Five different components are involved in normal hemostasis namely (blood vessels, platelets, plasma coagulation factors, their inhibitors, and the fibrinolytic system). The cornerstone assays for coagulation (secondary or coagulation phase of hemostasis) testing are the prothrombin time (PT) and activated partial thromboplastin time (APTT) combined with the results of the platelet count. Firstline tests of coagulation, or “clotting screen,” usually consist of PT, APTT, TT (thrombin time), fibrinogen assay, and platelet count.
Purpose
The prothrombin time assay has two purposes: to screen for inherited or acquired deficiencies in the extrinsic and common pathways of coagulation and to monitor oral anti-coagulant therapy.
Principle
The prothrombin time test measures the clotting time of re-caleified plasma in the presence of an optimal concentration of tissue extract (thromboplastin). Tissue factor in the thromboplastin preparation binds factor VIl in patient plasma to initiate coagulation. The prothrombin time is affected by decreased levels of fibrinogen, prothrombin (II), factors V, VII, or X. Since 3 of the 5 coagulation factors measured by the PT are vitamin K-dependent proteins (prothrombin, factors VII and X), the prothrombin time assay is useful in detecting vitamin K deficiency from any cause, including liver disease, malnutrition, or warfarin therapy. Prothrombin time does not measure factor XIII activity or components of the intrinsic pathway.
Equipment & Reagents:
1. Patient’s plasma
2. Control plasma
3. Thromboplastin containing tissue factor and phospholipid. (some thromboplastin combined with calcium chloride) stored at 4°C
4. CaCl₂ – 0.025 mol/L stored at 4°C
5. Citrate vial
6. Centrifuge
7. Cuvette / Plastic tube
8. Waterbaths
9. Refrigerator
10. Micropipettes
11. Stop watch
Sample
Citrated blood (9 volume blood : 1 volume 3.2% trisodium citrate)
Calibration Procedure
Steps of Procedure:
1. Reconstitute a vial of thromboplastin in accordance with the manufacturer’s instruction. Leave in water-bath at 37°C for 10 minutes.
2. Dispense 0.1 ml into a plastic tube and add 0.1 ml of pre-warmed calcium 1:2 chloride (or dispense 0.2 ml if the thromboplastin-calcium are already combined).
3. Add 0.1 ml of pre-warmed plasma and start the stop-watch.
4. Tilt the tube gently every other second, keeping it as much as possible under water to maintain the temperature. Record the appearance of a fibrin clot as the end-point.
5. Perform the test on the patient’s plasma in duplicate, and also in duplicate on the normal control plasma. Repeat the test if duplicate measurements differ by more than 5%.
Normal range
Normal values depend on the thromboplastin used, the exact technique, and whether a visual or instrumental endpoint reading is used. With most rabbit thromboplastins the normal range of the PT is between 11 and 16 sec, for recombinant human thromboplastin, it is somewhat shorter (10-12 sec).
Expression of Results
The results are expressed as the mean of the duplicate readings in seconds or as the prothrombin ratio (PR) of the mean patient’s plasma time to the mean normal control plasma time. The International Normalized Ratio (INR) was developed to standardize PT values via mathematical transformation, where PT is the prothrombin time, MNPT is the mean normal PT, and ISI is the International Sensitivity Index of the thromboplastin.
INR = (PT/MNPT)ISI = PRISI
MNPT is the geometric mean of PT.
MNPT = (PT1 × PT2 × PT3 x ….PTn) 1/n
Usually, plasma from at least 20 normal healthy individuals should be used to establish the MNPT. The average of such PT results in seconds MNPT. WHO reference thromboplastin has an ISI of 1.0.
Interpretation of Results
The common causes of prolonged PTs are as follows:
1. Administration of oral anticoagulant drugs (vitamin K antagonists)
2. Liver disease, particularly obstructive jaundice
3. Vitamin K deficiency
4. Disseminated intravascular coagulation
5. Factor VII, X, V or prothrombin deficiency or defect.
Interference & Cross-Reaction
Each preparation of Thromboplastin has a different sensitivity to clotting factor deficiencies and defects, in particular the defect induced by oral anticoagulants.
Sources of error in tests:
- Error in blood collection: incorrect ratio of blood to citrate.
- Wrong anticoagulant for blood collection.
- Delay in separating plasma from cells.
- Delay in performing tests.
- Tests were not performed with plasma and reagents kept at 37°C.
- Reagents deteriorating: incorrectly stored or out of date.
- Technique is not constant; it is essential to use the same technique as incubation time, temperature, and pipetting.
- Inability to detect the beginning of a clot and/or a slow response when using a stop-watch.
Quality Control
- Run control plasma daily. Take a mean of control values of 10 days, calculate standard deviation and CV%. The daily control value obtained subsequently should be within ±2SD of mean control value.
- When collecting samples in commercial citrate vials, check the clotting times obtained from the sample in the vials with the sample collected into the lab-prepared 3.2% sodium citrate for each batch of commercial vials.
Safety Precaution
Every test sample is regarded as potentially infectious material, so universal safety precautions have to be taken.