Prevalence and Epitope Specificity of Non-Neutralizing Antibodies in Haemophilia A Patients without Inhibitors Using a Multiplex x-MAP Technology.

Antibodies (Abs) directed against Factor VIII (FVIII) remain the main iatrogenic complication in haemophilia A (HA) patients. Anti-FVIII Abs neutralize procoagulant activity of FVIII. They are called inhibitors. Non-neutralizing antibodies (NNA) have also been described in hemophiliacs treated by FVIII concentrates. Their role and prevalence are still under debate. NNA could form immune complexes with FVIII and be responsible of an increased FVIII clearance inducing a shortened half-life. The aim of this retrospective study was to evaluate the prevalence of NNA in a large cohort of HA patients without inhibitors and to determine their epitope specificity against the two chains of FVIII or the B domain, using a x-MAP technology. Indeed, this approach has many advantages: it is fast, requires small volumes of plasma, and is suitable for multiplexing immuno-assay.

Samples of 187 patients (mean age 29 years, range [1-86]) with severe (n= 133), moderate (n= 31) or mild (n= 23) HA from three haemophilia centers were studied. All patients have been previously treated and were inhibitor-free at the time of the test (<0.6 Bethesda Units/mL). Among them 33% (n=61) were treated by plasma-derived FVIII while 67% (n=126) had received recombinant FVIII (rFVIII). Two assays based on x-MAP technology were used: (i) the first determined the NNA reactivity with the heavy chain (HC) or the light chain (LC) of FVIII (Lavigne-Lissalde et al. 2008); (ii) the second identified the NNA directed towards the B-domain, by differential binding to full-length rFVIII or B-domain deleted rFVIII.

The observed NNA prevalence in the 187 patients under study was 24% (n=45): 13.3% (n=6) of NNA were directed against the LC, 62.2% (n= 28) were directed against the HC and 24.5% (n=11) against both chains. In the NNA positive population, the proportion of NNA directed toward the B-domain was of 9% (n=4). No difference was observed in the prevalence between the 3 centers. The prevalence of NNA in patients grouped into four categories of age [1-15 yrs] (n=51, NNA prevalence: 27%), [16-30 yrs] (n=65; 20%), [31-45 yrs] (n=35; 20%) and [>45 yrs] (n=33; 27%) was not statistically different.

This retrospective study, using the x-MAP technology, found a prevalence of 24% NNA in HA patients, comparable to other reports in the literature. Most of the NNA were directed against the HC of FVIII. In 9% of the cases, NNA recognized the B domain. Further prospective studies will allow to better explain the role of NNA, overall on the FVIII pharmacokinetics and particularly those binding to the B domain. Other prospective studies could highlight a difference according to the type of treatment.

Lavigne-Lissalde G. et al. (2008). “Simultaneous detection and epitope mapping of anti-factor VIII antibodies.” Thromb Haemost99(6): 1090-6

No relevant conflicts of interest to declare.