HEMATOLOGY II LECTURES INTRODUCTION TO HEMOSTASIS

 2ND Century A.D – Hemophilia was first recognized

 12th Century A.D – Moises Maimonides described 2 male siblings who died because of excessive
bleeding after circumcision
 1803 – Clinical description of families with hemophilia was first published. The disorder was given
the name
 hemophilia which means “love of hemorrhage” by Schonlein
 1828 – the disorder was first described in a thesis published by Hoff
 1842 – Platelets were described
 1905 – Theory on Blood Coagulation by Paul Morawitz was accepted
 1913 – Lee and White whole blood clotting time was performed
 1930 – Prothrombin time was introduced by Quick
 1940 – Others tests for evaluating hemostatic mechanisms, like platelets count and bleeding time
were introduced
 1964 – “Cascade and Waterfall Theory” of coagulation was introduced

HEMOSTASIS
 Stoppage of blood flow (Gr.)
 Involves the interaction of blood vessels, platelets, the coagulation mechanism, fibrinolysis and
tissue repair.
 A complex process that:
• Produces a clot to stop the bleeding
• Keeps the clot confined
• Dissolves the clot as the wound heals
CELLULAR ELEMENTS OF HEMOSTASIS
 Extravascular Tissue Factor – tissue surrounding the vessel.
 Vascular intima – blood vessel through which the blood flows.
 Intravascular component – plasma proteins and platelets.
o e.g. coagulation factors, inhibitors of coagulation, inhibitors of fibrinolysis, Ca, vWF, platelets
STAGES OF HEMOSTASIS
a. Primary hemostasis – refers to the role of blood vessels (vascular system which includes the
arteries, veins and capillaries) and platelets in the primary formation of platelet plug in response
to vascular injury
Test: bleeding time
•Platelet adhesion
o Contact of platelets with the subendothelium and their adhesion to it (exposed collagen and
platelet in subendothelial)
o Platelet adheres to collagen
o Occurs in presence of vWF – (needed for normal platelet adhesion)
 Von Willebrand’s disease (GP1B)
 Bernard-Soulier disease
• Platelet activation
o Morphologic and functional changes in platelets (platelet shape change)
o From discoid to spherical with pseudopod formation
o Ca++, actin and myosin / thrombosthenin (a.k.a acronyosin)
o Agonists – substances that initiates activation
 EICOSMOID PATHWAY Arachidonic acid – (cyclooxygenase) – Thromboxane A2

o Thromboxane A2 – vasoconstrictor; stimulates platelet secretion

• Platelet secretion (release)
o Release of granules
ALPHA DENSE LYSOSOMAL GYLCOGEN MEMBRANE
(BFA2TP3F5C) (C2APAS) GRANULES PHOSPHOLIPIDS
B-thromboglobulin Calcium Neutral protease Glycogen – for Thromboxane A2
energy storage precursors
Fibronectin Catecholamine Acid hydrolase
Albumin ADP Bacteriocidal
enzyme
AAP (a2- Pyrophosphate
antiplasmin
thrombospondin ATP
Serotonin5-hyroxyindoleaceticacid
ALPHA (BFA2TP3F5C) DENSE (C2APAS)
 PF4 (platelet factor 4; heparin)  Serotonin(5-hyroxytryptamine)
 PDGF (platelet derived growth factor)  Magnesium
 Plasminogen
 FI (fibrinogen)
 FV
 FVIII
 vWF
 C1 esterase inhibitor

ROLE IN SUBSTANCE COMMENTS IN PRINCIPAL

HEMOSTASIS FUNCTION
1. Promote coagulation HMWK Contact activation of intrinsic
coagulation pathway
Fibrinogen Converted to fibrin for clot formation
FV Cofactor in fibrin clot formation
FVIII and vWF Assists platelet adhesionto
subendothelium to provide
coagulation surface
2. Promote aggregation ADP
Calcium Promote platelet aggregation
PF4
Thrombospondin
3. Promote Serotonin Promotes vasoconstriction
vasoconstriction Thromboxane A2
4. Promote vascular PDGF Promotes smooth muscle growth for
repair vessel repair
B-thromboglobulin Chemotactic for fibroblast to help in
vessel repair
5. Other system affected plasminogen Precursor to plasmin, which induces
clot lysis
A2-antiplasmin Plasmin inhibitor, inhibits clot lysis
C1 esterase inhibitor Complement system inhibitor
Release Disorders (Storage Pool Defects)
ALPHA GRANULES DEFICIENCY
GRAY PLATELET SYNDROME

DENSE GRANULES DEFICIENCIES

Hermansky-pudlak Chediak-higashi
Wiskott-Aldrich Syndrome
Platelet aggregation
o Aggregate platelet for oxygen exchange
o Requires- fibrinogen
■ Glanzmann's thrombasthenia
-platelet plasma membrane has no GPIIbIIIa (platelet receptor for fibrinogen)
b. Secondary Hemostasis - involves the enzymatic activation of series plasma proteins in the coagulation
system (coagulation factors) to form a fibrin meshwork (fibrin clot).
Test: clotting time

PRIMARY HEMOSTASIS
MEGAKARYOPOIESIS
 5 days
 Thrombopoietin is produced from the liver
 1/3 of the platelets can be found in the spleen while 2/3 of the platelets can be found in the
circulation
 N.V of platelet count:
 150,000 – 400,000 /ul
 150 – 400 x 109 / L
 Splenomegaly - decrease Platelet count
 Splenectomy – increase Platelet count
THROMBOPOIETIN (TPO)
• Is a glycoprotein hormone produced mainly by the liver and the kidney that regulates the
production of platelets by the bone marrow.
• It stimulates the production and differentiation of megakaryocytes. Thrombopoiesis -
production of platelets
 CELL CHARACTERISTICS
1. Megakaryoblast  20-50 um diameter
 Blue cytoplasm
 N/C ratio is about 10:1
 Multiple nucleoli
 Fine chromatin

