Hemostasis and Platelet Physiology ➢ Elastic fibers are found in larger

veins
4 Interrelated systems involved in hemostasis ➢ Fewer nerves distributed
➢ Venules – microscopically sized
● Blood Vessels
veins which connect capillaries
● Platelets
to veins.
● Blood Coagulation Factors
❖ Capillaries – thinnest walled and most
● Components of the Clot Degradation
numerous of blood vessels.
System (Fibrinolysis)
➢ Sinusoids – found in BM, Spleen
and Liver
➢ Composed of only one cell layer
HEMOSTASIS
of Simple Squamous
➢ Is the arrest of bleeding, by Epithelium.
○ Normal vasoconstriction (the
vessel walls closing
temporarily),
○ Abnormal obstruction (such as
a plaque) or
○ Coagulation or surgical means
(such as ligation).
➢ Primary Hemostasis, Secondary
Hemostasis and Fibrinolysis

Blood Vasculature
PLATELET PHYSIOLOGY
❖ Arteries – vessels that leave the heart Platelets Production:
➢ Tunica intima – forms the Hematopoietic stem cell
smooth surface of endothelium ↓
➢ Tunica media – thickest coat, Megakaryoblast - earliest recognizable stage
composed of smooth muscle ↓
and elastic fiber Promegakaryocyte
➢ Tunica adventitia – consist of ↓
fibrous connective tissue that Megakaryocyte
contains nerve endings and the ↓
vaso vasorum. Fragmentation of cytoplasm

➢ Arterioles - Microscopic Platelets

continuation of arteries that
gives off metarterioles –
capillaries
❖ Veins – vessels that return to the heart
➢ •Larger, more irregular lumen
➢ Relatively thin-walled with
weaker middle coat
What is a Platelet?
● Small 2-3 µm
● Disc-shaped/biconvex & Anuclear
● Reddish-purple granules/lavender and
granular
● 60% protein, 30% lipid, 8% carbs,
minerals, water and nucleotides
● Cytoplasm: light blue to purple
• Chromomere - granular, centrally located
• Hyalomere - surrounds the chromomere
● Surface has a sponge-like look, openings
are channels that extend deep within
cell
● MPV – 8 to 10 Fl Platelet Development
● Megakaryoblast
○ 10-15 µm
○ Increased nuclear: cytoplasmic
ratio
● Promegakaryocyte
○ 80 µm
○ Dense alpha and lysosomal
granules
● Megakaryocyte
● Platelets

Production of Platelets
● Made in Bone marrow
● Need dictates the amount of platelets
produced.
● Stimulus for production is the platelet
mass in circulating blood is 80 % and
megakaryocyte mass in bone marrow
● Platelets are released via sinuses of
bone marrow
• 2/3 peripheral blood
• 1/3 sequestered in spleen
 ❖ Thrombopoietin (TPO)
● Regulates platelet development
● Produced in the liver, kidney 1. Peripheral zone
and spleen ➢ Responsible for platelet adhesion and
● Influences all stages of aggregation
megakaryocyte production ○ Glycocalyx: Fluffy surface coat
● Levels are increased in ■ Contains glycoprotein
thrombocytopenia,and reduced receptors:
in thrombocytosis ■ GPIb binds von
Willebrand’s factor
How does TPO work? (TPO levels increases when needed for platelet
thrombocytopenia occurs and decreases in thrombocytosis)
adhesion to collagen
➢ Maintains a constant number of ■ GPIIb/IIIa binds
platelets in peripheral blood by binding fibrinogen needed for
Mpl (platelet receptor). Bound TPO can aggregation
not stimulate proliferation of bone ■ Bind ADP and thrombin,
marrow progenitor cells promoting aggregation
➢ The higher the platelet count, the more ■ Factors I, V, VIII on
TPO is bound and stimulation of bone surface, involved in 2o
marrow is decreased hemostasis
○ Plasma Membrane:
PLATELET CIRCULATION ■ Exposed on platelet
● Normal count is 250,000. activation
● Reference Range: 150-450 x 109 /L ■ Layer called PF3
● Normal life span 7-10 days. (platelet factor) surface
● About 1/3 are trapped in the splee for interaction of
plasma coagulation
Anatomy of a Platelet factors
■ Initiation of formation
Divided into 4 zones of thromboxane A2.
● Peripheral This stimulates
● Structural aggregation and
● Organelle vasoconstriction
● Membrane 2. Structural/ sol-gel zone
➢ Responsible for platelet
retraction/contraction functions and
platelet shape
○ Microtubules - Tubulin
○ Cytoskeleton
○ Binding
proteins/Microfilaments
■ Actin
■ Myosin
 • Dense tubular system(DTS) -
site of arachidonic acid
3. Organelle zone metabolism
➢ Responsible for storage and
platelet release functions STAGES
○ Granules 1. Vascular spasm
■ Dense bodies, 2. Platelet plug formation
alpha granules, ● Collagen exposure
lysosome,Mitoc ● Platelets adhesion
hondria ● ADP release; thromboxane
○ Glycogen release
● Platelet accumulation = plug
3. Clot formation
● Cascade reactions
○ Inactive precursors
○ Final stages
a. prothrombin
------> thrombin
- heparin
effects
b. fibrinogen
-------> fibrin
(clot)

Mucopolysacch. Coat: Glycoprotein content

which are important for interaction of platelets
with each other or aggregating agents.

- α Granules:

- Dense core:

Membrane Zone

• Responsible for secretion of granule contents

and storage of calcium

• Two systems

• Surface-connected open
canalicular system (OCS) -
release of granules, provides
direct communication between
intracellular and extracellular
compartments
Platelet plug formation: platelet Platelet plug formation: platelet
adhesion aggregation

Membrane Phospholipid
↓
Adhesion of platelet to subendothelial collagen. Arachidonic acid
↓ Cyclo-oxygenase
Dependent on the VW factor (Von Willebrand
Thromboxane synthetase ↓
part of Fact VIII). Also dependent on
Thromboxane A2
glycoproteins.

Platelet plug formation: platelet Thromboxane A2: Potentiase aggregation and

vasoconstrictor.
release/secretion action
Aggregation: Release ADP+thromboxane
A2 →aggregation. This is followed by more
secretion
→secondary aggregation.
→platelet mass or plug.

Collagen exposure results in the release of

granules contents (ADP, serotonin, fibrinoen).
Related Terms

• Thrombosis
• Petechiae
• Purpura
• Ecchymosis
• Menorrhagia
• Metrorrhagia
•Hematohidrosis
• Epistaxis
• Hematemesis
• Hemoptysis
• Hemarthrosis
• Hematochezia
• Melena
SECONDARY HEMOSTASIS
● A complex system of procoagulant
activities and control activities to
contain and limit clot formation.
● Coagulation factors – produced by the
LIVER except Factors III and IV.
(III produced by tissue and IV is
calcium)
● Zymogens – precursors (inactivated
form of coagulation)
● End point: Stable fibrin clot
Factor I: Fibrinogen (most concentrated)
● Essential for platelet aggregation.
● It is the precursor of fibrin, which forms
the resulting clot
Fibrinogenthrombin → fibrin monomers →
fibrin clot
Factor II: Prothrombin
• Prothrombin has a half-life of almost 3
days with 70% consumption during clotting
Thromboplastin will become thrombin
(activated form) with the help of calcium
Factor III: Tissue Factor
• Also called “Tissue
Thromboplastin/Thrombokinase”
• These tissues can be from the brain, lung,
vascular endothelium, liver, placenta, or
kidneys; these tissue types are capable of
converting prothrombin to thrombin
Factor IV: Ionized Calcium
• Ionized calcium is the physiologically active
form of calcium in the human body, and only
small amounts are needed for blood coagulation.
Factor V: Proaccelerin (accelerator
globulin)
• Also called “Labile factor/Thrombogen”.
• Factor V is an extremely labile globulin
protein. It deteriorates rapidly, having a half-life
of 16 hours. Factor V is consumed in the clotting
process and is essential to the later stages of
thromboplastin formation.
Factor VII: Proconvertin (stable factor)
• Action: Activation of tissue thromboplastin
and the acceleration of the production of
thrombin from prothrombin
Factor VIII: Antihemophilic factor A
• Also called “Antihemophilic globulin”.
• Is extremely labile, with a 50% loss within 12
hours at 4°C in vitro and a similar 50% loss in
vivo within 8 to 12 hours after transfusion.
Von Willebrand factor
• Has a receptor sited for both platelets and
collagen.
• Primary platelet surface receptor
Factor IX Christmas factor
• Also called “Antihemophilic factor B” or
Plasma Thromboplastin Component.
• Stable protein factor that is neither consumed
during clotting nor destroyed by aging at 4°C for
2 weeks.
Factor X: Stuart Prower Factor
• in the presence of calcium ions forms the final
common pathway through which the products of
both the extrinsic and intrinsic thromboplastin • Also called “Williams factor” or “Flaujeac
generating systems merge to form the ultimate factor”.
thromboplastin that converts prothrombin to
thrombin. ZYMOGENS (inactivated)
● Prothrombin
● Factor VII
● Factor IX
● Factor X
Factor XI: Antihemophilic Factor C ● Factor XI
• Also called “Plasma Thromboplastin ● Factor XII
Antecedent” ● Prekallikrein
● Factor XIII
• beta-globulin, can be found in serum because it
is only partially consumed during the clotting Cofactors (aids in the activation of
process. mechanism. zymogens to activate enzyme)
Factor XII: Hageman factor ● Tissue
● Factor
Start of coagulation cascade, it will interact in
● Factor V
negative charges of platelets and converted into
● Factor VIII
its activated form.
● HMWK
• Also called “Glass factor”

• Stable factor that is not consumed during the

coagulation process.

Factor XIII: Fibrin Stabilizing factor

• Also called “Fibrinase” or “Laki-Lorand
factor..

• in the presence of ionized calcium produces a

stabilized fibrin clot.

Prekallikrein
• Also called “Contact factor” or the “Fletcher
factor”.

High Molecular Weight Kininogen

 Inhibitors of Coagulation

Intrinsic Extrinsic Common Inhibitor Function

Pathway Pathway Coagulation Protein C Degrades Factor Va
Pathway and VIIIa
Protein S Degrades Factor Va
Factors: Factors: Factors: and VIIIa
Anti-thrombin III Inhibits factors Ixa,
XII III X Xa, Xia, kallikrein
XI VII V and plasmin
IX II Heparin cofactor II Inhibits thrombin
VIII I A2- macroglobulin Inhibits thrombin,
kallekrein and
plasmin
EPI (Extrinsic Inhibits VIIa-tissue
Pathway inhibitor) factor complex
Fibrinogen Prothrombin Contact and LACI
group group group (Lipoprotein
Factors: Factors: Factors: Associated
I II XII Coagulation
V VII XI Inhibitor)
VIII IX PK C1 inhibitor Inactivates XIIa and
XIII X HMWK kallekrein, factor Xia
and plasmin
A1-antitrypsin Inhibitor of
Thrombin, Xa and
XIa
von Willebrand Factor (vWF)

● Two major functions: Aids in platelet adhesion & carrier protein for Factor 8
● Two sites of Synthesis: Endothelial cells and Megakaryocyte
● Two sites of Storage: Weibel-Palade bodies (found in blood vessels) and Alpha granules (in
platelets)

Demarcation System (DMS)

● Delineates individual platelets during thrombocytopoiesis (platelet shedding)

ALPHA GRANULES :
● PLATELET FACTOR
● PLATELET DERIVED GROWTH FACTOR
● PLATELET FIBRINOGEN
● FACTOR V
● VWF
● B-THROMBOGLOBULIN
● THROMBOSPONDIN
● FIBRONECTIN
● PLATELET ALBUMIN

DENSE GRANULES :
● CALCIUM
● ADP
● SEROTONIN ( 5-HYDROXYTRYPTAMINE)

COAGULATION FACTORS:
Produced moslty in the Liver and circulate in an inactivate precursor form.

