Optimization of pre-analytical heat treatment for inhibitor detection in haemophilia A.

Abstract

Factor VIII (FVIII) antibody formation is the greatest clinical and laboratory challenge within the haemophilia centre. The Nijmegen-Bethesda assay (NBA) is the gold standard for inhibitor quantification, but affected by pre-analytical variables including a patient's FVIII activity (FVIII:C). Pre-analytical heat treatment (PHT) provides a methodology for inhibitor testing when measurable FVIII:C is present. We evaluated the effect of different PHT conditions (time/temperature) on FVIII:C as well as on potency of inhibitory activity in samples containing FVIII:C (endogenous pooled plasma and exogenous recombinant FVIII (rFL-FVIII) concentrate) or FVIII inhibitor. PHT of endogenous FVIII at 37°C, 47°C and 52°C resulted in declining measurable FVIII:C at 120minutes (69%, 57% and 13% of the original FVIII:C, respectively). Incubation at 56°C resulted in FVIII:C≤1IU/dL after 60minutes for endogenous FVIII and 120minutes for rFL-FVIII. Incubation at 58°C resulted in FVIII:C<1IU/dL at 15-30minutes for endogenous FVIII and at 30-60minutes for rFL-FVIII. No difference was seen for inhibitor detection following PHT (56°C or 58°C) by NBA or anti-FVIII IgG ELISA. PHT at 58°C for 30minutes demonstrated consistent reduction in FVIII:C<1IU/dL without appearing to affect inhibitor detection. Laboratory awareness of differences in thermostability of different sources of FVIII is important when choosing PHT conditions.