Category Archives: COAGULATION PROFILE

D-DIMER AND FIBRINOLYTIC SYSTEM


Fibrinolysis is the enzymatic process used by the body to remove a fibrin thrombus to restore normal blood flow once damaged endothelium is repaired.
During the clotting process, tissue plasminogen activator (t-PA) released from the blood vessel wall and the plasma proenzyme plasminogen bind to the fibrin thrombus. When activated, plasminogen is converted to plasmin which degrades the
fibrin network, causing the clot to dissolve. During this process, fibrin degradation products (FDPs), i.e. fragments called D-Dimers are produced.

Raised FDP levels in DIC

In Disseminated intravascular coagulation (DIC), activated procoagulants are released into the circulation. Platelets and coagulation factors are consumed and fibrin is deposited in small vessels, activating the fibrinolytic system. The plasmin formed degrades the fibrin (also some fibrinogen), resulting in a build-up of FDPs in the circulation. The FDPs act as anticoagulants interfering with platelet function and fibrin stabilization.
Laboratory tests are available to semi-quantify D-DIMER in plasma.

THROMBIN TIME (TT) TEST PROCEDURE

(Courtesy of Diagen Thrombin Test Time Kit. Test )

Test the patient’s plasma and
control plasma in duplicate.

  1. Pipette 200 l (0.2 ml) of plasma into a small glass tube. Incubate at 37°C for 1–2 minutes.
  2. Add (0.1 ml) of thrombin, mix and start the stop-watch. Hold the tube in the water bath and tilt the mixture back and forth, looking for clot formation. When a clot forms, stop the stopwatch and record the time in seconds.
  3. Report the patient’s TT (average of the duplicate tests) providing the TT of the control plasma is satisfactory.

What are the normal ranges for TT?

12–15 seconds

What are causes of prolonged TT?

  • DIC and other conditions which produce a low fibrinogen level
  • Abnormal fibrinogen
  • Treatment with heparin
  • Liver failure
  • Presence of inhibitors of thrombin such as FDPs

See also:

TT test principle

THROMBIN TIME (TT) TEST

Thrombin time (TT) test
The TT test is sensitive to a deficiency of fibrinogen or inhibition of thrombin. It measures the formation of a fibrin clot by the action of thrombin on fibrinogen.

Principle of test
Thrombin is added to citrated plasma at 37°C. The time taken for the mixture to clot is measured and the appearance of the
clot noted.

Reagent
The use of a thrombin time test kit is recommended.
This provides the correct concentration of thrombin to use in the test, i.e. that which gives a clotting time of 12–15 seconds with pooled normal plasma.

Collect venous blood into citrate
anticoagulant and centrifuge to obtain platelet poor plasma as described for the APTT test. Perform the test with as little delay as possible.

See also:

Procedure for TT test

PROTHROMBIN TIME (PT) TEST


The PT is a screening test for the extrinsic clotting system, i.e. factor VII. It will also detect deficiencies of factors, prothrombin, V, X, and fibrinogen. It is mainly used to monitor patients receiving warfarin
anticoagulation.

Principle of the test

Plasma or capillary blood is added to a thromboplastin and calcium chloride reagent at 37°C and the time taken for a clot
to form is measured. The clotting time in seconds is converted to the International Normalized Ratio (INR), usually by reference to a table provided by the manufacturer of the reagent OR by the formula:
INR= (PT PATIENT/PT CONTROL)^ISI
ISI is provided by reagent manufacturer

Reagents

Thromboplastin calcium chloride combined reagent.
Several different thromboplastin calcium combined reagents are available depending on the source of the thromboplastin and whether the test is to be performed using plasma or capillary whole blood. Some manufacturers supply a thromboplastin calcium reagent that can be used with both capillary blood and plasma.

Capillary blood PT testing:

This avoids the need to collect venous blood when monitoring patients being treated with warfarin. It should not be used
however when a patient is anaemic or polycythaemic. Plasma should then be used.

