HEMATOLOGY 2

PLATELET PRODUCTION, FUNCTION, AND STRUCTURE

MEGAKARYOPOIESIS / THROMBOPOIESIS / THROMBOCYTOPOIESIS

The maturation/differentiation of the cells of the megakaryocytic system has been divided into three stages.
Maturation sequence of megakaryoblast takes about 5 days. Platelets are produced directly from the
megakaryocyte cytoplasm. Platelets are fragment of megakaryocyte cytoplasm
A specific Hormone, Thrombopoietin (70,000daltons), is responsible for megakaryopoiesis
Thrombopoietin is produced by the liver
Megakaryocytic cells are unusual in that their nuclei are able to undergo multiple mitotic divisions without
cytoplasmic division, generating giant multinucleated or polyploidy cells. This is referred to as Endomitosis.
Endomitosis= a form of mitosis that lacks telophase and cytokinesis

(Maturation series From Barbara Brown)

Megakaryoblast
20-50 um
Blue cytoplasm
Multiple nucleoli
N:C ratio about 10:1
Fine chromatin
First recognizable stage

Promegakaryocyte
20-60 um
Less basophilic cytoplasm
Chromatin becomes coarse
Irregularly shaped nucleus, may show slight lobulation
N:C ratio of4:1 to 7:1

Granular megakaryocyte
30 to 90 um in diameter
Abundant, pinkish blue in color cytoplasm
Very fine and diffusely granular cytoplasm with irregular peripheral border
Multiple nuclei may be visible or the nucleus may show multi-lobulation
N:C ratio is 2:1 to 1:1

Mature megakaryocyte
40 to 120 um in diameter
Cytoplasm contains coarse clump of granules aggregating into little bundles, which bud off from the periphery to become
platelets
Nucleus is multilobulated
N:C ratio is 1:1

Platelet (thrombocyte)
1 to 4 um
Cytoplasm: light blue to purple, very granular
No nucleus

(Maturation from Steinenger)

Maturation stage Cytoplasmic Cytoplasmic tags Nuclear fragments Thrombocytes
granules visible
Megakaryoblast Absent Present Single nucleus, fine No
chromatin nucleus
Promegakaryocyte Few Present Double nucleus No
Megakaryocyte Numerous Usually absent Two or more nucleus No
Metamegakaryocte Aggregated absent Four or more nucleus Yes
Note : In differentiating the maturation stages of the megakaryocytic cells, emphasis should be placed on the cytoplasmic
appearance rather than the number of nuclei or the chromatic structure (Steinenger).

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RODAK’S ORDER OF MATURATION (from least to most mature)

Progenitors – resemble lymphocytes and cannot be distinguished by wright-stained light microscopy

Burst forming unit-Meg (BFU-Meg) = diploid and able to perform mitosis
Colony forming unit – Meg (CFU-Meg) = diploid and able to perform mitosis
Light density CFU-Meg (LD-CFU-Meg) = cannot divide but it retains DNA replication and cytoplasmic maturation for
endomitosis

Precursors
MK-1 / megakaryoblast = least mature precurosor
MK-II / promegakaryocyte
MK-III/ Megakaryocyte

FEATURES OF THE THREE TERMINAL MEGAKARYOCYTE DIFFERENTIATION STAGES

MK-I MK-II MK-III
% of precursors 20 25 55
Diameter 14 to 18um 15 to 40um 30 to 50um
Nucleus Round Indented Multilobed
Nucleoli 2 to 6 variable Not visible
Chromatin Homogenous Moderately condensed Deeply and variably condensed
N:C ratio 3:1 1:2 1:4
Mitosis Absent Absent Absent
Endomitosis Present Ends Absent
Cytoplasm Basophilic Basophilic and granular Azurophilic and granular
Alpha –granules Present Present Present
Dense granules Present Present Present
Demarcation system Present Present Present

Earlier influences on megakaryopoiesis include GM-CSF, IL-3, IL-6, IL-11, KIT ligand, and TPO.The stimulating hormonal factor
TPO (also known as MPL ligand), along with IL-11, controls the production and release of platelets. The liver is the main site of
production of TPO
Platelet Ultrastructure

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 PLATELETS / THROMBOCYTES

Diameter of 2 to4 um or 1 to 4 um with a volume approximately 6 to 7.5 fL and have a discoid shape.
With Wright’s stain, platelets have a light violet-purple granular appearance and look like “specks of dust”
Platelets are produced directly from the megakaryocyte cytoplasm
Each megakaryocyte produces between 2000 to 4000 platelets (Rodak’s), (1000-4000 Steinenger)
Average platelet counts are slightly higher in woman than in men
On a Wright-stained wedge-preparation blood film, platelets are distributed throughout the red blood cell monolayer at 8
to 20 cells per 100x field /OIF. (7 to 21 per OIF =RODAKS)
Maturation time: 5 days
Life span of 8 to 11 days or 9 to 12 days. At the end of their life span, platelets are phagocytized by the liver and spleen
and other tissues of the mononuclear phagocytic system.
2/3 of the platelets are on the circulation
1/3 of the platelets are found on the spleen
The platelet is composed about 60% protein, 30 % lipid, 8 % carbohydrate, various mineral, water and nucleotides.
Platelet is anatomically divided into four areas: 1. Peripheral zone, 2. Sol-gel zone, 3. Organelle zone, 4. Membranous
system

PLATELET STRUCTURE
Peripheral zone – composed of the membranes and is responsible for platelet adhesion and aggregation

Glycocalyx – outer surface, fuzzy coating, primarily composed of glycoproteins including coagulation factors V,
VIII and fibrinogen.

Plasma membrane- bilayer of asymmetrically distributed phospholipids

Submembranous area- where messages from external membrane are translated into chemical signals causing activation
and physical change in platelet
Glycocalyx
A Fluffy coat This glycocalyx is unique among the cellular components of the blood. It is composed of plasma proteins and
carbohydrate molecules that are related to the coagulation, complement, and fibrinolytic systems. The glycoprotein receptors of
the glycocalyx mediate the membrane contact reactions of platelet adherence, change of cellular shape, internal
contraction, and aggregation. (Turgeon)

Sol gel zone

Microfilaments –actin and myosin, which upon stimulation of platelet will interact to form actomyosin
(thrombosthenin), a contractile protein for platelet contraction

Microtubules – tubulin, maintains platelet disc shape

Directly beneath the cell membrane is a series of submembrane filaments and microtubules that form the cellular cytoskeleton.
In addition to providing the structure for maintaining the circulating discoid shape of the cell, the cytoskeleton also
maintains the position of the organelles. A secondary system of microfilaments is functional in internal organization and
secretion of blood coagulation products, such as fibrinogen.

Organelle zone

Mitochondria – for energy production /ATP Synthesis

Alpha granules
▪ 300 to 500 nm in diameter examples are Platelet factor 4, platelet derived growth factor, thrombospondin, vwf,
fibrinogen, fibronectin, factor V, etc.
▪ There are 50 to 80 alpha granules in each platelet

Dense granules / Delta granules

▪ 250 to 350 nm in diameter examples are Calcium, ADP, Pyrophosphate, ATP, Serotonin, and magnesium
▪ There are two to seven dense granules per platelet

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 Lysosomal type granules (lysosome)
▪ Contains Acid hydrolases, ACP, and Hydrolytic enzymes

Membranous system
Dense tubular system
▪ Derived from rough ER and sequesters or hold calcium for platelet activation process and prostaglandin
synthesis
▪ It is the “control center” for platelet activation

Surface connecting canalicular system

▪ An invagination of plasma membrane, act as a canal for the release of granule. Also involve in platelet
phagocytosis
▪ Allows for enhance interaction of the platelets with its environment
▪ It is this system that forms the invaginated, sponge-like portion of the cell that provides an expanded reactive
surface to which plasma clotting factors are selectively adsorbed

Circulating platelets These are platelets found in the circulation and ca be counted.
Circulating, resting platelets are biconvex, although the platelets in blood collected using the
anticoagulant ethylenediaminetetraacetic acid (EDTA, lavender closure tubes) tend to “round up.”
Reticulated platelets -Known as stress platelets
-Appear in compensation for thrombocytopenia
-They are larger than ordinary mature platelets
-Round up in EDTA, Cylindrical and beaded in citrated blood
-They carry free ribosomes and fragments of rough E.R
-They are prothrombic, and may be associated with increased risk of cardiovascular
disease
Proplatelet process Projections that resemble strings of beads, through or between the endothelial cells and into the
venous sinuses, releasing platelets from the tips of the processes into the circulation.

The proplatelet process sheds platelets, cells consisting of granular cytoplasm with a membrane
but no nuclear material, into the venous sinus of the bone marrow.
Thrombocytopoiesis A process where Platelets are released into the bone marrow through shedding
from megakaryocyte proplatelet processes

HEMOSTASIS
Hemostasis is derived from Greek meaning “The stoppage of blood flow”.
Process that retains the blood within the vascular system during periods of injury, localizes the reaction involved in the
site of injury, and repairs and re-establishes blood flow through the injured vessel.
The maintenance of circulatory hemostasis is achieved through the process of balancing bleeding (hemorrhage) and
clotting (thrombosis).

Excess hemostasis Clot extension and thrombosis

Inadequate hemostasis Poor wound healing

THE THREE HEMOSTATIC COMPONENTS

A. Extravascular Component
- Involves the tissues surrounding a vessel.
- The ability of the surrounding tissues to aid in hemostasis depends on the following factors:
a. Bulk or amount of the surrounding tissue
b. The type of tissue surrounding the injured vessel
c. The tone of surrounding tissue

B. Vascular Component
- Involves the blood vessels in which the blood flows.
a. Capillaries
b. Arteries
c. Veins

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 Substances Released from or Found on the Surface of Intact Endothelial Cells:
SUBSTANCE ACTION
Prostacyclin (PGI2) Inhibits Platelet activation
ATP Stimulates vasodilation
Thrombomodulin Endothelial surface receptor for thrombin
Binds and inactivates thrombin and enhances anticoagulant and fibrinolytic action of
protein C found in the plasma
Heparan Sulfate Coats the endothelial cell surface and weakly enhances activity of anti-thrombin III,
a plasma procoagulant
Tissue plasminogen activator (tPA) Converts plasminogen to plasmin, which plays important role in fibrinolysis
Von Willevbrand factor (vWF) Protein secreted by endothelium into subendothelium; required for platelet
adhesion to site of vessel injury
Storage site of vwf in endothelium- Weibel Palade
Storage site of vwf in platelets: alpha granules

C. Intravascular Component
- The key component in intravascular hemostasis are platelets and biochemical (procoagulants) in the plasma.

Hemostasis, the arresting of bleeding, depends on several components. The four major components are the vascular system,
platelets (thrombocytes), blood coagulation factors, and fibrinolysis and ultimate tissue repair. Three other, less important,
components are the complement and kinin systems as well as serine protease inhibitors. (Turgeon)

PRIMARY HEMOSTATIS

-Primary hemostasis is initiated by the exposure of platelets to the subendothelial connective tissue components blood vessels
(Collagen, microfilaments, basement membranes).

