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A Practical Guide to Laboratory Haemostasis
 

Thrombin Time


Introduction

The thrombin time (or thrombin clotting time) is widely performed test 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). The primary cleavage product, fibrinopeptide A is cleaved from fibrinogen after amino acid 16 and sometimes after amino acid 19, while a secondary cleavage product, fibrinopeptide B is produced by cleavage at amino acid 14.

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 the plasma is not necessary.

Reagent Explanation
Platelet Poor Plasma (PPP) See pre-analytical variables
Human or bovine thrombin (IIa) Historically bovine thrombin was used in this test but this is now frequently replaced by human thrombin

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 <1.0 g/L.

Abnormality Interpretation
Decreased Fibrinogen Congenital deficiencies of fibrinogen
Afibrinogenaemia or hypofibrinogenaemia.
Dysfibrinogenaemia (a dysfunctional fibrinogen) which may be present in normal or reduced amounts e.g. a hypo-dysfibrinogenaemia
Decreased Fibrinogen Acquired deficiencies of fibrinogen
DIC
Following thrombolytic therapy
Liver disease
Malignancy
Some anticoagulants will prolong the thrombin time Unfractionated heparin
(Low Molecular Weight Heparins (LMWHs) do not usually lead to prolongation of the thrombin time but may do so in if present in very high concentration e.g. an overdose
Hirudin
Argatroban

Warfarin has no effect upon the thrombin time
The thrombin time is not a recommended test for monitoring direct thrombin inhibitors.
Elevated levels of Fibrin(ogen) Degradation Products (FDPs) These interfere with fibrin polymerisation and can at high concentration lead to a prolonged thrombin time
Paraproteins May interfere with fibrin polymerisation leading to a prolonged thrombin time
Hypo-albuminaemia This can result in a prolongation of both the thrombin time and the reptilase time. The prolongation appears to be an in vitro phenomenon and can be corrected by raising the albumin concentration in vitro which corrects the prolonged thrombin and reptilase times.
These patients do not appear to be at increased risk of bleeding and there is some evidence that they may have hyperaggregable platelets rendering them at increased risk of thrombosis.
Amyloidosis Prolongation of the Thrombin time and the Reptilase time has been observed in patients with amyloidosis due to the inhibition of the conversion of fibrinogen to fibrin.
Following the use of bovine thrombin Patients exposed to bovine thrombin may develop inhibitors that prolong the bovine-based thrombin time. If the antibody cross-reacts with human thrombin, human-based thrombin times can also be prolonged.
The Reptilase time is normal with these inhibitors.
Pathological anticoagulants Heparin-like anticoagulants have been reported (rarely) in patients with malignancies or other disorders, leading to a prolonged thrombin time but a normal Reptilase time.
Hyperfibrinogenaemia Hyperfibrinogenaemia can on occasion be associated with a prolonged thrombin time (and reptilase time). The mechanism is unclear but may reflect interference with fibrin assembly by excess fibrinogen
Fetal fibrinogen The thrombin time in the neonate is often prolonged due to the presence of a fetal fibrinogen.
It is important to remember when undertaking haemostatic investigates in the neonate and in children to use the appropriate reference ranges

 

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.

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.

Summary of Correction Tests with Normal Plasma, Toludine Blue and Protamine Sulphate

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

Toludine blue is a dye that binds to and inhibits heparin - it is rarely used today. Protamine sulphate is a highly cationic protein that binds to unfractionated heparin neutralising its anticoagulant activity. In practice these correction reactions are rarely performed. The reptilase time is frequently used to exclude contamination with unfractionated heparin in cases in which there is a grossly prolonged thrombin time.

What Test Next

The following table above summarises the causes of a prolonged thrombin and/or reptilase time. This can be useful to guide what may be the next most appropriate investigation in a clinical scenario.

 
Thrombin Time
Reptilase Time
Presence of unfractionated heparin ↑   Normal
Presence of LMWH May show some prolongation Normal
Presence of direct thrombin inhibitors ↑ Normal
Warfarin Normal Normal
Decreased/absent fibrinogen ↑ ↑
Dysfibrinogenaemia ↑ ↑
DIC ↑ ↑
Liver disease ↑ ↑
Heparin-like anticoagulants ↑ Normal
Paraproteinaemias ↑ ↑
Thrombolytic therapy ↑ ↑
Neonate ↑ ↑
Amyloid ↑ ↑
Hyperfibrinogenaemia ↑ ↑
Hypoalbuminaemia ↑ ↑


Data Interpretation

Click HERE to go to the Data Interpretation Exercises.

Comments

1. Monitoring of unfractionated heparin using the thrombin time.

The thrombin time is a clot-based assay and historically was used by some laboratories to monitor patients receiving unfractionated heparin.
This 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.
However, the thrombin time is often too sensitive to monitor heparin anticoagulation and the assay is not standardized for this purpose. A recalcified thrombin time can be used when monitoring heparin therapy but the recommendations are to use the APTT to monitor UFH.

2. 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). In some patients in whom the APTT is already prolonged before going onto CPG e.g. severe Factor XII deficiency, the ACT can be misleading. In these situations measuring anti-Xa levels may be of value.

3. 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).

4. 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 Toludine blue test.

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

References

1. 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.

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

3. 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.

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

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

6. 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.

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Updated: 09-Nov-2011