SERINE PROTEASES: II, VII, IX, X, XI, XII, PREKALLIKREIN, HMWK

TRANSGLUTAMINASE : XIII
FIBRINOGEN: MOST CONCENTRATED CLOTTING FACTOR
VWF: PRODUCED BY MEGAKARYOCYTES AND ENDOTHELIAL CELLS

COAGULATION FACTORS GROUPS:

1. FIBRINOGEN GROUP (I,V,VIII,XIII)

- CALCIUM DEPENDENT
-COMPLETELY CONSUMED DURING COAGULATION
-PRESENT IN PLASMA BUT ABSENT IN SERUM

2. PROTHROMBIN GROUP ( IX, X, VII , II) (1972)

- CALCIUM AND VITAMIN K DEPENDENT
-HALF-LIFE: ( FROM LEAST) VII-IX-X-II
-ABSORBABLE FACTRS (REMOVED BY ADSORBING AGENT, EXAMPLE BaSO4, AI(OH)3
-PRESENT IN PLASMA BUT ABSENT IN ADSORBED PLASMA

3. CONTACT GROUP ( XII, XI, PREKALLIKREIN, HMWK)

- CALCIUM AND VITAMIN K INDEPENDENT
-INVOLVED IN THE CONTACT PHASE (ACTIVATION OF XII)
MODULE 1.3: FIBRINOLYSIS
HEMATOLOGY LEC
Fibrinolysis (thrombolysis)
➢ Process of breaking down fibrin clot
through hydrolysis of fibrin. (dissolves
the clot)
> If you leave the clot alone, it will get
bigger and bigger and press the local
structures of the body and occlude the
vessel that can cause heart attack.
➢ Starts a few hours after fibrin
polymerization and cross-linking
> PLASMIN ang bida - little monster or
eats the clot. Enzymes that break the
clot.
> When clot is broken down into
fragments which are the FDPs or
fibrinogen or fibrin degradation
products.
> And after, FDP will be cleared by the
sewage system of our body, the liver and
kidney.
> If you leave the clot alone, it will get
bigger and bigger and press the local
structures of the body and occlude the
vessel that can cause heart attack.
Fibrin degradation products (FDP) - water
soluble, they can be excreted by the kidneys.
- substances are remain in the
bloodstream after dissolving the blood
clot
- Used to diagnose DIC
- NV: < 10mg/dL
clot is degraded by plasmin → converts
fibrinogen/fibrin into x component → cleaves
into component y and b → component y cleaves
into component D and E
X and Y - early degradation product
D and E - late degradation product
Pathologic effects:
X and Y - anticoagulant activity
Y and D - inhibit fibrin polymerization
E - powerful inhibitor of thrombin
Pathologic Fibrinogen
● Primary Fibrinolysis
➢ refers to normal breakdown of clots
- degradation of FIBRINOGEN
- NO formation of fibrin
monomer, fibrin polymer, and
d-dimer (most important)
- Have negative protamine
sulfate test
- Excessive amount of
plasminogen activators from
damaged cells
- Converts plasminogen to
plasmin in the absence of fibrin
formation
➢ Secondary Fibrinolysis (can cause
severe bleeding)
➢ breakdown of blood cells due to medical
disorder, medicine or etc.
- Degradation of FIBRIN
- WITH formation of fibrin
monomer, fibrin polymer and
D-dimer
MODULE 1.3: FIBRINOLYSIS

HEMATOLOGY LEC

- Positive protamine sulfate test to fibrin (TANAN NALANG

- Disseminated Intravascular DAW PARA LIMPYO)
Coagulation (DIC)
○ uncontrolled ❖ Urokinase Plasminogen
inappropriate formation Activator
of fibrin - Secreted by epithelial cells,
○ MASS consumption of monocytes, macrophages
platelets - Circulates with little activity.
- infection - Digest extracellular matrix
- Neoplasm
- Snake bite
- HTR (Hemolytic Transfusion
Reaction)
Plasmin's Roles Comments
Components Activates Cleaves Fibrin
Fibrinolysis and Fibrinogen to
➢ Plasminogen - made by the liver; fibrin (ogen)
naturally present in plasma degradation
- Stored and transported in products X, Y
eosinophils and D-E (FDPs)
- Zymogen for plasmin (enzyme
precursor) Activates Factors
- increase concentration when Intrinsic XII----XIIa is
there is inflammation Coagulation amplified
- it gets incorporated into the Pathway (starts indirectly by
blood clot and TPA will add. the activation of plasmin
TPA binds to plasminogen and F12)
becomes plasmin.
Interferes with Destroys factors
intrinsic and VIII and V (to
❖ Tissue Plasminogen Activator
common prevent new clot
(Tpa)
pathways formation)
- Produced by
endothelial cells Block Thrombin FDP interfere
(injured) conversion of with thrombin
- Activates plasminogen fibrinogen to influence on
➢ Plasmin - degrades the clot fibrin fibrinogen
- Degrades proteins susceptible to
degradation.
- Relevant hemostatic target: ➢ Streptokinase
Fibrin, Fibrinogen, Factors V - Causes conformational change
and Factor VIII (and factor II to plasminogen when bound.
and XII) - Not localized to fibrin (bc this
> bc prothrombin (II) will enzyme comes from group b
become thrombin to fibrinogen beta hemolytic streptococci)
MODULE 1.3: FIBRINOLYSIS

HEMATOLOGY LEC

➢ Staphylokinase - Fibrin required for

activation of plasminogen

Outcomes: (fibrinolysis product)

❑ Fibrin Degradation products
- Fragment X
- Fragment Y, D,E

❑ D-dimers
- indicative of clot formation
- appear after clot dissolves
- important marker of clot
formation
Red arrows - inhibitors
a2 - antiplasmin - major inhibitor of
fibrinolysis bc first one to bind to
plasmin
a2 - macroglobulin - second to bind to
plasmin
a1 - antitrypsin - last major naturally
occurring defense against to plasmin

FDPs and D-dimer

Protein C degrade factor 5 and 8

Wound → thrombomodulin + thrombin

→activates protein C and w/ the help of protein
S, protein C degrades factor 5 and 8
→inactivates inhibitors of TPA → TPA is free or
TPA is activated→ go to plasminogen →
plasmin → eat the clot → lysis of clot → end Inhibitors
MODULE 1.3: FIBRINOLYSIS

HEMATOLOGY LEC

➢ Plasminogen Activator inhibitors

(PAIs)
a. PAI-1: inhibits TPA, scuPA and
Upa
b. PAI-2: located in placent
c. a and macrophages, inhibits tPA
and uPA

➢ Thrombin-activatable fibrinolysis
inhibitor (TFPI)
- inhibits binding and activation
of plasminogen

➢ A2 -Antiplasmin
- inhibitors of circulating plasmin
- Inhibits the clot-promoting
activities of plasma kallikrein
- Inhibits the serine proteases
Xlla, XIa, IIa andXa

➢ A2 -macroglobulin
- Inhibits component in both the
fibrinolysis and coagulation
systems
- Inhibits plasmin after alpha 2
anti-plasmindepletion

➢ A1 -Antitrypsin
- Inhibits XIa and inactivates
plasmin
MODULE 1.3: FIBRINOLYSIS
HEMATOLOGY LEC
Laboratory Diagnosis
➢ Whole Blood Clot Lysis Time
(WBCLT)
- Clot should remain intact for
approximately 48 hours at 37C
- Lysis ang bantayan
➢ Protamine Sulfate Dilution Test
- Detects the presence of fibrin
monomers in the plasma
- protamine sulfate is added to
plasma, it displaces the
secondary degradation product
from fibrin monomers and
primary FDP which will then
polymerize spontaneously
- Paracoagulation
➢ Ethanol Gelation Time Test
- Less sensitive compared to
protamine sulfate dilution test
but more specific
- in the presence of 50% solution
of ethanol, any soluble fibrin
monomer complexes present
will dissociate resulting in
polymerization of monomers
and subsequent gel formation.
➢ Latex D-dimer Assay
- Latex: mouse anti-human
D-dimer
- Latex particles coated with the
antibody against FDP D and E
or human fibrinogen are mix
with patient serum
- Macroscopic agglutination of
latex particles (end result) (naay
presence of FDP)
- NV: 200 ng/L
➢ Euglobulin Lysis Time
- Screening procedure for the
measurement of fibrinolytic
activity.
- does not detect FDP
- Plasma + water > acidified =
euglobulin (thrombin)
- Time for complete lysis: > 2
hours
- NV: Lysis should be observed
greater than 2 hours.
- Increased fibrinolytic activity -
lyse in less than 2 hour
(problem)
Fibrinogen
NV: 200 - 400mg/dL
Module 1.4: Laboratory Diagnostic Test for Primary and Secondary Hemostasis
HEMATOLOGY 2 LEC
PRIMARY HEMOSTASIS
● Bleeding time
● Platelet Count (Reese-Ecker, Guy &
Leake, BreckerCronkite) and Platelet
Estimate
● Clot retraction time
● Capillary Fragility Test
● Platelet Aggregation Test
Bleeding Time
● Screening test to detect disorders of
platelet functions.
● Time it takes for a wound to stop
bleeding.
● Invivo measure of primary hemostasis
Methods:
1. Duke method – NV: 2 to 4 mins.
2. Ivy method – NV: 1 to 7 mins.
3. Template Bleeding time - 2 to 9 mins.
4. Copley-lalitch method
- Isotonic, Light Microscopy,
Platelets: Bluish
2. Guy and Leake – Sodium oxalate,
formaldehyde, Crystal violet
- Isotonic, light microscopy, lilac
refractile objects
3. Brecker-Cronkite – 1% Ammonium
oxalate, hypotonic, Phase microscopy
Reasons why platelet count are hard to find:
● Platelets adhere to foreign substances
● Easily disintegrates
● Hard to differentiate from debris
● Unevenly distributed because of
clumping tendency.
NV: 150 – 450 x109L
Platelet Count Estimate
● To determine the approximate number
of platelets/field.
● Normal blood smear = 8 to 20
platelets/field.

Factors affected by: Platelet Estimate Report as:

1. Platelet count and function
2. Thickness and vascularity of skin 0 to 49,000/Ul Marked decrease
3. Quality of blood vessels
50,000 to 99,000/Ul Moderate decrease
4. Medications
100,000 to Slight decrease
Carbenicillin - most potent drug capable 149,000/Ul
affecting the platelet function
150,000 to Low normal
199,000/Ul
Aspirin - inactivates cyclooxygenase thus
resulting in no platelet aggregation. 200,000 to Normal
400,000/Ul
Arachidonic acid - a substrate that is converted
401,000 to Slight increase
into thromboxane A2 by cyclooxygenase.
599,000/Ul
Platelet Count
600,000 to Moderate increase
Counted using a hemocytometer Methods: 800,000/Ul
1. Reese-Ecker – Sodium citrate,
Above 800,000/Ul Marked increase
formaldehyde, Brilliant cresyl blue
Module 1.4: Laboratory Diagnostic Test for Primary and Secondary Hemostasis
HEMATOLOGY 2 LEC
Clot Retraction Time
● Principle: Normal blood clot contracts.
● Degree of clot retraction: measured on
the amount expressed in serum
- Takes 1hr after whole blood is allowed
to clot in a clean glass tube at 37 degrees
celsius the clot will begin to shrink and
retract from the walls of the tube.
- Clot retraction depends on the normal
number of platelets
- Normal number of contractile platelets:
(3 methods)
a. Calcium
b. ATP
c. Concentration of fibrinogen
A. Qualitative test
1. Hirshboek/Castor oil method –
NV: 15 to 45 mins (formation of
dimpling) or droplet like serum
on the surface of blood drop
B. Quantitative test
1. Stefanini method – spx: 3 to 5
mL, temp: 37C, NV: Begins
within 1 hour, complete within
18 to 24 hours (observe 1 hr,
2hrs, 16hrs, 18hrs to 24hrs DILI
BIYAAN)
2. McFarlane method – spx: 5 mL,
Incubation time: 1 hour, NV:
44-67%
𝑣𝑜𝑙 𝑜𝑓 𝑠𝑒𝑟𝑢𝑚
𝐶𝑙𝑜𝑡 𝑟𝑒𝑡𝑟𝑎𝑐𝑡𝑖𝑜𝑛 𝑡𝑖𝑚𝑒 = 𝑡𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒
𝑥 100
Capillary Fragility (Resistance) Test
● Principle: Partially obstructing the
venous blood, the capillary pressure is
increased. >>>>> Petechiae
Petechiae - purplish red pinpoint hemorrhagic
spots (< 3 mm) in the skin caused by loss of
capillary ability to withstand normal blood
pressure and trauma
> uses sphygmomanometer inflate 100 mmHg
for 5 mins. After 15-30 mins petechiae will
appear.
> capillary fragility test correlates with the
degree of thrombocytopenia
Method: Tourniquet test (Rumple Leede
Test/Hess test) other name
GRADE PETECHIAE
1+ 0-10
2+ 10-20
3+ 20-50
4+ >50
More petechiae, low platelet
Platelet Aggregation Test
● Assess the ability of platelets to
aggregate after the addition of
aggregating agents.
● Sample: Platelet rich plasma
● Aggregating agents: Epinephrine,
Collagen, ADP, Ristocetin, thrombin,
arachidonic acid, serotonin
How?
- Collect samples in the blue top tube and
add agonist. After, measure the
aggregation using optical density.
- Increased OD - no aggregation
- Decreased OD - with aggregation
SECONDARY HEMOSTASIS
● Clotting (Coagulation) Time
● Prothrombin Time (PT)
● Activated Partial Thromboplastin Time
(APTT)
Module 1.4: Laboratory Diagnostic Test for Primary and Secondary Hemostasis
HEMATOLOGY 2 LEC
● Stypven Time (Russel Viper Venom) International Normalized Ratio (INR) -
● Thrombin time
● Reptilase time
● Duckert’s Test (5M Urea Solubility
Test)
● Mixing studies

