Normal Hemostasis

By: Girum T.

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Outline
 Hemostasis
definition
 Components of Hemostasis
 Vascular Intima
 Platelets
 Tissue factor bearing cells
 The coagulation system
 Coagulation regulatory Mechanisms
 Fibrinolysis
 Coagulation pathways
 Laboratory investigation

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Overview of normal hemostasis
 A complex process that keeps blood fluid in the
circulation
 When an injury occurs, produces a clot to stop
the bleeding
 Keeps the clot confined to the site of injury
 Dissolves the clot as the wound heals
 When Hemostasis system is out of balance,
thrombosis(clotting) or Hemorrhage(bleeding)
can be life threatening.
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Overview…
 Elements of Hemostasis
 Cellular elements
 The vascular intima
 Platelets
 Extra vascular Tissue factor (TF) bearing cells
 Plasma components
 Coagulation proteins
 Fibrinolytic proteins
 Coagulation inhibitors
 Fibrinolytic inhibitors
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Vascular intima in Hemostasis
 Plays important role in Hemostasis
 The inner most lining monolayer is the
Endothelium
 It is smooth and unbroken surface that
promotes fluid passage
 Elastin and collagen surrounds the endothelium
 Sub-endothelial connective tissues are collagen
and fibroblasts as well as smooth muscle cells
in arteries
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Vascular intima…

Fig 1Normal blood flow in intact vessels

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Anticoagulant properties of intact
vascular intima
Composed of rhomboid cells presenting a smooth, contiguous
surface
Secretes the eicosanoid platelet inhibitor prostacyclin
Secretes vascular “relaxing” factor nitric oxide
Secretes the anticoagulant glycosaminoglycan heparan sulfate
Membrane ecto-ADPase
Secretes coagulation extrinsic pathway regulator tissue factor
pathway
inhibitor (TFPI)
Maintains cell membrane thrombomodulin, a protein C
coagulation control system activator

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Procoagulant properties of vascular Intima

During damage
Vasoconstriction in arteries and arterioles
Exposed sub-endothelial collagen binds and activates
platelets
Endothelial cells secrete VWF
On activation, endothelial cells secrete adhesion
molecules
Subendothelial cells expresses tissue factor which
activates the coagulation system

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 Fig. 2 Anti and pro coagulant role of Ecs (Rodaks Hem. P:644)
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Fibrinolytic properties of vascular
intima
 Secretion of tissue plasminogen activators
(TPA)
 Secretion of plasminogen activator
inhibitors 1 (PAI-1)
 Thrombin bound to thrombomodulin
activates thrombin-activatable fibrinolysis
inhibitor (TAFI)

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Platelets
Are produced from the cytoplasm of bone marrow
megakaryoctes (How?)
Though 2 to 3 µm In diameter, they are complex
Adhere : roll and cling to non platelet surfaces
Aggregate: platelets adhere to each other
Secrete: platelets discharge
the contents of their granules
In-depth description of platelet structure and
function?

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Tissue factor bearing cells

 TF is a transmembrane receptor found on

extravascular cells such as smooth muscles,
fibroblasts, neuroglia, epidermal cells.
 Monocyte and Endothelium also express TF
after activation
 TF can circulate in flowing blood on small
procoagulant particles (microparticles)

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Coagulation system
Occurs when the enzyme thrombin is
generated and proteolyses soluble plasma
fibrinogen, forming the insoluble fibrin
polymer or clot
In a tightly regulated fashion
And removal of the clot

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Nomenclature
 16 procoagulants (coagulation factors)
 8 are enzymes that circulate in an inactive
form(zymogens)
 Others are cofactors
 In 1958 named using roman numbers in the
order of their initial description or discovery

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Or we can group as:
 GLA domains
 II, VII, IX, X, PC, PS, PZ
 Non-GLA domais
 XI, XII, XIII, PK, TAFI
 Cofactors
 V, VIII, PS, PZ, VWF, HMWK, TF, TM
 Structural proteins
 Fibrinogen
 Inhibitors
 AT, TFPI, PZ dependent protease Inhibitors

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 Secondary hemostasis(coagulation)

Fig. 3 The coagulation cascade (PG Hem.

P:748
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Deficiencies of the coagulation cascade scheme
 Variation in clinical phenotype and deficiency
of components of the intrinsic pathway
 There are two independent pathways
 No recognition of the complex role of platelets
in clot formation
 Does not show that Thrombin formation
requires 3 consecutive phases namely:
Initiation,Amplification,Propagation

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Secondary hemostasis(coagulation)

The cell based coagulation model:

Initiation
Amplification
Propagation
Stabilization

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Secondary…
 Tissue factor initiates blood coagulation
 TF-FVIIa complex activates FX and FIX
 Traces of TF-FVIIa complex rapidily
inactivated by TFPI
 Fxa interacts with its cofactor Fva to form
prothrombinase comlex and generates small
amount of thrombin on TF surface.