VASCULAR RESPONSE
Vascular injury to a large or medium-size artery or vein requires rapid surgical intervention to prevent exsanguination.
When a smaller vessel, such as an arteriole, venule, or capillary, is injured, contraction occurs to control bleeding. This
contraction of the blood vessel wall is called vasoconstriction.

Minimal interactions leading to platelet activation or clot formation occur between the circulating blood and intact
endothelial surfaces. However, disrupted endothelial cells release thromboplastic substances that can initiate coagulation.
Collagen, in particular, initiates contact activation of factor XII, thereby initiating blood coagulation.

Blood vessel endothelial functions are

Angiogenesis = synthesis of stromal components
Coagulation = vascular tone regulation
Inflammation = special metabolic functions (blood vessel will undergoes vasodilation)
Immune responses

Vascular integrity or the resistance to vessel disruption requires three essential factors. These factors are circulating
functional platelets, adrenocorticosteroids, and ascorbic acid. A Lack these factors produces fragility of the vessels,
which makes them prone to disruption

Endothelial Prothrombotic-Antithrombotic Balance

Prothrombic Antithrombic
Platelet activating factor Prostacyclin
Tissue factor Thrombomodulin
Vwf Tissue plasminogen activator
Plasminogen activator Urokinase
Inhibitor-1 Heparin like molecules
Other coagulation factors (e.g factor V)

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 Endothelial Vasoconstrictor-Vasodilator Balance
Constrictor Dilator
Endothelin-1 Prostacyclin
Angiotensin-2 Nitric oxide
Vasoconstrictor
Prostaglandins

PLATELET RESPONSE
Platelets have an average diameter of 2 to 4 um, with younger platelets being larger than older ones. In contrast to
megakaryocytes platelets have no nucleus. The cytoplasm is light blue, with evenly dispersed, fine red-purple granules.
An inactive or unstimulated platelet circulates as a thin, smooth-surfaced disc. This discoid shape is maintained by the
microtubular cytoskeleton beneath the cytoplasmic membrane.
Platelets are extremely sensitive cells and may respond to minimal stimulation by forming pseudopods that spontaneously
retract. Stronger stimulation causes platelets to become sticky without losing their discoid shape; however, changes in
shape to an irregular sphere with spiny pseudopods will occur with additional stimulation. This alteration in cellular shape
is triggered by an increase in the level of cytoplasmic calcium.
Agonists that lead to platelet activation are varied and include a nucleotide (ADP), lipids (thromboxane A2, platelet-
activating factor), a structural protein (collagen), and a proteolytic enzyme (thrombin).

Platelet Adhesion When vascular injury occurs, platelet come in contact with subendothelium (collagen, fibronectin).
vWf binds to glycoprotein Ib/IIb or the GP Ib/IX/V complex on platelet surface
If vascular injury exposes the endothelial surface and underlying collagen, platelets adhere to the
subendothelial collagen fibers, spread pseudopods along the surface, and clump together
(aggregate). Platelet adhesion to subendothelial connective tissues, especially collagen, occurs
within 1 to 2 minutes after a break in the endothelium.
Platelet Activation Morphological and functional change in plates
Cyclooxygenase (from the platelets) metabolizes arachidonic acid to form prostaglandin
enderoperoxides, which are converted to thromboxane A2 (a vasoconstrictor and a platelet
stimulator, causing platelet secretion and aggregation)
Aspirin inhibits cyclooxygenase pathway (remission after 7 to 10days)
Platelet secretion Following activation, platelet undergoes a shape change most probably caused by contraction of
microtubules
From a disk shape to spherical shape with the extrusion of numerous pseudopods
Platelet granules move to the center of the platelets and fuse with the open canalicular system
connected to the outside of the platelet; in this way the content of the granules are extruded to the
outside
Platelet Simultaneously with platelet release, platelet stimulating agents (collagen, ADP, epinephrine,
aggregation thrombin) bind to the platelets, causing then to adherence to one another
Platelet stimulating agents such as collagen, ADP, epinephrine, thrombin binds to platelets, causing
them to adhere to one another. Fibrinogen binds to GP IIb/ IIIa receptors on adjacent platelets and
joins them together in the presence of ionized calcium
Fibrinogen is necessary as cofactor for platelet aggregation
Bridges formed by fibrinogen in the presence of calcium produce a sticky surface on platelets. This
results in aggregation. If these aggregates are reinforced by fibrin, they are referred to as a
thrombus

Platelet Plug Consolidation and Stabilization

Fibrinogen, under the influence of small amounts of thrombin, provides the basis for this consolidation and stabilization. This
process involves the precipitation of polymerized fibrin around each platelet. The result is a fibrin clot that produces an
irreversible platelet plug.

Vascular injury →exposes sub-endothelium and vaso-constriction→ platelet adhesion→ platelet aggregation→
platelet plug formation → consolidation of platelets →fibrin stabilization

PLATELET ACTIVATION PATHWAY

G proteins Controls the cellular activation for all cells at the inner membrane surface
Eicosanoid Called as prostaglandin, cyclooxygenase, or thromboxane pathway
It is triggered by G proteins
Inositol triphosphate Diacylglycerol Second G protein dependent activation
(IP3-DAG pathway)

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SUMMARY OF MOST IMPORTANT SUBSTANCES SECRETED BY PLATELETS AND THEIR ROLE IN HEMOSTASIS
ROLE IN SUBSTANCE SOURCE FUNCTION
HEMOSTASIS
Promote coagulation HMWK Alpha granules Contact activation of intrinsic coagulation pathway
Fibrinogen Alpha granules Converted to fibrin clot formation
Factor V Alpha granules Cofactor in fibrin clot formation
FactorVIII:Vwf Alpha granules Assist platelet adhesion to subendothelial to
provide coagulation surface
Promote aggregation ADP Dense granules Promote platelet aggregation
Calcium Dense granules Promote platelet aggregation
Platelet factor 4 Alpha granules Promote platelet aggregation Pro
Promote Serotonin Dense granules Promotes vasoconstriction at injury site
vasoconstriction
Thromboxane A2 Membrane phospholipids Same
Promote vascular repair Platelet derived growth Alpha granules Promotes smooth muscle growth for vessel repair
factor
Beta thromboglobulin Alpha granules Chemotactic for fibroblasts to help in vessel repair
Other systems affected Plasminogen Alpha granules Precursor to plasmin, which induces clot lysis
Alpha 2 antiplasmin Alpha granules Plasmin inhibitor; inhibits clot lysis
C1 esterase inhibitor Alpha granules Complement system inhibitor

SECONDARY HEMOSTATIS

COAGULATION FACTORS / CLOTTING FACTORS

NUMERAL PREFERRED NAME SYNONYMS
I Fibrinogen ---
II Prothrombin Prethrombin
III Tissue factor Tissue thromboplastin
IV Calcium Ionized calcium
V Proaccelerin Labile factor
Accelerator globulin
VII Proconvertin Stable factor
Serum prothrombin conversion accelerator (SPCA),
Autoprothrombin I
VIII Antihemophilic factor (AHF) Antihemophilic factor A
Antihemophilic globulin (AHG)
Platelet cofactor I
IX Plasma thrombopastin component (PTC) Christmas factor
Antihemophilic factor B
Platelet cofactor II
X Stuart-Prower factor Stuart factor
Prower factor
Autoprothrombin III
XI Plasma thromboplastin antecedent Antihemophilic factor C
XII Hagemen factor Glass factor
Contact Factor
XIII Fibrin stabilizing factor Laki- Lorand factor
Fibrinase
Plasma transglutaminase
Fibrinoligase
HMWK Fitzgerald factor Contact activation cofactor
Williams factor
Flaujeac Factor
Pre Kallikrein Fletcher factor

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 Involved Coagulation Factors according to function
Zymogens/Enzyme precursors Factor II, VII, IX, X, XI, XII, PK
Serine Proteases Factor IIa, VIIa, IXa, Xa, XIa, XIIa, Kallikrein
Cofactor Factor V, VIII, III, HMWK

COAGULATION MECHANISM
Thought of as a complex series cascading reactions involving development of enzymes from their precursor (zymogens,
procoagulants, proenzymes)
Most of the substances necessary for coagulation are present in an inert form and must be converted to an activated
state.
As one enzyme is formed it the becomes available to convert the next zymogen to its activated enzyme (serine protease)
Factors have been given a Roman-numerals. They are numbered in the approximate order of their discovery
When a procoagulant becomes activated, a lower case “a” appears behind the numeral; for instance, activated factor VII
is VIIa. Both zymogens and cofactors become activated in the coagulation process
The initiation of the coagulation process may occur via one of two pathways: the extrinsic pathway and the intrinsic
pathway. Regardless of the initiating pathway, the two pathways converge into a final common pathway. The outcome of
this process is the conversion of circulating insoluble coagulation factors into a gelatinous fi brin clot with entrapped blood
cells, a blood clot. As repair of damaged tissue takes place, the clot is lysed and the particulate matter is removed by the
mononuclear phagocytic system.

STAGES
Stage 1 Generation of prothrombinase complex
Stage 2 Conversion of prothrombin to thrombin
Stage 3 Conversion of fibrinogen to fibrin

EXTRINSIC PATHWAY
initiated by the entry of tissue thromboplastin into the circulating blood. Tissue thromboplastin is derived from
phospholipoproteins and organelle membranes from disrupted tissue cells. These membrane lipoproteins, termed tissue
factors, are normally extrinsic to the circulation.
Factor VII binds to these phospholipids in the tissue cell membranes and is activated to factor VIIa, a potent enzyme
capable of activating factor X to Xa in the presence of ionized calcium.

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INTRINSIC PATHWAY
involves the contact activation factors prekallikrein, HMWK, factor XII, and factor XI. These factors interact on a surface to
activate factor IX to IXa. Factor IXa reacts with factor VIII, PF 3, and calcium to activate factor X to Xa. In the presence of
factor V, factor Xa activates prothrombin (factor II) to thrombin, which in turn converts fibrinogen to fibrin.
Strong negatively charged solids that can participate in the activation of factor XII include glass and kaolin in vitro as well
as elastin, collagen, platelet surfaces, kallikrein, plasmin, and high–molecular-weight kininogen in vivo. Collagen exposed
by blood vessel injury greatly influences the rate of reaction.

FINAL COMMON PATHWAY

Once factor X is activated to Xa, the extrinsic and intrinsic pathways enter a common pathway. Factor II, prothrombin, is
activated to thrombin (factor IIa), which normally circulates in the blood as an inactive factor.
Following the activation of factor Xa, it remains platelet bound and activates factor V. The complex of factors Xa and Va
on the platelet surface is formed near platelet-bound factor II molecules. In turn, the platelet-bound Xa/Va complex
cleaves factor II into thrombin (factor IIa).
Thrombin plays a major role in converting factor XIII to XIIIa and in converting fibrinogen to fibrin. Fibrin formation
occurs in three phases: proteolysis, polymerization, and stabilization
Initially, thrombin, a protease enzyme, cleaves fibrinogen, which results in a fibrin monomer, fibrinopeptide A, and
fibrinopeptide B fragments. In the second step, the fibrin monomers spontaneously polymerize end-to-end due to
hydrogen bonding. Finally, the fibrin monomers are linked covalently by factor XIIIa into fi brin polymers. These polymers
form a meshy network, and the final fibrin solution is converted to a gel when more than 25% of the fi brinogen is
converted to fibrin.
Factor XIII is converted to the active form, factor XIIIa, in two steps. In the first step, thrombin cleaves a peptide from
each of the two alpha chains of factor XIII with formation of an inactive intermediate form of factor XIII. In the second
step, calcium ions cause factor XIII to dissociate, forming factor XIIIa.

