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Introduction: Monitoring replacement therapy with standard and extended half-life (EHL) products is challenging, since one-stage assay (OSA) and chromogenic substrate assay (CSA) results may differ significantly. Recent recommendations include local validation of each new product with recovery within 20-30%, depending on activity level., Aim: To validate factor VIII (FVIII) activity for monitoring products in clinical use on Atellica Coag and to correlate it with thrombin generation., Methods: Plasma samples spiked with Advate ® , Elocta ® , Adynovi ® , Nuwiq ® , NovoEight ® and Afstyla ® (0.05, 0.20, 0.50 and 0.80 IU/ml) were analysed using Atellica Coag 360 with CSA-1 (Coatest SP) and CSA-2 (FVIII chromogenic), and OSA (Actin FS). Thrombin generation was performed using two thrombin generation assays (TGA-1 (Thrombinoscope) and TGA-2 (Technothrombin)., Results: All products at levels above 0.05 IU/ml, except Adynovi, showed acceptable recovery using CSA-1, whereas measurements using CSA-2 gave more results outside the target level. All products, except Afstyla, showed acceptable recovery using OSA. Correlation between CSA-1 and OSA was excellent (r 2 =1.0) with biases of 6-3​2%, depending on FVIII product. A clear dose-response was seen for all thrombin generation parameters and products using both methods, except at low levels for lag time using TGA-1. With CSA-1 as an independent variable, the correlations to thrombin peak (measured with TGA-2) were good (r 2  = .8-.9)., Conclusion: Our data revealed good correlation and acceptable bias between CSA and OSA using our sets of reagents, methods and analyser in spiked samples. Thrombin generation gave good correlation to CSA-1 factor activity and is a possible complement to factor activity assays., (© 2021 John Wiley & Sons Ltd.)

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 fluorescence‐immunoassay (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 xFLI‐positive 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. [ABSTRACT FROM AUTHOR]