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 2mL EDTA + 0.5mL NSS/Citrate
Westergren ESR Ratio = 1:4 (Anticoagulant-to-Blood)
(Black top tube)
Citrate For coagulation and platelet studies
(Light blue top = Preserves labile factors V and VIII
tube) = Buffered 3.2% (0.109M) citrate
Inversion: 3-4x
Ratio = 1:9 (Anticoagulant-to-Blood)
Polycythemic Hct
patients 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
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 Lavender
tubes Pink
White
Royal blue
Tan
Skin puncture 1. Fingertips
2. Earlobe: less admixture w/ tissue juice, less pain, less free
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 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 19, 20, 21
(needle) Routine: 20g
Common length of 1-1.5 inches
needle
Color coded hub 18 = pink
(gauge) 21 = green
22 = gray
23 = blue/light blue/turquoise
25= orange
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 40-60 mmHg
tourniquet
Reassure the Crying = cell count
patient
Position the patient Lying down = hemodilution ( PCV by 8%, WBC)
Lying up = hemoconcentration
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
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 = Aspiration needle (University of Illinois sterna needle)
Posterior iliac crest 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
BM Cellularity Percentage of marrow space occupied by hematopoietic cells
compared w/ fat
Normocellular marrow (Adult): 30% - 70% hematopoietic cells
♫ 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/Juve Predominant cell (WBC) in adult BM (up to 32%)
nile 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
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 Prorubricyte
normoblast Intensely basophilic cytoplasm
N/C ratio = 6:1
Nucleoli usually not visible

3. Rubricyte
Polychromatophilic Blue-gray to pink-gray cytoplasm
normoblast 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

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 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
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
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 Rose-pink granules
neutrophil 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)

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 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
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
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
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
Thrombopoiesis 5 days (Megakaryoblast Platelets)
1. Megakaryoblast N/C ratio = 10:1
2. Nucleus: may show slight lobulation (Endomitosis)
Promegakaryocyte N/C ratio = 4:1 to 7:1
3. Granular
megakaryocyte Largest cell in BM
4. Mature Cytoplasm contains coarse clumps of granules aggregating into
megakaryocyte little bundles, which bud off from the periphery to become
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 platelets
Multiple nuclei
N/C ratio = <1:1
5. Disintegrated cell surrounded by platelet
Metamegakaryocyt
e
6. 1-4μm in diameter
Platelet/Thrombocy Light blue to purple
te 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 CO2
(ODC) Temperature
2,3-DPG
pH
Affinity of Hgb for O2
HbF
Shift to the right CO2
(ODC) Temperature
2,3-DPG
pH
Affinity of Hgb for O2

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
↓
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 (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.
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
(Pentose PO4 pathway) denaturation
G6PD deficiency: most common red cell enzyme
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 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
pathway functional
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)
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
Megaloblastic anemia Oval macrocytes
Howell-Jolly bodies
Hypersegmented neutrophils

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 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
Macroovalocytes immature nucleus
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
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
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 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
Schistocyte Fragmentation produced by damage of RBC by fibrin,
Schizocyte altered vessel walls, 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
Crescent cell released
♫ 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
Cabot rings Figure of 8
Remnant of microtubules of mitotic spindle
♫ Megaloblastic anemia
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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
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
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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)
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 Characteristics w/in 1+ 2+ 3+ 4+
Normal (per (per (per OIO) (per
Limits OIO) OIO) OIO)
(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 Agg. of 5- Numerous --
3-4 10 RBCs aggregate

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Pelger-Huet
Hypersegmentation
Cytoplasmic Abnormalities
Alder-Reilly granules
Toxic granules
Toxic vacuoles
Auer rods
Faggot cells
Chediak-Higashi granules
May-Hegglin inclusion
Dohle bodies
Dohle-Amato bodies
IT: Infections, Toxic states
Abnormalities in Function
Job’s syndrome
Lazy leukocyte syndrome
Chronic Granulomatous
RBCs s
Nuclear Abnormalities
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
≥ 6 lobes (neutrophil)
Abnormal DNA synthesis
♫ Undritz anomaly = hereditary hypersegmentation
♫ Megaloblastic anemia
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)
Large purple to black granules resembling ALR granules
♫ Infections
♫ Toxic states
Infections
Toxic states
Pink or red rod shaped structures
Fused primary granules (peroxidase positive)
Myeloid and monocytic series only
w/ mass of Auer rods
M3 (APL) = associated w/ DIC
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)
Pale blue inclusions derived from RNA
♫ May-Hegglin anomaly
= Autosomal Recessive
= Giant platelets
= Thrombocytopenia
Resemble Dohle bodies (IT)
Single or multiple blue inclusions
Aggregates of free ribosomes of rough ER
Resembles
♫ Infections
♫ Toxic states
Dohle bodies
Toxic granules
Toxic vacuoles
Normal random activity
Abnormal chemotactic activity
Abnormal random and chemotactic activity
Inability of phagocytes to kill ingested microorganisms
Page | 13
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)
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

