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Hematology

EDTA
(Lavender top)

Modified Westergren
ESR
(Black top tube)
Citrate
(Light blue top tube)

Polycythemic patients

Oxalate
Heparin

Order of Draw
(Henry 21st Edition)

Order of Draw
(Syringe method)

MUST TO KNOW IN HEMATOLOGY


Greek:
-Haima = Blood
-Logos = Study/science
Chelates calcium
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
2mL EDTA + 0.5mL NSS/Citrate
Ratio = 1:4 (Anticoagulant-to-Blood)
For coagulation and platelet studies
= Preserves labile factors V and VIII
= Buffered 3.2% (0.109M) citrate
Inversion: 3-4x
Ratio = 1:9 (Anticoagulant-to-Blood)
Hct
Excess Citrate = PT, APTT
Remedy: Reduce the volume of citrate
Amount of citrate = [(100-Hct)(595-Hct)] x mL WB
Double/balanced oxalate (Ratio = 2:3): Maintained cell structures
a. Potassium oxalate (Paul-Hellers) = shrink cells
b. Ammonium oxalate (Wintrobes) = swell cells
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
Evacuated tube:
1. Sterile blood culture tube
2. Citrate (blue)
3. Nonadditive tube (red)
4. Heparin (green)
5. EDTA (lavender)
6. Fluoride (gray)
1. EDTA
2. Other anticoagulated tubes
3. Nonadditive tube
Page | 1

EDTA containing tubes

Skin puncture

Venipuncture

Common gauge
(needle)
Common length of
needle
Color coded hub
(gauge)
Angle
Tourniquet
BP cuff as tourniquet
Reassure the patient
Position the patient
IV line

Hematopoiesis
Mesoblastic period

Hepatic period

Myeloid period

Lavender
Pink
White
Royal blue
Tan
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
Veins in the arms (antecubital region):
1. Median cubital = preferred, most stable
2. Cephalic (lateral)
3. Basilic (medial)
19, 20, 21
Routine: 20g
1-1.5 inches
18 = pink
21 = green
22 = gray
23 = blue/light blue/turquoise
Venipuncture: 150
BB: 450 10-200 once in the skin
3-4 inches above the site (7.5-10cm)
Not exceed 1min/2mins
Prolonged application hemoconcentration
40-60 mmHg
Crying = cell count
Lying down = hemodilution ( PCV by 8%, WBC)
Lying up = hemoconcentration
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
Cellular formation, proliferation, differentiation and maturation of
blood cells
19th day of gestation
Yolk salk = Erythropoiesis
Embryonic hemoglobins:
a. Gower 1 = Zeta2 + Epsilon2
b. Portland = Zeta2 + Gamma2
c. Gower 2 = Alpha2 + Epsilon2
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
Between 5th & 6th month of gestation persist throughout life
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Adults
Neonates
Marrow specimens

Posterior iliac creast


M:E ratio

BM Cellularity

Yellow BM
Red BM
Marrow differential
Metamyelocyte/Juvenil
e granulocyte
Stem cells
Osteoblasts
Osteoclasts
CD2, CD3
CD19, CD20
CD34
CD16, CD56
CD10
Erythropoietin
Thrombopoietin
Erythropoiesis
IL-3
1. Pronormoblast

BM = 1 source of cell production (Hematopoiesis)


Sternum = principal source of hematopoiesis in adults
HbA1 = 95%
HbA2 = 1.5-3%
HbF = <2%
HbF = 60-80%
HbA = 20-40%
1. Trephine (Core) Biopsy
= Trephine biopsy needle (Jamshidi needle)
2. Aspiration
= Aspiration needle (University of Illinois sterna needle)
Safest site for BM aspirate/biopsy
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
Percentage of marrow space occupied by hematopoietic cells
compared w/ fat
Normocellular marrow (Adult):
Fat = 10-50%
Hematopoietic elements = 40-60% (Ave. 50%)
Fats
Hematopoietic cells
Recommended that at least 500, preferably 1000 cells be counted
for a marrow differential
Predominant cell (WBC) in adult BM (up to 32%)
<1% cells in BM
Bone forming cells
Confused w/ plasma cells
Waterbug or comet appearance
Bone destroying cells
Confused w/ megakaryocytes
T cells
B cells
Stem cell marker (lymphoid and myeloid precursor)
NK cells
CALLA (Common ALL Antigen)
Produced by the kidney
Primary regulator of erythropoiesis
Produced by kidney and liver
Regulator of thrombopoiesis
1 stimulus = Hypoxia
Multi-CSF (Colony Stimulating Factor)
Stimulates hematopoietic cells
Proerythroblast/rubriblast
N/C ratio = 8:1
Nucleoli = 1-2
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2. Basophilic
normoblast

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
normoblast

Rubricyte
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
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)
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5. Band

6a. Segmented
neutrophil

6b. Eosinophil

6c. Basophil

Monopoiesis
Monocyte

Lymphopoiesis
Lymphocyte

Indented/kidney-shaped nucleus
Predominant WBC in BM
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
Rose-pink granules
Nucleus: 2-5 lobes
Diurnal variation (PM)
Specific granules:
a. Lysozyme
b. Lactoferrin
c. Collagenase
d. Plasminogen activator
e. Aminopeptidase
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
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
1. Monoblast
2. Promonocyte
3. 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)
1. Lymphoblast
2. Prolymphocyte
3. Lymphocyte
Small = 8-10m (Size = RBC)
Medium = 10-12m
Large = 12-16m (Rare)
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T lymphocytes
B lymphocytes
Null lymphocytes
Plasma cells
differentiation
Plasma cell

Thrombopoiesis
1. Megakaryoblast
2. Promegakaryocyte
3. Granular
megakaryocyte
4. Mature
megakaryocyte
5. Metamegakaryocyte
6.
Platelet/Thrombocyte

2/3 (67%)
1/3 (33%)
Endomitosis
2000-4000
1 heme molecule
1 hemoglobin
Mitochondria
141 amino acids
146 amino acids
Chromosome 11
Chromosome 16
Oxyhemoglobin
Dissociation Curve
Shift to the left (ODC)

Cytoplasm: bluish (Robins egg blue)


