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.)
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.
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.
|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.
The Thrombin time will, in general be prolonged when functional fibrinogen levels are <1.0 g/L.
|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
Following thrombolytic therapy
|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
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
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 to 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.
Hemoclot® Thrombin Inhibitor Assay
The Hemoclot® Thrombin Inhibitor Assay is a quantitative, clotting-based assay for the measurement of Hirudin and other direct thrombin inhibitors [DTI] including Argatroban and Dabigatran. The assay involves mixing pre-diluted test plasma [the precise dilution depending upon the predicted concentration of the DTI e.g. High concentration then dilute 1:20, low concentration then dilute 1:8] with a normal human plasma pool. Clotting is then initiated by adding a fixed but excess of α-thrombin and measuring the clotting time. The time to clot formation is directly proportional to the concentration of the direct thrombin inhibitor present in the plasma. A calibration curve is constructed from a series of plasma calibrants of known DTI [e.g. Dabigatran] concentration. On linear graph paper, the concentration of the DTI is plotted on the X-axis and the clotting time in seconds, on the Y-axis. From this graph the concentration of the DTI can be determined.
Summary of Correction Tests with Normal Plasma, Toludine Blue and Protamine Sulphate
|Thrombin Time - Corrects with:|
|Normal Plasma||Toludine Blue||Protamine Sulphate||Interpretation|
|Variable||No||Yes||High levels of FDPs|
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.
|Presence of unfractionated heparin||↑||Normal|
|Presence of LMWH||May show some prolongation||Normal|
|Presence of direct thrombin inhibitors||↑||Normal|
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