Mixing-based inhibitor screening in haemophilia A: challenges in interpretation

: Inhibitor development in haemophilia A patients is a dreaded complication of factor VIII (FVIII) replacement therapy. With increasing use of FVIII replacement therapy, there is an imperative need for cost-effective and standardized screening. To evaluate the efficacy of mixing-based inhibitor screening (MBIS) in the detection of FVIII inhibitors and to assess the best cut-off values for MBIS. Forty inhibitor positive and 40 inhibitor negative haemophilia A patients, diagnosed by standard criteria, with detailed clinical, haematological and on-demand treatment records were included. MBIS was evaluated in all 80 cases and a classical Bethesda assay and Nijmegen modification of Bethesda assay (NBA) were used as gold standards for inhibitor diagnosis. Classical Bethesda assay missed eight cases, most with low titres, which were confirmed by NBA. A systematic analysis of cut-offs for MBIS using a receiver operating characteristic curve fixed the cut-off at more than 5 s. MBIS detected 36 out of 40 inhibitor positive haemophilia A patients with a sensitivity, specificity, PPV and NPV of 90.0, 95, 94.7, 90.5%, respectively, whereas at the conventional cut-off of more than 10 s, MBIS detected only 25 of 40 cases with a low sensitivity of 62.5%. The likelihood ratio of a positive test was 11. The false-negative haemophilia A patients had low titres from 1.6 to 4.2 BU/ml. MBIS at a cut-off of 5 s can be considered as an effective screening test in low-resource situations. In clinical situations and in cases with clinical evidence of inhibitors we recommend that a direct NBA should be done.