CLOTTING PROFILE MEASUREMENT

Objectives :-

At the end of the practical, the student should be able to,1. Explain how to perform the following clotting profile measurements

i. Bleeding time (Duke’s method and Ivy’s method)ii. Clotting time

iii. Prothrombin time (PT)iv. Activated Partial Thromboplastin time (APTT)

2. Interpret the results of these tests and identify the clotting pathways they represent.

3. Give the normal values of these tests and identify common disorders (conditions) where these values become abnormal.

Bleeding Time (BT)

Duke's Method Ivy's Method

- Wipe the subject's earlobe with surgical spirit and allow to dry

- Using a sterile lancet, make a 4mm deep puncture in the earlobe

- Next, using a clean filter paper, blot the drops of blood appearing on the earlobe every 15 seconds.

- Should be done till bleeding ceases.- Count the number of dots on the filter

paper and multiply by 15 sec. To arrive at the bleeding time.

- Normal :- 2 - 7 minutes

- Clean the anterior surface of the forearm with surgical spirit and allow to dry.

- Put a sphygmomanometer cuff around the arm.

- Raise the pressure to 40mmHg and maintain this pressure till the end of the experiment.

- Using a sterile lancet, make a 2mm deep puncture in the forearm.

- Remove the drops of blood using a clean filter paper, every 15 seconds

- Normal : - 3 - 6 minutes

Practical No : 06

Clotting Time (CT)

Lees & White Method :-

- 3 ml of blood is collected by venepuncture.- Timing is begun with a stopwatch, from the time the blood sample is withdrawn.- The sample is transferred to 3 glass tubes. (1 ml each)- These tubes are placed in a water bath at 37oC- Tilt the glass tubes every 30 seconds and check for clots.- The average time taken to form firm clots in all three tubes is taken as the clotting

time.

Results :-Eg.

- Tube A - 3.5 minutes- Tube B - 4 minutes- Tube C - 4 minutes

Average value = 3.5 + 4 + 4 3

= 11.5 3

= 3.7 minutes

Normal Range is 4 - 10 minutes

Prothrombin Time (PT)

- This measures the clotting time of plasma after the addition of brain extract containing tissue thromboplastin.

- This will test the extrinsic clotting pathway involving factors V, VII, X and Fibrinogen.

Method :-

- 4.5 ml of venous blood is collected into a bottle containing 0.5 ml of 3.1% sodium citrate, and mixed well (anticoagulation).

- This mixture is centrifuged.- Using a micropipette, take 0.1 ml of

separated plasma into a glass tube (citrated plasma).

- This tube is then placed in a water bath at 37oC.

A B C

- A tube containing 0.2 ml of Tissue Thromboplastin and CaCl2 solution is also kept separately in this water bath to warm it to 37oC.

- The contents of the tubes are quickly mixed, and a stopwatch started.- The tube is watched with the lower end just below the surface of the water for the

appearance of a fibrin clot.- The appearance of the clot marks the end point, and stopwatch is de-activated. - The time thus obtained is recorded as the prothrombin time of that sample.

- At the same time, a control sample is also prepared and the time recorded under similar conditions.

Results :-Prothrombin time of the control sample - X sec.Prothrombin time of the test sample - Y sec.

Interpretation :-Normally, Y should equal X 1Normal Range is 15 - 20 seconds

Abnormal values :-

Prolongation of the Prothrombin Time is seen in,Liver cell dysfunctionVitamin K deficiencyWarfarin therapyDisseminated Intravascular Coagulation (DIC)

Activated Partial Thromboplastin Time (APTT)

This will test for defects in the extrinsic pathway.The clotting factors are Factors, XII, XI, IX, VIII, X, V, II and I

Method :-

- 4.5 ml of venous blood is collected into a bottle containing 0.5 ml of 3.1% sodium citrate, and mixed well (anticoagulation).

- This mixture is centrifuged.- Using a micropipette, take 0.1 ml of separated plasma into a glass tube (citrated

plasma).- This tube is then placed in a water bath at 37oC.- A tube containing Thrombofax reagent (Kaolin) is also kept in the water bath and

allowed to warm.- The contents of the tubes are quickly mixed, and a stopwatch started.

- The tube is watched with the lower end just below the surface of the water for the appearance of a clot.

- The appearance of the clot marks the end point, and stopwatch is de-activated. - The time thus obtained is recorded as the activated partial thromboplastin time of

that sample.

- As for the earlier practical, a control is also made and the time recorded.

Results :-APTT of the control sample - A sec.APTT of the test sample - B sec.

Interpretation :-Normally, B should equal A 5Normal Range is 30 - 40 seconds

Abnormal Values :-

Prolongation of APTT is seen in,Haemophilia A and B (Factors VIII and IX)Von Willebrandt's Disease (stabilizing factor for factor VII ed)Other factor deficiencies (XII, XI)Liver failureDisseminated Intravscular Coagulation.

Whole Blood Clotting Time

Is a very crude test done to determine whether there is any clotting defect.

Method :-- 5 ml of blood is collected by venepuncture- This is put in a glass tube and timing is started- The glass tube is tilted at intervals to check if the blood has clotted.- The time thus taken is the whole blood clotting time.

Normally the time is approx. 20 minutes.

Importance :-This test is performed as a rough guide to whether systemic envenomation has occurred following a snake bite.