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-Hellers) = shrink cells
b. Ammonium oxalate (Wintrobes) = 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 Romanowskys 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
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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
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
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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
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)

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1. Pronormoblast
2. Basophilic normoblast
Stimulates hematopoietic cells
Proerythroblast/rubriblast
N/C ratio = 8:1
Nucleoli = 1-2
Can produce up to 16 RBCs per 1 pronormoblast/rubriblast
Prorubricyte
Intensely basophilic cytoplasm
N/C ratio = 6:1
Nucleoli usually not visible

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 m 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
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

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 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
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
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Monocyte Largest cell in PB
14-20 m 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 = 8-10m (Size = RBC)
Medium = 10-12m
Large = 12-16m (Rare)
Cytoplasm: bluish (Robins 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 differentiation 1. Plasmablast
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
3. Granular megakaryocyte
4. Mature megakaryocyte Largest cell in BM
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 1-4m 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

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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

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+

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 (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
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 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)
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 = Fanconis anemia
Acquired = radiation, chemical (benzene), drugs (chloramphenicol)

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 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
Ineffective erythropoiesis (pancytopenia)
Vitamin B12 (Cobalamin) w/ CNS problems
deficiency 1. Pernicious anemia
= Deficient in intrinsic factor (produced by parietal cells) for B 12 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 Wrights 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
Codocyte Greek: Kodon = bell
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Target cell Central hemoglobinized area (bulls 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

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
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Teardrops Squeezing and fragmentation during splenic passage
MMM
Hypersplenism
Microspherocyte Severe burns
Pyropoikilocyte Hereditary microspherocytosis
Hereditary pyropoikilocytosis
= sensitivity to temperature
= RBCs fragment at 45C (Normal: 49C)
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
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 (4)
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
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 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/ Wrights 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 226 Glu Val
HbC 226 Glu Lys
HbE 2226 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
-thalassemia Alpha-globin chain is or absent
a. Adult = 4 = HbH (deletion of 3/4 alpha genes)
b. Neonate = 4 = Hb Barts (deletion of 4/4 alpha genes)
-thalassemia Beta-globin chain is or absent
HbF and HbA2

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Cooleys anemia Thalassemia major
Homozygous -thalassemia
Cooleys trait Thalassemia minor
Heterozygous -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 -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: 50C 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 m) 0-5 5-10 10-20 20-50 >50
Microcytes (<9 m) 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 3-4 RBCs Agg. of 5-10 RBCs
Numerous --
aggregates
Nuclear Abnormalities
Pelger-Huet Hyposegmentation (neutrophil)
Bilobed nucleus: Dumb-bell shaped/spectacle/peanut-shaped/Pince-nez
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Hypersegmentation
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
Jobs syndrome
Lazy leukocyte syndrome
Chronic Granulomatous
Disease (CGD)
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
Cytoplasmic Abnormalities
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
Abnormalities in Function
Normal random activity
Abnormal chemotactic activity
Abnormal random and chemotactic activity
Inability of phagocytes to kill ingested microorganisms
Impaired NADPH oxidase
Impaired oxidative metabolism/respiratory burst
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 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 = Turks 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)
Gauchers disease Accumulation of glucocerebroside
(-) glucocerebrosidase/-glucosidase
Wrinkled/crumpled cytoplasm (Chicken scratch)
Niemann-Pick disease Accumulation of sphingomyelin
(-) sphingomyelinase
Foamy cytoplasm
Foam cells w/ sphingomyelin
Tay Sachs disease Accumulation of glycolipid and ganglioside
(-) Hexosaminidase A
Vacuolated cytoplasm
Sandhoffs 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
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 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
Dutchers bodies Intranuclear protein inclusions
Platelet Abnormalities (Morphologic)
Giant platelet Bernard-Soulier syndrome
May-Hegglin anomaly
Small/micromegakaryocyte Myelodysplastic syndromes
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
Chronic leukemia Variable
Minimum of 1 or 2 years
Predominantly mature cells
Leukemic leukemia WBC >15,000/L
Subleukemic leukemia WBC <15,000/L
(+) Abnormal and immature cells in PB
Aleukemic leukemia WBC <15,000/L
(-) 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 results,
Acute Leukemias 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 children 80% ALL
20% ANLL
Tests to differentiate ALL 1. MPO: Myeloperoxidase
from ANLL = (+) AML
= (-) ALL
2. SBB: Sudan Black B
= (+) AML
= (-) ALL
3. TdT: Terminal Deoxyribonucleotidyltransferase
= Marker for immature lymphocyte