Nucleus: compact
60-80%
Long-lived (4-10 years)
10-20%
Short-lived (3-4 days)
Can differentiate into plasma cell or memory B cells
Large granular lymphocyte
10%
1. Plasmablast
2. Proplasmacyte
3. Plasmacyte/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
5 days (Megakaryoblast Platelets)
N/C ratio = 10:1
Nucleus: may show slight lobulation (Endomitosis)
N/C ratio = 4:1 to 7:1
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
Disintegrated cell surrounded by platelet
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
Circulating platelets
Platelets stored in the spleen
Nuclear division w/o cytoplasmic division
# of platelets a megakaryocyte can produce
1 mol O2
4 mol O2
Early and late heme synthesis
Alpha
Beta, Gamma, Delta, Epsilon, Zeta
Alpha, Zeta
Beta, Gamma, Delta, Epsilon
Normal = Sigmoid in shape
X-axis = Hgb concentration in g/dL | Y-axis = OD
CO2
Temperature
2,3-DPG
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Shift to the right (ODC)

Heme synthesis

Arterial blood
Venous blood
P50
Bohr effect
Oxyhemoglobin (HbO2)
Deoxyhemoglobin
(HbCO2)
Carboxyhemoglobin
(HbCO)

pH
Affinity of Hgb for O2
HbF
CO2
Temperature
2,3-DPG
pH
Affinity of Hgb for O2
Succinyl coenzyme A + Glycine + Pyridoxal PO4

(ALA synthase)

D-Aminolevulinic acid

(ALA dehydrase)

Porphobilinogen

(Uroporphyrinogen synthase)
(Uroporphyrinogen cosynthase)

Uroporphyrinogen

(Uroporphyrinogen decarboxylase)

Coproporphyrinogen

(Coproporphyrinogen oxidase)

Protoporphyrinogen

(Protoporphyrinogen oxidase)

Protoporphyrin IX + Fe2+

(Ferrocheletase)

HEME
O2 saturation = 95%
pO2 = 95 mmHg
O2 saturation = 70%
pO2 = 40 mmHg
pO2 = 26.6 mmHg
pH = Hgb affinity for O2 --- (L)
pH = Hgb affinity for O2 --- (R)
Arterial blood
Blood: Bright red color
Venous blood
Blood: Purplish red color
Blood: Cherry red color
Reversible
Cannot bind and carry O2
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Methemoglobin/
Hemiglobin (Hi)

Sulfhemoglobin

Erythrocyte membrane

Embden-Meyerhoff
pathway
Hexose
monophosphate shunt
(Pentose PO4 pathway)
Rapoport-Luebering
pathway
Methemoglobin
reductase pathway
Pitting
Culling
RBC lysis
Extravascular
hemolysis

Intravascular hemolysis

CO has 200x or 210x greater affinity for Hgb compared to O2


Smoking, gas, etc.
Blood: Chocolate brown color
Reversible
Fe2+ Fe3+
Methemoglobin reductase deficiency
Exposure to chemicals/drugs
Exposure to nitrite, chlorine and nitrate
HbM (Abnormal hemoglobin)
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
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
Major pathway
90% glycolysis (anaerobic)
Glucose Lactic acid = 2 ATP
PK deficiency: common enzyme deficiency
10% glycolysis (aerobic)
Provides reduced glutathione to prevent Hgb denaturation
G6PD deficiency: most common red cell enzyme deficiency
= (+) Heinz bodies: denatured Hgb (Supravital stain: RHH)
= (+) Bite cells: due to pitting
Generates 2,3-DPG that regulates Hgb affinity for O2
Maintains Hgb iron in ferrous (Fe2+) state to be functional
Removal of inclusion by the spleen
Removal of senescent/aged RBCs by the spleen
RBC age = Enzymes, ATP, Size, Density
1% of RBCs/day broken down by the mononuclear phagocytic
system (MPS)
90% aged red cell destruction
w/in the RES
when C is not/incompletely activated
Unconjugated bilirubin
Urine and fecal urobilinogen
10% aged red cell destruction
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Anisocytosis
RDW
Anisochromia
MCV
MCH
MCHC
Normocytic
normochromic anemia
Microcytic hypochromic
anemia

Macrocytic,
normochromic anemia
Aplastic anemia
TIBC
Degree of Hypochromia

Megaloblastic anemia

Vitamin B12
(Cobalamin) deficiency

Folic acid (Vit. B9)


deficiency
Polychromasia

w/in the blood vessels


when C is completely activated
Haptoglobin
Hemopexin
(+) Hemoglobinemia
Variation in size
Numerical expression that correlates w/ the degree of anisocytosis
NV = 11.5-14.5%
Variation in Hgb content (Ex. Dimorphic anemia)
NV = 80-100fL (Normocytic)
<80fL = Microcytic
>100fL = Macrocytic
NV = 27-32 pg
NV = 31-36% (Normochromic)
<31% = Hypochromic
>36% = Hyperchromic
1. Acute blood loss
2. Hemolytic anemia
3. Aplastic anemia
1. Chronic blood loss = most common cause
2. Thalassemia = N-RDW
3. IDA = Iron, TIBC (Ex. Hookworm infections) | RDW
4. Sideroblastic anemia
5. Chronic disease
1. Megaloblastic anemia = Vitamin B12/Folate deficiency
2. Nonmegaloblastic anemia = Liver disease, alcoholism
Congenital = Fanconis anemia
Acquired = radiation, chemical (benzene), drugs (chloramphenicol)
Pancytopenia = WBCs, RBCs, Retics Plts
Differentiates IDA (TIBC) from other microcytic, hypochromic
anemia
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
Oval macrocytes
Howell-Jolly bodies
Hypersegmented neutrophils
Ineffective erythropoiesis (pancytopenia)
w/ CNS problems
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
w/o CNS problems
1. Pregnancy
2. Dietary deficiency
3. Steatorrhea, sprue
Reticulocytosis
Page | 9

Spherocytosis
Microcytic,
hypochromic
Poikilocytosis
Macrocytes
Oval macrocytes
Macroovalocytes
Acanthocyte
Spur cell
Thorn cell
Echinocyte
Burr cell
Sea urchin cell
Crenated RBCs

