Prothrombin Time (Pt) Test (Test to Measure The Extrinsic System)

Prothrombin Time (Pt) Test (Test to Measure The Extrinsic System)

TEST TO MEASURE THE EXTRINSIC SYSTEM,

Prothrombin Time (Pt) Test (Test to Measure The Extrinsic System)

PROTHROMBIN TIME (PT) TEST

•  PT Reagents

• Specimen 

• PT Technique
  CALCULATION
  Interpretation 

PROTHROMBIN TIME (PT) 

This test measures the extrinsic pathway of coagulation. When tissue thromboplastin and calcium ions are added to plasma, extrinsic clotting factors are activated, resulting in the generation of thrombin and the formation of fibrin clot. Thus, the test indicates functions of factors II, V, VII and X.

A tissue extract serves as a source of thromboplastin. The reagent most popularly employed is the saline extract of human brain.

Note

Rabbit brain is now used instead of human brain because of the risk of HIV infection.

PT Reagents 

1.    Thromboplastin Remove the meninges, cer ebellum and blood vessels from a fresh human (or rabbit) brain removed at post mortem. Wash thoroughly in running water, drain and blot dry. Cut into small pieces and emulsify in saline using a pestle and mortar or in a blending machine at a low speed. 

2.    Centrifuge the emulsion at 70g for 20 min. Decant the supernatant in a separate container. Prepare dilutions (from 1:2 to 1:10) and determine prothrombin time of a normal plasma, using each dilution. Select the dilution which gives a clotting time of 12 seconds. Dilute the bulk in 0.5% phenol saline (0.5 8 phenol in 100ml saline) to the desired concentration, distribute in suitable aliquots and store at 4°C.

Note: 

Brain thromboplastin is commercially available

2. 0.025M Calcium chloride Dissolve 2.7g of calcium chloride in distilled water and make up to one litre. 

Specimen 

Collect blood into a tube containing 3.8% sodium citrate in the ratio of 9: 1. Plastic tube and syringes should be used wherever possible. 

Mix the blood gently with citrate by inverting it several times. 

The test should be performed within 2-3 hours of blood collection since the labile factor (factor V) is destroyed on standing at room temperature.

Collect a control blood sample in the same way, 

PT Technique 

(1) Centrifuge both the test and the control blood samples at 1000 g for 15 minutes to obtain platelet-poor plasma

(2) Add 0.1 ml thromboplastin in two glass tubes and place them at 37°C water bath.

(3) Add 0.1 ml of normal control plasma to each tube and incubate for one minute.

(4) Add 0.1 ml of calcium chloride to one tube, and start a stop watch.

(5) Tilt the tube repeatedly until a clot forms. Note the time.

(6) Repeat with the second control tube and take the average time as: The reading for the control.

(7) Repeat the procedure with the test sample. Express the prothrombin time in seconds as the mean of the two readings. 

Normal range 

Control time should be 13-15 seconds, though each laboratory should establish its own range.

The test sample should form a clot within two seconds of the control

CALCULATION

calculation of prothrombin time test

Interpretation 

Prolongation of prothrombin time occurs in liver disease, congenital deficiencies of coagulation factors in the extrinsic pathway and in oral anticoagulant therapy.