Revealing and IgG4 analysis to factor VIII in haemophilia-A patients with and without inhibitors.

Introduction: The acquisition of factor VIII inhibitors poses major management challenges for haemophilia A (HA) patients. Most (Factor VIII) inhibitors are immunoglobulin G4 (IgG4) and G1 (IgG1) subclasses, with IgG4 being the most prevalent. The Nijmegen Bethesda Assay (NBA) was used to quantify inhibitors. However, the requirement for a large sample volume, accompanying costs, and required technical expertise complicate NBA, particularly in developing countries. ELISA-based screening proved to be more viable in a resource-constrained scenario.

Aim: This study aimed to standardise and evaluate an in-house IgG4 ELISA for the detection of haemophilia A inhibitors.

Methods: This study enrolled thirty HA patients with inhibitors, thirty three HA without inhibitors, and 33 healthy controls. Standardisation of in-house IgG4 ELISA was performed. The checkerboard method was employed to optimise plasma-derived Factor VIII concentrations (HEMOFIL M Baxalta US Inc.), sample dilutions, and anti-human IgG4-HRP conjugate (Southern Biotechnology, USA). The samples were evaluated three times, and the mean optical density (OD) was used to determine the cutoff.

Results: Using a cutoff OD (mean±2SD) of 0.502 in our in-house ELISA, we could differentiate healthy controls and HA without inhibitors from HA with inhibitors with 93.3 % sensitivity, 97.0 % specificity, 97 % NPV, and 93.3 % PPV, respectively. However, the accuracy was 95.83 %. The two-way mixed-effects model, interclass correlation (ICC) derived by Cronbach's Alpha was 0.912 (p = 0.001) and close to perfect agreement.

Conclusions: IgG4 ELISA is an effective method for detecting neutralising or functionally significant FVIII inhibitors, particularly in resource-constrained settings, following which patients may be referred to referral laboratories for quantification of inhibitors.