BOARD REVIEW IN HEMATOLOGY

HEMOSTASIS o Hyalomere – surrounds the

chromomere, nongranular and clear to
Definition: The process by which the body stops bleeding light blue
and maintains blood in the fluid state within the vascular • Discoid-shape
compartment.
NOTES:
5 General Components: • Endomitosis – nuclear division without cytoplasmic
1. Vascular system division.
2. Platelets • 1 megakaryocyte = 2,000-4,000 platelets
3. Coagulation Factors • 8-11 days – platelet life-span
4. Fibrinolytic Factors • Circulating platelets represents 2/3 and the remaining
5. Naturally Occuring Inhibitors 1/3 are stored in the spleen.
• Thrombopoietin – increases platelet production
2 Phases:
A. Primary Hemostasis PLATELET STRUCTURE
• Reversible A. Peripheral Zone
• Response of blood vessels and platelets to 1. Glycocalyx
tissue injury.

B. Secondary Hemostasis
• Irreversible
• Involves participation of clotting factors.

MEGAKARYOCYTE-PLATELET SYSTEM

Platelets: Maturation sequence of megakaryoblast takes

about 5 days. Platelets are produced directly from the
megakaryocyte cytoplasm. As the megakaryocyte
matures, clusters of granules aggregate to form platelets.

1. Megakaryoblast
• 20-50 um diameter
• blue cytoplasm
• N/C ratio is about 10:1
• Multiply nucleoli
• Fine chromatin • Amorphous exterior coat or outer
2. Promegakaryocyte membrane
• 20-60 um diameter • Contains glycoproteins that acts as a
• less basophilic cytoplasm receptor and useful in aggregation and
• chromatin becomes coarse adhesion.
• irregularly shaped nucleus, may show slight 2. Plasma Membrane
lobulation • Consist of 30 or more glycoproteins
• N/C ratio is 4:1 to 7:1 • Phospholipid bilayer
• Demarcating Membrane System (DMS) 3. Submembranous Area
appears B. So-Gel Zone
3. Mature Megakaryocyte 1. Microtubules
• 40-120 um in diameter • Tubulin – maintains platelet shape
• cytoplasm contains coarse clumps of granules • For the discoid shape of the platelet
aggregating into little bundles, which bud off 2. Microfilaments
from the periphery to become platelets • Contains the contratile protein known as
• multiple nuclei are present thrombosthenin (15%).
• no nucleoli is visible • Thrombosthenin: Actin and myosin or
• N/C ratio is less than 1:1 ctinomyosin – responsible for clot
4. Metamegakaryocyte retraction.
• Disintegrated cells surrounded by platelets C. Organelle Zone
• Platelet producing stage 1. Alpha Granules – amplifys reaction to injury;
5. Platelet or Thrombocyte 20-200/platelet.
• 1-4 um diameter • PF4 – inhibits heparin
• light blue to purple, very granular • Beta-thromboglobulin (BTG) – inhibits
o Chromomere – granular and located heparin
centrally
1
 • Platelet-derived Growth Factor (PDGF) – • Collagen Type I and III
promotes growth and proliferation of o Synthesized by smooth muscle
smooth muscles. o Located in deeper regions of blood vessel
• Thrombospondin (TSP) – (+) aggregation o Promotes adh esion
• Chemotactic Factor o Facilitates aggregation and release
• Permeability Factor • Collagen Type IV and V
• Bactericidal Factor o Synthesized by endothelial cells
• Acid Hydrolases o Located below the endothelium
• Fibrinogen (FGN) o Promotes adhesion
• FVIII:vWF o Cannot facilitate aggregation
• FV • Thrombin – increases ADP release; TXA2 synthesis;
2. Dense Granules/Bull’s Eye Granules and coagulation.
• Calcium • Aspirin inhibits cyclooxygenase and prolong BT.
• ADP - (+) aggregation
• Serotonin - vasoconstriction PRIMARY HEMOSTASIS
• ATP I. Vascular System
• Catecholamines (EP/NEP) A. Arteries
D. Membranous System B. Veins
1. Dense Tubular System C. Capillaries
• Site of arachidonic acid metabolism II. Platelets: Formation of Primary Hemostatic Plug
• Site of prostaglandin synthesis and Ca++ A. Adhesion
sequestration • Process where platelet stick to foreign
• Derived from smooth ER of megakaryocyte surface receptors:
2. Open Canalicular System o GpIb – receptor for vWF
• For release of granules o vWF – bridge between platelet and
collagen.
PLATELET CYCLOOXYGENASE PATHWAY o Gp1a – directly binds collagen in less
extent.
Arachidonic acid B. Activation
↓ cyclooxygenase • Morphologic and functional changes in
Prostaglandin Endoperoxide platelets.
↓ thromboxane synthetase C. Secretion
Thromboxane A2 • Release of granules that promotes
aggregation.
PLATELET LIPOOXYGENASE PATHWAY D. Aggregation
A. Blood Vessel • Interaction and adhesion of platelets to one
another to form initial platelet plug.
Linoleic Acid • Occurs in the presence of calcium and
↓ lipooxygenase fibrinogen
Monohydroperoxide • GpIIb/IIIa – receptor for fibrinogen
↓ peroxidase
13 HODE (13-hydroxyoctadecadrenoic acid) SECONDARY HEMOSTASIS
– Involves coagulation factors
13-HODE
• Inhibits adhesion • Blood factors are classified as:
• Released by intact blood vessel into endothelial 1. Substrate, substance on which enzyme acts
lining 2. Zymogen, enzyme precursor
• chemorepellant 3. Cofactor, component that aids in the activation of
zymogen to active enzyme
B. Platelet 4. Calcium
• In conversion of zymogens to enzymes, either they
Arachidonic Acid are serine proteases (II, VII, IX, X, XI, XII), which
↓ kipooxygenase uses serine as the active site and cleave peptide
Monohydroperoxide bonds, or they create covalent bonds as
↓ peroxide transaminases.
12-HETE (hydroxyeicosatetraenoic acid) • Blood factors are produced mostly in the liver and
circulate in an inactive precurson form.
12-HETE – promotes adhesion
Serine Proteases: II, VII, IX, X, XI, XII,
NOTES: Prekallikrein, HMWK
• Mitochondria – source of ATP for platelet function
(10-60/platelet) Cofactor: V and VIII
• TXA2 – promotes aggregation and vasoconstriction
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 COAGULATION FACTORS XII Serine Pretease Intrinsic No Yes
Numeral Name Synonym XIII Transglutaminase Common No Yes
Prekal Serine Pretease Intrinsic No Yes
I Fibrinogen HMWK Serine Pretease Intrinsic No Yes
II Prothrombin Prethrombin
III Tissue Factor Tissue Thromboplastin DISORDERS OF COAGULATION CAUSING CLOTTING
IV Calcium FACTOR DEFICIENCIES
V Proaccelerin Accelerator globulin Inherited Coagulopathies
Acquired
(aCg) Factor Inheritance
Coagulopathy Coagulopathy
Pattern
VII Proconvertin Stable factor, SPCA
VIII:C Antihemophilic Antihemophilic globulin I Autosomal Afibrinogenemia Severe liver Disease
recessive DIC
Factor Antihemophilic Factor A Autosomal Dysfibrinogenemia Fibrinolysis
Platelet Cofactor A dominant
IX Plasma Christmas Factor II Autosomal Prothrombin Liver Dse.
Thromboplastin AHF-B recessive deficiency Vit. K Deficiency
Component (PTC) Platelet Cofactor B Anticoagulant
Therapy
X Stuart-Prower Factor Stuart Factor
V Autosomal Factor V Def. Severe liver Disease
Prower Factor recessive (OWRENS’s Dse.) DIC
Autoprothrombin III Fibrinolysis
XI Plasma AHF-C VII Autosomal Factor VII Def. Liver Dse.
Thormboplastin recessive Vit. K Deficiency
Antecedent (PTA) Anticoagulant
Therapy
XII Hageman Glass Factor VIII X-linked Hemophilia A Severe liver Disease
Contact Factor recessive DIC
XIII Fibrin Stabilizing Laki-Lorand Factor Autosomal vWD Fibrinolysis
Factor Fibrinase dominant
Plasma transglutamase IX X-linked Hemophilia B Liver Dse.
recessive Vit. K Deficiency
Fibrinoligase Anticoagulant
Prekallikrein Fletcher Factor Therapy
HMWK Fritzgerald Factor X Autosomal Factor X Def. Liver Dse.
Contact activat’n Cofac. recessive Vit. K Deficiency
Williams Factor Anticoagulant
Therapy
Flaujeac Factor
XI Autosomal Hemophilia C *
recessive (common in Eastern
INHIBITORS OF COAGULATION European Jewish
descent/Ashkenazi
- major site of inhibition: endothelium and platelet Jews)
1. Protein C – degrades FVa and FVIIIa XII Autosomal Factor XII Def. *
2. Protein S – degrades FVa and FVIIIa recessive
3. ATIII – major inhibitor of thrombin, also inhibits FIXa, XIII Autosomal Factor XIII Def. Liver Dse.
recessive Vit. K Deficiency
Xa, Xia, XIIa, Kallikrein, and Plasmin.
Anticoagulant
4. Heparin Cofactor II – inhibit thrombin Therapy
5. α2-macroglobulin – forms a complex with thrombin, Prekall Autosomal Fletcher Trait *
kallikrein and plasmin, thus inhibiting their activities. recessive
6. Extrinsic Pathway Inhibitor (EPI) and Lipoprotein HMWK Autosomal Fritzgerald Trait *
Assoc. Coagulation Inhibitor (LACI) – inhibits the recessive
* Unclear whether any acquired disorders cause FXI and XII deficiencies or
VIIa-tissue factor and Kallikrein, it also inhibits FXIa Prekallikrein and HMWK deficiency.
and plasmin.
7. C1 Inhibitor – inactivator of FXIIa and Kallikrein, it CONDITIONING MOST OFTEN ASSOC. WITH
also inhibits FXIa and plasmin. PHYSIOLOGIC VARIATIONS IN COAGULATION AND
8. α1-antitrypsin – inhibitor or thrombin, Xa and Xia. FIBRINOLYTIC FACTORS
Condition Factor Increases Factor Decreases
CHARACTERISTICS OF COAGULATION FACTORS Stress, Tissue Necrosis, I
Present Inflammation
Pathway Vitamin K in BaSO4 Pregnancy I, VIII, IX, X XIII, XI, ATIII
Factor Active Form
Paticipation Dependent Adsorbed
Oral Contraceptives I, VIII, VII, IX, X
Plasma
Hypermetabolism I, VII, plasminogen
I Fibrin clot Common No Yes Vigorous Exercise VIII, XI, XII
II Serine Pretease Common Yes No Chronic Thrombocytopenia VIII
V Cofactor Common No Yes Hypothyroidism IX, XI, plasminogen
VII Serine Pretease Extrinsic Yes No Child birth I, VIII
VIII:C Cofactor Intrinsic No Yes Surgical operations,
IX Serine Pretease Intrinsic Yes No Trauma
X Serine Pretease Common Yes No Myocardia Infarction
XI Serine Pretease Intrinsic No Yes Acute illness

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 PLATELET DISORDERS II. Quantitative Platelet Disorders

PRIMARY HEMOSTASIS DISORDERS A. Thrombocytopenia

• Decreased platelet count due to:
I. Bleeding Disorders (Vascular Defects) a. Generalized BM suppression/Aplastic
Anemia
A. Hereditary Connective Tissue Defects – decrease in all cell types
– acquired thru ionizing radiation and
1. Ehler-Danlos Syndrome chemotherapeutic agents
• (+) hyperextensible joints and – Fanconi’s Anemia (congenital) –
hyperplastic skin renal glucosuria, mental retardation
• defect in peptidase enzyme that converts – Caused by chloramphenicol,
procollagen to collagen. benzene
• Increase vascular fragility b. Decrease platelet survival due to
increase platelet destruction
Note: Marfan Syndrome – increase vascular fragility. – DIC – mass consuption of platelet
– ITP – formation of AutoAbs for plt.
c. Increase platelet sequestration by the
2. Pseudoxanthoma Elasticum lesion
• Connective tissue elastic fibers in small – Splenomegaly
arteries are calcified and structurally 2/3 (70-80%) platelet – circulation
abnormal. 1/3 (20-30%) platelet - spleen
d. Selective Suppression of
B. Acquired Connective Tissue Defects Megakaryocytes
1. Scurvy (Vitamin C Deficiency) – Associated with chlorothiazide
• Deficiency in ascorbic acid which is e. TAR Syndrome
important in the formation of the intact – Seen in neonates
vascular basement membrane. – Absent or decreased and abnormal
• (+) gingival bleeding and hemorrhage into megakaryocyte in BM
subcutaneous tissues and muscles. – Congenital deformities of arm
• Vit. C is responsible for hydroxylation of f. Myelophthisic Process
lysine and proline during collagen – Space-occupying lesions in BM like
formation. fibrosis, leukemia etc.
2. Senile Purpura g. Ineffective thrombopoiesis assoc. with
• Degeneration of collagen, elastin, and ethanol abuse, sever IDA, and PNH
subcutaneous fat. h. May Hegglin Anomaly
– Wiskott-Aldrich Syndrome - Triad
C. Hereditary Alteration of Vessel Wall • Immune platelet destruction
1. Hereditary Hemorrhagic Telangiectasia a. Isoimmune/Post-Transfusion Purpura
• ESLER-WEBER-RENDU – Lack platelet-specific antigen
• Dilated small blood vessels with poor wall located in platelet membrane
support and decrease ability to contract glycoprotein III.
• Most common vascular wall defect b. Neonatal Isoimmune Thrombocytopenia
• Walls are thinner - caused by transplacental passage
• Telangiectasias are permanent of maternal IgG against fetal
2. Congenital Hemangiomata platelet Ags.
• KASABACH-MERRITT Syndrome c. Patients Refractory to Platelet
• Assoc. with tumors composed of vessels Tranfusion
that commonly swell and bleed at the - Characterized by failure to increase
surface. the platelet count after transfusion
d. Immune Thrombocytopenic Purpura
D. Endothelial Damage e. Dug0induced Thrombocytopenia
1. Autoimmune Vascular Purpura - Associated with quinine, quinidine,
a. Drug-induced Purpura sulfonamides, rifampicin,
• Aspirin, coumarin, penicillin, chloraquin.
sulfonamides, sedatives
b. Allergic Purpura B. Thrombocytosis (Splenectomy)
c. Henoch-Schoenlein Purpura a. Primary (Autonomous)
• Associated with abdominal pain • Increased in platelet; uncontrolled
secondary to GI bleeding proliferation of platelet
• Henoch – “abdominal” pain • Myeloproliferative (CML, PV, MMM,
secondary to GI bleeding ET)
• Schoenlein – “joint” pain b. Secondary (Reactive)
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 • Moderate increase platelet; – Lack alpha and dense
asymptomatic granules
• Ex. Splenectomy; after blood loss
Granules
Syndrome
III. Qualitative Platelet Disorders Alpha Dense
A. Hereditary
Gray-Platelet - +
1. Adhesion Defects
Wiskott-Aldrich + -
a. Bernard-Soulier Syndrome
Hermansky-Pudlack + -
• Lack of GpIb (receptor for vWF)
• (+) “giant platelets” (>20 um) Chediak-Higashi - -
b. vWD
• most frequently inherited
coagulopathy b. Granule Release Defects (Aspirin-like)
• lacks vWF • Prostaglandin Enzyme Deficiency
– Defect in cyclooxygenase;
Platelet Aggregation Test abnormal TXA2 activity
EAC Ristocetin – Irreversible
Bernard Soulier Normal Abnormal B. Acquired
vWD Normal Abnormal a. Aspirin (Acetyl Salicylic Acid) – inhibits
Glanzzman’s T. Abnormal Normal cyclooxygenase thru irreversible acetylation
of the enzyme’s active site.
Note: The interaction of vWF with the platelet is primarily
through the GpIb receptor, and both the vWF and SECONDARY HEMOSTASIS DISORDERS
GpIb must be present for normal platelet adhesion. I. Inherited Disorders of Coagulation
In vwD, it is the plasma component that is A. Intrinsic
defective, whereas Bernard-Soulier, it is the 1. Factor XII (Hageman) / HMWK
platelet. (Fritzgerald) / Prekallikrein (Fletcher)
Deficiencies
2. Aggregation Defects • DON'T suffer from bleeding disorder
a. Glanzmann’s Thrombasthenia (GT) 2. Factor XI (Hemophilic C/Rosenthal’s
• Lack of GpIIb/IIIa (receptor of Syndrome) Deficiency
fibrinogen) 3. Factor VIII:C Deficiency (Classic
• Failure of platelet to aggregate Hemophilia or Hemophilia A)
even with stimulus (EAC) • MOST severe
• (-) clot formation • Sex or crosslinked
b. Afibrinogenemia • Therapy:
• Similar to GT but fibrinogen is a. Cryoprecipitate (Prothrombin
lacking Complex Concentrates)
• Treated with cryoprecipitate o Contains FII, VII, IX, X
o Bypasses the need for
3. Secretion Defects FVIII:C
a. Storage Pool Defects o Reserve for life threatening
• Gray Platelet Syndrome situations
– Lacks alpha-granules b. DDAVP
• Wiskott-Aldrich Syndrome o An analogue of ADH that
– Characterized by triad of maybe substituted for blood
thrombocytopenia, recurrent components
infection, and eczema. o Increase plasma FVIII:C by
• Hermansky-Pudlak causing its release from
– Characterized by triad of endogenous stores.
tyrosine-positive o intravenous
oculocutaneous albinism, 4. Factor IX Deficiency (Hemophilia B or
accumulation of ceroid-like Christmas Disease)
pigment in macrophage, and • Therapy: FFP or FIX Concentrate
bleeding tendency. 5. vWF Deficiency
– Lack dense granules • deficiency of FVIII:C and vWF
• Chediak Higashi Anomaly • MOST inherited coagulopathy
– Characterized by albinism, • Therapy: Cryoprecipitate (the only
recurrent infection and giant component that contains FVIII:C)
lysosomes in all granule
contg. cells.