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Secondary…
 Duringamplification, Small amount of
thrombin generated from initiation phase is
responsible for:
 Activating platelets
 Activating FV
 Activating FVIII
 Dissociating factor FVIII from VWF
 Activating FXI

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Secondary…
 Thrombin generation is propagated on the
platelet surface
 FV, FVIII, FIX and FXI will be localized to the
surface of platelets
 FXI is activated by priming amount of
thrombin bypassing the need for FXII
 FXIa activates FIX to FIXa
 Once platelet tenase complex is assembled, FX
is activated to support thrombin burst
21FXIII stabilizes the fibrin strands
 Fig.4 The cell based coagulation model (Rodaks Hem.
22 Page: 656)
Physiological anticoagulants
 AT
 Heparin cofactor II
 TFPI
 PC-PS pathway

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Fig. 5 Regulatory points (Rodaks Hem. Page: 657)
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 Tertiary hemostasis( fibrinolysis)
 The final effector is plasmin
 Plasmin is formed from plasminogen
 Plasmin is involved in breaking down fibrin into
soluble fragments (fibrinolysis)

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Fibrinolysis…

 Inhibitors of fibrinolysis
 Plasminogen activator inhibitor type 1
 α2- Antiplasmin

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Fig. 6 Fibrinolysis and its modulation (PG hem, P. 752)
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Laboratory investigation of bleeding and
coagulation disorders
 It is an attempt to mimic in vitro process that
normally occur in vivo
 May give misleading results
 Prolonged APTT in complete deficiency of
FXII
 Normal screening test do not necessarily imply
that the patient has entirely normal hemostasis
 It is a multistep process (first-line, Second-line)
 No single test for definitive diagnosis

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Laboratory…

A practical algorithm to assist the diagnosis of

29 hemostasis disorders
Equipments
 Water bath
 Refrigerators and freezers
 Centrifuge
 Reagents and buffers
 Plastic and glass tubes
 Pipettes
 Automated coagulation analyzers

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Pre analytical variables
 The quality of the test result is only as good as
the quality of the test sample
 Venous blood sample should be obtained
whenever possible
 Patients should be relaxed and in a warm
surroundings
 Light pressure using tourniquet (for <1 min)
 Not from indwelling line or catheter

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Pre analytical variables
 Sample collection
 Anticoagulant - 3.2% sodium citrate
 Brand of the blood tube
 Blood to citrate ratio- 9:1
 Adjusting anticoagulant volume for hematocrit
(see Dacie and Lewis Page 391 or Rodack
Hemat. 5th ed. Page 764)
 Use plastic or siliconized glass collection tubes
 Venipuncture collection directly into evacuated
tubes containing anticoagulant is recommended
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Pre analytical variables
 Transportation
 Separation of plasma from blood cells
immediately
 Safely capped in a vertical position
 Assurance of stable conditions of temperature
 Avoidance of excessive light exposure
 Deliverance to the reference laboratory as
soon as possible etc…

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Pre analytical variables
 Centrifugation
 Preparation of PPP
 At 1500g for 15 minutes
 Within 2 hrs of collection
 Without disturbing the buffy coat
 To achieve Platelet count <10,000

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Common technical errors
 Faulty collection of sample
 Under-filling or over-filling
 High or low hematocrit
 Unsuitable anticoagulant
 Sample collection through a line that is in
contact with heparin
 Delay in sample analysis

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Analytical considerations
 Methods:
 Clot detection
 Optical
 Mechanical
 Turbidity
 Chromogenic
 Chemiluminescence
 Clot waveform analysis (CWA)

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Calibration and quality control
 Internationalstandards
 Reference standards
 Normal pool (at least 20 donors)

 See page 393 of Dacie and Lewis

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Post-analytical consideration
 Post-analytical sample treatment and storage
 Reflex testing
 Result interpretation

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The CBC:

 For platelet count

 EDTA anticoagulated blood is obtained for
analysis in an automated particle counter
 Pseudo thrombocytopenia or EDTA-
induced thrombocytopenia should be
considered in asymptomatic patients
 Does not measure platelet function

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The PT(prothrombin time) Assay:
 To screen deficiencies in the extrinsic and
common pathways of coagulation
 To monitor oral anticoagulant therapy
 Detect vitamin K deficiency due to liver
disease, malnutrition and warfarin therapy
 The PT is affected by decrease in level of
fibrinogen, prothrombin, FV, FVII or FX

