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%.

Freezing of plasma and recovery of factor VIII.

The freezing procedure and its influence on the plasma quality was studied with a new plasma container and freezing bath and compared with the results from those obtained with commonly used equipment. Cryoprecipitation was performed on plasma pools frozen at three different rates, and quality and recovery of the cryoprecipitates were determined by analysis of factor VIII, factor VIII-related antigen (vWF:Ag), and fibrinogen. The plasma freezing time was 95 and 65 percent shorter with a new -40 degrees C freezing bath as compared to -25 and -80 degrees C freezing boxes, respectively. A further 30 percent reduction of the plasma freezing time was gained by the introduction of a new flat 750-ml plasma container. Rapid plasma freezing prevented substantial loss of factor VIII in frozen plasma. Cryoprecipitate purity measured as factor VIII-to-fibrinogen ratio increased from 0.50 to 0.82 (IU/mg) when the freezing time was decreased from 10 hours to 45 minutes, although the recovery of factor VIII increased less. In summary, freezing of plasma in small flat containers in an effective ethanol bath resulted in rapid freezing with high recovery of factor VIII in plasma, and increased purity and recovery of subsequently processed cryoprecipitate. This freezing concept, adapted at Swedish blood banks, has contributed to higher source plasma quality and increased self-sufficiency.

Screening of factor VIII:C levels in blood donors.

A new chromogenic peptide substrate method, modified for assay with semi-micro tubes, Cobas Bio centrifugal analyser and microplates, was used for screening of F VIII:C in blood donors. The precision of the assays is high and the costs are reasonable. The assay with microplates is especially suitable for selection of donors for a plasma programme and for quality control in blood banks.

Improvement of plasma quality as raw material for factor VIII:C concentrates. Storage of whole blood and plasma and interindividual plasma levels of fibrinopeptide A.

Blood collected into different anticoagulants was stored in small tubes at +4 degrees C for up to 26 h. Seven blood coagulation analyses were performed under standardized conditions. High yield and stability of factor VIII:C were found for ACD and CPD-adenine. No changes could be found in the other six parameters tested. Whole blood in blood bags could be stored for 2-4 h at +4 degrees C with maximal yield of F VIII:C, with blood stored overnight the recovery was 65%. In plasma F VIII:C was stable for at least 2 h at room temperature. F VIIIR:Ag and F VIIIR:RCoF were stable in both whole blood and plasma. No activation by plasmin as measured by B beta 15-42 could be demonstrated. The initial FPA levels, reflecting thrombin activation, in the donated blood differed individually and in some blood bags very high concentrations were found. The levels of FPA were not correlated to the time for collection of a bag of blood.