HEMATOLOGY 2

PLATELET PRODUCTION, FUNCTION, AND STRUCTURE

MEGAKARYOPOIESIS / THROMBOPOIESIS / THROMBOCYTOPOIESIS
The maturation of the cells of the megakaryocytic system has been divided into four stages. (1) Megakaryoblast , (2)
Promegakaryocyte, (3) Megakaryocyte , (4) Metamegakaryocyte
Maturation sequence of megakaryoblast takes about 5 days. Platelets are produced directly from the megakaryocyte
cytoplasm.
.

A specific Hormone , Thrombopoietin (70,000daltons) , is responsible for megakaryopoieisis

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
As endomitosis proceeds, megakaryocyte progenitors leave the
(Maturation series From Barbara Brown) proliferation phase and enter terminal differentiation (maturation)
1. Megakaryoblast
20-50 um
Blue cytoplasm
Multiple nucleoli
N;C ratio about 10:1
Fine chromatin
First recognizable stage

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

3. 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 visibleor the nucleus may show multilobulation
N:C ratio is 2:1 to 1:1

4. 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

5. Platelet (thrombocyte)
1 to 4 um
Cytoplasm: light blue to purple , very granular
No nucleus
(Steinenger et al.,)
Maturation stage Cytoplasmic granules T Cytoplasmic tags Nuclear fragments T Thrombocytes visible
tr

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).

RODAKS ORDER OF MATURATION (from least to most mature)

bone marrow : Osteoclast
-

resembles Mega Karyocyte

Progenitors – resemble lymphocytes and cannot be distinguished by wright stained

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

Precursors
a. MK-1 / megakaryoblast
b. MK-II / promegakaryocyte
c. MK-III/ Megakaryocyte

SYBIL ROSE POL SIBAL, RMT

 FEATURES OF THE THREE TERMINAL MEGAKARYOCYTE DIFFERENTIATION STAGES
MK-I MK-II MK-III
% of precursors 20 25 55
Diameter 14 to 18 um 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 stage
Absent
last to
Ehdomitosis undergo

Cytoplasm Basophilic Basophilic and granular Azurophilic and granular

Alpha –granules Present Present Present
Dense granules Present Present Present
Demarcation system Present Present Present

Platelet UltraStructure
Micromegakaryocytes

-Dwarf megakaryocytes

-Lost their ability to undergo endomitosis

-Myeloproliferative disorders

-Resemble lymphocytes

-Distingushing feature: Cytoplasmic blebs

PLATELETS / THROMBOCYTES
Also known as thrombocytes with a 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 platelet(Rodaks), (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. ( 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 nucletotides.
Platelet is anatomically divided into four areas: 1. Peripheral zone, 2. Sol-gel zone, 3. Organelle zone, 4. Membranous system
Personal ) PSOM
reps
1. Peripheral zone – composed of the membranes and is responsible for platelet adhesion and aggregation
a. Glycolayx – outer surface, fuzzy coating , primarily composed of glycoproteins including coagulation factors V,VIII and
fibrinogen. 5. 8,1
b. Plasma membrane- bilayer of asymmetrically distributed phospholipids
c. Submembranous area- where messages from external membrane are translated into chemical signals causing activation and
physical change in platelet Phsphatidylserine- Platelet membrane phospholipid flips from the inner surface to the plasma
shape surface on activation and serves as the assembly point for coagulation factors
contraction
Glycocalyx scam aggregation
membrane contact rxn

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)
shape contraction
actomyosin
-
-

organelles
2. Sol gel zone tubulin Shape -_

a. Microfilaments –actin and myosin, which upon stimulation of platelet will interact to form actomyosin (thrombosthenin), a
contractile protein for platelet contraction
b. 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.
White clots: Largely composed of Platelets and Von williebrand factor

Microparticles: Bud off of platelets after their exposure to strong agonists

SYBIL ROSE POL SIBAL, RMT

 3. Organelle zone granules
a. Mitochondria – for energy production /ATP Synthesis
b. Alpha granules
- 300 to 500 nm in diameter examples are Platelet factor 4, platelet derived growth factor, thrombospondin, vwf, fibrinogen,
fibronectin, factor V, etc. PM CASA HE
- There are 50 to 80 alpha granules in each platelet Phosphate
c. Dense granules / Delta granules Magnesium
Calcium
- 250 to 350 nm in diameter examples are Calcium , ADP, Pyrophospate, ATP, Serotonin, and magnesium ADP
- There are two to seven dense granules per platelet Serotonin
d. Lysosomal type granules (lysosome) ATP
Histamine
- Contains Acid hydrolases, ACP, and Hydrolytic enzymes Epinephrine

4. Membranous system CD activation

Calcium fatprostaglandin synthesis
a. Dense tubular system
- derived from rough ER and sequesters or hold calcium for platelet activation process and prostaglandin sysnthesis
- It is the “control center” for platelet activation
pd lysosomes
granules
b. 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

These are platelets found in the circulation/peripheral blood.

Circulating platelets Circulating, resting platelets are biconvex, although the platelets in blood collected using the anticoagulant
ethylenediaminetetraacetic acid (EDTA, lavender closure tubes) tend to “round up.”
-Known as stress platelets
-Appear in compensation for thrombocytopenia
-They are larger than ordinary mature platelets
Reticulated 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
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.
Proplatelet process
.
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.
A process where Platelets are released into the bone marrow through shedding
Thrombocytopoiesis
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).

