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Tests to measure fibrin clot. General Approach in Investigation of Haemostasis. Lecture 9:. Tests to Measure Fibrin formation. Thrombin Time TT Reptilase Time Fibrinogen Activity assay. Thrombin Time (TT). Also Called Thrombin Clotting Time (TCT).

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tests to measure fibrin clot

Tests to measure fibrin clot

General Approach in Investigation of Haemostasis

Lecture 9:

tests to measure fibrin formation
Tests to Measure Fibrin formation

Thrombin Time TT

Reptilase Time

Fibrinogen Activity assay

thrombin time tt
Thrombin Time (TT)

Also Called Thrombin Clotting Time (TCT)

  • The thrombin time (TT) is the time required for thrombin to convert fibrinogen to an insoluble fibrin clot.
  • It does not measure defects in the intrinsic or extrinsic pathways.
  • The test is affected by
    • Abnormal levels of fibrinogen (usually less than 100 mg/dl.) and dysfibrinogenemia
    • The presence of antithrombins such as heparin and direct thrombin inhibitors such as hirudin and FDPs.
principle of tt
Principle of TT

Commercially prepared bovine thrombin reagent at 2 NIH units/mL cleaves fibrinopeptides A and B from plasma fibrinogen to form a detectable polymer

1 WHO unit = 0.56 NIH unit

1 NIH unit = 0.324 +/- 0.073 µg

reptilase atroxin time
Reptilase (Atroxin)Time
  • The Reptilase time is a modification of the thrombin time in which the purified enzyme Reptilase is used to replace thrombin.
  • It is a thrombin-like enzyme, isolated from the venom of Bothropsatrox, that catalyzes the conversion of fibrinogen to fibrin in a manner similar to thrombin.
  • Reptilase cleaves fibrinogen releasing fibrinopeptide A (FpA) generating fibrin. In contrast thrombin cleaves both fibrinopeptide A and fibrinopeptide B from fibrinogen to generate the fibrin clot.
  • All the congenital dysfibrinogenemias have an infinite reptilase time.
  • The reptilasetime is also infinitely prolonged in cases of congenital afibrinogenemia.
  • In states of hypofibrinogenemia, the reptliasetime may be variable, depending on the levels of fibrinogen present.
  • The reptilase time is moderately prolonged in the presence of FDPs and is unaffected by heparin

In the presence of heparin, thrombin is inhibited through the interaction of antithrombin (AT-III). However, heparin does not interfere with the ability of reptilase to cleave fibrinopeptide A from fibrinogen

Ancrod a similar enzyme from Agkistrodonrhodostoma can also be used to replace thrombin in the thrombin clotting time test.

  • Fibrinogen concentration can be measured in 3 ways. Fibrinogen concentration is usually reported in milligrams per deciliter (mg/dl).
    • Heat precipitation:(Heating at 56o C will precipitate fibrinogen only)
    • Clotting method - thrombin clot time:
    • Immunologic assays: Fibrinogen antigen levels may be assayed by means of radial immunodiffusion (RID) or nephelometry
fibrinogen activity test rolf greiner biochemica
Fibrinogen activity test (Rolf Greiner // Biochemica)


  • Fibrinogen assays are quantitative techniques to measure the amount of functional fibrinogen present in the plasma.
  • The assay is based on the Clauss assay, which is the reference method.
  • This fibrinogen assay measures the time required for thrombin to convert fibrinogen to fibrin.

The procedure is a determination based on fibrinogen activity, but results are converted to concentration (mg/dL) by comparison with control plasma results.

  • In the fibrinogen procedure, thrombin is added to various dilutions of known concentrations of fibrinogen to produce a thrombin-clotting time in seconds.
  • The clotting times are then plotted on a graph, with the known concentrations on the x-axis, versus the clotting time on the y-axis.
  • The clotting times are performed using controls and the patient sample at a 1:10 dilution.
  • An excess amount of thrombin reagent is added and the time it takes for the specimen to clot is recorded in seconds.