Codocyte
Target cell
Mexican hat cell

Leptocyte
Spherocyte
Ball
Bronze cell

AIHA vs. HS
Stomatocyte

Elliptocyte

Visible on Wrights stain


Blue-gray coloration, pink cytoplasm
Indicates young RBCs
Erythropoietic activity
a. Hemorrhage
b. Hemolysis
EOFT
EOFT
Variation in shape
ESR = No rouleaux formation
Megalocytes: Vit. B12 or folic acid deficiency
Asynchronous development: mature cytoplasm but immature
nucleus
Irregular spikes/spicules
Abnormal L:S ratio
Abetalipoproteinemia
Liver diseases
Echinocyte: artifactual
Burr cell: pathologic
RBCs w/ projections of equal length and distribution
ATP
Exposure to hypertonic solution
Artifact in air drying
Anemia associated w/ renal insufficiency
Greek: Kodon = bell
Central hemoglobinized area (bulls eye)
Scanning EM: Bell/tall hat shaped
Surface membrane to volume ratio
Cholesterol & phospholipid
Hemoglobinopathies
Thalassemia
Liver disease, postsplenectomy, IDA
Thin variant of a codocyte
Spheroid
Biconvex
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 = OFT, MCHC, (+) DAT
HS = OFT, MCHC, (-) DAT
Greek: Stoma = mouth
Mouth, slit-like pallor area
Bowl-shaped
Permeability to Na+ (Normal: permeability to K+)
Hereditary stomatocytosis
Rhnull disease
Rod/cigar shaped
Narrower than ovalocytes
Defect in cytoskeleton
Page | 10

Ovalocyte

Schistocyte
Schizocyte
Fragmentocyte

Keratocyte
Knizocytes
Pinch cells
Dacryocyte
Teardrops
Microspherocyte
Pyropoikilocyte

Semilunar bodies
Half-moon cell
Crescent cell
Burns
Drepanocytes
Sickle cells
Menisocytes
Howell-Jolly bodies
Basophilic stippling
Punctate basophilia
PICA
Cabot rings

Membrane protein band 4.1


Egglike/oval-shaped
Wider than elliptocytes
Bipolar arrangement in Hgb
Cholesterol
Hereditary ovalocytosis
Megaloblastic BM
Myelodysplasia

Fragmentation produced by damage of RBC by fibrin, altered vessel


walls, prosthetic heart valves
Microangiopathic hemolytic anemia
= Presence of fibrin strands in small blood vessels clothesline-like
effect
Burns
TTP
DIC
Schistocyte w/ hornlike projections
Triangular cells
2 palor areas
Teardrop/pear-shaped
Squeezing and fragmentation during splenic passage
MMM
Hypersplenism
Severe burns
Hereditary microspherocytosis
Hereditary pyropoikilocytosis
= sensitivity to temperature
= RBCs fragment at 45C (Normal: 49C)
Large, pale pink staining ghost of the red cell
Membrane remaining after the contents have been released
Malaria
Spherocytes
Schistocytes
Microspherocytes
Crescent-shaped cell
Holly-leaf
Polymerization of deoxygenated Hgb
Sickle cell anemia
Sickle cell disease
Nuclear remnants of DNA (from karyorrhexis)
Feulgen (+)
Megaloblastic anemia
Precipitation of ribosomes and RNA
1. Fine stippling = polychromatophilia ( production of RBCs)
2. Course stippling = Lead poisoning
Pyrimidine-5-nucleotidase deficiency = Basophilic stippling
In children = Lead poisoning
In adults = IDA
Figure of 8
Remnant of microtubules of mitotic spindle
Page | 11

Heinz bodies

HbH inclusions
HbCC crystals

HbSC crystals
Ringed Sideroblast

Siderocyte

Pappenheimer bodies

Malaria

Babesia
Agglutination
Rouleaux

Megaloblastic anemia
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
Tetramer of beta globin chains (4)
Requires Supravital stain (RHH)
Bar of Gold
Clam shell appearance
Hexagonal w/ blunt ends and stain darkly
Solubility
Washington monument shape
Dark-hued crystal of condensed Hgb distorts the RBC membrane
Solubility
Nucleated RBC (immature) that contains nonheme iron particles
Requires Prussian blue stain
Sideroblastic anemia (Iron overload)
MDS
Non-nucleated RBC (mature) containing iron granules
(hemosiderin)
Sideroblastic anemia
MDS
Iron granules visible w/ Wrights stain
Resembles basophilic stippling
= PB: Periphery
= BS: Homogeneous
Unused iron deposits
Sideroblastic anemia
MDS
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
MOT: Tick bite
Maltese cross
Resemble P. falciparum rings
Cold agglutinin disease (anti-I)
Primary atypical pneumonia (anti-I)
RBCs in stack of coins arrangement
plasma globulin
Multiple myeloma: proliferation of Ig-producing plasma cells
(ESR)
Macroglobulinemia
Page | 12

Drop of NSS
Hemoglobinopathies
HbS
HbC
HbE
Sickle cell anemia
Sickle cell trait
Thalassemia
-thalassemia
-thalassemia
Cooleys anemia
Cooleys trait
Cellulose acetate Hgb
electrophoresis

Citrate agar Hgb


electrophoresis

Screening test for HbS

HbA2
HbF

Disperses rouleaux formation


True agglutination remains intact
Hemoglobinopathies
Qualitative defect in Hgb
Ex. Substitution
226 Glu Val
226 Glu Lys
2226 Glu Lys
80/90-100% HbS
HbF & HbA2
55-60% HbA1 |40-45% HbS
Thalassemia
Quantitative defect in Hgb
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)
Beta-globin chain is or absent
HbF and HbA2
Thalassemia major
Homozygous -thalassemia
Thalassemia minor
Heterozygous -thalassemia
Alkaline pH: 8.6
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)
Acid pH: 6.0-6.3
Migration: (Cathode > Anode)
F > A |Origin| O > S > C
E
D
G
1. Sodium metabisulfite = (+) Sickling of cells
2. Solubility test
= Sodium thiosulfite
= (+) Turbidity
in -thalassemia
Quantitation: Anion exchange microchromatography
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
Page | 13