FUNCTIONS OF THROMBIN
Procoagulant ✓ induces platelet activation and aggregation
✓ activates cofactor VIII to VIIIa
✓ activates factor XIII to XIIIa
✓ converts prothrombin to thrombin via autocatalysis
Coagulation Inhibitor ✓ The coagulation inhibition activity displayed by thrombin is the binding of AT-III to inhibit
serine proteases and binding to thrombomodulin to activate protein C.
✓ Promotion of endothelial cell release of t-PA (Tissue plasminogen activator).
Tissue repair ✓ Thrombin mediates tissue repair by inducing cellular chemotaxis and stimulation of
proliferation of smooth muscle and endothelial cells.

COMMON CHARACTERISTICS OF COAGULATION FACTORS

Proteins that are clotting factors have four characteristics in
common. These characteristics are as follows:

1. A deficiency of the factor generally produces a bleeding tendency

disorder with the exception of factor XII, prekallikrein (Fletcher
factor), and high– molecular weight kininogen (HMWK; Fitzgerald
factor).
2. The physical and chemical characteristics of the factor are known.
3. The synthesis of the factor is independent of other proteins.
4. The factor can be assayed in the laboratory.

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 NOMENCLATURE FOR FACTOR VIII of the International Committee on Thrombosis and Hemostasis
VIII/vWF
➢ The entire molecule as it circulates in the plasma. Composed of VIII:C and VIII: vWF
VIII: vWF
➢ Portion of molecule responsible for binding to endothelium and supporting normal platelet adhesion and function.
Tested by bleeding time
VIII: C
➢ Portion of molecule acting in intrinsic system as cofactor to factor IXa (with Ca++) in the conversion of factor X to Xa.
Tested by APTT.
VIIIC:Ag
➢ Antigenic property of procoagulant portion as measured by immunologic monoclonal antibody techniques
VIIIR:Ag
➢ Factor VIII-related antigen, which is a property of the large vWF portion of the molecule and measured by immunologic
techniques of Laurel rocket or immunoradiometeric assay.
VIII: RCo
➢ Ristocetin (an antibiotic no longer used therapeutically) cofactor activity, which is factor VIII-related activity required
for aggregation of human platelets with ristocetin in in vitro aggregation studies.

Factor VIII can be subdivided into various functional components. The total molecule, consisting of both a high–molecular-
weight fraction and a low–molecular-weight fraction, is described by the nomenclature VIII/vWF. Factor VIII/vWF consists of
two major moieties. The high– molecular-weight moiety consists of the vWF, VIIIR:RCo, and VIIIR:Ag components. The low–
molecular-weight moiety consists of the VIII:C and VIIIC:Ag components.
Factor VIII:C has procoagulant activity as measured by clotting assay techniques.
Factor VIII/vWF multimers form ionic bonds with factor VIII:C and transport VIII:C in the circulation.
Factor VIIIC:Ag is a procoagulant antigen as measured by immunological techniques using antibodies for factor VIII:C.
Factor VIIIR:Ag is a related factor VIII antigen that has been identifi ed using immunological techniques employing
heterologous antibodies to VIII/vWF.
Factor VIIIR:RCo demonstrates ristocetin cofactor activity, which is required for the aggregation of human platelets
induced by the antibiotic ristocetin

ADDENDUM
Factor IX is a stable protein factor that is neither consumed during clotting nor destroyed by aging at 4°C for 2 weeks

Factor XIII, an acute-phase reactant, is consumed during the clotting process and is not found in serum. Factor VIII 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. In addition, factor VIII can be falsely decreased in the presence of lupus anticoagulant (LA).

Factor V is an extremely labile globulin protein. It deteriorates rapidly, having a half-life of 16 hours
A large quantity of thrombin is consumed during the process of converting fibrinogen to fibrin. A unit of thrombin will coagulate
1 mL of a standard fibrinogen solution in 15 seconds at 28°C.

Tissue thromboplastin is the term given to any non-plasma substance containing lipoprotein complex from tissues.
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 VII, a beta-globulin, is not an essential component of the intrinsic thromboplastin-generating mechanism. It is not
destroyed or consumed in clotting and is found in both plasma and serum, even in serum left at room temperature for up to 3
days. The action of factor VII is the activation of tissue thromboplastin and the acceleration of the production of thrombin from
prothrombin. This factor is reduced by vitamin K antagonists

SUBSTITUTION/ MIXING STUDIES

Fresh Plasma All coagulation factors are present
Aged Plasma Factors V and VIII are absent
Fresh Serum Factors I, V, VIII and XIII are absent, only ≤20% Factor II is detected
Aged serum Factors I, II, V, VIII, and XIII are absent
Adsorbed plasma Factors IX, X, VII, II are absent

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 Deficiency PT APTT TT SUBSTITUTION STUDIES
NORMAL ABSORBED AGED SERUM
PLASMA PLASMA
I Abn Abn Abn C C NC
II Abn Abn N C NC NC
V Abn Abn N C C NC
VII Abn N N C NC C
VIII N Abn N C C NC
IX N Abn N C NC C
X Abn Abn N C NC C
XI OR XII N Abn N C C C
*Abn= abnormal, N=normal

MUST KNOW IN ORDER TO ASSESS COAGULATION SUSBSTITUTION STUDIES

Factors according to pathway

Common pathway =

Intrinsic pathway =

Extrinsic pathway=

Factors/Pathway according to different coagulation test

PT =

APTT=

Reptilase and Thrombin time (TCT)=

Stypven time=

EXAMPLE CASES:
a. Fibrinogen deficiency
b. Factor IX deficiency
c. Factor VII deficiency
d. Factor XIII deficiency

1. PT abnormal, PTT normal, TCT normal =

2. PT abnormal, PTT abnormal, TCT abnormal =
3. PT normal, PTT abnormal, TCT normal =
4. PT normal, PTT normal, TCT normal =

MIXING STUDIES
a. Lupus anticoagulant / circulating anticoagulant
b. Fibrinogen deficiency
c. Factor II deficiency
d. Factor VII deficiency

1. PT Abnormal, PTT normal, Adsorbed plasma not corrected, Aged serum corrected, Fresh plasma corrected=
2. PT Abnormal, PTT Abnormal, Adsorbed plasma not corrected, Aged serum not corrected, Fresh plasma corrected =
3. PT Abnormal, PTT Abnormal, Adsorbed plasma corrected, Fresh serum not corrected, fresh plasma corrected =
4. PT Abnormal, PTT Abnormal, Fresh plasma not corrected, Fresh serum not corrected, Adsorbed plasma not corrected =

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 COAGULATION CASCADE

Fibrinogen group/thrombin Vitamin K dependent group Contact group

sensitive group Prothrombin Group

✓ Factor I, V, VIII, XIII ✓ Factor VII, IX, X, II ✓ Factor XII, XI, HMKW, PK
✓ Consumed during coagulation ✓ Requires vitamin K ✓ Not consumed during
✓ Absent in serum ✓ Not consumed during coagulation
✓ Not absorbed by barium sulfate coagulation ✓ Not absorbed by barium sulfate
or aluminum hydroxide ✓ Absorbed by barium sulfate or or aluminum hydroxide
✓ Increases in inflammation, aluminum hydroxide
pregnancy, stress and fear, oral ✓ Increases in pregnancy, and
contraceptives oral contraceptives

NOTE ☺ Vitamin K catalyzes an essential posttranslational modification of the prothrombin group proteins:
gamma-carboxylation of amino-terminal glutamic acids

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 INHIBITORS OF COAGULATION
Protein C Major inhibitor of blood coagulation. Activated protein C is a strong anticoagulant and
degrades factor Va and VIIIa and stimulates fibrinolysis by inactivating plasminogen
activator inhibitors
Protein S Servers as cofactor with protein C. 40 % - free form and active, 60% - bound to
complement c4 Binding protein and not active
Antithrombin C Major inhibitor of thrombin. Enhanced by heparin
Heparin cofactor II Also inhibits thrombin
Alpha 2 macroglobulin Forms complex with thrombin, kallikrein, and plasmin thus inhibiting their activities
Extrinsic pathway inhibitor Also called as lipoprotein associated coagulation inhibitor (LACI) , inhibits the VIIa-tissue
(EPI) factor complex
C1-inhibitor Principle inactivator of factor XIIa and plasma kallekrein
Alpha 1 antitrypsin Weak inhibitor of thrombin and factor Xa and Xia
Activated protein C inhibitor Inhibit the activity of protein C
Thrombin + thrombomodulin Modifies the action of thrombin to act more as inhibitor by inactivating protein C
(endothelial surface)

ASSAYS FOR DETERMINATION OF PROTEIN C

Antigenic Measures amount of material present
Does not measure function
Chromogenic Measures some but not all functions
Clotting Measures all functions of the protein C molecule

NOTE : PROTEIN C AND PROTEIN S ARE ALSO VITAMIN K DEPENDENT

FIBRINOLYSIS
Primary purpose is to digest fibrin clots as they are formed in order to keep vascular system free of deposited fibrin and
fibrin clots
Occurs when plasminogen is converted to plasmin, which dissolves the fibrin or fibrinogen into smaller fragments termed
FDP (Fibrin degradation product) or FSP (Fibrin split product)
Plasmin – a serine protease that systematically digest fibrin polymer by the hydrolysis of arginine-related and lysine
related peptide bonds
Free plasmin- capable of digesting plasma fibrinogen, factor V, Factor VIII, and fibronectin.

Plasminogen is a 92,000 Dalton plasma zymogen produced by the liver. It is a single-chain protein possessing five
glycosylated loops termed kringles. Kringles enable plasminogen, along with activators TPA and UPA, to bind the lysine
moieties on the fibrin molecule during the polymerization process. This fibrin-binding step is essential to fibrinolysis.

Fibrin-bound plasminogen becomes converted into a two-chain active plasmin molecule when cleaved between
arginine at position 561 and valine at position 562 by neighboring fibrin-bound TPA or UPA.

