Mapping of Conformational Epitopes, and Analyze of the Distribution of Ig Isotypes and Subclasses Using a Semi-Quantitative Multiplex Assay in 6 Cases of Postpartum Acquired Hemophilia.

Background and objectives: Acquired hemophilia (AH) is a severe life-threatening autoimmune disease due to the development of polyclonal autoantibodies (autoAbs) which neutralize the procoagulant activity of coagulation factor VIII (FVIII). The most common epitopes for autoAbs to FVIII are located in the A2, A3 and C2 domains of FVIII. We have recently developed a multiplex assay based on the Luminex technology that allows simultaneously the detection and epitope mapping of anti-FVIII antibodies (Lavigne-Lissalde et al, Thromb; Haemost. 2008). This technology is fast and requires only 100 μl of plasma and we showed that most autoAbs to FVIII are monospecific and preferentially bind to the light chain (LC) of FVIII. The aim of the present study was to evaluate the epitope specificity and IgG isotype/subclasses of autoAbs to FVIII detected in the post partum period.

Methods: Acquired hemophilia was diagnosed in six women (median age: 28 years) included in a French cohort (SACHA) and who exhibited prolonged activated partial thromboplastin time, low FVIII levels (range = 0.5–21 IU/dL) and inhibitor titers between 1.5 and 40 Bethesda units (BU). The epitope mapping was studied in each patient by testing 5 plasma samples collected (at inclusion and after 1, 3, 6 and 12 months of follow up) using a multiplex assay as previously described (Lavigne-Lissalde et al, Thromb; Haemost. 2008). A panel of monoclonal antibodies that bind to LC, heavy chain (HC) and the C2 domain of FVIII was employed and the use of specific immunoconjugates also allowed to define the isotype (IgG, A, M) and subclass (IgG₁ to IgG₄) distributions of autoAbs to FVIII. Results were expressed in mean fluorescence intensity (MFI).

Results: The autoAbs detected in the post partum in the six women studied recognized both and simultaneously HC and LC with predominant epitopes located in the LC and the C2 domain in 5 cases. At the inclusion, the autoAbs were mixtures of IgG₁ and IgG₄ in all patients, but the latter subclass was predominant in most cases. In addition, IgM Abs were also present in 3 women. No correlation between the titer of inhibitor (in BU) and the level of autoAbs binding (in MFI) could be evidenced. The evolution was good in all patients but one case for whom an increase in IgG₄ binding to the FVIII C2 domain was demonstrated whereas inhibitor titer decreased (from 40 to 2.6 BU). Importantly, no modification in the epitope specificity between inclusion and 12 months of follow-up was found.

Conclusion: The study shows that autoAbs to FVIII developed in the context of pregnancy are mainly IgG₄ specific to the C2 domain. In addition, the epitope mapping of anti-FVIII antibodies by multiplex approach could contribute to a better understanding of the pathophysiology of acquired hemophilia.