Page | 193
 = (+) 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
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)
Naegelis leukemia
20% to <80% monocytic cells
M5a Acute monoblastic leukemia w/o maturation
Schillings leukemia
>80% monocytic cells (>80% monoblasts)
M5b Acute monoblastic leukemia w/ maturation
>80% monocytic cells (<80% monoblasts)
M6 Erythroleukemia
Erythremic myelosis
Di Guglielmos 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)
Page | 194
2. Myelofibrosis w/ myeloid Fibrosis and granulocytic hyperplasia of BM, w/ granulocytic and
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
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
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/Gaisbo ck 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

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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-Hodgkins lymphoma
= proliferation of neoplastic lymphocytes
= Rappaport classification
b. Hodgkins lymphoma
= proliferation of cells reacting to neoplasm
= (+) Reed-Sternberg cell: large cell w/ large nucleoli (Owls 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
-Naphthyl Acetate Nonspecific esterase
esterase Marker for monocytes, megakaryocytes and plasma cells
(+) AMoL [M5] = inhibited by fluoride
(+) AMegL [M7]
(-) AML
Page | 196
-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%
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
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 (+) 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 | 198
 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 Features Thrombocytes
Granules Tags Visible
Megakaryoblast (-) (+) Single nucleus (-)
Fine chromatin
(+) Nucleoli
Promegakaryocytes Few (+) 2 nucleus (-)
Megakaryocytes Numerous Usually (-) 2 or more nuclei (-)
Metamegakaryocytes Aggregated (-) 4 or more nuclei (+)
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
Page | 199
(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
Platelet Secretion Release of granules
a. Alpha granules
= Platelet factor
= Platelet derived growth factor
= Fibrinogen
= Factor V
= vWF
= -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 ( & dense granule deficiency)
Important Substances Secreted by Platelets & Their Role in Hemostasis
Promote coagulation HMWK ()
Fibrinogen ()
Factor V ()
Factor VIII:vWF ()
Promote aggregation ADP (d)
Calcium (d)
Platelet factor 4 ()
Thrombospondin ()
Promote vasoconstriction Serotonin (d)
Thromboxane A2 precursors (mb.PL)
Promote vascular repair Platelet-derived growth factor () = promotes smooth muscle growth
-thromboglobulin () = chemotactic for fibroblasts
Other systems affected Plasminogen ()
2-antiplasmin ()
Page | 200
 C1 esterase inhibitor ()
Platelet aggregation Platelet attachment to each other
Requires fibrinogen and Ca2+
Glanzmanns (-) 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
Marfans syndrome
Pseudoxanthoma elasticum Elastic fibers are calcified & structurally abnormal
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 # of units transfused
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
Page | 201
 Platelet aggregation test:
= Normal: Ristocetin
= Abnormal: Epinephrine, Collagen, ADP
3. Storage pool defects
= Gray platelet (), 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. Fonios
c. Olefs
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/L Markedly decreased
Page | 202
50,000-100,000/L Moderately decreased
100,000-150,000/L Slightly decreased
150,000-199,000/L Low normal
200,000-400,000/L Normal
401,000-599,000/L Slightly increased
600,000-799,000/L Moderately increased
>800,000L 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
Coagulation factors All are produced in the liver except Factor VIII
Page | 203
 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
Stage I: Generation of Intrinsic: XIIXIIXVIII (Cofactor)
Thromboplastin Extrinsic: IIIVII
Common: XV (Cofactor)
----> Common thromboplastin/Prothrombinase (Va-Xa-Ca2+-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

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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 Duckerts
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
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
Adsorbed
Fresh Plasma Aged Plasma Fresh Serum Aged Serum
Plasma
I + + + - -
II + + - + (<20%) -
V + - + - -
VII + + - + +
VIII + - + - -
IX + + - + +
X + + - + +
XI + + + + +
XII + + + + +
XIII + + + - -
Prothrombin:
Page | 205
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 Owrens disease Liver disease
Labile factor deficiency DIC
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 deficiency Common inherited coagulation factor deficiency
a. Hemophilia A/Classic hemophilia/Royal disease = Queen Victorias 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