Clotting Time develop to standardize the difference

● A measure of the ability of the blood to sensitivity in individual thromboplastin
clot and is not influenced by the platelet
reagent and the effect on PT assays?
functions. It also measures only the time
required for the formation of thrombin
● Range: 2.0 to 3.0
sufficient to produce a visible clot.
● More sensitive thromboplastin has <1.0
and less sensitive reagents have an index
Methods:
of >1.0.
A. Micro method
The more sensitive reagent, the longer
1. Slide/Drop method - : NV – 2 to
the PT. Less sensitive reagent, the
4 mins
shorter PT
B. Macro method
ISI - found in PT reagent bottle assigned
1. Lee and White (Whole Blood
by manufacturers
CT) – NV: 7 to 15 mins.
INR - recommended for monitoring oral
anticoagulant therapy
Prothrombin time (PT)
● Assess Extrinsic and Common Pathway
Activated Partial Thromboplastin Time
● Specimen requirement: citrated (blue
(APTT)
top) platelet poor plasma for 15 mins. At
2500x and process within 24 hours.
● Assess Intrinsic and Common Pathway
Ratio of sodium citrate anticoagulant to
● Specimen requirement: citrated platelet
blood - 1:9
poor plasma for 15 mins. At 2500xg and
● Reagent: Simplastin = thromboplastin +
processed within 4 hrs ONLY
CaCL
● Reagent: Phospholipid (Platelet
● Uses: To monitor patients in warfarin
substitute) and CaCl
therapy AND to detect coagulation
● Activators: Silica, kaolin, celite,
factor deficiency
bentonite, ellagic acid
Warfarin - oral anticoagulant, it inhibits
● Uses: To monitor patients in heparin
the coagulation factors 9, 10, 7, 2
therapy AND to detect coagulation
(Vitamin K dependent coagulation
factor deficiency
factors)
● Reference range: 35 to 45 seconds
● Reference range: 10 to 13 seconds
● Epoxide reductase (VKORC1)
Module 1.4: Laboratory Diagnostic Test for Primary and Secondary Hemostasis
HEMATOLOGY 2 LEC
Therapeutic Anticoagulants Oral
anticoagulant (Coumarin drugs)
➢ Warfarin – inhibit synthesis of clotting
factors II, VII, IX, X (1972).
Intravenous anticoagulant
➢ Heparin – most commonly used
Stypven Time (Russel Viper Venom Time)
● Determines problem in common
pathway
● Used snake venom (Vipera russeli)
● Reagent: platelin + chloride (calcium
chloride according saiya ingon)
● Specimen: Platelet poor plasma • NV: 6
to 10 seconds.
dRVVT – diluted
- Venom came from Vipera Russelli or
Daboira Russelli. This venom directly
activated factor 10.
- Assess the common pathway
Thrombin time
● Prolonged in fibrinogen deficiency,
when function of fibrinogen is impaired,
and in the presence of FDPs and
thrombolytic agents such as
streptokinase and heparin.
● NV: 15 to 18 seconds
● Spx: Platelet poor plasma (PPP)
● Reagent: Thrombin + CaCl
● End point: Clot formation
Fibrinogen NV: 200 - 400mg/dL
Heparin Therapy - prolonged thrombin time
Concentration of immunoglobulin - high
thrombin time
> Multiple myeloma
Reptilase Time
● Not affected by heparin
● Uses enzymes found in the venom of
Bothrops atrox snake.
Bothrops atrox - snake (diri makita si
reptilase)
- Thrombin life in nature
● NV: 10 to 15 seconds
● Spx: PPP
● Reagent: Atroxin
● End point: Clot formation
Reptilase time is similar in function to thrombin
time - measure the final step in coagulation
RT is NOT affected by heparin unlike thrombin
time.
RT is prolonged in:
- Fibrinogen deficiency
- Presence of FDP
- Thrombolytic agent (streptokinase)
Duckert’s test (5M Urea Solubility test)
● Test for Factor XIII (fibrin stabilizing
factor)
● Reagent: 5M Urea (1%
monochloroacetic acid or 2% acetic
acid)
● Normal result: insoluble to urea when
incubated for 24 hrs. (24 degrees
celsius)
- Specimen should not be
dissolve
TRIVIA:
The symbol of medicine is not a snake, it is a
parasite.
● Dracunculus medinensis - fiery serpent
of Israelite
Substitution test (Mixing Studies)
● Identify specific factor deficiency
● Uses four reagents:
1. Fresh plasma – contains ALL
clotting factors
2. Aged plasma – contains ALL
clotting factors except Factor V
& VIII
3. Aged serum – contains ALL
clotting factors except Factors I,
V, VIII, XIII, II
Module 1.4: Laboratory Diagnostic Test for Primary and Secondary Hemostasis
HEMATOLOGY 2 LEC
4. Adsorbed plasma – contains
ALL clotting factors except
factors I, IX, VII, II

Important Considerations
● Avoid traumatic venipuncture.
● Use plastic or silicone coated tubes
● Ratio of anticoagulant to blood
● Cold temperature causes: precipitation
of vWF, activation of Factor VII and XI
and destruction of platelets
● Factors V and VIII are labile factors
● EDTA is the anticoagulant for platelet
count.
HEMATOLOGY 2: ADDITIONAL NOTES stimulates platelet aggregation. If there's no
thromboxane A2, there is no aggregation
Primary Hemostasis and thus resulting in no platelet plug.
● About platelet
● End result - platelet plug PLATELET SECRETION DISORDERS

STEPS: ➢ ALPHA GRANULE DEFICIENCY

1. Vasoconstriction - PLATELETS ARE LARGE
2. Platelet adhesion AND GRAY OR BLUE-GRAY
3. Platelet activation IN COLOR
4. Platelet secretion - DENSE TUBULAR SUSTEM
5. Platelet aggregation WHICH IS STORAGE SITE
6. Platelet plug FOR CALCIUM AND
CYCLOOGENASE IS
Secondary Hemostasis ABNORMAL.
● About coagulation factors
● End result - fibrin clot ➢ DENSE GRANULE DEFICIENCES
- HERMANSKY-PUDLAK-
Fibrin Mesh - cross linking of fibrin TRIAD OF
APTT - monitor the Intrinsic and common TYROSINASE-POSITIVE
pathway OCULOCUTANEOUS
PT - monitors Extrinsic and common ALBINISM,
pathway ACCUMULATION OF
Vascular Endothelial Growth factors - CEROID-LIKE PIGMENT IN
regenerate a new endothelial cells MACROPHAGES AND
BLEEDING TENDENCIES.
Fribrinolysis - CHEDIAK-HIGASHI-
● Plasmin - enzymes that break the CHARACTERIZED BY
clot ALBINISM, RECURRENT
● Plasminogen is made by the liver INFECTIONS AND GIANT
(synthesize plasminogen) LYSOSOMES IN ALL
Zymogen - enzyme precursor GRANULE-CONTAINING
CELLS.
Hemostasis
1. Vasoconstriction ➢ WISKOT-ALDRICH SYNDROME
2. Temporary platelet plug - TRIAD OF
3. Coagulation cascade THROMBOCYTOPENIA,
4. Fibrinolysis RECURRENT INFECTIONS
AND ECZEMA
Aspirin - can cause prolonged bleeding - PLATELETS ARE SMALL
time. Because due to platelet dysfunction it AND HAVE ABNORMAL
activates cyclooxygenase which has an CELLULAR MEMBRANES
arachidonic acid where it is a substrate that
is converted into thromboxane A2 which
BASIC TERMINOLOGY FOR CLINICAL IN
BLEEDING DISORDERS

1. Petechiae - purplish red pinpoint

hemorrhagic spots (< 3 mm) in the
skin caused by loss of capillary
ability to withstand normal blood
pressure and trauma
2. Purpura - hemorrhage of blood (1
cm) into small areas of skin, mucous
membranes
3. Ecchymosus - form of purpura (> 3
cm) 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. Hematoma - swelling or tumor in the
tissues
8. Hematuria - red blood cells in urine
9. Hemoglobinuria - hemoglobin in
urine
10. Melena - stool with dark red or black
blood
11. Menorrhagia - excessive menstrual
bleeding
MODULE 1.1: PLATELET COUNT

HEMATOLOGY LAB

Platelet Count ➢ The whole scale is divided into 9 big

➢ Platelets are small, colorless, moderately squares.
refractile bodies. They appear as azure ➢ Each square is 1 mm long and 1 mm
granules with scanty light blue wide.
cytoplasm when stained. They are
characterized by a tendency to aggregate
and adhere to foreign surfaces. To obtain
reproducible and representative result,
the specimen should be collected in a
plastic syringe and immediately mixed
with EDTA.

Objectives:

• To demonstrate how to count platelets using

the manual methods

• To compute the platelet count and report the

result using conventional and SI units

• To identify the causes of thrombocytosis and

thrombocytopenia

Materials:
❖ Anticoagulated blood
❖ Aspirator
❖ RBC pipette
❖ Microscope Rees-Ecker diluting fluid
❖ Improved Neubauer CC
❖ Unopette WBC and Platelet test
❖ Thick coverslip
❖ Tissue paper

NEUBAUER’S SLIDE
➢ It is the name given to a thick glass
slide. In the center of the slide, there is
an H- shaped groove. On the two sides
of the central horizontal bar, there are
scales for counting the blood cells.
➢ The depth of the scales is 1/10 mm or
0.1 mm.
➢ Each scale is 3 mm wide and 3 mm
long.
MODULE 1.1: PLATELET COUNT

HEMATOLOGY LAB
MODULE 1.1: PLATELET COUNT

HEMATOLOGY LAB
MODULE 1.1: PLATELET COUNT

HEMATOLOGY LAB
MODULE 1.1: PLATELET COUNT

HEMATOLOGY LAB

The platelets can be counted in all the small

squares of the central square.BUT FOR
CONVENIENCE WE WILL COUNT
PLATELET IN THE CORNERS SPECIFIED
FOR RBCS. (5 of the tertiary squares = total of
80 smallest squares)
MODULE 1.1: PLATELET COUNT

HEMATOLOGY LAB

Volume of one smallest square = 1/20 x 1/20 x

1/10 = 1/4000mm³

CALCULATION OF VOLUME OF
PLATELET SQUARE

Length of one smaller square = 1/5 mm Width

of one smaller square = 1/5 mm Depth of one
smaller square = 1/10mm Volume of one
smaller square = 1/5 x 1/5 x 1/10 = 1/250mm³

Each of the smaller squares is further

subdivided into sixteen smallest squares.

Length of one smallest square = 1/5 x 1/4 =

1/20mm
Width of one smallest square = 1/20mm
Depth of one smallest square = 1/10mm
MODULE 1.1: PLATELET COUNT

HEMATOLOGY LAB

Counting Rule
• Do not count cells touching
• Bottom line
• Right line

➢ This is to avoid double counting.

MODULE 1.1: PLATELET COUNT

HEMATOLOGY LAB

DIFFERENCES BETWEEN RBC AND

WBC PIPETTE

RBC pipette WBC pipette

1 It has a red bead It has a
white bead
2 It has It has
graduations graduations
upto mark upto mark 11
101
3 Size of bulb Size of bulb
is larger is smaller
4 Size of lumen Size of lumen
is smaller is larger

RBC PIPETTE
MODULE 1.1: PLATELET COUNT

HEMATOLOGY LAB

WBC PIPETTE
MODULE 1.1: PLATELET COUNT

HEMATOLOGY LAB
MODULE 1.1: PLATELET COUNT

HEMATOLOGY LAB

For Platelet counting

1 part of blood is mixed in 100 parts of Platelet
fluid (Rees Ecker) , so the dilution factor for
platelet counting is 100.

PLATELET COUNTING

Total no. of Cells in 80(5x16) smallest squares = X

No. of Cells in one smallest square = X/80
Volume of one smallest square = 1/4000mm³
No. of Cells in 1/4000mm³ = X/80
No. of Cells in 1mm³ = X/80 x 4000
No. of Cells in 100 times diluted blood
= X/80 x 4000
No. of Cells in undiluted blood
= X/80 x 4000 x 100/mm³
= X x 32,000,000/mm³
MODULE 1.1: PLATELET COUNT

HEMATOLOGY LAB

FOCUSING
• 40X for platelet counting
MODULE 1.1: PLATELET COUNT

HEMATOLOGY LAB
MODULE 1.1: PLATELET COUNT

HEMATOLOGY LAB

40x magnification
MODULE 1.1: PLATELET COUNT

HEMATOLOGY LAB

Rees-Ecker Method 2. Perform procedure 2-6 of Rees-Ecker

Method
Diluting fluid:
Brilliant cresyl blue (stain) ---- 0.1 gm Note: Platelets are seen as colorless,
40% formalin (preservative) ---- 0.2 mL refractile bodies.
Sodium citrate (prevents clumping)
----100.0 mL
Computation:
Procedure:
𝑇𝑜𝑡𝑎𝑙 𝑛𝑜. 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑
𝑁𝑜. 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠/𝑚𝑚3 = 𝐴𝑟𝑒𝑎 𝑥 𝐷𝑒𝑝𝑡ℎ 𝑥 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛
1
1. Draw blood up to the 0.5 mark of the
RBC pipette. =
𝑇𝑜𝑡𝑎𝑙 𝑛𝑜. 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑
1 𝑥 1/10 𝑥 1/100
2. Dilute blood with Rees-Ecker diluting
fluid up to the 101 mark. This makes = Cells counted x 1,000
1:200 dilution.
3. Shake the pipette for 1-5 minutes. Other Methods of Platelet Count
4. Discard 5-6 drops and charge the I. Direct Methods
counting chamber. A. Nygard’s
5. Place the counting chamber on a Petri B. Guy and Leake’s
dish with a wet filter paper to prevent Diluting Fluid:
evaporation. Let it stand for 10-15 1. Sodium citrate
minutes to allow the platelets to settle. 2. Crystal violet (stain)
6. Count the platelets in all 25 tertiary 3. 40% formalin
squares of the central secondary square
with a 1 mm2 area. Note: Platelets are C. Brecker-Cronkite
stained light blue. ● Equipment:
1. Neubauer counting
Computation: chamber
2. Phase contrast
𝑇𝑜𝑡𝑎𝑙 𝑛𝑜. 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑
𝑁𝑜. 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠/𝑚𝑚3 = 𝐴𝑟𝑒𝑎 𝑥 𝐷𝑒𝑝𝑡ℎ 𝑥 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 microscope
● Diluting Fluid: 1% ammonium
=
𝑇𝑜𝑡𝑎𝑙 𝑛𝑜. 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 oxalate in distilled water
1 𝑥 1/10 𝑥 1/100
● Source of Errors:

