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A Practical Guide to Haemostasis


Thrombin Time

Introduction

The Thrombin Time (or Thrombin Clotting Time) is a widely performed test although not necessarily part of the basic screening profile.

Principles

The Thrombin Time involves only the addition of Bovine or Human Thrombin to citrated platelet poor plasma [PPP]. 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, whilst a secondary cleavage product, Fibrinopeptide B is produced by cleavage at amino acid 14.

Strucutre of Fibrinogen


Methodology

Human Thrombin (or Bovine Thrombin) is added to platelet poor plasma [PPP] 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 - shown in blue.


Clotting cascade highlighting the key role of Thrombin and the proteins invovled in the Thrombin Time

and more diagrammatically...

Illustration of the Thrombin Time


Interpretation

The Thrombin time will, in general be prolonged when functional Fibrinogen levels are <1.0 g/L.

Abnormality Interpretation
Inherited causes of a decreased Fibrinogen that lead to a prolonged Thrombin Time 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
Acquired causes of a decreased Fibrinogen that lead to a prolonged Thrombin Time 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 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 e.g. Dabigatran.
Dabigatran Conventional Thrombin Times are very sensitive to the presence of Dabigatran with >10-fold prolongation at peak levels.
A normal Thrombin Time can be used to exclude the presence of clinically relevant levels of Dabigatran.

Apixaban and Rivaroxaban do not prolong the Thrombin Time.
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, then a human-based Thrombin Time 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 of 13-15s.


Modifications of the Thrombin Time


1. The High Dose Thrombin Time [HiTT].

The HiTT is a modified Thrombin Time that uses a higher concentration of Thrombin to assay UFH at the doses employed 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 Activated Clotting Time [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.

2. Modifications of the Thrombin Time to measure the Direct Thrombin Inhibitors [DTIs]:
  i. Hemoclot® Thrombin Inhibitor Assay
  ii. Quantitative Thrombin Time [QTT]
  iii. HemosIL Dilute Thrombin Inhibitor [DTI] assay

i. The HemoclotŪ 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 a 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.

ii. The Quantitative Thrombin Time [QTT] is very similar and involves a 1:10 dilution of patient plasma with human Fibrinogen and clotting is then initiated by the addition of human Thrombin. A standard curve is generated using dilutions of the relevant anticoagulant e.g. Dabigatran, Argatroban - added to a normal plasma pool and measuring the clotting times.  From this the concentration of the relevant DTI by comparison to a reference curve, can be derived.
The QTT appears insensitive to heparin, lupus anticoagulants, low fibrinogen, clotting factor concentrations and FDPs and appears to be suitable for monitoring the DTIs.

iii. The HemosIL DTI Assay is a modified Thrombin time test performed on citrated patient plasma. Patient plasma is diluted in pooled normal plasma to minimise pre-analytical variables. A fixed concentration of bovine Thrombin is added to the diluted patient sample. The presence of Dabigatran in the patient sample will have an inhibitory effect on the activity of the exogenous Thrombin. The clotting time is recorded and from comparison with a Dabigatran reference curve constructed using a known reference plasma standard, the concentration of Dabigatran in the patient plasma sample can be established.


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

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 in the laboratory. The Reptilase Time is frequently used to exclude contamination with unfractionated heparin in cases in which there is a grossly prolonged Thrombin Time.

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

 

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.

Cause
Thrombin Time
Reptilase Time
Unfractionated heparin ↑   Normal
LMWHs May show some prolongation Normal
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

 

The Figure below may help with the investigation of a prolonged Thrombin Time.

Investigation of a Prolonged APTT