2. Promegakaryocyte  20-60 meter

 Less basophilic cytoplasm
 Chromatin becomes coarse
 Irregularly shaped nucleus, may show slight
lobulation
 N/C ratio is 4:1 to 7:1

3. Granular megakaryocytes
4. Mature megakaryocyte
 40-120 um diameter
 Cytoplasm contains coarse clumps of granules
aggregating into little bundles which bud off
from the periphery to
become platelets
 Multiple nuclei are present
 No nuclei is visible
 N/C ratio is less than 1:1

5. metamegakaryocyte
6. platelet/ thrombocyte  1-4 um diameter
 Light blue to purple, very granular
 Chromosome- granular and located centrally
 Hyalomere- surrounds the
chromomere, nongranular and clear to light
blue

DIFFERENTIATION OF THE FOUR STAGES OF THE MEGAKARYOCYTE MATURATION SERIES

MATURATION CYTOPLASMI CYTOPLASMC NUCLEAR THROMBOCYTES
STAGE C GRANULES TAGS FEATURES VISIBLE
Megakaryoblast Absent Present No
Single nucleus, fine
chromatin, nucleoli
Promegakaryocyte Few Present Double nucleus No
Megakaryocyte
Usually absent Two or more nuclei No
Numerous
Metamegakaryocyte Aggregated Absent Four or more nuclei YES
 Note:
• Promegakaryocyte is the stage where DMS (Demarcating Membrane System) is first
formed.
• In differentiating the maturation stages of the megakaryocytic cells, emphasis should be
placed on the cytoplasmic appearance rather than the number of nuclei or chromatin
structure, as is usually the rule in evaluating other hematologic cells, (Steininger)

PLATELET SHEDDING
To facilitate the release of platelets into the bone marrow sinus, cytoplasmic pseudopodia of the
megakaryocytes protrude through the extravascular side of the endothelium, making an opening into the bone
marrow sinus. This opening facilitates the flow of more megakaryocytic cytoplasm containing new platelets
into the sinus. Eventually, these cytoplasmic outflows separate from the body of the megakaryocytes, resulting
in the release of an abundance of platelet fragments. These cytoplasmic fragments undergo further dissolution
within the sinus from which individual platelets evolve.
PLATELET STRUCTURE
 Anucleate
 Diameter: 2-5 um
 MPV: 8-10fL
 Shape: disk shaped or circular to irregular, lavender, and granular under Wright-stained wedge
preparation
 N.V: 150,000 – 400,000 /ul
 150 – 400 x 109 / L
 Composed of 60% protein, 30% lipid, 8% carbohydrate, various minerals, water and nucleotides
 Divided anatomically into four areas: peripheral zone, sol-gen zone, organelle and membranous system
AREA CONTENT
1. Peripheral zone  Glycocalyx
 Platelet (plasma) membrane
 Submembranous are (region)
2. Sol-gel zone  Microfilament:
Protein: Actin and Myosin
-upon stimulation of the platelet, these two
will interact to form actomyosin
(thrombosthenin) a contractile protein,
important in clot retraction

 Microtubules
 Protein: Tubulin – maintains the platelets
disc shape

3. Organelle zone  Alpha granules, dense granules, lysosome.

Glycogen, mitochondria

4. Membranous system  OCS (open canalicular system) /surface

connecting system
 DTS (dense tubular system)
IMPORTANT TERMS
1) Petechiae -purplish red pinpoint hemorrhagic spots in the skin caused by loss of capillary ability to
withstand normal blood pressure and trauma
2) Purpura - hemorrhage of blood into small areas of skin, mucous membranes, and other tissues
3) Ecchymosis - form of purpura in which blood escapes into large areas of skin and mucous
membranes, but not into deep tissues
4) Epistaxis - nosebleed
5) Hemarthrosis - leakage of blood into joint cavities
6) Hematemesis - vomiting of blood
7) Hematochezia - "red stool"; associated with lower GIT bleeding
8) Hematoma - swelling or tumor in the tissues or a body cavity that contains clotted blood
9) Hematuria - RBC in urine
10) Hemoglobinuria - hemoglobin in urine
11) Hemorrhage - Is a severe form of bleeding that requires immediate intervention and transfusion
12) Melena- stool containing dark red or black blood; "black tarry stool"; associated with upper GIT
bleeding
13) Menorrhagia - excessive menstrual bleeding

PATTERNS OF CLINICAL BLEEDING IN DISORDERS OF HEMOSTASIS

CHARACTERISTICS PRIMARY HEMOSTASIS SECONDARY HEMOSTASIS
(PLATELET/VASCULAR (COAGULATION FACTOR
PROBLEM) PROBLEM)
ONSET Spontaneous, immediate after Delayed after trauma
trauma
SITES Skin, mucous membrane Deep tissues
FORM Petechiae, ecchymosis Hematomas
MUCOUS MEMBRANE Common (nasal, oral, Less common
gastrointestinal genitourinary)
OTHER SITES Joint, muscle, CNS, retroperitoneal
CLINICAL EXAMPLES Thrombocytopenia, platelet defects, Factor deficiency, liver
von Willebrand disease, scurvy disease, acquired inhibitors

MANIFESTATIONS OF HEMOSTATIC DISORDERS

• Easy bruisability
• Subcutaneous bleeding
• Mucosal bleeding:
 Epistaxis (>15 mins)
 Gingival bleeding
 GI bleeding
 Menorrhagia
 Hematuria
 A. DISORDERS OF PRIMARY HEMOSTASIS
PRIMARY PURPURA
Comprises disorders that result in bruising but
are not associated with any specific disease
Simple purpura (Devil’s pinches) Occurs as a result of skin fragility
Mechanical purpura Occurs as a result of sudden increase in capillary
pressure and usually manifests as petechiae
Senile purpura Seen in older individuals, or in individuals
undergoing corticosteroid therapy, purpuric
lesions occur on the hand and arms
Factitious purpura Is caused by self-induced trauma and usually is
found on areas of the body that are easily
accessible
Schamberg’s (progressive pigmentary Characterized by the development of cayenne
purpura) pepper petechiae on a background of
hyperpigmented brown or orange oval patches
often seen in tibial regions bilaterally as a
chronic
eruption

