A simple and accurate microplate assay for the determination of factor VIII activity.
Based on the CoatestR 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 improvement 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 environmental 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 greater than 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%.
D001780:Blood Coagulation Tests / Q000379:methods; D002863:Chromogenic Compounds; D005069:Evaluation Studies as Topic; D005169:Factor VIII / Q000032:analysis* / Q000378:metabolism; D015951:Factor Xa; D006801:Humans; D008722:Methods; D011933:Reagent Kits, Diagnostic; D012697:Serine Endopeptidases / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
D002863:Chromogenic Compounds; D011933:Reagent Kits, Diagnostic; D005169:Factor VIII; D012697:Serine Endopeptidases; D015951:Factor Xa