Page | 14
Dutcher’s bodies
Platelet Abnormalities (Morphologic)
Giant platelet
Small/micromegakaryocyte ♫ Myelodysplastic syndromes
Large megakaryocyte
Mononuclear
megakaryocyte
Vacuolated megakaryocyte
Leukemia
Acute leukemia
Subacute leukemia
Chronic leukemia
Leukemic leukemia
Subleukemic leukemia
Aleukemic leukemia
French-American-British
(FAB) Classification of
Acute Leukemias
Acute leukemia
Acute leukemia in children
Tests to differentiate ALL
from ANLL
Acute Lymphoblastic Leukemias (ALL)
L1
L2
L3
Acute Nonlymphocytic Leukemias (ANLL)
M1
Intranuclear protein inclusions
♫ Bernard-Soulier syndrome
♫ May-Hegglin anomaly
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
Days to 6 months
Predominantly immature cells (blasts and “pro” stages)
2 to 6 months
Variable
Minimum of 1 or 2 years
Predominantly mature cells
WBC >15,000/μL
WBC <15,000/μL
(+) Abnormal and immature cells in PB
WBC <15,000/μL
(-) Abnormal and immature cells in PB
Divides acute leukemias into lymphoblastic and
monoblastic
= Subdivided according to cellular morphology,
cytochemical staining results, cytogenetic studies and T
& B lymphocytes marker results
Normocytic, normochromic RBCs
FAB = ≥30% blasts
Henry: WHO (Now the standard for diagnosis) = ≥20%
80% ALL
20% ANLL
1. MPO: Myeloperoxidase
= (+) AML
= (-) ALL
2. SBB: Sudan Black B
= (+) AML
= (-) ALL
3. TdT: Terminal Deoxyribonucleotidyltransferase
= Marker for immature lymphocyte
= (+) ALL
= (-) ANLL
Lymphoblasts are small and homogeneous (vary little in
size)
Childhood ALL
Lymphoblasts are large and heterogeneous (vary in
size)
Adult ALL
Burkitt-type
Rare
Lymphoblasts are large but homogeneous, and
vacuolated
Acute myeloblastic leukemia w/o maturation (AML w/o
Page | 15
 mat)
BM:
>30% blasts
<10% granulocytic cells
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/ myeloid Fibrosis and granulocytic hyperplasia of BM, w/
metaplasia (MMM) granulocytic and 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
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 production In patients w/ pulmonary/cardiac disease
of EPO RBCs, WBCs, Platelets
Page | 16
 EPO
2’ Absolute polycythemia In patients w/ tumors of kidney, liver, brain, adrenal and
w/ inappropriate pituitary gland
production of EPO RBCs, N-WBCs, N-Platelets
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 <1% <5% Mildest type
(RA)
RA w/ ringed sideroblast <1% <5% RS: Ringed
(RARS) >15% RS sideroblast
RA w/ excess blast <5% 5-20%
(RAEB)
RAEB in transformation >5% 20-30%
(RAEBt)
Chronic Myelomonocytic <5% 5-20% Persistent
Leukemia (CMML) monocytosis
Lymphoproliferative disorders
LPD Proliferation of cells derived from lymphoid stem cell
T/B cells
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
Myeloperoxidase (MPO)
Sudan Black B (SBB)
Sezary cells: w/ cerebriform nucleus (Brainlike)
Special Stains
Marker for primary granules and Auer rods
(+) AML
(-) ALL
Stain should only be done on fresh specimens
Marker for phospholipids and lipids
(+) AML
(-) ALL
Can be done on stored specimens
Parallels MPO for interpretation

