INFLUENCE OF PHOSPHOLIPIDS ON THE ASSESSMENT OF FACTOR-VIII ACTIVITY

In view of reported discrepancies between different factor VIII assays , the influence of phospholipids on the performance of one-stage clott ing (OS) and chromogenic substrate (CS) assays was evaluated. The 13 d omain deleted recombinant factor VIII, rVIII SQ, two full-length recom binant products and a plasma derived factor VDI concentrate were each diluted into severe haemophilia A plasma and assayed against a plasma standard. The one-stage activity was 50, 80, 75 and 106%, respectively , of the chromogenic result. Variations in the phospholipid concentrat ion did not affect the chromogenic assay, except at very low levels wh ere the apparent activity increased. In contrast, dilution of the phos pholipid reagent had a substantial influence on the activity measured by OS assays, especially in the case of rVIII SQ. At low levels of pho spholipid, the one-stage activity of rVIII: SQ exceeded the chromogeni c result. When mixtures of phosphatidylserine (PS) and phosphatidyl-ch oline (PC) were used as a source of phospholipid, the OS results for r VIII SQ agreed well with the CS activity as long as the content of PS was below 10%, i.e., closer to the physiological level. At higher leve ls of PS, as in most commercial APTT reagents, the OS activity decreas ed. When the APTT reagent was replaced by platelets in the OS assay, t he results compared well with those obtained by the CS assay for both t-VIII SQ and full-length factor VIII products.