[Analysis of factors which affect the measurement of factor VIII inhibitor by Bethesda method]

Factor VIII neutralizing antibody (factor VIII inhibitor) sometimes develops in the patients with hemophilia A who are treated with factor VIII concentrates and more rarely in non-hemophilic individuals as auto antibody. Factor VIII inhibitor titer is most commonly measured by Bethesda method in which the sample is mixed with normal pooled plasma as a source of factor VIII for 2-hour incubation followed by measurement of residual factor VIII activity. Because of its simplicity and accessibility of normal pooled plasma, Bethesda method is widely used in clinical laboratories. However, the reproducibility of the assay is not generally considered to be satisfactory and measurement of inhibitor units varies among laboratories. In the present study, the factors which affect the measurement of Bethesda method were assessed. The reproducibility of Factor VIII activity showed better in a factor VIII dose dependent manner and varies according to clotting threshold of the sample that is analyzed by an automated coagulation equipment and a desktop computer. The reproducibility of inhibitor measurement was worse in the inter-assay than in the intra-assay. The assay fluctuation of the measurement of lower inhibitor units tends to be more evident when the sample of 0.39 to 2.39 BU/ml were tested. The factor VIII content in the normal pooled plasma did not affect the measurement of factor VIII inhibitor units in the range of 0.8 to 1.2 U/ml. The buffers retained factor VIII in the control sample during the 2-hour incubation better in the order of imidazole buffer, bernard buffer, Tris-HCl buffer and saline.