Page | 17
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
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
phosphatase (LAP)/ activity
Neutrophil ALP (NAP) Found in 3’ granules (Metamyelocytes)
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
Page | 18
(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%
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

HEMOSTASIS
Primary hemostasis Involves blood vessels and platelets
Formation of platelet plug
Test: Bleeding time
Platelets Functions:
Adhesion
Activation
Release
Aggregation
Secondary Involves the coagulation factors
hemostasis 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 Thrombocyte
Granules Tags Features s Visible
Megakaryoblast (-) (+) Single nucleus (-)
Fine chromatin
(+) Nucleoli
Promegakaryocytes Few (+) 2 nucleus (-)
Megakaryocytes Numerous Usually (-) 2 or more (-)
nuclei
Metamegakaryocyt Aggregated (-) 4 or more (+)
es nuclei
Platelet structure 60% proteins
30% lipids
Page | 19
 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 (-) gpIb = receptor for vWF
syndrome
(Giant platelet
syndrome)
Von Willebrand (-) vWF = for platelet adhesion
disease
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
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)
Page | 20
 Important Substances Secreted by Platelets & Their Role in Hemostasis
Promote HMWK (α)
coagulation Fibrinogen (α)
Factor V (α)
Factor VIII:vWF (α)
Promote ADP (d)
aggregation Calcium (d)
Platelet factor 4 (α)
Thrombospondin (α)
Promote Serotonin (d)
vasoconstriction Thromboxane A2 precursors (mb.PL)
Promote vascular Platelet-derived growth factor (α) = promotes smooth muscle
repair growth
β-thromboglobulin (α) = chemotactic for fibroblasts
Other systems Plasminogen (α)
affected α2-antiplasmin (α)
C1 esterase inhibitor (α)
Platelet Platelet attachment to each other
aggregation Requires fibrinogen and Ca2+
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 Most common inherited vascular disorder
hemorrhagic Blood vessels are thin & lack smooth muscle
telangiectasia
(Oslwer-Weber-
Rendu disease)
Congenital Tumor composed of blood vessels
hemangiomata
(Kasabach-Merritt
syndrome)
Ehler-Danlos vascular fragility
syndrome
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 Immunologic damage to endothelial cells
purpura
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
Page | 21
 Units transfused = Thrombocytopenia
Multiple transfusion: stored blood contains nonviable platelets
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

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
Page | 22
(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
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 Aggregating agents:
aggregation test a. Epinephrine
b. Collagen
c. ADP
d. Ristocetin
Sample: Citrated blood Centrifuge at 60-100g for 10mins
PRP + Agonist O.D. monitored (Aggregometer)
Platelet Determination of in vitro platelet adhesion
adhesiveness Plt ct. 1 = routine collection method
(Salzmann) 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 Measures capillaries to resist pressure
test Correlates w/ the degree of thrombocytopenia
Touriquet test 100 mmHg for 5 mins After 15-30mins, count petechiae
Rumpel-Leedes test (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
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:

Page | 23
 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 Christmas factor
component Antihemophilic factor B
Platelet cofactor 2
X Stuart-Prower factor Stuart factor
Prower factor
Autoprothrombin III
XI Plasma thromboplastin Antihemophilic factor C
component
XII Hageman factor Glass factor
Contact factor
XIII Fibrin stabilizing factor Laki-Lorand factor
Fibrinase
Plasma transglutaminase
Fibrinoligase
- Prekallikrein Fletcher factor
- High-molecular weight Fitzgerald factor
kininogen Contact activation cofactor
Williams factor
Flaujeac factor
Stage I: Generation Intrinsic: XIIXIIX—VIII (Cofactor)
of Thromboplastin Extrinsic: IIIVII
Common: X—V (Cofactor)
----> Common thromboplastin/Prothrombinase (Va-Xa-Ca2+-PL)
Stage II: Conversion II ---(Prothrombinase)---> Thrombin
of prothrombin to
thrombin
Stage III: I ---(Thrombin)---> Fibrin clot
Conversion of XIII---(Thrombin)---> XIIIa
fibrinogen to fibrin Fibrin clot ---(XIIIa)---> Stable fibrin clot
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