= Cells counted x 2,000

1. Platelet clumps imply
platelet aggregation and
clotting.
Ammonium Oxalate Technique
2. Imperfect sources of
Procedure:
blood e.g., skin
1. Draw blood up to the 1.0 mark of the puncture.
RBC pipette and ammonium oxalate up 3. Blood in EDTA kept at
to the 101 mark producing a 1:100 20 degrees Celsius is
dilution. satisfactory only within
five hours after
collection.
MODULE 1.1: PLATELET COUNT

HEMATOLOGY LAB

5. Examine under OIO and count the

II. Indirect Methods RBCs and platelets until 1,000 RBCs are
A. Damahesk counted.

Procedure: C. Olef’s – this is considered the best indirect

1. Place a drop of diluting fluid
over the puncture wound and
gently press the finger so that a
small amount of blood wells up
into the drop of diluting fluid.
The proportion of blood to the
diluting fluid should use 1:5
2. Transfer the mixture into a
coverslip and place it on top of a
slide.
3. Allow the platelets to settle for
15-45 minutes.
method.
Note: Confirmation of the platelet count
can be done on the basis of the
occurrence of platelets in the peripheral
smear.
> 1 platelet/OIO field – decrease in the number
of platelets
5-20 platelets/OIO field (with occasional
clumping) – adequate supply of platelet
<25 platelets/OIO field – increase in the
number of platelets
Normal Value

4. Count the RBCs and platelets Platelet Count = 150 – 450 x 109 /dL
under OIO until 250 RBCs have
been counted.

B. Fonio’s
Diluting fluid:

14% magnesium sulfate

Giemsa or Wright’s stain

Procedure:

1. Place a large drop of diluting fluid on

the site of puncture. Then press blood
from the puncture site.

2. Mix one part blood and five parts

magnesium sulfate.

3. Make a smear using the mixture.

4. Allow the smear to dry, then apply

staining fluid.
MODULE 1.2: HEMATOLOGY 2 LAB
EXPLAINING MECHANISM OF
PRIMARY HEMOSTASIS AND
ROLE OF PLATELET TO
VASCULAR INJURY
Platelet Aggregation
● Simultaneously with platelet release,
platelets stimulating agents (collagen,
ADP, epinephrine, thrombin) bind to the
platelets, causing them to adhere to one
another.
ADP and thromboxane A2 - stimulate the
platelet to adhere in the injury site (manawag
silag platelet)
● Fibrinogen is necessary as cofactor for
platelet aggregation.
GPIIb/IIIa will bind to fibrinogen causing the
platelet to attach each other or aggregate
● Glanzmann’s Thrombasthenia =
decrease or absence of the platelet
membrane glycoprotein IIb -IIIa
(GpIIb-IIIa) complex, which acts as
fibrinogen binding site on the platelet
surface.
GpIIb-IIIa kay defective thus no platelet
aggregation as it can't bind to fibrinogen.
Aspirin - common cause of prolonged bleeding
time.
- It inhibits cyclooxygenase (produce
thromboxane A2) if there will be no
thromboxane A2 there's no platelet
aggregation resulting to no platelet plug
Primary Hemostasis TEST
1. Platelet count
2. Bleeding Time
3. Platelet Aggregation
Disease associated are those mucocutaneous or
superficial bleeding (mabaw na samad) ex.
Bruise, epistaxis
Secondary Hemostasis TEST
1. APPT - measures intrinsic and common
pathway
2. PT - monitor extrinsic and common
pathway
3. Clotting Time
Disease associated are those anatomical or deep
tissue bleeding
Platelets have 2 parameter:
1. Platelet number - platelet count
● Thrombocytopenia - low count
2. Platelet function - bleeding time and
platelet aggregation (aggregometry)
● Thrombasthenia - BSS and GT
Platelet formation:
1. Adhesion - requires VWF, receptor GpIb
2. Activation
3. Secretion - alpha and dense granules
4. Aggregation - add certain agonist
- Aim is to measure the determine
the platelet function to assess
the primary the hemostasis
- Requires normal plt membrane,
normal plt activation pathways
and normal plasma fibrinogen
Platelet Aggregometry Test
Methods: Classic Platelet, Whole blood, Light
Scattering
NORMAL: Two Ways
1. Primary Aggregation - direct interaction
to agonist
2. Secondary Aggregation - secretion of
dense granules
Optical Light Density (resistant of the light
passing)
- Increased - if daghan nagkatag katag
(gamay ra maka pass through na light)
- Decreased - if the platelets aggregate
(naa sa certain areas so maka pass
through ra)
MODULE 1.2: HEMATOLOGY 2 LAB

Platelet aggregometry is the GOLD - Thrombosthenin - protein that gives

STANDARD to determine platelet function the platelet strength
whether adhesion, secretion or aggregation - It also helps the clot
retract and release the
Ristocetin (RIPA) - induced platelet serum.
aggregation this RIPA forces the Gp1b and - Deficient in Glanzmann
vWBF to bind together Thrombasthenia.
- RIPA is abnormal to patients with vWD ● Etiology - same in BSS
and Bernard Soulier Syndrome (Gp1b ● Epidemeology - infant/child -
defective) after circumcision
- Drugs: Desmopressin - forces the VWF ● Pathophysiology - defective
to be expressed so that Gp1b can bind GpIIb/IIIa
- RIPA is normal in Glanzman ● Clinical manifestation -
thrombasthenia superficial bleeding (primary
hemostasis)
Bernard Soulier Syndrome - defective GpIb ● Diagnosis
thus this leads to defective platelet adhesion and - platelet count is
cant proceed to others step that leads to NORMAL
bleeding. - Bleeding time increase
- Platelet number decreases - PBS is normal size
- Platelet in PBS is giant size ● PLATELET AGGREGATION
● Etiology - congenital or autosomal TEST
recessive. - ADP, collagen,
● Epidemiology - Infant or Child epinephrine and
- Defective GpIb or GpIb-IX-V arachidonic acid
complex decreases
● Diagnosis - Ristocetin NORMAL
- Decrease platelet Same treatment
- Prolonged bleeding time
- Giant platelet (RBC size) How do we make the platelet to aggregate?
● Agonist (PLATELET AGGREGATION - Take blood sample (blue stopper)
TEST) (whole blood or platelet rich plasma
- ADP, Epinephrine, collagen, samples), add certain agonist (ADP,
arachidonic acid is NORMAL epinephrine, collagen, ristocetin and
- Ristocetin is ABNORMAL arachidonic acid)
● Treatment:
- Platelet transfusion PPT CONTINUATION..
- Desmopressin (DDAVP) ● Primary assay to determine alterations in
platelet function
Glanzmann Thrombasthenia - platelet ● In vitro test to determine the ability of
aggregation is the main problem because of the platelets to aggregate with certain
defective receptor GpIIb/IIIa agonist
● Citrated blood
MODULE 1.2: HEMATOLOGY 2 LAB

● Platelet Aggregometry: PRP; centrifuge Disorder of Platelet Adhesion - BSS

@ 50 g for 30 minutes. Disorder of Platelet Aggregation - GT
a) Epinephrine Disorder of Platelet Secretion - Storage Pool
b) Collagen Defect
c) ADP
d) Ristocetin

PRP + agonist → O. D. monitored or

Absorbance monitored

O.D - increased if no aggregation

O.D - decreased

Platelet Aggregation Studies

Common symptoms of Patients for Platelet

aggregation studies:
● excessive bleeding
● excessive bruising
● bleeding from the nose or gums
● excessive menstrual bleeding
● blood in the urine or stool
● BernardSoulierSyndrome & von
Willebrand Disease
Abnormal: Ristocetin
Normal: ADP, Collagen,
Epinephrine
● Glanzmann’s Thrombasthenia
Abnormal: ADP, Collagen,
Epinephrine
Normal: Ristocetin
● Storage Pool Defect
Abnormal: ADP, Collagen,
Epinephrine, Ristocetin

*Platelet Aggregation
*Blood film taken from a capillary puncture has
sometimes been referred to as the “poor man’s
aggregation test” (Fresh capillary blood)

REVIEW:
HEMATOLOGY LAB: Module 1.3
DISCUSSING EVENTS IN
SECONDARY HEMOSTASIS
Bleeding Time
- Measures the ability of small blood
vessels to control free flow of blood
after injury. The duration of bleeding is
a measure of the function of platelets as
well as the integrity of the blood vessel
wall.
- Determine platelet function but platelet
aggregation is the gold standard
- Assess primary hemostasis
Learning Objectives:
● Perform bleeding time determination
accurately.
● Describe the clinical significance of
prolonged bleeding time.
Materials:
➢ 70% isopropyl alcohol
➢ Cotton
➢ Blood
➢ Lancet
➢ Filter paper
➢ Timer - most important
GENERAL NORVAL VALUE FOR
BLEEDING TIME: 2 - 9 minutes
Duke’s Method (most common)
Procedure:
1. Cleanse the earlobe or the 3rd or 4th
finger with 70% isopropyl alcohol and
allow it to dry.
2. Make a relatively deep puncture with a
sterile blood lancet and start timing.
3. Blot the blood using filter paper every
30 seconds.
Note: Do not allow the filter paper to touch the
wound as this will hasten the bleeding time.
4. Stop timing as bleeding ceases and
record the bleeding
Normal Value: 2 – 4 minutes
Other Methods of Measuring
Bleeding Time
A. Ivy’s
Procedure:
1. Apply a sphygmomanometer
cuff on the patient’s upper arm.
Inflate it at 40 mmHg. Maintain
this pressure during the entire
procedure
2. Cleanse an area on the volar
surface of the forearm with
70% alcohol and allow it to dry.
The selected area should be free
from visible veins.
3. Make three successive
punctures in the form of a
triangle o a depth of 2 – 3 mm
using a disposable lancet. Start
timing
4. Blot the blood with filter paper
at 30 second intervals until the
bleeding stops.
5. Record the time when the
bleeding stops.
Normal Value: 1 – 7 minutes
B. Copley-Lalitch Method
Procedure:
1. Cleanse the fingertip with
alcohol and allow it to dry.
2. Make a puncture to a depth of 6
mm
3. Immerse the punctured finger in
sterile physiologic saline
solution warmed at 37 degrees
celsius until the bleeding stops.
4. Record the bleeding time
Normal Value: < 3 minutes
(A normal bleeding time can't predict the safety
of a surgical procedure. A shortened bleeding
time is not clinically significant.)
HEMATOLOGY LAB: Module 1.3
Disorder that prolong bleeding time:
Thrombocytopenia
> aplastic anemia
Thrombasthenia:
> Von Willebrand Disease
> GT
> BSS
LIver disease
Uremia
Agammaglobulinemia
Antiplatelet drugs: Aspirin
Coagulation or Clotting Time
- Measures the period required for blood
to clot or solidify after it has been
extracted from the body.
- Assess the secondary hemostasis
Learning Objectives:
● Perform different methods of
coagulation time determination.
● Discuss the advantages and
disadvantages of the different
techniques.
● Explain the significance of prolonged
coagulation time
Materials:
➢ 70 % isopropyl alcohol
➢ 37 degrees Celsius water bath
➢ Cotton
➢ Syringe with needle
➢ Capillary tube (blue)
➢ Gum label
➢ Wassermann tube (3)
➢ Timer
Whole Blood or Lee and White Method
Procedure:
1. Label three tubes (1, 2, 3). Place the
clean, dry tubes in a 37 degrees Celsius
water bath.
2. Obtain 3 mL (1mL per tube) venous
blood in a plastic syringe, preferably
using the two-syringe method.
3. Place 1 ml of blood into each of the
tubes starting with tube 1.
4. Start timing as blood is delivered into
tube 1. Put the tubes back into the water
bath.
5. Gently tilt tube 3 every 30 seconds until
blood solidifies. Handle tube 2 in the
same way. Finally, tilt tube 2 until blood
forms a solid clot.
6. Stop timing and record the coagulation
time.
Normal Value: 7–15 minutes
Other Methods of Coagulation Time
I. Capillary Blood Methods
A. Drop or Slide - common in lab
Procedure:
1. Perform a skin puncture.
Discard the first drop of blood.
2. Place a drop of blood on a clean
glass slide. Start timing.
3. Draw the tip of the lancet
across the drop of blood at 30 -
second interval until fibrin
threads cling to the tip.
Normal Value: 2 – 4 minutes
Capillary Tube or Dale and Laidlaws
Method
Procedure:
1. Make a skin puncture. Wipe off
the first drop of blood.
2. Fill a non - heparinized capillary
tube ¾ with blood.
3. Start timing as soon as blood
enters the tube.
4. Set the capillary tube aside in a
horizontal position for two
minutes.
5. Break off about 1 cm of the
capillary tube at 30 - second
HEMATOLOGY LAB: Module 1.3

