Factor VIII inhibitor profile (Nijmegen-Bethesda & ELISA)

FVIII inhibitors are progressive so dilutions of test plasma (i.e. reducing concentrations of inhibitor) are incubated for 2 hours at 37°C in the presence of a fixed amount of exogenous FVIII in the Bethesda assay. One unit of inhibitor is defined as the amount of inhibitor that will neutralise 50% of a normal plasma containing 100% FVIII after the 2 hour incubation. The amount of residual FVIII in each tube is used to calculate the inhibitor level.

Our laboratories employ a modified Nijmegen-Bethesda assay which uses buffered normal plasma and dilutions in 4% BSA to reduce pH drift and maintain constant protein concentration, that can otherwise falsely suggest the presence of a weak inhibitor.

FVIII antibodies can also be detected using indirect enzyme-linked immunosorbent assay (ELISA). FVIII antibodies in patient sera, whether inhibitory or 'immune', are captured in microtitre plates coated with recombinant FVIII. Unbound material is then washed off and a solution of antibody to human immunoglobulin that is conjugated to an enzyme is added to ‘tag’ onto any captured FVIII antibodies. Unbound conjugate is washed off and a substrate for the enzyme is added, the product of the enzyme-substrate reaction being coloured. Colour intensity is in direct proportion to the degree of conjugate-binding, itself proportional to the amount of FVIII antibody capture. The assay is not quantitative and is reported as either positive or negative based on whether the colour intensity exceeds a kit-specific cut-off value.