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. The bulk or amount of surrounding tissue.
b. The type of tissue surrounding the injured vessel.
c. The tone surrounding tissue.

B.
Vascular Component
- Involves the blood vessels in which the blood flows.
a. Capillaries
b. Arteries
c. Veins PATH TV
Substances Released from or Found on the Surface of Intact Endothelial Cells:
SUBSTANCE ACTION
Prostacyclin (PGI2) Prostaglandin inhibit Inhibits Platelet activation /✓
ATP vasodilator Stimulates vasodilation
inactivate Endothelal surface receptor for thrombin
thrombin
Thrombomodulin Binds and inactivates thrombin and enhances anticoagulant and fibrinolytic action of protein C found in
the plasma ↳ pit activation 4 inactivates 5948A

enhances Coats the endothelial cell surface and weakly enhances activity of antithrobim – III, a plasma
Heparan Sulfate Ill
procoagulant
Tissue plasminogen activator (tPA) Converts plasminogen to plasmin, which plays important role in fibrinolysis
antithrombin
Plasminogen →
Plasmin
Protein secreted by endothelium into subendothelium; required for platelet adhesion to site of vessel
injury
Von Willevbrand factor (vWF) aw
Adhesion Storage site of vwf in endothelium- Weibel Palade
Storage site of vwf in platelets: alpha granules

Thrombomodulin + Thrombin =
1. Protein C: inactivates 5a and 8a
2. TAFI (Thrombin Activatable Fibrinolysis Inhibitor): antifribinolytic
SYBIL ROSE POL SIBAL, RMT
 C.
Intravascular Component
- The key component in intravascular hemostatis 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 hemostatis 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. I Intrinsic ) collagen
t

X11
Blood vessel endothelial functions are
a. Angiogenesis = synthesis of stromal components
b. Coagulation = vascular tone regulation
c. Inflammation = special metabolic functions (blood vessel will undergoe vasodilation) ✓ ✓ ✓
functional
d. Immune responses platelets

CAP
Adreno corticosteroids
ascorbic acid
Apc
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 Prostacylin
Tissue factor Thrombomodulin
Vwf Tissue plasminogen activator
Plasminogen activator Urokinase
Inhibitor-1 Heparin like molecules
Other coagulation factors (e.g factor V)

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 mm, 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. DTS
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). ATTC ttnbrombin
TXAZ
collagen

Platelet Adhesion When vascular injury occurs, platelet come in contact with subendothelium (collagen, fibronectin). vWf binds
↳ SPH b surface
Plt -2
Endothelial to glycoprotein Ib/IIb or the GP Ib/IX/V complex on platelet surface ↳ TXAZRADP
releases Prothrombin
surface ↳ Matt .ofdzml6Pla/Ha )
for collagen
V Wf
GPIBHXIV
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 CAPT
cyclooxygenase L pit ) Cyclooxygenase(from the platelets) metabolizes arachidonic acid to form prostaglandin enderoperoxides,
tr
Arachidonic acid which are converted to thromboxane A2 (a vasoconstrictor and a platelet stimulator, causing platelet
t
secretion and aggregation)
prostaglandin
↳ TXA2 Aspirin inhibits cyclooxygenase pathway(remission after 7 to 10days)
Platelet secretion Following activation,platelets will 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

SYBIL ROSE POL SIBAL, RMT

 Thrombin PARI & PA Ra C Plt ) COAT p It
injury TF
=
BV → →

Bz (6101lb 11119) It agg

Lil b fibrinogen & VWF =p .

Platelet Simultaneously with platelet release, platelet stimulating agents (collagen, ADP, epinephrine, thrombin) CATE
aggregation Plt →
Plt bind to the platelets, causing then to adherence to one another
GP 1lb 11119 platelet stimulating agents such as collagen, ADP, epinephrine , thrombin bind to platelets, causing them
Fibrinogen
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 subendothelium and vasoconstriction→platelet adhesion→ platelet aggregation→

platelet plug formation → consolidation of platelets →fibrin stabilization

SUMMARY OF MOST IMPORTANT SUBSTANCES SECRETED BY PLATELETS AND THEIR ROLE IN HEMOSTASIS
ROLE IN HEMOSTASIS SUBSTANCE SOURCE FUNCTION
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 vasoconstriction Serotonin Dense granules Promotes vasoconstriction at injury site
Thromboxane A2 Membrane Same
phospholipids
Promote vascular repair Platelet derived growth factor Alpha granules Promotes smooth muscle growth for vessel repair
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

PLATELET ACTIVATION PATHWAYS

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 Second G protein dependent activation
triphosphate-
Diacylglycerol