Page | 24
 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’ N N N N N
hemostasis
Fibrinogen N* N
deficiency
Prothrombin N N N
deficiency
Parahemophilia N N N
Factor VII deficiency N N N N N
Hemophilia A N N N N N
von Willebrand N N N N
disease
Hemophilia B N N N N N
Factor X deficiency N N N
Hemophilia C N N N N N
Factor XII deficiency N N N N N
Factor XIII N N N N N Abn
deficiency
DIC Abn
*BT may be prolonged in afibrinogenemia
Adsorbed
Fresh Plasma Aged Plasma Fresh Serum Aged Serum
Plasma
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
Page | 25
 Parahemophilia Fibrinolysis
VII Autosomal recessive Factor VII deficiency Liver disease
Vit. K deficiency
Anticoagulant therapy
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 Common inherited coagulation factor deficiency
deficiency a. Hemophilia A/Classic hemophilia/Royal disease = Queen
Victoria’s son
b. vWD = most frequently inherited coagulation disorder
Major sites of Endothelium
coagulation Platelet
inhibition
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
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
thromboplastin common pathway
time PPP + APTT rgt:
1. Activators:
a. Micronized silica
b. Ellagic acid
c. Celite
d. Kaolin
2. Phospholipids: substitute for platelets
3. CaCl2
Page | 26
 Begin timing for clot formation on the addition of CaCl2 rgt
NV = 25-35 secs
Stypven East Indian viper venom Vipera russelli: directly activates factor
time/Russel viper 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 Rgt: 5M Urea
test 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 Prolonged APTT and PT not corrected
anticoagulants
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 fibrin clot formation depends on in light scattering
detection of clot associated w/ conversion of fibrinogen fibrin
formation ♫ 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 1. Intrinsic activators
activators - XIIa
- Kallikrein
- HMWK
2. Tissue type
Page | 27
 - Urokinase-like PA
3. Therapeutic activators = treatment for thromboemboli
- Streptokinase
- Urokinase
- Tissue-like PA
Inhibitors of α2-antiplasmin = Major inhibitor
fibrinolysis α2-macroglobulin
Thrombospondin
PA inhibitor 1 and 2
Degradation of Crosslinked fibrin (urea insoluble)
cross-linked fibrin ↓
by plasmin (Plasmin)
↓
Complex DD/E | Complex YD/DY | Complex YY/DXD
(D-dimer)
Degradation of non- Fibrinogen or
crosslinked fibrin Noncrosslinked fibrin (urea soluble)
and 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 DIC = uncontrolled, inappropriate formation of fibrin w/in the
fibrinolysis 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 Clot should remain intact for approximately 48 hrs at 37’C
lysis time (WBCLT) Clot lysis prior to 48 hrs indicates excessive systemic fibrinolysis
Euglobulin lysis Euglobulins: proteins that ppt. when plasma is diluted w/ H2O &
time 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 Detects the presence of fibrin monomers (2’-DIC) in the plasma
Page | 28
test Plasma + Protamine sulfate + ETOH (+) gel-like clot
(paracoagulation)
Ethanol gelation Detects the presence of fibrin monomers (2’-DIC) in the plasma
test Plasma + NaOH (pH) + ETOH (+) pptn/gel
Latex D-dimer Most specific test for DIC
assay
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 | 29
 HEMATOLOGY PROCEDURES
Brightfield Examine blood films
microscopy
Oil immersion Erythrocyte morphology & leukocyte differential
microscopy
Phase-contrast For manual platelet counts
microscopy
Fluorescence ANA and T/B cell studies
microscopy
Electron microscopy Observation of fine ultrastructures of cells (100,000x
magnification)
Basic component of Diopter rings: adjust for focusing differences between eyes
Standard light Rubber eyeguard: adjust for comfort
microscope 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 1g Hgb = 1.34mL O2
Slyke)
Chemical 1g Hgb = 3.47mg Fe2+
(Kennedy’s,
Wong’s)
SG method CuSO4 method (See BB notes)
Cyanmethemoglobi Manual and automated
n/ Dilution = 0.02mL blood: 5mL rgt (1:251)
Hemiglobincyanide Reagents:
method Drabkin’s reagent (Brown container)
Page | 30
 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)
Micromethod Capillary tube:
(Adam) 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 Hct = only computed
methods Hct = MCV x RBC count

RBC Count
RBCs AM, PM
Page | 31
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) + H2O
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)
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