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
Characteristics
Normal
1+
2+
3+
Limits
(per OIO)
(per OIO)
(per OIO)
(OIO)
Macrocytes (>9 m)
0-5
5-10
10-20
20-50
Microcytes (<9 m)
0-5
5-10
10-20
20-50
Hypochromia
0-2
3-10
10-50
50-75
Poikilocytosis
0-2
3-10
10-20
20-50
Polychromatophilia
-1-5
6-10
>10
Agg.
of
3-4
Agg.
of
5-10
Numerous
Rouleaux
-RBCs

Pelger-Huet

Hypersegmentation

Alder-Reilly granules

Toxic granules
Toxic vacuoles
Auer rods
Faggot cells
Chediak-Higashi
granules

May-Hegglin inclusion

RBCs

aggregates

4+
(per OIO)
>50
>50
>75
>50
---

Nuclear Abnormalities
Hyposegmentation (neutrophil)
Bilobed nucleus: Dumb-bell shaped/spectacle/peanutshaped/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
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
Page | 14

Dohle bodies
Dohle-Amato bodies

IT: Infections, Toxic


states
Jobs syndrome
Lazy leukocyte
syndrome
Chronic Granulomatous
Disease (CGD)

LE cell

Tart cell

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
Test: NBT dye test
Cells Exhibiting Phagocytosis
Neutrophil w/ large purple homogeneous round inclusion
Believe to be a neutrophil that ingested another neutrophil
Buffy coat
Smooth and evenly stained
SLE
Monocyte w/ ingested lymphocyte
Rough and unevenly stained

Abnormalities Involving Lymphocytes


a. Type I
= Turks irritation cell
= Plasmacytoid lymphocyte w/ large block of chromatin
b. Type II
= 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)
Page | 15
Reactive lymphocyte
Atypical lymphocyte
Stimulated lymphocyte
Variant lymphocyte
Downey cell

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
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/micromegakaryo Myelodysplastic syndromes
cyte
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
Acute Leukemias
staining results, cytogenetic studies and T & B lymphocytes marker
results
Page | 16

Acute leukemia
Acute leukemia in
children
Tests to differentiate
ALL from ANLL

L1
L2
L3

M1

M2

M3

M4
M5a
M5b
M6

M7

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
Acute Lymphoblastic Leukemias (ALL)
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 Nonlymphocytic Leukemias (ANLL)
Acute myeloblastic leukemia w/o maturation (AML w/o mat)
BM:
>30% blasts
<10% granulocytic cells
Acute myeloblastic leukemia w/ maturation (AML w/ mat)
BM:
>30% blasts
>10% granulocytic cells
Acute promyelocytic leukemia (APL)
>30% blasts
>10% granulocytic cells
>30% or >50% promyelocytes
(+) Faggot cells = Associated w/ DIC
Acute myelomonocytic leukemia (AMML)
Naegelis leukemia
20% to <80% monocytic cells
Acute monoblastic leukemia w/o maturation
Schillings leukemia
>80% monocytic cells (>80% monoblasts)
Acute monoblastic leukemia w/ maturation
>80% monocytic cells (<80% monoblasts)
Erythroleukemia
Erythremic myelosis
Di Guglielmos syndrome
>30% blasts
>50% erythrocytic precursors
Acute megakaryocytic leukemia
>30% blasts
>30% megakaryocytic cells
Page | 17

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
(+) Philadelphia chromosome: t(9+;22-) - both long arms
Myelogenous Leukemia If (-) Ph chromosome = poor prognosis
(CML)
Similar to leukomoid reaction, to differentiate:
a. Chromosome studies
b. LAP = ( in Leukomoid reaction, in CML)
2. Myelofibrosis w/
Fibrosis and granulocytic hyperplasia of BM, w/ granulocytic and
myeloid metaplasia
megakaryocytic proliferation in the liver and spleen
(MMM)
(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
BM: Panmyelosis
(PV)
PB: Pancytosis/Pancythemia
RBCs, WBCs, Plts
LAP (Other polycythemia: N-LAP)
Polycythemia
1 Absolute
Other names: Polycythemia Vera, Polycythemia Rubravera, Vaquez
polycythemia
Osler disease, Panmyelosis
RBC mass ( Hct)
RBCs, WBCs, Platelets
Erythropoietin (EPO)
2 Absolute
In response to hypoxia
polycythemia w/
In patients w/ pulmonary/cardiac disease
appropriate production
RBCs, WBCs, Platelets
of EPO
EPO
2 Absolute
In patients w/ tumors of kidney, liver, brain, adrenal and pituitary
polycythemia w/
gland
inappropriate
RBCs, N-WBCs, N-Platelets
production of EPO
EPO
Relative polycythemia
Spurious/Gaisbck 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
<5%
RS: Ringed
<1%
sideroblast (RARS)
>15% RS
sideroblast
RA w/ excess blast
<5%
5-20%
Page | 18

(RAEB)
RAEB in transformation
(RAEBt)
Chronic
Myelomonocytic
Leukemia (CMML)
LPD
1. T/B cell leukemia
2. Lymphoma

3. Hairy cell leukemia

4. Mycosis fungoides
and Sezary syndrome
Myeloperoxidase (MPO)

Sudan Black B (SBB)

Terminal
deoxyribonucleotidyl
Transferase (TdT)
Periodic Acid-Schiff
(PAS)

Naphthol AS-D
Chloroacetate esterase

-Naphthyl Acetate

>5%

20-30%

<5%

5-20%

Persistent
monocytosis

Lymphoproliferative disorders
Proliferation of cells derived from lymphoid stem cell T/B cells
-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
Leukemic reticuloendotheliosis
Originally B cells w/ hairlike projections
(+) TRAP: Tartrate-resistant acid phosphatase
Neoplastic T cells
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
DNA polymerase immunoperoxidase
Marker for immature lymphocytes
(+) ALL
(-) AML
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
Specific esterase
Marker for mature and immature neutrophils and mast cells
(+) AML
(-) AMoL [M5]
(-) ALL
Lasts for months
Nonspecific esterase
Page | 19

esterase

-Naphthyl Butyrate
esterase
Acid phosphatase

Tartrate resistant acid


phosphatase (TRAP)
Leukocyte alkaline
phosphatase (LAP)/
Neutrophil ALP (NAP)