interval until fibrin thread

bridges the broken ends of the
capillary tube.
6. Record the coagulation time.
Normal Value: 2 – 4 minutes
MODULE 1.4: PT and APTT
Hematology Laboratory 2
Prothrombin Time (PT)
● It is the plasma clotting time obtained
when excessive thromboplastin and
optimum calcium are added to citrated
plasma under standardized conditions.
● It is essentially a test of the extrinsic
pathway of clotting and the common
pathway.
● It detects deficiencies in factors I, II, V,
VII, and X.
● It is least sensitive to factor II and
insensitive to factor IX and to other
factors involved in the intrinsic
thromboplastin generation.
● It is frequently used to monitor oral
anticoagulant therapy (warfarin).
(Factors II, V, VII, and X are inhibited
by oral anticoagulants)
● It should be performed within two (2)
hours after blood collection. (4hrs
stable)
● Prolonged PT is seen in Vitamin K
deficiency, certain liver diseases,
specific coagulation deficiencies,
disseminated intravascular coagulation
(DIC), some dysproteinemias, and the
presence of circulating anticoagulants
and fibrin/fibrinogen split products. (F
9, 10, 7, 2 Vit K dependent)
● Sodium citrate is the preferred
anticoagulant.
Monitor the warfarin - oral anticoagulant
★ Learning Objectives
● Perform Prothrombin Time Test
accurately.
● Demonstrate the proper way of
preparing blood samples for
coagulation studies.
● Explain the significance of
prolonged prothrombin time.
★ Materials:
➢ Two-way needle
➢ Wassermann tube (2)
➢ Citrated evacuated tube
➢ 37 degrees Celsius water bath
➢ Centrifuge
➢ Thromboplastin reagent
➢ 0.1 mL serological pipette
➢ Tissue paper
➢ 0.2 mL serological pipette
➢ Timer
★ Preparation of Specimen for PT:
1. Collect venous blood in a
citrated tube.
2. Centrifuge at 1,500 rpm for five
minutes.
3. Separate the plasma by placing
it into a clean dry tube.
Hemostat Prothrombin Time
➢ Intended Use
Hemostat Thromboplastin SI
(PT-SI) is a high-sensitivity reagent that
is used to perform the one-stage
prothrombin time (PT). Prolongation of
PT indicates either acquired or
congenital disorders that affect
coagulation factors I, II, V, VII, and X.
The PT has been widely accepted as the
means to monitor patients on oral
anticoagulant therapy due to the
reduction in the activity of vitamin
K-dependent clotting factors (II, VII,
IX, X, Protein C, and Protein S).
HemoStat Thromboplastin SI can be
used to assay coagulation factors in the
extrinsic and common pathways of
coagulation.
➢ Principle
The one-stage PT measures the
clotting time of plasma after adding a
Transes: Febrienne Felise Molina
MODULE 1.4: PT and APTT
Hematology Laboratory 2
source of tissue factor (thromboplastin)
and calcium. The recalcification of
plasma in the presence of tissue factor
generates activated factor X, with the
consequent formation of thrombin and
ultimately a fibrin clot.
➢ Reagent
● Thromboplastin reagent (stored
@ 2-8 degrees Celsius)
● Lyophilized extract of rabbit
brain
● Calcium chloride
(If mangutana unsay reagent sa
PT: Thromboplastin and
calcium chloride)
➢ Reagent Preparation
● Reconstitute with exactly 2 mL
distilled water. Agitate gently
until the solution is complete.
● After reconstitution, the reagent
is stable for seven days when
stored at 2-8 degrees Celsius, 24
hours @ 15-25 degrees Celsius
and 24 hours @ 37 degrees
Celsius. Storing the reagent
between 2-8 degrees Celsius
when it is not in use is
recommended.
➢ Test Procedure
1. Perform samples and controls in
duplicates.
2. Prewarm the HemoStat
Thromboplastin-SI reagent at 37
degrees Celsius.
3. Pipette 0.1 ml of plasma/control
into a prewarmed test tube.
4. Incubate at 3-5 minutes at 37
degrees Celsius.
5. Add PT-SI reagent.
6. Start timer with the addition of
reagent.
7. Record the time required for
clot formation. (end result)
Normal Value: 10-14 seconds
Activated Partial Thromboplastin Time
● It is a routine test for screening
coagulation disorders in the intrinsic
pathway by measuring factors VIII, IX,
XI, XII, and Prekallikrein (PK) or
Fletcher factor but not platelets and
factor XIII. (plt count, platelet
aggregometry, f13 - 5M urea solubility
test)
● It is also used to detect the presence of
circulating anticoagulants or inhibitors,
and to monitor heparin therapy.
(warfarin in PT)
● It should be performed within two (2)
hours after blood collection.
● Sodium citrate is the preferred
anticoagulant
★ Learning Objectives
● Perform the determination of
activated partial thromboplastin
time accurately.
● Describe the principle involved
in the said test.
● Explain the significance of
prolonged activated partial
thromboplastin time .
★ Materials:
● Two-way needle
● Citrated evacuated tube
● Centrifuge
● 0.3 mL serological pipette
● 0.4 mL serological pipette
● Calcium chloride
● Wassermann tube (2)
● 37 degrees Celsius water bath
● aPTT reagent
Transes: Febrienne Felise Molina
MODULE 1.4: PT and APTT
Hematology Laboratory 2
● Tissue paper
● Timer
★ Preparation of Specimen for aPPT:
1. Collect venous blood in a
citrated tube.
2. Centrifuge at 1,500 rpm for five
minutes.
3. Separate the plasma by placing
it into a clean dry tube.
Hemostat Activated Partial
Thromboplastin Time
(only APTT has an activator)
Determination of Activated Partial
Thromboplastin Time Utilizing Ellagic Acid
Activator
The activated partial
thromboplastin time (aPTT) is a simple
and versatile test which is sensitive to
deficiencies of all plasma clotting
factors except factor VII. However, it is
principally used to detect deficiencies
in stage 1 of the coagulation mechanism,
namely factors VIII, IX, XI, and
Prekallikrein (or Fletcher factor).
➢ Test Principle
The HemoStat aPTT-EL test is
performed by adding aPTT reagent
containing a plasma activator and
phospholipids to the test specimen; the
phospholipids serve as a substitute for
platelets (factor 3). This mixture is
incubated for three minutes at 37
degrees Celsius for optimum activation.
The incubated mixture is then
recalcified with a calcium chloride
solution and clot formation is timed.
The aPTT-EL reagent can also
be used to perform quantitative factor
assays.
➢ Contents and Reagent Preparation
1. HemoStat aPTT-EL
*Chloroform extract of rabbit
brain
*Ellagic acid
2. CaCl2
*Calcium chloride (0.02 mol/L)
3 APTT Reagents:
> Activator - optional, if duha lang
gipangayo but tulo jud kabuok actually
> Phospholipid
> Calcium chloride
➢ Test Procedure
1. Perform samples and controls in
duplicates.
2. Prewarm the HemoStat CaCl 2
(0.02 M) to 37 degrees Celsius.
3. Pipette into 0.1 mL of
plasma/control prewarmed test
tube.
4. Incubate for 1-2 minutes at 37
degrees Celsius.
5. Add 0.1 mL aPTT-EL reagent.
6. Incubate for 3 minutes at 37
degrees Celsius.
7. Add 0.1 ml of prewarmed
CaCl2 . (3rd addition of CaCl2
start timer)
8. Start timer with addition of
CaCl2 .
9. Record time required for clot
formation.
Calculate the mean time of duplicate aPTT
determinations for each test plasma and report to
the nearest 0.1 seconds.
Normal Reference Range: 23.4 – 36.2 seconds
GENERAL NRR: 25 - 35 seconds
Transes: Febrienne Felise Molina
Module 1.4: Laboratory Diagnostic Test
HEMATOLOGY 2 LEC
MIXING STUDIES APTT + Fresh Plasma = C 10, 5, 2, 1,
APTT + Absorbed Plasma = C 5, 1,
- Done to identify factor deficiencies. APTT + Aged Serum = NC 5, 1,
SPECIFIC FACTOR.
- EXCEPT FACTOR 3 bc it comes from Circulating Anticoagulants
the tissue cells, it doesn’t synthesize by Actions:
the liver. 1. Inactivate and activated coagulation
factor
EXTRINSIC 2. Blocks interaction between coagulation
- PT: Prolonged factors and platelets
- APTT: Normal Examples:
Lupus inhibitor
INTRINSIC Non specific anticoagulant
- APTT: Prolonged Platelet neutralization procedure
- PT: Normal Xa-Va-Calcium-Platelet Phospholipid

COMMON DIC (Disseminated intravascular

- Both PT and APTT are Prolonged coagulation) - mass consumption of platelets
- Thrombin time: Prolonged - Hemostasis is out of control
- Wide spread clot formation = organ
NORMAL/FRESH PLASMA = CORRECTED ischemia
ALL
ABSORBED PLASMA = CORRECTED ALL Serious medical conditions:
EXCEPT 9, 10, 7 and 2 (Prothrombin group) Sepsis
AGED SERUM = CORRECTED ALL except 1, Malignancy
5, 8, 13 and 2 (Fibrinogen group except 2, Trauma
include 2 if walay fresh serum sa option) Obstetric complications
FRESH SERUM = CORRECTED ALL except Intravascular hemolysis
1, 5, 8, 13 and 2
= These conditions can lead to Procoagulants
Examples: - Trigger formation of new clots
PT - N; APTT - P = INTRINSIC 12, 11, 8, 9 - Depletion of platelets and CF
APTT + Fresh Plasma = C 12, 11, 8, 9 - Increased FDPS = Interferes with clot
APTT + Absorbed Plasma = C 12, 11, 8 formation
APTT + Aged Serum = NC 8
Factor 8 = deficient LAB findings
- Decreased plts and fibrinogen
PT - N; APTT - P = 12, 11, 9, 8 - PT and APTT - Prolonged
APTT + Fresh Plasma = C 12, 11, 9, 8 Many D-dimer
APTT + Absorbed Plasma = NC 9
APTT + Aged Serum = C 9
Factor 9

PT - P; APTT - P = 10, 5, 2, 1

Transes: Febrienne Felise Molina

ANSWER KEY (QUIZZES)
HEMATOLOGY 2 LEC&LAB
1. A newly admitted patient has the APPT- ABNORMAL
following coagulation results: ABSORBED PLASMA- CORRECTED
AGED SERUM- NOT CORRECTED
PT: 12.9 seconds (N = 12-14 seconds) - FACTOR VIII DEFICIENCY
aPTT: 84 seconds (N = 25-35 seconds)
Platelet Count: 200 x 109/L (N = 150-450 x 7. PT- NORMAL
109/L) APPT- ABNORMAL
ABSORBED PLASMA- NOT CORRECTED
A mixing study was performed due to the AGED SERUM- CORRECTED
abnormal aPTT test results. The mixing - FACTOR IX DEFICIENCY
study demonstrated the following:
aPTT was corrected by normal plasma, 8. PT- ABNORMAL
factor IX deficient plasma, but not by APPT- ABNORMAL
factor VIII deficient plasma. ABSORBED PLASMA- NOT CORRECTED
What factor assay should be performed AGED SERUM- NOT CORRECTED
next? - FACTOR II DEFICIENCY
- Factor VIII assay
9. PT- ABNORMAL
2. Which of the following tests could APPT- ABNORMAL
be used to determine whether an ABSORBED PLASMA- NOT CORRECTED
abnormal screening coagulation test AGED SERUM- CORRECTED
result (PT or aPTT) is caused by a - FACTOR X DEFICIENCY
factor deficiency or an inhibitor?
- Mixing studies 10. PT- ABNORMAL
APPT- ABNORMAL
3. PT- ABNORMAL ABSORBED PLASMA- CORRECTED
APPT- NORMAL AGED SERUM- NOT CORRECTED
ABSORBED PLASMA- NOT CORRECTED - FACTOR I DEFICIENCY
AGED SERUM- CORRECTED
- FACTOR VII DEFICIENCY 11. PT- NORMAL
APPT- ABNORMAL
4. PT- ABNORMAL ABSORBED PLASMA- CORRECTED
APPT- ABNORMAL AGED SERUM- CORRECTED
ABSORBED PLASMA- CORRECTED - FACTOR XI DEFICIENCY
AGED SERUM- NOT CORRECTED
- FACTOR V DEFICIENCY 12. IF A PATIENT HAS A PROLONGED PT
AND APPT BUT BOTH ARE
5. PT- NORMAL CORRECTED BY AGED PLASMA AND
APPT- ABNORMAL SERUM BUT NOT CORRECTED WITH
ABSORBED PLASMA- CORRECTED ADSORBED PLASMA, THE MOST
AGED SERUM- CORRECTED LIKELY DEFICIENCY IS FACTOR:
- FACTOR XII DEFICIENCY - X

