Preparation and use of a Combined Reagent for the Two-Stage Assay of Factor VIII Procoagulant Activity

Activated serum, bovine factor V and phospholipid, diluted In barbitone sodium saline pH 7.3 were prepared as a pooled reagent and freeze-dried in aliquots. Normal plasma with CPD as anticoagulant was used as the substrate. The reagent was easily prepared, is convenient to use and has been assessed in three independent trials. Repeated assays performed using the International standard for factor VIII and the British Standards for plasma factor VIII and concentrate factor VIII provided combined potency estimates which showed no significant difference from the values stated for these standards. In a group study, where seven participants performed two-stage assays using their normal reagents and techniques, the potency estimates obtained by RPL using the combined reaqent were in close agreement with those obtained by workers with other assay methods. Quality control measurements of procoagulant activity in factor VIII concentrate performed in duplicate at RPL and plasma Fractionation Laboratory PFL , Oxford over a period of several months provided a satisfactory comparative assessment of the combined reagent assay and the two-stage factor VIII assay used at PFL. Use of the combined reagent in a simplified two-stage assay has been studied and its suitability in automatic techniques has beer shown to be acceptable.