5
 B. Extrinsic 3. Protein S Deficiency
1. Factor VII Deficiency • Destroys Factor V and VIII
• Influenced by warfarin therapy
C. Common 4. Homocytinuria
1. Factor X Deficiency (Stuart-Prower)
• Therapy: FFP or PT Complex B. Secondary
Concentrate 1. Lupus Anticoagulant
2. Factor V Deficiency (Owren’s Dse) • Associated with high risk of throbosis due
3. Factor II Deficiency to prostacyclin inhibition.
4. Factor I Deficiency
5. Factor XIII Deficiency Factor Coagulation Tests
Deficiency
BT CT PT APTT Stypven TT Ducket’s
II. Acquired Disorders of Coagulation and
Fibrinolysis 1o Hemostasis ↑ N N N N N N
Fibrinogen N ↑ ↑ ↑ ↑ ↑ N
A. Hepatic Disease Prothrombin N ↑ ↑ ↑ ↑ N N
• Liver is the primary site of procoagulant, Parahemophilia N ↑ ↑ ↑ ↑ N N
fibrinolytic and inhibitory synthesis. Factor VII N ↑ ↑ N N N N
B. Vitamin K Deficiency Hemophilia A N ↑ N ↑ N N N
• Due to: decrease dietary intake vWD ↑ ↑ N ↑ N N N
Hemophilia B N ↑ N ↑ N N N
absence of normal flora in GIT Factor X N ↑ ↑ ↑ ↑ N N
obstructive jaundice Hemophilia C N ↑ N ↑ N N N
anatagonist drug (courmarin) Factor XII N ↑ N ↑ N N N
• Prolong APTT and PT Factor XIII N N N N N N Abnormal
C. Circulating Anticoagulant DIC ↑ ↑ ↑ ↑ ↑ ↑ Abnormal
NOTE: Factor VIII Deficiency – negative for bleeding tendency
• Majority are antibodies which is stimulated
by blood transfusion. PT = Extrinsic / Common
D. Therapeutic Anticoagulation APTT = Intrinsic / Common
• Coumarin (oral) – discovered in Wisconsin SUBSTITUTION STUDIES
• Warfarin – most frequently used; interferes SUBSTITUTION STUDIES
with the carboxylation of the Vit K- DEFICIENCY PT APTT TT Normal Adsorbed Aged
dependent plasma factors in the liver by Plasma Plasma Serum
interrupting the enzymatic phase which I Abn Abn Abn C C NC
II Abn Abn N C NC NC
results into non-functional proteins V Abn Abn N C C NC
circulating plasma – reffered to as Protein VII Abn N N C NC C
Induced by Vitamin K Anatagonist (PIVKA) VIII N Abn N C C NC
• Heparin (IV anticoagulant) IX N Abn N C NC C
X Abn Abn N C NC C
E. MaIIssive Transfusion Effects XI or XII N Abn N C C C
• Increase citrate (binds to calcium which
result to bleeding) Factor Fresh Aged Adsorbed Fresh Aged
Plasma Plasma Plasma Serum Serum
• Decrease labile factors and platelet in
stored blood. I     
F. DIC II    <20% 
• Increase: TCT, PT, APTT, FSP V     
• Decrease: Platelet count, FGN, ATIII VII     
• (+) D-Dimer Test VIII     
• Treatment: FFP, Platelet Concentrate, IX     
Cryoprecipitate
X     
XI     
XII     
THROMBOTIC DISORDERS (lack of inhibitors, intact BV)
XIII     
A. Primary Prothrombin Stypven
1. Antithrombin III Deficiency Time Time
• Recurrent venous thromboembolism Common (X, V, II, I) ↑ ↑
• Treated with heparin Extrinsic (VII) ↑ Normal

2. Protein C Deficiency NOTES:

• Recurrent superficial or deep vein • Factor V and VIII – decreased in storage
thrombophlebitis and frequent pulmonary • Factor II – consumed during coagulation; <20% residual prothrombin
• TT, normal Reptilase = patient on heparin therapy
emboli
• All coagulation factors are produced by liver except Tissue Factor,
• Influenced by warfarin therapy Calcium, and Factor VIII complex.
• Types I – decrease Ag level and activity • vWF are produced by megakaryocyte and endothelial cells
• Factor VIII:C (Factor VIII:Coagulant)
II – normal Ag level, dec. activity • Factor VIII Complex – a large multimer composed of FVIII:C which is
low MW portion and the FVII:vWF which is high MW portion;
noncovalently bound; composed of FVII:C, FVIII:C Ag, FVII:vWF,
FVIIIR Ag, FVIIIR:RCo.
6
STAGES OF SECONDARY HEMOSTASIS or 2. Tissue Type
COAGULATION CASCADE • Urokinase-like PA
I. Generation of Thromboplastin 3. Therapeutic Activators
• Intrinsic: XII – XI – IX – VIII • Streptokinase
• Extrinsic: III – VII • Urokinase
• Common: X–V • Tissue-like PA
• Common Thromboplastin/Prothrombinase
↓ INHIBITORS OF FIBRINOLYSIS
II. Factor II ---------------------------------- Thrombin 1. Alpha 2 Antitrypsin
• Major inhibitor of plasmin
thrombin • Neutralizes plasmin thus inhibiting it from
III. Factor I ------------------------ Fibrin (stabilized by binding to thrombin
XIII) • For slow and orderly dissolution of the clot and
adequate time for tissue repair.
CONTACT GROUP (XII, XI, HMWK, Prekallikrein) • Also inhibits XIIf, XIa, Thrombin, Kallirein, tPA
• Ca++ and Vintamin K independent 2. Alpha 2 Macroglobulin
• Initial phase of the intrinsic activation of ciagulation • Inhibits both coagulation and fibrinolysis
• NOT consumed during coagulation 3. Alpha 1 Antitrypsin
• NOT absorbed by BASO4 • Binds plasmin when both alpha-2-antiplasmin
and alpha-2-macroglobulin are saturated.
FIBRINOGEN GROUP (I, V, VIII, XIII) 4. Thrombospondin
• Also known as High Molecular Weight Group 5. PA inhibitor 1 and 2
• NOT absorbed by BaSO4
• Ca++ dependent PLASMIN
• Vitamin K independent • Main mediator of fibrinolysis hence it is also called
• Completely consumed during coagulation fibrinolysin
• Factor V and VIII are least stable thus NOT present in • Active form of the zymogen plasminogen
stored plasma • A broad spectrum endopeptidase that acts
• (+) plasma, (-) serum nonspecifically but has a strong affinity for fibrin.
• Cannot distinguish between fibrinogen and fibrin.
PROTHROMBIN GROUP (II, VII, IX, X) • Functions:
• Ca++ dependent o Destroy fibrin and fibrinogen
• Vitamin K dependent o Production of FDP
• ABSORBED by BASO4 o Destroy FV and VIII and others
• Not consumed during coagulation except Factor II o Indirectly enhace the conversion of FXII
• Halflife: (1st) VII – IX – X – II (last) o Stimulate the conversion of Prekallikrein to
kallikrein, liberating kinins from kininogen
CIRCULATING ANTICOAGULANTS o Cleaves C3 into fragments
• Prolonged PT and APTT not corrected
• Inactivate an activated coagulation factor or block PATHOLOGIC FIBRINOLYSIS
interaction between coagulation factors and platelets. 1. Primary Fibronolysis
• Example: Lupus Anticoagulant • Excessive amounts of plasminogen activators
o Non-specific anticoagulant from damaged cells/malignant cells.
o IgG, IgM, IgA which interfere with • Converts plasminogen to plasmin in the
phospholipid complex of the absence of fibrin formation.
prothrombinase complex. • Example: Prostatic CA – NO fibrin monomer
o Platelet neutralization procedure, Dilute NO Fibrin polymers
Russell Viper venom time NO D-Dimer
2. Secondary Fibrinolysis
FIBRINOLYSIS • DIC: uncontrolled, inappropriate formation of
fibrin within the blood vessels
Definition: clot dissolution; the physiologic process of Infection
removing unwanted fibrin deposits; a gradual progressive Neoplasm
enzymatic cleavage of fibrin to soluble fragments which Snake Bite
are then removed by the RES; begin with the activation of HTR
plasminogen to plasmin. • WITH fibrin monomer
WITH fibrin polymers
PLASMINOGEN ACTIVATORS WITH D-Dimer
1. Intrinsic Activators
• Factor XIIa
• Kallikrein
• HMWK

7
 ANTICOAGULANT THERAPY 2. 2o Hemostasis – clotting factors of blood – end point
Warfarin/Coumadin is clot formation.
Heparin (IV)
Coumarin (Oral)
Action Inhibit thrombin Vit. K antagonist Factors that promotes premature activation of
coagulation prior to testing:
Neutralized Protamine Sulfate Vit. K and/or FFP
1. Tissue thromboplastin
Monitoring APTT PT
• Potent clot-activating substance
• Found in fluids that escaped from injured cells
INTERNATIONAL NORMALIZED RATIO (INR) and tissue spaces
• Formula: 2. Contact with glasses
INR = PT of Patient ISI • Contact factors (Prekallikrein, HMWK, XII, XI)
Normal PT are activated prematurely
where; ISI – International Sesitivity Index Note: Recommended materials – plastic,
- Calibrated PT rgt. with Manchester rgt. polyesterene or silicone-coated glass
(derived from human brain 3. Temperature
thromboplastin) • Affect labile factors (V and VIII) by deterioration
• 2-3 INR = prevent MI, embolism, and thrombosis when left at room temperature
• 2.5-3.5 INR = mechanical/prothetic heart valves • Activate factors VII and XI at cold temperature
4. Hemolysis – release of Hgb from ruptured red cells
BASIC TERMINOLOGY FOR CLINICAL FINDINGS ON into the plasma
BLEEDING DISORDERS - Do have thromboplastin-like activity
1. Petechiae – purplish red pinpoint hemorrhagic spots in
the skin caused by loss of capillary ability to withstand Anticoagulants
normal blood pressure and trauma. a. Citrate (Trisodium Citrate) – blue tube
2. Purpura – hemorrhage of blood into small areas of - 3.2% buffered Na citrate – standard
skin, mucous membranes, and other tissues. anticoagulant for coagulation studies
3. Ecchymosis – form of purpura in which blood escapes - Preserves labile factors V & VIII
into large areas of skin and mucous membranes, but - 6 hours; unopened – if not analyzes at the same
not into deep tissues. time
4. Epistaxis – nosebleed. - Doesn’t inhibit clotting factors
5. Hemarthosis – leaking of blood into cavities - Satisfactory for platelet aggregation studies
6. Hematemesis – vomiting of blood - Ratio of anticoagulant and blood → 9:1
7. Hematoma – swelling or tumor in the tissues or a body - 3.8% Na Citrate (0.129 M) & 3.2% Na Citrate (0.109
cavity that contains clotted blood. M)
8. Hematuria – RBC in urine - Excess citrate (prolonged PT & APTT - ↑ Hct at
9. Hemoglobinuria – Hb in urine about >50 L/L
10. Melena – stool containing dark red or black blood Remedy:
11. Menorrhagia – excessive mentrual bleeding.  Adjust the volume of anticoagulant
 ↑ amount fo Ca++ during recalcification
LABORATORY EVALUATION OF PLATELETS  ↓ the concentration of the anticoagulant
from 3.8% - 3.2% Na Citrate
Introduction: The goal of a phlebotomist is drawing b. EDTA – inhibits the fibrinogen-thrombin reaction
blood by doing a non-traumatic venipuncture - Not used due to instability of Factor V
- Recommended for platelet counting
3 Major Components of Hemostasis c. Sodium Oxalate – not recommended due to
1. Extravascular (tissue surrounding the blood vessel) production of complexes or
2. Intravascular (platelets; clotting factos) precipitate upon recalcification.
3. Vascular (arterioles, venules, veins, arteries) d. Heparin – acts with anti-thrombin III
- Inhibits reactions of all stages of coagulation
• Alteration of any of the components (esp. the - Anticoagulant of choice for platelet retention test
intravascular & vascular) may lead to various
problems regarding the hemostasis of an individual, Collection of Blood
which usually leads to bleeding tendency. Two-Syringe Technique
• Problems in Coagulation & Fibrinolysis will lead to 1. 1mL to 3mL of blood – withdrawn into first syringe;
bleeding episodes in an individual. may be discarded or used for serum-required test
• To pinpoint which among the components are altered, 2. Sterile gauze is placed beneath the hub (before
specific laboratory test are perform for platelets, blood switching of syringes)
vessels, coagulation & fibrinolysis. 3. 1st syringe is carefully detached and replaced with
2nd syringe
Two Phases of Hemostasis 4. Blood is transferred without delay into appropriate
1. 1o Hemostasis – platelets, blood vessels, tubes.
fibrinogen, vWF – end point is
platelet plug.
8
Using Evacuated Tubes g. Note: A grade 2+ above is indicative of
1. Multiple-sample Leur adapter is screwed into a capillary weakness as in: thrombocytopenia,
needle holder (initial venipuncture) thrombasthenia, vascular purpura, senile
2. Needle enters the vein, tourniquet is released purpura, scurvy, firbrinogenopenia, and
3. Draw blood into red-top tube hemorrhagic fever.
4. Citrated plasma are drawn next
2. Quick’s Method
Holding Pre-testing Samples a. Apply pressure cuff to upper forearm and
1. Centrifugation inflate to a pressure midway systolic and
• PPP – heavy spin for 10 – 15 minutes diastolic (100 mmHg) for 5 minutes.
• PRP – light spin for 5 minutes b. Release pressure cuff and inspect for
2. The test should be done within 6 hours after petechiae in arm, wrist and hand after 15-30
collection, because dramatic change in pH after 6 mins.
hours will produce discrepancies. c. Normal value: 0 – 5 petechiae in a 5 cm.
3. Frozen Samples circular.
• Samples should not be frozen if testing can be d. Note: More than 5 petechiae indicates
done within 2 hours fragility.
• Freezing is necessary – done rapidly at -20oC e. NOTE: can only reassessed after 7 days
or lower
• Refrigeration – destroys clotting factors VII & XI 3. Gothlin’s Method
Note: Fibrinogen is stable for at least 4 hours after a. Apply pressure cuff on both arms and inflate
thawing to 35 mmHg for 15 minutes.
b. Release pressure cuff and examine for
Samples Used for Platelet Function petechiae in both arms
a. No hemolysis (because ADP will prematurely c. Multiply the number of petechiae in both
activates platelet. arms by two.
b. Citrate is required except in glass bead analysis d. Normal value: 0 – 8 petechiae
c. PRP (Platelet-Rich-Plasma) is used e. Note: 8 – 12 petechiae is equivocal. More
d. Platelets are stable between 30 minutes to 3 hours than 12 petechiae indicates fragility.
e. Temperature storage at Room Temperature.
B. Suction Test or Petechiometer Method
PRIMARY HEMOSTATIS TESTING Principle: Application of negative pressure of
sufficient degree to the skin and indirectly to the
TEST FOR VASCULAR INTEGRITY vascular tissue causes extravasation of blood, the
A. Tourniquet test extent of which will provide measure for
Principle: This test the stability of capillaries under permeability.
increased hypoxia and hydrostatic pressure..
Normal value: 0 – 2 petechiae at 200 mmHg
1. Rumple-Leede Test/Tourniquet/Capillary Note: 4 – 8 petechiae is equivocal and test is
Resistance Test repeated on another site. Presence of more than 8
Procedure: petechiae indicates capillary fragility.
a. Determine patient’s blood pressure
b. Apply pressure cuff on the upper forearm TEST FOR PLATELETS
and inflate to pressure midway systolic & - the 1st logical step in investigating problems in
diastolic and maintain this pressure for 5 hemostasis is to Quantify platelets.
minutes.
c. Remove the cuff and examine the presence Platelet Count
of petechiae on the volar surface of the arm
and on the dorsal of hands and fingers. Platelet Estimate of Report Platelet Estimate as:
d. 15 minutes after release of pressure cuff, 0 – 49,000/uL Marked decreased
choose and area 1-inch diameter anywhere 50,000 – 99,000/uL Moderate decreased
on the areas mentioned above.
100,000 – 149,000/uL Slight decreased
e. Count the number of petechiae formed
150,000 – 199,000/uL Low normal
within the 1-inch diameter area and grade as
follows: 200,000 – 400,000/uL Normal
# of petechiae Grade 401,000 – 500,000/uL Slight increase
600,000 – 800,000/uL Moderate increase
0 – 10 +1 Above 800,000/uL Marked increase
10 – 20 +2
20 – 50 +3
> 50 +4

