Finding out Prothrombin Time is helpful in the detection of possible coagulation defect. It is a laboratory procedure helpful in identifying and diagnosing health problems. PT is often prolonged in case of lack of Vitamin K absorption or lack of its synthesis in hepatocellular disease.
Prothrombin Test (PT) is useful for the diagnosis of congenital & acquired deficiency of extrinsic pathway involving factors like II, V, VII, and X. It is also helpful in the monitoring of oral anticoagulant therapy.(1)
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Prothrombin Time Test Manual Procedure- How PTT Blood Test is done?
The mixture of tissue thromboplastin and calcium is added to citrated plasma, to trigger stage 2 of the coagulation mechanism in the presence of factor VII. The time taken to form a solid clot is recorded.
Since factors XII, XI, VIII, and platelets are bypassed, the results depend on the activity of remaining factors i.e. I, II, V, VII, and X. In case there is a deficiency in any of these factors, the clot formation time is prolonged.(2)
How is Primary Sample Collected for Prothrombin Test?
- Only plasma needs to be used as a specimen for the test
- 7 mL of venous blood in 3.2 % trisodium citrate vacutainer should be collected
- The sample should be centrifuged for at least 15 minutes at 4,000 rpm
- Plasma should be immediately separated in the autoclaved test tube
- A sample can be stored at 2 to 6oC until it is processed
- Samples are to be processed within 1 hour of collection
What are the Consumables / Chemicals / Reagents used for Prothrombin Test?
- Thromboplastin Reagent
- Laboratory Centrifuge
- 37oC water bath
- Autoclaved test tubes (of the size 12 x 100 mm)
- Plastic centrifuge tubes
Above is the list of items required to perform the Prothrombin Time Test.
What is the Procedure of Prothrombin Time Test?
Following is the step-by-step guide to performing the Prothrombin Time Test:
- Blood samples are to be collected as per the requirements
- Reagents are to be prepared as per the requirements
- Warm up the instrument for at least 5 minutes
- Prewarm PT reagent to 37oC incubator
- Pipette 0.1 mL test plasma into the test tube
- After incubating, add 0.2 mL pre-warmed thromboplastin reagent to the plasma
- Pipette out prepared PT reagent, add 0.2 mL each of this reagent to the tubes labeled as T1 and T2.
- Place the tubes in the water bath
- Position T Tube in a water bath for at least 5 minutes
- Add 0.1 mL of plasma from T tube into the test tube marked T1. Start the Stop-watch simultaneously.
- Remove T1 from water batch at the end of 12 seconds
- Quickly wipe outer surface of the tube with a clean tissue
- Tilt the tube back and forth gently until clot forms. Stop the stop-watch soon after the solid clot is formed.
- Repeat the above steps with T2 tube.
- Average of T1 and T2 should be noted and is given as a result of PT of the patient’s sample.
- Repeat the entire process on any normal plasma labeled as C, C1, and C2. Control PT is calculated as an average of C1 and C2.
- Repeat the above test for any abnormal values
- Note down the results
- Perform platelet count on the plasma and note down the results
How are Results Interpreted in Prothrombin Time Test?
MNPT is critically required in the derivation of INR i.e. International Normalized Ratio. MNPT has to be derived from each laboratory from 10 normal patients for given PT reagent. (4)
Prothrombin Time results are to be reported only in INR format. Results of this test are to be reported to the nearest 10th of a second.
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What are the Precautions to be taken while doing (PTT) PROTHROMBIN TIME TEST?
- All reagents are to be used carefully because they are dangerous when came in contact with skin, mouth, and eyes
- Samples are to be used carefully because they can be potentially infectious
- Used reagents and samples are to be discarded in a proper way
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What are the Potential Sources of Variability in Prothrombin Test
The test results are often influenced by method and time of plasma storage, type of anticoagulant used, collection technique, pH, temperature and clot detection method adopted.
Inaccurate or invalid results might be obtained when there is the presence of heparin or EDTA as an in-vitro anticoagulant.
In case of presence of factors I, II and V, the results of the Prothrombin Time test may be inaccurate. (5)
What is the Normal / Standard / Reference Range for Prothrombin Time Test?
Normal Range: 11 to 15 seconds