This time is then converted to mg/dL of fibrinogen by comparing these results to results obtained on a fibrinogen standard curve.

  • Patient results may be read directly off of the standard curve graph, or off of a data chart prepared from the graph that already converts time in seconds to mg/dL.
  • The time it took for the specimen to clot is inversely proportional to the fibrinogen concentration in mg/dL.
  • For instance, a prolonged fibrinogen clotting time means the fibrinogen level (mg/dL) is low.
reagents and equipment
Reagents and Equipment
  • Test tubes
  • Commercial fibrinogen determination kit:
      • Thrombin, 100 National Institutes of Health (NIH) units/mL, bovine lyophilized (reconstitute with 2 ml Distilled water)
  • Fibrinogen standard
  • Imidazole buffer,
  • Control (with a known fibrinogen concentration)
  • Collect blood by clean venipuncture technique according to recommended procedures previously described.
  • Process and store plasma samples following recommended guidelines.
  • Reconstitute the thrombin reagent according to the manufacturer's directions.
  • This assay is commonly performed on a coagulation analyzer.

Preparation of Calibration Curve

The calibration curve is prepared from the reference standard .

Make dilutions of the fibrinogen standard with Imidazole buffer as follows: 1:5, 1:10, 1:20 and 1:40. Make all transfers from the first test tube.


Perform determinations on each dilution of the fibrinogen standard as follows:

  • Incubate 0.1 mL of fibrinogen standard dilution at 37°C for at least 2 minutes but no more than 5 minutes.
  • Add 0.05 mL of thrombin reagent.
  • Measure the clotting time. performed in duplicate, and average the results.
sample assay
Sample Assay**
  • Prepares a 1: 10 dilution of each patient PPP and control with Imidazole buffer.
  • Incubate 0.1 ml of the patient dilution at 37°C for at least 2 minutes but no more than 5 minutes.
  • Add 0.05 mL of thrombin reagent.
  • Measure the clotting time. run in duplicate.
  • Reference range: 200-400 mg/dL
  • Prolonged clotting times may indicate either
      • A low fibrinogen concentration
      • The presence of inhibitors such as heparin or circulating FDPs.
  • Some manufacturers include a heparin neutralizer in the fibrinogen reagent that will negate any interference by therapeutic levels of heparin.

The effect of heparin may also be excluded by

      • treatment of the sample with a heparin-digesting enzyme
      • performing the reptilase time, because reptilase is unaffected by heparin.
clinical significance
Clinical Significance:
  • There are several causes for a deficiency of fibrinogen.
    • Severe hemorrhaging may result in any case.
    • congenital deficiencies may be due to **
      • Afibrinogenemia (a lack of fibrinogen)
      • a dysfibrinogenemia (abnormal fibrinogen)
    • Acquired deficiencies may be due to
      • liver disease
      • disseminated intravascular coagulation (DIC)
      • fibrinolysis

High fibrinogen levels are seen

    • During pregnancy
    • In women taking oral contraceptives.
    • In patients in a hypercoagulable state such as with thrombosis.
    • Fibrinogen is considered an acute-phase reactant, and, therefore, high levels may be seen in states of acute infection, neoplasms, collagen disorders, nephrosis, and hepatitis along with other conditions causing physical stress.
  • For fibrinogen values out of the linearity range (46-700 mg/dL for this fibrinogen standard curve) a 1:10 dilution of the plasma will not work and a different dilution must be used.
      • For extremely high fibrinogen levels (>700 mg/dL) a 1:20 dilution of the plasma is used for the procedure. However, due to the change in dilution, the result read off of the fibrinogen data table must be multiplied by a factor of 2 (since our 1:20 dilution is 2 times the 1:10 dilution originally meant for the data table).
      • For extremely low fibrinogen levels (<46 mg/dL) a 1:5 dilution of the plasma is used for the procedure. The result read off of the data table must then be divided by a factor of 2 (since our 1:5 dilution is half of the 1:10 dilution originally meant for the data table).