Page | 32
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
22mm x 22mm Square coverslip
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 Wright’s
stains 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 Thick films
stain Prolonged staining time
Inadequate washing
Too high alkalinity of stain
Diluents tends to cause excessive basophilia
Excessively pink Insufficient staining
stain 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 WBCs are counted from the tail toward the head of the smear
method
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
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

Page | 33
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
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 ARC = (% Retics ÷ 100) x RBC count (1012/L) x 1,000
reticulocyte count NV = 25-75 x 109/L
Corrected CRC = % Retics x (Patient Hct ÷ Normal Hct [0.45L/L])
reticulocyte count NV = 1
Reticulocyte General indicator of the rate of erythrocyte production increase
production above normal in anemias
index/Shift Indicates BM response to anemia
correction RPI = CRC ÷ Maturation time of retics in the blood
NV = 1 (Hct: 45%)
Maturation time of 1.0 day = Hct: 45 ± 5%
retics 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

Page | 34
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
Eosinophilia Allergic reactions
Parasitic infections
Brucellosis
Leukemias
Eosinopenia Hyperadrenalism (Cushing’s disease)
Shock
Administration of ACTH
Eosinophil diluting Composition:
fluids 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 Values affected:
centrifuge = Hct
= MCV
= MCHC
MCHC >38% does Incorrect calculation
not occur (+) cold agglutinins
MCHC will not fall Lipemia
<22% (+) HbS & HbC

Erythrocyte Sedimentation Rate (ESR)

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
Page | 35
 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 & Requires smaller amount of blood
Landsberg 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 Anticoagulant: 2mL EDTA + 0.5mL NSS/Citrate
Westergren
Zeta Sedimentation Not affected by anemia
Ratio (ZSR) Major disadvantage: requires special capillary tubes and
Zetafuge
ZSR = (%Hct ÷ %Zetacrit) x 100
Erythrocyte Anticoagulant: Heparin
Osmotic Fragility % NaCl = # drops NaCl x 0.02
test Add RBCs, stand for 2hrs at room temp
(Griffin and Sanford Check for hemolysis (pink/red supernatant)
method) 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 Acquired disorder in w/c red cells are abnormally sensitive to
nocturnal complement
hemoglobinuria (-) DAF
(PNH)
Sucrose hemolysis Screening test for PNH
test Patient RBCs + ABO compatible serum + sucrose solution
Page | 36
 Normal = (-) Hemolysis
PNH = (+) Hemolysis
Ham’s acidified Confirmatory test for PNH
serum 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 Patient w/ PNH + blood transfusion ---(Ham’s test)---> 
received normal Hemolysis
RBCs
PCH IgG autoanti-P = biphasic hemolysin
- Cold = attaches to RBCs
- Warm = RBC lysis
Donath-Landsteiner Test for PCH
test 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
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 Causes of Spurious
Increase Decrease
WBC count Cryoglobulin Clotting
Cryofibrinogen Smudge cells
Heparin Uremia plus
Monoclonal proteins immunosuppressants
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 Cryoglobulin Autoagglutination
Page | 37
(automated) Cryofibrinogen Clotting
Giant platelets Hemolysis
WBC >50,000/μL Microcytic RBCs
Hyperglycemia >600mg/dL
Hematocrit Hyponatremia Excess EDTA
(microhct) Plasma trapping Hemolysis
Hypernatremia
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 Blood cells when subject to light will create forward & side light
scattering scatters w/c are detected by photodetector
Forward LS = cell size
Side LS/900/right angle scatter = cell granularity
Ex. Technicon autoanalyzer
Electrical Blood cells are nonconductors of electricity. they create
impedance 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
Histograms RBCs, WBCs, plts

X-axis
- Horizontal/abscissa
- Size of cells
Y-axis
- Vertical/ordinate
- Number of cells
Page | 38
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 % of RBCs that are polychromatophilic
grading Slight = 1%
1+ = 3%
2+ = 5%
3+ = 10%
4+ = >11%
Normocytic, 1. Defective formation of RBCs or the presence of tumor
Normochromic RBCs cells in BM:
*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
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. Intracorpuscular causes: immune hemolytic anemias
*Isoimmune antibodies: incompatible blood transfusion, HDN
*Autoimmune antibodies: warm/cold reacting, drug-induced
4. Miscellaneous
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*Anemia of liver disease
*Sulfhemoglobinemia
*Porphyrias
*Methemoglobinemias

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