Professional Documents
Culture Documents
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)
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
Page | 2
Adults
Neonates
Marrow specimens
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
2. Basophilic
normoblast
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)
Page | 4
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)
Page | 5
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)
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
Page | 7
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
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
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
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
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
HbA2
HbF
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
LE cell
Tart 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
(RAEB)
RAEB in transformation
(RAEBt)
Chronic
Myelomonocytic
Leukemia (CMML)
LPD
1. T/B cell leukemia
2. Lymphoma
4. Mycosis fungoides
and Sezary syndrome
Myeloperoxidase (MPO)
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
Toluidine blue
Modified NBT
Feulgen reaction
BCB
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
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
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)
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)
Capillary resistance
test
Touriquet test
Rumpel-Leedes test
Bleeding time
Coagulation factors
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
Contact group
Fibrinogen group
Prothrombin group
Diseases
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
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
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)
(Plasmin)
Fragment X
(Plasmin)
Fragment Y | Fragment D
(Plasmin)
Fragment D | Fragment D
No fibrin monomer
Page | 31
Secondary fibrinolysis
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)
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
% 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
ESR
Stages of ESR
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
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
Page | 42
MCHC
Cryoglobulin
Cryofibrinogen
Heparin
Clotting
Hemolysis
Autoagglutination
Defibrinated blood
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
Page | 43
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
Page | 44
Page | 45