In this article, we will describe the assays used, as well as their
development, pitfalls in testing such as inter-laboratory variability and false negative/positive results, as well as some strategies for overcoming these pitfalls and potential alternative test approaches. The inter-laboratory coefficient of variation often approaches (and sometimes exceeds) 50%, as evidenced by various external quality assessment groups, and this variability has not improved over recent years. Additional important considerations include appropriate interpretation of test results, repeat testing for confirmation, Everolimus clinical trial and assessment of recovery as part of the diagnostic process. The Bethesda assay (BA) for factor(F)VIII inhibitors, described in 1975 by Kasper and colleagues [1], was the first method yielding
an acceptable degree of standardization, using normal pooled plasma (NPP) as Palbociclib FVIII source and imidazole buffer as reference sample. The BA replaced the Oxford assay [2], which lacked reliability as it used FVIII concentrate (cryoprecipitate) as FVIII source. Since the late 1980s, inhibitor assays were performed with increasing frequency, largely driven by multicentre studies of new purified and recombinant FVIII products. The BA soon appeared to be rather non-specific, yielding many false positive results [3]. Its sensitivity and specificity were improved
by modification in the Nijmegen assay (NA), by buffering the NPP (BNPP) and replacing the imidazole buffer with inhibitor-free Sodium butyrate FVIII-deficient plasma as reference sample [4]. Additional recommendations, including heating of test and reference plasma to remove residual FVIII [5], the appropriate use of FVIII-deficient reference plasma [6], the use of 4M imidazole solution instead of solid imidazole for buffering the incubation mixture and the determination of residual FVIII activity with chromogenic substrates, continue to improve the sensitivity and specificity of the assay. Nevertheless, inter-laboratory coefficients of variation (CV) approaching (and sometimes exceeding) 50% are evidenced by various EQA groups, inclusive of ECAT (External quality Control of Assays and Tests) Foundation, NEQAS (UK National External Quality Assurance Scheme) and RCPA (Royal College of Pathologists of Australasia) (Fig. 1) [7-10]. This variability has not improved over the years. Reasons for such high variability largely derive from assay variability, which can occur at any stage within the assay, including choice and source of laboratory reagents, buffering (yes, no, how), NPP, etc.