For patients with hemophilia A, researchers have developed a diagnostic approach for measuring neutralizing inhibitors and non-neutralizing antibodies (NNAs) to factor VIII (FVIII) based on the Luminex xMAP-based fluorescence immunoassay (xFLI). Development and validation of the method were described in a report in the journal Haemophilia.

The presence of inhibitors can hinder management of hemophilia using factor replacement therapy, and the presence of NNAs may be predictive of inhibitor development. However, according to the researchers, methods used to identify inhibitors and NNAs have shown a lack of standardization and variation in results between laboratories measuring them. The researchers set out to develop an improved diagnostic method for measuring inhibitors and NNAs.

The diagnostic approach developed by the researchers involved an xFLI system that they prepared in the laboratory. With this approach, the researchers used commercially available FVIII concentrates coupled to dye-labeled magnetic beads, which could then be used for immunoassays. The FVIII concentrates were a full-length product (Advate) and a product lacking in B domain (NovoEight). The researchers compared xFLI-based results with those obtained using an enzyme-linked immunosorbent assay (ELISA) and another approach called the chromogenic Bethesda-Nijmegen Assay (CBA).

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Measurements of precision between and within assays were reported as coefficients of variation (CVs). With xFLI-based assays, imprecision was below a CV of 6% across all analyses. With xFLI using NovoEight FVIII concentrate, the researchers were able to calculate a lowest limit of quantification of 0.084 ng/mL.

In analyses including 112 samples obtained from patients with hemophilia A and inhibitors, the xFLI system using Advate FVIII concentrate showed a statistically significant correlation with the ELISA system in detecting anti-FVIII antibodies (Spearman’s correlation coefficient [rs], 0.9144). In analyses of 86 samples, the xFLI system using Advate also showed a significant correlation with results from the CBA system for detecting anti-FVIII antibodies (rs, 0.8052). In analyses comparing the xFLI system with NovoEight FVIII concentrate, significant correlations were also seen with the ELISA approach (rs, 0.8677) and with the CBA approach (rs, 0.8261).

Analyses using ELISA and CBA methods demonstrated a concordance of 69.8% with each other. Overall concordance between the xFLI system and the ELISA approach was calculated to be 82.1%, and between the xFLI system and the CBA approach it was 77.9%. Sensitivity also appeared improved with xFLI than with both ELISA and CBA approaches.

“Our optimised xFLI assay exhibits excellent sensitivity, reproducibility, recovery and linearity and is a reliable method to detect both NNAs and inhibitory antibodies,” the researchers wrote in their report. They concluded that the xFLI approach showed greater sensitivity and reproducibility than seen with both ELISA and CBA approaches.

Disclosures: Some authors have declared affiliations with or received grant support from the pharmaceutical industry. Please refer to the original study for a full list of disclosures.


Martin M, Augustsson C, Lind V, et al. 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). Haemophilia. Published online October 3, 2022. doi:10.1111/hae.14669