Development of a new modified Bethesda method for coagulation inhibitors: the Osaka modified Bethesda method

Abstract

The Nijmegen assay for the factor VIII (F-VIII) inhibitor is recommended by the International Society on Thrombosis and Haemostasis/Scientific and Standardization Committee. However, due to cumbersome and complicated preprocessing, it is presently difficult to introduce this assay into hospital laboratories. We used buffered plasma that was made by addition of 1 volume of 1 mol/l HEPES buffer at pH 7.35 to 9 volumes of plasma to form the test samples. The inhibitor titer was calculated by the remaining rate of F-VIII coagulation activity (F-VIII:C), using the ratio of actual value to the theoretical value. Five hundred microliters of the buffered test plasma and the control (30 mmol/l HEPES buffered saline at pH 7.35) were each mixed with equal volumes (500 μl) of normal pooled plasma in a test tube (11 mm internal diameter and 6.5 ml volume capacity), and incubated at 37°C for 2 h. In our modified Bethesda method, there were no significant changes in pH and F-VIII:C of control and test mixtures after incubation tests for stability. With the modified method, the inhibitor titers (mean, SD) from examining three hemophilia A plasma samples (F-VIII:C, <1-3%) and 40 normal samples (F-VIII:C, 34.5-168.3%) were 0.032, 0.057 and -0.009, 0.057, respectively. By our method, the F-VIII inhibitor titer of type I inhibitor-positive samples was higher than the Nijmegen method, and for type II inhibitor-positive samples, the titer was similar. We believe that our method can be applied to not only the type I inhibitor, but also to assays of type II inhibitor, without cumbersome and complicated preprocessing.