Nonneutralizing FVIII-specific antibody signatures in patients with hemophilia A and in healthy donors

Schweiger, Helmut; Rejtő, Judit; Hofbauer, Christoph J.; Berg, Verena; Allacher, Peter; Zwiauer, Karl; Feistritzer, Clemens; Schuster, Gerhard; Ay, Cihan; Reipert, Birgit M.; Pabinger, Ingrid

doi:10.1182/bloodadvances.2021005745

Cited by 11 publications

(13 citation statements)

References 75 publications

(115 reference statements)

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“…Indeed, 1 BU/mL only showed a borderline result when spiked into plasma NP #1. On the other hand, however, plasma NP #2 showed a positive result also for the unspiked sample, most probably reflecting the well‐known presence of non‐neutralizing FVIII‐specific antibodies in healthy individuals 21 . Further FVIII‐spiking studies showed only marginal impact of FVIII at levels around 0.15 IU/mL (Figure S1).…”

Section: Resultsmentioning

confidence: 93%

“…Indeed, such a strategy would address the described presence of low-titer non-inhibitory FVIII-antibodies in healthy individuals. 21,25 In order to standardize the detection of FVIII-bAb, determination of SCPs for the FVIII-bAb assay was done at both sites. In this regard, the specificity was pre-defined as 95%, necessitating the need for a confirmatory assay setting whose specificity was set to 99.9%, yielding a 0.1% false positive rate for the final sample results.…”

Section: Fviii-bab Assaymentioning

confidence: 99%

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Two‐center validation of assays for the detection of binding and neutralizing anti‐factor VIII antibodies

Müller,

Neimanis,

Kahle

et al. 2023

Haemophilia

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IntroductionPatients with hemophilia A treated with coagulation Factor VIII (FVIII) products are at risk for developing anti‐FVIII antibodies. The ABIRISK Consortium aimed to provide knowledge on the formation and detection of anti‐drug antibodies against biopharmaceutical products, including FVIII. Accordingly, standardized and validated assays for the detection of binding (total) and neutralizing antibodies are needed.AimTwo‐center validation of an ELISA for the detection of total FVIII‐binding IgG‐antibodies and Nijmegen‐Bethesda assays for the quantification of FVIII‐neutralizing antibodies according to consensus validation guidelines.MethodsValidation of assays at both sites was done according to published recommendations and included preanalytics, the determination of key assay parameters, including cut‐points, assay sensitivity, precision, and FVIII interference.ResultsThe validated assays reproducibly detected FVIII‐binding and ‐neutralizing antibodies with comparable performance in both laboratories. Floating screening cut‐points were established for both assays. Determined mass‐based sensitivity of both assays (all values ≤66 ng/mL) complied with the minimum sensitivity for the detection of anti‐drug antibodies as recommended by the FDA (<100 ng/mL). Intra‐ and inter‐assay coefficients of variation did not exceed 25%. Assay validation further revealed that pre‐analytical heat treatment led to potentially false‐positive ELISA results, while up to 0.15 IU/mL, residual FVIII showed no significant impact. Overall, good agreement of results was found for patient samples analyzed at both study sites.ConclusionComprehensive validation of different anti‐FVIII‐antibody assays in two laboratories gave novel insights into the impact of pre‐analytical sample treatment as well as the comparability of test results generated by the use of methodically different assays.

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Section: Resultsmentioning

confidence: 93%

Section: Fviii-bab Assaymentioning

confidence: 99%

Two‐center validation of assays for the detection of binding and neutralizing anti‐factor VIII antibodies

Müller,

Neimanis,

Kahle

et al. 2023

Haemophilia

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“…26 Another limitation of the study is that no detection of IgG subclasses was performed as in the recent publication by Schweiger et al since this was not the aim of the current study. 27 All methods were not performed in all samples and especially CBA results were lacking and could not be performed due to lack of sample, since the amount of plasma needed for xFLI and ELISA is much smaller.…”

Section: Discussionmentioning

confidence: 99%

Methods for anti‐factor VIII antibody levels in haemophilia A patients – validation of a multiplex immunoassay and comparability with assays measuring non‐neutralising and neutralising antibodies (inhibitors)

