Development of a simple chromogenic factor VIII assay for clinical use

The aim of this study was the development of a simple chromogenic factor VIII assay for practical clinical use. The criteria that the assay fulfils are: (1) The method is so sensitive that even 1 % factor VIII in human plasma is easily detected. (2) The method is linear in the amount of factor VIII from 0 to 200% in plasma. (3) The pipetting scheme is very simple; two reagents are prepared, reagent 1 (factor IXa, thrombin, Ca2+ and phospholipids) and reagent 2 (factor X). Then we pipet at t = 0 s, 100 μl diluted plasma + 100 μl reagent 1 in a reaction tube; at t = 30 s, 100 μl reagent 2 in the same tube and at t = 90 s, 200 μl of the reaction mixture in a cuvette with 700 μl EDTA buffer (stop buffer) and the formed factor Xa is measured with a chromogenic substrate. (4) The reaction components are stable during at least a whole working day. Factor VIII was measured in an assay using bovine clotting factors, so one avoids the risk of viral infections, which one might catch by working with clotting factors isolated from human plasma.