6. PT- NORMAL

Transes: Febrienne Felise Molina

ANSWER KEY (QUIZZES)
HEMATOLOGY 2 LEC&LAB
13. BASED ON THE FOLLOWING DATA, 21. GIVE ATLEAST TWO ACTIONS OF
WHAT IS THE MOST LIKELY FACTOR CIRCULATIONG ANTICOAGULANTS
DEFICIENCY? - 1. Inactivated and activated
coagulation factor
PT - NORMAL - 2. Blocks interaction
APTT- PROLONGED between coagulation factors
APTT+NORMAL PLASMA - CORRECTED and platelets
APTT + ADSORBED PLASMA - NOT
CORRECTED
APTT + AGED SERUM - CORRECTED
- IX

14. THE COMBINATION OF PROLONGED

APTT AND A PROLONGED TEST WITH
THE MIXING STUDY PROCEDURES
INDICATES THE PRESENCE OF :
- CIRCULATING INHIBITOR

15. WHICH OF THE FOLLOWING

FACTORS IS NOT PRESENT IN BaSO4
ADSORBED PLASMA?
- II

16. WHAT ARE THE FACTORS THAT ARE

VITAMIN K DEPENDENT?
- 9, 10, 7, 2

17. WHAT ARE THE FACTORS THAT ARE

NOT PRESENT IN BASO4 ADSORBED
PLASMA?
- 9, 10, 7, 2

18. WHAT IS THE MEANING OF DIC?

- Disseminated Intravascular
Coagulation
- DISSEMINATED COAGULATION
DISORDER

19. OTHER NAME FOR MIXING STUDIES?

- Substitution Studies

20. EXPLAIN THE MECHANISM OF THE

DIC

Transes: Febrienne Felise Molina

ANSWER KEY (QUIZZES)
HEMATOLOGY 2 LEC&LAB
1. Modified True or False. Protein S
and Protein C are both Vitamin K
dependent factors.
- True
2. Modified True or False. PT detects
deficiency in factor I
- True
3. Modified True or False. PT is least
sensitive to factor IX.
- II
4. Modified True or False. PT is used to
monitor heparin therapy.
- warfarin
5. Modified True or False. PT is used
to monitor oral anticoagulant
therapy.
- True
6. Modified True or False. Shortened
PT is seen in Vitamin K deficiency.
- Prolonged
7. Modified True or False.
Thromboplastin reagent should be
stored at 2-3 degrees Celsius.
- 2-3 - 2-8
8. Modified True or False. It detects
deficiency in factor VII.
- True
9. Preparation of Specimen for PT:
11. Modified True or False. PT is the
plasma bleeding time obtained
when excessive thromboplastin and
optimum calcium are added to
citrated plasma under standardized
conditions.
- clotting
12. Modified True or False. PT detects
deficiency in factor II.
- True
13. Modified True or False. Prolonged
PT is seen in DIC.
- True
14. Modified True or False. PT is
insensitive to factor II.
- IX
15. Modified True or False. PT is a test
of the intrinsic pathway of clotting.
- extrinsic
16. Modified True or False. Prolongation
of PT indicates both acquired or
congenital disorders.
- either

● Prepare a two-way needle

for ETS.
● Collect venous blood in a
citrated tube.
● Centrifuge at 1,500 rpm for
five minutes.
● Separate the plasma.
● Placing plasma into a dry
tube.
10. Modified True or False. Factors II, V,
VII, and X are enhanced by oral
anticoagulants
- inhibited

Transes: Febrienne Felise Molina

ANSWER KEY (QUIZZES)
HEMATOLOGY 2 LEC&LAB
1. Modified True or False. Using the
Copley-Lalitch's method, you should
start timing after making a
puncture to a depth of 3 mm.
- 6
2. What bleeding time method uses
sphygmomanometer?
- Ivy’s method
3. Modified True or False. Using the
Duke's method, the earlobe or the
3rd or 4th finger can be used.
- True
4. Modified True or False. Using the
Ivy's method, you should start
timing after making the puncture.
- True
5. What is the normal value using
Duke's Method? *according to the
laboratory manual
- 2-4 minutes
6. Modified True or False. Using the
Capillary Tube's method, a
heparinized capillary tube should be
used.
- non-heparinized
7. What is the normal value using
Copley-Lalitch's Method? *according
to the laboratory manual
- < 3 minutes
8. What is the normal value using
Whole Blood's Method? *according to
the laboratory manual
- 7 - 15 minutes
9. What measures the ability of small
blood vessels to control free flow of
blood after injury?
- Bleeding time
10. What is the other name for the
Capillary Tube method used to
measure clotting time?
- Dale and Laidlaws Method
11. What is the normal value using Ivy's
Method? *according to the
laboratory manual
- 1-7 minutes
12. Modified True or False. Using the
Copley-Lalitch's method, immerse
the punctured finger in sterile
physiologic saline solution warmed
at 37 degrees Celsius until the
bleeding stops.
- True
13. Modified True or False. Using the
Ivy's method, make three successive
punctures in the form of a square
to a depth of 2 – 3 mm using a
disposable lancet.
- triangle
14. What is the other name for the
Whole Blood method used to
measure clotting time?
- Lee and White Method
15. Modified True or False. Using the
Duke's method, blot the blood using
filter paper every 60 seconds.
- 30
16. Modified True or False. When using
sphygmomanometer cuff to
measure bleeding time, it should be
inflated at 40 mmHg and
maintained during the entire
procedure.
- True
17. What serves as a measure of the
function of platelets as well as the
integrity of the blood vessel wall?
- Bleeding Time
18. Modified True or False. Do not allow
the filter paper to touch the wound
as this will prolong the bleeding
time.
- hasten
19. What is the normal value using Drop
or Slide's Method (capillary blood
method)? *according to the
laboratory manual
- 2-4mins
Transes: Febrienne Felise Molina
ANSWER KEY (QUIZZES)
HEMATOLOGY 2 LEC&LAB
20. What measures the period required
for blood to clot or solidify after it
has been extracted from the body?
- Clotting time

Transes: Febrienne Felise Molina

ANSWER KEY (QUIZZES)
HEMATOLOGY 2 LEC&LAB
1. Modified True or False. In
performing HemoStat aPTT-EL test,
start the timer after the addition of
calcium chloride.
- T
2. Modified True or False. The aPTT-EL
reagent cannot be used in
performing quantitative factor
assays.
- can
3. Modified True or False. In
performing HemoStat aPTT-EL test,
HemoStat CaCl2 should be
prewarmed to 30 degrees Celsius.
- 37
4. Modified True or False. HemoStat
aPTT-EL consists of the chloroform
extract of rabbit brain and ellagic
acid.
- T
5. Modified True or False. In
performing HemoStat aPTT-EL test,
only 0.1 mL aPTT-EL reagent is
added.
- T
6. Modified True or False. APTT is
principally used to detect
deficiencies in stage 2 of the
coagulation mechanism.
- 1
7. Modified True or False. In
performing HemoStat aPTT-EL test,
the normal value ranges from 23.4 –
36.3 seconds
- 36.2
8. Modified True or False. APTT cannot
detect deficiency in factor XIII.
- T
9. Modified True or False. In
performing HemoStat aPTT-EL test,
samples and controls should be
performed in triplicates.
- duplicates
10. Modified True or False. APTT is a
routine test for screening
coagulation disorders in the
extrinsic pathway.
- intrinsic
11. Modified True or False. APTT
monitors coumadin therapy
- heparin
12. Modified True or False. In
performing HemoStat aPTT-EL test,
only 0.1 ml of unwarmed CaCl2 is
added.
- prewarmed
13. Modified True or False. APTT
detects deficiency in Fletcher
factor.
- T
14. Modified True or False. APTT
monitors coumarin therapy.
- heparin
15. Modified True or False. APTT can
detect deficiency in platelet.
- cannot
16. Modified True or False. In
performing HemoStat aPTT-EL test,
the incubated mixture is then
recalcified with a calcium chloride
solution.
- T
17. Modified True or False.
Platelet-Rich Plasma is used during
APTT testing.
- Platelet-Poor Plasma
18. Modified True or False. Ammonium
citrate is preferred as an
anticoagulant.
- sodium
19. Modified True or False. APTT should
be performed within 1 hour.
- 2 hours
20. Modified True or False. In
performing HemoStat aPTT-EL test,
phospholipids serve as a substitute
for thrombocytes.
Transes: Febrienne Felise Molina
ANSWER KEY (QUIZZES)
HEMATOLOGY 2 LEC&LAB
- T
21. Modified True or False. APTT is used
to detect the presence of
circulating anticoagulants.
- T
22. Modified True or False. APTT
detects deficiency in Prekallikrein.
- T
23. Modified True or False. APTT is used
to detect the presence of
circulating inhibitors.
- T
24. Modified True or False. APTT
monitors warfarin therapy.
- heparin
25. Modified True or False. In
performing HemoStat aPTT-EL test,
the mixture is incubated for five
minutes at 37 degrees Celsius.
- three