Toluidine blue

Cooper & Cruickshank


Nitroblue tetrazolium
test (Original)

Modified NBT

Feulgen reaction

Marker for monocytes, megakaryocytes and plasma cells


(+) AMoL [M5] = inhibited by fluoride
(+) AMegL [M7]
(-) AML
Nonspecific esterase
Identifies monocytes, promonocytes and monoblasts
(+) AMoL [M5] = inhibited by fluoride
(-) AML
Present in all hematopoietic cells and found in lysosomes
(+) Hairy cell leukemia = TRAP (+)
(+) T cell leukemia
(-) Non-T cell leukemia
Cannot be stored
Excellent marker for hairy cell leukemia
Neutrophil is the only leukocyte that contains this activity
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
Binds w/ acid mucopolysaccharides in blood cells
Strongly metachromatic
Useful for recognition of mast cells and tissue basophils
(metachromatic granules)
CGL: Chronic granulocytic leukemia
Stain for basophils
Reagent: Toluidine blue
Test for CGD
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%
w/ stimulating agent
Test:
Buffy coat (WBC) + Bacterial suspension ---(NBT)---> (+)
Formazan
NV = 80-100% formazan
CGD = <50% formazan
Specific reaction for DNA
Page | 20

Prussian blue stain


Supravital stain (RHH)

BCB

Howell-Jolly bodies = Feulgen (+)


Stain for hemosiderin
(+) Sideroblast
(+) Siderocytes
RHH = NCB
Reticulum = New methylene blue
Heinz bodies = Crystal violet
Hemoglobin H = Brilliant cresyl blue
Stain for automated cell counter

Page | 21

Primary hemostasis
Platelets

Secondary hemostasis
Arteries
Veins
Capillaries
Maturation Stage
Megakaryoblast
Promegakaryocytes
Megakaryocytes
Metamegakaryocytes
Platelet structure

Platelet Adhesion

Bernard-Soulier
syndrome

HEMOSTASIS
Involves blood vessels and platelets
Formation of platelet plug
Test: Bleeding time
Functions:
Adhesion
Activation
Release
Aggregation
Involves the coagulation factors
Formation of stable fibrin clot
Test: Clotting time
Carry blood from the heart to the capillaries
Primary Hemostasis
Return blood from the capillaries to the heart
Injured vessel: vasoconstriction
= initiated by serotonin and thromboxane A2 derived from platelets
and endothelial cells
Cytoplasmic
Cytoplasmic
Nuclear
Thrombocyte
Granules
Tags
Features
s Visible
(-)
(+)
Single nucleus
(-)
Fine chromatin
(+) Nucleoli
Few
(+)
2 nucleus
(-)
Numerous
Usually (-)
2 or more
(-)
nuclei
Aggregated
(-)
4 or more
(+)
nuclei
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 adherence to exposed subendothelial surface (collagen)
Occurs in the presence of von Willebrand factor
In vivo: collagen
In vitro: glass
(-) gpIb = receptor for vWF
Page | 22

(Giant platelet
syndrome)
Von Willebrand disease
Platelet Activation

(-) vWF = for platelet adhesion


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
Serotonin (d)
vasoconstriction
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 ()
C1 esterase inhibitor ()
Page | 23

Platelet aggregation
Glanzmanns
thrombasthenia
Petechiae
Purpura
Ecchymosis
Epistaxis
Hemarthrosis
Hematemesis
Hematoma
Hematuria
Hemoglobinuria
Melena
Menorrhagia
Hereditary hemorrhagic
telangiectasia
(Oslwer-Weber-Rendu
disease)
Congenital
hemangiomata
(Kasabach-Merritt
syndrome)
Ehler-Danlos syndrome
Marfans syndrome
Pseudoxanthoma
elasticum
Senile purpura
Scurvy
Henoch-Schonlein
purpura
Thrombocytopenia

Thrombocytosis

Platelet adhesion

Platelet attachment to each other


Requires fibrinogen and Ca2+
(-) gpIIb/IIIa complex: receptor for fibrinogen
Pinpoint hemorrhagic spots
Hemorrhage of blood into small areas of skin
Blood escapes into large areas of skin
Nosebleed
Leakage of blood into joint cavities
Vomiting of blood
Swelling or tumor in the tissues or a body cavity that contains
clotted blood
RBC in urine
Hgb in urine
Stool containing dark red or black blood
Excessive menstrual bleeding
Vascular Disorders
Most common inherited vascular disorder
Blood vessels are thin & lack smooth muscle
Tumor composed of blood vessels

vascular fragility
Elastic fibers are calcified & structurally abnormal
Degradation of collagen & elastin
(-) Vitamin C = for collagen synthesis
Defective synthesis of collagen
Immunologic damage to endothelial cells
Quantitative Platelet Disorders
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
Reactive = moderate increase, asymptomatic (after hemorrhage,
splenectomy)
Autonomous = marked increase, associated w/
thrombotic/hemorrhagic complications (Ex. ET: platelet function is
abnormal)
Qualitative Platelet Disorders
1. vWD = (-) vWF
Platelet aggregation test:
= Normal: Epinephrine, Collagen, ADP
= Abnormal: Ristocetin
Page | 24

Acquired

Platelet count

Unopette

Platelet estimate
(Wedge smear)
WBC estimate (HPF)

2. BSS = (-) gpIb


(+) Giant platelets
Platelet aggregation test:
= Normal: Ristocetin
= Abnormal: Epinephrine, Collagen, ADP
3. Storage pool defects
= Gray platelet (), HP, WAS, CHS (d)
Uremia: toxic metabolites
Paraproteinemias: coating of platelet membrane w/ abnormal
protein
AML: abnormal megakaryocytes
MPD
Drugs: Aspirin = inhibits COX

Laboratory Tests for Primary Hemostasis


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
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
8-20 platelets/OIO
Factor: 20,000
Factor: 2,000
Platelet Estimates (Smear)
Page | 25

Platelet Estimate of
0-49,000/L
50,000-100,000/L
100,000-150,000/L
150,000-199,000/L
200,000-400,000/L
401,000-599,000/L
600,000-799,000/L
>800,000L
N-Plt count, BT
Plt count, N-BT
Plt count, BT
Platelet aggregation
test

