Haematology Watch

Haematology Watch, Vol 3, Issue 2.

STUDENTS' CORNER

Prothrombin Time 

Mehmood

INTRODUCTION:

One of the important functions of human body is Haemostasis i.e. maintaining a fluid state of blood in normal conditions and securing bleeding points by localized plug made of platelets and fibrin.

This mechanism is composed of five components: coagulation factors, fibrinolysis, antifibrinolysis, platelets, and vessel wall. Coagulation factors help in making fibrin threads after activated by exposed collagen (Intrinsic pathway) and /or by expression of tissue thromboplastin (Extrinsic pathway). Either of the pathways culminate in conversion of Factor X, V, II, and I into activated forms leading to formation of Fibrin (Common pathway).

In a laboratory, assessment of function of coagulation factors is routinely done by Prothrombin Time test (assessing Extrinsic and Common pathway), and Activated Partial Thromboplastin Time test (assessing Intrinsic and Common pathway). These tests are helpful in investigating status of haemostatic system in an acutely bleeding patient, a person with suspected bleeding tendency or as a precaution before an invasive procedure.

Prothrombin time is a misnomer: initially it was suspected to be affected by (and determining) the activity of Prothrombin in plasma, thus named as Prothrombin time; later it was found to be dependent on many other factors.

PRINCIPLE:

Prothrombin time (PT) is the time taken by citrated plasma to clot  after adding thromboplastin, and calcium chloride, and correlates with an overall function of Extrinsic and Common pathway of coagulation cascade. When Thromboplastin is added to plasma, extrinsic pathway of coagulation is stimulated which, in the presence of added Calcium chloride, culminates in the formation of Fibrin, visible as an off-white clot in test tube. The time elapsed between adding the reagent and just appearance of the clot is noted, and termed Prothrombin Time.

Compared to normal control plasma, the PT test is prolonged in deficiency of coagulation factors VII, X, V, II, I. If a coagulation factor of Common Pathway is deficient, Activated Partial Thromboplastin Time (APTT) test is also prolonged.

REQUIREMENTS:

     EQUIPMENTS:

    Waterbath, Glass Test tubes, Stop watch, Micropipette with tips, Tube rack, Gloves.

     REAGENTS:

    Platelet-poor plasma of patient and of control, Thromboplastin with Calcium chloride.

PROCEDURE:

1. Deliver 100 µL plasma of patient into a glass test tube placed in waterbath at 37° C.

2. Start stop watch.

3. After 2 minutes, add 200 µL Thromboplastin with Calcium reagent (contains 100 µL each).

4. Make a layer of plasma by tilting the tube near horizontal, avoiding losing any sample, and dip the tube in water until stop watch reads ~3 seconds below control value (e.g. if control is 12 sec, start noting end-point at 9 seconds).

5. By holding the top of the test tube, bring it out of the rack and dip its bottom in the water gently in a to-and-fro motion at a rate of ~1 dip/sec.

6. Carefully detect the end-point i.e. appearance of off-white jelly-like clot; the later it forms, the thinner it appears. Do not look at the stop watch while observing the end-point detection.

7. When clot is formed (i.e. End-point detected), without looking at the watch, press the stop button immediately, and record the time.

8. Run the test in duplicate, and take a mean to report the PT test result.

 

RESULT:

The result of the mean of the duplicate tests is expressed in seconds compared to a normal value. Normal range is a test value falling in ±2 seconds compared to the normal control value. For example, if normal value is 12 seconds, 10 – 14 is normal range; a PT test of 10, or 13, or 14 is normal in this case, while a value of 9 or 15 is abnormal.

 

INTERPRETATION:

 

A falsely prolonged PT test result can be seen in following conditions:

1. Pre-analytical variables:

       - Increased citrate:blood ratio (Under filled citrate tube, Haematocrit > 55%)

       - Wrong labeling

       - Delay in transport

2. Analytical variables:

       - Testing the sample > 2 hrs after venepuncture (Lazy technician).

       - Delay in reading the end-point (Poorly-trained technician)

       - Expired reagents

3. Post-analytical variables:

       - Wrong entry of the result (Lack of concentration of technician)

       - Inability to compare the difference from normal control (Poorly trained physician)

 

CONCEPT OF INTERNATIONAL NORMALIZED RATIO (INR):

 

Thromboplastin, the reagent used in PT test, is made worldwide by different companies from different sources. Thus the results of a same person at same time differ in different laboratories.

To eliminate the difference, a reagent should be compared to a standard reagent made by W.H.O. laboratory. The difference is expressed as International Sensitivity Index (I.S.I.) i.e. difference of the extent of the sensitivity of Thromboplastin to FVII in plasma compared to that made in W.H.O. laboratory. If both are equal, I.S.I. of the Thromboplastin is scored 1.0; if its sensitivity is less, it is scored 1.1, 1.2, and so on according to the difference. Thus, the lesser, the better!

Old concept:

Whatever is the ISI, the INR will remain same.

New concept:

Using a reagent with high ISI will result in variations in INR.

Background:

Suppose you receive a specimen of a patient who is also tested recently on same therapy from another lab with a different ISI than yours, then your INR will be similar (not same*) to the other lab because the PT of control and test both will be affected to a similar extent.

But if difference in ISI is great between two labs, the difference in INR will be significant.

NOTE:

1. INR between labs is compared when their reagents have similar ISI values.

2. ⁠We should select a reagent whose ISI is closest to 1.0. According to WHO, it should be within 0.9-1.7 range.

3. ⁠If we use a reagent having a high ISI eg 2.0, the INR will be falsely elevated leading to reduction/stoppage in anticoagulant therapy and chances of thrombosis.

This value of I.S.I. is incorporated in a formula as shown:

              INR= (Patient’s PT/Control PT)^ISI

e.g. if PT of a patient in 17, control value is 12, and I.S.I. value of the Thromboplastin reagent used is 1.1, INR is calculated as following:

             (17/12)^1.1

             =1.46.

This can be performed in a scientific calculator as following:

     Enter test value

     Click sign of division

     Click =

     Click x^y

     Enter 1.1 (or whatever ISI value of the reagent is mentioned)

     Click =

     Record the value now.

If the same sample is sent to another laboratory for PT and is tested using a reagent with I.S.I. of 1.3, his INR will be 1.46 although his PT will differ from the previous laboratory result. Thus the difference of PT test due to difference in reagent in two laboratories will be corrected by measuring I.N.R..

Normal range for INR is 0.8 - 1.2.

Remember: For APTT, not any INR is being used.

USES:

1. Values of PT test between different laboratories are correlated in terms of I.N.R..

2. I.N.R. should be 2 – 3 in patients receiving Warfarin; reduce dose if I.N.R. is >3, and increase dose if I.N.R. is <2.