f. Normal value: Grade +1

9
3 Ways to Quatify Platelets • Light scatter from platelet suspension
A. Platelet Estimate from PBS (Indirect) detected – voltage amplitute proportional
10-40 RBC / 1 platelet to particle concentration
100 RBC / 3-10 platelet • Ex. Technicon
200 RBC / 5-20 platelet 4. Electronic Impedance
• Hydrodynamic focusing
Indirect Platelet Count
- 5 – 10 fields Platelet Count BT
- Constant – 20,000 (n/mm3) → SI = N x 10 9/L Aspirin intake
- In 1 OIO field, there should be 3-5 platelets vWD N
10 vascular problem
Formula:
Autoimmune
N
thrombocytopenia
Quali and Quanti
deficient
B. Direct Manual Count
Platelet Adhesion Test
1. Tocantin’s Method / Rees-Ecker Method (Light
A. Bleeding Time
Microscopy)
- The time required for a standardize skin wound
- citrate buffer formaldehyde with BCB
to stop bleeding
(Brilliant Cresyl Blue)
- It measures the 1o Hemostasis
- Dilution 1:200
- If patient is taking aspirin - ↑ BT
- Plt./sq. mm X 200 (dil.) X 10 (DF)
- It is dependent on: platelet plug formation,
Plt/uL = ---------------------------------------------
vasoconstriction, and coagulation
1 (area correction)
1. Ivy Method
2. Brecher and Cronkite (Phase Contrast)
Materials
- Uses siliconized syringe then placed in ice
 Sphygmomanometer (blood pressure cuff)
water
 70% alcohol sponge
- Diluent: ammonium oxalate
 Sterile gauze
- Plt./sq. mm X 200 (dil.) X 10 (DF)
 No. 11 Bard-Parker blade (sterile)
Plt/uL = ---------------------------------------------
 Filter paper
0.2 (area correction)
 Stopwatch
-
Procedure
3. Unopette System
a. Apply pressure cuff on the patient’s upper
- Uses diluting fluid 1% ammonium oxalate
forearm and inflate (40 mmHg), maintain
- Ratio of blood and diluting fluid → 1:100
pressure throughout the procedure.
- Normal Value: 140-440 x 109/L – adult
b. Disinfect the area (approximately 3 finger-
150-350 x 109/L – children
widths below the bent of the elbow) with
70% alcohol sponge. Dry.
Quantitative Platelet Disorder
c. Hold skin tightly grasping the underside of
• Thrombocytopenia – aplastic anemia, ITP
the arm firmly.
(Idiopathic thrombocytopenic
d. Make 2 skin incisions (2mm deep and 2mm
purpura), HUS, TTP, heparin-
long). Avoid any subcutaneous veins.
induced.
e. Star timer
• Thrombocytosis – myeloproliferative
f. Blot the drop of blood from each puncture
disorders.
site on 2 separate filter paper every 30
seconds. The filter paper should not touch
B. Automated – optical or electrical methods (2-20 fL)
the wound at any time.
1. Impedance
g. Stop time as soon as the bleeding stops.
• As platelet pass 50 um aperture,
h. Release the pressure cuff
electronic impedance signal is generated,
i. Record the bleeding times of the 2
with pulse height proportional to volume
puncture sites and report the average of
of platelets
the 2 results.
• Ex. Coulter
j. Normal Value: 2-4 minutes
2. Laser Light Scattering
• Differentiates platelet from RBC by
2. Duke Method
refractive index
- Finger (ideal site) & for pediatric patients
• Ex. Ortho
Materials
3. Optical Particle Counting
 70% alcohol sponge
• RBC are lysed by urea
 Sterile gauze
 Disposable blood lancet (sterile)
 Filter paper
10
  Stop watch - TAR – neonates
Procedure - Chediak Higashi - albinism
a. Disinfect site of puncture (most preferably e. Vascular disorders
the earlobe) with 70% alcohol sponge. Dry.
b. Puncture to the depth of 3mm with a B. Glass Bead Retention Test (Salzmann’s Test)
sterile, disposable blood lancet. Principle: An in-vitro designed to measure the
c. Start the timer as soon as the 1st drop of ability of the platelet to adhere on glass surfaces,
blood appears. when they passed through a column of glassbead
d. Blot the drop of blood with filter paper at a constant rate; some of the platelets will be
every 30 seconds. Make sure that the filter retained by the glass bead.
paper does not touch the wound. Formula:
e. Stop timer as soon as the bleeding stops.
f. Normal Value: 2-4 minutes
3. Copley-Lalitch Immersion Test
a. Disinfect the finger with 70% alcohol and Normal value: 26-60% platelet retention
wipe dry with sterile gauze. • Defective in-vitro platelet adhesion
b. Puncture (6mm depth) • Collection
c. Start timer immediately Platelet CT 1: Routine Collection System
d. Immerse puncture finger in a beaker of Platelet CT 2: Glass Bead Collection System
NSS prewarmed at 37oC water bath.
e. Observed for flowing out of red blood Decrease Platelet Increase Platelet
against the colorless NSS Retention Retention
f. Stop timer as soon as blood stops flowing. Glanzmann’s Thrombotic disorders
g. Normal Value: 3-6 minutes thrombasthenia
4. Alderson-Crossby Immersion Test
vWF disease Hyperlipidemia
- similar to the Copley-lalitch Immersion Test
Bernard – Soulier Carcinoma
5. Aspirin Tolerance Test (Quick’s Test)
Syndrome
Principle: Overdosage of aspirin prolongs
Chediak Higashi Oral Contraceptive Intake
bleeding time so that 4 tablets are enough
to prolong the bleeding time of a normal Myeloproliferative Pregnant
individual and just 2 tablets are enough to Plasma Cell Dyscrasia
prolong the bleeding time of a patient with Uremia
vWB disease and thrombopathia. Aspirin – ingestion
Note: Patient should abstain from aspirin and
its derivatives at least 3 days before the test. Clot Retraction Time
- problems in platelet & clotting factors; especially
Procedure: intrinsic and common pathway
a. Determine patient’s first bleeding time Principle: The normal blood must clot after the first
using Duke’s method hour & be firm & should retract from the walls of
b. If bleeding time is > 6 minutes, don’t do the the tube. Complete retraction occurs within 24o
test anymore because it’s useless. by which the clot occupies about half of the
c. Give patient 2 tablets of aspirin and a glass original blood volume.
of water.
d. Determine patient’s 2nd bleeding time using A. Qualitative Test (Presence/Absence of Retraction)
duke’s method after 2 hours 1. Hirshboeck Method (Castor Oil Method)
e. Normal value: Difference between 1st and Procedure:
2nd bleeding should not be more than 2 a. Add 1 big drop of fresh blood (w/out
minutes. anticoagulant) on the test tube with castor
6. Modified Template / Simplate – uses spring oil and start timer.
loaded blade producing 5mm x b. Observe for “Dimpling” or extrusion of
1mm incision. droplet-like serum on top portion of the
drop of blood.
Factors that prolonged BT: c. Stop as soon as “dimpling” of serum is
a. Platelet number ↓ about < 100,000/mm3 observed.
b. Aspirin intake or anti-histamine drugs d. Normal Value: 15-45 minutes
c. vWF disease, afibrinogenemia or e. Note: Dimpling is less than 15 minutes is
hypofibronogenemia interrupted as thrombotic tendency and
d. Platelet disorder dimpling after 45 minutes is interrupted as
- Glanzmann’s thrombosthenia (GpIIb/IIIa) hemorrhagic tendency.
- Bernard Soulier (GpIb) f. Clot is reported as normal or
- Hermansky Pudlack – dense granules defective/poor.
- Wiskott-Aldrich – thrombocytopenia, Comment: Poor Clot Retraction occurs in:
immune infections & eczema
11
 a. Platelet count of <100,000/mm3 or g. Measure the volume of expressed serum
100 x 109/L directly against the calibration of the tube.
b. Glanzmann’s thrombosthenia h. Compute for clot retraction as follows:
In DIC the clot appears small & ragged.
Rapid dissolution of clot is evidence of
increase fibrinolytic activity.

Normal Characteristic of Extruded Serum:

- Milky in cases of leukemias, diabetes, and i. Normal value: 44-67% CR
after meals. j. Note: Clot which have retracted by the end
- Dark yellow in jaundice of the 1st hour of incubation may be freed
- Cloudy in multiple myeloma. by carefully passing a fine stiff wire around
the inside of the tube. Tube should then be
B. Quantitative Test (Estimate the amount/degree of reincubated for another hour.
retraction) Clot retraction is actually a complicated
1. Stefanini Method (Test Tube Method) process that involves the interaction of at
a. Draw 3-5 mL fresh venous blood from least 4 effects:
patient. * Presence of platelets
b. Transfer blood to Wasserman tube. * Concentration of fibrinogen
c. Incubate at 37’C (1 hour) after which let * Activity of a retraction promoting
tube stand at room temperature and principle in the serum
observe for retraction to take place. Clot * The nature of the tube surface
retraction starts within 1 hour after blood Clot retraction is a useful index of platelet
extraction and is usually complete within activity. Clot retraction is poor when platelet
18-24 hours. (1/2/16/18/24 hours) count is below 100,000/mm3 and when there
d. Record results as follows: is thrombopathia.
Normal or Complete Retractility
Partial Retractility Platelet Factor Assays
Poor Retractility A. PF3 Availability – uses kaolin or Epinephrine
Very Poor Retractility Principle: The platelets provide phospholipids or
e. Normal Value: begins within 1 hour, PF3 to facilitate the activation of clotting factors.
complete within 18-24 hours PF3 is released when platelets are activated by a
f. Note: if the tube is not very clean the clot stimulus such as kaolin or celite
may adhere to the wall of the tube and
present a flase result. In such case, ring Procedure:
the clot with a stiff straight wire to separate a. Obtain 9mL of citrated blood
it from the wall of the tube. If the blood is b. Prepare PPP & PRP & control (normal plt.)
normal, retraction will take place in a short c. The determine the clotting time of the ff. 3
time. assays:
• Control -0.1mL platelin + activator + 0.1mL
2. Mac Farlene Method PPP + 0.025 M CaCl2
a. Place 5mL fresh venous into calibrated • PRP – 0.1mL PRP + celite + 0.025 M CaCl2
tube. • PPP – 0.1mL PPP + celite + 0.025 M CaCl2
b. Place a glass rod inside the tube with the Incubate at 37oC for 5 minutes
expansion immersed in the column of
blood. Result: A normal concentration of PF3 is obtained
c. Fit a cork into the glass rod by boring a when the clotting time of the PRP is near that
hole at the center of the cork and letting the of the control. A low level of PF3 is obtained
projecting tip of the glass rod pass through when the PRP is near the clotting time of
the hole, thus covering the tube in the PPP.
process. (+) gel formation
d. Place the tube in 37oC water bath and Normal Value: 37-51 seconds for PRP
observe every 5-10 minutes for B. PF4 and BTG-RIA
coagulation.
e. 1 hour after firm clotting, allow tube to Platelet Aggregation Test
stand just at room temperature for - it is studied by means of platelet aggregometer (a
retraction to take place. (Retraction has photo-optical instruments)
taken place if the clot has shrunk and has - If PRP or PPP is used – more turbid
attached itself onto the glass rod)
f. After retraction has taken place, remove PRP – centrifuge at 60-100g for 10mins
the cork to remove the glass rod and
attached clot.

12
 PRP + agonist: (monitored O.D.) o Normal Value: 7-15 minutes or 5-15 minutes
• Epinephrine o Note: A coagulation time is more than 15 minutes
• ADP is abnormal.
• Collagen
• Ristocetin Tubes used in this test – washed with saline solution
or may be siliconized. The sensitivity of the test can
A. Platelet Aggregometry be increased by silicone-coating the tubes so that the
Principle: This test used to measure the capability of normal clotting time is prolonged to 20-40 minutes.
the platelet to aggregate normally by placing Use of siliconized tubes will allow detection of
substances that induce platelet aggregation such as hemophiloid diseases which may be overlooked due
collagen, ADP, Epinephrine, thrombin, serotonin, to a normal clotting time obtained if ordinary method
arachidonic acid, ristocetin (aggregating agents). is used.
Other substance that can induce platelet
aggregation. o Increased in factor deficiency except for factor
VII & XIII
Note: Requirements Platelet Aggregation o Used in monitoring heparin therapy
• PRP is used (citrate is used as anticoagulant) o Increased in the presence of heparin & other
• No hemolyzed sample. circulating anticoagulant
• Patient must be on fasting (8-12 hours)
• Test are performed at 37oC at pH 6.5-8.5 B. Plasma Recalcification Time – uses either PRP/PPP
• No anti-inflammatory drugs taken 1 week prior or both
to test. Principle: The test used to measure the intrinsic
coagulation pathway. This test is based on the fact
Platelet unresponsive to aggregating agent: that all clotting factors except Ca++ is present on the
• ADP – 3 concentration used: PRP which is necessary to form a fibrin clot.
High concentration Therefore upon the addition of Calcium
Low concentration (recalcification) to PRP it will form a clot at specified
Optimum concentration given time.
If no aggregation,
→ Glanzmann’s Thrombastenia Procedure:
a. Prepare PPP & PRP
•Ristocetin, if no aggregation, b. Add CaCl2 to both PPP & PRP
o Bernar-Soulier Syndrome c. Wait for clot formation
o vWF disease
o Idiopathic Thrombocytopenic purpura Normal Value: PRP – 100-150 seconds
vWF Assay – uses ristocetin PPP – 130-240 seconds
Normal value: 45-140% of Normal Activity
o The clotting time of PRP must be at least 20
TESTS FOR COAGULATION or seconds faster than PPP
SECONDARY HEMOSTASIS o Used for heparin theraphy monitoring;
indicative also to identify defects of platelets.
Test for Intrinsic and Common Pathways
A. Lee and White Whole Blood Clotting Time C. Activated Clotting Time
- is the length of time required for blood to clot in a - uses diatomite
glass test tube which is used to measure the - used also in monitoring heparin therapy
overall activity of coagulation factors of the Principle: The whole blood is allowed to clot in a
intrinsic and common pathway tube that contains activators such as
diatomite/diatomaceous earth to hasten the
Procedure: formation of clot.
o Place three numbered test tubes in 37oC water
bath Normal Values: 72-120 seconds
o Obtain 5 mL venous blood with a plastic syringe 140-185 seconds target range
using the 2-syringe method during heparin treatment
o Place 1mL blood into each test tube, starting with *Increase ACT
tube 1 o Deficiency of Intrinsic & Common
o Start timer as soon as blood enters tube 1 Coagulation Factors
o Gently tilt tube 3 every 30 seconds and observe o Presence of circulating anticoagulants
for coagulation. After coagulation is observed into
tube 3, observe tube 2. When coagulation is D. Dale and Laidlaw’s Capillary Clotting Time
observed in tube 2, observe tube 1. - done if not obtain venous blood; uses blue
o When no more flow of blood is observed on tilting capillary tube
of tube 1 then stop timer. Coagulation time is the Procedure:
time it takes blood to coagulate in tube 1.
13
 a. Collect blood into 2 capillary tube and start timer Normal Value: 11-14 second
as soon as the 2nd drop of blood appears. 12-16 seconds (other books)
b. Break off ¼ of an inch every 30 second.
c. Stop timer as soon as fibrin strands are seen B. Stypven Time
bridging the 2 broken ends of the tube. - Used to differentiate FX and FVII Deficiencies

Normal Values: 2-4 minutes Principle: This test uses an extract from Vipera
russelli called Russel’s viper venom to cleave
E. Activated Partial Thromboplastin Time (APTT) factors X & Xa by passing the common pathway.
Principle: This test is used to measure all CF
except VII & XIII. Using PPP with the addition of Procedure:
platelet phospholipids (derived drom plant & a. 4.5mL blood – PRP + 0.1mL of venom + 0.02 M
animal or brain extracts) substitutes & APTT rgt CaCl2 – clot
plus Ca++, clot will form.
*Extrinsic - ↑PT, normal ST
Procedure: *Common - ↑PT, ↑ST
a. PPP + APTT rgt (obtain pL, activator (Kaolin, * ? – normal PT, ↑ST (impossible to happen)
celite, elagic acid, micronized silica)
b. Incubate for 3-5 mins @ 37oC C. PT-Proconvertin Time / P and P Test
c. Then recalcified by adding 0.025 M CaCl2, then
start timing. D. Thrombin Time
d. Check for gel/clot formation and record time. • Prolonged in:
Normal Value: 20-45 seconds o fibrinogen deficiency
o presence of FDP/FSP
Hypofibrinogenemia o presence of thrombolytic agent
Anticoagulants will prolong PTT o presence of Heparin
Under heparin therapy • PPP + Thrombin + CaCl2
Intrinsic & common PW
Note: E. 5M Urea Solubility Test (Duckert’s Test)
About 83% of Stage I disorders are due to FVIII • for FXIII deficiency
deficiency (Classic Hemophilia A). Prolonged APTT • Can be substituted with 1% monochloro HOAc or
can be corrected completely by adsorbed plasma. 2% HOAc
About 14% of Stage I disorders are due to FIX • NV: clot insoluble to 5M Urea within 24 hours
deficiency (X’mas Disease or Hemophilia B).
Prolonged APTT can be corrected almost Indirect Test for Coagulation Abnormalities
completely by serum.
FXI deficiency or Hemophilia C (rare) may be A. Two Stage Prothrombin Time
corrected partially by serum and plasma as both
contain FXI B. PT Consumption Test/Serum Prothrombin Time
Principle: The serum contg. the residual
Test for the Extrinsic and Common Pathways prothrombin, will be mixed with simplastin A which
contain factor XI, IV, II, and thromboplastin, to allow
A. Prothrombin Time (Quick’s Method) clot formation. This test is used to determine if there
- used for monitoring oral anticoagulant therapy is any abnormality in thrombogenesis action of the
- is the time required to form fibrin clot when PPP blood.
is added to a thromboplastin-calcium mixture
Procedure
Procedure: a. Obtain serum
a. Centrifuge citrated blood (heavy spin) & obtain b. Add 0.1mL serum to 0.2 Simplastin A.
plasma free from platelets c. Then wait for clot or gel formation
b. Incubate PPP & Simplatin @ 37oC
c. PPP (0.1mL) + Simplatin (0.2mL) Normal Value: > 20 - 25 seconds
d. Wait for Gel formation
e. Record Time ↑factor II – impaired clotting ability
Simplastin rgt. – used to determine problems in
Simplastin = Thromboplastin + CaCl2 thromboplastinogenesis which is
responsible of converting factor II to
Oral anticoagulant thrombin + platelets & intrinsic
• Coumarin/Warfarin pathway
- inhibits Vit K (Factors II, VII, IX, X)
- Vitamin K antagonist

14
 Note: E. Reptilase Time
o This test is a screening test for thrombopenia Principle: This test uses reptilase enzyme (derived
thrombopathic thrombocytopenia and from, Bothrox atrox) that assumes a thrombin-like
Hemophilia A & B enzyme acitivity. Therefore reptilase has the
o In hemophilia – SPT shorter than plasma capability to cleave fibrinogen peptide to form fibrin
prothrombin time, usually 8-9 seconds for the monomers. This test is not influenced by heparin or
SPT compared to about 13 seconds for plasma circulating anticoagulant but highly affected by
prothrombin time. abnormality in fibrinogen like dysfibrinogenemia
o Test detects the entire IF & platelets & Factor X which is more affected.
activity
Procedure:
C. Thromboplastin Generation Test (TGT) a. Citrated PPP + reptilase
- Reagents are the one used in mixing studies b. Wait for clot formation
c. The record the time
EXCEPT
Adsorbed II, IV, IX, X Normal Value: 10-15 seconds
Plasma
Serum I, II, V, VIII, Comparison of Results Between TT and RT
XIII Thrombin Reptilase
Normal Plasma Ca++ Time Time
D. Thrombin Clotting Time Heparin Therapy Prolong Normal
Principle: It is the time required for thrombin to Immunologic
Prolong Normal
convert fibrinogen to an insoluble fibrin clot. The Antithrombin
formation of fibrin is triggered by the add’n of Dysfibrinogenemia Greatly
Prolong
thrombin to plasma thereby by passing prior to prolong
steps in the coagulation. This test is not used to Hypofirbrinogenemia Prolong Prolong
measure the Intrinsic and Extrinsic Pathways but FDS’s/FSP’s Prolong Prolong
measuring the levels of fibrinogen,
dysfibrogenemia, the presence of circulating F. Modified Thrombin Clotting Time
anticoagulants. (heparin, plasmin, FDPs)
immunologic antithrombin Inhibitor Studies
A. Screening Tests
- determine problems of fibrinogen: - add normal plasma, if prolonged PT/APTT
o afibrinogenemia
o dysfibrogenemia B. Tissue Thromboplastin Inhibition Test
o hypofibrogenemia
Procedure: C. Platelet Neutralization Test
a. Prepare PPP & control plasma (normal) - Screens the presence of lupus anticoagulant; if
b. Mix 0.1mL of TRIS buffer + 0.1mL of PPP into prolonged, add freeze thawed platelets.
tube D. Agarose Plasma Gel Technique
c. Incubate it @ 37oC for 1 minute (+) halo
d. Exactly after 1minute add 0.1mL of thrombin rgt.
e. Wait for clot to form, record the time. E. Bethesda Inhibitor Assay
- factor VIII inhibitor
Plasma + thrombin → gel
(FI converted to fibrinogen) Factor Assays
A. One Stage Clotting Assay
Protamine sulphate – affects heparin therapy Normal Result: 50-150% of normal activity

Normal Value: 15-20 seconds B. FVIII Immunologic Assay

8-9 sec, 15-20 sec, <24 sec, 20-25
secs. C. Ristocetin Agglutination Test
Normal Result: 50-150% of normal activity
- This is the most sensitive indication for the
presence of DIC (FDPs) D. FXIII Assay
- If the TT is corrected by adding normal plasma – E. Fletcher Factor Assay
molecular abnormality of the fibrinogen is indicated Principle: FF facilitates in the contact phase of the
- It can be used in monitoring heparin therapy intrinsic pathway for the confirmation of the
- Prolonged TT in hypofibrinogenemia (<100mg/dL) deficiency of this factor usually indicates an initial
prolong PTT but then corrected after adding
activators like celite/kaolin but not ellagic acid.

