ASSAYS OF COAGULATION FACTORS

ASSAYS OF COAGULATION FACTORS

1. Fibrinogen

Principle During the process of coagulation, the enzyme thrombin converts soluble fibrinogen into insoluble fibrin. The time required for this conversion is proportional to the concentration of fibrinogen in plasma. By plotting a graph of known concentrations of fibrinogen and their clotting time, the concentration of fibrinogen in the test sample can be determined.

Reagents

(i) Veronal buffer (pH 7.35) Dissolve 5.9 g of sodium barbital and 7.1 g of sodium chloride in 2.5 ml of 0.1 N hydrochloric acid and 785 ml of distilled water. It is stable for a long time at 4°C.

(ii) Thrombin 100 NIH (Nilhydril) units/ml

(iii) Standard fibrinogen A lyophilised fibrinogen with a known concentration is commercially available.

Specimen Collect 4.5 ml of whole blood in 0.5 ml of 3.8% sodium citrate.

Collect a normal control blood in the same way.

Separate the plasma by centrifuging at 1000 g for 15 minutes.

Technique

Step 1:To obtain a calibration curve using the fibrinogen standard (1) Set up three tubes and prepare 1:5, 1:15 and 1:40 dilutions of the fibrinogen standard as follows:

(3) Add 0.1 ml of the thrombin solution to tube 1. Start the stop watch.

(4) Determine the clotting time at the first sign of clot when a fibrin strand is observed with a hooked nichrome wire.

(5) Repeat the test with the second tube of the same dilution and record the average as the clotting time.

(6) Determine the clotting time for tubes 2 and 3 in the same way.

(7) Plot the clotting time of each dilution against the fibrinogen concentration.

Step II : To determine the clotting time for the test and control plasma 

(1) Dilute the test and control plasma 1:10 with the veronal buffer.

(2) Determine the clotting time of each in duplicate in the same way as in step I.

(3) Read the concentration of fibrinogen in the 1:10 dilution of the test sample and the control from the calibration curve.

(4) Multiply by 10 to get the actual concentration. Normal range 200 to 400mg/dl

2. Factor Assays Based on the Prothrombin Time (Assay of factor V, VII, X and 11)

In the assay of these factors, substrate plasmas deficient in the factors to be assayed are used.

The factor deficient substrate plasma is mixed with serial dilutions of pooled normal plasma. Prothrombin time (PT) of each dilution is determined to plot a calibration curve. The same procedure is repeated with the test plasma. The percentage of the factor is then read from the normal curve.

This basic assay can be used for all factors, except fibrinogen, which is involved in the prothrombin time test. A plasma substrate deficient for the factor should be used, all other reagents are con stant.

Reagents

(i) Thromboplastin prepared as for the prothrombin time test or obtained commercially.

(ii) Calcium chloride (0.025M) :2.77g of calcium chloride is dissolved in one litre of distilled water.

(iii) Normal pooled plasma, prepared at least from five normal specimens. The concentration of the factor under assay is considered to be 100% in this plasma.

(iv) Factor-deficient substrate plasma

(v) Veronal buffer, pH 7.35 as for fibrinogen assay. Specimen Citrated blood collected as for prothrombin time ( 9 parts whole blood + 1 part 3.8 % sodium citrate)

Technique

Step 1: To prepare a normal activity curve using normal pooled plasma with factor deficient plasma: (1) Prepare serial dilutions of the normal pooled plasma ranging from 1:10 to 1:320 as follows:

(2) Mix 0.1 ml of 1:10 dilution of the normal plasma with 0.1 ml of the factor deficient substrate plasma. Keep at 37°C for two minutes.

(3) Add0.2 ml of pre-warmed thromboplastin solution and 0.1 ml of 0.025 M calcium chloride solution. Start the stop watch and record the clotting time.

(4) Repeat the test with the same dilution and record the average clotting time.

(5) Determine the clotting time for each dilution of the normal plasma in the same way.

(6) Plot the dilutions against the clotting time (seconds) on a graph paper. The graph should be a straight line. 

Step II: To determine the PT of test plasma

(1) Dilute the test plasma 1:10 and 1:20 with the veronal buffer.