SECONDARY PURPURA
Infectious- inflammatory response to an  Purpura fulminans-applies to any purpura
infection, bacterial toxins or direct injury by the on rapid onset
infectious agent
 Septic emboli- may be seen in
endocarditis Ecthyma gangrenosum

Allergic type- this is a syndrome characterized  Henoch-Schonlein purpura- result of

by a relatively distinctive purpuric eruption in allergic vasculitis which involves the skin
association with various constitutional and GIT kidneys, heart and CNS
localized symptoms. This disorder is the result of
an autoimmune process or allergic vasculitis  Drug-induced-this is a purpura induced by
iodides, quinine, procaine, penicillin, and
aspirin

Metabolic- caused by biochemical or hormonal  Scurvy- vitamin c deficiency

abnormalities
 Cushing’s syndrome- caused by
corticosteroid excess results in purpura

 Diabetes mellitus- increase glucose in

blood
 Protein c deficiency
PURPURA SECONDARY TO
DYSPROTEINEMIA
Waldenstrom’s purpura Is a disorder of women that presents with
recurrent purpura on the lower extremities and
resultant hemosiderin staining of the skin similar
to Schamberg’s
Cryoglobulinemia caused by production of cryoprecipitable serum
proteins or protein complexes
Hyperviscosity Syndrome results from hypergammaglobulinemia owing to
an increase in plasma viscosity.
Amyloidosis bleeding is caused by deposition of amyloid
protein around small blood vessels, resulting in
vessel fragility

CONNECTIVE TISSUE DISORDERS

Ehlers-Danlos Syndrome An autosomal-dominant, recessive or x linked
disorder characterized by hyper distensible joints
and fragile skin; bleeding is due to abnormalities
of collagen in blood vessel walls leading to
vascular fragility
Pseudoxanthoma elasticum is an autosomal recessive disorder affecting
elastic fibers of connective tissue of skin and
arteries
Marfan Syndrome is an autosomal-dominant genetic disorder due to
mutation of the gene for fibrillin resulting in
abnormalities of connective tissues and risk for
bleeding and bruising

Osteogenesis imperfecta is a rare autosomal dominant disorder caused by

Hemoptysis-bloody mutation of genes which code for peptides of
sputum type 1 collagen; individuals may demonstrate
easy bruising, epistaxis, hemoptysis and
intracranial bleeding

Hereditary Hemorrhagic Telangiectasia Involves vessels throughout the body, which are
(osler- weber-rendu disease) dilated, tortuous and disorganized; associated
with iron-deficiency anemia
Congenital hemangiomata (kasabach- Tumors composed of vessels that swell and bleed
meritt syndrome) at the surface
 B. PLATELET DISORDERS
1. QUALITATIVE PLATELET DISORDERS
a. Bernard-Soulier Syndrome
 Hereditary
 Problem in platelet adhesion\
 Inherited disorder of the platelet GPIb/IX/V complex characterized by
thrombocytopenia giant platelets >20 um in diameter and a failure of the platelets
to bind GPIb ligands
 In 1948, Bernard and Soulier described two children from a consanguineous
family who had a severe bleeding disorder characterized by mucocutaneous
hemorrhage
 In 1975, Nurden and Caen identified abnormality in platelet GPIb as the cause of
the functional defect
Features of Bernard-Soulier syndrome:
 Thrombocytopenia
 Von Willebrand’s factor
 Abnormal platelet interactions with thrombin
 Abnormal platelet coagulant activity
 Abnormal platelet interactions with P-selectin
 Abnormal platelet interactions with leukocyte integrins amb2

b. GLANZMANN’S THROMBASTHENIA
 Hereditary
 Problem in platelet aggregation
 absence or deficiency of the membrane GPIIbIIIa complex
 1918 Eduard Glanzmann, a swiss pediatrician, described a group of patients with
hemorrhagic symptoms and a defect on platelet function
 In the mid-1970, Nurden, Caen, Philipps and colleagues discovered that
thrombasthenic platelets are deficient in both IIb and IIIa
Features of Glanzmann's thrombasthenia:
 Bleeding is most common from mucosal surfaces
 Facial petechiae and sub conjugal hemorrhages seen in infants associated with crying

Difference between Bernard Soulier syndrome and Glanzmann’s thrombasthenia

LABORATORY BERNARD- GLANZMANN’S
TESTS SOULIER THROMABSTHENIA
SYNDROME
Platelet count Decreased Normal
platelet morphology Giant platelet normal
Bleeding time Prolonged Prolonged
Platelet aggregation:
ADP N A
Thrombin A A
Collagen N A
Epinephrine N A
Ristocetin A N
Clot retraction N A
 c. VON WILLEBRAND DISEASE
 Hereditary
 Problem in platelet adhesion
 Associated with either quantitative deficiency (type 1 and type 3) or qualitative
abnormalities of vWF (type 2)
 Uncommon type 3 variant is the most severe form
 In 1926, Eric von Willebrand described a bleeding disorder in 24-66 members of a
family from the Aland Islands.
Von Willebrand disease: treatment
o DDAVP (1-desamino-8-D-arginine vasopressin or desmopressin - initial treatment of choice
o Humate P - contains intact vWF
o Cryoprecipitate - used in unresponsive to DDAVP
o In type 3 vWF disease: Factor VIII products or cryoprecipitate

CLASSIFICATION OF VON WILLEBRAND DISEASE

TYPE DESCRIPTION
1 Partial quantitative deficiency of von Willebrand
factor (vWF)
2 Qualitative deficiency of VWF
2A Decreased platelet-dependent vWF function
which selective deficiency of high-
molecular weight multimers
2B Increased affinity for platelet glycoprotein Ib
2M Decreased platelet-dependent vWF function with
high- molecular-weight multimers present
2N Markedly increased binding of factor VIII to vWF
3 Complete deficiency of vWF

