Antibodies to factor VIII in plasma of patients with hemophilia A and normal subjects

Abstract

Non-neutralizing factor VIII (FVIII) antibodies (FVIII-Ab) in hemophilia A may be associated with an abnormal clinical response to FVIII concentrates. Patients with FVIII inhibitors may develop noncoagulation FVIII-Ab after the induction of immunotolerance. Natural FVIII-Ab may be detected in the plasma of some healthy subjects. The aim of this study was to analyze the presence of FVIII-Ab in the plasma of 53 normal blood donors and 124 patients with hemophilia A (18 patients had a previous history of FVIII inhibitor, but only 12 had inhibitor at the moment this study was performed). FVIIII inhibitor was measured using the Bethesda method. FVIII-Ab were analyzed by a specific ELISA assay using purified FVIII from a monoclonal concentrate and a standard plasma containing 26 Bethesda units (BU) of FVIII inhibitor. Purified FVIII was used to coat wells of a microtiter plate and was incubated with dilutions of plasma to be tested. Bound human IgG FVIII-Ab were detected by incubation with polyclonal sheep anti.human IgG alkaline phosphatase conjugate, and the OD405 was quantitated. A linear fit was obtained (by plotting FVIII-Ab positivity [OD 405nm] versus BU titer) when serial dilutions of this standard inhibitor plasma, containing titers of 0.5 BU or higher, were used. Four different levels of FVIII-Ab positivity [OD 405nm] were distinguished in this assay: Negative levels (-) were obtained with dilutions of the standard inhibitor containing < 0.5 BU. Mild levels (+) were obtained with dilutions of 0.5-5 BU. Moderate levels (+2) were obtained for dilutions ranging from 5-25 BU. Maximum positivity (+3) was obtained for dilutions of titers > 25 BU. FVIII-Ab positivity was detected in eight of the normal subjects (15%): three were found to be moderately positive (+2) and five mildly positive (+). No inhibitory activity was detectable when whole plasma was used. All the hemophilic patients with a presence of FVIII inhibitor at the time of the study were found to be positive for FVIII-Ab. In addition, the level of positivity correlated with the corresponding BU. Four of the six patients who had a history of inhibitory were negative and two positive. Twenty additional patients (16.12%) in whom no inhibitory activity was detected were found to be positive for FVIII-Ab: 16 + and four +2. The mean age of patients with FVII-Ab positivity was significantly higher than that of patients of the FVIII-Ab negative group (p < 0.005). In conclusion, FVIII-Ab positivity in patients with hemophilia A was 17.7% higher than the level of positivity detected by an inhibitory assay. We propose that this method for FVIII-Ab analysis could be used for patients with hemophilia A, at least to complement the functional inhibitor assay. FVIII recovery or half-life should be assessed in patients who test positive for FVIII-Ab and who show no evidence of inhibitor.

Quantitation of anti–factor VIII antibodies in human plasma

“…The incidence of autoantibodies in the healthy population examined by our assay is at the low end of reported frequency (0%-36%) for healthy individuals. [41][42][43][44][45][46] The high incidence of antibodies in healthy donors reported by others might arise due to the lack of nonspecific binding controls in the detection assays used to analyze the samples. Inclusion of a binding control in our assay largely eliminates from analyses signals from the nonspecific binding of plasma proteins.…”

Efficacy and safety of rVIII-SingleChain: results of a phase 1/3 multicenter clinical trial in severe hemophilia A

“…This rate of ADA development is in line with other recently approved rFVIII products, 7 and the incidence of non-neutralizing antibodies against FVIII in healthy individuals and in patients with hemophilia. 22,23 Eight patients had non-inhibitory ADAs prior to dosing with rVIII-SingleChain, 7 of whom remained ADA positive at the end of the study.…”

Nonneutralizing antibodies against factor VIII and risk of inhibitor development in severe hemophilia A

“…In previous reports, NNA prevalence ranged from 2% to 3% in healthy individuals 16,18,21,24 and from 12.2% to 53.8% in patients with hemophilia A. [13][14][15][16][17][18][19][20][21][22]24,25,29,30 Several factors, particularly that all previous studies predominantly included patients with multiple transfusions, explain this large variation and the gaps in knowledge about their potential clinical role.…”

“…[13][14][15][16][17][18][19][20][21][22]24,25,29,30 Several factors, particularly that all previous studies predominantly included patients with multiple transfusions, explain this large variation and the gaps in knowledge about their potential clinical role. For instance, the intron 22 inversion of FVIII mutation was associated with an increased frequency of NNAs in patients who had had multiple transfusions in 1 study 29 that was not confirmed in another study.…”

“…Their prevalence is approximately 2% to 3% in healthy individuals, 16,18,21,24 but estimates in patients who have hemophilia with different degrees of severity vary widely from 12% to 54%. [13][14][15][16][17][18][19][20][21][22]24,25,[29][30][31] Although NNAs and inhibitors cannot be distinguished on the basis of their isotypes, clonality, and epitopes, 32 recent data published by Hofbauer et al 16 indicate that anti-FVIII immunoglobulin G (IgG) with inhibitory activity has an up to 100-fold higher affinity for FVIII than IgG without inhibitory activity. On the basis of cross-reactivity studies, it has also been suggested that FVIII inhibitors in hemophilia A patients originate from the expansion of a natural anti-FVIII clone of B lymphocytes that exists before any treatment with FVIII and secretes anti-FVIII antibodies similar to the natural antibodies found in healthy individuals.…”