15
 TEST FOR PHYSIOLOGIC INHIBITORS Primary Secondary
Fibrinolysis Fibrinolysis
A. Antithrombin III WBCLT < 48 hrs < 48 hrs
Normal Value: 20-80 mg/dL or 80-120% of normal
Euglobulin Clot
activity < 2 hrs < 2 hrs
Lysis
Protamine
B. Protein C - +
Sulfate
Normal Value: 3-4 ug/mL
Ethanol Gelation - +
C. Protein S D-dimer - +
Normal Value: approximately 1 ug/mL
SUMMARY OF MOST IMPORTANT SUBSTANCE
TEST FOR FIBRINOLYSIS SECRETED BY PLATELETS AND THEIR ROLE IN
HEMOSTATSIS
Lysis Time
A. Whole Blood Clot Lysis (WBCLT) Conmments
Role in
Substance Source on Principal
Principle: This test is based on the fact that WB will Hemostasis
Function
clot spontaneously when collected in a glass
tube w/o anticoagulant this clot should Promote HMWK Alpha Contact
remain intact for about 48-37oC. Dissolution coagulation granules activation of
of clot before 48 hours is indicative of intrinsic
excessive systemic fibrinolytic activity. pathway
1. Dilute WBCLT Fibrinogen Alpha Converted to
granules fibrin clot
2. Plasma Clot Lysis Time
formation
Factor V Alpha Cofactor in
Normal Value: clot lysis starts after 48 hours
granules fibrin clot
formation
B. Euglobulin Lysis Time FVIII:vWF Alpha Assists platelet
Principle: Euglobulin are proteins that precipitate granules adhesion to
after the plasma is diluted with H2O & then subendotheliu
acidified. Euglobulin fraction contains plasminogen, m to provide
plasmin, finrinogen, plasminogen activators while coagulation
the supernatant contains inhibitors if fibrinolysis surface
(anti-plasmin & anti-plasminogen) Promote ADP Dense Promote
Aggregation granules platelet
Procedure: Calcium Dense aggregation
a. Citrated PPP + water + acid. granules
b. Then refrigerate Platelet Alpha
c. Euglobulin will ppt. & the supernatant is Factor 4 granules
decanted Thrombospo Alpha
ndin granules
INSTRUMENTATION FOR TESTS OF HEMOSTASIS Promote Serotonin Dense Promotes
vasocontricti granules vasocontriction
A. Visual detection of fibrin clot formation on TXA2 Membrane
• Tilt tube method precursors phospholip
B. Electromehcanical detection of fibrin clot formation id
• Fibrin strand formation is detected using a wire Promote PDGF Alpha Promotes
loop or hook; has been incoporated into a semi- vascular granules smooth muscle
automated mechanical instrument. repair growth for
vessel repair
• Instrument: FIBROMETER - impedance
Beta- Alpha Chemotactic
C. Photo-optical detection of fibrin clot formation
thromboglob granules for fibroblast to
• Detection of fibrin clot formation depends on the
ulin help in vessel
increase in light scattering associated with the repair
conversion of soluble fibrinogen molecules to Other Plasminogen Alpha Precursor to
the insoluble polymerized fibrin clot. systems granules plasmin, which
• Semi-automated instruments: Electra 750 and affected induces clot
750A, Fibrintimer series, and FP 910 lysis
Coagulation Analyzer Alpha-2- Alpha Plasmin
• Automated instruments: Ortho Koagulab 16S antiplasmin granules inhibitor;
and 40A, the Coag-A-Mate X2 and XC, and the inhibits clot
MLA Electra 700 and 800. lysis
C1 esterase Alpha Complement
inhibitor granules system inhibitor
16
 RED BLOOD CELLS The synthesis of heme begins in the mitochondria with the
formation of D-ALA from glycine and succinyl coenzyme A
Erythropoiesis: process by which erythroid precursor
cells differentiate to become mature RBC. The primary Biosynthesis of Heme
regulator of this process is erythropoietin. Succinyl coenzyme A + Glycine
In normally takes 3-5 days for the production of
reticulocytesfrom pronomoblasts. The reticulocytes Pyridoxal phosphate D-ALA synthetase
remain in BM for 1-2 days before being released to the (Vit. B6)
circulation. In the peripheral circulation, the reticulocyte Delta-Aminolevulinic Acid
continues to mature for one more day.
D-ALA dehydrase
MATURATION
1. Pronormoblast / Rubriblast Porphobilinogen
• 14-20 um diameter Uroporphyrinogen III
• Deeply basophilic cytoplasm cosynthetase
• Non-granular Uroporphyrinogen III
• N/C ratio is 8:1 Uroporphyrinogen
• Fine chromatin decarboxylase
• Usually 1-2 nucleoli Coproporphyrinogen III
2. Basophilic Normoblast / Prorubricyte Coproporphyrinogen
• 12-17 um diameter oxidase
• intensely basophilic cytoplasm Protoporphyrinogen IX
• N/C ratio is 6:1 Protoporphyrinogen
• Chromatin slightly coarse oxidase
• Nucleoli are usually not visible Protoporphyrin IX
3. Polychromatophilic Normoblast / Rubricyte Ferrochelatase Fe++
• 10-15 um diameter
• blue-gray to pink-gray cytoplasm Heme molecule
• N/C ratio is 4:1
• Last stage of mitosis The globin portion og Hgb is produced on specific
4. Orthochromatophilic Normoblast / Metarubricyte ribosomes in the cytoplasm of the RBC. The globin in
• 7-12 um diameter each Hgb molecule consists of four polypeptide chains
• pink cytoplasm which determine the type of hemoglobin formed.
• N/C ratio is 2:1
• Small pyknotic nucleus NOTES:
• Last nucleated stage • 1 heme = 1 mole of O2
5. Reticulocyte • 1 Hgb = 4 moles of O2
• 7-10 um diameter • Hgb production begins at polychromatophilic
• pink to slightly pinkish gray cytoplasm normoblast stage and ends at reticulocyte stage.
• contains fine basophilic reticulum RNA, which is • Genes: alpha & zeta – Chr. 16
only demostrated by supravital stain (NMB) beta, delta, epsilon, gamma – Chr. 11
6. Mature RBC
• 6-8 um diameter Hemoglobin Globin Chain
• pink in color Embryonic Gower I 2 zeta + 2 epsilon
• non-nucleated, round, biconcave Portland 2 zeta + 2 gamma
Gower II 2 alpha + 2 epsilon
NOTES: Fetal Hb F 2 alpha + 2 gamma
• 1 rubriblast = 16 RBCs
Adult Hb A1 2 alpha + 2 beta
• last stage capable of mitosis – rubricyte
Hb A2 2 alpha + 2 delta
• last nucleated stage – metarubricyte
• Index of BM activity or effective erythropoiesis –
EFFECTS OF VARIOUS FACTORS ON
Reticulocyte count
OXYHEMOGLOBIN DISSOCIATION CURVE
• Lifespan of RBC – 120 days
SHIFT CAUSED BY
Hemoglobin Structure and Synthesis FACTOR
INCREASE DECREASE
Primary function of the RBC is to manufucture Hgb, which Blood Temp. R L
in turn, transports oxygen to the tissues and CO2 from pH L R
tissue to lungs. 2,3 - DPG R L
CO2 R L
The Hgb molecule is composed of four subunits, each Hb F admixture L n/a
containing heme and globin. A shift to the left (L) is associated with increased affinity to O2. A shit to the
right (R) is associated with decreased addinity to O2.

17
OXYGEN DISSOCIATION CURVE ERYTHROCYTE MEMBRANE
• Sigmoid in shape 1. Protein (50%)
• Arterial blood 95% = 95 mmHg • Integral Protein contains:
• Venous blood 70 % = 40 mmHg o Glocophorin A (contains sialic acid
• p50 = 26.6 mmHg responsible for the negative charge)
o Component A
Shift to the Left Shift to the Right • Peripheral Protein contains actin and myosin for
the maintenance of the shape of RBC.

x
↑ affinity of Hb to O2 ↓ affinity of Hb to O2
↓ delivery of O2 to tissues ↑ affinity of O2 to tissues 2. Lipid (40%)
• External surface: Phospholipids
2,3-DPG 2,3-DPG
Phosphatidylcholine
pH CO2 pH CO2
Glycolipid
Temp. Temp.
Sphingomyelin
• Internal surface: Phosphatidylethanolamine
Bohr Effect Phosphatidylinositol
• Increase pH = Increase Hb affinity to O2 (R) Phosphatidylserine
• Decrease pH = decrease Hb affinity to O2 (L) • Cholesterol content of the membrane depends
upon the concentration of plasma cholesterol,
Normal Hemoglobin – transport of O2 and CO2 bile acids, and the activity of the enzyme LCAT.
3. Carbohydrate (10%)
Oxyhemoglobin Reduced Hb
(HbO2) (HbCO2) METABOLISM OF RED BLOOD CELL
Arterial blood Venous blood
Bright red Purplish red 1. Embden-Meyerhof Pathway (major pathway)
• 90% glycolysis; anaerobic
Abnormal Hemoglobin • every glucose broken down to lactic acid
produced 2 ATPs
• Hb bound to carbon • Control flow of Na+/K+
monoxide • Prevents oxidation of membrane lipid
• Hb affinity for CO is 2. Hexoze Monophopsphate Shunt or PPP
200X greater than its • 10% glycolysis; aerobic
CarboxyHb (HbCO) • provides reduced glutathione to prevent
affinity to O2
• Tobacco smokers: 1- oxidation denaturation of Hb.
10% 3. Rapaport-Leubering Pathway
• REVERSIBLE • Generates 2,3-DPG
• Hb iron is in Fe+3 state 4. Methemoglobin Reductase Pathway
• Inherited: methHb • main Hb iron is “Ferrous”
reductase deficiency
• Acquired: exposure to BREAKDOWN OF RBC
chemicals like Chlorate,
MethHb/Hemiglobin (Hi) nitrite/nitrate As RBC ages, there is decrease in its enzymes, ATPs,
• Px is cyanotic; blood is size, and increase in density.
chocolate brown
• Treatment: methelene Approximately 1% of the RBC leaves the circulation each
blue or ascorbic acid day and are broken down by the mononuclear phagocytic
• REVERSIBLE system (MPS).
• Caused by chemical or
drugs like aromatic 1. Extravascular (90% aged RBC destruction)
amines or sulfonamides • within RES; when complement is not activated
Sulfhemoglobin or incompletely activated.
• Can combine with CO to
form carboxysulfHb • Increase B1, urine and fecal urobilinogen
• IRREVERSIBLE