DIFFERENCE BETWEEN BERNARD SOULIER, VON WILLEBRAND DISEASE AND

GLANZMANN’S THROMBASTHENIA
PLATELET BERNARD- VON GLANZMANN’S
AGGREGATION SOULIER WILLEBRAND THROMBASTHENIA
TEST SYNDROME DISEASE (R)
(ECA) (ECA)
Epinephrine N N A
Collagen N N A
ADP N N A
Ristocetin A A N

DIFFERENCE BETWEEN HEMOPHILIA A AND VON WILLEBRAND DISEASE

LABORATORY TESTS HEMOPHILIA A VON WILLEBRAND
DISEASE
Bleeding time N A
Clot retraction time N N
Glass bead N A
Platelet count N N
Ristocetin aggregation N A
PT N N
APTT A A
VIII:C activity N A
vWFR: Co N A
vWF: Ag N A
 d. STORAGE POOL DEFECTS
 Hereditary
 Problem in platelet secretion
 Conditions:
o Gray platelet syndrome
o Wiskott-Aldrich Syndrome
o Hermansky-Pudlak Syndrome
o Chediak-Higashi anomaly
o Prostaglandin enzyme deficiency

OTHERS

Alpha-gamma storage pool  Rare disorders that is characterized by

def. Deficiency platelet P- moderate to severe defects in both alpha and
selectin gamma granules
 Decreased platelet P-selectin (CD62P) a
point of distinction from other patients with
the disorder and patients with gray platelet
syndrome
 Clinical and laboratory features are similar
to those of y-storage pool def.
Quebec platelet disorder  Originally described as Factor V Quebec
 Features: severe bleeding after trauma, mild
thrombocytopenia, decreased functional
platelet factor 5 and normal plasma factor V
 Diagnosis: analysis of platelet urokinase-
type plasminogen activator or the
identification of degraded granule
proteins by immunoblot
analysis
Scott Syndrome  Impaired ability of platelets to
promote coagulation
 Platelets have a defect in the
translocation of PS to the platelet outer
membrane leaflet
 Hemorrhagic manifestations are not
primarily mucocutaneous
 Laboratory results
o Bleeding time: Normal
o Serum prothrombin time:
Prolonged
o Platelet aggregation: Normal
Acquired  Uremia
 Paraproteinemia
 AM1L
 Myeloproliferative disorders
 Drugs (ex. Aspirin - inhibits
cyclooxygenase)
2. QUANTITATIVE PLATELET DISORDERS
THROMBOCYTOPENIA
Decreased platelet production  Aplastic anemia
 Suppression of megakaryocyte
 TAR syndrome
 Myelopthisic process
 Ineffective
 Leukemia
 Pernicious anemia
 Gaucher's disease
 Sometimes following chemotherapy and radiation

Platelet destruction (decreased survival  Immune platelet destruction

time) o PTP (Post-transfusion Purpura)
o Neonatal Isoimmune
thrombocytopenia
o Platelet refractoriness
o ITP (Immune Thrombocytopenic
Purpura)
o Secondary Immune
Thrombocytopenia
 Non-immune platelet destruction
o DIC (Disseminated Intravascular Coagulation)
o BUS (Hemolytic Uremic Syndrome) and TTP
(Thrombotic Thrombocytopenic Purpura) -
anemia, reticulocytosis, decreased platelet
count, schistocytes, anisocytosis,
hemoglobinemia, decreased haptoglobin,
increased LDH and increased bilirubin

Abnormal platelet distribution  Splenomegaly (increased platelet

sequestration by spleen)
Dilution of platelet count  Extensive blood transfusion often is accompanied
by thrombocytopenia, the degree of which is directly
proportional to the number of units
transfused

THROMBOCYTOSIS Increased platelet production

Classifications:
Reactive – moderately increased, asymptomatic
Autonomous – marked increased, associated with
thrombotic/hemorrhagic complications
o Conditions:
Polycythemia vera
o EssentialThrombocytosis
o Idiopathic
thrombocythemia AML
o Splenectomy
LABORATORY TESTS FOR PRIMARY HEMOSTASIS
Note:
Specimen collection is preanalytic variable that may have serious implications in laboratory testing.
According to studies done in recent years 32-75% of testing errors occur during the pre-analytic phase.

A. QUANTITATIVE PLATELET EVALUATION

Specimen: EDTA anticoagulated whole blood
PLATELET SATELLITOSIS
 A form of pseudo-thrombocytopenia
 Antibodies directed against GPIIb-IIIa react with the leukocyte Fe gamma receptor III and attach the
platelets to neutrophils and monocytes, inducing the phenomenon.
 Platelets form a rosette around the periphery of leukocytes
 Neutrophils are the most frequently involved; occasionally monocytes
 Naturally occurring, but exposure of antigen on EDTA-treated platelets and leukocytes may trigger
the phenomenon.
 Using sodium citrate as an anticoagulant should correct this problem. Because of the dilution in the
citrate tubes, it is necessary to multiply the obtained platelet count b 1.1 (Rodak)

1.Direct platelet count

• Platelets are counted in a hemocytometer as in erythrocytes and leukocytes
A. Reese-Esker
 Diluent: Isotonic
o Sodium citrate-prevent platelet aggregation
o Formaldehyde –preservative
o BCB (Brilliant Cresyl Blue) – stain / dye
 Microscopy: Light Microscopy
 Appearance of platelets: bluish

B. Guy and Leake

 Diluent: Isotonic
o Sodium oxalate –prevent platelet aggregation
o Formaldehyde –preservative
o CV (Crystal Violet) – stain / dye
 Microscopy: Light microscopy
 Appearance of platelets: lilac refractile object

C. Brecker-Cronkite – reference method

 Diluent: Hypotonic= 1% ammonium oxalate / (N114)2C204
 Microscopy: Phase-Contrast Microscopy

D. Unopette
 Diluent: 1% ammonium oxalate
 Dilution: 1:100
2. Indirect platelet count
• Platelets are counted in their relationship to red cells on a fixed-stained smear. This method is not
reliable because the results depend upon the distribution of platelets and on the red cell count
factor = x20,000
A. Fonio's
 14% magnesium sulfate
 Wright's stain