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 Labile factor
Proaccelerin Ouren's disease/ Parahemophilia
Accelerator globulin
VII Stable factor
Proconvertin,
Serum prothrombin conversion accelerator (SPCA), Autoprothrombin I
VIII Antihemophilic factor A
Antihemophilic factor (AHF) Hemophilia A/ Classic hemophilia Antihemophilic globulin (AHG)
Platelet cofactor I
IX Christmas factor
Plasma thrombopastin component (PTC) Antihemophilic factor B
Hemophilia B/ Christmas disease Platelet cofactor II
X Stuart factor
Stuart-Prower factor Prower factor
Autoprothrombin III
Hemophilia C/ Rosenthal syndrome
XI Plasma thromboplastin antecedent (PTA) Ashkenazi Jews Antihemophilic factor C
XII Glass factor
Hagemen factor FXII: No bleeding tendency
Contact Factor
XIII Laki- Lorand factor
Fibrinase
Fibrin stabilizing factor 5M Urea Solubility Test/ Duckert's test
Plasma transglutaminase
Fibrinoligase
Contact activation cofactor
HMWK Fitzgerald factor Williams factor
Flaujeac Factor
Pre Kallikrein Fletcher factor
FIBRINOGEN
Most concentrated
<100mg/dL = Prolong PT and APTT
Platelet aggregation
Absorbed, transported and released by Alpha granules
Increase 10 mg/dL/decade in the elderly

SYBIL ROSE POL SIBAL, RMT

 Involved the coagulation factors
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 3. 5. 8. HMWK
XIII: Transglutaminase
COGULATION MECHANIMS
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.

1. 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.

2. 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.

3. 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– molecularweight 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.

SYBIL ROSE POL SIBAL, RMT

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 thqe large vWF portion of the molecule and measured by immunologic
techniques of Laurell 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.

SUBSTITUTION/ MIXING STUDIES

Defiiency 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

Fresh Plasma . coagulation factors are present

All
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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signing x
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SYBIL ROSE POL SIBAL, RMT

MUST KNOW IN ORDER TO ASSESS COAGULATION SUSBSTITUTION STUDIES
1. Factors according to pathway
A. Common pathway = 10, 5, 2, 1
B. Intrinsic pathway = 12, 11, 9, 8
C. Extrinsic pathway= 3 and 7

2. Factors/Pathway according to different coagulation test

A. PT = Extrinsic and Common Pathway
B. APTT= Intrinsic and Common Pathway
C. Reptilase and Thrombin time (TCT)= Fibrinogen Reptilase time
D. Stypven time= Common Pathway -unaffected by Heparin

EXAMPLE CASES:

i*
a. Fibrinogen deficiency
b. Factor IX deficiency
c. Factor VII deficiency
d. Factor XIII deficiency 13

ex 3,7
1. PT abnormal, PTT normal , TCT normal = C
2. PT abnormal , PTT abnormal , TCT abnormal = a
3. PT normal, PTT abnormal, TCT normal = b
4. PT normal, PTT normal, TCT normal = d
↳
in
MIXING STUDIES timing
:
①
:p
a. Lupus anticoagulant / circulating anticoagulant
b. Fibrinogen deficiency :c
:¥¥÷÷÷
c. Factor II deficiency :
. .

d. Factor VII deficiency t 10171/2,9

1. Adsorbed plasma not corrected, Aged serum corrected , Fresh plasma corrected= d
2. Adsorbed plasma not corrected, Aged serum not corrected, fresh plasma corrected = 1017,2019C .

3. Adsorbed plasma corrected, Fresh serum not corrected, fresh plasma corrected = 115,8113 b .

4. Fresh plasma not corrected, Fresh serum not corrected, Adsorbed plasma not corrected = a .

COAGULATION CASCADE

?①
Absqybed
Fibrinogen Vitxk Seyon
L prothrombin ✓ V ✓ V except 2

contact X X

Fibrinogen group/thrombin sensitive

Vitamin K dependent group Contact group
group
1972
Factor VII, IX ,X, II 21719,10
.

Factor I, V, VIII, XIII 1583 Factor XII, XI, HMKW, PK

Consumed during coagulation Requires vitamin K Not consumed during coagulation
Absent in serum Not consumed during coagulation Not absorbed by barium sulfate
Not absorbed by barium sulfate Absorbed by barium sulfate or or aluminum hydroxide
or aluminum hydroxide aluminum hydroxide
Increases in inflammation, Increases in pregnancy, and oral
pregnancy, stress and fear, oral contraceptives
contraceptives

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

INHIBITORS OF COAGULATION
1. 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
2. Protein S – servers as cofactor with protein C. 40 % - free form and active , 60% - bound to compelement c4 Binding protein and
not active
3. Antithrombin C – major inhibitor of thrombin. Enhanced by heparin . Inhibits main serine protease
4. Heparin cofactor II- inhibits also thrombin
5. Alpha 2 macroglobulin- forms complex with thrombin, kallikrein, and plasmin thus inhibiting their activities
6. Extrinsic pathway inhibitor (EPI) – Also called as lipoprotein associated coagulation inhibitor (LACI) , inhibits the VIIa-tissue factor
complex
7. C1-inhibitor- principle inactivator of factor XIIa and plasma kallekrein
8. Alpha 1 antitrypsin- weak inhibitor of thrombin and factor Xa and Xia
9. Activated protein C inhibitor- inhibit the activity of protein C
10. Thrombin + thrombomodulin(endothelial surface) - modifies the action of thrombin to act more as inhibitor by inactivating
protein C