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 Degradation of Fibrinogen and Non Degradation of Crosslinked Fibrin (stabilized clot)
crosslinked fibrin

Primary Fibrinolysis Secondary Fibrinolysis

Excessive amount of plasminogen activator from DIC: uncontrolled, Inappropriate formation of fibrin
damaged cells or malignant cells within the blood vessels
Examples are from malignancy like prostatic Examples are seen in infection and HTR
carcinoma (+) D DIMER
NO D-DIMER (+) FIBRIN MONOMER
NO FIBRIN MONOMER (+) FIBRIN POLYMER
NO FIBRIN POLYMER Decrease fibrinogen
Decrease Fibrinogen Decreased platelet count
Normal platelet count

FIBRIN DEGRADATION PRODUCTS

Fragment X is described as the central E domain with the two D domains (D-E-D), minus some peptides cleaved by plasmin
Fragment Y is the E domain after cleavage of one D domain (D-E).
The D-D fragment, called D-dimer, is composed of two D domains from separate fibrin molecules (not fibrinogen) crosslinked
by the action of factor XIIIa

PLASMINOGEN ACTIVATORS INHIBITORS OF FIBRINOLYSIS

1. Intrinsic factors= factor XIIa, Kallikrein, and HMWK 1. Alpha 2 antiplasmin – primary inhibitor of plasmin
2. Tissue type urokinase- secreted by the kidney, 2. Alpha 2 macroglobulin
activates plasminogen 3. Thrombospondin – released by the platelets, inhibits
3. Therapeutic activators such as treatment for activation of fibrin-bound plasminogen
thromboemboli 4. Plasminogen activator inhibitor 1 (PAI-1) and
4. Streptokinase, urokinase, Tissue like PA Plasminogen activator inhibitor 2 (PAI-2)
5. Tissue plasminogen activator 5. Alpha 1 antitrypsin

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 SPECIMEN COLLECTION AND PROCESSING FOR HEMOSTATIS TESTING

Methods of collection
Two syringe method
Evacuated tube technique
These two methods are preferred because it requires that a few volume of blood should be discarded first then the next
volume of blood is collected, this is done to prevent possible contamination of tissue fluid that contains thromboplastin
which somehow alters results obtain from clotting test

Special considerations and Information

Anticoagulant- 0.105 to 0.109M (3.2%) buffered Sodium citrate
Ratio of Blood to anticoagulant – (9:1)
Citrate is the anticoagulant of choice for platelet function testing except for the glass bead retention test, which
require heparin.
Specimen processing for hemostasis testing should be done at 37 ‘C
Labile factors (V and VIII) deteriorate at RT and increased pH
Factors VII and XI prematurely activates when place at a cold temperature (4’C)

Aspirin and other NSAID’s inhibit cyclooxygenase pathway

o Platelet function test: 1week abstinence from the following drugs
o Bleeding time test: 24 hours aspirin intake is prohibited

Platelet poor plasma (PPP)

Centrifuged at 2000 x g for 10 minutes (Steinenger),
Centrifuged at 1500 x g for 15 minutes (Rodak’s)
centrifuged at 2500 x g for 20 minutes (Turgeon)

Platelet rich Plasma (PRP)

Centrifuged at 60-100 x g for 10 minutes (steinenger),
Centrifuged by 50 x g for 30minutes (Rodak’s)

Coagulation specimen should be centrifuged within 1 hour of obtaining the sample

Specimen processing for PT is valid within 24 hours
Specimen processing for APTT is valid for 4 hours
The PT and APTT, TCT are prolonged when Fibrinogen level is ≤100mg/dl (Rodak)
The size of glass test tube used for PT and APTT is 12 x75mm (Steinenger), or 13x 100mm (Brown)

PRP Can be obtained by centrifuging WB collected from 3.2% Sodium Citrate at 50g x for 30
(Platelet Rich Plasma) minutes
It contains 200,000 to 300,000 /ul platelets (200 to 300 x109/L platelets)
It is used for platelet aggregometry or platelet function test
PPP Can be obtained by centrifuging WB collected from 3.2% Sodium Citrate at 1500g x for 15
(Platelet Poor Plasma) minutes
It contains less than 10,000/ul platelets
It is used for clot-based coagulation test (E.g PT and APTT)

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 FACTORS THAT INTERFERE WITH THE VALIDITY OF CLOT BASED TEST RESULTS
Problem Solution
Blood collection volume less than specified PT falsely prolonged; recollect specimen
minimum
Hematocrit >55% (polycythemia) Adjust anticoagulant volume using formula and recollect specimen using new
anticoagulant volume
Clot in specimen All results are affected unpredictably; recollect specimen
Visible hemolysis PT falsely shortened; recollect specimen
Icterus or lipemia Measure PT using a mechanical coagulometer.
Heparin therapy Use reagent known to be insensitive to heparin or one that includes a heparin
neutralizer such as polybrene
Lupus anticoagulant PT result is invalid; use chromogenic factor X assay instead of PT.
Incorrect calibration, incorrect dilution of Correct analytical error and repeat test.
reagents

LABORATORY TESTS FOR PRIMARY HEMOSTASIS

1.PLATELET COUNT

Reference value= 150-400 x10 9 /L, 150 to 450 x10 9 (Rodak’s)

❖ Thrombocytosis: PV, idiopathic thrombocythemia, CML, splenectomy
❖ Thrombocytopenia: thrombocytopenia purpura, aplastic anemia, acute leukemia, pernicious anemia, Gaucher’s disease
Dilution factor 1:100
Squares counted Count 25/25 squares or 10 tertiary squares of the center square
Ideal microscope Phase contrast microscope

Formula: Platelet count x Dilution factor

Area counted x 0.1 cm (depth)

MANUAL PLATELET COUNT PROCEDURE (RODAK’S 5th Ed.)

1. Make a 1:100 dilution by placing 20 mL of well-mixed blood into 1980 ul of 1% ammonium oxalate in a small test tube.
2. Mix the dilution thoroughly and charge the chamber. (Note: A special thin, flat-bottomed counting chamber is used for phase-
microscopy platelet counts.)
3. Place the charged hemacytometer in a moist chamber for 15 minutes to allow the platelets to settle.
4. Platelets are counted using the 40x objective lens (400x total magnification). The platelets have a diameter of 2 to 4 um and
appear round or oval, displaying a light purple sheen when phase-contrast microscopy is used. The shape and color help
distinguish the platelets from highly refractile
dirt and debris. “Ghost” RBCs often are seen in the background.
5.Count the number of platelets in the 25 small squares in the center square of the grid. The area of this center square is 1
mm2. Platelets should be counted on each side of the hemacytometer, and the difference between the totals should be less
than 10%.
6.Calculate the platelet count
7.The accuracy of the manual platelet count should be verified by performing a platelet estimate on a wright-stained peripheral
blood.

NOTE
A. If fewer than 50 platelets counted on each side = repeat procedure and dilute blood to 1:20
B. If more than 500platelets counted on each side = repeat procedure and dilute blood to 1:200

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DETERMINATION
A. Direct
o Reese ecker/ Toncantin’s method – sodium citrate, bicarbonate, brilliant cresyl blue, formalin
o Guy and Leake – sodium oxalate, bicarbonate, crystal violet
o Brecker –Cronkite – 1% sodium oxalate, uses phase contrast microscope
o Unopette – 1 % ammonium oxalate as diluting fluid to lyse RBC and allows platelets to form pseudopods.
- dilution factor is 1:100
-platelet count = initial platelet count x 1000

B. Indirect (Smear)
o Dameshek
o Fonio’s
o Olek’s
NOTE
1 platelet per 10-40 RBCs
3-10 platelets per 100 RBCs (or in 1 OIF)
5-20 platelets per 200 RBCs
A normal blood smear should demonstrate approximately 8 to 20 platelets per field

SMEAR: platelet estimate factor ( x20,000), counted on 10 OIF

Platelet estimate Report platelet as
0-49 000 / Ul Marked decreased
50,000-99,000 / ul Moderate decreased
100,000-149,000 / ul Slight decreased
150,000-199,000 / ul Low normal
200,000-400,000 /ul Normal
401,000-599,000 /ul Slight increased
600,000-800,00 /ul Moderate increased
Above 800,000 /ul Marked increased

Manual Platelet count

Normal Platelet count + prolonged BT
✓ Qualitative platelet abnormality
✓ Primary vascular abnormality
✓ Von Willebrand’s syndrome
Lowplatelet count + Normal BT
✓ Autoimmune thrombocytopenia
Low Platelet count + very prolonged BT
✓ Simultaneous quantitative and qualitative platelet deficiency

Significant Platelet Values

-less than 100,000/ul = abnormally low
-30,000-50,000/ul = bleeding possible with trauma
-less than 30,000/ul = spontaneous bleeding possible
-less than 5,000/ul = severe spontaneous bleeding

2. PLATELET AGGREGATION TEST

❖ In vitro test to determine the ability of platelet to aggregate with certain agonist
❖ Platelet aggregation is the gold standard test to determine platelet function
❖ The principle of the test is that platelet-rich plasma is treated with a known aggregating agent. If aggregated,
cloudiness or turbidity can be measured using a spectrophotometer. Depending on the type of aggregating agent
used, a curve that can be used to assess platelet function is obtained.
❖ Uses PRP (Platelet aggregometry): centrifuged at 50g for 30 minutes
❖ PRP + agonist ---------→ checked for O.D
❖ In a normal specimen, after the agonist is added, the shape of the suspended platelets changes from discoid to spherical,
and the intensity of light transmittance initially (and briefly) decreases, then increases in proportion to the degree of
shape change
❖ Poor man’s aggregation test: blood film taken from capillary puncture

AGONISTS= EPINEPHRINE, ADP, COLLAGE, THROMBIN, RISTOCETIN, ARACHIDONIC ACID

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 Aggregometer An instrument designed to measure platelet aggregation in a suspension of citrated whole blood or PRP
(Platelet Rich Plasma)
ADP and Agonists that are stored at –20° C, reconstituted with physiologic saline, and used immediately after
thrombin reconstitution
Epinephrine Agonist that is stored at 1° C to 6° C and reconstituted with distilled water immediately before it is used
Collagen Agonist that is stored at 1° C to 6° C and used without further dilution. It cannot be frozen
Arachidonic Agonist that is readily oxidized and must be stored at –20° C in the dark or dark bottle. It must be diluted
acid with a solution of bovine albumin for immediate use.
ADP The most commonly used agonist, particularly in aggregometry systems that measure only aggregation and
not luminescence. It Produces Biphasic aggregation
Thrombin The agonist that has the disadvantage that it often triggers coagulation (fibrin formation) simultaneously with
aggregation

3.PLATELET ADHESIVENESS (Salzmann)

❖ Measure ability of platelets to adhere in glass surfaces
❖ Decreased in VwD PC1 - PC2 x 100
❖ PC 1: routine procedure, collection %PA = PC1
❖ PC 2: collected through glass bead collection system
❖ Reference Value: 26-60%

4.CLOT RETRACTION TIME

❖ The contractile abilities of platelets also result in the contraction of formed clots. Normal clot retraction requires a normal
number of functioning platelets, calcium, ATP and fibrinogen
❖ Actomyosin or thrombostenin is responsible for clot retraction
❖ Clot retraction reflects the number and quality of platelets, fibrinogen concentration, fibrinolytic activity, and packed red
cell volume (Turgeon)
❖ The degree of clot retraction is directly proportional to the number of platelets and inversely proportional to the
hematocrit and the level of the blood coagulation factor fibrinogen. (Turgeon)

a. CASTOR OIL / HIRSCHBOEK

o NV: 15-45 minutes
o Formation of a dimpling/droplet like serum on the surface of the blood

b. STEFANINI
o 3-5 ml blood (37 oC)
o 1/2/16/18/24 hours
o NV: appreciable within 1 hour, complete within 18 to 24 hours

c. MacFarlane
o 5 mL of blood, 370C (1hour)
o NV= 44-67%
o CRT = Volume of serum x 100
TV

