TEST TO MEASURE THE EXTRINSIC SYSTEM/ PROTHROMBIN CONSUMPTION INDEX (PCI)/TEST FOR INTRINSIC SYSTEM Activated Partial Thromboplastin Time (APTT)

TEST TO MEASURE THE EXTRINSIC SYSTEM:
 

ONE-STAGE PROTHROMBIN

TIME (PT) This test measures the extrinsic pathway of coagulation. When tissue thromboplastin and calcium ions are added to plasma, extrinsic clotting factors are activated, resulting in the generation of thrombin and the formation of fibrin clot. Thus, the test indicates functions of factors II, V, VII and X.

A tissue extract serves as a source of thromboplastin. The reagent most popularly employed is the saline extract of human brain.

Note

Rabbit brain is now used instead of human brain because of the risk of HIV infection.

Reagents 

1.    Thromboplastin Remove the meninges, cer ebellum and blood vessels from a fresh human (or rabbit) brain removed at post mortem. Wash thoroughly in running water, drain and blot dry. Cut into small pieces and emulsify in saline using a pestle and mortar or in a blending machine at a low speed.

2.    Centrifuge the emulsion at 70g for 20 min. Decant the supernatant in a separate container. Prepare dilutions (from 1:2 to 1:10) and determine prothrombin time of a normal plasma, using each dilution. Select the dilution which gives a clotting time of 12 seconds. Dilute the bulk in 0.5% phenol saline (0.5 8 phenol in 100ml saline) to the desired concentration, distribute in suitable aliquots and store at 4°C.

Note: Brain thromboplastin is commercially available

2. 0.025M Calcium chloride Dissolve 2.7g of calcium chloride in distilled water and make up to one litre. 

Specimen Collect blood into a tube containing 3.8% sodium citrate in the ratio of 9: 1. Plastic tube and syringes should be used wherever possible. 

Mix the blood gently with citrate by inverting it several times. The test should be performed within 2-3 hours of blood collection since the labile factor (factor V) is destroyed on standing at room temperature.

Collect a control blood sample in the same way, 

Technique 

(1) Centrifuge both the test and the control blood samples at 1000 g for 15 minutes to obtain platelet-poor plasma

(2) Add 0.1 ml thromboplastin in two glass tubes and place them at 37°C water bath.

(3) Add 0.1 ml of normal control plasma to each tube and incubate for one minute.

(4) Add 0.1 ml of calcium chloride to one tube, and start a stop watch.

(5) Tilt the tube repeatedly until a clot forms. Note the time.

(6) Repeat with the second control tube and take the average time as: The reading for the control.

(7) Repeat the procedure with the test sample. Express the prothrombin time in seconds as the mean of the two readings. Normal range Control time should be 13-15 seconds, though each laboratory should establish its own range.

The test sample should form a clot within two seconds of the control

CALCULATION

Interpretation Prolongation of prothrombin time occurs in liver disease, congenital deficiencies of coagulation factors in the extrinsic pathway and in oral anticoagulant therapy.

TEST FOR PROTHROMBIN CONSUMPTION INDEX (PCI)

About 95% of prothrombin-is utilised by activation to thrombin when whole blood clots. Citrated plasma is clotted in the absence of platelets and much less prothrombin is consumed. Thus, by comparing the clotting time of serum and plasma in the presence of fibrinogen, the contribution of platelets to the coagulation process can be determined.

Specimens Collect 1.8 ml of blood in a tube containing 0.2 ml of 3.8% sodium citrate and 2.0 ml blood in a plain tube. Keep the plain tube at 37°C for one hour and centrifuge to separate the serum. Centrifuge the citrated blood as for prothrombin time (1000 g for 15 minutes). 

Reagents

1. Brain thromboplastin 

2. 0.025 M Calcium chloride 

3. Fibrinogen: Human fibrinogen, 150-200 mg in 100 ml saline. 

Technique 

(1) Add 0.1 ml of serum to two glass tubes and keep at 37°C.

(2) Add 0.1 ml of brain thromboplastin to one tube, start a stop watch.

(3) At the end of one minute, add 0.1 ml calcium chloride and mix.

(4) After two minutes, remove any fibrin formed with an applicator stick; and add 0.2 ml fibrinogen. Note the time required for clot formation at 37°C.

(5) Repeat the same procedure with the second tube, record average time.

(6) Repeat the same procedure with citrated plasma.

Calculation

Prothrombin Consumption Index = Plasma clotting time/Serum clotting time x 100

Normal range <30% Interpretation

PCI may be prolonged due to

1. Thrombocytopenia

2. Platelet coagulation defects

3. Platelet aggregation defects

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. 

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. 

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. 

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.

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. 

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.