Platelet adhesiveness
(Salzmann)

Clot retraction time

Capillary resistance
test
Touriquet test
Rumpel-Leedes test

Bleeding time

Coagulation factors

Report Platelet Estimate as


Markedly decreased
Moderately decreased
Slightly decreased
Low normal
Normal
Slightly increased
Moderately increased
Markedly increased
Qualitative platelet abnormality
Primary vascular abnormality
vWD
Autoimmune thrombocytopenia
Simultaneous quantitative and qualitative platelet deficiency
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)
Determination of in vitro platelet adhesion
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
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%
Measures capillaries to resist pressure
Correlates w/ the degree of thrombocytopenia
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
In vivo measure of primary hemostasis
a. Duke method = fingertip/earlobe
b. Ivy method = uses blood pressure cuff (40mmHg) puncture
forearm
Secondary Hemostasis
All are produced in the liver except Factor VIII
Page | 26

Numeral
I
II
III
IV
V
VII
VIII:C

IX
X
XI
XII
XIII

Stage I: Generation of
Thromboplastin
Stage II: Conversion of
prothrombin to
thrombin
Stage III: Conversion of
fibrinogen to fibrin clot
Fibrinogen
Calcium

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
Preferred Name
Synonyms
Fibrinogen
Prothrombin
Prethrombin
Tissue factor
Tissue thromboplastin
Calcium
Proaccelerin
Labile factor
Accelerator globulin (aCg)
Proconvertin
Stable factor
Serum prothrombin conversion
accelerator (SPCA)
Antihemophilic factor
Antihemophilic factor globulin
(AHG)
Antihemophilic factor A
Platelet cofactor 1
Plasma thromboplastin
Christmas factor
component
Antihemophilic factor B
Platelet cofactor 2
Stuart-Prower factor
Stuart factor
Prower factor
Autoprothrombin III
Plasma thromboplastin
Antihemophilic factor C
component
Hageman factor
Glass factor
Contact factor
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
Intrinsic: XIIXIIXVIII (Cofactor)
Extrinsic: IIIVII
Common: XV (Cofactor)
----> Common thromboplastin/Prothrombinase (Va-Xa-Ca2+-PL)
II ---(Prothrombinase)---> Thrombin
I ---(Thrombin)---> Fibrin clot
XIII---(Thrombin)---> XIIIa
Fibrin clot ---(XIIIa)---> Stable fibrin clot
Most concentrated
Involved in all stages of coagulation except contact phase
Page | 27

Contact group

Fibrinogen group

Prothrombin group

Diseases

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
I, V, VIII, XIII
Ca2+ dependent
Vit. K independent
Completely consumed during coagulation
(+) in plasma
(-) in serum
II, VII, IX, X
Ca2+ and Vit. K dependent
First: VII IX X II: Last
Adsorbable factors: removed by adsorbing agents [BaSO 4, Al(OH)3]
(+) in plasma
(-) in serum
BT
PT
APTT
Stypven
TT
Duckert
s
N
N
N
N
N

Disease of 1
hemostasis
Fibrinogen deficiency
N*
Prothrombin deficiency
N
Parahemophilia
N
Factor VII deficiency
N
Hemophilia A
N
N
von Willebrand disease
N
Hemophilia B
N
N
Factor X deficiency
N
Hemophilia C
N
N
Factor XII deficiency
N
N
Factor XIII deficiency
N
N
DIC
*BT may be prolonged in afibrinogenemia
Fresh Plasma

Aged Plasma

I
+
+
II
+
+
V
+
VII
+
+
VIII
+
IX
+
+
X
+
+
XI
+
+
XII
+
+
XIII
+
+
Prothrombin:
80% is consumed during coagulation
<20% residual prothrombin

N
Adsorbed
Plasma
+
+
+
+
+
+

N
N
N
N
N
N
N

N
N
N
N
N
N
N
N
N
N
Abn
Abn

N
N
N
N
N
N
N
N
N
N

Fresh Serum

Aged Serum

+ (<20%)
+
+
+
+
+
-

+
+
+
+
+
-

Page | 28

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
Endothelium
coagulation inhibition
Platelet
Protein C
Degrades factor Va and VIIIa
Protein S
Vit. K dependent glycoprotein
Antithrombin
Major inhibitor of thrombin
Tests for Secondary Hemostasis
Clotting time
Measure the period required for free form of blood to clot after it
has been removed from the body
a. Capillary blood method
= Drop or slide
= Capillary tube/Dale and Laidlaw
= Blue capillary tube
NV = 2-4mins
b. Whole blood/Lee and White
Page | 29

Prothrombin time

Activated partial
thromboplastin time

Stypven time/Russel
viper venom time

Thrombin time

Reptilase time

Duckerts test
5M urea solubility test

Circulating
anticoagulants
Lupus inhibitor
Tilt tube method

NV = 7-15mins
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
Detect coagulation factor deficiencies involving intrinsic and
common pathway
PPP + APTT rgt:
1. Activators:
a. Micronized silica
b. Ellagic acid
c. Celite
d. Kaolin
2. Phospholipids: substitute for platelets
3. CaCl2
Begin timing for clot formation on the addition of CaCl 2 rgt
NV = 25-35 secs
East Indian viper venom Vipera russelli: directly activates factor X
Detect coagulation factor deficiencies involving common pathway
PPP + Stypven rgt: platelin-CaCl2 rgt
NV = 6-10 secs
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
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
For factor XIII deficiency
Rgt: 5M Urea
Substitutes for urea:
- 1% monochloroacetic acid
- 2% acetic acid
Normal = Clot is insoluble to urea (24 hrs)
F. XIII def. = Clot is soluble to urea (24 hrs)
Prolonged APTT and PT not corrected
Against the phospholipid portion of prothrombinase complex (VaXa-Ca2+-PL)
Instrumentation for Tests of Hemostasis
Visual detection of fibrin clot formation
Page | 30

Fibrometer

Photo-optical detection
of clot formation

Fibrinolysis
Plasminogen activators

Inhibitors of fibrinolysis

Degradation of crosslinked fibrin by plasmin

Degradation of noncrosslinked fibrin and


fibrinogen by plasmin

Primary fibrinolysis

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

(Plasmin)

