Introduction
The prothrombin time (PT) assay is a blood test that measures how long it takes for a blood clot to form. It is one of the most common blood tests performed in the world, and it is used to diagnose and monitor a variety of bleeding and clotting disorders.
The PT assay works by measuring the amount of time it takes for prothrombin to be converted to thrombin. Prothrombin is a protein that is produced by the liver, and it is essential for blood clotting. Thrombin is an enzyme that converts fibrinogen to fibrin, which is the main component of blood clots.
The PT assay is performed by mixing a sample of the patient’s blood with a reagent that contains tissue factor and calcium. Tissue factor is a protein that activates the coagulation cascade, which is the series of events that leads to blood clot formation. Calcium is also required for blood clotting.
The time it takes for a clot to form is measured in seconds. The normal range for the PT assay is 10 to 13 seconds. A prolonged PT assay indicates that it is taking longer for the blood to clot than normal. This can be caused by a variety of factors, including:
- Vitamin K deficiency
- Liver disease
- Kidney disease
- Certain medications, such as warfarin and heparin
- Bleeding disorders, such as hemophilia and von Willebrand disease
The PT assay is a valuable tool for diagnosing and monitoring bleeding and clotting disorders. It is also used to monitor the effectiveness of blood thinners, such as warfarin.
Principle of Prothrombin Time (PT)
The prothrombin time (PT) assay is based on the principle that the conversion of prothrombin to thrombin is the final step in the extrinsic coagulation pathway.
The extrinsic coagulation pathway is activated by tissue factor, which is released from damaged tissues. Tissue factor activates factor VII, which in turn activates factor X. Factor X then activates factor IX, which in turn activates factor II (prothrombin). Prothrombin is converted to thrombin in the presence of calcium.
Thrombin is a protease that cleaves fibrinogen to fibrin. Fibrin is a protein that forms a mesh-like network that traps blood cells and other elements of the blood, forming a clot.
Method differs slightly according to the manufacturer’s protocol.
Materials
- #Platelet poor plasma (PPP) of patient and control. PPP is prepared by centrifuging the peripheral blood at 2000 g for 15 minutes at room temperature.
- *Thromboplastin
- *0.025 mol/L calcium chloride (CaCl2)
- *Glass tubes 5 ml
- *Water bath
- Stop watch
- Pipettes 100 – 200 uL
- Pipette tips
*These items must be at 37°C.
#PPP must be kept at room temperature to prevent activation of factor VIII which may cause an inaccurate timing result.
Protocol
This test must be performed in the water bath at all times apart from the observation of the clot formation step.
- Add 0.1 mL plasma of both patient and control into a test tube, respectively and label accordingly. Incubate for 1 – 3 minutes.
- Add 0.1 mL thromboplastin into each test tube. Incubate for 1 – 3 minutes.
- Get the stopwatch ready, add 0.1 mL CaCl2 and immediately start the timer.
- Gently mix the solution in the test tubes by tilting the test tubes to 45° angle and agitate lightly.
- Intermittently take the tubes out of the water bath to observe for the first sign of clot formation.
- Stop the timer and record the time at the first sign of clot formation.
- Test both patient and control plasma in duplicates and record the average time.
*Some manufacturers have added the thromboplastin reagent to CaCl2, thus step 3 can be omitted in such cases and just proceed with recording the time after step 2.
Interpretation
PT reference range:
| Parameter | Time | Unit |
| Rabbit thromboplastin | 11 – 16 | seconds |
| Human thromboplastin | 10 – 12 | seconds |
A prolonged PT assay indicates that it is taking longer for the blood to clot than normal. This can be caused by a variety of factors, including:
- Vitamin K deficiency
- Liver disease
- Kidney disease
- Certain medications or oral anticoagulation drugs, such as warfarin and heparin – vitamin K antagonists
- Factor VII, X, V or prothrombin deficiency or defect.
- Disseminated intravascular coagulation (DIC)
Isolated prolonged PT assay could indicate Factor VII deficiency while shortened PT could be found following treatment with rVIIa.
Disclaimer: This protocol is intended for informational purposes only and may need to be modified depending on the specific laboratory procedures and patient circumstances. Always consult with a qualified healthcare professional for guidance. See additional information.
