A Rapid Screening Method To Increase Efficiency In Assaying Plasma Levels Of Factor VIII Inhibitors

The plasma level of naturally acquired inhibitors to clotting Factor VIII activity (F.VIII: C) was assessed by the Bethesda Inhibitor Assay (BIA). At inhibitor concentrations outside the BIA working range (25% to 75% residual F.VIII: C), patient plasmas had to be diluted. However, when a rough estimate of the inhibitor concentration was unavailable, the choice of dilution was arbitrary and testing became tedious and inefficient. In an effort to improve the BIA, a screening test was devised to predict the appropriate plasma dilution. It is based on the partial thromboplastin time (PTT) of the incubated sample and is performed just prior to preparing dilutions for the F.VIII: C assay. Test plasmas were diluted serially in multiples of two, from 1:2 to 1:1024, and then incubated according to the BIA. After 90 min, samples were removed for PTT determinations to screen for the appropriate dilution to use in the F.VIII: C assay. The first two test dilutions to produce PTT values 21 sec (± 4 sec, 95% confidence interval) greater than the control resulted in a residual F.VIII: C for the BIA within the 25% to 75% acceptable range. When the log ratio of patient-to-control PTT determinations (specific PTT) for a given dilution was plotted as a function of log residual F.VIII: C at that test dilution, in 81 Bethesda assays a linear relationship was observed (p> .25; with r2 = .81). The screening test was 89% effective in predicting an appropriate dilution (range 0 to 1020 Bethesda units/ml). The accuracy can be enhanced further if, based on the first of three dilution screen tests in which the specific PTT >21 sec, the F.VIII: C assay is performed using the middle dilution value first. Depending on whether that dilution yields an acceptable residual F.VIII: C, either the higher or lower dilution is chosen in the second F.VIII: C assay.