5.CAPILLARY FRAGILITY TEST/RUMPEL- LEEDE TEST/HISS METHOD

❖ Inflate BP cuff to a point hallway between the systolic and diastolic pressure (never exceed 100mmHg), maintain pressure
for 5 minutes
❖ Remove BP cuff and wait for 5 to 10 minutes before proceeding, count petechiae
❖ Positive test is found on thrombocytopenia, decreased fibrinogen and in vascular purpura

Grade Description Numbers of petechiae

1+ Few petechiae on the anterior part of the forearm 0-10
2+ Many petechiae on the anterior part of the forearm 10-20
3+ Multiple petechiae over the whole arm and back of the hand 20-50
4+ Confluent petechiae on the arm and back of the hand 50 and above

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 6.GLASS BEAD RETENTION TEST

❖ An in vitro test for platelet adhesion

❖ Principle: as the anticoagulated blood passes through a glass bead column, normal platelets and vWF adhere to the glass
bead surface that will result to a decrease in platelet count from the original platelet count.
❖ Uses 3 anticoagulated blood

DECREASED PLATELET RETENTION INCREASED PLATELET RETENTION

-Glanzmann’s thrombasthenia -Thrombotic disorders- such as venous thrombosis and
-Von Willebrand’s disease pulmonary embolism
-BSS -Pregnancy
-Chediak Higashi -Hyperlipidemia
-Aspirin administration

7.BLEEDING TIME ( Test for Platelet Adhesion)

❖ In vivo measure of primary hemostasis

❖ The bleeding time test was first described by Duke
❖ This test provides an estimate of the integrity of the platelet plug and thereby measures the interaction between the
capillaries and platelets.
❖ The bleeding time test was the original test of platelet function, although it is now largely replaced by near-patient
analysis of platelet function using the PFA-100
❖ Test is affected if the platelet count is <75,000 x 109/L
❖ As the platelet count drops below 100 × 109/L, the bleeding time increases progressively from a normal of
3 to 8 minutes to more than 30 minutes. (Turgeon)
❖ A prolonged bleeding time in a patient with a platelet count greater than 100 × 10 9/L indicates either impaired platelet
function or a defect of subendothelial factor.

❖ The blood is blotted on the filter paper every 30 seconds

Method Reference value

Duke method-skin puncture 2 to 4 minutes
Ivy method – uses BP cuff inflated at 40mmHg 3 to 6 minutes
Modified template method 6 to 9 minutes

LABORATORY TEST FOR SECONDARY HEMOSTASIS

1.CLOTTING TIME
❖ Measures period required for free formation of blood to clot after it has been removed from the body
❖ Capillary blood method N.V = 2-4 minutes
-Slide and drop
-Capillary tube/ Dale and Laidlaw’s

❖ Whole blood/ Lee and White method (Tilt tube method) N.V = 5-15 minutes
-uses glass tube 13x100mm in size

2.PROTHROMBIN TIME
❖ NV 10-12 Seconds / 12.6 to 14.6 seconds (Rodak’s)
❖ Determine extrinsic and common pathway factor deficient
❖ Citrated blood →Centrifuged →PPP

❖ PPP + PT reagent
PT reagent: tissue thromboplastin + CaCl2 (calcium chloride)

❖ Begin the time for clot formation after the addition of calcium chloride
❖ The test is done at 37 ‘C

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 ❖ If the procedure is performed in duplicate, the duplicate values must be within 10% of their mean or the test is
repeated for a third time
❖ It is used to monitor Warfarin/Coumadin therapy

-PT reagents, often called thromboplastin or tissue thromboplastin, are prepared from recombinant or affinity-purified tissue
factor suspended in phospholipids mixed with a buffered 0.025 M solution of calcium chloride. A few less responsive
thromboplastins are organic extracts of emulsified rabbit brain or lung suspended in calcium chloride.

-Recently, however, newer generation thromboplastin reagents have been developed that are based on purified,
recombinant human tissue factor that has been reconstituted into phospholipid vesicles

Results are reported as International Normalized Ratio (INR)

INR = (PATIENT’S PT / CONTROL PT)ISI

INTERNATIONAL SENSITIVITY INDEX (ISI)

Index comparing the sensitivity of a given thromboplastin preparation with the sensitivity of the international reference
preparation (from WHO) as determined using orthogonal regression. The international sensitivity index is used as an
exponent in the equation for calculating the international normalized ratio.
The closer the ISI is to 1, the more sensitive the reagent is the higher the ISI, the less sensitive the reagent

INR CONDITIONS
2.0-2.5 DVT, high risk surgery
2.0-3.0 Hip surgery, femur fracture
2.0-3.0 DVT, Pulmonary embolism, transient ischemic attack
2.5-3.5 Mechanical/ prosthetic heart valves
2.0-4.5 Recurrent DVT and pulmonary embolism, myocardial infarction, arterial disease

The ISI is a calibration parameter that defines the responsiveness of the reagent relative to a World Health Organization (WHO)
International Reference Preparation, which by definition has an ISI of 1.0.

3.ACTIVATED PARTIAL THROMBOPLASTIN TIME

❖ Reference Value of 20-45 seconds
❖ Determines Intrinsic and common pathway factor deficient
❖ Monitors unfractionated heparin therapy
❖ Citrated blood →centrifuged →PPP
❖ PPP + APTT reagent

PTT reagent contains = Activator, Calcium chloride, and Phospholipids

❖ Activator such as kaolin, celite, silica and ellagic acid
❖ Phospholipids = which was historically extracted from rabbit brain, is now produced synthetically

❖ Begin the time for clot formation after the addition of calcium chloride
❖ The test is done at 37 ‘C
❖ If the PTT is performed manually, the test should be done in duplicate, and the two results must match within
10%.
❖ It is standard method for monitoring unfractionated heparin therapy
❖ The test is affected and will give a prolong results in the presence of specific inhibitors/Lupus anticoagulant, Fibrin
degradation products or paraproteinemia such as Multiple myeloma

The PTT reagent contains phospholipid (previously called partial thromboplastin) and a negatively charged particulate activator
such as silica, kaolin, ellagic acid, or celite in suspension. The phospholipid mixture, which was historically extracted from rabbit
brain, is now produced synthetically

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 5.THROMBIN TIME / THROMBIN CLOTTING TIME
❖ NV 10-14 seconds / 15 to 20 seconds (Rodak’s 5th edition)
❖ Test for fibrinogen deficiency
❖ PPP + Thrombin calcium chloride
❖ TCT Reagent: Commercially prepared bovine thrombin reagent at 5 National Institutes of Health (NIH) units/mL
❖ Principle: thrombin reagent cleaves fibrinopeptides A and B from plasma fibrinogen to form a detectable fibrin polymer
❖ The test is done at 37 ‘C
❖ Prolonged in the presence of FDP/FSP, Heparin, paraproteins and of thrombolytic agent such as streptokinase
❖ Prolonged when fibrinogen is below 100 mg/dl, and when the function of fibrinogen is impaired.
❖ Hypofibrinogenemia, Afibrinogenemia (absence of fibrinogen) and dysfibrinogenemia (presence of fibrinogen that is
biochemically abnormal and nonfunctional) also cause a prolonged TCT

4.STYPVEN TIME/ RUSSEL VIPER VENOM TIME

❖ NV 6-10 Seconds
❖ From the viper (Vipera ruselli ) venom / from the Daboia russelii viper
❖ It bypasses coagulation by directly activating factor X
❖ Determines coagulation factor deficiency in the common pathway
❖ PPP + Stypven reagent (platelin-calcium chloride)
❖ It is now an obsolete test
❖ The diluted russel viper venom test (DRVVT) is used to detect and confirm lupus anticoagulant

6.REPTILASE TEST
❖ Reference value: 10-15seconds
❖ Reptilase is an enzyme found in the venom of Bothrops athrox snake, capable of converting fibrinogen to fibrin
❖ Test for fibrinogen deficiency/ fibrinogen abnormality
❖ PPP + ATROXIN (reptilase)
❖ Principle: reptilase catalyzes the conversion of fibrinogen to fibrin. In contrast to thrombin, this enzyme cleaves only
fibrinopeptide A.
❖ Prolonged in the presence of FDP/FSP, Streptokinase, and paraproteins
❖ It is NOT AFFECTED by Heparin / Heparin therapy
❖ The reagent is reconstituted with distilled water and is stable for 1 month when stored at 1° C to 6° C

7.DUCKERT’S TEST / 5 M UREA SOLUBILITY TEST

❖ Screening test for factor XIII deficiency
❖ Alternative reagents used can be:
1 % monochloroacetic acid
2 % acetic acid

❖ PPP + 5M urea
❖ Normal clot is insoluble to urea for 24 hours
❖ Factor XIII deficiency – the clot is dissolved in less than 24 hours

LABORATORY TEST FOR FIBRINOLYSIS

1.Whole Blood Clot Lysis time (WBCLT)
❖ Test for increased fibrinolysis
❖ Clot should remain intact for approximately 48 hours at 37oC
❖ Clot lysis prior to 48 hours is indicative of excessive systemic fibrinolysis

2. Euglobulin lysis time

❖ Screening procedure for the measurement of fibrinolytic activity
❖ Euglobulin are proteins that precipitate when plasma is diluted with water and acidified
❖ Euglobulin is made of plasminogen, plasmin, fibrinogen and plasminogen activators
❖ This fraction is precipitated with 1% acetic acid and resuspended in a borate solution. The euglobulins are then clotted
by the addition of thrombin. The clot is incubated and the time of lysis is reported.
❖ Normally, clot lysis does not occur in less than 2 hours, <1hour (Brown)
❖ If the lysis time is <2 hours – increase fibrinolytic activity

Normal fibrinolysis Lysis time is more than 1 or 2 hours

Increase fibrinolysis Lysis time is less than 1 or 2 hours

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3.Protamine sulfate test
❖ Detects the presence of fibrin monomers in the plasma
❖ Formation of fibrin strands or gel like clot (PARACOAGULATION)
❖ Normally, there should be no fibrin monomer present in plasma
❖ Normal result: NO gel formation

4.Ethanol gelation test

❖ Detects presence of fibrin monomers in the plasma
❖ Less sensitive but more specific than protamine sulfate
❖ Screening procedure to be utilized as an aid in the diagnosis of DIC and is distinguishing this condition from primary
fibrinolysis
❖ Normal result: No gel formation

5.Latex D dimer assay

❖ Presence of crosslinked D-dimer indicates that a stable fibrin clot has been lysed and will be found in the pulmonary
embolism, deep vein thrombosis, DIC with secondary fibrinolysis and sickle cell disease
❖ D-dimer test is positive in DIC as soon as 4 hours after onset. Fibrinogen levels may decrease in 4 to 24 hours,
platelets decrease up to 48 hours after onset.