Martin¹,

Augustsson²,

Lind³

et al. 2022

Haemophilia

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Introduction:The development of neutralising (inhibitors) and non-neutralising antibodies (NNAs) is a complication to factor replacement therapy in haemophilia. The diagnostic methods available lack standardisation, have high inter-laboratory variation, and false-negative as well as false-positive results may affect treatment.Both functional inhibitors and NNAs may be detected with higher reproducibility, sensitivity and specificity using the immunological Luminex xMAP-based fluorescenceimmunoassay (xFLI).Aim: Validation of our xFLI and comparability with enzyme-linked immunosorbent assay (ELISA) and chromogenic Nijmegen-Bethesda assay (CBA) for anti-FVIII antibodies in haemophilia A (HA) patients. Methods:The xFLI method was developed with full-length and B-domain deleted factor coupled to magnetic beads, optimised and validated for performance characteristics. Comparability with ELISA and CBA was evaluated in HA patient samples (n = 112), serial samples in six inhibitor patients and reference interval and decision-limits in healthy donors (n = 44). Results:The intra-and inter-assay precision (CV%) for the xFLI method was below 6% and detection limit (LLOQ) .084 ng/mL (NovoEight). All ELISA-positive samples were positive with either Advate or NovoEight. Additionally, 10.7%-14.3% were xFLIpositive and ELISA-negative. All but one CBA-positive sample was above 3SD with xFLI; one was between 2 and 3SD. 29.1% were xFLI-positive and CBA negative. The overall concordance between xFLI and ELISA was 82.1% and xFLI and CBA 77.9%. Conclusion:The anti-FVIII antibody xFLI method is adaptable to clinical practice and more sensitive and reproducible than ELISA and CBA. Actual NNA titers are determined to both full-length and B-domain deleted FVIII. The xFLI is thus valuable for confirmation of all anti-FVIII antibodies.

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“…It is important to recognize that non-inhibitory anti-FVIII antibodies can also be present in patients with hemophilia A and even in healthy individuals [ 11 , 12 ]. For some hemophilia A patients, these non-inhibitory antibodies can influence the half-life of FVIII in circulation [ 13 ] and impact the efficacy of replacement therapy, although to a smaller magnitude.…”

Section: The Development Of Anti-factor VIII Neutralizing Antibodies ...mentioning

confidence: 99%

Immunogenicity of Current and New Therapies for Hemophilia A

et al. 2022

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Anti-drug antibody (ADA) development is a significant complication in the treatment of several conditions. For decades, the mainstay of hemophilia A treatment was the replacement of deficient coagulation factor VIII (FVIII) to restore hemostasis, control, and prevent bleeding events. Recently, new products have emerged for hemophilia A replacement therapy, including bioengineered FVIII molecules with enhanced pharmacokinetic profiles: the extended half-life (EHL) recombinant FVIII products. However, the main complication resulting from replacement treatment in hemophilia A is the development of anti-FVIII neutralizing alloantibodies, known as inhibitors, affecting approximately 25–30% of severe hemophilia A patients. Therefore, the immunogenicity of each FVIII product and the mechanisms that could help increase the tolerance to these products have become important research topics in hemophilia A. Furthermore, patients with inhibitors continue to require effective treatment for breakthrough bleedings and procedures, despite the availability of non-replacement therapy, such as emicizumab. Herein, we discuss the currently licensed treatments available for hemophilia A and the immunogenicity of new therapies, such as EHL-rFVIII products, compared to other products available.

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Nonneutralizing FVIII-specific antibody signatures in patients with hemophilia A and in healthy donors

Cited by 11 publications

References 75 publications

Two‐center validation of assays for the detection of binding and neutralizing anti‐factor VIII antibodies

Two‐center validation of assays for the detection of binding and neutralizing anti‐factor VIII antibodies

Methods for anti‐factor VIII antibody levels in haemophilia A patients – validation of a multiplex immunoassay and comparability with assays measuring non‐neutralising and neutralising antibodies (inhibitors)

Immunogenicity of Current and New Therapies for Hemophilia A

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