A simple and accurate microplate assay for the determination of factor VIII activity

Abstract Based on the Coatest R Factor VIII kit, a simple and accurate microtiter plate assay has been developed. The method has been simplified through the combination of the bovine factors IXa + X, phospholipid and calcium chloride into one reagent. A further improvment was obtained by generating F Xa to a stable plateau level, thus minimizing influences from variations in time and temperature. The substrate hydrolysis was terminated with 1 M citrate buffer, pH 3.0, which reduced the environmetal effects as compared to acetic acid. Overall a fourfold reduction in reagent consumption was achieved. A high correlation with clotting assays was obtained with various types of factor VIII concentrates as well as with plasma samples from blood donors and hemophilia A patients (r>0.85). The accuracy was proven over the whole investigated range of factor VIII activities. Finally, the described microplate assay allows a high turnover of samples in a short period of time, still maintaining a coefficient of variation below 5%.