Assay of Factor VIII:C with a Chromogenic Substrate

Abstract

A chromogenic method for the determination of factor VIII:C in plasma and concentrate has been developed. Bovine factor X a is generated in a system containing bovine factors IX a and X, Ca 2+ and phospholipid. The amount of factor X a formed is directly proportional to the amount of factor VIII:C. Thus, after hydrolysis of the factor X a selective chromogenic substrate S‐2222, the colour produced by released p‐nitroaniline is linearly related to the concentration of factor VIII:C in the sample. Hydrolysis of the substrate by thrombin is prevented by including the thrombin inhibitor I‐2581. A major methodological advantage is the exclusion of hemophilia A plasma or artificially prepared factor VIII deficient plasma. The detection limit is 1 per cent factor VIII:C, and the CV is lower than 5 per cent in both the earlier defined ranges, 1–20 per cent and 15 ‐ 100 per cent factor VIII:C and in the recently introduced extended range 20–150 per cent factor VIII:C. The resolution in the 15–100 per cent range is about 0.008 absorbance units per per cent factor VIII:C, allowing a good discrimination between samples containing e.g. 30–70 per cent factor VIII:C. Comparative studies between the chromogenic assay and clotting assays have been performed in other laboratories with both plasma and concentrate samples and the correlation coefficient was in all cases ≥ 0.92.