NOMENCLATURE AND ABSORPTION MAXIMA OF

HEMOGLOBINS
Absorption Absorption Absorption
Term Symbol
Peak 1 Peak 2 Peak 3
Hemoglobin Hb 431 (140) 555 (13.04)
OxyHb HbO2 415 (131) 542 (14.37) 577 (15.37)
CarboxyHb HbCO 420 (192) 539 (14.36) 568.5 (14.31)
MetHb Hi 406 (162) 500 (9.04) 630 (3.70)
CyanmetHb HiCN 421 (122.5) 540 (10.99)
The wavelength in nanometers is followed by the extinction
coefficient placed in parenthesis.
18
 2. Intravascular (10% aged RBC destruction) III. CELL SHAPE
• When complement is completely activated
• Decrease haptoglobin and hemopexin Poikilocytosis – variation in shape ; decrease ESR
• (+) hemoglobinuria
A. Poikilocytes Secondary to Membrane
Abnormalities
1. Acanthocyte/Spur Cell/Thorn Cell/Spike Cell
• Spheroid with 3-12 irregular spikes or club-
like spicules
• Abnormal lipid ratios of membrane lecithins
and sphingomyelins
• IRREVERSIBLE
• Clinical Significance:
RED BLOOD CELL ABNORMALITIES Abetalipoproteinemia
Alcoholic cirrhosis with HA
I. CELL SIZE Hepatitis of newborn
• Anisocytosis – variation in size Pyruvate kinase deficiency
• RDW – numerical expression that correlates with Malabsorption states
the degree of anisocytosis. (NV: 11.5-14.5%) Postsplenectomy state
Severy HA assoc. with cirrhosis and
1. Normocytic (MCV 80-100 fL) metastatic liver disease (spur cells)
• Clinical Significance: 2. Echinocyte/Burr Cell/Crenated RBC/Sea
o Normal condition Urchin cell
o Pathologic condition: • Regular 10-30 scalloped short projections
Acute Post Hemorrhagic Anemia evenly distributed
Hemolytic Anemia • Caused by: depletion of ATP
Aplastic Anemia Exposure to hypertonic sol’n
2. Microcytic (MCV <80 fL) Artifact in drying
• Clinical Significance: • Clinical Significance:
Iron Deficiency Anemia Renal insufficiency (HUS)
Thalassemia 3. Codocyte/Target Cell/Mexican Hat Cell
Anemia of Chronic Disease • Bell-shaped
3. Macrocytic (MCV >100 fL) • Perioheral rim of Hb surrounded by clear
• Clinical Significance: area and central hemoglobinized area
Megaloblastic Anemia (bull’s eye)
Alcoholism • Increases in cholesterol and phospholipid
• Excess of surface membrance to volume
Liver Disease
ratio; decrease OFT
• Clinical Significance:
II. HEMOGLOBIN CONTENT
Hemoglobinopathies SS, CC, DD, EE
Thalassemia
Anisochromia – variation in hemoglobin
Obstructive liver disease
1. Normochromic (HCHC=31-36%)
Postsplenectomy state
• Seen in normal and pathologic conditions, A
Iron deficiency anemia
H A.
4. Spherocyte/Bronze Cell
2. Hypochromic
• Decrease surface area:volume ratio
• Central pallor area exceeds 1/3 of the
• A. Spherocytosis – involves AutoAbs and
diameter of the cell.
hemolytic anemia
• Clinica Significance:
• H. Spherocytosis – deficient spectrin
Thalassemia
(hallmark)
Iron Deficiency Anemia • Increase OFT
Chronic Blood Loss • Clinical Significance:
3. Hyperchromic (MCHC= >36%) Hereditary Spehrocytosis
• No central pallor <1/3 Isoimmune and Autoimmune HA
• C/C: SPHEROCYTOSIS Sever BURNS
4. Polychromasia Stored blood for long time
• Blue-gray coloration NOTE: after splenectomy in a px with HS,
• Indicates young RBC spherocytes persists, indicating that the
• Increased erythropoietic activity or abonrmality involves the RBC membrane itself
reticulocytosis (ex. hemorrhage and rather than splenic damage to the cells.
hemolysis) 5. Stomatocyte
• Mouth or slit-like pallor area, bowl-shaped
in wet preparation.
19
 • High cellular uptake of sodium and low C. Poikilocytes Secondary to Abnormal
potassium content. Hemoglobin Content
• Abnormal Na+-K+ transport ratio 1. Drepanocytes/Sickle Cells
• Clinical Significance: • Crescent-shaped
Hereditary Stomatocytosis • Polymerization of deoxygenated Hb
Rh Null disease • Clinical Significance:
Alcoholism Sickle Cell Anemia
Obstructive liver disease SC Disease
Cirrhosis
6. Elliptocyte/Oat Cell D. Developmental Macrocytosis or Macrovalocyte
• Rod or cigar-shaped, pencil or sausage- 1. Macrocytes/Oval Macrocytes
shape, narrower than ovalocytes • Due to Vitamin B12 and Folate Deficiency
• Protein band 4.1 deficiency (hereditary • Asynchronous development of RBC
elliptocytosis) because nucleus is still immature (nRBC)
• NOT associated with Hemolysis • Impaire DNA synthesis
• Decrease lifespan but functions normally
• OFT is normal MEGALOBLASTIC ANEMIA
7. Ovalocyte Vitamin B12 deficiency Folic Acid Deficiency
• Egg-like or oval shaped, wider than • Pernicious Anemia – • Pregnancy
elliptocyte decrease intrinsic factor • Dietary deficiency
• Bipolar arrangement of Hb • D. latum • Sprue or steatorrhea
• Reduction of membrane cholesterol • Vegetarian diet
• Clinical Significance: • Sprue or steatorrhea
Megaloblastic BM NON-MEGALOBLASTIC ANEMIA
Myelodysplasia
Alcoholism, Chemotherapy, Hypothyroidism
B. Poikilocytes Secondary to Trauma
1. Schistocyte/Schizocyte/Keratocyte/Helmet/Bite
Cell NOTES:
• Fragmentation produced by damage of •Keratocyte – horn-like projections
RBC by fibrin, altered vessel walls, •Blister cell – eccentric nucleus
prosthetic heart valves. •Krizocyte or Pinch cell – triangular with 2 pallor
• HALLMARK of HA secondary to red cell edges.
fragmentation •Ineffective Erythropoiesis:
• NOT Hereditary o decrease reticulocyte, WBC and Platelet
• Clinical Significance: o (+) Howell Jolly bodies
DIC, TTP, BURNS o (+) Hypersegmented Neutrophil
Microangipathic Hemolytic Anemia
2. Dacryocyte/Dacrocyte (Teardrop Cell) OFT MCHC DAT
• Pear-shaped with blunt pointed projection Hereditary Spherocytosis ↑ ↑ (-)
• Clinical Significance: Acquired Spherocytosis or
Myelofibrosis with Myeloid Metaplasia ↑ ↑ (+)
AIHA
(MMM)
Myelophthisic Anemia NOTES:
Pernicious anemia • Abetalipoproteinemia – Acanthocyte
Tb • Chronic Renal Disease – Burr cell
Tumor in BM • Hemoglobinopathies/Thalssemia – Codocyte
B-thalassemia • MAHA (“clothes-line effect”) – Schistocyte
3. Microspherocytes/Pyropoikilocytes • Myeloid Metaplasia – Dacryocyte
• Increase fragmentation at 45’C instead of • Rh Null Disease – Stomatocyte
49’C • Burns – Spherocyte, Schistocyte, and Dacryocyte
• Decrease MCV
• Clinical Significance: DESCRIPTIVE TERMS:
Severe BURNS • Variation in Size, Shape, or Color Content per OIO
Hereditary Pyropoikilocytosis Slight (<5%)
4. Semilunar bodies/Half-moon or Crescent Cell) Moderate (5-15%)
• Large, pale-pink staining ghost of the red Marked (>15%)
cell – the membrane remaining after the • Particular Size variation such as Macrocytes,
contents have been released. Poikilocytes, or Schistocytes
• Always acquired Occasional (<1%)
• Seen in MALARIA Few (1-5%)
Frequent (5-10%)
Many (>10%)
20
SAMPLE CRITERIA FOR ERYTHROCYTE 5. Hemoglobin C Crystals
MORPHOLOGY EVALUATION • Tetragonal, rectangular rod shaped dense
Morphologic
WNL 1+ 2+ 3+ 4+ staining crystals (bar of gold, clam shell)
Characteristics • Seen in Hb C disease
Macrocytes >9 um 0-5 5-10 10-20 20-50 >50 6. Hemoglobin SC Crystals
Microcytes <9 um 0-5 5-10 10-20 20-50 >50 • “washington monument” shape
Hypochromia 0-2 3-10 10-50 20-75 >75 7. Ringed Sideroblast
Poikilocytosis (generalized 0-2 3-10 10-20 20-50 >50
• nRBC that contains nonheme iron particles
variations in shape)
Burr cells 0-2 3-10 10-20 20-50 >50 • excessive iron overload in mitochondria of
Acanthocytes <1 2-5 5-10 10-20 >20 normoblast
Schistocytes <1 2-5 5-10 10-20 >20 • due to defective heme synthesis
Dacryocytes 0-2 2-5 5-10 10-20 >20 • Hemosiderin – needed for the identification
Codocytes 0-2 2-5 5-10 10-20 >20 • Clinical Significance:
Spherocytes 0-2 2-5 5-10 10-20 >20 Sideroblastic anemia
Ovalocytes 0-2 2-5 5-10 10-20 >20 MDS
Stomatocytes 0-2 2-5 5-10 10-20 >20
Abs Report as 1+ to indicate
8. Siderocyte
Sickle cells • Non-nucleated cell containing iron granules
presence, do not quantitate
Polychromatophilia • Seen in sideroblastic anemia and MDS.
Adult <1 2-5 5-10 10-20 >20 9. Pappenheimer Bodies
Newborn 1-6 7-15 15-20 20-50 >50 • Basophilic inclusions that aggregate in small
Basophilic Stippling 0-1 1-5 5-10 10-20 >20 clusters near periphery with Wright’s stain.
Howell-Jolly bodies Abs 1-2 3-5 5-10 >10
• Unused iron deposits.
Siderocytes (Pappernheirmer Abs 1-2 3-5 5-10 >10
bodies) • Clinical Significance:
Guidelines for semiquantitation are ecpressed in number of Sideroblastic anemia
occurences per OIO (applies to fields of approx. 200-250 cells per MDS
100 OIO. WNL- within normal limits. Thalassemia
HA
IV. INCLUSIONS Defective erythropoiesis
1. Howell-Jolly Bodies 10. Malaria
• Coarse densely stained purple granules, • Protozoan transmitted by bite of female
eccentrically located on periphery of membrane Anopheles mosquito
• Nuclear remnants of DNA – (+) Fuelgen 11. Babesia
• Clinical Significance: • Protozoan inclusion (B. microti), transmitted
Megaloblastic Anemia from deer to humans by tick bite.
Severe hemolytic process
Thalessemia V. MISCELLANEOUS
Accelerated erythropoiesis 1. Autoagglutination – clumping of RBC
2. Basophilic Stippling • Cold agglutinin syndrome
• Dark blue granules o PAP by M. pneumonia – anti I
• Precipitation of ribosomes and RNA • AIHA
• Clinical Significance: 2. Rouleaux – RBCs in stack of coin arrangement
Lead poisoning • Multiply Myeloma
Pyrimidine-5-nucleotidase deficiency • Waldenstrom’s Macroglobulinemia
Heavy metal poisoning
Thallasemia MORPHOLOGIC CLASSIFICATION OF ANEMIA
3. Cabot Ring
• Rings, loops, figure of eight, red to purple A. Macrocytic, Normochromic
• Remnant of microtubules of mitotic spindle 1. Vitamin B12 and Folic acid Deficiency
• Clinical Significance: Pernicious anemia
Dyserythropoiesis Sprue
Megaloblastic anemia Following gasterctomy
4. Heinz Bodies Diatery
• Deep purple irregularly shaped inculsions Abnormal intrinsic factor
• Precipitated, denatures Hb due to oxidative Tapeworm infection
injury 2. Disease of the Liver
• CANNOT be seen in Wright’s stain
• Requires supravital stain B. Normocytic, Normochromic
• Clinical Significance: 1. Defective formation of the blood cells or the
Hereditary defects in Hexose presence of tumor cells in thr BM.
Monophosphate Shunt Aplastic anemia
G6PD deficiency Leukemia
Unstable Hemoglobins Hodgkin’s disease
Multiple myeloma
21
 Leukoerythroblastosis 2. Extracorpuscular causes: Nonimmune and
Matastatic cancer Immune HA
Anemia assoc. with renal and
endocrine disease Extracorpuscula Causes
Anemia assoc. with inflammatory dse. Nonimmune Immune
2. Abnormal Hb, increased destruction of RBCs. • Chemicals, toxins, • Isoimmune Abs
Acquire HA venoms o Incompatible BT
PNH • Physical trauma – o HDN
Sickel cell disorders causing • Autoimmune Abs
HDN fragmentation o Warm reacting
o Burns o Cold reacting
Anemia of chronic renal insufficiency o Cardiac replacement o Drug-induced
valves
C. Microcytic, Hypochromic o MAHA
1. Thalassemia o HUS
2. Iron Deficiency anemia
3. Chronic Blood loss 3. Miscellaneous
4. Sideroblastic anemia • Anemia of liver disease
• Sulfhemoglobinemia
CLASSIFICATION OF ANEMIAS ACCORDING TO • Porphyrias
CAUSE • Methemoglobinemias

A. Decreased or impaired production of RBCs C. Acute Blood Loss

1. BM damage, infiltration, atrophy
• Leukemia HEMOGLOBINOPATHIES
• Leukoerythroblastosis
• Aplastic anemia Qualitative Defect in Hemoglobin (Substitution)
• Lymphoma • Alpha2 Beta2 6th glu→val – Hb S
• Multiple myeloma • Alpha2 Beta2 6th glu→val – Hb C
• Myelofibrosis • Alpha2 Beta2 26th glu→lys – Hb E
• Pure red cell aplasia
2. Decreased EPO A. Sickle Cell
• Inflammatory process – anemia of chronic 1. Sickle Cell Anemia
disease • 90-100% Hb S
• Chronic renal disease • some Hb A2 and Hb F
• Hypothyroidism 2. Sickle Cell Trait
3. Vitamin and Mineral deficiency • 55-60% Hb A
• IDA • 40-45% Hb S
• Vit B12 and Folate deficiency
4. Defect in Globin Synthesis B. Thalassemia
• Thalassemia 1. Alpha Thalassemia
5. Iron Overload • Decrease of absert alpha globin chain
• Sideroblastic anemia • Adult 4 Beta → Hb H
• Hemochromatosis Neonate 4 Gamma → Bart’s
6. Ineffective Erythropoiesis 2. Beta Thalassemia
• Chronic dyserythropoietic anemia • Decrease or absent beta globin chain
• Increase Hb A2 and Hb F
B. Increased RBC Destruction (Hemolytic Anemias) • Types:
1. Intrinsic defects within RBCs a. Cooley’s Anemia
Hereditary Acquired o Thalassemia major
o Homozygous beta-thalassemia
Membrane Defects Paroxysmal
Spherocytosis Nucturnal
b. Cooley’s Trait
isa
Elliptocytosis
s tatb
ban uin Hemoglobinuria o Thalassemia minor
o Heterozygous beta-thalassemia
lip
Acanthocytosis aAkitapram (PNH)
Stomatocytosis
Rh Null Disease stomatastes
Enzyme Defect
G6PD
Pyruvate kinase Def.
Hemoglobinopathies
Sickle Cell disease
Hb C disease
Ustable Hb Disease
Hb E disease
Defective Globin Synthesis
Thalassemia

22
HEMATOPOIESIS: cellular formation, proliferation, • Fat (yellow marrow); hematopoietic cell (red
differentiation and maturation of blood cells. marrow)
• Normocellular marrow for adult:
STAGES OF HEMATOPOIESIS Fat – 10-50%
1. Mesoblastic (Mesenchymal) Hematopoietic cells – 40-60%
• As early as 19th day gestation in the blood • Child under 2 years has 100% red marrow.
islands of the yolk sac of the human embryo. • Hypercellular/hyperplasia – increase in one or more
• Blood islands remain active for 8 to 12 weeks cell lines due to compnesation.
• Hematopoietic activity is confined to • Hypocellular/hypoplasia – loss of cellularity or
erythropoiesis. incomplete development in one or more cell lines.
• Embryonic hemoglobins produced: Gower I,
Portland, and Gower II MARROW DIFFERENTIAL
2. Hepatic (Extramedullary) • Recommended that at least 500, and preferably
• 3rd month, yolk sac discontinues its role, fetal 1000 cells be counted for marrow differential.
liver becomes active (erythrocytes and
granulocytes in production) Guidelines for Adult BM Differential in
• by the end of 4th month, primitive cells are Concentrated Smears, 1000-Cell Counts
disappearing, with an increase in the more (Steininger)
definitive erythroblast, granulocytes, and CELL TYPE RANGE (%)
megakaryocytes. Erythroblast 18-24
• Also active are the spleen, thymus, and
Myeloblast, Type I 0-1
lymphnodes.
Myeloblast, Type II 0-2
• Hemoglobin produced: HbF, HbA1, HbA2
3. Myeloid (Medullary) Promyelocytes 1-4
• Between 5th and 6th month gestation, the BM Neutrophils and precursors 53-63
becomes the primary site of hematopoiesis. Monocytes 0-2
• At birth, BM becomes the primary source of Eosinophils and precursors 1-3
cell production. Basophils and precursors 0-1
• Hematopoiesis occurs in most bones but Lymphocytes 8-12
primarily in the flat bones of the sternum, ribs, Plasma cells 0-2
vertebrae, skull and pelvis.
• In ADULT, the principal source of production in NORMAL MARROW CELLS
the STERNUM and other flat bones. In addition to the developing hematopoietic cells, several
other cell types are present in a normal marrow:
Adult Neonates • Macrophages
• Mast cells
HbA1 - ≥ 95% HbF – 60-80%
• Osteoblast – waterbug or comet appearance
HbA2 – 1.5 to 3% HbA – 20-40%
(confuse with plasma cells); bone-forming cells
HbF - < 2% • Osteoclast (misidentified with megakaryoblast)
BONE MARROW GRANULOCYTES
• Granulopoiesis is the orderly production of mature
Specimen Collection granulocytes. It takes 14 days from the blast stage to
1. Trephine Biopsy or Core Biopsy the release of mature granulocytes into the peripheral
• Use of Trephine biopsy needle (Jamshidi blood.
needle)
2. Aspiration Granules
• Aspiration needle (University of Illinois sternal
Neutrophil Eosinophil Basophil
needle)
ACP, acid hydrolase, Peroxidase, Histamine,
MYELOID TO ERYTHROID RATIO (M:E Ratio) muramidase and ACP, and heparin,
• Numeric expression comparing the relative number lactoferrin, which are other and
of granulocytic precursors with the relative erythroid essential for the proteolytic chonroitin
precursors in the BM. phagocytic function of enzymes but sulfates
• Specimen used: BM aspirate the cell do not contain
• Normal M:E ratio: 2:1 to 4:1 (average 3:1) ALP
• Example of various M:E ratio:
Infection 6:1 1. Myeloblast
Leukemia 25:1 • 15-29 um diameter
• Basophilic cytoplasm
BONE MARROW CELLULARITY • Round or slightly oval nucleus
• Percentage of marrow sapce occupied by • N/C ratio of 4:1
hematopoietic cells compared with fat • Fine chromatin
• 2-5 nucleoli
23
 2. Promyelocyte 2. Promonocyte
• 15-21 um diameter • 14-18 um diameter
• Basophilic cytoplasm • Blue-gray cytoplasm
• Contains primary or nonspecific granules • N/C ratio 3:1 to 2:1
Myeloperoxidase 3. Monocyte
Acid phosphatase • 14-20 um diameter
Acid hydrolase • blue-gray cytoplasm
Muramidase (lysozyme) • many fine azurophilic granules (“ground-glass
Cationic bactericidal protein cytoplasm”)
• N/C ratio or 3:1 to 2:1 • round, kidney-shaped nucelus, may show slight
• 2-3 nucleoli lobulationl it may be forled showing brainlike
• chromatin pattern slightly coarser convolutions.
3. Myelocyte • NO nucleoli are visible
• 12-18 um diameter
• Last stage that undergo Mitosis LYMPHOCYTES
• Contains secondary or specific granules
(lactoferrin, lysozyme, collagenase) 1. Lymphoblast
o Neutrophil myelocyte – rosepink granules • 10-18 um diameter
o Eosinophil myelocyte – orange-red granule • Moderate to dark blue cytoplasm
o Basophil myelocyte – dark purple granules • N/C/ ratio is 4:1
• Decreased N/C ratio • 1-2 nucleoli
• Generally shows no nucleoli 2. Prolymphocyte
• Coarse chromatin • May be the same size as the lymphoblast or
4. Metamyelocyte (Juvenile) smaller
• 10-15 um diameter • Moderate to dark blue cytoplasm
• decreased N/C ratio • Round or oval nucleus
• Indented or kidney-shaped nucleus • May contain 1-2 nucleoli
• Chromatin pattern is coarse and clumped 3. Mature lymphocyte
• Last stage capable of mitosis • Small lymphocyte 8-10 um
5. Band / Stab/ Staff • Medium lymphocyte 10-12 um
• 9-15 um diameter • Large lymphocyte 12-16 um
• elongated or band-shaped nucleus
• chromatin pattern is coarse and clumped (C or BLAST
S shape) • 15-20 um
• youngest cell in the series to normally appear • basophilic cytoplasm
on peripheral blood • absence of granules
6. Mature Granulocyte • large nucleus
a. Segmented neutrophil • INCREASE N/C ratio
• 9-15 um diameter • Presence of 2-5 nucleoli
• Pink to rose-violet specific granules • Fine chromatin (most reliable to identify)
• Normally 2-5 lobes (nucleus)
• Coarse, clumped chromatin pattern WBC ABNORMALITIES
b. Eosinophil
• 9-15 um diameter A. NUCLEAR
• Reddish-orange granules 1. Hypersegmentation (≥ 6 lobes)
• Usually 2 lobes (nucleus) a. Hereditary – autosomal dominant
• Coarse, clumped chromatin pattern b. Acquired – Megaloblastic anemia
c. Basophil 2. Hyposegmentation
• 10-16 um diameter - Neutrophil is bilobed showing a dumbell,
• dark purple to blue-black water soluble specktacle-like, peanut-shape, or “Pince
granules nez” shape
• generally unsegmented or bilobed, rarely a. Pelger Huet Anomaly
has 3 or 4 lobes (nucleus) • Autosomal dominant
b. Pseudo Perger Huet (acquired)
MONOCYTES • Myeloproliferative disorder like CML