(2) Determine the clotting time for each dilution in the same way as in step I.

(3) The percentage factor activity of the test plasma is indicated by the point at which the clotting time of 1:10 dilution intercepts the normal i.e. 100% concentration curve. The results obtained with 1:20 dilution are multiplied by 2 to obtain the percentage deficiency. The average of the two values should be reported.

3. Factor Assays Based on the Partial Thromboplastin Time (Assay of Factors VIII, IX and XII)

Principle this test detects deficiencies of factors in the intrinsic pathway using the APTT method and substrate deficient plasma.

Reagents

(i) Phospholipid reagent (brain extract), as for APTT.

(ii) Factor-deficient substrate plasma for the factor under investigation.

(iii) Calcium chloride, 0.025M-Dissolve 2.77 g per litre distilled water.

Reagents

(i) Phospholipid reagent (brain extract), as for APTT.

(ii) Factor-deficient substrate plasma for the factor under investigation.

(iii) Calcium chloride, 0.025M-Dissolve 2.77 g per litre distilled water.

(iv) Veronal buffer, pH 7.35. Specimen Citrated blood sample (9 parts whole blood + 1 part 3.8% sodium citrate). Collect a pooled control plasma in the same way.

Note

(i) For factors VIII and IX assays, it is essential to keep the test and control plasmas frozen if any delay is expected in carrying out the test. Moreover, while carrying out the test the diluted plasma specimens should be kept at 4°C or on crushed

ice until required for the test. 

(ii) For the assay of factor XI, only fresh

test samples should be used. Storage in the refrigerator may increase

factor XI activity. 

(iii) For factors XI and XII assays, the test

plasma must not come in contact with any glass surface during collection and testing.

(iv) Veronal buffer, pH 7.35.

Specimen Citrated blood sample (9 parts whole blood + 1 part 3.8% sodium citrate). Collect a pooled control plasma in the same way.

Note

(i) For factors VIII and IX assays, it is essential to keep the test and control plasmas frozen if any delay is expected in carrying out the test. Moreover, while carrying out the test the diluted plasma specimens should be kept at 4°C or on crushed ice until required for the test. 

(ii) For the assay of factor XI, only fresh test samples should be used. Storage in the refrigerator may increase factor XI activity. 

(iii) For factors XI and XII assays, the test plasma must not come in contact with any glass surface during collection and testing.

Technique

Step 1: To prepare a normal activity curve using control plasma with factor deficient plasma: (1) Dilute the control plasma 1:5, 1:10, 1:20 and 1:40 with veronal buffer as follows:

(2) In a 37°C water bath, mix 0.1 ml volumes of phospholipid reagent, factor deficient plasma and diluted control plasma from tube 1. Start the stop watch as soon as the control plasma is added.

(3) Exactly at the end of three minutes, add 0.1 ml of 0.025 M calcium chloride solution to the mixture.

(4) Mix and start another stop watch or start counting the time freshly at this point.

(5) Leave the tube undisturbed at 37°C for 40 seconds.

(6) Remove the tube and tilt back and fourth quickly till the clot is formed. Record the time.

(7) Repeat the test for the same dilution. Record the average clotting time.

(8) Repeat the same procedure for tubes 2, 3 and 4.

(9) Plot a curve of factor concentration against clotting time in seconds. 

Step II: To determine the partial thromboplastin time of the test plasma with factor-deficient plasma Repeat the same technique as in step I using the test plasma instead of the control plasma. Draw a separate curve for the test plasma plotting the expected factor concentration at each dilution against clotting time.

The degree of correction can be determined by finding out the concentration in the control plasma at the clotting time of 1:5 dilution of the test plasma. For example, clotting time for tube 1 of test plasma is 70 seconds whereas the control plasma curve shows a factor concentration of 30% at 70 seconds clotting time, the factor activity of the test plasma is said to be 30%. 

Normal range

Factors VIII, IX and XI   50–150 % 

Factor XII 40-150 %

Other Tests for Coagulation Disorders

In a specialized coagulation laboratory other tests are performed such as Russell viper venom time, reptilase time, assays of other factors not described above and the assays for inhibitors.