BASIC TERMINOLOGY
Petechiae Purplish red pinpoint hemorrhagic spots in the skin caused by loss of capillary ability to withstand
normal blood pressure and trauma.
Size: 1 mm or less than 3mm
Purpura Hemorrhage of blood into small areas of the skin, mucous membranes, and other tissue.
Size:3mm to 1cm
Ecchymosis Form of purpura in which blood escapes into large areas of skin and mucous membranes, but not into
deep tissues.
It is caused by leakage of a small amount of blood in the tissue around the puncture site
Size: more than 1cm
Epistaxis Nosebleed that are recurrent, bleed from both nostrils, last longer than 10 minutes, or require physical
intervention
Hemarthrosis Leakage of blood into joint cavities
Hematemesis Vomiting of blood
Hematoma Swelling of tumor in the tissue or a body cavity that contains clotted blood.
Results when leakage of a large amount of blood around the puncture site causes the area to
rapidly swell.
Hematuria RBC in urine
Hemoglobinuria Hemoglobin in urine
Melena Stool containing dark red or black blood
Menorrhagia Excessive menstrual bleeding

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 PLATELET DISORDERS
QUANTITATIVE PLATELET DISORDERS

THROMBOCYTOPENIA
Decreased platelet production

Decreased megakaryocyte in the bone marrow.

Congenital hypoplasia
Aplastic anemia
Fanconi syndrome
Ineffective thrombopoiesis
Infiltration of the bone marrow by malignant cells
TAR syndrome (Thrombocytopenia with absent radii)
Acquired hypoplasia of megakaryocytes due to drugs, radiation, chemotherapy, or infectious agents
Hereditary macrothrombocytopenia (e.g., Alport’s syndrome)

Decreased platelet survival time due to increased consumption or destruction

Immunologic/Idiopathic thrombocytopenic purpura (ITP)

- Associated with increase platelet destruction
- Majority of Acute ITP cases is due to viral infection and is common in children
- Platelets are large
- Platelet destruction in chronic ITP is the result of an immunologic process. The offending antibodies attach to
platelets, and as a result, the antibody-labeled platelets are removed from the circulation by reticuloendothelial cells,
primarily in the spleen. Autoantibodies that recognize platelet surface glycoproteins such as glycoprotein IIb (GP IIb)
and GP IIIa (aIIb/b3), GP Ia/IIa, and others.

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 Drug induced immunologic thrombocytopenia
- Cause by antibiotics, hypnotics, analgesics, heavy metals, diuretics, etc.
- Generally both the drugs and the antibody must be present in the system at the same time for destruction of platelets
Post transfusion purpura
- Occurs 7 to 10 days after transfusion, due to anti –PIA or anti-HPA-1a(platelet antibodies). The recipient’s plasma is
found to contain alloantibodies to antigens on the platelets or platelet membranes of the transfused blood product,
directed against an antigen the recipient does not have.

Isoimmune neonatal thrombocytopenia/Neonatal autoimmune thrombocytopenia

- A rare hemolytic disease of newborn. Result of transplacental transfer of maternal antiplatelet antibodies produced in
response to fetal antigen inherited from the father. It is associated to a mother with ITP or SLE
-
Non immunologic: Thrombotic thrombocytopenic purpura (TTP)
- The development of TTP seems to be directly related to the accumulation of ultra-large von Willebrand factor
(ULVWF) multimers in the plasma due to deficiency of the von Willebrand factor cleaving protease known as a
disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS-13).

Purpura fulminans
- Unique and devastating thrombotic disorder, often acute and fatal, that manifests as large irregular areas of blue-
black cutaneous bleeding that rapidly progress to necrosis of superficial skin and deeper soft tissues. The identification
of the cause of PF depends on the patient’s age and context of presentation. Occurring most commonly in neonates
and children.
- Can be the presenting feature of acute sepsis resulting from bacterial infections with Neisseria meningitidis,
Streptococcus pneumoniae, group A and B streptococci, and, less commonly, Haemophilus influenzas or
Staphylococcus aureus

Decreased platelet count due to dilutional effect

- Extensive blood transfusion or massive blood transfusion often is accompanied by thrombocytopenia, the degree of
which is directly proportional to the number of units transfused.
-
Decreased platelet count due to abnormal distribution
Increase platelet sequestration to the spleen such as in cases of splenomegaly, or any big spleen syndrome
Hypothermia or lower body temperature (less than 25’C)
- Results in a transient but mild thrombocytopenia secondary to platelet sequestration in the spleen and liver.

THROMBOCYTOSIS
1. Reactive (secondary) thrombocytosis
Moderate increase of platelets for a limited time, usually asymptomatic
Generally responds when the underlying disorder is treated like recovery from major surgery, splenectomy, after
childbirth and acute blood loss
The term reactive thrombocytosis is used to describe an elevation in the platelet count secondary to
inflammation, trauma, or other underlying and seemingly unrelated conditions.
Reactive thrombocytosis is not associated with thrombosis, hemorrhage, or abnormal thrombopoietin levels.

2. Autonomous thrombocytosis
Marked increase, associated with thrombotic/ hemorrhagic complication
Primary or autonomous thrombocytosis is a typical finding in four chronic myeloproliferative disorders (CML,
Polycythemia vera, Essential thrombocythemia, and Primary Myelofibrosis)

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 QUALITITAVE PLATELET DISORDERS

Qualitative platelet disorders can be attributed to adhesion, aggregation, or secretion defects. Release defects are the largest
group of platelet function disorders. This condition is caused by abnormalities of signal transductase from membranes,
abnormal internal metabolic pathways, or abnormal release mechanisms.
-Platelet Dysfunction can be categorized as Hereditary, Acquired, or Drug-induced.

SUMMARY OF QUALITATIVE PLATELET DEFECTS (RODAK’S 5th Ed.)

Platelet adhesion problems Hereditary defects of platelet adhesion
Bernard-Soulier syndrome
Von Willebrand disease

Acquired defects of platelet adhesion

Myeloproliferative, lymphoproliferative disorders, dysproteinemias
Antiplatelet antibodies
Cardiopulmonary bypass surgery
Chronic liver disease
Drug-induced membrane modification

Platelet aggregation problems Hereditary defects of platelet aggregation

Glanzmann thrombasthenia
Hereditary afibrinogenemia
Essential athrombia

Acquired defects of platelet aggregation

Acquired von Willebrand disease
Acquired uremia

Platelet Secretion Storage pool diseases

(release reactions) problems Thromboxane pathway disorders
Hereditary aspirin-like defects:
- Cyclooxygenase or thromboxane synthetase deficiency
Drug inhibition of the prostaglandin pathways
Drug inhibition of platelet phosphodiesterase activity
Changes in Membrane Scott syndrome
Phospholipid Distribution Stormorken syndrome
Hyperactive prothrombic platelets ---

NOTE PAARAL IF ANONG YUNG FUNCTION ANG AFFECTED AT PAARAL DIN IF HEREDITARY OR ACQUIRED

HEREDITARY / INHERITED QUALITATIVE PLATELET PROBLEMS

1. PLATELET ADHESION PROBLEMS

Von Willebrand’s disease (Vwd) -With five major subtypes Normal aggregation with:_________
-Inherited as both an autosomal
dominant types and autosomal
recessive trait Abnormal aggregation with:________

Bernard – Soulier syndrome -Autosomal recessive trait Normal aggregation with:__________

(Giant platelet Syndrome) -Platelets lack gp1b or the
gp I/IX/V complex Abnormal aggregation with:________
-Presence of giant platelets

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 CLASSIFICATION OF CONGENITAL VON WILLEBRAND’S DISEASE
Type Description
1 Partial quantitative deficiency of vWF
2 Qualitative deficiency of Vwf
2A Decreased platelet-dependent vwF function and selective deficiency of high molecular weight multimers
2B Increased affinity to platelet glycoprotein1b
2M Decreased platelet dependent vWF function with high molecular weight multimers present
2N Markedly decreased binding of factor VII to vWF
3 Complete deficiency of wWF

THE ACQUIRED VON WILLEBRAND’S DISEASE

Acquired VWF deficiency, with symptoms similar to those of congenital VWD, has been described in association with
hypothyroidism; autoimmune, lymphoproliferative, and myeloproliferative disorders; benign monoclonal gammopathies; Wilms
tumor; intestinal angiodysplasia; congenital heart disease; pesticide exposure; and hemolytic uremic syndrome

As in congenital VWD, the diagnosis is based on a finding of diminished VWF activity (ristocetin cofactor [VWF:RCo] assay) and
diminished VWF antigen (VWF:Ag) by immunoassay

2. PLATELET AGGREGATION DEFECTS

Glanzmann’s thrombasthenia

Autosomal recessive trait

Decrease or absence or abnormality of GP IIb-IIIa complex
Abnormal clot retraction results
Heterozygotes are clinically normal, whereas homozygotes have serious bleeding problems

Laboratory Features
1. Normal platelet count.
2. Normal platelet morphology
3. Normal response to ristocetin in the platelet aggregation test
4. Abnormal or no response to Epinephrine, Collagen, ADP, and Thrombin in the platelet aggregation test
5. Decrease platelet factor 3 test /platelet procoagulant activity test

3. PLATELET SECRETION / STORAGE POOL DEFECTS

Alpha granules deficiency Dense/Delta granules deficiency

*Gray platelet syndrome *Wiskott Aldrich syndrome
- Gray appearance of platelets in Wright’s stain -triad of eczema, thrombocytopenia with smallest
- Characterized by large platelets platelets and immunodeficiency

*Quebec platelet disorder *Hermansky pudlak

-an autosomal dominant bleeding disorder that results from a
deficiency of multimerin (a multimeric protein that is stored *Chediak Higashi syndrome
complexed with factor V in a-granules)
*Thrombocytopenia with absent Radius
(TAR syndrome)

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 ACQUIRED PLATELET PROBLEMS
Uremia
Paraproteinemias such as Multiple myeloma and Waldenstrom macroglobulinemia
AML
Myeloproliferative disorders such as CML, PV, ET, Myelofibrosis
Paroxysmal nocturnal hemoglobinuria
Sideroblastic anemia
Drugs – such as aspirin (inhibits cyclooxygenase)
Chronic liver disease
Anemia – such as PERNICIOUS ANEMIA
Cardio pulmonary bypass surgery
Autoimmune diseases – such as collagen vascular disease

UREMIA
Uremia is commonly accompanied by bleeding caused by platelet dysfunction It is proposed that circulating guanidinosuccinic acid
or hydroxy phenolic acid interferes with platelet function. Dialysis often corrects or improves platelet function. Other mechanisms
of altered platelet function in uremia, including altered prostaglandin metabolism, have been proposed

DISORDERS WITH GIANT PLATELETS

Bernard-Soulier syndrome, ITP, May-Hegglin Anomaly, Alport Syndrome, Gray platelet syndrome, Giant platelets with
velocardiofacial syndrome, Giant platelets with abnormal surface glycoproteins and mitral valve insufficiency, Familial
macrothrombocytopenia with GP IV abnormality, Montreal platelet syndrome, Fechtner syndrome, Sebastian syndrome, Hereditary
macrothrombocytopenia, Epstein syndrome, Mediterranean macrothrombocytopenia

DISORDERS WITH HYPERAGGREGABLE PLATELETS

Platelets from these patients tend to aggregate at lower concentrations of aggregating agents than do platelets from individuals
without these conditions

Patients with a variety of disorders associated with thrombosis or increased risk for thrombosis, including hyperlipidemia, diabetes
mellitus, peripheral arterial occlusive disease, acute arterial occlusion, myocardial infarction, and stroke, have been reported to
have increased platelet reactivity.