1. Monoblast B. CYTOPLASM
• 12-20 um diameter a. Alder Reilly Inclusions
• Basophilic cytoplasm • Accumulation of degreaded
• Non-granular mucopolysaccahrides resembling toxic
• N/C ratio is 4:1 to 3:1 granulation.
• 1-2 nucleoli • Ex. Alder-Reilly Anomaly
Hunter’s / Hurler’s Syndrome
24
 b. Auer Rods • Type III
• Fused primary granule; peroxidase (+) – Vacuolated
• Seen in a: – “Swiss Cheese or Moth Eaten”
myeloblast in AML appearance
monoblast in AMML b. Basket Cell / Smudge Cell
• FAGGOT CELLS are cells with mass of Seen in:
Auer rods usually seen in M3. • Pressure in wedge smear preparation;
c. Chediak-Higashi Inclusion usually the remedy is the addition of
• Large azurophilic or purplish-red granules BOVINE ALBUMIN.
• “lysozyme defect” • Chronic Lymphocytic Leukemia (CLL)
• seen in lymphocyte, neutrophil, monocyte where in there is an increase in Smudge
• autosomal recessive cell.
• associated with ALBINISM c. Hairy Cells
• platelet lack DENSE granules • “salt and pepper chromatin”
d. May-Hegglin Inclusion • Lymphocyte with hair-like cytoplasmic
• Pale blue inclusion derived from RNA projections surrounding nucleus, thought to
(confused with Dohle Bodies) be of B cell origin.
• May Hegglin Anomaly – autosomal • (+) Tartrate Resistant Acid Phosphatase
dominant; associated with d. Sezary Cells
thrombocytopenia and GIANT PLATELETs. • Lymphocyte with cerebriform nucleus
e. Dohle Amato Bodies (brain-like nucleus)
• Aggregates of free ribosomes of rough ER • Seen in : Mycosis Fungoides and Sezary
seen in severe infections and toxic states. Syndrome
• Single multiple blue inclusions
f. Toxic granules F. PLASMA CELL ABNORMALITIES
• Large purple to black granules a. Flame Cell
• Seen in infections and toxic states • Red or pink cytoplasm
g. Toxic Vacuoles • Increase cytoplasmic Ig (usually IgA)
• Seen in severe infection and toxic states • Seen in Multiply myeloma
b. Russel Bodies
C. FUNCTION • Individual globules of Ig
a. Job’s Syndrome c. Grape / Berry / Morula Bodies
• NORMAL random activity • MOTT CELL – accumulation or Russel
• ABNORMAL chemotactic activity Bodies; seen in Multiple Myeloma
b. Lazy Leukocyte Syndrome d. Dutcher’s Bodies
• ABNORMAL both random and chemotatic • Intranuclear protein inclusions
activity
c. Chronic Granulomatous Disease (CGD) G. ABNORMAL MONOCYTES / MACROPHAGE /
• Mobility of neutrophil to kill ingested HISTIOCYTES
microorganism. a. Gaucher’s Disease
• Impaired NADPH Oxidase / Oxidative • Deficient GLUCOCEREBROSIDASE or
Metabolism / Respiratory Burst BETA-GLUCOSIDASE
• Test: Nitroblue Tetrazolium Test (NBT) • Accumulation of glucocerebroside
• Characterized by: “wrinkled paper or
D. CELLS EXHIBITING PHAGOCYTOSIS crumpled cytoplasm”
a. LE Cell – a NEUTROPHIL with homogenous b. Niemann-Pick
round body; smooth and evenly stained • Deficient SPHINGOMYELINASE
b. Tart Cell – a MONOCYTE with ingested • Accumulation of sphingomyelin
lymphocyte; rough and unevenly stained • FAOMY cytoplasm
*buffy coat is needed c. Tay-Sach’s
• Deficient HEXOAMINIDASE A
E. LYMPHOCYTE ABNORMALITIES • Accumulation of glycolipid and ganglioside
a. Atypical Lymphocyte / Reactive / Variant / • Vacuolated cytoplasm
Stimulated Lymphocyte / Virocyte / Downey d. Sandhoff
• Type I • Deficient HEMOAMINIDASE A and B
– Turks irritation or Plasmacytoid • Accumulation of glycolipid and ganglioside
Lymphocyte • Vacuolated cytoplasm
– With large block of chromatin e. Sea Blue Histiocytosis
• Type II • Unknown deficiency
– Associated with IM • Blue-green cytoplasm
– With round mass of chromatin
– “Ballerina-skirt” appearance

25
 PLATELET ABNORMALITIES c. M2 (Acute Myeloblastic Leukemia with
Maturation)
A. Giant Platelet (> 20 um) – seen in Bernard Soulier and • > 30% blast ; > 10% granulocytic cells
May Hegglin Anomaly. d. M3 (Acute Promyelocytic Leukemia)
B. Mononuclear Megakaryocytes • > 30% blast
C. Small (Micromegakaryocytes) seen in MDS > 10% granulocytic cells
D. Large Megakaryocyte > 50% promyelocytes
E. Vacuolated Megakaryocyte • involves chromosomal translocation of
Chromosome 15 and 17
LEUKEMIA • assciated with DIC
• presence of FAGGOT CELLS
CLASSIFICATION: e. M4 (Acute Myelomonocytic Leukemia)
A. Duration of untreated disease • “Naegeli”
1. Acute – days to 6 months • 20 to less than 80% monocytic cells
2. Subacute – 2 months to 6 months f. M5 (Acute Monocytic/Monoblastic Leukemia)
3. Chronic – minimum of 1 to 2 years a. M5a (Schilling’s)
B. Type of cell seen • Without differentiation
1. Acute – predominant immature cell particularly • > 80% monocytic cells
Blasts and “Pro” stages • > 80% monoblastic cells
2. Chronic – predominant mature cells b. M5b
C. Number of WBCs • With differentiation
1. Leukemic Leukemia • > 80% monocytic cells
• WBC >15,000/uL • < 80% monoblastic cells
2. Subleukemic Leukemia g. M6 (Erythroleukemia)
• WBC <15,000/uL • “Di Guglielmo’s”
• PRESENCE of abnormal and immature • > 30% blast
cells in PB. > 50% Erythroid precursors
3. Aleukemic Leukemia h. M7 (Acute Megakaryocytic Leukemia)
• WBC <15,000/uL • > 30% blast
• ABSENCE of abnormal and immature > 30% megakaryocytic cells
cells in PB.
D. Cell Lineage AML ALL
1. Myeloid Leukemia Myeloperoxidase (MPO) + -
2. Lymphocytic Leukemia
Sudan Black B (SBB) + -
Terminal deoxyribonucleotidyl
NOTES: - +
Transferase (TdT)
• CALLA (Common Acute Lymphoblastic
Leukemia Antigen) – produced by immunization of
MYELODYSPLASTIC SYNDROME
rabbits with surface Ig (Sig) for identification B
• dysmelopoietic syndrome
lymphocytes and E-rosette for identification of T
• clonal abnormality of hematopoietic cell
lymphocytes.
• “preleukemia” – can progess to ANLL
• < 30% blast
FAB CLASSIFICATION OF ACUTE LEUKEMIA
% Blast
ACUTE – minimum of 30% blast PB BM
Refractory Anemia (RA) < 1% < 5%
A. Acute Lymphoblastic Leukemia (ALL) < 5%
Refractory Anemia with
< 1% 15% ringed
Ringed Sideroblast (RARS)
LYMPHOBLAST sideroblast
L1 L2 L3 Refractory Anemia with
< 5% 15-20%
Childhood ALL Adult ALL Burkitt’s Excess Blast (RAEB)
small large large Refractory Anemia with
and and and Excess Blast in > 5% 20-30%
homogenous heterogenous homogenous Transformation (RAEBIT)
(uniform in size) (varies in size) (stippled) Chronic Myelomonocytic < 5%
with persistent 5-20%
Leukemia (CMML) monocytosis
B. Acute Non-Lymphoblastic Leukemia (ANLL)
a. M0 (Acute Undifferentiated Leukemia)
• > 30% blast
b. M1 (Acute Myelobalstic Leukemia without
Maturation)
• > 30% blast ; < 10% granulocytic cells

26
 LYMPHOPROLIFERATIVE DISORDERS Philadelphia Chromosome
• One long arm of chromosome 22 is
• proliferation of cells derived from lymphoid cells (B and translocated to chromosome 9.
T cells) • Absence of this in CML patients
indicates POOR PROGNOSIS.
A. T and B Cell Leukemia B. Myelofibrosis with Myeloid Metaplasia
B. Lymphoma • Fibrosis and granlocytic hyperplasia of the BM
• Malignancy of lymphoid tissue. • Granulocytic and Megakaryocytic proliferation
• Types: in the liver and spleen.
a. Non Hodgkin’s • MB aspirate CANNOT be performed instead
• Characterized by predominance of BM biospy.
malignant lymphocytes • (+) Dacrocytes
• Associated with EBV C. Essential Thrombocythemia
• Classification: Pappaport • Platelet count >1000 X 109/L
• Lymphosarcoma – aleukemia • Platelets are functionally abnormal.
lymphoma D. Polycythemia vera
• Types: Small Lymphocyte Lymphoma • “PANMYELOSIS”
Lymphoblastic Lymphoma • Increase BM production: RBC, WBC, platelet
Follicular Lymphoma • Prolonged PT and PTT
Burkitts Lymphoma • Decrease EPO
b. Hodgkin’s
• Predominance of cells reacting to the POLYCYTHEMIA
neoplasm
• Reed-Sternberg Cell characterized A. Absolute Polycythemia
by “owl’s eye appearance”, usually a. Primary Absolute Polycythemia
large and has large nucleoli. • Polycythemia vera
• Diagnosed by Lymph Node Biopsy • Polycythemia rubra vera
• Classifications: • Vasquez-Osler Disease
o Rye – based on histologic b. Secondary Absolute Polycythemia with
appearance on lymph node Appropriate Production of EPO
biopsy • Response to HYPOXIA
o Ann Arbor – staging; extent of • Seen in patients with pulmonary and
localization of tissue biopsy cardiac disorders
C. Hairy Cell Leukemia • In BM: ↑ RBC, ↑ WBC, ↑ platelet
• “Leukemic Reticuloendotheliosis” • Increase EPO
• originally B Cells with hairlike projections c. Secondary Absolute Polycythemia with
• (+) TRAP Inappropriate Production of EPO
D. Mycosis Fungoides / Sezary Syndrome • Seen in patients with tumors of kidney,
• Presence of neoplastic T-cells liver, brain, adrenal, and pituitary.
• Sezary cells • In BM: ↑ RBC, Normal WBC and Platelet
• Epidermis is involved, with clusters of • Increase EPO
lymphocyte forming “Pautrier’s microabscess”.
Such primary skin appearance is referred to as B. Relative Polycythemia
“mycosis fungoides” (no fungal infection • Increase Hematocrit due to decrease of plasma
present) volume.
E. Chronic Lymphocytic Leukemia • Associated with DEHYDRATION; stress and
• Increase in SMUDGE cells anxiety
• Lymphocytosis (> 1000 X 109/L) • “GAISBOCK or SPURIOUS POLYCYTHEMIA)

MYELOPROLIFERATIVE DISORDER NOTES:

• Multiple Myeloma
• Proliferation of cells derived from myeloid stem cells o (+) Mott cell, Flame cell, Multinucleated cells
(RBC, granulocutes, monocytes, and platelets) o “punch-out” defect
o Renal Insufficiency: Bench Jone Protein
A. Chronic Myelogenous Leukemia o M protein
• Involves RBCs, granulocytes, monocytes, and o Rouleaux formation
platelets.
• PHILADELPHIA Chromosome (Chromosome Myeloblast Lymphoblast
22 and 9)
• Decreased Leukocyte Alkaline Phosphatase Esterase + -
• Same blood picture with Leukomoid Reaction MPO + -
PAS - +
(except M6)
TdT - +
27
 BASIC MICROSCOPY 2. Gasometric (Van Slyke Oxygen Capacity
Method)
Brightfield Microscopy • 1 g of Hb = 1.34 mL of O2
• Most common type; in hematology, used to examine 3. Specific Gravity (CuSO4 Method)
blood films. • Specific gravity of CuSO4 = 1.053
• Object appear darker than the illuminated field of view. • A 30mL container with CuSO4 is good for 25
tests.
• The distance of blood to the solution should
KOEHLER method of illumination – ensures be 1 cm.
optimum contrast and resolution for the maximum • The acceptable drop is that it should sink
definition of specimen details by precisely focusing within 15 secs which is equal to Hb ≥ 12.5
and centering the light path and spreading the light g/dL.
uniformly over the field of view. 4. Chemical (Kennedy’s, Wong’s)
• 1 g of Hb = 3.37 mg of Iron

Oil immersion Microscopy Cyanmethemoglobin or Hemiglobincyanide (HiCN)

• For observation of RBC morphology and differentiate • test usually used by automated systems.
WBCs. • Modified Drabkin’s Reagent:
a. K+ ferricyanide
Phase Contrast Microscopy ferrous to ferric
• For MANUAL PLATELET COUNTS Hb ------------------------→ metHb / hemiglobin
• Precautions: b. K+ cyanide
metHb / Hemiglobin ----→ cyanmetHb / Hbcyanide
o Debris may result to confusing data
c. Monoionic Detergent
o Halo effect may conceal useful data causing
• Improves lysis of RBC
falsely decreased platelet count.
• Decreases turbidity
d. Dihydrogen Potassium Phosphate (KH2PO4)
Fluorescence Microscopy
• Allows the test solution to be read after
• Used in ANA and T-cell and B-cell studies.
3 mins instead of 15 mins (NaHCO3 –
old Drabkin’s reagent)
Electron Microscopy
• Read at 540 nm means all forms of Hb
• For observation of fine ultrastructure of cells with a
except suflhemoglobin.
magnification of approximately X 100,000.
• Over anticoagulant does NOT affect
result.
HELATOLOGY PROCEDURES
TURBIDITY: False Increase in Hb
COMPLETE BLOOD COUNT Sources Remedy

A. HEMOGLOBIN ↑ WBC Centrifuge then read supernatant

Dilute 1:1 with water then multiply
• NV: At birth: 15-20 g/dL HbC / HbS
result by 2.
Women: 12-16 g/dL
Prepare patient blank plasma + HiCN
Men: 13-18 g/dL Lipemia
Rgt.
• Higher in morning and lower in evening
• Increased in strenous exercise B. HEMATOCRIT
• The higher the altitude, the greater the hemoglobin • NV: At birth: 45-60%
• Slightly higher in smokers Female: 36-48%
• Decreased when patients is lying down Male: 40-55%
• Slight decrease after 50 years old
Determination: • Decreased in anemia
1. Colorimetric • Increased in polycythemia
a. Direct/Visual
• Acid Hematin 0.1 N HCl RULE OF THREE:
– Matching the brownish yellow • Apply only to normocytic and normochromic
color of the solution to the RBCs.
standard comparator block. 3 X RBC = Hb
• Alkali Hematin 0.1 N NaOH 3 X Hb = Hct ± 3 (%)
– NOT for neonates because their
RBCs are resistant to alkali. Determination:
– HbF (alkali resistant). 1. Manual
b. Indirect Macrohematocrit
• Cyanmethemoglobin or a. Wintrobe
Hemiglobincyanide • Uses double oxalate
• Manual or Automated • Used for Hct and ESR

28
 • Length = 11.5mm ; diameter = RBC Pipet
3mm Blood : TV
• Centri: 2000-2,300g for 30 mins 0.5 : 100
1 : 200
• Layes of Spun Hematorit
1 Fatty layer (barely visible) Dilution Factor = 200
2 Plasma Depth Factor = 10 (constant)
3 Buffycoat
4 Packed cell volume (bottom) 3
RBC/mm = # RBC X AF X DF X DilF
• Plasma:
= # RBC X 5 X 10 X 200
o Pink/red = Hb → hemolysis = # RBC X 10,000
o Icteric = ↑ Bili → Jaundice
o Milky = ↑ TAG → Lipemia (for every RBC misses = 10,000 less)
• Buffycoat:
1 mm = 10,000 WBC/mm3 In polycythemia:
b. Van Allen – uses Na oxalate
c. Haden – uses Na oxalate Blood : TV RBC/mm3 = RBC X AF X DF X DilF
0.3 : 100 = RBC X 5 X 10 X 333.33
d. Sanford-Magath – uses Na oxalate 1 : 333.33 = RBC X 16, 667
e. Bray – uses Heparin
In Oligocythemia:
Microhematocrit (Adams)
• Use of capillary tube o 3
Count in 10 3 sq. RBC/mm = RBC X AF X DF X DilF
o Red – heparin 1:200 DilF = RBC X 2.5 X 10 X 200
o Length = 70-75mm (7-7.5 cm) = RBC X 5,000
o Bore = 1mm Count in 10 3o sq. RBC/mm3 = RBC X AF X DF X DilF
• Centri: 10,000-15,000g for 15mins 1:100 DilF = RBC X 5 X 10 X 100
• Layers = RBC X 5,000
1 Plasma
2 Platelet D. WHITE BLOOD CELL COUNT
3 WBC • NV At birth: 10.0 – 30.0 X 109/L
4 Retics and nRBC Female/Male: 4.0 – 11.0 X 109/L
5 Mature RBCs • Elevated in: bacterial infections, leukemia,
6 Clay seal appendicitis, pregnancy, HDN, ulcers
• Advantages: • Drop below normal in: viral diseases such as
o Better packing of cells measles, brucellusis, typhoid fever, infectious
o Less time consuming hepatitis, RA, cirrhosis of the liver, and SLE.
o Venipuncture NOT necessary • Slightly higher in the afternoon than in the
• Trapped plasma: amount of plasma that morning. Increase following strenous exercise,
still remains in RBC portion after the emotional stress and anxiety.
microhematocrit has been spun. • Diluting Fluids: (hypotonic)
Increased in macrocytic anemias, o 1-3% HOAc
spherocytosis, thalassemia, o 1% HCl
hypochromic anemia, and sickle cell o Turk’s solution (gential violet, glacial HOAc,
anemia. and water)

2. Automated WBC Pipet

• Hematocrit is only computed: Blood : TV
Hct = MCV X RBC count 0.5 : 10
1 : 20
C. RED BLOOD CELL COUNT *allow lysis of RBC for 3mins
• NV At birth: 5.0 – 6.5 X 1012/L
Female: 3.6 – 5.6 X 1012/L Dilution Factor = 20
Male: 4.2 – 6.0 X 1012/L Depth Factor = 10 (constant)
• Highest in the morning and at its lowest in the
WBC/mm3 = WBC X AF X DF X DilF
evening = WBC X ¼ X 10 X 20
• Increased in polycythemia vera and secondary = WBC X 50
polycythemia due to dehydration and the effect of = WBC/mm3 X 0.001 = SI unit
altitude
• Decreased in anemia In Leukocytosis: 1:200 using RBC pipet
• Diluting Fluids used: (isotonic)
o NSS WBC/mm3 = WBC X AF X DF X DilF
= WBC X ¼ X 10 X 200
o 3.8% Na citrate = WBC X 500
o Dacie’s or formol citrate – “best”
o Hayem’s , Toisson’s , Bethell’s , Gower’s
29
 In Leukopenia: 1:10 dilution RBC Count
3
o Center square with 25 3o square with
WBC/mm = WBC X AF X DF X DilF 16 small square.
= WBC X ¼ X 10 X 10
= WBC X 25
25 X 16 = 400
5 X 16 = 80
*nRBC are not lysed by WBC diluting fluid and it o Depth: 0.1mm
falsely counted as WBCs. o Depth Factor: 10

WBC Correction TV = A X D X CA
• Formula: = 9 mm2 X 0.1mm X 2
Uncorrected WBC X 100 = 1.8 mm3 (0.9 mm2)
Corrected WBC ct. = --------------------------------
100 + nRBC
• Criteria: R
o Adult: ≥5 nRBC/100 WBC Diff.
o Newborn: ≥10 nRBC/100 WBC Diff.