Transes: Febrienne Felise Molina

ANSWER KEY (QUIZZES)
HEMATOLOGY 2 LEC&LAB
1. It detects the presence of fibrin
monomers in the plasma.
- Protamine sulfate test
2. The test reagent in APTT contains
which of the following substance(s)?
1.Citrated plasma
2.Calcium ions
3.Tissue thromboplastin
4.Phospholipids
- 2,4
3. Explain the mechanism of
fibrinolysis.
4. A standard 4.5-mL blue-top tube
filled with 3.0 mL of blood was
submitted to the laboratory for PT
and APTT tests. The sample is from
a patient undergoing surgery the
following morning for a
tonsillectomy. Which of the
following is the necessary course of
action by the technologist?
- Reject the sample and
request a new sample
5. IDENTIFY: COMPLETE ANSWER Russel
Viper test uses the venom from the
snake of _____________.
- Vipera russeli
6. Which coagulation test(s) would be
abnormal in a vitamin K–deficient
patient?
- PT and APTT
7. Which protein is the primary
inhibitor of the fibrinolytic system?
- α2-Antiplasmin
8. Which of the following statements
are true of the fibrinolytic system?
Plasmin digests fi brin and fi brinogen
The active enzyme of the system is plasmin
Inactive plasminogen circulates in the
plasma until an injury occurs
All of the above
None of the above
9. A content of the platelet reagent
that prevents platelet aggregation.
- Sodium citrate
10. Which of the following is correct
regarding the international
normalized ratio (INR)?
- standardizes PT results
11. IDENTIFY: A specific fragment
generated from two cross-linked
fibrin molecules after a clot has
formed.
- D-dimer
12. A modification of which procedure
can be used to measure fibrinogen?
- Thrombin time
13. IDENTIFY: The end result of
thrombin time is ___________.
- Clot formation
14. Using the Reese-ecker method, the
platelets will appear
________________ under the
microscope.
- Bluish
15. Which ratio of
anticoagulant-to-blood is correct
for coagulation procedures?
- 1:9
16. IDENTIFY: COMPLETE ANSWER
Reptilase time uses the enzyme
found in the venom of
_______________.
- Bothrops atrox
17. Which of the following is not a
plasminogen activator?
tPA
uPA
Kallikrein
XIIa
All of the above
None of the above
18. Which of the clotting factors will be
inhibited in cases of coumadin
therapy?
- Stuart-Prower factor
Transes: Febrienne Felise Molina
ANSWER KEY (QUIZZES)
HEMATOLOGY 2 LEC&LAB
19. A platelet count estimate of
165,946/uL is reported as:
- Low normal
20. Inhibitor of fibrinolysis that
prevents the binding and activation
of plasminogen.
- TAFI
21. Which of the following is referred
to as an endogenous activator of
plasminogen?
- Tissue plasminogen
activator
22. IDENTIFY: The key enzyme in
fibrinolysis that destroys the fibrin.
- Plasmin
23. IDENTIFY: Sample of choice for PT
and APTT.
- Platelet poor plasma
24. Explain the action and the
importance of warfarin and heparin
anticoagulants in patient's therapy.
25. IDENTIFY: The end result to be
observed in Hirschboek or castor oil
method is the _____________.
- Dimpling
26. Which results would be expected
for the prothrombin time (PT) and
activated partial thromboplastin
time (APTT) in a patient with
polycythemia?
- Both prolonged
27. Which of the following initiates in
vivocoagulation by activation of
factor VII?
- Tissue factor
28. The anticoagulant of choice for
most routinecoagulation studies is?
- Sodium citrate
29. All of the following are tests to
evaluate the coagulation cascade
except one:
Stypven test
PT
Clotting time
Thrombin time
All of the above
None of the above
30. What reagents are used in the PT
test?
- thromboplastin and calcium
31. Which of the following clotting
factors are measured by the APTT
test?
- XII, XI, IX, VIII, X, V, II, I
32. IDENTIFY: Sample of choice for
evaluating the platelet's ability to
aggregate to each other.
- Platelet rich plasma
33. A patient with a severe decrease in
factor X activity would demonstrate
normal ____________________.
- Bleeding time
34. In primary fibrinolysis, the
fibrinolytic activity results in
response to:
- Spontaneous activation of
fibrinolysis
35. Heparin inhibits the clotting of
blood by neutralizing the effect of
____________________.
- thrombin
36. IDENTIFY: The inhibitor of free
plasmin in fibrinolysis.
- A2-antiplasmin
37. Which statement about the
fibrinogen/fibrin degradation
product test is correct?
- It detects late degradation
products (D and E
38. It refers to the test that involves
the counting of the formed
petechiae in the patient's arm.
- Capillary Fragility
(Resistance) Test
Transes: Febrienne Felise Molina
ANSWER KEY (QUIZZES)
HEMATOLOGY 2 LEC&LAB
1. NORMAL : ADP, COLLAGEN,
EPINEPHRINE
ABNORMAL : RISTOCETIN
- BERNARD- SOULIER AND VWD
2. ABNORMAL : ADP, COLLAGEN,
EPINEPHRINEABNORMAL :
RISTOCETIN
NORMAL : RISTOCETIN
- GLANZMANN'S
THROMBASTENIA
3. LACKS GLYCOPROTEIN IIB/IIA
- GLANZMANN'S
THROMBASTENIA
4. LACKS GLYCOPROTEIN IB
- BERNARD- SOULIER
5. ASSOCIATED WITH LOW PLATELET
COUNT AND PROLONGED BLEEDING
TIME
- BERNARD- SOULIER
6. ASSOCIATED WITH NORMAL
PLATELET COUNT AND PROLONGED
BLEEDING TIME
- GLANZMANN'S
THROMBASTENIA
7. IN THE PERIPERHAL BLOOD SMEAR ,
THE PLATELETS ARE SEEN AS "GIANT
PLATELETS
- BERNARD- SOULIER
8. CAN RESULT IN A PLATELET
DYSFUNCTION BY IRREVERSIBLE
INACTIVATED OF THE
CYCLOOXYGENASE ENZYME AND THE
RESULTANT DECREASE IN TAX2
FORMATION.
- ASPIRIN
9. SUPERFICIAL BLEEDING (EXAMPLE :
BRUISING, EPIXTASIS & PETECHIAE)
- PRIMARY HEMOSTASIS
10. ANATOMICAL ( DEEP TISSUE
BLEEDING)
- SECONDARY HEMOSTASIS
11. LAB TEST TO MEASURE PLATELET
FUNCTION
- PLATELET AGGREGATION
12. THE FOLLOWING ARE AGONIST IN
PLATELET AGGREGATION TEST
EXCEPT:
- DDVAP
13. TRUE OR FALSE : THE GREATER THE
PLATELET AGGREGATION, THE
HIGHER THE OPTICAL DENSITY, THE
GREATER THE AMOUNT OF LIGHT
PASSING
- FALSE
14. TRUE OR FALSE : THE GREATER THE
PLATELET AGGREGATION, THE
LOWER THE OPTICAL DENSITY, THE
LESSER THE AMOUNT OF LIGHT
PASSING
- FALSE
15. TRUE OR FALSE : THE GREATER THE
PLATELET AGGREGATION, THE
LOWER THE OPTICAL DENSITY, THE
GREATER THE AMOUNT OF LIGHT
PASSING
- TRUE
16. TRUE OR FALSE: RIPA IS NORMAL IN
BSS AND VWD
- False
17. TRUE OR FALSE: RIPA IS NORMAL IN
GLANZMAN THROMBASTENIA.
- True
18. PURPLISH RED PINPOINT
HEMORRHAGIC SPOTS (<3mm) IN
THE SKIN CAUSED BY LOSS OF
CAPILLARY ABILITY TO WITHSTAND
NORMAL BLOOD PRESSURE AND
TRAUMA
- PETECHIAE
19. FORM OF PURPURA ( >3mm) IN
WHICH BLOOD ESCAPES INTO LARGE
AREAS OF THE SKIN AND MUCOUS
MEMBRANES, BUT NOT INTO DEEP
TISSUES
- ECCHYMOSIS
20. ALPHA GRANULE DEFICIENCY
- GRAY PLATELET SYNDROME
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ANSWER KEY (QUIZZES)
HEMATOLOGY 2 LEC&LAB

21. DIFFERENTIATE BERNARD SOULIER

AND GLANZMANN'S
THROMBASTHENIA
22. EXPLAIN THE PLATELET
AGGREGATION TEST

Transes: Febrienne Felise Molina

ANSWER KEY: QUIZZES LEC&LAB
HEMATOLOGY2
1. Fibrinogen interacts with which of factors in extrinsic, intrinsic and
the following receptors? common pathway
- GB IIb/IIIa - factor 3

2. What is adhesion? 12. Correct order of coagulation

- interaction between platelets and factors:
endothelial surface - prothrombin- thrombin - fibrinogen
- fibrin
3. Which of the following is involved in
the final phase of blood clotting? 13. The Antihemophilic Factor
- formation of fibrin - VIII

4. Calcium ions were the __________
substance discovered to be involved
in the process of blood clotting.
- 4 th
5. Which of the following is the
correct sequence of events leading
to blood clotting?
- vasoconstriction, platelet
aggregation, coagulation
6. Which drug inhibits production of
thromboxane A2, a signal for
platelet platelet aggregation?
- aspirin
14. It is the earliest recognizable stage
of megakaryopoiesis
- megakaryoblast
15. What is secondary hemostasis?
- formation of the fibrin clot
16. the initial aggregation of platelets
is caused by the release of (blank)
from the adhering platelet
- ADP
17. Each of the following is involved in
hemostasis EXCEPT:
- Red cells

7. Labile factors 18. Calcium and Vitamin K Independent

- V, VIII - Contact group

8. The Extrinsic system is monitored 19. Which of these forms an insoluble

by ? clot?
- Prothrombin Time - fibrin

9. platelet adheres to collagen with 20. The Intrinsic system is monitored by

the help of (blank) and (blank) ?
- Gp1b and VWF - APPT

10. What is primary hemostasis? 21. What is the normal value of

- formation of the platelet plug platelets?
- 150 - 400 x 10^9/L
11. helps in the thrombin formation
thru the activation of clotting

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ANSWER KEY: QUIZZES LEC&LAB
HEMATOLOGY2
22. A clot made mainly of __________
is the final product of secondary
hemostasis.
- Fibrin
23. A form of anticoagulation therapy:
- Heparin
24. The fibrin clot is initiated when
which factor is exposed to Tissue
Factor, which is normally confined
to the subendothelial space?
- VIIa
25. The Plasma Thromboplastin
Antecedent
- XI
26. What are the steps in platelet plug
formation?
- adhesion, activation, secretion,
aggregation
27. Why is D-dimer significant ?
- Major indicator that there is clot
formation occuring
28. Why is vasoconstriction important in
the hemostasis process?
- the muscles contract and it narrows
to reduce blood flow and allows
platelets to stick to each other and
the collagen
29. life span of platelets
- 7 - 10 days
30. overall function of the platelets
- maintains vascular integrity
- formation of platelet plug
- stabilize the plug by activating the
fibrin or clot formation
31. What converts fibrinogen to fibrin?
- Thrombin
32. What is aggregation?
- interaction between platelets
33. Clots form to stop the “leakage” of
blood from a damaged vessel. After
the damaged vessel has healed, the
clot is no longer needed and goes
through a process of dissolution.
Which of the following is involved in
the dissolution of a clot?
- plasmin
34. Cyclooxygenase is essential in the
formation of
- all of the above
35. Present in plasma but absent in in
adsorbed plasma
- Prothrombin group
36. 2 growth factors involved in
megakaryopoiesis
- CFU-Meg
- thrombopoietin
37. Calcium dependent and Vitamin K
Independent
- Fibrinogen group
38. A series of complex processes by
which the body spontaneously stops
bleeding and maintains blood on its
fluid state within the blood vessel.
- homeostasis
- hemostasis
39. Which of the following is the
platelet receptor needed for
platelet adhesion?
- GP IIb/IIIa
40. Which of the following is the
platelet receptor needed for
platelet adhesion?
- GP Ib
Transes: Febrienne Felise Molina
ANSWER KEY: QUIZZES LEC&LAB
HEMATOLOGY2

41. Completely consumed during

coagulation
- Fibrinogen group

Transes: Febrienne Felise Molina

MODULE 2.1: Thrombin Time
HEMATOLOGY LAB
DESCRIBING DEFECTS OF PLASMA
CLOTTING FACTORS AND
LABORATORY TESTS FOR BLEEDING
DISORDERS
Thrombin Time
- Is used to measure the availability of
functional fibrinogen. It is prolonged in
cases of low levels of fibrinogen, when
fibrinogen function is impaired, and
there is the presence of heparin,
fibrin/fibrinogen products, and
thrombolytic agents like streptokinase.
- Actual goal is to measure the final step
of coagulation
Reptilase time - same function as thrombin time
but not affected by heparin
Materials
● Two- way needle
● Wassermann tube (2)
● Citrated evacuated tube
● 37 degrees Celsius water bath
● Centrifuge
● aPTT reagent
● 0.5 mL serological pipette
● Calcium chloride
● 0.6 mL serological pipette
● Timer
● Tissue paper
Preparation of specimen for TT
1. Collect venous blood in a citrated tube.
2. Centrifuge at 1,500 rpm for five
minutes.
3. Separate the plasma by placing it into a
clean dry tube.
Normal Value: 8 - 14 seconds
HEMOSTAT THROMBIN TIME
Clot Retraction Time
Thrombin Reagent for the Manual and
Automated Determination Thrombin Time
- Fibrinogen (Factor I) is a soluble plasma
protein that is instrumental in the normal
coagulation process. Following trauma
or injury, fibrinogen is converted into an
insoluble fibrin clot by a two-stage
process. In stage one, thrombin cleaves
fibrinogen to form a fibrin that are
recognized as the end product in
thrombin clotting assays.
- fibrin - insoluble
★ Test Principle
- The thrombin time is a simple test to
screen for conditions that can interfere
with the conversion of fibrinogen into
fibrin. A low-potency thrombin is added
to undiluted plasma and clot formation
is timed.
★ Thrombin Time Reagent
● Thrombin time Reagent
(bovine)
● Lyophilisate for 1mL
★ Test Procedure
1. Perform samples and controls in
duplicates.
2. Pipette 0.2mL plasma/control
into a powdered test tube.
3. Incubate for 3 minutes at 37
degrees Celsius.
4. Add 0.1mL of the thrombin
time reagent .
5. Start timer with addition of
reagent.
6. Record the time required for
clot formation.
- When a normal blood clot contracts; it
expresses the serum. The degree of clot
retraction can be measured on the basis
Transes: Febrienne Felise Molina
MODULE 2.1: Thrombin Time
HEMATOLOGY LAB
of the amount of serum expressed.
Retraction is directly parallel to platelet
count.
Materials
● Castor oil
● Wassermann tube
● Sahli’s pipette
● Cotton
● 70% isopropyl alcohol
● Timer
● Blood lancet
Castor Oil or Hirschboeck Method
Procedure:
1. Pour castor oil into a Wassermann tube
up to about 1 inch from the rim.
2. Make a Skin puncture. Discard the first
drop of blood.
3. Use a Sahli’s pipette to aspirate blood up
to the 20 mm3 mark.
4. Expel the blood into the central surface
of the castor oil by touching the tip of
the pipette into the oil. Note the time.
5. Place the tube on a rack and observe for
the formation of dimpling or nipple-like
protrusion on the surface of the drop of
blood. Note the time. This indicates the
end point of the test.
NOTE: Clot retraction is reported as delayed
after one hour.
Other Methods of Clot Retraction Time
I. Quantitative
A. Stefanini or Test Tube Procedure:
1. Perform the venipuncture and
place 3-5mL of blood in a
chemically clean Wassermann
tube.
2. Close the tube with a cotton
plug and place it in a water bath
set at 37 degrees Celsius.
3. Observe the tube after one hour,
two hours, 16 hours, 18 hours,
and 24 hours.
4. The observation can be reported
either as follows: no retraction,
partial retraction, or complete
retraction.
NOTE: Occasionally, blood may adhere to the
wall of the tube. If this condition persists, rim
the clot with a stiff straight wire to detach the
clot from the wall of the tube.
Normal Value: Retraction begins within one
hour and usually ends within 18-24 hours.
B. McFarlane Method
Procedure:
1. Fill a calibrated centrifuge tube with
freshly drawn blood up to the 5mL
mark.
2. Place a glass rod into the tube and
immerse in the column of blood.
3. Fit a cork into place over the projecting
end of the glass rod.
4. Place the tube in a water bath set at 37
degrees Celsius and examine the blood
for evidence of coagulation at 5-10
minute intervals.
5. Remove the tube from the water bath
one hour after a firm cloth as formed. If
retraction has taken place, the clot will
shrink and attach to the glass rod.
6. Pull the glass rod and the clot will be
removed together with the rod.
7. Read the volume of the serum directly in
the graduated scale of the tube.
Normal Value: 44% - 67%
Transes: Febrienne Felise Molina
 LEARNING OUTCOME
Discuss the different quantitative and qualitative
platelet disorders and explain the principles and
procedures of the laboratory tests in primary
hemostasis.
 Patterns of Clinical Bleeding in
Disorders of Hemostasis
CHARACTERISTICS PRIMARY HEMOSTASIS SECONDARY HEMOSTASIS
Onset Spontaneous, immediate after Delayed after trauma
trauma
Sites Skin, mucous membranes Deep tissues
Form Petechiae, ecchymosis Hematomas
Mucous membranes Common (nasal, oral, Less common
gastrointestinal, genitourinary)
Other sites Rare Joint, muscle, central nervous
system, retroperitoneal
Clinical examples Thrombocytopenia, platelet defects, Factor deficiency, liver disease,
vWF defects, scurvy acquired inhibitors
 PLATELET DISORDERS
A. Qualitative Platelet Disorders
1. Bernard-Soulier Syndrome
2. Glanzmann’s Thrombasthenia
3. Von Willebrand Disease
4. Storage Pool Defects
B. Quantitative Platelet Disorders
1. Thrombocytopenia
2. Thrombocytosis
 Bernard-Soulier Syndrome
• Problem in platelet adhesion
• Inherited disorder of the platelet GPIb/IX/V complex characterized by
thrombocytopenia, giant platelets (>20 um) and a failure of the
platelets to bind GPIb ligand.
• Features:
• Thrombocytopenia
 Glanzmann’s Thrombasthenia
• Problem in platelet aggregation
• Absence of deficiency of membrane GPIIb/IIIa complex
• In 1918, Edward Glanzmann, 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:
Bleeding is the most common from mucosal surfaces
Facial petechiae abd subconjugal haemorrhage in
infants
 Differences between Bernard-Soulier
and Glanzmann’s Thrombasthenia
LABORATORY TESTs BERNARD-SOULIER GLANZMANN’S
SYNDROME THROMBASTHENIA
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
 Von Willebrand Disease
• Problem in platelet adhesion
• Associated with either quantitative deficiency (type 1 and type 3) or
qualitative abnormalities of Vwf.
• In 1926, Eric von Willebrand described a bleeding disorder in 24-66
members of a family from the Aland islands.
• Von Willebrand disease: Treatment
DDAVP (1-desamino-8-D-arginine vasopressin)
Humate P – contains intact Vwf
Cryoprecipitate – used in unresponsive DDAVP
Classification of von
Willebrand Disease
Platelet Aggregation Test Bernard-Soulier Von Willebrand Disease Glanzmann’s
Syndrome Thrombasthenia

