A variation of the Nijmegen-Bethesda assay using heat or a novel heat/cold pretreatment for the detection of FIX inhibitors in the presence of residual FIX activity

Introduction:The inclusion of a heat treatment step has improved the classic Nijmegen-Bethesda assay for detection of factor VIII (FVIII) inhibitors in the presence of residual FVIII activity (FVIII:C). However, information regarding heat-modified Nijmegen-Bethesda assays for the detection of FIX inhibitors is still limited. Methods:Three methods to measure FIX inhibitors in the presence or absence of residual FIX activity (FIX:C) using three different activated partial thromboplastin time (aPTT) reagents were investigated. These included the standard Nijmegen-Bethesda assay (method 1), a heat-modified assay (method 2) and a novel heat/cold-modified assay (method 3). Results:In the absence of FIX:C, all methods measured similar levels of FIX inhibitor, while FIX inhibitor titres varied widely in the presence of residual FIX:C. Using method 1, inhibitors were not accurately measured in the presence of residual circulating FIX:C. Using method 2, detection was improved, especially at higher inhibitor titres. Using method 3, some additional sensitivity was obtained. The choice of aPTT reagent did not affect the detection of inhibitors. Conclusion:Heat pretreatment is recommended for detecting FIX inhibitors in samples with residual FIX:C. The heat/cold modification improved the sensitivity of the Nijmegen-Bethesda assay, resulting in higher tolerance for residual FIX:C.