Page | 206
 = 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 CaCl 2 rgt
NV = 10-12 secs
Activated partial Detect coagulation factor deficiencies involving intrinsic and common pathway
thromboplastin time 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 CaCl 2 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
Duckerts 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)
Page | 207

Circulating anticoagulants
Lupus inhibitor
Tilt tube method
Fibrometer
Photo-optical detection of
clot formation
Fibrinolysis
Plasminogen activators
Inhibitors of fibrinolysis
Degradation of cross-linked
fibrin by plasmin
Degradation of non-
crosslinked fibrin and
fibrinogen by plasmin
F. XIII def. = Clot is soluble to urea (24 hrs)
Prolonged APTT and PT not corrected
Against the phospholipid portion of prothrombinase complex (Va-Xa-Ca 2+-PL)
Instrumentation for Tests of Hemostasis
Visual detection of fibrin clot formation
Manual technique
End point = clot formation
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
Detection of fibrin clot formation depends on in light scattering 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
Digestion of fibrin clot
Occurs when plasminogen plasmin
1. Intrinsic activators
- XIIa
- Kallikrein
- HMWK
2. Tissue type
- Urokinase-like PA
3. Therapeutic activators = treatment for thromboemboli
- Streptokinase
- Urokinase
- Tissue-like PA
2-antiplasmin = Major inhibitor
2-macroglobulin
Thrombospondin
PA inhibitor 1 and 2
Crosslinked fibrin (urea insoluble)
(Plasmin)
Complex DD/E | Complex YD/DY | Complex YY/DXD
(D-dimer)
Fibrinogen or
Noncrosslinked fibrin (urea soluble)
(Plasmin)
Fragment X
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Primary fibrinolysis
Secondary fibrinolysis
Whole blood clot lysis time
(WBCLT)
Euglobulin lysis time
Protamine sulfate test
Ethanol gelation test
Latex D-dimer assay
Test
WBCLT
Euglobulin clot lysis
Ethanol gelation
Protamine sulfate
D-dimer
Heparin
(Plasmin)
Fragment Y | Fragment D
(Plasmin)
Fragment D | Fragment D
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
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
Clot should remain intact for approximately 48 hrs at 37C
Clot lysis prior to 48 hrs indicates excessive systemic fibrinolysis
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
Detects the presence of fibrin monomers (2-DIC) in the plasma
Plasma + Protamine sulfate + ETOH (+) gel-like clot (paracoagulation)
Detects the presence of fibrin monomers (2-DIC) in the plasma
Plasma + NaOH (pH) + ETOH (+) pptn/gel
Most specific test for DIC
Primary Fibrinolysis Secondary Fibrinolysis
< 48 hrs < 48 hrs
< 2 hrs < 2 hrs
- +
- +
- +
Injected
Action: inhibits thrombin
Monitoring: APTT = sensitive, method of choice (CAP)
Neutralize w/ protamine sulfate
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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

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 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
Chemical 1g Hgb = 3.47mg Fe2+
(Kennedys, Wongs)
SG method CuSO4 method (See BB notes)
Cyanmethemoglobin/ Manual and automated
Hemiglobincyanide method Dilution = 0.02mL blood: 5mL rgt (1:251)
Reagents:
Drabkins reagent (Brown container)
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 a. Potassium ferricyanide = ferrous ferric
b. Potassium cyanide = Hi HiCN
c. Nonionic detergent = lysis of red cells, decreases turbidity
d. Sodium bicarbonate (Orig. Drabkins) = result is read after 15mins
e. Dihydrogen potassium phosphate (Mod. Drabkins) = 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 patients 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 (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
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 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.) Hayems
5.) Toissons
6.) Bethells
7.) Gowers
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.) Turks diluting fluid: Gentian violet + glacial acetic acid (solid at 17C) + H 2O
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
Fuchs 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
Speirs 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:
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 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/Spinners technique = Automated: Hemaspinner
25 (30-40 )
0 0
Angle between 2 slides
22 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
Romanowskys stains Wrights
Giemsa = preferred stain for blood parasites
Modified Wrights-Giemsa
Leishman
Jenner
May-Grunwald
------------------------------------------------------------------------------------------------------
Contains:
= Methylene blue (or Azure B - oxidized): basic
= Eosin: acidic
w/in 2-3 hours of specimen Time blood smears should be stained
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