SYBIL ROSE POL SIBAL, RMT

 Excess fibrinolysis: Bleeding & Poor wound healing

Inadequate fibrinolysis: Clot extension, Thrombosis

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
/FSP
Acitvated by Plasminogen – a single chain glycoprotein found in the plasma in concentration of 20 to 40 mg/dL and in all other fluids in a
TPA and UPA
lesser amounts. Produced by the liver, Stored and transported by eosinophils ~ Plaminogen Activator Inhibitor (PAI)
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. ~ Alpha antiplasmim

Degradation of cross-linked fibrin Degradation of fibrinogen and non-crosslinked fibrin

Plasmin - Lyse fibrin - D-dimer Plasmin - Lyse fibrinogen - no D-dimer

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 carcinoma Examples are seen in infection and HTR
NO D-DIMER (+) D DIMER
NO FIBRIN MONOMER (+) FIBRIN MONOMER
NO FIBRIN POLYMER (+) FIBRIN POLYMER
Normal platelet count Decreased platelet count
schist
ocyte

Degradation of FIBRINOGEN Degradation of CROSS LINKED FIBRIN

PROTAMINE SULFATE TEST - Detection of Fibrin monomer and Fibrin degradation products
PLASMINOGEN ACTIVATORS
1. Intrinsic factors= factor XIIa, Kallikrein, and HMWK 12 PK HMWK , ,

UPA 2. Tissue type urokinase- secreted by the kidney, activates plasminogen Urinary tract epithelial cells,
3. Therapeutic activators such as treatment for thromboemboli monocytes and macrophages
- Streptokinase, urokinase, Tissue like PA
TPA 4. Tissue plasminogen activator

INHIBITORS OF FIBRINOLYSIS
1. Alpha 2 antiplasmin – primary inhibitor of plasmin
2. Alpha 2 macroglobulin
3. Thrombospondin – released by the platelets, inhibits activation of fibrin-bound plasminogen
4. Plasminogen activator inhibitor 1 (PAI-1) and Plasminogen activator inhibitor 2 (PAI-2) - Principal inhibitor of plasminogen
5. Alpha 1 antitrypsin
6. Thrombin-Activatable Fibrinolysis Inhibitor (TAFI) - antifribinolytic
Epsilon aminocaproic acid (EACA)

-Specific inhibitor of Plasmin

-Used to turn off inappropriate lysing that occurs in PRIMARY FIBRINOGENOLYSIS

-DIC Fibrinolysis: Appropriate body response to systemic clotting

-If administered, clots that form will not be lysed

SYBIL ROSE POL SIBAL, RMT

 SPECIMEN COLLECTION AND PROCESSING FOR HEMOSTATIS TESTING
I. Method of collection
a. Two syringe method
b. 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

II. Special considerations and Information

a. Anticoagulant- 0.105 to 0.109M (3.2% )buffered Sodium citrate
b. Ratio of Blood to anticoagulant – ( 9:1)
c. Citrate is the anticoagulant of choice for platelet function testing except for the glass bead retention test, which require
heparin.
d. Specimen processing for hemostasis testing should be done at 37 ‘C
e. Labile factors (V and VIII) deteriorate at RT and increased pH 500 is mins
f. Factors VII and XI prematurely activates when place at a cold temperature (4’C)
g. Aspirin and other NSAID’s inhibit cyclooxygenase pathway 50 30 MIN
- Platelet function test : 1 week abstinence from the following drugs
- Bleeding time test : 24 hours aspirin intake is prohibited
h. Platelet poor plasma (PPP) –centrifuged at 2000 x g for 10 minutes (steinenger), Centrifuged at 1500 x g for 15
minutes ( Rodaks ) , centrifuged at 2500 x g for 20 minutes (Turgeon)
i. Platelet rich Plasma (PRP) – centrifuged at 60-100 x g for 10 minutes (steinenger),Centrifuged by 50 x g for
30minutes(Rodaks)
j. Coagulation specimen should be centrifuged within 1 hour of obtaining the sample
k. Specimen processing for PT is valid within 24 hours
l. Specimen processing for APTT is valid for 4 hours
m. The PT and APTT , TCT are prolonged when Fibrinogen level is ≤100mg/dl(Rodak) , or less than 80mg/dl(Brown)
n. The size of glass test tube used for PT and APTT is 12 x75mm(Steienenger) , or
13x 100mm(Brown)

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

FACTORS THAT INTERFERE WITH THE VALIDITY OF CLOT BASED TEST RESULTS
Problem Solution
Blood collection volume less than specified minimum PT falsely prolonged; recollect specimen
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 reagents Correct analytical error and repeat test.
short draw :
riffraff FT
:

Hemolysis : pre activation of cells : Ft

SYBIL ROSE POL SIBAL, RMT

 LABORATORY TESTS FOR PRIMARY HEMOSTASIS
1.PLATELET COUNT
NV= 150-400 x10 9 / L , 150 to 450 x10 9 (Rodak’s )
PICS Thrombocytosis : PV, idiopathic thrombcythemia ,CML, splenectomy
Thrombocytopenia: thrombocytopenia purpura, aplastic anemia, acute leukemia, pernicious anemia, Gaucher’s disease
Dilution : 1:100
Squares: count 25/25 squares or 10 tertiary squares of the center square
Microscope: Phase contrast microscope
20 UL
Formula : Platelet count x Dilution factor
Area counted x 0.1 cm (depth) 1980hL
Manual platelet count Procedure (RODAK’S 5th Ed.)
u of well-mixed blood into 1980 ul of 1% ammonium oxalate in a small test tube.
1. Make a 1:100 dilution by placing 20 mL
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