B. Dameshek
 BCB (Brilliant Cresyl Blue)
 Wright's stain

C. Olef's
PLATELET ESTIMATE
PLATELET ESTIMATE REPORTING
>800,000 Marked increased
600,000-800,000 Moderate increase
401.000-599,000 Slight increase
200,000-400,000 Normal
150,000-199,000 Low normal
100,000-149,000 Slight decrease
50,000-99,000 Moderate increased
0-49,000 Marked increased

TIPS:
Increasing order by: 200,000
Decreasing order by:
50,000
Starting at: 200,000

3. Automated platelet count

• red cells must first be removed from whole blood, either by sedimentation or by controlled
centrifugation
1. Voltage pulse counting
2. Electro-optical counting SIGNIFICANT PLATELET LEVELS Less than 100,000/ul – abnormally
low
30,000-50,000/ul – bleeding possible with trauma Less than 30,000/ul – spontaneous bleeding possible Less
than 5,000/ul – severe spontaneous bleeding Note:
• Normal platelet count + prolonged BT: Qualitative platelet abnormality, Primary vascular
abnormality and von Willebrand's syndrome
• Low platelet count + normal BT: Autoimmune thrombocytopenia
Low platelet count + very prolonged BT: Simultaneous quantitative and qualitative platelet deficiency

B. PLATELET AGGREGATION TEST

• Assess the ability of platelets to aggregate after the addition of aggregating agents
• Sample: PRP (Platelet Rich Plasma)
• Aggregating agents (Agonists):
o Epinephrine
o Collagen
o ADP
o Ristocetin
o Also: thrombin, arachidonic acid, serotonin

• Test considerations:
o No hemolysis
o Fasting: 8 hours
o pH: 6.5-8.5
o No NSAIDS Within 3 hours

C. PLATELET RETENTION (ADHESIVENESS) TEST

o Principle: The adhesiveness of platelets may be measured in vitro by their ability to adhere on glass
surfaces with beads
o Salzmann Method: test of the retention of platelets within glass bead column
Abnormal platelet retention test
DECREASED PALETLET RETENTION INCREASED PLAETELET RETENTION
Bernard-Soulier Glanzmann thrombasthenia vWD
Chediak-Higashi Myeloproliferative disorders Uremia
Aspirin administration Thrombotic disorders Hyperlipidemia Carcinoma
Oral contraceptives Pregnancy
D. CLOT RETRACTION TIME
• Measures the entire platelet function
• Detects: Entire function of platelets
• Methods:
1. Qualitative Test
a. Hirshboeck or castor oil method
NV: 15-45 mins
Formation of dimpling/droplet like serum on the surface of blood drop
b. Single tube method
2. Quantitative Test
a. Stefanini method — similar with single tube method
• Specimen: 3-5 mL blood
• Temperature: 37°C
• 1/2/16/18/24 hours
• Normal: begins within 1 hour, complete within 18 to 24 hours
b. Macfarlane method.
• Specimen: 5 mL blood
• Temperature: 37°C
• Incubation time: 1 hour
• N.V.: 44-67%
E. CAPILLARY FRAGILITY (RESISTANCE TEST)
1. Tourniquet test (Rumpel-leede test or Hess test or Positive pressure technique)
• Measure capillaries to resist pressure
• Correlates with the degree of thrombocytopenia
Principle: By partially obstructing the venous blood, the capillary pressure is increased. This will give rise to
intravasation of blood which will be manifested in the form of small hemorrhage called petechiae.
• Apply pressure (100mrnlig for 5 mins.) after 15-30mins., count petechiae

GRADE PETECHIAE
1+ 0-10
2+ 10-20
3+ 20-50
4+ >50

a. Quick’s Method
b. Gothlin’s method
2. Suction cup method (Petchiometer method or Negative pressure technique)
Principle: employs the use of a modified da silva melle instrument

F. BLEEDING TIME
• In vivo measures of primary hemostasis
• Determines both congenital and acquired platelet disorder
Factors which affects bleeding time:
Elasticity
Ability of the blood vessel to constrict and retract
Mechanical and chemical action of platelets in the formation of hemostatic plug
Methods for bleeding time:
Duke’s method (Template bleeding time) N.V: 6-10 mins
Modified Ivy’s method- best method to assess platelet’s adhesiveness, N.V: 3-6 mins; Pressure: 40-45
mmHg Coply lalitch method
Adelson-Crosby method
Macfarlane’s method- same principle with Adelson-Crosby method but it only uses ear lobe as the site of
puncture Aspirin tolerance test- assesses the effect of a standard dose of aspirin on the Duke’s bleeding time
Abnormal bleeding time Thrombocytopenia Hypofibrinogenemia vWF disorder
Connective tissue disorder

SECONDARY HEMOSTASIS

Factors involved: Coagulation factor

Main objective: Formation clot (irreversible)

Blood factors can be classified as:

1. Substrate, substance on which enzyme acts
2. Zymogen, enzyme precursor
3. Cofactor, component the aids in the activation of zymogen to active enzyme
4. Calcium

COAGULATION
• Is a process whereby, on vessel injury, plasma proteins, tissue factors, and calcium interact on the
surface of platelets to form a stable fibrin clot Platelets also interact with fibrin to form a stable platelet-
fibrin clot.
• This mechanism consisting of a series of cascading reactions involving development of enzymes
from their precursors (zymogen) which will further be converted to an activated state (serine protease).
• In conversion of zymogens to enzymes, either they are serine proteases (H, VII, IX, X, XI, XII),
which uses serine as the active site and cleave peptide bonds, or they create covalent bonds as
transaminases.
• Blood factors are produced mostly in the liver, and circulate in an inactive precursor form
FACTOR PREFERED SYNONYMS ACTIVE FORM PATHWAY PARTICIPATIO N
VITAMIN K DEPENDEN T PRESENT IN BaSO4 ADSORBE
D PLASMA
Factor I Fibrinogen Fibrin clot Common No Yes
Factor II Prothrombin Prethrombin Serine protease Common Yes No
Factor III Tissue factor Tissue thromboplastin Extrinsic No Yes
Factor IV Calcium Intrinsic, extrinsic and common No Yes
Factor V Proaccelerin Labile factor, Accelerator globulin (aCg) Cofactor Common No
Yes
Factor VII Preconvertin Stable factor, Serum Serine protease extrinsic Yes No

