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The thrombin time (or thrombin clotting time) are widely performed tests although not necessarily part of the basic screening profile. The Reptilase Time is a frequently performed test and is similar to the thrombin time but utilises a different activator (a snake venom rather than thrombin.)

Principles

The thrombin time involves only the addition of bovine or human thrombin to platelet poor plasma. It, therefore, reflects the conversion of fibrinogen to fibrin but is also sensitive to the presence of inhibitors that may be present in the plasma e.g. heparin.

Thrombin cleaves fibrinogen, releasing fibrinopeptide A (FpA) and fibrinopeptide B (FpB) from fibrinogen and converting fibrinogen into a fibrin clot.

Method

Human thrombin (or bovine thrombin) is added to platelet poor plasma at 37°C and the time taken for the formation of a fibrin clot recorded. Recalcification of plasma is not necessary

The diagram below shows the stages involved in the Thrombin Time.

Interpretation

The Thrombin time will, in general be prolonged when functional fibrinogen levels are <1g/L.

Reference Ranges

Each laboratory must establish its own reference range but in general, the reference range for the thrombin time is in the region on 13-15s.

Comments

1. Monitoring of unfractionated heparin by thrombin time.

The thrombin time is a clot-based assay and historically was used by some laboratories to monitor unfractionated heparin therapy.

This assay is performed by adding a known concentration of thrombin to platelet-poor plasma and measuring the time to clot formation. Under the appropriate assay conditions, heparin produces a dose-dependent prolongation of the thrombin time, which is semi-logarithmic.

The thrombin time is often too sensitive to monitor heparin anticoagulation and the assay is not standardized for this purpose. It is recommended that a recalcified thrombin time be used when monitoring heparin therapy.

For patients on cardio-pulmonary bypass and receiving unfractionated heparin the high concentrations of heparin (8-10 IU/mL) mean that both the thrombin time and the APTT are unclottable and other methods must be used to monitor the degree of anticoagulation. This is usually the Activated Clotting Time (ACT).

2. The thrombin time is often used to establish the presence or absence of unfractionated heparin in a sample prior to undertaking more complex tests of coagulation.

In a sample in which the thrombin time is prolonged, a normal reptilase time would (in most cases) be consistent with the presence of heparin unfractionated heparin).

3. Other tests that can be used to establish the presence of unfractionated heparin in a sample with a prolonged thrombin time include:

The protamine sulphate correction test
The Toluidine blue test.

Protamine sulphate is a positively charged reagent which neutralises heparin (and FDPs). In contrast, Toluidine Blue neutralises heparin but not FDPs. Toluidine Blue will also correct the prolonged thrombin time in some cases of dysfibrinogenaemia.

Thrombin Time - correction with			Interpretation
Normal Plasma	Toludine Blue	Protamine Sulphate
No	Yes	Yes	Heparin present
Yes	No	No	Fibrinogen deficiency
Variable	No	Yes	High levels of FDPs
Variable	No	Yes	Some dysfibrinogenaemias

Specific enzymes that neutralise heparin e.g. heparinase can also be used to establish the presence (or absence) of heparin.

High-Dose Thrombin Time [HiTT] 

The HiTT is a modified thrombin time that uses a larger dose of thrombin to assay UFH at the doses used during cardio-pulmonary bypass [CPB.] The HiTT assays the final common pathway of coagulation i.e. the conversion of fibrinogen to fibrin and may, therefore, be less susceptible to the variables that affect the ACT. In contrast to the ACT, the HiTT test appears to be unaffected by antifibrinolytic drugs, hypothermia, haemodilution, a minor decrease in fibrinogen or the high levels of fibrin degradation products (FDPs). However, the test cannot be used to measure baseline values in a non-anticoagulated samples as the high thrombin concentration results in such a short clotting time that it is unmeasurable. This limitation can be overcome by performing a standard Thrombin Time that contains a low thrombin concentration.

What Test Next?

The following table summarises the abnormalities of the thrombin time and reptilase time. This can guide the the most appropriate investigation

The finding of a prolonged thrombin time should raise the possibility of heparin - therefore look at the APTT and consider performing a reptilase time.

The table above and the comments section - list the possible causes of a prolonged TT and therefore, a logical way path of investigation.

Useful Links & References

For a discussion of fibrinogen structure and sites of the thrombin (and plasmin) cleavage sites - see: www.ebi.ac.uk/interpro/potm/2006_11/Page1.htm.

1. Krammer, B., et al., Screening of dysfibrinogenaemia using the fibrinogen function versus antigen concentration ratio. Thromb Res, 1994. 76(6): p. 577-9.

2. Lee, M.T., et al., Transient hemorrhagic diathesis associated with an inhibitor of prothrombin with lupus anticoagulant in a 1 1/2-year-old girl: report of a case and review of the literature. Am J Hematol, 1996. 51(4): p. 307-14.

3. Abshire, T.C., et al., The prolonged thrombin time of nephrotic syndrome. J Pediatr Hematol Oncol, 1995. 17(2): p. 156-62.

4. Gandrille, S., et al., A study of fibrinogen and fibrinolysis in 10 adults with nephrotic syndrome. Thromb Haemost, 1988. 59(3): p. 445-50.

5. Toulon, P., et al., Fibrin polymerization defect in HIV-infected patients--evidence for a critical role of albumin in the prolongation of thrombin and reptilase clotting times. Thromb Haemost, 1995. 73(3): p. 349-55.

Data Interpretation

Click HERE to go to the Data Interpretation Exercises.