DETERMINATION
A. Direct
o Reese ecker/ Toncantin’s method – sodium citrate, bicarbonate, brilliant cresly 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 98mL 1.
ammonium oxalate
0.02mL
-platelet count = initial platelet count x 1000 blood

B. Indirect (Smear)
o Dameshek
o Fonio’s
o Olek’s

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
-50,000 150,000-199,000 / ul Low normal
200,000-400,000 /ul Normal
+200,000 401,000-599,000 /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 qualitatitve 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

SYBIL ROSE POL SIBAL, RMT

Principle: Change in OD

As platelets aggregate, the OD of the PRP decreases

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. Light transmittance aggregometry
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 RACE PLATELET LUMIAGGREGOMETRY

-measures aggregation and ATP release

a. Epinephrine
}
-Whole blood diluted with Saline

b. Collagen Normal: Bernard Soulier, vWD -ATP release: measured by addition of Luciferin (from fireflies)

c. ADP -Aggregation: measured by Impedance

d. Ristocetin Normal: Glanzmann's thrombasthenia TEA CAR
e. Thrombin Biphasic : ADP monophonic :
Arachidonic acid
phasic collagen
f. serotonin
:
mono
Thrombin Collagen
biphasic :
ADP Epinephrine Ristocetin

An instrument designed to measure platelet aggregation in a suspension of citrated whole blood or PRP(Platelet Rich
Aggregometer
Plasma)
ADP and Agonists that are stored at –20° C, reconstituted with physiologic saline, and used immediately after reconstitution
thrombin
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 Monophasic aggregation
Arachidonic Agonist that is readily oxidized and must be stored at –20° C in the dark or dark bottle. It must be diluted with a
acid solution of bovine albumin for immediate use.
The most commonly used agonist, particularly in aggregometry systems that measure only aggregation and not
ADP
luminescence. It Produces Biphasic aggregation
The agonist that has the disadvantage that it often triggers coagulation (fibrin formation) simultaneously with
Thrombin
aggregation
Collagen: Only aggregating agent that includes a single wave response preceded by a lag phase
3. PLATELET ADHESIVENESS (Salzmann)
Measure ability of platelets to adhere in glass surfaces Adhesion
Decreased in VwD Salzmann

PC 1: routine procedure, collection

PC 2: collected through glass bead collection system

PC1 - PC2 X 100

%PA = PC1

NV: 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) Degree of clot retraction

Directly proportional

a. CASTOR OIL / HIRSCHBOEK Platelets

o NV: 15-45 minutes

Inversely proportional

o Formation of a dimpling/droplet like serum on the surface of the blood Hematocrit

Fibrinogen
b. STEFANINI
o 3-5 ml blood (37 oC)
o 1/2/16/18/24 hours
Abnormal Clot Retraction

o NV: appreciable within 1 hr, complete within 18 to 24 hrs Thrombocytopenia

Abn fibrinogen

c. MacFarlane Paraproteins ei. MM

o 5 mL of blood, 370C (1hr) Glanzmann's thrombasthenia

o NV= 44-67% Castor Oil/ Hirschboek

o CRT = Volume of serum x 100 Abnormal characteristic of extruded serum

TV 1. Dark Yellow - Jaundice

2. Cloudy - Plasma cell myeloma

3. Milky - Diabetes, Leukemia, After meals

SYBIL ROSE POL SIBAL, RMT

 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 proceding, 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 wholearm and back of the hand 20-50
4+ Confluent petechiae on the arm and back of the hand 50 and above

6. GLASS BEAD RETENTION TEST

Anticoagulant: Heparin
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 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 × 109/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-skinpuncture Earlobe 2 to 4 minutes
Ivy method – uses BP cuff inflated at 40mmHg 3 to 6 minutes
Modified template method 6 to 9 minutes

SYBIL ROSE POL SIBAL, RMT

 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 (Rodaks 5th Ed.) Wdr
EX
PT

Determine extrinsic and common pathway factor deficient

Citrated blood →Centrifuged →PPP

PPP + PT reagent
PT reagent: tissue thromboplastin + CaCl2

Begin the time for clot formation after the addition of calcium chloride
The test is done at 37 ‘C
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 PT x 100

Control PT
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 DIP 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
NV 20-45 seconds
10-12
Determines Intrinsic and common pathway factor deficient
20-45
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 rabbit
Phospholipids = which was historically extracted from rabbit brain, is now produced synthetically brain

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

4. STYPVEN TIME/ RUSSEL VIPER VENOM TIME

NV 6-10 Seconds
From the viper (Vipera ruselli ) venom / from the Daboia russelii viper(Rodak’s 5th edition)
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

SYBIL ROSE POL SIBAL, RMT

 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

6. REPTILASE TEST
Reference value : 10-15seconds
Reptilase = 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) 2
days
Test for increased fibrinolysis
Clot should remain intact for approximately 48 hours at 37 oC
Clot lysis prior to 48 hours is indicative of excessive systemic fibrinolysis