Sticky platelet syndrome an autosomal dominant, thrombophilic disorder that is associated with venous and arterial
thromboembolic events. The disorder is characterized by hyperaggregable platelets in response to ADP, epinephrine, or
both. In these patients venous and/or arterial thrombotic events are often associated with emotional stress

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 VASCULAR DISORDERS (PRIMARY HEMOSTASIS DISRODER)
Generally, the platelet count is normal, as are most platelet function and coagulation studies. The bleeding time and tourniquet
test are usually abnormal

Hereditary hemorrhagic Most common inherited vascular disorder

telangiectasia Inherited as autosomal dominant trait
(Osler-Weber-Rendu Syndrome) The vascular defect of this disorder is characterized by thin-walled blood vessels
with a discontinuous endothelium, inadequate smooth muscle, and inadequate or
missing elastin in the surrounding stroma
Localized dilation of the walls of the small blood vessels of skin and mucous
membranes; walls of the affected blood vessels are thin and lack of smooth
muscle.
Telangiectasias are thin, dilated vessels. With a lesion ranges from pinpoint to 3mm
in size and are red violet in color. They are most obvious on the face, lips, tongue,
conjunctiva, nasal mucosa, fingers, toes, and trunk and under the tongue.
There are several other disorders and conditions in which telangiectasias are
present, including cherry-red hemangiomas (common in older men and women),
ataxia-telangiectasia (Louis-Bar syndrome), and chronic actinic telangiectasia; they
also are seen in association with chronic liver disease and pregnancy.
The diagnosis of hereditary hemorrhagic telangiectasia is based on the
characteristic skin or mucous membrane lesions, a history of repeated hemorrhage,
and a family history of a similar disorder

Hereditary Hemangioma Disorder associated with tumors composed of blood vessels that commonly swell
(Kasabach – Meritt syndrome) and bleed at the surface described the association of a giant cavernous
hemangioma (vascular tumor), thrombocytopenia, and a bleeding diathesis.
Other well-recognized features of Kasabach-Merritt syndrome include acute or
chronic disseminated intravascular coagulation and microangiopathic
hemolytic anemia.

Ehlers Danlos Syndrome May be transmitted as an autosomal dominant, recessive, or X-linked trait. It is
manifested by hyperextensible skin, hypermobile joints, joint laxity, fragile tissues,
and a bleeding tendency, primarily subcutaneous hematoma formation.
Increased vascular fragility
Hyperextended or hypermobile joints and hyperplastic skins
Autosomal dominant
The defect may lie in a peptidase enzyme that converts procollagen to
collagen

Marfan syndrome Increased vascular fragility

Autosomal dominant
Long extremities (long arm and legs)
Characterized by skeletal and ocular defects

Pseudoxanthoma elasticum Autosomal recessive

Connective tissue elastic fibers in small arteries are calcified and structurally
abnormal
Subarachnoid and gastrointestinal bleeding are the most common causes of death

Senile Purpura Loss of elasticity of the skin

Usually due to aging, (elderly persons)
The aging process brings about a degeneration of collagen, elastin, and
subcutaneous fat

Scurvy Deficiency of ascorbic acid (Vitamin C)

Acquired defect in the synthesis of collagen and hyaluronic acid

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Acquired Allergic purpura Immunologic damage to endothelial cells (specific allergic purpura)
(Henoch-Schonlein purpura) Gastrointestinal hemorrhage and joint swelling occur with purpuric rash
Most common in children and often follows upper respiratory infections
Allergic purpura has been associated with certain foods and drugs, cold, insect
bites, and vaccinations
The skin lesions are urticarial and gradually become pinkish, then red, and finally
hemorrhagic. The appearance of the lesions may be very rapid and accompanied
by itching. The lesions have been described as “palpable purpura,” in
contrast to the perfectly flat lesions of thrombocytopenia and most other
forms of vascular purpura.

VASCULAR DISORDERS -RODAK’S

Hereditary Vascular Disorders Hereditary hemorrhagic telangiectasia (Rendu-Osler-Weber syndrome)
Hemangioma-thrombocytopenia syndrome (Kasabach-Merritt syndrome)
Ehlers-Danlos syndrome and other genetic disorders
Acquired Vascular Disorders Allergic purpura (Henoch-Schönlein purpura)
Paraproteinemia and amyloidosis
Senile purpura
Drug-induced vascular purpuras
Vitamin C deficiency (scurvy)
Purpuras of Unknown Origin Purpura simplex (easy bruisability)
Psychogenic purpura

PRINCIPAL HEREDITARY AND ACQUIRED BLEEDING DISORDERS ASSOCIATED WITH VASCULAR ABNORMALITIES
ABNORMALITY HEREDITARY ACQUIRED
Connective Tissue defects Ehlers-Danlos syndrome Vitamin C deficiency (Scurvy), Senile purpura, Corticosteroid
Pseudoxanthoma elasticum purpura, Aging, and Cushing’s disease

Altered vessel wall structure Hemorrhagic telangiectasia Diabetes Mellitus

Congenital Hemangiomata Amyloidosis

Endothelial Damage Infectious purpura

-Bacterial: TB, Scarlet fever, typhoid fever, diphtheria,
endocarditis, others
-Viral: small pox, influenza, measles, others
-Ricketssial: Rocky mountain spotted fever
-Protozoal: malaria

Autoimmune vascular purpura

-Allergic purpuras: Henoch-Schonlein
-Drug induced purpuras: Quinine, Procaine, Penicillin, Aspirin,
sulfonamides, sedatives, coumarins
Miscellaneous abnormalities Waldenstrom’s macroglobulinemia, Kaposi’s sarcoma, Certain
causing purpura secondary to skin diseases, Hemochromatosis, Snake venom
vessel damage

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 COAGULATION DISORDERS
A.CLOTTING FACTOR DEFICIENCIES
FACTOR INHERITED COAGULOPATHIES ACQUIRED
COAGULOPATHY
INHERITANCE COAGULOPATHY
PATTERN
I Autosomal recessive Afibrinogenemia Severe liver disease
DIC
Autosomal dominant Dysfibrinogenemia Fibrinolysis
II Autosomal recessive Prothrombin deficiency Liver disease
Vitamin K deficiency
Anticoagulant therapy
V Autosomal recessive Factor V deficiency Severe liver disease
(Owren’s disease/ Parahemophilia) DIC
Fibrinolysis
VII Autosomal recessive Factor VII deficiency Liver disease
Vitamin K deficiency
Anticoagulant therapy
VIII X-linked recessive Hemophilia A DIC
autosomal dominant Fibrinolysis
IX X-linked recessive Hemophilia B (Christmas Disease) Liver disease
Vitamin K deficiency

X Autosomal recessive Factor X deficiency Liver disease

Vitamin K deficiency
Anticoagulant therapy
XI Autosomal recessive Hemophilia C (Rosenthal syndrome)
-common in Eastern European, Jewish descent /
Ashkenazi Jews
XII Autosomal recessive Factor XII deficiency
XIII Autosomal recessive Factor XIII deficiency Liver disease
DIC
Fibrinolysis
Pre-Kallikrein Autosomal recessive Fletcher trait
HMWK Autosomal recessive Fitzgerald trait

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 CLINICAL MANIFESTATIONS OF COAGULATION FACTOR DEFICIENCIES
Type of Bleeding Coagulation factor deficiency
Easy bruising Factors II, VIII, IX
Hematoma Factors II, VII, IX
Mucosal bleeding Factors II, VII, IX, XI
Hemarthrosis Factors X, VII, IX
Post- surgical bleeding Factors I, II, V, VII, VIII, IX, X, XI, XIII
Intracranial bleeding Factors VII, VIII, IX, XIII
Delayed wound healing Fibrinogen, Factor XIII
Umbilical cord bleeding Factors X, XIII
Miscarriage Fibrinogen, Factor XIII
Thrombosis ABNORMAL FIBRINOGENS
Asymptomatic/ No bleeding tendencies Factor XII, Pre-kallikrein, HMWK

Bleeding associated with Primary Hemostasis defect Petechia, purpura, Ecchymoses, Epistaxis (nosebleed), Gingivail/gums
bleeding, Hematemesis (vomiting of blood), Menorrhagia
Bleeding associated with Secondary Hemostasis defect Hemarthroses, Intracranial hemorrhage, hematomas, Post-
(Clotting factor def.) surgical bleeding

Mucocutaneous (systemic) hemorrhage Associated with thrombocytopenia (platelet count lower than 150,000/ul),
qualitative platelet disorders, or vascular disorders
Anatomic (soft tissue) hemorrhage Seen in acquired or congenital defects in secondary hemostasis, or plasma
coagulation factor deficiencies (coagulopathies).

Most common congenital deficiencies VWD, factor VIII and IX deficiencies (hemophilia A and B), and platelet function disorders
Rare congenital deficiencies Inherited deficiencies of fibrinogen, prothrombin, and factors V, VII, X, XI, and XIII

B.CIRCULATING ANTICOAGULANTS AND INHIBITORS

➢ Can lead to Prolonged APTT and PT and NOT CORRECTED by the addition of normal fresh serum or plasma
➢ Inactivate an activated coagulation factor or block interaction between coagulation factors and platelet
➢ They can be IgM, IgG, or IgA

Two group:
a. The specific inhibitors against specific coagulation factors and
b. Non-specific inhibitors such as the LUPUS INHIBITOR – anti phospholipid antibody, Paraproteins and FDP’s.
Tests:
a. Platelet neutralization test
b. Dilute Russel viper venom test (DRVVT)
c. silica-based partial thromboplastin time (PTT),
d. kaolin clotting time (KCT)
e. dilute thromboplastin time (DTT, also named tissue thromboplastin inhibitor test, TTI).

ANTI-PHOSPHOLIPID ANTIBODIES (APLAs)

Antiphospholipid antibodies comprise a family of immunoglobulins that bind protein-phospholipid complexes
APLAs arise as immunoglobulin M (IgM) or IgG isotypes
Because they may bind a variety of protein-phospholipid complexes, they are sometimes called nonspecific inhibitors
Most APLAs arise in response to a bacterial, viral, fungal, or parasitic infection or to treatment with one of a variety of
drugs and disappear within 12 weeks.
Autoimmune APLAs are part of the family of autoantibodies that arise in collagen vascular diseases; 50% of patients with
systemic lupus erythematosus have autoimmune APLAs. Autoimmune APLAs are also detected in patients with rheumatoid
arthritis, scleroderma, and Sjögren syndrome but may arise spontaneously, a disorder called primary APS
An LAC (Lupus anticoagulant) is suspected because of an unexplained prolonged partial thromboplastin time (PTT) that
does not correct in mixing studies.
The mixing study is an important first step in the LAC laboratory workup to establish the presence of an LAC because it
can differentiate an LAC from a factor deficiency.

