A More Uniform Measurement of Factor VIII Inhibitors

Abstract

A group of hematologists, involved with hemophilia research and care in the U.S.A., met under the sponsorship of the Division of Blood Diseases and Resources of the National Heart and Lung Institute. In order to improve future communication among ourselves, we decided to alter our individual methods of measurement of inhibitors to the extent necessary to permit a uniform, although arbitrary, description of inhibitor units. We agreed to the following standards: (1) The incubation mixture consists of one part citratecl patient plasma, undiluted or diluted, plus an equal part of citrated pooled normal human plasma. (2) A control incubation mixture consists of equal parts of normal pooled plasma and imidazole buffer, as formulated by Dr. Biggs. (3) The mixtures are incubated at 37° C for two hours. (4) Assays specific for Factor VIII are then performed and the Factor VIII activity in the patient mixture is divided by the Factor VIII activity in the control mixture to determine the percent residual Factor VIII activity. (5) A patient plasma giving a residual Factor VIII activity of 50 percent in this test is said to contain one “Bethesda unit” of inhibitor per ml. (6) On a graph, the log percent residual Factor VIII activity is plotted against inhibitor units. If the residual Factor VIII activity of the incubation mixture is between 75 and 25 percent, the inhibitor units are read from the graph. Plasmas containing strong inhibitors are diluted with imidazole buffer before being placed in the incubation mixture. A dilution is sought which will result in a residual Factor VIII activity between 75 and 25 percent. The units of inhibitor read from the graph are then multiplied by the dilution factor to determine the number of Bethesda units of inhibitor per ml of undiluted patient plasma.We invite interested colleagues to join us in the use of this method, and we invite discussion of better methods of describing inhibitor potency.