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The PT assay…
 Mixing the patient plasma with thromboplastin
 Thromboplastin is a commercial tissue factor/
phospholipid /calcium preparation
 Tissue factor binds FVII in the patient plasma to
initiate coagulation
 Clot formation is measured by mechanical or
photo-optical end points that detect fibrin
formation.
 Thromboplastin preparations varies in their
sensitivity resulting in different clotting time .
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The international normalized ratio(INR)
 Developed to standardize PT values via the
mathematical transformation
ISI
 INR= PT
MNPT
Where, PT = prothrombin time, MNPT = mean
normal PT,
and ISI = International Sensitivity Index of the
thromboplastin

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The PT assay…
 The typical reference range of PT is 10-15sec.
 Prolonged PT in:
 Deficiency of one or more coagulation
factors in the extrinsic pathway: i.e., factors
VII, X, V, and II or I
 Vit K deficiency
 Certain liver diseases
 Circulating anticoagulants
 Anticoagulant therapy (e.g. Coumarin)
 DIC (disseminated intravascular coagulation)

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The PT assay…
 Shortened PT in:
 Poor quality venipuncture (activated sample)
 Chronic disseminated intravascular
coagulation (in vivo activation)
 Cold activation of the sample
 Administration of recombinant factor VIIa

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The APTT Assay
 To screen deficiencies of the intrinsic pathway
 to detect the lupus anticoagulant
 to monitor heparin therapy
 patient plasma is pre-incubated with the APTT
reagent(phospholipid and a surface-activating
agent such as silica or kaolin)
 Calcium is then added to the pre-incubation
mixture

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The APTT Assay…
 A typical PTT reference range is 25–36 sec.
 APTT prolonged in:
 Decreased levels of intrinsic pathway
components (factors VIII, IX, XI, XII, pre-
kallikrein, and high-molecular-weight
kininogen)
 Shortened APTT in:
 Poor-quality venipuncture (activated sample),
 Chronic disseminated intravascular
coagulation (in vivo activation),
46 Increased factor VIII levels
The Mixing Test
 To distinguish between deficiency and
inhibitor
 Mixing patient plasma with normal plasma in a
1:1 ratio
 Followed by repeat assay of the PT or APTT
immediately after mixing and repeated 1–2
hours later
 Corrected to normal at both intervals suggest
that the original abnormal result was due to
factor deficiency
 Fail to correct to normal at one or both
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The Thrombin Time Assay
 Measures the conversion of fibrinogen to fibrin
 Addition of purified thrombin to patient plasma
 The resulting clotting time is a function of
fibrinogen concentration and activity
 A screening test for the presence of heparin in
a plasma sample
 Quantitative deficiency of fibrinogen,
qualitative abnormality of fibrinogen
(dysfibrinogenemia) prolongs TT
 RR=<20 sec
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Reptilase Time
 A modification of the thrombin time in which the
purified enzyme Reptilase is used to replace
thrombin
 Reptilase is isolated from the snake Bothrops
atrox
 Ancrod a similar enzyme from Agkistrodon
rhodostoma can also be used to replace thrombin
 The snake venoms are not inhibited by heparin
and will be normal in the presence of heparin
 But remains raised in the presence of raised FDP
or abnormal or reduced fibrinogen
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Fibrinogen Assays
 Methods:
 The clause technique
 PT-derived method
 NR= 200-400mg/dL
 May be decreased in liver disease or due to
consumption of fibrinogen when there is
accelerated intravascular clotting

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Fibrinogen Assays
 Clause technique
 Make dilution of calibration plasma in veronal buffer to give
a range of fibrinogen concentration
 0.2 ml of each dilution and 0.1ml of thrombin performed in
duplicae
 Plot the clotting time in seconds against the fibrinogen
concentration in g/l on log/log graph paper
 1 in 10 concentration considered to be 100%
 There should be straight line connection b/n 5 and 50 sec.
 Make 1 in 10 dilution of each patient’s sample an dclot o.2ml
of the dilution with 0.1ml of thrombin
 Read fibrinogen level directly from the graph

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Fibrinogen Assays
PT-derived method
The fibrinogen concentration is proportional to the total
change in optical signal observed during the PT assay
Derived directly from the PT assay without additional
time or expense
FDPs generated by thrombolysis do not interfere with
these
assays
But, not recommended for routine use in the clinical
laboratory

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Interpretation of first line tests
 http://www.practical-haemostasis.com/Data Interpretation

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