IN VIVO, THEY ARE ASSOCIATED WITH ARTERIAL AND VENOUS THROMBOSIS/ THEY ARE PROTHROMBIC.
IN VITRO, THEY ARE ANTICOAGULANT.

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C.LIVER DISEASE
The bleeding associated with liver disease may be localized or generalized, mucocutaneous or anatomic.
Associated with multiple factor deficiency without the presence of D-dimer
Liver is one of the major sites for coagulation factor synthesis
Liver disease predominantly alters the production of the vitamin K–dependent factors II (prothrombin), VII, IX, and X and
control proteins C, S, and Z. Vitamin K catalyzes an essential posttranslational modification of the prothrombin group
proteins: gamma-carboxylation of amino-terminal glutamic acids. In liver disease, these seven factors are produced in
their des-y-carboxyl forms, which cannot participate in coagulation
Platelet count is decreased (<150,000cells/ul) due to sequestration and shortened platelet survival associated with portal
hypertension and resultant hepatosplenomegaly
Platelet aggregation and secretion is affected and suppressed
Factor VII is the first coagulation factor to exhibit decreased activity in liver disease
Factor V is more specific marker of liver disease because it is not affected by Vitamin K deficiency
Prothrombin time (PT) Serves as a sensitive early marker for mild liver disease that can detect factor VII
deficiency and Vitamin K deficiency
The factor V activity assay, performed in conjunction with the factor VII assay, may be used to distinguish liver disease
from vitamin K deficiency
The PT, PTT, thrombin time, fibrinogen concentration, platelet count, and D-dimer concentration are used to characterize
the hemostatic abnormalities in liver disease.
For hemostatic treatment to resolve liver disease, If the fibrinogen level is less than 50 mg/dL, spontaneous bleeding is
imminent, and cryoprecipitate or fibrinogen concentrate may be selected for therapy

Stage of Liver disease Fibrinogen level

Early, mild liver disease >400mg/dl
Moderate to severe liver disease <200mg/dl causing hypofibrinogenemia and dysfibrinogenemia
End stage liver disease <100mg/dl which is a hallmark of liver failure

Factor V and VII assays may be used in combination to differentiate liver disease from vitamin K deficiency
Normal Factor V and Decreased Factor VII Dietary Vitamin K deficiency
Decreased Factor V and Decreased Factor VII Liver disease

D.DIC/ Consumptive coagulopathy/ Defibrination syndrome

A complication or intermediary phase of many diseases such as liver disease, renal disease, and
lymphoproliferative disorders
It can also be triggered by infections, trauma, shock, hypothermia, AMI, and eclampsia.
There is a continuous and uncontrolled activation of both coagulation and fibrinolytic system
Decrease platelet count, prolong all coagulation test
Positive for D-Dimer assay (most specific test)

In acute, uncompensated DIC 1. The PT, PTT, and thrombin time are prolonged
2. The fibrinogen level is reduced to less than 100 mg/Dl
3. Fibrin degradation products, including D-dimers, are significantly
increased
In chronic, compensated DIC The only elevated result may be the D-dimer assay

Acute seen in association with obstetric emergencies, intravascular hemolysis, septicemia, viremia, burns, acute inflammation,
DIC crush injuries, dissecting aortic aneurysms, and cardiac disorders
Chronic associated with vascular tumors, tissue necrosis, liver disease, renal disease, chronic inflammation, use of prosthetic
DIC devices, and adenocarcinoma

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 COMPARISON OF PRIMARY AND SECONDARY HEMOSTASIS DISORDERS
ACQUIRED BLEEDING DISORDERS CONGENITAL BLEEDING DISORDERS
✓ LIVER disease ✓ Congenital Von willie brand disease
✓ Kidney failure (most common congenital bleeding disorder)
✓ Nephrotic syndrome
✓ Chronic infection ✓ Coagulation factor deficiency such as associated
✓ Autoimmune disorders with HEMOPHILIA
✓ Vitamin C deficiency
✓ Vitamin K deficiency ✓ Platelet function disorders
✓ Blunt or penetrating trauma
✓ Inflammatory disorders with bleeding
✓ DIC
✓ ACOTS (Acute coagulopathy of trauma-shock) = most fatal
and most common acquired bleeding disorder
✓ Acquired Von willie brand disease

INSTRUMENTATION FOR TESTS OF HEMOSTASIS

Visual Detection of Fibrin ❖ Tilt tube method
Clot Formation
Electromechanical Detection ❖ Fibrin strand formation is detected using a wire loop or hook; has been incorporated
of Fibrin clot formation into a semi-automated mechanical instrument
❖ Instrument: FIBROMETER
Photo- optical Detection of ❖ Detection of fibrin clot formation depends on the increase in light scattering,
Fibrin Clot Formation associated with the conversion of some fibrinogen molecules to the insoluble
polymerized fibrin clot
❖ SEMI AUTOMATED: Electra 750 and 750A, Fibrintimer series, FP 910 Coagulation
analyzer
❖ AUTOMATED: Ortho Koagulab 16s and 40A, Coag-A-Mate X2 and XC, MLA Electra 700

WARNING FLAGS AVAILABLE ON COAGULOMETERS

SAMPLE QUALITY FLAGS

1. Clotted: will cause falsely shortened clotting times because of premature activation of coagulation factors and
platelets that generate FVIIa and thrombin

2. Lipemia: may cause falsely prolonged clotting times on OD instruments because of interference with light
transmittance

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 3. Hemolysis: may cause falsely shortened clotting times because of premature activation of coagulation factors and
platelets that generate Factor VIIa and thrombin

4. Icterus (bilirubinemia): indicates liver dysfunction that may lead to prolonged clotting times because of inadequate
clotting factor production; also may interfere with OD instruments

5. Abnormal clot formation: may lead to falsely elevated clotting times because of instrument inability to detect an end-
point

6. No end-point detected: indicates that the instrument was unable to detect clot formation; the specimen may need to
be tested using an alternate methodology

ANTICOAGULANT THERAPY
1. Heparin therapy
❖ Used to prevent of thrombi in veins or to prevent propagation of previously formed thrombi in veins and arteries
❖ Continuous IV infusion has now become the most popular method of administration
❖ ACTION: inhibit thrombin
❖ MONITORED BY APTT or ACT (Activated clotting time)
❖ NEUTRALIZED WITH: ___________________

2. Warfarin / Coumadin / Coumarin

❖ Oral anticoagulant
❖ Gold standard anticoagulant therapy
❖ Prevent activation of the clotting sequence by altering hepatic synthesis of vitamin K dependent procoagulant
❖ INR for PT is used to monitor the effects of oral coumarin therapy, and the therapeutic range is universally
established at an INR of 2 to 3
❖ The onset of action of most warfarin derivatives is between 8 and 12 hours. The maximum effect occurs in
approximately 36 hours, and the duration of action is approximately 72 hours
❖ ACTION: Vitamin K antagonist
❖ MONITORED BY: ___________
❖ NEUTRALIZED WITH: ______________________
❖ The major limitations of treatment with warfarin are a narrow therapeutic window, that is, INR, and the slow
onset of effects an offset of action.

In recent years, the new oral anticoagulants (NOAs) factor Xa inhibitors apixaban, thrombin inhibitor abigatran, an factor Xa
inhibitor rivaroxaban and factor Xa inhibitor edoxaban have revolutionized anticoagulation therapy.

3. OTHERS
❖ Examples are Hiruden, Lepirudin (Refludan), Argatroban, Danaparoid (Orgaran), Fondaparinux (Arixtra)
❖ Lepirudin levels can be monitored by the Aptt, ECARIN clotting time, or a chromogenic assay based on the
inhibition of thrombin. The Aptt is the most widely used method of monitoring patients. The target range for
anticoagulation is 1.5 to 2.5 times the baseline Aptt
❖ Argatroban binds to thrombin directly and acts as an anticoagulant by blocking the active site on the thrombin
molecule. This drug is monitored by the Aptt. The therapeutic level is 1.5 to 3.0 greater than the baseline Aptt.
❖ Danaparoid is a mixture of heparinoids which only accelerates the binding of factor Xa to antithrombin and
possesses no anticoagulant activity of its own. It is monitored by chromogenic assay.
❖ Fondaparinux is a synthetic pentasaccharide that accelerates the binding of antithrombin to activated factor Xa.
It has no antithrombin activity. Although this drug usually does not require monitoring, it is recommended that it
be assayed by a system based on the inhibition of factor Xa, if monitoring is needed.

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 LABORATORY TESTS of vWD
Definitive diagnosis 1. Family history of mucocutaneous bleeding
2. Laboratory demonstration of decreased VWF activity
initial VWD workup 1.
2.
3.
standard VWD test panel 1. Quantitative VWF test (VWF:Ag assay)
2. VWF activity test / VWF:RCo assay
3. Factor VIII activity assay
It Identifies vWD subtype 2B Low-dose ristocetin induced platelet aggregometry (RIPA), also called the ristocetin response curve
differentiates between VWD VWF multimer analysis by sodium dodecyl sulfate–polyacrylamide gel electrophoresis
subtypes2A and 2B

Disorder BT CT PT PTT TCT RT ST DUCKERT’S

Primary hemostasis (Platelet and Ab N N N N N N N
vascular problems)
Factor I deficiency N* Ab Ab Ab Ab Ab Ab N
Factor II deficiency N Ab Ab Ab N N Ab N
Factor III deficiency N Ab Ab N N N N N
Factor V deficiency N Ab Ab Ab N N Ab N
Owren’s disease /Parahemophilia
Factor VII deficiency N Ab Ab N N N N N
Factor VIII deficiency/Hemophilia A N Ab N Ab N N N N
Vwd(Von Willebrand’s disease) Ab N/Ab* N Ab N N N N
Factor IX deficiency/Hemophilia B N Ab N Ab N N N N
Factor X deficiency N Ab Ab Ab N N Ab N
Factor XI deficiency/Hemophilia C N Ab N Ab N N N N
Factor XII deficiency N Ab N Ab N N N N
Factor XIII deficiency N N N N N N N Ab
HMWK AND PK DEFICIENCY N Ab N Ab N N N N
DISSEMINATED INTRAVASCULAR Ab Ab Ab Ab Ab Ab Ab --
COAGULATION (DIC)
*positive for D-Dimer
*Increase FDP/FSP
*positive fibrin monomers
*Decrease platelet count

“RISE ABOVE THE STORM AND YOU WILL FIND THE SUNSHINE”

Disclaimer: This is intended for seminars and in-house lectures and is not the full copy of my notes

R.M.T
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