2. Euglobulin lysis time 2 hours

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

3.Protamine sulfate test N NO GEL

:

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
Monoclonal antibodies against D-dimers
5.Latex D dimer assay Negative Predictive value
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
Ecchymosis Bruise Form of purpura in which blood escapes into large areas of skin and mucous membranes, but not into deep tissues. Size: ≥1cm
Epistaxis Nosebleed
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
Hematuria RBC in urine
Hemoglobinuria Hemoglobin in urine
Melena Stool containing dark red or black blood
Menorrhagia Excessive menstrual bleeding

SYBIL ROSE POL SIBAL, RMT

 PLATELET DISORDERS
I. QUANTITATIVE PLATELET DISORDERS
A. THROMBOCYTOPENIA

1. Decreased platelet production-

Decreased megakaryocyte in the bone marrow.
Congenital hypoplasia
Aplastic anemia
Fanconi syndrome
Ineffective thrombopioesis
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)

2. Decreased platelet survival time due to increased or decreased consumption

Destroy Increased platelet destruction : Immunologic : Idiopathic thrombocytopenic purpura (ITP) – majority cases is
due to viral infection

Immunologic

Idiopathic thrombocytopenic purpura (ITP)

Drug induced immunologic thrombocytopenia

Post transfusion purpura (PTP)

Isoimmune neonatal thrombocytopenia

Non immunologic

Thrombotic thrombocytopenic purpura (TTP)

DIC

HUS

Drug induced immunologic thrombocytopenia- 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 – 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.

consume Increased platelet consumption : Non immunologic: Thrombotic thrombocytopenic purpura (TTP) – rare
disorder ,the exact cause is unkown. 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 factorcleaving protease known as a disintegrin and metalloproteinase with a thrombospondin
type 1 motif, member 13 (ADAMTS-13).

DIC
HUS

3. Increased platelet sequestration on the spleen

4. Dilution of platelet count by multiple blood transfusions

o Extensive blood transfusion often is accompanied by thrombocytopenia, the degree of which is directly
proportional to the number of units transfused

SYBIL ROSE POL SIBAL, RMT

 B. THROMBOCYTOSIS
1. Reactive (secondary) thrombocytosis
o Moderate increase, usually asymptomatic
o Generally responds when the underlying disorder is treated like recovery from major surgery, splenectomy,
after childbirth and acute blood loss
o 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.
o Reactive thrombocytosis is not associated with thrombosis, hemorrhage, or abnormal thrombopoietin levels.

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

II. 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.

A. HEREDITARY / INHERITED PLATELET PROBLEMS

1. Platelet adhesion problems
Von Willebrand’s disease (Vwd)
With five major subtypes Normal with ______
ADP, Collagen, Epinephrine
Inherited as both an
autosomal dominant types and
autosomal recessive trait Abnormal aggregation with
______
Ristocetin

Bernard – Soulier syndrome Autosomal recessive trait Normal with ________________

Platelets lack gp1b or the gp
I/IX/V complex
Presence of giant platelets
ADP, Collagen, Epinephrine
Abnormal aggregation with
______________________
Ristocetin

CLASSIFICATION OF 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

SYBIL ROSE POL SIBAL, RMT

 2. Platelet Aggregation problems
o Glanzmann’s thrombasthenia – autosomal recessive trait
Decrease or absence or abnormality of GP IIb-IIIa complex
Plate aggregation: Normal with restocetin.
ri
Abnormal with Epinephrine, collagen, and ADP
Abnormal clot retraction results
Heterozygotes are clinically normal, whereas homozygotes have serious bleeding problems

LAB FEATURES OF GLANZMANN’S THROMBASTHENIA

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 problems

o Storage pool defects
Alpha granules deficiency Dense/Delta granules deficiency
Gray platelet syndrome = gray appearance of *Wiskott Aldrich syndrome CHeWT
platelets in Wright’s stain *Hermansky pudlak
*Chediak Higashi syndrome
*Quebec platelet disorder = an autosomal *Thrombocytopenia with absent Radius (TAR
dominant bleeding disorder that results from a syndrome)
deficiency of multimerin (a multimeric protein that is
stored complexed with factor V in a-granules)

B. ACQUIRED PLATELET PROBLEMS

1. Uremia
2. Paraproteinemias such as Multiple myeloma and Waldenstrom macroglobulinemia
3. AML
4. Myeloproliferative disorders such as CML
5. Drugs – such as aspirin (inhibits cyclooxygenase)
6. Chronic liver disease

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

Platelet adhesion problems
Platelet Secretion
(release reactions) problems
Platelet aggregation problems Glanzmann thrombasthenia
Changes in Membrane
Phospholipid Distribution
Severe coagulation factor deficiencies
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
Storage pool diseases
Thromboxane pathway disorders
Hereditary aspirin-like defects:
Cyclooxygenase or thromboxane synthetase deficiency
Drug inhibition of the prostaglandin pathways
Drug inhibition of platelet phosphodiesterase activity
Essential athrombia
Hereditary afibrinogenemia
Acquired defects of platelet aggregation:
Acquired von Willebrand disease
Acquired uremia
Scott syndrome
Stormorken syndrome

Afibrinogenemia

Factor VIII:C deficiency

Factor IX:C deficiency

SYBIL ROSE POL SIBAL, RMT

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

Most common inherited disorder

Inherited as autosomal dominant trait
Hereditary hemorrhagic
Localized dilation of the walls of the small blood vessels of skin and mucous membranes; walls of
telangiectasia / Osler-
the affected blood vessels are thin and lack of smooth muscle.
Weber-Rendu Syndrome
Telangiectasias are thin, dilated vessels. With a lesion ranges from pinpoint to 3mm in size and are
red violet in color. .