Prothrombin Conversion
Accelerator (SPCA)
Factor VIII Antihemophili c Antihemophilic factor A,
Platelet cofactor 1 Cofactor Intrinsic No Yes
Factor IX Plasma Thromboplasti n
Component (PTC) Christmas factor, Antihemophilic factor B, Platelet
cofactor 2 Serine protease Intrinsic Yes No
Factor X Staurt-Prower Factor Autoprothromb in III, Stuart factor, Prower
factor Serine protease Common Yes No
Factor XI Plasma Thromboplasti n
Antecedent Antihemophilic factor C Serine protease Intrinsic No Yes
Factor XII Hageman Factor Glass factor, Contact factor Serine protease Intrinsic No
Yes
Factor XIII Fibrin Stabilizing Factor Laki-Lorand factor, Fibrinase, Plasma transglutaminas
e, Fibrinoligase Transglutamina se Common No Yes
Prekallekrei n Fletcher factor Serine protease Intrinsic No Yes
HMWK
(high molecular
weight kininogen) Fitzgerald factor William's factor, Flaujeac factor, Contact activation.
cofactor Serine protease intrinsic No Yes

THREE PATHWAYS THAT MAKEUP THE CLASSICAL BLOOD COAGULATION PATHWAY

DISORDERS OF CAOGULATION CAUSING CLOTTING FACTOR DEIFICIENCIES

• Liver disease — the most common acquired clotting factor deficiency; treatment: FFP
• Hemophilia H—the most common inherited clotting factor deficiency

FACTOR INHERITED COAGULAPATHIES ACQUIRED COAGULOPATHY

Inheritance pattern Coagulopathy
I Autosomal recessive Afibrinogemia Severe liver disease Diffuse intravascular coagulation
Fibrinolysis
Autosomal dominant Dysfibrinogenemia
II Autosomal recessive Prothrombin deficiency Liver disease Vitamin K deficiency
Anticoagulant therapy
V Autosomal recessive Factor V deficiency (Owren’s disease or Labile factor deficiency or
parahemophilia) Severe liver disease Diffuse intravascular coagulation Fibrinolysis
VII Autosomal recessive Factor VII deficiency Liver disease Vitamin K deficiency
Anticoagulant therapy
VIII X-linked recessive Hemophilia A (classic hemophilia or Royal’s disease) Trivia: the
disorder of Queen Victoria’s son Diffuse intravascular coagulation Fibrinolysis
Autosomal dominant vWD
IX X-linked recessive Hemophilia B (Christmas disease)
Trivia: named after a person with a surname
“Christmas”

Liver disease Vitamin K deficiency

Anticoagulant therapy
X Autosomal recessive Factor X deficiency Liver disease Vitamin K deficiency
Anticoagulant therapy
XI Autosomal recessive Hemophilia C (Rosenthal deficiency)
Trivia: common in Eastern European Jewish descent/
Ashkenazi Jews
XII Autosomal recessive Factor XII deficiency
XIII Autosomal recessive Factor XIII deficiency Liver disease Diffuse intravascular
coagulation Fibrinolysis
Prekallekrein Autosomal recessive Fletcher trait
HMWK Autosomal recessive Fitzgerald trait

CLINICAL MANIFESTATIONS OF COAGULATION FACTOR DEFICINECIES

TYPE OF BLEESING COAGULATION FACTOR DEFICIENCY

Easy bruising FII, VIII, FIX
Hematomas FII, VII, FIX
Mucosal bleeding FII, VII, FIX, FXI
Hemarthrosis FIII, FIX, FX
Postsurgical bleeding Fibrinogen, FII, FV, FVII, FVIII, FIX, FX, FXI, FXIII
Intracranial bleeding FVII, FVIII, FIX, FXIII
Delayed wound healing Fibrinogen, FXIII
Umbilical cord bleeding FX, FXIII
Miscarriage Fibrinogen, FXIII
Thrombosis Abnormal fibrinogens
Asymptpomatic FXII, prekallekrein, hmwk

CONDITIONS MOST OFTEN ASSOCIATED WITH PHYSIOLOGIC VARIATIONS IN

COAGULATION AND FIBRINOLYTIC FACTORS
CONDITION FACTOR INCREASES FACTOR DECREASES
Stress I
Tissue necrosis I
Inflammation I
Pregnancy I, VIII, IX, X XIII, XI, AR-III
Oral contraceptives I, VIII, VII, IX, X
Hypermetabolism (e.g. hyperthyroidism) I, VIII, plasminogen
Vigorous exercise VIII, XI, XII
Chronic thrombocytopenia VIII
Hypothyroidism IX, XI, plasminogen
Childbirth I, VIII
Surgical procedures I, VIII
Trauma I, VIII
Myocardial infarction I, VIII
Acute illness I, VIII

INHIBITORS OF COAGULATION
Major site of inhibition: endothelium and platelet
INHIBITOR FUNCTION
Protein C Degrades factor Va and VIIIa
Protein S Degrades factor Va and VIIIa
Anti-thrombin III Major inhibitor of thrombin, also inhibits factors IXa, Xa, XIa, XIIa, kallikrein and
plasmin
Heparin cofactor II Inhibit thrombin
A2macroglobulin Forms a complex with thrombin, kallikrein and plasmin, thus inhibiting their activities
EPI (Extrinsic Pathway Inhibitor) and LACI (Lipoprotein Associated Coagulation Inhibitor) Inhibits the
VIIa-tissue factor complex
C1 inhibitor Inactivator of factor XIIaa and kallikrein, it also inhibits factor Xia and plasmin
A1antitrypsin Inhibitor of thrombin

