmex counter, Coulter counter
Coulter counter Triplicate count (3x)
a. Blood is diluted 1:6250 (isotonic)
♫ RBCs = 36-360fL
♫ Plts = 2-20fL
b. Blood is diluted 1:251 (hypotonic)
♫ Lymphocytes = 35-90fL
♫ Monocytes = 90-160fL
♫ Granulocytes = 160-450fL
Page | !53
Histograms RBCs, WBCs, plts
X-axis
- Horizontal/abscissa
- Size of cells
Y-axis
- Vertical/ordinate
- Number of cells
Ohm’s law V=IxR
Where:
V = voltage
I = current
R = resistance

Positive error ! Count: “BEA”

♫ Bubbles
♫ Extraneous electrical pulses
♫ Aperture plug
Negative error " Count
♫ Improper setting of aperture error

Polychromasia grading % of RBCs that are polychromatophilic

Slight = 1%
1+ = 3%
2+ = 5%
3+ = 10%
4+ = >11%
Normocytic, 1. Defective formation of RBCs or the presence of tumor cells in BM:
Normochromic RBCs *Aplastic anemia
*Leukemia
*Hodgkin’s disease
*Multiple myeloma
*Leukoerythroblastosis
*Metastatic cancer
*Anemia of renal & endocrine disease
*Anemia of inflammatory disease
2. Abnormal hemoglobin, increased destruction of RBCs
*Certain acquired hemolytic anemia
*PNH
*Sickle cell anemia
*HDN
*Anemia of chronic renal insufficiency

Page | !54
Hemolytic anemias 1. Intrinsic defects w/in RBC
a. Hereditary – membrane defects
**Spherocytosis
**Elliptocytosis
**Acanthocytosis
**Stomatocytosis
**Rh null disease
b. Hereditary – enzyme defects
**G6PD
**PK
c. Hereditary – hemoglobinopathies
**Sickle cell disease
**Hemoglobin C disease
d. Unstable hemoglobin disease
**Hemoglobin E disease
e. Hereditary – defective globin synthesis
**Thalassemia
f. Acquired
**PNH
2. Extracorpuscular causes: nonimmune acquired hemolytic anemias
*Chemicals, toxins, venoms
*Physical trauma: disorders causing fragmentation (burns, cardiac
replacement valves, MAHA, HUS)
3. Extracorpuscular causes: immune hemolytic anemias
*Isoimmune antibodies: incompatible blood transfusion, HDN
*Autoimmune antibodies: warm/cold reacting, drug-induced
4. Miscellaneous
*Anemia of liver disease
*Sulfhemoglobinemia
*Porphyrias
*Methemoglobinemias

Page | !55