Fragment Y | Fragment D

(Plasmin)

Fragment D | Fragment D
No fibrin monomer
Page | 31

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

Warfarin
Coumarin
Coumadin
INR

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/ H 2O &
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
Oral
WARF: Wisconsin Alumni Research Foundation
Action: Vit. K antagonist, inhibits II, VII, IX, X
Monitoring: PT (reported in INR)
Neutralize w/ Vitamin K, FFP
International normalized ratio
INR = (Patient PT Normal PT)ISI
INR = 2-3
- Prevents MI, embolism & thrombosis
Page | 32

ISI

INR = 2.5-3.5
- For patients w/ mechanical heart valves
International Sensitivity Index (PT rgt)
PT rgt is calibrated w/ Manchester rgt = from human brain
thromboplastin

Page | 33

Brightfield microscopy
Oil immersion
microscopy
Phase-contrast
microscopy
Fluorescence
microscopy
Electron microscopy
Basic component of
Standard light
microscope

Total magnification

Hemoglobin

Acid hematin
Alkali hematin
Gasometric (Van Slyke)
Chemical
(Kennedys, Wongs)
SG method
Cyanmethemoglobin/

HEMATOLOGY PROCEDURES
Examine blood films
Erythrocyte morphology & leukocyte differential
For manual platelet counts
ANA and T/B cell studies
Observation of fine ultrastructures of cells (100,000x
magnification)
Diopter rings: adjust for focusing differences between eyes
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
TM = Ocular x Objective
(10)(LPO: 10) = 100x
(10)(HPO: 40) = 400x
(10)(OIO: 100) = 1,000x
Hemoglobin Determination
AM, PM
Strenuous muscular activity
Altitude = Hgb
Lying down
Rgt: 0.1N HCl
Comparing the brownish yellow color of solution to standard
comparator block
Rgt: 0.1N NaOH
Not for newborns (HbF: alkali resistant)
1g Hgb = 1.34mL O2
1g Hgb = 3.47mg Fe2+
CuSO4 method (See BB notes)
Manual and automated
Page | 34

Hemiglobincyanide
method

Rule of three

Macromethods

Wintrobe tube

Micromethod (Adam)

Dilution = 0.02mL blood: 5mL rgt (1:251)


Reagents:
Drabkins reagent (Brown container)
a. Potassium ferricyanide = ferrous ferric
b. Potassium cyanide = Hi HiCN
c. Nonionic detergent = lysis of red cells, decreases turbidity
d. Sodium bicarbonate (Orig. 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)
3 x RBC = Hgb
3 x Hgb = Hct
Apply only to normocytic, normochromic red cells
Hematocrit Determination
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
Length = 11.5cm
Bore = 3.0mm
Calibration = 0-100
a. Left: 0-100 (ESR)
b. Right: 100-0 (Hct)
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
Page | 35

Automated methods

RBCs
RBC diluting fluids

RBC count
WBCs
WBC Diluting fluids

WBC count
Fuchs Rosenthal

Speirs Levy

Improved Neubauer

RBC count

3rd: Leukocytes
4th: Retics & nRBCs
5th: Mature RBCs
Bottom: Clayseal (4-6mm)
Hct = only computed
Hct = MCV x RBC count
RBC Count
AM, PM
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/mm3 = # RBC x AF x DCF x DF = # RBC x 5 x 10 x 200 = #
RBC x 10,000
WBC Count
AM, PM
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) + 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/mm3 = # WBC x AF x DCF x DF
Counting Chamber
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
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
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
Center square:
w/ 25 3 square
Each 3 square w/ 16 small squares
Page | 36

WBC count
Nucleated RBCs

25 x 16 = 400
5 (counted) x 16 = 80 small squares
4 corners:
Each 2 square w/ 16 3 squares
4 x 16 = 64 3 squares
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
0
0
25 (30-40 )
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
Time blood smears should be stained
specimen collection
pH 6.8
Blood and bone marrow staining
pH 7.2
Malarial parasite staining
Excessively blue stain
Thick films
Prolonged staining time
Inadequate washing
Too high alkalinity of stain
Diluents tends to cause excessive basophilia
Excessively pink stain
Insufficient staining
Prolonged washing time
Mounting coverslips before they are dry
Page | 37

Cross-sectional or
crenellation method
Longitudinal method
Battlement method
WBC Counting

PV patients

Anemic patients

Neutrophils
Lymphocytes
Monocytes
Eosinophils
Basophils
Neutrophilia
Eosinophilia

Lymphocytosis

Monocytosis

Too high acidity of the stain


Buffer may cause excessive acidophilia
Blood film is moved from side to side
WBCs are counted from the tail toward the head of the smear
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
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)
Thinner smear:
- smaller blood drop
- slow spread
- low angle
Thicker smear:
- larger blood drop
- fast spread
- increase angle
Relative = 47-77%
Absolute = 1.8-7.8 x 109/L
Relative = 20-40%
Absolute = 1.0-4.8 x 109/L
Relative = 2-10%
Absolute = 0.01-0.8 x 109/L
Relative = 0-6%
Absolute = 0-0.6 x 109/L
Relative = 0-1%
Absolute = 0-0.2 x 109/L
Appendicitis
Myelogenous leukemia
Bacterial infections
Allergies
Scarlet fever
Parasitic infections (T. spiralis)
Eosinophilic leukemia
Viral infections
Whooping cough
IM
Lymphocytic leukemia
Lymphoma
Brucellosis
Tuberculosis
Monocytic leukemia
SBE
Typhoid
Rickettsial infections
Collagen disease
Hodgkins disease
Page | 38

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

Thorns test

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
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
Assess adrenocortical function
Sample 1: fasting specimen
Page | 39