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 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
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
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Monocytosis
Shift to the left
Shift to the right
Reticulocyte count
% Retics
Absolute reticulocyte count
Corrected reticulocyte
count
Reticulocyte production
index/Shift correction
Maturation time of retics in
the blood
RPI > 3
RPI < 2
Miller disk
Eosinophil count
Eosinophilia
Eosinopenia
Eosinophil diluting fluids
Brucellosis
Tuberculosis
Monocytic leukemia
SBE
Typhoid
Rickettsial infections
Collagen disease
Hodgkins disease
Gauchers disease
(+) immature granulocytic cells
Leukemia
Bacterial infections
Hypersegmented neutrophils (6 lobes)
NV:
Adult = 0.5-1.5% (Ave: 1.0%)
Newborn = 2-6%
[# Retics # RBC (1000)] x 100
ARC = (% Retics 100) x RBC count (1012/L) x 1,000
NV = 25-75 x 109/L
CRC = % Retics x (Patient Hct Normal Hct [0.45L/L])
NV = 1
General indicator of the rate of erythrocyte production increase above normal
in anemias
Indicates BM response to anemia
RPI = CRC Maturation time of retics in the blood
NV = 1 (Hct: 45%)
1.0 day = Hct: 45 5%
1.5 days = Hct: 35 5%
2.0 days = Hct: 25 5%
2.5 days = Hct: 15 5%
Adequate response of BM to anemia
- Chronic hemolysis
- Recent hemorrhage
- Response to therapy
Inadequate response of BM to anemia
- Aplastic anemia
- Ineffective erythropoiesis (megaloblastic anemia)
% Retics = Retics (A) [RBC (B) x 9] x 100
NV = 50-350 x 106/L
Allergic reactions
Parasitic infections
Brucellosis
Leukemias
Hyperadrenalism (Cushings disease)
Shock
Administration of ACTH
Composition:
a. Phloxine/eosin/neutral red iodide = stains eosinophils
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 b. Propylene glycol = lyses RBCs
c. Na2CO3 = lyses WBCs except eosinophils, intensifies staining of granules
d. Heparin = prevents clumping
Diluting fluids:
1. Pilots = phloxine
2. Manners = phloxine
3. Randolphs = phloxine
4. Hinkleman = eosin
5. Tannens = neutral red iodide
Thorns 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: m3)
MCH MCH = (Hgb RBC) x 10
NV = 27-32 pg (old: g)
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 occur Incorrect calculation
(+) cold agglutinins
MCHC will not fall <22% Lipemia
(+) 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
*1-globulin
*2-globulin
*-globulin
*-globulin
*Cholesterol
Technical factor:
*Tilting = 30 angle = 30% error
*Temp.
ESR Erythrocytes:
*Microcytes
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 *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
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 screening G6P + NADP ---(RBC: G6PD)---> 6-phosphogluconate + NADPH (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 complement
hemoglobinuria (PNH) (-) DAF
Sucrose hemolysis test Screening test for PNH
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 Patient RBCs + ABO compatible serum + sucrose solution
Normal = (-) Hemolysis
PNH = (+) Hemolysis
Hams acidified serum test Confirmatory test for PNH
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 normal Patient w/ PNH + blood transfusion ---(Hams test)---> Hemolysis
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 37C for 30mins incubate at 37C for 30mins
Test: Patient WB incubate at 4C for 30mins incubate at 37C 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 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/L Microcytic RBCs
Hemoglobin HbCO >10% Clotting
Cryoglobulin Sulfhemoglobin
Cryofibrinogen
Hemolysis
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 Heparin
WBC >50,000/L
Hyperbilirubinemia
Lipemia
Monoclonal proteins
Hematocrit (automated) Cryoglobulin Autoagglutination
Cryofibrinogen Clotting
Giant platelets Hemolysis
WBC >50,000/L Microcytic RBCs
Hyperglycemia >600mg/dL
Hematocrit (microhct) Hyponatremia Excess EDTA
Plasma trapping Hemolysis
Hypernatremia
MCV Autoagglutination Cryoglobulin
WBC >50,000/L Cryofibrinogen
Hyperglycemia Giant platelets
Reduced red cell deformability Hemolysis
Microcytic RBCs
Swollen RBCs
MCHC Autoagglutination WBC >50,000/L
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
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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
Ohms 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, Normochromic 1. Defective formation of RBCs or the presence of tumor cells in BM:
RBCs *Aplastic anemia
*Leukemia
*Hodgkins 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
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**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

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