Epinephrine N N A

Collagen N N A

ADP N N A

Ristocetin A A N
STORAGE POOL DEFECTS
• Problems in Platelet Secretion
• Conditions:
Gray Platelet Syndrome
Wiskott-Aldrich Syndrome
Hermansky-Pudlak Syndrome
Chediak-Higashi Anomaly
TAR syndrome
 Scott OTHERS
syndrome
• Impaired ability of platelets to • AML
promote coagulation
• Laboratory results: • Uremia
BT: Normal • Paraproteinemias
PT: Prolonged
• Drugs
Platelet aggregation: Normal
 Quantitative Platelet Disorders
THROMBOCYTOPENIA
Decreased platelet Production Platelet Destruction
• Aplastic anemia • Immune platelet destruction
• TAR syndrome  Post transfusion purpura
 Platelet refractoriness
• Acute leukemia  Immune Thrombocytopenic purpura
• Pernicious anemia  Secondary immune Thrombocytopenia
• Gaucher’s disease • Non-immune platelet destruction
• Sometimes after chemotherapy and  Disseminated Intravascular coagulation
radiation  HUS and TTP

Abnormal platelet distribution Dilutional Thrombocytopenia

• Splenomegaly - Seen after Multiple Transfusion.
Quantitative Platelet Disorders
THROMBOCYTOSIS
Increased platelet production
It could be reactive and autonomous.
Conditions:
• Polycythemia vera
• Essential thrombocytosis
• Idiopathic thrombocythemia
• CML
• Splenectomy
 Platelet Aggregation Studies

Aggregating agents Normal Response Abnormal response

A, C, E BSS Glanzmann
Vwd Thrombasthenia

Ristocetin Glanzmann BSS

thrombasthenia vWD
VASCULAR DISORDERS
General Laboratory results:
NORMAL: platelet count, platelet function test, coagulation test
ABNORMAL: Bleeding time, Rumple-Leede test
Hereditary Hemorrhagic Ehlers-Danlos Syndrome
Telangiectasia
- Cutis Hyperelastica
- Also called Osler Weber Rendu
- Characterized by hyperextensible
disease
joints, hyperplastic skin
Marfan’s Syndrome
- Characterized by skeletal
defects and arachnodactyly
Kasabach-Meritt Syndrome
- Also called Congenital
Hemangiomata
- Associated with tumors of
vessels that commonly swell
and bleed at the surface
Pseudoxanthoma elasticum
• Autosomal recessive disorder
• Characterized by elastorrhexia or progressive calcification or
fragmentation of elastic fibers in the skin, retina and cardiovascular
system.
Henoch-Schonlein Purpura Senile purpura
- Also called Allergic Purpura or -Characterized by the presence
Nonthrombocytopenic purpura
of bruised areas on the
- Vascular abnormalities forearms of elderly persons
- Characterized by gastrointestinal
haemorrhage and joint swelling
Drug-induced vascular
purpura
- Development of antibodies,
immune complexes, and
changes in vessel wall
permeability
Purpura Fulminants
- Characterized by hemorrhagic
manifestation that develop after a
streptococcal infections
Scurvy
- Also known as Vitamin C
deficiency
- Characterized by defects in the
synthesis of collagen and
hyaluronic acid
Paraproteinemia and
Amyloidosis
- Paraproteins present in MM and
Waldenstroms macroglobulinemia.
END…
 LEARNING OUTCOME
•Describe the common clotting factor
disorders and the mechanism of
Disseminated Intravascular
Coagulation.
 TOPIC CLASSIFICATION

Primary • Haemophilia
• von Willebrand
(Inherited) Disorder

Secondary • Vitamin K
Deficiency
(Acquired) • Hepatic Failure
 HAEMOPHILIA
A group of blood disorders in which thereis defect in clotting
factors
70% are X-linked recessive disorder. 30% spontaneous
mutation.
The bleeding patterns of haemophilia are similar.
Types :
 A:Deficiency in factor VIII (classic haemophilia)
 B: Deficiency in factor IX (Christmas disease)
 C: Deficiency in factor XI
 CLINICAL MANIFESTATION
Haemarthrosis (spontaneous bleeding in
muscle or joints - painful)
Illiapsoas bleeding
Joint Swelling
Easy bruising
Epistaxis
Haematuria
Intracranial hemorrhage
 HAEMOPHILIA A
a. Sex-linked disorder transmitted on the X chromosome by carrier women
to their sons
b. Accounts for 80% of the hemophilias; second most common hereditary
bleeding disorder
c. Clinical: Bleeding symptoms are proportional to the degree of the factor
deficiency. Spontaneous bleeding occurs often and is especially bad in joint
regions (hemarthrosis).
d. Laboratory: Prolonged aPTT only, factor VIII:C assay to confirm
e. Treatment: Cryoprecipitate and factor VIII concentrates are used; in
mild cases, DDAVP can be used to stimulate the release of VIII:C and vWF
from stored reserves.
 HEMOPHILIA B
• Christmas disease) deficiency
a. Sex-linked recessive trait
b. Accounts for 20% of the hemophilias; third most common
hereditary bleeding disorder
c. Clinical: Bleeding symptoms are similar to those seen in hemophilia
A.
d. Laboratory: Prolonged aPTT only; factor IX assay to confirm
e. Treatment: Fresh frozen plasma (FFP) or factor IX concentrates
 HEMOPHILIA C
• Factor XI (hemophilia C) deficiency
• a. Mainly seen in the Ashkenazi Jewish population
• b. Characterized by clinical bleeding that is asymptomatic
until surgery or trauma
• c. Laboratory: Prolonged aPTT only; factor XI assay to
confirm
 VON WILLEBRAND DISORDER
Most common hereditary deficiency caused abnormality in
von Willerbrand protein.
Functions on both primary & secondary
homestasis.
1. To act as bridge between subendothelial collagen and
platelets
2. Bind and protect factor VIII from rapid clearance then
delivers it to site of injury.
 TYPES
Type I (70%-80%) – Quantitative,
Partial decrease in quantity vWF
Mild clinical symptoms
Type 2 (15%-20%)– Qualitative,
Decrease affinity toward Factor VIII and platelet
Type 3 – Quantitative,
Absence of von Willerbrand factor
Severe clinical symptoms
Lack of response to Desmorphine (DDAVP)
 CLINICAL
MANIFESTATION
Asymptomatic
Mucous membranebleeding
Epistaxis
Cutaneus bleeding
Gingival bleeding
Menorrhagia
 LABORATORY
Full Blood Count – platelet normal
aPTT PROLONGE or normal
Factor VIII LOW or normal.
von Willerbrand Factor activity (ristocetin cofactor)
Ristocetin, an antibiotic that causes vWF to bind to platelet taken from
plasma.
In healthy people, platelet rapidly agglutinate.
von Willerbrand Factor antigen
Measure vWF protein and binding sites.
 TREATMENT
Desmopressin (DDAVP) – Treatment of choice for patients
with vWD types 1 and 2 .
Concentrate of von Willerbrand Factor (Humarate P)
when high levels of vWF are needed but cannot achieved
with DDAVP (type 3)
Contraceptive for menorrhagia
 VITAMIN K DEFICIENCY
3 main types of VK are
K-1, phylloquinone, derived from plants;
K-2, menaquinone, produced by the intestinal flora
K-3, menadione which is a synthetic, water-soluble form
used for treatment.
Required for synthesis of Plasma factor II, VII, IX,
and X
Hemorrhagic disease in infant that breastfeed
exclusively.
 HEPATIC FAILURE
Severe impairment of hepatic functions or
severe necrosis of hepatocytes in the
absence of pre-existing liver disease
Fatal for most affected children.
The mortality rate may reach 80-90% in the
absence of liver transplantation.
FIBRINOLYTIC DISORDER
 DISSEMINATED INTRAVASCULAR COAGULATION
(DIC)
Disorder characterized by coagulation pathway activation leading to diffuse
fibrin deposition in the microvasculature and consumption of coagulation
factors and platelets.

 Occurs as secondary complication of variety diseases.

Caused by the systemic activation of coagulation pathways, leading to

formation of thrombi throughout the microcirculation and widespread
thromboses. There is consumption of platelets and coagulation factors and
secondarily activation of fibrinolysis. As consequence, there is depletion of the
elements required for hemostasis ( consumptive coagulopathy)

 Initiated through the tissue factor pathway.

Disseminated Intravascular Coagulation

• A hemorrhagic disorder, in which there is an

uncontrolled inappropriate formation and lysis of fibrin
within the blood vessels.

• As fibrin is formed, several clotting proteins as well as

naturally occurring inhibitors and platelets are
consumed faster than they are synthesized.

A secondary group of symptoms that is always

triggered by a primary condition that does not
necessarily involve coagulation.
Disseminated Intravascular Coagulation
TYPES OF
DIC
A c u t e D IC
severe and often life- threatening
Rapid, and both fibrinogen and platelets may be
depleted

C h r o n ic D I C
may have mild manifestations of the disorder or be
recognizable only by laboratory data
Disseminated Intravascular Coagulation

CAUSES

Release or entry of tissue factors that act as coagulants

into the bloodstream

Extensive endothelial damage

Complications of pregnancy

Septicemia
Disseminated Intravascular Coagulation
Disseminated Intravascular Coagulation
C L I N I C A L M A N I F E S TAT I O N

Organ damage
• Skin, bone and bone
• marrow necrosis may be
seen
Disseminated Intravascular Coagulation
LAB FINDINGS
Test Normal Range DIC
D- dimer 0- 100 ng/ mL > 500 ng/ mL
Fibrinogen 200- 400 mg/ dL < 200 mgl dL
Platelet count 200- 400 × 109/ L <200- 400 × 109/ L

• Peripheral blood smear------ Fragmented RBC

• Platelet Count--------------------------- Decreased
• Fibrinogen ------------------------------- Decreased
• Thrombin time ------------------------- Prolonged
• Reptilase time---------------------------Prolonged
 Disseminated Intravascular
Coagulation
a. Triggering events include gram-negative septicemia, acute promyelocytic
leukemia (FAB M3), obstetrical complications, massive tissue damage.
b. Fibrinogen group factors (I, V, VIII, XIII) and platelets are consumed in clotting.
c. Laboratory
1) PT, aPTT, and thrombin time are prolonged.
2) Platelet count, antithrombin, and fibrinogen concentrations are decreased.
3) Fibrin and fibrinogen degradation products are present (abnormal D-dimer and FDP tests).
4) Schistocytes form when RBCs are fragmented by intravascular clots.
e. Clinical: A systemic thrombotic event causes multiple organ failure; systemic
lysis ultimately leads to severe hemorrhage.
f. Treatment: Treat the underlying condition with FFP, platelet transfusions,
antithrombin concentrates, and heparin to stop systemic clotting.
END…