See also

Prothrombin time test procedure

PROTHROMBIN TIME (PT) TEST PROCEDURE

  1. Pipette 0.25 ml of the thromboplastin/calcium reagent into a small glass tube. Place in a 37°C water bath for 1–2 minutes.
  2. Using a calibrated capillary or delivery pipette, add (0.05 ml) of capillary blood or plasma, mix, and start the stop-watch. Hold the tube in the water bath and tilt the mixture back and forth looking for clot formation. When a clot forms, stop the stop-watch and record the time
  3. Convert the clotting time to the INR using the table provided by the manufacturer. Separate INR tables are provided for capillary blood and plasma.

The INR conversion table provided by the manufacturer is specific for The batch of thromboplastin supplied with it.

What is the normal range for Prothrombin time?

11 – 16 seconds

What are Causes of Prolonged PT?

  • Treatment with oral anticoagulant drugs (vitamin K antagonists) such as warfarin.
  • Liver disease
  • DIC
  • Haemolytic disease of the newborn
  • Rarely a deficiency of factor VII, X, V, or prothrombin.

See also

The principle of prothrombin time test

DISORDERS OF BLOOD COAGULATION

Can result in uncontrolled haemorrhaging into joints, muscles and deep tissues with the formation of haematoma. Coagulation disorders may be:

Hereditary

  • haemophilia A due to a deficiency of clotting factor VIII (commonest hereditary coagulation disorder),
  • haemophilia B (Christmas disease) due to a deficiency of factor IX
  • Von Willebrand’s disease caused by a deficiency or abnormality of the von Willebrand factor resulting in a defect in platelet adhesion. Haemophilia is carried by the female and affects males.

Acquired

  • Vitamin K deficiency
  • Severe liver disease, and disseminated intravascular haemolysis (DIC) associated with infections.
  • Obstetric complications (septic abortion, eclampsia, fetal retention, ruptured uterus).
  • Haemorrhagic disease of the newborn.
  • Snake envenomation.
  • Malignancies.
  • Overdose of anticoagulant drugs, e.g. warfarin.

Laboratory investigations

ACTIVATED PARTIAL THROMBOPLASTIN TIME (APTT)

The APTT is a screening test of the intrinsic clotting system. It will detect the inhibition or deficiency of one or more of the following factors: prothrombin, V, VIII (antihaemophilic factor), IX, X, XI, XII and fibrinogen. The APTT is also used to monitor patients being treated with heparin.

Principle of test

Kaolin (surface activator) and platelet substitute (phospholipid) are incubated with citrated plasma at 37°C for the time
specified in the test method. Calcium chloride is added and the time taken for the mixture to clot is measured.

Reagents

Kaolin/platelet substitute mixture
Purchase from a reliable manufacturer in lyophilized form and reconstitute as instructed.

Calcium chloride, 0.025 mol/l (25 mM)
This is best obtained ready made unless the laboratory has facilities to make the reagent accurately and standardize it.

See also:

APTT TEST PROCEDURE

APTT MEASUREMENT USING PLATELET SUBSTITUTE MIXTURE

Test the control plasma and patient’s plasma in duplicate.

1. Pipette 0.2 ml of well-mixed kaolin/platelet substitute in a small glass tube.

2. Add 0.1 ml of plasma, mix, and incubate at 37° C for exactly 2 minutes (tilting the tube at intervals).

3. Add 0.1 ml 0.025 mol/l calcium chloride, mix and start the stop-watch. Hold the tube in the water bath and tilt the mixture back and forth, looking for clot formation. When a clot forms, stop the stop-watch and record the time.

4. Report the patient’s APTT (average of the duplicate tests) providing the APTT of the normal control plasma is satisfactory.

Reference APTT range

36–50 seconds

What causes prolonged APTT?

  • DIC (involving several clotting factors)
  • Deficiency of clotting factors: prothrombin, V, VIII, IX, X, XI or XII due to vitamin K deficiency, liver disease, heparin or warfarin anticoagulation, or less commonly an inherited coagulation disorder.

See also:

The Principle of APTT test