RBC indices
MCV
MCH
MCHC
Defective centrifuge

MCHC >38% does not


occur
MCHC will not fall
<22%
ESR
ESR

ESR

Stages of ESR

Wintrobe & Landsberg

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)
Classify anemia according to RBC morphology
MCV = (Hct RBC) x 10
NV = 80-100 fL (old: m3)
MCH = (Hgb RBC) x 10
NV = 27-32 pg (old: g)
Rarely used
MCHC = (Hgb Hct) x 100
NV = 31-36% (31-36 g/dL)
Values affected:
= Hct
= MCV
= MCHC
Incorrect calculation
(+) cold agglutinins
Lipemia
(+) HbS & HbC
Erythrocyte Sedimentation Rate (ESR)
Nonspecific measurement used to detect & monitor an
inflammatory response to tissue injury
Erythrocytes:
*Macrocytes
*Anemia
Plasma composition: most important determinant
*Fibrinogen
*1-globulin
*2-globulin
*-globulin
*-globulin
*Cholesterol
Technical factor:
*Tilting = 30 angle = 30% error
*Temp.
Erythrocytes:
*Microcytes
*Poikilocytes
*Polycythemia
*Anisocytes
Plasma factor: most important determinant
*Albumin
*Lecithin
Technical factor:
*Overanticoagulation = EDTA = shrinkage of RBC = Hct, ESR
10 mins = 1. Initial rouleaux
40 mins = 2. Rapid settling of RBCs
10 mins = 3. Final sedimentation of RBCs
60 mins = Total
Requires smaller amount of blood
Involves no dilution
Length: 11.5cm (115mm)
Page | 40

Standard/Original
Westergren

Modified Westergren
Zeta Sedimentation
Ratio (ZSR)
Erythrocyte Osmotic
Fragility test
(Griffin and Sanford
method)

Ascorbate cyanide
screening

G6PD fluorescent
screening
Paroxysmal nocturnal
hemoglobinuria (PNH)
Sucrose hemolysis test

Hams acidified serum


test

Patient has received


normal RBCs
PCH
Donath-Landsteiner
test

Internal bore: 3.0mm


Anticoagulant: Double oxalate
Most sensitive
Requires more blood
Length: 300mm
Internal bore: 2.65 0.15mm
Anticoagulant: Citrate (black)
Anticoagulant-to-Blood ratio = 1:4
Anticoagulant: 2mL EDTA + 0.5mL NSS/Citrate
Not affected by anemia
Major disadvantage: requires special capillary tubes and Zetafuge
ZSR = (%Hct %Zetacrit) x 100
Anticoagulant: Heparin
% NaCl = # drops NaCl x 0.02
Add RBCs, stand for 2hrs at room temp
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%)
Detects deficiencies in the pentose phosphate pathway:
- G6PD
- glutathione peroxidase
- glutathione reductase
Rgts:
- Na ascorbate
- Na cyanide
Normal = red
(-) Enzyme = brown
G6P + NADP ---(RBC: G6PD)---> 6-phosphogluconate + NADPH
(fluorescence)
Normal: Max fluorescence at 10mins
G6PD def: Little or no fluorescence
Acquired disorder in w/c red cells are abnormally sensitive to
complement
(-) DAF
Screening test for PNH
Patient RBCs + ABO compatible serum + sucrose solution
Normal = (-) Hemolysis
PNH = (+) Hemolysis
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 w/ PNH + blood transfusion ---(Hams test)---> Hemolysis
IgG autoanti-P = biphasic hemolysin
- Cold = attaches to RBCs
- Warm = RBC lysis
Test for PCH
Ctrl: Patient WB incubate at 37C for 30mins incubate at 37C for
Page | 41

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
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/L
Microcytic RBCs
Hemoglobin
HbCO >10%
Clotting
Cryoglobulin
Sulfhemoglobin
Cryofibrinogen
Hemolysis
Heparin
WBC >50,000/L
Hyperbilirubinemia
Lipemia
Monoclonal proteins
Hematocrit
Cryoglobulin
Autoagglutination
(automated)
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
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MCHC

Cryoglobulin
Cryofibrinogen
Heparin
Clotting
Hemolysis
Autoagglutination
Defibrinated blood

Optical light scattering

Electrical impedance

Coulter counter

Histograms

Ohms law

Microcytic RBCs
Swollen RBCs
WBC >50,000/L
Spuriously low Hgb
Spuriously high Hct

Autoagglutination
Clotting
Hemolysis
Spuriously high Hgb
Spuriously low Hct
Increased: WBC count, RBC count, Platelet count, Hgb, Hct
Decreased: MCV
Increased: WBC count, Hgb
Decreased: Platelet count
Increased: MCHC
Decreased: WBC count, RBC count, Platelet count, Hgb, Hct
Increased: Hgb, MCHC, Platelet count
Decreased: RBC count, Hct, MCV
Increased: MCV, MCHC
Decreased: RBC count, Hct
Blood Glass Beads/clips
Tests: OAA
- OFT
- Autohemolysis test
- Acidified serum test
Automated Cell Counter
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
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
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
RBCs, WBCs, plts
X-axis
- Horizontal/abscissa
- Size of cells
Y-axis
- Vertical/ordinate
- Number of cells
V=IxR
Where:
V = voltage
I = current
R = resistance
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Positive error

Negative error
Polychromasia grading

Normocytic,
Normochromic RBCs

Hemolytic anemias

Count: BEA
Bubbles
Extraneous electrical pulses
Aperture plug
Count
Improper setting of aperture error
% of RBCs that are polychromatophilic
Slight = 1%
1+ = 3%
2+ = 5%
3+ = 10%
4+ = >11%
1. Defective formation of RBCs or the presence of tumor cells in
BM:
*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
1. Intrinsic defects w/in RBC
a. Hereditary membrane defects
**Spherocytosis
**Elliptocytosis
**Acanthocytosis
**Stomatocytosis
**Rh null disease
b. Hereditary enzyme defects
**G6PD
**PK
c. Hereditary hemoglobinopathies
**Sickle cell disease
**Hemoglobin C disease
d. Unstable hemoglobin disease
**Hemoglobin E disease
e. Hereditary defective globin synthesis
**Thalassemia
f. Acquired
**PNH
2. Extracorpuscular causes: nonimmune acquired hemolytic
anemias
*Chemicals, toxins, venoms
*Physical trauma: disorders causing fragmentation (burns, cardiac
replacement valves, MAHA, HUS)
3. Extracorpuscular causes: immune hemolytic anemias
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*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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