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A Practical Guide to Laboratory Haemostasis
 

Prothrombin Time [PT]


Introduction

The prothrombin time was described by Quick in 1935 and the test was often referred to as 'Quick's Prothrombin Time.' The prothrombin time was developed to measure Prothrombin (Factor II) and hence its name. However, it subsequently became clear that it was sensitive to abnormalities of factors VII, X, V, II and fibrinogen.
The Prothrombin Time (PT) in contrast to the APTT measures the activity of the so-called extrinsic and common pathways of coagulation. The division of the clotting cascade into the intrinsic, extrinsic and common pathways is medieval and has little in vivo validity but nevertheless remains a useful concept for interpreting the results of laboratory investigations.
The prothrombin time is a one-stage test based upon on the time required for a fibrin clot to form after the addition of Tissue Factor (TF) (historically known as tissue thromboplastin), phospholipid and calcium to decalcified, platelet poor plasma.
The term 'Thromboplastin' was originally used to describe a substance in plasma that converted prothrombin to thrombin. Historically thromboplastins were extracted from brain and other organs and these contained significant amounts of tissue factor [TF] and phospholipid [Pl]. TF is species specific and most laboratories now use a recombinant human TF with an ISI close to 1 and which is relipidated to provide a source of phospholipid. Animal thromboplastins are usually derived from rabbit brain.
TF was originally designated Factor III when the nomenclature of the clotting proteins was undertaken.

Principles

The PT measures the activity of the so-called extrinsic and common pathways of coagulation and therefore, is dependent on the functional activity of factors VII, X, V, II (Prothrombin) and fibrinogen. The diagram below shows the clotting cascade and the factors that affect the prothrombin time.

Method

Platelet poor plasma [PPP] is mixed with Tissue Factor (TF) (containing phospholipid) at 37°C and an excess of calcium chloride (25mM) is added to initiate coagulation. In the manual technique at the same time as the calcium is added, a stopwatch is started and stopped when the clot forms. The time taken from the addition of calcium to the formation of the fibrin clot is known as the Prothrombin Time or PT. In an automated system the formation of the clot is detected electronically but the principles are identical.

Reagent Explanation
Platelet Poor Plasma (PPP) See pre-analytical variables
Tissue Factor [containing phospholipid] Tissue Factor binds to FVII and initiates coagulation.
Exogenous phospholipid is used to replace platelet phospholipid

Calcium Required for re-calcification

 

Interpretation

The PT is usually performed as part of a series of tests which will include the APTT and sometimes the measurement of fibrinogen levels and possibly a thrombin time.

Abnormality Interpretation
Isolated Prolonged PT Factor VII deficiency
Prolonged PT in association with other coagulation abnormalities Vitamin K deficiency
Vitamin K antagonists e.g. warfarin, phenindione, rodenticides
Liver disease
Malabsorption (leading to vitamin K deficiency)
High concentrations of unfractionated heparin
Direct thrombin inhibitors e.g. Lepirudin, argatroban
Afibrinogenaemia and dysfibrinogenemia
Dilutional coagulopathy e.g. massive blood transfusion
Multiple clotting factor deficiencies e.g. FV and FVIII deficiency
Abnormalities of the vitamin K cycle e.g. mutations within the VKORC1 gene
Chromosomal aberrations - the F7 and F10 genes are located on the long arm of chromosome 13 - deletions of which are associated with reduced FVII and FX levels.
Shortened PT Following treatment with rVIIa

 

Reference Ranges

The reference range depends upon a number of variables including:
 - Source of Tissue Factor e.g. human, rabbit etc
 - The exact technique used e.g. manual or automated
 - Method of end-point determination e.g. optical or mechanical

Each laboratory should establish its own normal range but in general the prothrombin time for a normal plasma sample, lies between 13-15 seconds.

What Test Next

In cases in which there is an isolated prolongation of the PT and the remainder of the screening tests (APTT, TT and Fibrinogen) are normal - the next most logical test is a Factor VII assay.
Factor VII deficiency is rare and it is more common to find a prolonged PT in combination with other abnormalities of the screen e.g. a prolonged APTT. In these cases - consult the table above for the possible differential diagnoses and therefore, how to proceed. The history including a drug history and the examination are of fundamental importance
Remember - Warfarin and other oral Vitamin K antagonists will significantly prolong the PT but may prolong the APTT by only a few seconds (except in overdose)

Data Interpretation

Click HERE to go to the Data Interpretation Exercises.

Comments

1. The Prothrombin time forms the basis for monitoring oral vitamin K antagonists e.g. warfarin and phenindione (see INR).

2. The PT can be used as a screening test for lupus anticoagulants [LA] (see Tissue Thromboplastin Inhibition Test.) In general, however, the PT is relatively insensitive to a LA as the high PL concentration in the assay neutralises the anti-PL antibody (i.e. the LA).

3. The prothrombin time forms the basis for the 1-stage assay used for assaying factors VII, V, X and II.
The PT can be relatively insensitive to minor reductions in some clotting factors.

4. Some FVII variants (FVII Padua, FVII Yamomoto) can give rise to differing FVII assay results depending upon the source of the tissue factor (see FVII assays).

5. A normal PT does not exclude a significant underlying coagulopathy e.g. the PT is normal in severe haemophilia A, B and factor XI deficiency.

6. The relationship between the PT and a factor deficiency is not linear but prolongs exponentially at lower factor concentrations.

7. A shortened PT can be seen in some/many patients following the administration of rVIIa [NovoSeven].

8. Historically the PT was also performed using dilute Russel Viper venom with the RVV replacing the original brain suspension. A normal RVV-PT but a prolonged 'Brain'-PT indicated FVII deficiency.

9. The PT will be prolonged with the new oral direct Xa and IIa inhibitors.

10. The Prothrombin Index is derived from measuring [Prothrombin Time Control Plasma/ Prothrombin Time Patient Plasma] x 100. For example in a patient in whom the Prothrombin Time is 24s and the PT of the control plasma is 12s the Prothrombin Index would be 12/24 x 100 = 50%.

11. The Owren method for measuring the PT is diluted before estimation and the sample volume in the final reaction mix is 5%.

References

1. Girolami, A., et al., Factor VII Padua 2: another factor VII abnormality with defective ox brain thromboplastin activation and a complex hereditary pattern. Blood, 1979. 54(1): p. 46-53.

2. James, H.L., et al., The dysfunction of coagulation factor VIIPadua results from substitution of arginine-304 by glutamine. Biochim Biophys Acta, 1993. 1172(3): p. 301-5.

3. Marchetti, G., et al., Detection of two missense mutations and characterization of a repeat polymorphism in the factor VII gene (F7). Hum Genet, 1992. 89(5): p. 497-502.

4. Takamiya, O., et al., Factor VII activity and antigen in a patient with abnormal factor VII. Clin Lab Haematol, 1988. 10(2): p. 159-65.

5. Quick AJ. The Thromboplastin Reagent for the Determination of Prothrombin. Science. 1940;92(2379):113-4.

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Updated: 29-Apr-2012