PT APTT Technical Information

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PT & aPTT

Manish Pandey

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Hemostasis Is a Balance Between Clot Formation & Clot Dissolution.

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Normal Hemostasis

Clot formation(Coagulation)

Clot dissolution(Fibrinolysis)

PTaPTT

Thrombin TimeFibrinogen

Individual factor tests

FDP D-Dimer

vWFHMWK

Prekallikrein

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Thrombosis

Abnormal Hemostasis is Thrombosis

Formation of Blood Clot (thrombus) in normal blood vessels

Thrombotic occlusion of a vessel after relatively minor injury

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Hemostasis involves the interaction of:

Vascular EndotheliumPlateletsCoagulation Factors and Fibrinolytic Proteins

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Hemostasis has 2 main functions:

1. Induce a rapid & localized hemostatic plug at the site of vascular injury (clot formation)

2. Maintain Blood in a fluid, clot-free state after the injury is healed (clot dissolution)

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Primary Hemostasis

Injury

Endothelial Cells

Exposure of thrombogenic surface (subendothelial extracellular matrix)

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Platelets adhere and get activated

Change shape

Release secretory granules (e.g. ADP, TXA2)

Attract other platelets and Aggregate

Hemostatic plug or Primary Platelet Plug

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Secondary HemostasisFibrin is required to stabilize the primary platelet plugFibrin is formed by two coagulation pathways i.e. Extrinsic & IntrinsicExtrinsic Pathway is initiated when Tissue Factor (III) present in damaged organ comes in contact with Blood Intrinsic Pathway is initiated when Factor XII binds to a negatively charged “foreign” surface exposed to Blood

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Coagulation Factors

Factor Trivial Name Pathway

Prekallikrein Fletcher factor Intrinsic

HMWK Contact activation cofactor

Intrinsic

I Fibrinogen Both

II Prothrombin Both

III Tissue Factor Extrinsic

IV Calcium Both

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V Proaccelerin, Labile factor

Both

VI (Va) Accelerin Both

VII Proconvertin Extrinsic

VIII Antihemophilic factor A Intrinsic

XI Antihemophilic factor B Intrinsic

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XII Hageman Factor Intrinsic

XIII Fibrin Stabilizing factor Both

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PT and aPTT testing

PT (Prothrombin Time) test is done for deficiency of factors of extrinsic pathway

aPTT (activated Partial Thromboplastin Time) test is done for deficiency of factors of Intrinsic pathway

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Effects of Hereditary or Acquired Factor Deficiency on the PT & aPTT

aPTT prolonged, PT normal Deficiencies of intrinsic pathway Factor(s)

VIII, IX, XI or XII

PT prolonged, aPTT normalDeficiency of extrinsic Pathway factor VIIOccasionally, mild to moderate deficiency

of common pathway factor(s) fibrinogen, II, V or x

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Both PT and aPTT ProlongedDeficiency of common pathway factor(s)

fibrinogen, II, V, or X Multiple factor deficiencies

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Mixing studies in PT

Treat specimen with heparinase (degrades heparin)

PT normal, prolongation due to heparin PT prolonged

PT mixing study (1:1 mix of patient and normal plasma

PT normalizes

Factor Deficiency;Measure factorsI, II, V, VII, X

PT initially shortens and then prolongs

Factor V inhibitor (rare)

aPTT remains prolonged

Inhibitor (specific factor inhibitor, rare)

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Mixing studies in aPTT

Treat specimen with heparinase (degrades heparin)

aPTT normal, prolongation due to heparin aPTT prolonged

aPTT mixing study (1:1 mix of patient and normal plasma

aPTT normalizes

Factor Deficiency;Measure factorsVIII, IX, XI, XII

aPTT initially shortens and then prolongs

Factor VIII inhibitor

aPTT remains prolonged

Inhibitor, most commonly Lupus Anticoagulant

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Fibrinolytic MechanismStable Fibrin Clot

Activation of Protein C and Protein S

Secretion of t-PA by endothelial cells

Plasminogen Plasmin

Stabilized fibrin clot

X, Y fragments

Plasmin Degrades D-E-D fragments

E fragment + D dimer

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Fibrinolysis

As soon as the injury is healed clot dissolution starts, to restore the normal flow of Blood

