Characterization of Anti-FVIII Antibodies in Acquired Hemophilia A Patients Using a bead-based multiplex immunoassay

Acquired hemophilia A (AHA) is an autoimmune disorder characterized by bleeding due to neutralizing antibodies against coagulation factor VIII (FVIII). The Nijmegen Bethesda assay (NBA) is the gold standard for detection of neutralizing antibodies (inhibitors). Whereas both, inhibitors and non-neutralizing antibodies can be detected by immunoassays.

The in-house multiplex Luminex™ bead-based domain-epitope binding assay (LumiTope) comprised full-length (FL) and B-domain deleted (BDD) FVIII, along with nine purified FVIII single or multi-domains as previously described1. These proteins were coupled to magnetic beads to detect domain specific Immunoglobulin (IgG (IgG1-4), IgA, IgM and IgE) anti-FVIII antibodies in a large cohort of AHA patients. Data were acquired as Mean Fluorescence Intensity (MFI).

The study included a total of 80 AHA patients. Demographic and baseline characteristics are detailed in Table 1. All patients were screened for anti-FVIII antibodies of IgG, IgM, IgA, and IgE isotypes, with no detection of anti-FVIII antibodies of the IgE subtype. Overall, the LumiTope assay demonstrated high sensitivity and specificity, with all ELISA-positive patients also testing positive in LumiTope. The BDD-FVIII bead exhibited higher MFI values compared to the FL-FVIII bead for anti-FVIII antibodies of the IgG isotype. MFI values for IgA and IgM antibodies were lower compared to IgG antibodies. AHA patients exhibited distinct differences in IgG subclasses and domain specificity of anti-FVIII antibodies compared to congenital hemophilia A (HA) patients. Analysis of IgG subtypes revealed that similar to HA patients IgG4 antibodies were predominant (96%). However, all other IgG subclasses were equally detected among AHA patients (IgG3 (60%), IgG1 (55%), and IgG2 (44%)). Interestingly, a comparison of the overall domain specificity of anti-FVIII antibodies in congenital and AHA patients showed that the majority of AHA patients had anti-FVIII antibodies directed towards the light-chain beads, specifically the C1C2 and C2 domain beads, whereas congenital H A patients primarily had antibodies directed towards the A2 and A2a2 bead.

LumiTope assay provides a sensitive and specific method for detection but also domain specification of anti-FVIII-antibodies in AHA patients. Different domain specificities of the identified anti-FVIII antibodies may be associated with disease outcomes.

The study was supported by Octapharma Germany. There is no other conflict of interest.