Hereditary Hemangioma
Disorder associated with tumors composed of blood vessels that commonly swell and bleed at the
/ Kasabach – Meritt
surface
syndrome
Increased vascular fragility
Hyperextended joints and hyperplastic skins
Ehlers Danlos Syndrome
Autosomal dominant
The defect may lie in a peptidase enzyme that converts procollagen to collagen
Increased vascular fragility
Marfan syndrome Autosomal dominant
Long extremities (long arm and legs)
Autosomal recessive
Pseudoxanthoma elasticum Connective tissue elastic fibers in small arteries are calcified and structurally abnormal
Subarachnoid and gastrointestinal bleeding are the most common causes of death
Loss of elasticity of the skin
Senile Purpura Usually due to aging, (elderly persons)
The aging process brings about a degeneration of collagen, elastin, and subcutaneous fat
Deficiency of ascorbic acid
Scurvy
Acquired defect in the synthesis of collagen and hyaluronic acid
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

PRINCIPAL HEREDITARY AND ACQUIRED BLEEDING DISORDERS ASSOCIATED WITH VASCULAR ABNORMALITIES
ABNORMALITY HEREDITARY ACQUIRED
Vitamin C deficiency (Scurvy)
Senile Purpura
Ehlers-Danlos syndrome
Connective Tissue defects Corticosteroid purpura
Pseudoxanthoma elasticum
Aging
Cushing’s disease
Altered vessel wall Hemorrhagic telangiectasia Diabetes Mellitus
structure Congenital Hemangiomata Amyloidosis
Infectious purpura
-Bacterial: TB, Scarlet fever, typhoid fever, diphtheria, endocarditis, others
-Viral: small pox, influenza, measles, others
-Ricketssial: Rocky mountain spotted fever
-Protozoal: malaria
Endothelial Damage
Autoimmune vascular purpura
-Allergic purpuras: Henoch-Schonlein
-Drug induced purpuras: Quinine, Procaine, Penicillin, Aspirin, sulfonamides,
sedatives , coumarins
Waldenstrom’s macroglobulinemia
Miscellanoues
Kaposi’s sarcoma
abnormalities causing
Certain skin diseases
purpura secondary to
Hemochromatosis
vessel damage
Snake venom

SYBIL ROSE POL SIBAL, RMT

 COAGULATION DISORDERS

A. CLOTTING FACTOR DEFICIENCIES

FACTOR INHERITED COAGULOPATHIES ACQUIRED COAGULOPATHY
INHERITANCE COAGULOPATHY
PATTERN
Autosomal recessive Afibrinogenemia bleeding -

Severe liver disease

I DIC
Autosomal dominant Dysfibrinogenemia Fibrinolysis
Liver disease
II Autosomal recessive Prothrombin deficiency Vitamin K deficiency
Prothrombin 20210 mutation: 2nd most common hereditary
thrombotic disorder
Anticoagulant therapy
Severe liver disease
Factor V deficiency
V Autosomal recessive DIC
(Owren’s disease)
Most common hereditary thrombotic disorder Fibrinolysis
Liver disease
VII Autosomal recessive Factor VII deficiency Vitamin K deficiency
Anticoagulant therapy
x-linked recessive Hemophilia A
DIC
VIII
Fibrinolysis
autosomal dominant vWD
Liver disease
IX x-linked recessive Hemophilia B Vitamin K deficiency
.

Liver disease
X Autosomal recessive Factor X deficiency Vitamin K deficiency
Anticoagulant therapy
Hemophilia C
XI Autosomal recessive (common in eastern European, jewish descent /
Ashkenazi jews)
XII Autosomal recessive Factor XII deficiency
Liver disease
XIII Autosomal recessive Factor XIII deficiency DIC
Delayed bleeding at the incision site Fibrinolysis
PreKallikrein Autosomal recessive Fletcher trait Increased risk of thrombosis
HMWK Autosomal recessive Fitzgerald trait

CLINICAL MANIFESTATIONS OF COAGULATION FACTOR DEFICIENCIES

Type of Bleeding Coagulation factor deficiency
Easy bruising Factors II, VII, IX 2 it 9

)
,

Hematoma 1972
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 X 3 U 12 , ,

Intracranial bleeding Factors VII, VIII, IX, XIII 7 8 9 13 , . ,

Delayed wound healing Fibrinogen, Factor XIII 13 I

,

Umbilical cord bleeding Factors X, XIII 10 13 ,

Miscarriage Fibrinogen, Factor XIII lil 3

Thrombosis ABNORMAL FIBRINOGENS PK
Asymptomatic/ No bleeding tendencies Factor XII, Prekallikrein, HMWK