LABORATORY TESTS FOR SECONDARY HEMOSTASIS

CLOTTING (COAGULATION) TIME
• it measure of the ability of the blood to clot and is not influenced by the platelet functions other than
PF3, it also measures only the time required for the formation of the traces of thrombin sufficient to produce
a visible clot.
a. Micro methods N.V: 2-4 mins.
Slide (drop) method
Capillary tube method (Dale & Laidlaw’s)
b. Macro method N.V: 7-15 mins
• Whole blood method (Lee & White)
Equipment’s: water bath (37^C) glass test tubes (13x100mm) stopwatch, plastic syringe (10 mL) and 20-
gauge needle
PT (PROTHROMBIN TIME)
• Extrinsic and Common Pathway
Test of choice: to monitor warfarin therapy
Reagent: Simplastin = thromboplastin (replace tissue factor) + calcium chloride
Equipments: test tubes (13 x 100mm), 0.1mL patient's plasma and 0.2mL (200gL) PT reagent

Centrifugation: 2000g for 10 min.

N.V.: 10-12 sec.

INR = (PT patient/Mean of normal)ISI

*INR - International Normalized Ratio

*ISI - International Standardized Index
The closer the ISI is to 1, the more sensitive the reagent is; the higher the ISI, the less sensitive the reagent.
APTT (ACTIVATED PARTIAL THROMBOPLASTIN TIME)
• Intrinsic and Common Pathway
Test of choice: to monitor heparin therapy Specimen: Platelet Poor Plasma (PPP) Activators:' Silica, kaolin,
ellagic acid, celite
Reagent: platelet substit4te + activators + 0.025M calcium chloride Equipments:
• (Brown): 13x100mm tubes, 0.2 mL PPP, 0.2mL APTT reagent and 0.2mL CaC12
• (Steininger): 12 x 75 tubes, 0.1mL PPP, 0.1mL APTT reagent and 0.1m1 CaC12 N.V.: 25-35 sec.
End-point: clot formation
STYPVEN TIME (RUSSEL VIPER VENOM TIME)
• Uses snake venom (Vipera russeli, Common name: East Indian Viper)
• Reagent: platelin +'chloride
• Specimen: Platelet Poor Plasma (PPP)
• Determines problem in common pathway N.V: 6-10 sec.
THROMBIN TIME
Prolonged in fibrinogen deficiency, presence of FSP, FM', thrombolytic agents (ev.: streptokinase) and
heparin Affected by: heparin
N.V.: 10-14 sec
Specimen: Platelet Poor Plasma (PPP)
Reagent: thrombin + calcium chloride End-point: clot formation
REPTILASE TIME
Prolonged in fibrinogen deficiency Unaffected by heparin
Practical to assess the fibrinogen of patient receiving heparin
Uses enzyme found in the venom of Bothrops atrox snake (resembles thrombin)
N.V. 10-15 sec
Specimen: Platelet Poor Plasma (PPP) Reagent: atroxin
End-point: clot formation
DUCKERT’S (5M UREA SOLUBILITY TEST)
Test for FXIII
Reagent: 5M Urea (Substitutes: 1% monochloroacetic acid or 2% acetic acid) Normal result: insoluble to
urea when incubated for 24 hours
Abnormal result: soluble to urea when incubated for 24 hours

SPECIMEN CONSIDERATIONS IN COAGULATION TESTING

1. Non-traumatic venipuncture
2. Order of draw
3. Use plastic or silicone-coated glass tubes (noncontact surface)
4. Ratio of blood to anticoagulant
5. Specimen-processing – recommendations include processing within 4 hours for APTT and 24 hours
for PT.
6. Temperature- testing must be performed at 37^C, labile factor V and VII will breakdown at
temperature above 37^C. factors VII and XI will be activated at cold temperature

DIFFERENTIAL DIAGNOSIS OF ABNORMAL COAGULATION SCREENING TEST

ABNORMAL PARTIAL THROMBPLASTIN TIME (PTT) ALONE
Associated with bleeding: VIII, IX, XI, defects
Not associated with bleeding: XII, prekallekrein, (PK), hmwk, lupus anticoagulants
ABNORMAL PROTHROMBIN TIME (PT) ALONE
Factor VII defects
COMBINED ABNORMAL PTT AND PT
Medical conditions: anticoagulants, DIC, liver disease, vitamin K deficiency, massive transfusion Rarely
dysfibrinogenemia: factors X, V and II defects

FAMILIES OF COAGULATION PROTEINS

Contact group XII, XI, PK, HMWK
Prothrombin group X, IX, VII, II
Fibrinogen group I, V, VIII, XIII

BLOOD COMPONENTS AND THEIR CORRESPONDING COAGULATION FACTORS WHICH THEY

LACK
Fresh plasma: ALL COAGULATION FACTORS PRESENT
Aged plasma: V, VIII Adsorbed plasma: X, IX, VII, II Fresh serum: I, V, VIII, XIII Aged Serum: I, II, V,
VIII, XIII

OTHER SIGNIFICANT INFORMATION

Prothrombin group: Vitamin K dependent factors/Absent in adsorbed plasma (BaSO4)/ Calcium dependent
factors Fibrinogen group: Consumed during coagulation/Absent in serum/Thrombin-sensitive coagulation
proteins Surface-bound zymogen: All Contact group EXCEPT HMWK
Cofactors/Substrates: HMWK, all Fibrinogen group EXCEPT FXIII

SUBSTITUTION STUDIES
DEFICEINCY PT APTT TT SUBSITUION STUDIES
Normal Plasma Adsorbed Plasma Aged Serum
I A A A C C NC
II A A N C NC NC
V A A N C C NC
VII A N N C NC C
VIII N A N C C NC
IX N A N C NC C
X A A N C NC C
XI or XII N A N C C C

CIRCULATING ANTICOAGULANTS
• Prolonged APTT and PT not corrected
• Inactive as activated coagulation factor or block interaction between coagulation factors and platelets
Example: Lupus inhibitor
o Nonsp anticoagulant
o IgG, IgM, and IgA which interfere with phospholipids portion of the complex: Xa-Va-calcium-plt
phospholipids
Platelet neutralization procedure Dilute Russel Viper Venom Time