Plasminogen is converted to the active form Plasmin by 2 distinct Plasminogen Activators (PAs):

tissue plasminogen activator (t-PA) from injured endothelial cells

Urokinase from Kidney endothelial cells and plasma

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Fibrinolysis

or Kallikrein from Intrinsic PathwayPlasminogen can also be activated by the

bacterial product (e.g. Streptokinase) – having significance in certain Bacterial Infections

Free Plasmin is neutralized by α2- plasmin inhibitor (PAI)

t-PA activity is also blocked by PAI

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Endothelial cells modulate the coagulation / anticoagulation balance by releasing PAIs

PAIs block fibrinolysis by inhibiting t-PA binding to fibrin as it is most active when bound to fibrin

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Fibrinolysis

Three types of Natural Anti-coagulants regulate clotting:

1.antithrombin III – inhibit thrombin activity and Factors IXa, Xa, XIa and XIIa

2.Protein C and Protein S – Vitamin K dependent proteins, inactivate Factors Va and VIIIa

3.Plasmin

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Fibrin Degradation Products or FDP’s include:

fragments X and Y – early splits and D and E – late splitsD-Dimer is the smallest cross-linked FDP

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D-Dimer are specific FDP formed only by Plasmin activity on fibrin clot and not on intact fibrinogen

Thus the presence of D-Dimer indicates that fibrin has been formed and so is a marker for an ongoing in vivo thrombotic condition

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Clinical Significance of Hemostasis

Hemophilia A: Caused by the deficiency of Factor VIII

Hemophilia B: Caused by the deficiency of Factor IX

Vitamin K deficiency(PIVKA’s) Protein Induced Vitamin K

Antagonism can be used in thrombotic conditions

Vitamin K dependent factors are II, V, IX, & X

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Liver DysfunctionFibrinogen and Factor XIII deficiencyFactor XI and Contact Activation Antithrombin Deficiency leads to DVT and

PEvon Willebrand Disease: Deficiency of von

Willebrand Factor

Clinical Significance of Hemostasis

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DIC (Disseminated Intravascular Coagulation)

Massive Injury or Sepsis

Massive release of Tissue Factor III

Excessive Activation of Thrombin

Coagulation becomes systemic

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High consumption of Platelets, coagulation factors

Over production of fibrin clot

Fibrin clot “disseminates” or spreads throughout the microcirculation

Obstructing the blood flow to capillaries, smaller vessels

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Lack of blood supply leads to tissue injury (decreased oxygenation, organ infarction

& necrosis)

Once again release of Tissue Factor

Second time coagulation activation

More consumption of coagulation factors and platelets

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Continuous thrombi formation

Production capacity of Bone Marrow and Liver reaches its maximum level

Activation of fibrinolysis at first site

High consumption of Plasmin, antithrombin, Protein C and Protein S

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Generation of Thrombin & Plasmin at the same time

Plasmin acts on Fibrin Clots and produces FDPs and D-Dimer

FDPs interfere with platelet function and impair fibrin clot formation

Further bleeding results

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Thus an initial thrombotic disorder gets converted into a serious bleeding disorder

Whenever there is a widespread activation of Thrombin the chances are that it may lead to DIC

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Pharmacological Intervention

Most commonly patients are on OAC (oral anti-coagulant) therapy:

warfarin – over dose can lead to Vit. K deficiency

heparin

Fibrinolysis: AspirinStreptokinase, urokinase injectionst-PA injections

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INR & ISI values

All PT reagents are calibrated against WHO IRP (International Reference Preparation)

International Normalisation Ratio is intended to make comparison similar irrespective of the type of PT reagent used worldwide

International Sensitivity Index is a measure of the sensitivity of particular PT reagent

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Insensitive PT reagents will give prolonged results only when factor levels are very low

Sensitive PT reagent give prolonged results even when there is a mild change in factor level

Insensitive PT reagents have higher ISI values

Sensitive PT reagents have ISI value as close to 1.0 as possible

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Calculation of PT results

INR = (Ratio)ISI

Patient PT Time (secs) Mean Normal PT (MNPT)

MNPT = Mean PT Time of at least 20 known normal samples

INR =

ISI

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Thanks

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Priyank Dubey Ph: 09999990845priyank.exp@gmail.com Application Specialist