B. CIRCULATING ANTICOAGULANTS AND INHIBITORS

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.
Test: against phospholipid portion of

a. Platelet neutralization test prothrombin complex

as e

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).
C. LIVER DISEASE
Associated with multiple factor deficiency without the presence of D-dimer
Liver is one of the major site for coagulation factor synthesis
Factor VII is the first coagulation factor to exhibit decreased activity in liver disease
A 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
In end-stage liver disease, the fibrinogen level may fall to less than 100mg/dl which is a mark of liver failure

SYBIL ROSE POL SIBAL, RMT

 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 activation of both coagulation and fibrinolytic system
Decrease platelet count, prolong all coagulation test
Positive for D-Dimer assay (most specific test)
Consumption of Factors 1, 2, 5, 8, 13
1. the PT, PTT, and thrombin time are prolonged
In acute, uncompensated
2. the fibrinogen level is reduced to less than 100 mg/dL
DIC
3. fibrin degradation products, including D-dimers, are significantly increased
In chronic, compensated DIC The only elevated result may be the D-dimer assay

COMPARISON OF PRIMARY AND SECONDARY HEMOSTASIS DISORDERS

ACQUIRED BLEEDING DISORDERS CONGENITAL BLEEDING DISORDERS
LIVER disease Congenital Von willie brand disease (most common
Kidney failure congenital bleeding disorder)
Nephrotic syndrome Coagulation factor deficiency such as associated with
Chronic infection HEMOPHILIA
Autoimmune disorders Platelet function disorders
Vitamin C deficiency
Vitamin K deficiency von Willie brand

hemophilia
Blunt or penetrating trauma function disorder
platelet
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 Clot
Tilt tube method
Formation
Fibrin strand formation is detected using a wire loop or hook; has been incorporated into
Electromechanical Detection
a semi-automated mechanical instrument
of Fibrin clot formation
Instrument: FIBROMETER ,
BBL

Detection of fibrin clot formation depends on the increase in light scattering , associated
Photo- optical Detection of with the conversion of some fibrinogen molecules to the insoluble polymerized fibrin clot
Fibrin Clot Formation 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

RODAKS 5TH EDITION

Level Description Examples
-All reagents and specimens are transferred manually by the operator. -Tilt tube
-Temperature is maintained by a water bath or heat block; -Wire loop
manual -External measurement by operator may be required.
-End-point is determined visually by the operator.
-Timer is initiated and stopped by the operator.
-All reagents and specimens are transferred manually by the operator -Fibrometer
-Instrument usually contains a device for maintaining constant 37° C -STart 4
temperature -Cascade M and M-4
Semiautomated -Analyzer may internally monitor temperature -BFT-II
-Instrument has mechanism to initiate timing device automatically on -KC1
addition of final reagent and mechanism for detecting clot formation and -KC4
stopping the timer
ACL TOP,
-All reagents are automatically pipetted by the instrument STA-R Evolution,
-Specimens may or may not be automatically pipetted STA Compact, and Compact CT
Automated -Analyzer contains monitoring devices and internal mechanism to maintain Sysmex CA-530, CA-560,CA-620, CA-660,
and monitor constant 37° C temperature throughout testing sequence CA1500,CA-7000
-Timers are initiated and clot formation isdetected automatically. BCS XP,
CoaLAB
Chromogenic

-Depends on cleavage of synthetic substrates by an active serine protease

-Antithrombin, Plasminogen, Protein C, Heparin

SYBIL ROSE POL SIBAL, RMT

 DEE lot
Inc.
= Lipemia
=

shIort-hemoly.si,
Erolong
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
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
goal :
1. Heparin therapy
no clot Used to prevent of thrombi in veins or to prevent propagation of previously formed thrombi in veins and arteries
I Continuous IV infusion has now become the most popular method of administration
bleeding Monitoring: _____________________
Activated Prothrombin Time
ACTION: inhibit thrombin
NEUTRALIZED WITH: ___________________
Protamine sulfate

2. Warfarin / Coumadin / Coumarin

Oral anticoagulant
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 PT seconds: Dependent on reagent and instrument used

ACTION: Vitamin K agonist 41972 Protein

,
Cfs INR: Recommended by WHO. Independent on reagent and instrument used
MONITORING: Prothrombin
___________ time
NEUTRALIZED WITH: ______________________
Vit K supplements
Fresh frozen plasma
Coagulation factor concetrates
ADDENDUM
Kinin system and complement Kinin sytem is activated
. by both coagulation and fibrinolytic system
system Both are associated with hemostasis
Normal pregnancy beginning at the time of conception is associated with increased
Pregnancy associated thrombosis
concentrations of coagulation factors VII, VIII, and X and von Willebrand factor
Bethesda assay A test for factor VIII inhibitor
Normal concentration = 200 to 400 mg/dl
Fibrinogen levels Normal titer: 1:128 to 1:256
Abnormal titer: less than 1:64

LABORATORY TEST of vWD

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

Disorder BT CT PT PTT TCT RT ST DUCKERT’S

Primary hemostasis(Platelet and vascular Ab N N N N N N N
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 COAGULATION Ab Ab Ab Ab Ab Ab Ab --
(DIC)
*positive for D-Dimer
*Increase FDP/FSP
*positive fibrin monomers
*Decrease platelet count

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

SYBIL ROSE POL SIBAL, RMT