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 (Dukes method and !"s method#
ii. $lotting time
iii. %rothrombin time (%&#
i!. Acti!ated %artial &hromboplastin time (A%&&#
'. nterpret the results of these tests and identif" the clotting pathwa"s the"
represent.
(. )i!e the normal !alues of these tests and identif" common disorders (conditions#
where these !alues become abnormal.
Bleeding Tie !BT"
Duke*s +ethod !"*s +ethod
, -ipe the sub.ect*s earlobe with
surgical spirit and allow to dr"
, /sing a sterile lancet, make a 0mm
deep puncture in the earlobe
, 1ext, using a clean filter paper, blot
the drops of blood appearing on the
earlobe e!er" 12 seconds.
, 3hould be done till bleeding ceases.
, $ount the number of dots on the filter
paper and multipl" b" 12 sec. &o arri!e at
the bleeding time.
, 1ormal 4, ' , 5 minutes
, $lean the anterior surface of the
forearm with surgical spirit and allow to
dr".
, %ut a sph"gmomanometer cuff
around the arm.
, 6aise the pressure to 07mm8g and
maintain this pressure till the end of the
experiment.
, /sing a sterile lancet, make a 'mm
deep puncture in the forearm.
, 6emo!e the drops of blood using a
clean filter paper, e!er" 12 seconds
, 1ormal 4 , ( , 9 minutes
Cl#tting Tie !CT"
%ractical 1o 4 79
:ees ; -hite +ethod 4,
, ( ml of blood is collected b" !enepuncture.
, &iming is begun with a stopwatch, from the time the blood sample is withdrawn.
, &he sample is transferred to ( glass tubes. (1 ml each#
, &hese tubes are placed in a water bath at (5
o
$
, &ilt the glass tubes e!er" (7 seconds and check for clots.
, &he a!erage time taken to form firm clots in all three tubes is taken as the clotting
time.
6esults 4,
Eg.
, &ube A, (.2 minutes
, &ube B , 0 minutes
, &ube $ , 0 minutes
A!erage !alue < (.2 = 0 = 0
(
< 11.2
(
< (.5 minutes
1ormal 6ange is 0 , 17 minutes
P$#t%$#bin Tie !PT"
, &his measures the clotting time of plasma after the addition of brain extract
containing tissue thromboplastin.
, &his will test the extrinsic clotting pathwa" in!ol!ing factors >, >, ? and
@ibrinogen.
+ethod 4,
, 0.2 ml of !enous blood is collected into a
bottle containing 7.2 ml of (.1A sodium
citrate, and mixed well (anticoagulation#.
, &his mixture is centrifuged.
, /sing a micropipette, take 7.1 ml of
separated plasma into a glass tube (citrated
plasma#.
, &his tube is then placed in a water bath at
(5
o
$.
, A tube containing 7.' ml of &issue &hromboplastin and $a$l' solution is also kept
separatel" in this water bath to warm it to (5
o
$.
A B C
, &he contents of the tubes are Buickl" mixed, and a stopwatch started.
, &he tube is watched with the lower end .ust below the surface of the water for the
appearance of a fibrin clot.
, &he appearance of the clot marks the end point, and stopwatch is de,acti!ated.
, &he 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.
6esults 4,
%rothrombin time of the control sample , ? sec.
%rothrombin time of the test sample , C sec.
nterpretation 4,
1ormall", C should eBual ? 1
1ormal 6ange is 12 , '7 seconds
Abnormal !alues 4,
%rolongation of the %rothrombin &ime is seen in,
:i!er cell d"sfunction
>itamin D deficienc"
-arfarin therap"
Disseminated ntra!ascular $oagulation (D$#
Activ&ted P&$ti&l T%$#b#'l&stin Tie !APTT"
&his will test for defects in the extrinsic pathwa".
&he clotting factors are @actors, ?, ?, ?, >, ?, >, and
+ethod 4,
, 0.2 ml of !enous blood is collected into a bottle containing 7.2 ml of (.1A sodium
citrate, and mixed well (anticoagulation#.
, &his mixture is centrifuged.
, /sing a micropipette, take 7.1 ml of separated plasma into a glass tube (citrated
plasma#.
, &his tube is then placed in a water bath at (5
o
$.
, A tube containing &hrombofax reagent (Daolin# is also kept in the water bath and
allowed to warm.
, &he contents of the tubes are Buickl" mixed, and a stopwatch started.
, &he tube is watched with the lower end .ust below the surface of the water for the
appearance of a clot.
, &he appearance of the clot marks the end point, and stopwatch is de,acti!ated.
, &he time thus obtained is recorded as the acti!ated partial thromboplastin time of
that sample.
, As for the earlier practical, a control is also made and the time recorded.
6esults 4,
A%&& of the control sample , A sec.
A%&& of the test sample , B sec.
nterpretation 4,
1ormall", B should eBual A 2
1ormal 6ange is (7 , 07 seconds
Abnormal >alues 4,
%rolongation of A%&& is seen in,
8aemophilia A and B (@actors > and ?#
>on -illebrandt*s Disease (stabiliEing factor for factor > ed#
Fther factor deficiencies (?, ?#
:i!er failure
Disseminated ntra!scular $oagulation.
(%#le Bl##d Cl#tting Tie
s a !er" crude test done to determine whether there is an" clotting defect.
+ethod 4,
, 2 ml of blood is collected b" !enepuncture
, &his is put in a glass tube and timing is started
, &he glass tube is tilted at inter!als to check if the blood has clotted.
, &he time thus taken is the whole blood clotting time.
1ormall" the time is approx. '7 minutes.
mportance 4,
&his test is performed as a rough guide to whether s"stemic
en!enomation has occurred following a snake bite.