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Background: An inhibitor can develop in congenital hemophilia A (HA) patients against exogenous infused factor (F)VIII, whereas in acquired HA (AHA) inhibitors initially develop against endogenous FVIII. Inhibitors can be detected with the Nijmegen Bethesda Assay (NBA), which has an international cut-off level of 0.60 Nijmegen Bethesda Units/mL (NBU/mL). Thereby, very low-titer inhibitors may remain undetected., Aim: To describe the design and validation of the Nijmegen ultra-sensitive Bethesda Assay (NusBA) for the detection of very low-titer inhibitors., Methods: The NusBA is a modification of the NBA in which the ratio of patient plasma to normal pooled plasma is changed from 1:1 to 9:1. Analytical validation was performed according to the CLSI EP10 guideline in order to determine trueness and reproducibility. Clinical validation was performed in two cohorts of congenital HA patients (82 adults) with pharmacokinetic data and four AHA patients. The limit of quantitation (LOQ) was determined by measuring plasma samples spiked with inhibitor levels in the low range (0.05-0.80 NBU/mL)., Results: The LOQ for the NusBA was 0.10 NusBU/mL, with a coefficient of variation of 24.2 %. Seven (8.5 %) congenital HA patients had a positive NusBA result, of which only one was detected with the NBA. There was no correlation between NusBA and FVIII half-life. In three of the AHA patients the NusBA remained positive, when the NBA became negative., Discussion: The NusBA is able to detect very low-titer FVIII inhibitors of ≥0.10 NBU/mL. Thereby, it may have added value in early inhibitor detection and therapy adjustments in patients with congenital HA and AHA., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: MV and DM received a research grant from Sobi. WvH received unrestricted grants from Bayer, Shire, Novo Nordisk and CSL Behring. WvH is the founder and CSO of Enzyre BV, a Radboudumc spinoff company. SS received a research grant from Bayer. JOD has served on the speaker's bureau for Baxter, Bayer, Novo Nordisk, Sobi, Boehringer Ingelheim, Leo Pharma, Takeda and Octapharma. He has also served on the advisory boards of Baxter, Sobi, Bayer, Octapharma CSL Behring, Daiichi Sankyo, Boehringer Ingelheim, Takeda and Pfizer. JOD has also received research grant funding awards from 3M, Baxter, Bayer, Pfizer, Shire, Takeda, 3M and Novo Nordisk. BR was a full-time employees of Baxalta Innovations GmbH at the time when the laboratory part of the study was conducted. PT is full-time employee of Baxalta Innovations GmbH, a member of the Takeda group of companies, and shareholder of Takeda Pharmaceutical Company Limited. LV, BS, RP, NB, JJ, HT, EE have no conflict of interests to declare., (Copyright © 2023. Published by Elsevier Ltd.)