INSTRUMENTATION FOR TESTS OF HEMOSTASIS

WAY OF DETECTION Visual detection Electromechanical detection Phot-optical detection
INSTRUMENTS Tilt tube method Fibrometer Semi-automated instruments:
- Electra 750 and 75a
- Fibrintimer series
- FP 910 Coagulation Analyzer
Automated instruments:
- Ortho Koagulab 16s and 40A
- Coag-A-Mate X2 and XC
- MLA Electra 700 and 800
DESCRIPTIONS Fibrin strand formation is detected using a wire loop or hook, hasileen
incorporated into a semi-automated mechanical instrument Detection of fibrin clot formation depends on
the increase in light scattering associated with the conversion of soluble fibrinogen molecules to the
insoluble polymerized fibrin clot

FIBRINOLYSIS
FIBRINOLYSIS
It is a system whereby the temporary fibrin clot is systematically and gradually dissolved as the vessel heals
in order to restore normal blood flow.

COMPONENTS OF FIBRINOLYTIC SYSTEM

a. Plasminogen activators
• Intrinsic activators - FXIIa, kallekrein, HMWK
• Tissue type - urokinase-like PA
• Therapeutic activators (treatment for thromboemboli) - t-PA (tissue-like PA), single chain
urokinase,streptokinase
b. Plasminogen (profibrinolysin) - comes from the liver
c. Plasmin (fibrinolysin) -proteolytic enzyme
d. Inhibitors of Fibrinolysis- neutralize the activity of plasmin.
• Alpha2 antiplasmin - major inhibitor of plasmin
• Alpha). antitrypsin - it is present in the plasma and also in platelets
• Alpha2 macroglobulin - inactivates the plasmin that is not inhibited by alpha2 antiplasmin
• Thrombospondin - released by platelets, it inhibits activation of fibrin-bound plasminogen
• Plasminogen activator inhibitor1 (PSI-1) & Plasminogen activator inhibitor2 (PAI-2) -both are
naturally occurring. They come from endothelial cells and platelets. They inhibit free plasmin.
• TAFI (Thrombin-Activatable Fibrinolysis Inhibitor) - they inhibit bound plasmin

Note:
• Fragment X = D-D-D or Y-D
• Fragment Y = D-D or D-dimer
• Products of the degradation of cross-linked fibrin by plasmin: Fragment X and Fragment Y
• Products of the degradation of fibrinogen and non-crosslinked fibrin by plasmin: Fragment X,
Fragment Y and Fragment D

PATHOLOGIC FIBRINOLYSIS
Primary Fibrinolysis
• Excessive amounts of plasminogen activators from damaged cells/malignant cells
• Converts plasminogen to plasmin in the absence of fibrin formation

Secondary Fibrinolysis
• uncontrolled, inappropriate formation of fibrin within the blood vessels
o Infection
o Neoplasm
o Snake bite
o HTR
LABORATORY TESTS FOR FIBRINOLYSIS
WBCLT (WHOLE BLOOD CLOT LYSIS TIME)
N.V: Clot should remain intact for approximately 48 hours at 37^C
• Clot lysis prior to 48 hours is indicative of excessive systemic fibrinolysis
EUGLOBULIN LYSIS TIME
• The euglobulin clot lysis time is a screening test for the measurement of fibrinolytic activity. It is a
more sensitive test than clot lysis time
• Plasma is diluted with water then acidified which leads to the formation of a protein precipitate
(euglobulin)
• Euglobulin is clotted by adding thrombin
• Time required for complete lysis: greater than 2 hours
• Composition of euglobulin: plasminogen, plasmin, fibrinogen and plasminogen activators
• N.V: lysis should be observed greater than 2 hours but if the lysis occurs less than 2 hours is
indicative of excessive (increased) fibrinolytic activity
PROTAMINE SULFATE DILUTION TEST
• Detects the presence of fibrin monomers in the plasma
• Distinguish primary and secondary fibrinolysis
ETHANOL GELATION TIME TEST
• Detects the presence of fibrin monomers in the plasma
• Distinguish primary and secondary fibrinolysis
• less sensitive compared to protamine sulfate dilution test but more specific
LATEX D-DIMER ASSAY
• D-dimer is a specific fragment from fibrin degradation
• Latex: mouse anti-human D-dimer
• N.V: 200ng/mL
• The D-dimer test is positive in DIC as soon as 4 hours after onset. Fibrinogen levels may decrease in
4 to 24 hours: platelets decrease up to 48 hours after onset.

Note:
• Increased levels of fibrin (oven) split degradation products (FSP or FDP), as seen in DIC and lytic
disorders, exert an anticoagulant effect. Laboratory procedures utilized to evaluate this process include latex
FSP agglutination test, measurements of fibrin monomers, platelet counts, fibrinogen levels, APTT and PT.

ANTICOAGULANT THERAPY
Heparin (Monitoring: APTT) was the first agent administered as an anticoagulant
• Used for
Venous thrombosis
Pulmonary embolism
Active thrombophlebitis
Arterial thrombosis
• LMW heparin — from porcine mucosa
• BMW heparin — from bovine lung
• Thrombocytopenia on the first 21st day after administration
• Reversed by: protamine sulfate
---anti-histamine, digitalis, nicotine, penicillin, phenothiazines and tetracycline
ADMINISTRATION:
• Intermittent bolus injection
• Continuous infusion
• Minidose subcutaneous injection
• Pulsed IV injection of 5000U every 4-6 hours
ORAL ANTICOAGULANTS
• Dicumarol
• Warfarin / Coumadin / Coumarin (Monitoring: PT with INR)
• Prevents the activation of Vitamin-K dependent factors

References:
Rodak's Hematology Clinical Principles and Application 5th Edition Hematology: Principles and Procedures
by Barbara A. Brown ...
Clinical Hematology : Cheryl A.Lotspeich- Steininger