Low‐titre inhibitors, undetectable by the Nijmegen assay, reduce factor VIII half‐life after immune tolerance induction

Abstract

ing complications compared with the low-dose treatment. This finding of bleeding discrepancy between the two dose regimens persisted even in the post-ITI prophylactic phase (although not significant, P = 0.088), during which the inhibitors were not detectable with the established assays [5]. In our Haemophilia Treatment Centre in Nijmegen, we also recorded an abnormal high need for FVIII supply to prevent bleedings in the early post-ITI phase with formerly inhibitorpositive patients. We hypothesized that patients in the early post-ITI phase still have low-titre inhibitors that cannot be detected with the established assays, but contribute to rapid disappearance from the circulation of FVIII. The putative lowtitre inhibitors may, therefore, be responsible for the higher bleeding risk in this stage of treatment. In order to test this hypothesis we developed a low-titre inhibitor assay that is 20 times more sensitive than the commonly used (Nijmegen) assay. The method is based on concentration of the test plasma by selective protein filtration and quantification of the inhibitor titre by a mixing assay as described below. At first, residual FVIII that may interfere with the test was removed from both the test and reference (FVIII-deficient plasma) samples by heating the plasma samples for 1.5 h at 58 � C followed by a short centrifugal step (2 min at 18 000 ·g). This procedure also inactivated all other coagulation factors. Thereafter, 0.5 mL of the supernatant plasma was centrifuged for 45 min at 4200 ·g at room temperature in an Ultra-Free � -4 centrifugal filter unit with a nominal molecular weight limit of 100 kD (Millipore � ,