Activated Partial Thromboplastin Time (APTT) (TEST FOR INTRINSIC SYSTEM )

TEST FOR INTRINSIC SYSTEM 

Activated Partial Thromboplastin Time (APTT), Partial Thromboplastin Time with Kaolin (PTTK) or Kaolin Cephalin Clotting Time (KCCT)

These tests demonstrate deficiencies within the intrinsic pathway, and the final common pathway. That is, the clotting time is affected by all coagulation factors except factor VII and platelet factor 3

In all the three tests, a brain extract is used to provide platelet factor 3 activities.A rough surface such as kaolin or glass, is used to trigger the intrinsic system. 

  APTT Specimen 

The citrated plasma is centrifuged at 1500-2000 g for 15 minutes to remove platelets. The plasma sample used for prothrombin time can also be used.

Plasma from a healthy person should be used as control. 

APTT Reagents

1. Kaolin Suspend 0.5 g kaolin in 100 ml saline. 

2. Phospholipid (brain extract or partial thromboplastin) Place about 100 g washed brain in a large mortar and cover with acetone. Mash with a pestle for about two minutes.

When acetone becomes turbid, decant and replace with fresh acetone. Continue until the residual brain is granular. Spread it on a filter paper and allow it to dry at room temperature. This is the acetone-dried brain. Store in perforated containers in a dessicator.

Weigh I g acetone dried brain and add 20 ml acetone. Mix continuously for two hours. Centrifuge for five minutes at 1500 g. Discard the supernatant and add 20 ml chloroform. Mix again for two hours. Filter through a Whatman No.1 filter paper. 

Evaporate the chloroform in the filtrate using a stream of warm air. Emulsify the residue in 10 ml normal saline by mixing for up to 48 hours.

Standardise the partial thromboplastin thus obtained by selecting a dilution (e.g. 1:100) which gives optimum clotting time (e.g. 35-43 seconds) with control plasma.

This reagent is commercially available. 3. 0.025M calcium chloride Dissolve 2.77 g in one liter distilled water. 

APTT Technique 

(1) Mix 0.1 ml of well mixed kaolin suspension and 0.1 ml of phospholipids in a test tube. Keep it at 37°C for 15 seconds to warm.

(2) Add 0.1 ml of control plasma, previously warmed to 37°C.

(3) Incubate for exactly two minutes.

(4) At two minutes, add 0.1 ml of 0.025 M calcium chloride solution and start a stop watch.

(5) Leave the tube for 30 seconds, and then remove.

(6) Check for clotting by tilting the tube. 

(7) Record the time taken to clot.

(8) Repeat the same procedure with the test plasma. 

APTT Normal range 

Control plasma should clot between 35-43 seconds.

Prolongation of the clotting time by seven seconds or more than that of the control is significant. 

APTT Interpretation 

The activated partial thromboplastin time may be prolonged due to 

1. Defects in the intrinsic system: e.g. factors VIII: c, IX, XI, XII etc. 

2. Defects in the common pathway e.g. factors X, V, II, I. 

3. Inhibitory action of heparin, specific inhibitors of factors etc.