HEMOCYTOMETER
1. Fuchs-Rosenthal R R
• 2 counting area
• each CA with 16 1-mm2 squares R
• Depth: 0.2mm
• Depth Factor: 5 R R
• Depth Factor is the reciprocal of depth;
where depth = 2 → 10 = 5 E. DIFFERENTIAL CELL COUNT
10 2
Preparation for the Blood Smear
TV = A X DF X CA 1. Cover glass smear (Ehrlich)
= 16mm2 X 5 X 2 2. Cover glass and slide (Beacom)
= 6.4mm2 (3.2mm3) 3. Wedge Smear
2. Speirs-Levy • Angle between the 2 slides: 25o-40o
• 4 counting area 4. Spun Smear (Spinner’s Method: automated)
• each CA with 10 1-mm2 squares (HEMASPINNER)
• Depth: 0.2mm 5. Buffycoat Smear:
• Depth Factor • For patient with WBC ct. <1 X 109/L
• Demostration of LE cell
TV = A X D X CA 6. Thick Blood Smear:
= 10mm2 X 0.2 X 4 • For demonstration of blood parasite which
= 8 mm2 (2 mm3) is stained using GIEMSA.
3. Improved Neubauer
• 2 primary square Staining for the Blood Smear
• Each 1o square with 9 1mm2 2o squares • Contains: Methylene blue/Azure B (basic)
Eosin (acidic)
WBC Count • pH 6.8 – for blood and BM smear
o 4 corner squares pH 7.2 – for malarial smear
o Each 2o square with 16 3o square • Fixative: methanol
4 X 16 = 64 tertiary squares • For best results, blood smears should be
stained 2-3 hours of specimen collection.
W W • Types:
a. Wright’s
b. Giemsa
W W c. Modified Wright-Giemsa
d. Jenner
e. May-Grunwald

W W W W Relative DC (%) Absolute DC (X109/L)

W W W W Segmenters 47 - 77 1.8 – 7.8
W W W W Bands 0-7 0 – 0.7
Eosinophils 0-6 0 – 0.6
W W W W Basophils 0-1 0 – 0.2
Monocytes 2 – 20 0.01 – 0.8
Lymphocytes 20 - 40 1.0 – 4.8

30
Relative WBC Count – expressed in % Notes:
• Neutrophilia – appendicitis, myelogenous
Absolute WBC Count (more accurate) leukemia, bacterial infection
Abs WBC ct = relative WBC ct. X Total WBC ct. • Eosinophilia – allergies, scarlet fever, parasitic
infection, eosinophilic leukemia
Thick Smear Thin Smear • Lymphocytosis – viral infection, whooping cough,
Large drop Small drop IM, lymphocytic leukemia
Fast speed Slow speed • Monocytosis – brucellosis, tuberculosis, monocytic
↑ angle ↓ angle leukemia, SBE, typhoid, rickettsial infections,
collagen disease, Hodgkin’s disease, Gaucher’s
disease.
Counting Methods:
• During infancy and childhood, a mild lymphocytosis
1. Cross-sectional or Crenellation
may be present. Adult normal values are reached by
• WBCs are counted in consecutive fields as
the age of 21.
the blood film is moved from side to side.
• Shift to the Left: presence of immature granulocytic
2. Longitudinal Method
• WBCs are counted in consecutive fileds cells (leukemia, bacterial infections, increase band
from the tail toward the head of the smear. cell)
3. Battlement Method • Shift to the Right: increased number of
• Uses a pattern of consecutive fileds hypersegmented neutrophil.
beginning near the tail on a horizontl edge:
count three consecutive horizontal edge CAUSES OF REACTIVE NEUTROPHILIA
fields, count two fields towards the center of Absolute Neutrophil Count >7 or 8 X 109/L
the smear, count two fields vertically to the • Pathologic
edge. • Infections (pyogenic bacteria, virus, actinomyces
fungi, spirochetes/rickettsial organisms)
• Inflammatory responces to tissue destruction
Routine 100 Cell Differential • Serosal / Visceral
• 50 cells patient WBC <1.0 X 109/L • Blood cell destruction
• 200 cells Over 10% eosinophils • Posttraumatic (surgical/accidental)
Over 2% basophils • Thermal injury
• Chemicals/drugs/venoms
Over 11% monocytes • Parasitic infection
More lympho than neutro • Neoplastic growth
(except in children) • Metabolic disorders
• Acute hemorrhage
EOSINOPHIL COUNT • Corticosteroids
• Lithium
• Diluent: Randoph
• Physiologic response to therapy
Pilot phloxine • Pseudoneutrophilia caused by emotional/physical
Manner stimuli
Hinkleman - eosin CAUSES OF NEUTROPENIA
Tannen – neutral red Neurophil Count <1.75-1.8 X 109/L
• Component: • Decreased neutrophil production
o Phloxine – to stain eosinophil • Increased neutrophil destruction
o Neutral red iodide and Eosin • Inherited/acquired stem cell disorders
o Propylene glycol – lyse RBC • Chemical toxicity like benzene
o Na2CO3 – lyses WBC except eosinophil • Marrow replacement
• Nutritional deficiencies
o Heparin – to prevent clumping • Cytotoxic drugs
• Use of WBC pipet: 1:10 dilution • Infections (bacterial, viral)
• NV: 50-350 X 106/L • Immune reactions (isoimmune, autoimmune)
• Drug-induced
a. Modified Neubauer • Sequestration
• Pseudoneutropenia
Eo X AF X DF X DilF • Malignant myeloproliferative disorder
Eo X 1/9 X 10 X 10
Eo X 11.11 CHANGES IN EOSINOPHIL NUMBER
A. Eosinophilia (>7.0 X 109/L)
b. Fush-Rosenthal • Infestation by tissue-invading parasites like
Trichinella sp.
Eo X AF X DF X DilF • Respiratory (asthma, hay fever)
Eo X 1/16 X 5 X 10 • Skin disease (psoriasis, eczema)
Eo X 3.125 • Allergic reactions and certain poisons
• Leoffler’s syndrome
c. Speirs-Levy • Pulmonary infiltrates with eosinophil (PIE)
• Tropical eosinophilia
Eo X AF X DF X DilF • Hypersensitivity
Eo X 1/40 X 5 X 10 • Malignancies of myeloid cells
Eo X 1.25 • Familial, postirradiation, periarteritis nodosa
31
 B. Eosinopenia (<0.05 X 109/L) RETICULOCYTE COUNT
• Decreased production • NV Adult: 0.5 – 1.5% (ave. 1%)
• Acute bacterial infection Newborn: 2 – 6%
• ACTH administration • Index of BM production
• Reticulum RNA is demonstrated by Supravital stain
CAUSES OF MONOCYTOSIS (NMB)
(>0.9 X 109/L) • Increased in: hemolytic anemias, individuals with IDA
• Bacterial infection receiving iron therapy, thalassemia, sideroblastic
• Tuberculosis anemia, and in acute and chronic blood loss.
• SBE • Decreased in: aplastic anemia and in conditions in
• Syphilis
• Inflammatory process
which BM is not producing RBCs.
• Surgical trauma
• Tumors % Retics (NV: 0.5-1.5%)
• Collage vascular diseases
• GI diseases number of retics
• Recovery from neutropenia (relative) % retics = --------------------------------- X 100
• Myeloproliferative disorders number of RBC (1000)

Absolute Reticulocyte Count (ARC)

PLATELET COUNT NV: 25-75 X109/L
• NV: 150,000 – 400,000/uL
150 – 400 X 109/L % retic
• Thrombocytosis: polycythemia vera, idipathic ARC = ------------- X RBC count (1012/L) X 1000
thrombocythemia, CML, splenectomy 100
• Thrombocytopenia: ITP, aplastic anemia, acute
leukemia, pernicious anemia, Gaucher’s disease, Corrected Reticulocyte Count (CRC) NV: 1
following chemotherapy and radiation. Patient Hct (L/L)
CRC = ------------------------------
Determination Normal Hct (0.45L/L)
I. Manual
A. Direct (Hemocytometer) Reticulocyte Production Index or Shift Correction
1. Reese-Ecker (Na citrate, HCHO, BCB) NV: 1
2. Guy and Leake (Na citrate, HCHO, CV) • General indicator of the rate of erythrocyte
3. Brecker-Cronkite (Phase Contrast) production
• 1% ammonium oxalate • Increase above normal in anemias; BM response
• stand for 15 mins before counting to anemia
• Reference range: 1 when the Hct is 0.45L/L
3
Platelet/mm = platelet X AF X DF X DilF • PRI >3 Adequate response of the BM to anemia:
= platelet X 1 X 10 X 200 chronic hemolysis, recent hemorrhage, and
= platelet X 2,000
response to therapy
• RPI <2 Inadequate response of the BM to
#s 1,2,3 – uses RBC pipet (1:200)
anemia: BM failure ex. Aplastic anemia,
4. Unopette
ineffective erythropoiesis ex. Vit B12 or folate
• 1% ammonium oxalate
deficiency
• diluent 1.98mL; blood 0.02mL
• dilution = 1:100
Hematocrit Maturation Retics (Blood)
Platelet/mm3 = platelet X AF X DF X DilF 45 – 36% 1.0 day (normal)
= platelet X 1 X 10 X 100 35 – 26% 1.5
= platelet X 1000
25 – 16% 2.0
B. Indirect <15% 2.5
1. Dameshek
2. Fonio’s CRC
RPI = ---------------------------------------------
3. Olef’s
Maturation time of retics (blood)

# of platelet Miller Disk

Platelet count = ---------------------- X RBC count
1000 RBCs Retic (A)
% Retics = --------------------- X 100
II. Automated Retics (B) X 9
• 2 – 20 fL – counted as platelet

32
THORN’S TEST (assess adrenocortical function) ERYTHROCYTE SEDIMENTATION RATE (ESR)
• Nonspecific measurement used to detect and
Eo count #1: fasting specimen monitor an inflammatory response to tissue injury.
Eo count #2: 4 hours after ACTH administration
Elevated ESR Decreased ESR
Normal: Eo ct. #2 is lower than Eo ct. #1 by 50% • Pregnancy (after 3rd month) • Polycythemia
Hyperadrenalism: Eo count #1 = Eo count #2 • Acute/chronic infection • CHFhypofibrinogenemia
• Rheumatic fever • Presenceof RBC
ERYTHROCYTE INDICES (Wintrobe’s Indices) • MI abnormalities
1. Mean Corpuscular Volume (MCV) • Nephrosis (poikilocyteosis,
• NV: 80-100 fL / um3 • Acute Hepa spherocytes, sickle cells)
< 80 fL = microcytic • Menstruation
• Tuberculosis
> 80 fL = macrocytic
• Macroglobulinemia
• Increase (Macrocytic): Megalobalstic anemia, • Cryoglobulinemia
nonmegaloblastic anemia (liver disease, • Hypo/hyperthyroidism
hypothyroidism)
• Decrease (Microcytic): IDA, defective iron FACTOR THAT INFLUENCE ESR
utilization (chronic disease) and Thalassemia INCREASE DECREASE
Hct (%) RBCs Macrocytes Microcytes
MCV = ---------------------- X 10 Anemia Poikilocytes
Anisocytes
RBC ct (1012/L)
Polythemia
Plasma Fibrinogen Albumin
2. Mean Cell Hemoglobin (MCH)
composition Alpha 1 globulin
• NV: 27 – 31 pg / uug Alpha 2 globulin
• Increase: macrocytic anemia because RBCs
are larger and carry more Hb Technical
o
Tilting (3 ≈ 30% error) Increase EDTA
• Decrease: found in hypochromic anemias and Increase temperature (shrinkage of cell)
microcytic anemia unless RBCs are also
spherocyte. Stages:
• This is rarely used, MCV and MCHC are more • Initial rouleaux formation 10 mins
common. • Rapid settling of RBCs 40 mins
• Final sedimentation of RBCs 10 mins
Hb (g/dL) 60 mins
MCH = ------------------------X 10
RBC ct (1012/L) Methods:
1. Standar/Original Westergren
3. Mean Cell Hemoglobin Concentration (MCHC) • Anticoagulant: Citrate (black) 1:4
• NV: 31 – 36% or g/dL • Length tube: 30 cm
>36% - hyperchromic • Bore: 2.5 mm
<36% - hypochromic 2. Modified Westergren
• Increase (Hyperchromic): Spherocytes • Anticoagulant: EDTA (2mL EDTA + 0.5mL
• Decrease (Hypochromic): IDA, Thalssemia, NSS/Citrate)
defective iron utilization 3. Wintrobe and Landsberg
• Anticoagulant: double oxalate or balance
Hb (g/dL) oxalate
MCHC = ------------------- X100 • Length: 11.5 cm
Hct (1012/L) • Bore: 2.0 mm
MCHC above 38% does not occur, possible errors Normal Values: (in mm)
are: Male Female
o Incorrect computation Children
<50 >50 <50 >50
o Presence of cold agglutinin; remedy –
warm Westergren 0-15 0-20 0-20 0-30 0-10

MCHC will not fall below 22%, possible causes Wintrobe 0-9 0-20 0-13
are:
o Lipemia
o Presence of abnormal Hb (HbS or HbC)

Defective Centrifuge affects: MCV and MCHC

(usually high Hct reading)

33
 Increase ESR Normal ESR QUANTITATION OF HEMOGLOBIN F
Inflammatory conditions Polycythemia A. Betke Method of Alkali Denaturation (NaOH)
Acute/chronic infections Spherocytes B. Singer Method of Alkali Denaturation (KOH)
Rheumatic fever Sickle cells
RA and MI Sickle cell anemia UNSTABLE HEMOGLOBIN (detection of Hb H)
Nephrosis Hb CC disease A. Heat Precipitation Test
Tuberculosis Hereditary spherocytosis B. Isopropanol Test
MM
Waldenstrom’s macro. G6PD SCREENING
SBE; Hepa A. Ascorbate Cyanide Screening Test
Henstruation • Nonspecific procedure for detecting deficiencies
Pregnancy in the Pentose Phosphate Pathway: G6PD
deficiency, glutathione peroxidase, glutathione
reductase
ZETA SEDIMENTATION RATE
• Patient RBC + Na cyanide + Na ascorbate
• Dependent on the concentration of fibrinogen and
gammaglobulins • Normal: red
• NOT affected by anemia • Abnormal/enzyme deficient: brown
• Same reference range for male and femal B. G6PD Fluorescent Screening
• NV: 40-51% • G6P + NADP
G6PD ↓ RBCs
• Disadvantage: uses special instrument (zetafuge)
Uses special capillary tubes 6phosphogluconate + NADPH (fluores)
• Normal: max. fluorescence at 10mins
Hct (%) • Abnormal: no fluorescence
ZSR = ------------------ X 100 C. Blood alone (48 hours) 0.2 to 2%
Zetacrit (%) Blood + glucose → 0 to 0.8%
Blood + ADP → 0 to 0.9%
OSMOTIC FRAGILITY TEST a. D6PD deficiency
• Anticoagulant: HEPARIN • Corrected by glucose only
b. Pyruvate kinase deficiency
• Increased (decrease resistance): found in hemolytic
• Corrected by ADP only
anemia and hereditary spherocytosis, and whenever
c. Hereditary Spherocytosis
spherocytes are found
• Corrected by glucose and ADP
• Decreased (increase resistance): seen in
D. Supravital Stain
splenectomy and liver disease, thalassemia and in
conditions in which target cells are present
PAROXYSMAL NOCTURNAL HEMOGLOBINURIA
• Reticulocytes shows decreased OFT; the older the
• Acquired disorder in which patient’s RBCs are
red cells are also more fragile.
abnormally sensitive to destruction by normal
constituents of plasma like complement; deficiency in
Griffin and Sanford (uses 12 tubes)
DAF and HRF.
25 24 23 22 21 20 19 18 17 16 15 14 A. Sucrose Lysis Test (Screening)
0.5%
25 24 23 22 21 20 19 18 17 16 15 14
• RBCs + ABO compatible Serum + Sucrose
NaCl
H2O
solution
0 1 2 3 4 5 6 7 8 9 10 11
% • Normal: No hemolysis; PNH: hemolysis
NaCl B. Ham’s Acidified Serum Test
drop .50 .48 .46 .44 .42 .40 .38 .36 .34 .32 .30 .28
X • Tube 1: px RBCs + normal serum + 0.2 N HCl
0.02 Tube 2: px RBCs + pt own serum + 0.2 N HCl
+ Blood (RT, 2 hours) Tube 3: px RBCs + normal activated serum + 0.2
N HCl
NV: Initial Hemolysis Tube 21 or 22 (0.42-0.44%)
Complete Hemolysis Tube 16 or 17 (0.32-0.34%) Normal: No hemolysis in 3 tubes
PNH: Hemolysis in 3 tubes
Spherocytes: increase OFT, decrease tendency to swell
in hypotonic solution (HIS) PAROXYSMAL COLD HEMOGLOBINURIA
Target and Sickle cells: decrease OFT, increase • Presence of IgG auto Anti-P which cause biphasic
tendency to swell in hypotonic Solution hemolysis: cold temp: it attaches to RBCs
Warm temp: it lyses RBCs
SICKLE CELL TEST (detection of Hb S) • Donath-Landsteiner Antibody Test
A. Sodium metabisulfite Control: Px WB Test: Px WB
• Formation of sickle shape red cells (+HbS) 30 mins 37oC 4 oC
• (+) sickling of cells
30 mins 37oC 4 oC
Normal No hemolysis No hemolysis
B. Sodium Dithionite Test (Solubility Test)
PCH No hemolysis Hemolysis
• Turbid appearance (+HbS)
34
Tests That Require Defibrinated Blood
• OFT
• Autohemolysis Test
• Acidified Serum Test

HEMOGLOBIN ELECTROPHORESIS
A. Cellulose Acetate Electrophoresis (pH 8.6)

(-) C S F A1 Bart I H (+)

E D
A2

Slowest ←--------------------------------------→ fastest

Note: this cannot measure Hb F

B. Citrate Agar Electrophoresis (pH 6-6.3)

F A origin O S C
(-) E D (+)
G

35
AUTOMATED CELL COUNTERS
In Coulter:
A. Optical Light Scattering • Blood is directed to 2 channels:
• Blood cell creates forward and side light scatters Channel 1 (1:6250)
which are detected by photodetectors. a. RBC 36-360 fL
• Example: Technicon Analyzer b. Platelet 2-20 fL
a. Forward Light Scatter Channel 2 (1:251)
• Cell size a. Hb (Hemoglobincyanide)
b. Side Light Scatter b. WBC 35-90 fL – lymphocyte
• Cell granularity; right angle or 90o 90-160 fL – monocyte
160-450 fL - granulocyte
B. Electrical Impedance • Test is 2 times repeated before results are
• Blood cells asre non-conductors of electricity. released
They create impedance or resistance of current • Hematocrit is only computed:
when passed in a solution that conducts Hct = MCV X RBC
electricity.
• Ex. Coulter and Sysmex

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 immunosuppresants
Monoclonal proteins
nRBCs
Platelet clumping
Unlysed RBCs
RBC Count Cryoglobulin Autoagglutination
Cryifibrinogen Clotting
Giant platelets Hemolysis (in vitro)
High WBC (>50,000/uL) Microcytic red cells
Hemoglobin HbCO (>10%) Clotting
Cryoglobulin Sulfhemoglobin
Cryofibrinogen
Hemolysis (in vitro)
Heparin
High WBC (>50,000/uL)
Hyperbilirubinemia
Lipemia
Monoclonal proteins
Hematocrit Cryoglobulin Autoagglutination
(Automated) Cryofibrinogen Clotting
Giant platelets Hemolysis (in vitro)
High WBC (>50,000/uL) Microcytic red cells
Hyperglycemia (>600mg/dL)
Hematocrit Hyponatremia Excess EDTA
(MicroHct) Plasma trapping Hemolysis (in vitro)
Hypernatremia
MCV Autoagglutination Cryoglobulin
High WBC (>50,000/uL) Cryofibrinogen
Hyperglycemia Giant platelets
Reduced red cell deformability Hemolysis (in vitro)
Microcytic red cells
Swollen RBCs
MCHC Autoagglutination High WbC (>50,000/uL)
Clotting Spurious low Hb
Hemolysis (in vitro) Spurious high Hct
Hemolysis (in vivo)
Spurious high Hb
Spurious low Hct
Platelet Count Cryoglobulin Clotting
Cryofibrinogen Giant platelets
Hemolysis (in vitro and in vivo) Heparin
Microcytic red cells Platelet clumping
Red cell inclusions Platelet satellitism
White cell fragments

36
 SPECIAL STAINS

CONSTITUENTS IMPORTANT
NAME TYPE INTERPRETATION
STAINED INFORMATION
Myeloperoxidase (MPO) Enzyme Marker for primary Peroxidase activity Myeloperoxidase
granules and Auer rods produces dark brown enzyme deteriorates.
granules in cytoplasm Stain should be done
of granulocytes and only on fresh
monocytes specimens.
Sudan Black B (SBB) Nonenzyme Marker for phospholipids Dark purple-black Can be done on stored
and lipids granules in neutrophil specimens. Parallels
precursors. myeloperoxidase for
Lymphoblasts are interpretation.
negative.
Naphtol AS-D Enzyme Marker for mature and Enzyme activity results Known as specific
Chloroacetate Esterase immature neutrophils in bright red granules in esterase. Stable
(+) ganulocytic and mast cells cytoplasm of enzyme that lasts for
(-) monocytic neutrophils, neutrophil months.
precursors and mast
cells.
α-Naphthyl Acetate Enzyme Marker for monocytes, Monocytes stain red- Known as nonspecific
Esterase megakaryocytes, and brown. esterase (NSE)
(-) granulocytic plasma cells
(+) MS
α-Naphthyl Butyrate Enzyme Identifying monocytes, Enzyme activity results Known as nonspecific
Esterase promonocytes, and in dark red precipitates esterase (NSE)
(-) granulocytic monoblasts in cytoplasm
(+) MS
Acid phosphatase Enzyme Present in all Activity is indicated by Cannot be stored
(+) Hairy Cell Leukemia (TRAP) hematopoietic cells and purple to red granules
(+) T-cell Leukemia found in lysosomes
(-) B-cell Leukemia
Tartrate Resistant Acid Enzyme Marker for hairy cells Activity is indicated by Excellent marker for
Phosphatase leukemia purple to dark red hairy cell leukemia
granules in cytoplasm
Leukocyte Alkaline Enzyme Neutrophil is the only 100 cell count is done. Used to differentiate
Phosphatase (LAP) leukocytes that contains Neutrophils are scored CML from leukomoid
this activity form with no activity to reaction. CML has
4 with a large amount of decreased activity.
activity.
Periodic Acid-Schiff Nonenzyme Marker for glycogen, Activity results in bright Lymphoblastic
(PAS) glycoproteins, fushcia pink leukemia (ALL) shows
*All blood cells are negative mucoproteins and high blocky or chunky
except for Erythroblast. molecular weight Pattern of staining pattern.
carbohydrates varies with each cell L1 and L2 block
type. pattern.
L3 negative
Erythroblasts in M6
leukemia positive
Terminal Enzyme DNA polymerase Is present in 90% cases Used to differential
Deoxyribonucleotidyl immunoperoxidase of ALL AML from ALL
Transferase ((TdT) Marker for immature
lymphocytes
Toluidine Blue Nonenzyme Binds with acid Strongly metachromatic Useful for recognition
mucopolysaccharides in of mast cells and
blood cells tissue basophils

37
RED CELL INCLUSIONS
Red Cell Type Morphologic Appearance Defect or Change Associated Conditions
Heinz bodies Deep purple irregular Represent precipitated, Hereditary defects i9n
shaped inclusions 2 to 3 um denatured hemoglobin due hexose monophosphate
Found on RBC inner to oxidative injury shunt
surface of membrane G6PD deficiency
Unstable hemoglobin
Basophilic stippling Round dark blue granules Represents impaired Lead poisoning
uniformly distributed erythropoiesis Pyrimidine-5-nucleotidase
deficiency
Pappenheimer bodies Small 2 to 3 um irregular Unused iron deposites Sideroblastic anemia
basophilic inclusions that Defective erythropoiesis
aggregate in small clusters
near periphery with Wright’s
stain
Ringed Sideroblast Nucleated RBC that Excessive iron overload in Sideroblastic anemia
contains nonheme iron mitochondria of normoblasts
particles arranged in ring Due to defective heme
form synthesis
Siderocyte Non-nucleated cell Same as above Same as above
containing iron granules

Miscellaneous RBC abnormalities

Red Cell Type Morphologic Appearance Defect or Change Associated Conditions
Autoagglutination Clumping of RBCs Presence of antibody Cold agglutinin
Autoimmune hemolytic
anemia
Rouleaux Alignment of RBCs linear Caused by increased Multiple myeloma
appearing as stack of coins concentration of globulin Waldenstrom’s
macroglobulinemia

WBC ABNORMALITIES

Abnormalities Associated with Granulocytes

Description Morphologic Appearance Defect or Change Associated Conditions
Alder-Reilly granules Large purple-black coarse Accumulation of Alder-Reilly anomaly
cytoplasmic granules mucopolysaccharides Hunter’s syndrome
Auer rods Pink or red rod shaped Fused primary granules AML
cytoplasmic structures Found in myeloid and
monocytic series only
Chediak-Higashi granules Giant red, blue to grayish Large cytoplasmic inclusions Chediak-Higashi syndrome
round inclusions in of fused primary granules Albinism
cytoplasm that are deficient in enzymes
for phagocytosis
Hypersegmented >5 lobes Abnormal DNA synthesis Megaloblastic anemia
neutrophil
Pelger-Huet Single or bilobed nucleus Decreased segmentation Pelger-Huet anomaly
LE cell Neutrophil with large purple Three factors needed to Lupus erythematosus
homogenous round produce LE cell:
inclusion with nucleus Antinuclear antibodies, cell
wrapped around nuclei, phagocytes with
ingested material
Döhle bodies Single of multiple blue Aggregates of free Severe infections
cytoplasmic inclusions in ribosomes of rough Toxic states
neutrophil endoplasmic reticulum
Toxic granules Large purple to black Primary granules Infections, toxic states
azurophilic granules
Toxic vacuoles Large empty white areas Represent end stage Septicemia
within cytoplasm phagocytosis Severe infections
Toxic states
38
 RED CELL ABNORMALITIES
Red Cell Size
Red Cell Type Morphologic Appearance Defect or Change Associated Conditions
Normocytic Normal sized biconcave disc RBCs, NA NA
6-8 um
Microcytic Smaller RBCs, less than 6 um Abnormal size due to the Iron deficiency anemia
MCV<80fl failure of hemoglobin Thalassemia
synthesis
Macrocytic Larger RBCs Impaired DNA Synthesis Megaloblastic anemia
MCV>100fL Stress erythropoiesis Liver disease
Excess surface membrane Alcoholism

Red Cell Hemoglobin Content

Red Cell Type Morphologic Appearance Defect or Change Associated Conditions
Normochromic Normal in color NA NA
Hypochromic Central pallor exceed 1/3 of Reduced hemoglobin Iron deficiency anemia
the diameter of the cell content (MCHC) Thalassemia

Hyperchromic No central pallor Greater than normal MCHC Spherocytosis

Red Cell Shape

Red Cell Type Morphologic Appearance Defect or Change Associated Conditions
Acanthocyte Spheroid with 3-12 irregular Increased ratio of End stage liver disease
spikes cholesterol to lecithin Pyruvate kinase deficiency
Abetalipoproteinemia
Echinocyte Regular 10-30 scalloped Depletion of ATP Uremia
short projections Exposure to hypertonic Cirrhosis
solution Hepatitis
Artifact in drying Chronic renal disease
Codocyte Peripheral rim of Excess in surface Hemoglobinopathies
hemoglobin surrounded by membrane to volume ratio Thalassemia
clear area and central Post-splenectomy
hemoglobinized area (bull’s
eye)
Dacryocyte Teardrop or pear-shaped Squeezing and Myeloid metaplasia
with single elongated point fragmentation during splenic Hypersplenism
or tail passage
Drepanocyte Crescent shape cell that Polymerization of Sickle cell anemia
lacks zone of central pallor deoxygenated hemoglobin
Elliptocyte Rod or cigar shaped Polymerization of Hereditary elliptocytosis
hemoglobin
Schistocyte Fragments of RBCs varying Extreme fragmentation Disseminated intravascular
in size and shape produced by damage of coagulation (DIC)
RBCs by fibrin, altered Microangiopathic hemolytic
vessel walls and prosthetic anemia
heart valves
Spherocyte Smaller in diameter than Lowest surface area to Hereditary spherocytosis
normal RBC with volume ration Immune hemolytic anemia
concentrated hemoglobin Defective membrane
content
No visible central pallor
Stomatocyte Normal sized cell with slit Known to have increased Hereditary stomatocytosis
like area in center permeability to sodium Rh null disease

39
Red Cell Inclusions

Red Cell Type Morphologic Appearance Defect or Change Associated Conditions

Coarse round densely Nuclear remnants Megaloblastic anemia
stained purple 1-2 um containing DNA Accelerated erythropoiesis
granules eccentrically
Howell-Jolly bodies
located on periphery of
membrane
May be single or double
Rings, loop or figure of Remnants of microtubules dyserythropoiesis
Cabot ring
eight; red to purple of mitotic spindle

ABNORMALITIES ASSOCIATED WITH LYMPHOCYTES / PLASMA CELLS

Red Cell Type Morphologic Appearance Defect or Change Associated Conditions
Degenerated nucleus or Lymphocytes that are fragile Chronic lymphocytic
Basket cell ruptured cell in form of and break upon smearing leukemia
smudge of basket Small numbers are artifact
Lymnphocyte with hair-like Thought to be of B cell origin Hairy cell leukemia
Hairy cell cytoplasmic projections
surrounding nucleus
Round lymph cell with Represents leukemic phase Seźary
Seźary cell nucleus that is grooved or of mycosis fungoides Mycosis fungoides
convoluted T-cell characteristics
Plasma cell with red to pink Associated with increased in Multiple myeloma of IgA
Flame cell
cytoplasm IgA nature
Plasma cell that contains Large protein globules Multiple myeloma
Grape cell small colorless vacuoles giving the appearance of
grapes

LEUKOCYTE ACID PHOSPHATASE NITROBLUE TETRAZOLIUM TEST (NBT)

• Differentiates Leukomoid reaction from CML. • CGD: Chronic Granulomatous Disease
• 100 neutrophils are counted. o Inability of neutrophil to kill ingested
• Grading: 0 – no stain microorganism.
o Impaired NADPH Oxidase/Oxidative
Metabolism/Respiratory Burst.
o NV: 80-100% formazan
4+ - intense reaction <50% indicates CGD
• NV: 30-185 LAP Score
NBT Dye
Example: Clear pale yellow -------------------→ blue ppt.
0 X 10 =0 (original color) (Formazan)
1+ X 20 = 20
2+ X 20 = 40 Modified NBT (use of heparinized plasma)
3+ X 20 = 60
4+ X 30 = 120 Toxic oxygen
100 240 LAP score (increase)
production of
Leukomoid reaction CML
Buffycoat + bacterial ---------→ blue Formazan
MMM CGL suspension

Polycythemia vera PNH NOTES:

• Esterase – differentiates cells from monocytic cells
3rd Trimester pregnancy - differentiates ANLL from ALL.
• To differentiate Monocyte from positivity with
NOTES: plasma cell and megakaryocyte – it is inhibited by
• PAS – all blood cells are (-) except for erythroblast. Fluoride
• Neutrophil Alkaline Phosphatase (NAP) – believed • Toluidine Blue - (+) basophils and mast cells
to be 3o granules which appears as (purplish red); seen in CGL (Chronic Granulocytic
metamyelocyte. Leukemia)
40
Red Cell Inclusions

Red Cell Type Morphologic Appearance Defect or Change Associated Conditions

Coarse round densely Nuclear remnants Megaloblastic anemia
stained purple 1-2 um containing DNA Accelerated erythropoiesis
granules eccentrically
Howell-Jolly bodies
located on periphery of
membrane
May be single or double
Rings, loop or figure of Remnants of microtubules dyserythropoiesis
Cabot ring
eight; red to purple of mitotic spindle

ABNORMALITIES ASSOCIATED WITH LYMPHOCYTES / PLASMA CELLS

Red Cell Type Morphologic Appearance Defect or Change Associated Conditions
Degenerated nucleus or Lymphocytes that are fragile Chronic lymphocytic
Basket cell ruptured cell in form of and break upon smearing leukemia
smudge of basket Small numbers are artifact
Lymnphocyte with hair-like Thought to be of B cell origin Hairy cell leukemia
Hairy cell cytoplasmic projections
surrounding nucleus
Round lymph cell with Represents leukemic phase Seźary
Seźary cell nucleus that is grooved or of mycosis fungoides Mycosis fungoides
convoluted T-cell characteristics
Plasma cell with red to pink Associated with increased in Multiple myeloma of IgA
Flame cell
cytoplasm IgA nature
Plasma cell that contains Large protein globules Multiple myeloma
Grape cell small colorless vacuoles giving the appearance of
grapes

LEUKOCYTE ACID PHOSPHATASE NITROBLUE TETRAZOLIUM TEST (NBT)

• Differentiates Leukomoid reaction from CML. • CGD: Chronic Granulomatous Disease
• 100 neutrophils are counted. o Inability of neutrophil to kill ingested
• Grading: 0 – no stain microorganism.
o Impaired NADPH Oxidase/Oxidative
Metabolism/Respiratory Burst.
o NV: 80-100% formazan
4+ - intense reaction <50% indicates CGD
• NV: 30-185 LAP Score
NBT Dye
Example: Clear pale yellow -------------------→ blue ppt.
0 X 10 =0 (original color) (Formazan)
1+ X 20 = 20
2+ X 20 = 40 Modified NBT (use of heparinized plasma)
3+ X 20 = 60
4+ X 30 = 120 Toxic oxygen
100 240 LAP score (increase)
production of
Leukomoid reaction CML
Buffycoat + bacterial ---------→ blue Formazan
MMM CGL suspension

Polycythemia vera PNH NOTES:

• Esterase – differentiates cells from monocytic cells
3rd Trimester pregnancy - differentiates ANLL from ALL.
• To differentiate Monocyte from positivity with
NOTES: plasma cell and megakaryocyte – it is inhibited by
• PAS – all blood cells are (-) except for erythroblast. Fluoride
• Neutrophil Alkaline Phosphatase (NAP) – believed • Toluidine Blue - (+) basophils and mast cells
to be 3o granules which appears as (purplish red); seen in CGL (Chronic Granulocytic
metamyelocyte. Leukemia)
40