Department of Hematology, Radboud University Medical Center, Nijmegen, the Netherlands; Hemophilia Treatment Center, Nijmegen-Eindhoven-Maastricht, the Netherlands.
Department of Hematology, Radboud University Medical Center, Nijmegen, the Netherlands; Hemophilia Treatment Center, Nijmegen-Eindhoven-Maastricht, the Netherlands; Laboratory of Hematology, Department of Laboratory Medicine, Radboud University Medical Center, Nijmegen, the Netherlands.
Thromb Res. 2023 Nov;231:112-120. doi: 10.1016/j.thromres.2023.10.007. Epub 2023 Oct 11.
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.
To describe the design and validation of the Nijmegen ultra-sensitive Bethesda Assay (NusBA) for the detection of very low-titer inhibitors.
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).
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.
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.
先天性血友病 A (HA) 患者在输注外源性因子 (F)VIII 时会产生抑制剂，而获得性 HA (AHA) 患者的抑制剂最初是针对内源性 FVIII 产生的。抑制剂可以通过尼梅亨 Bethesda assay (NBA) 检测到，其国际截断值为 0.60 尼梅亨 Bethesda 单位/ml (NBU/ml)。因此，非常低滴度的抑制剂可能无法被检测到。
描述用于检测非常低滴度抑制剂的尼梅亨超敏感 Bethesda assay (NusBA) 的设计和验证。
NusBA 是 NBA 的一种改良，其中患者血浆与正常混合血浆的比例从 1:1 改为 9:1。根据 CLSI EP10 指南进行分析验证，以确定准确性和可重复性。在具有药代动力学数据的 82 名成年先天性 HA 患者和 4 名 AHA 患者的两个队列中进行了临床验证。通过测量低浓度 (0.05-0.80 NBU/ml) 抑制剂水平的血浆样本来确定定量限 (LOQ)。
NusBA 的 LOQ 为 0.10 NusBU/ml，变异系数为 24.2%。7 (8.5%)名先天性 HA 患者的 NusBA 结果呈阳性，其中只有 1 名患者的 NBA 结果呈阳性。NusBA 与 FVIII 半衰期之间没有相关性。在 3 名 AHA 患者中，当 NBA 结果转为阴性时，NusBA 仍呈阳性。
NusBA 能够检测到≥0.10 NBU/ml 的非常低滴度的 FVIII 抑制剂。因此，它可能在先天性 HA 和 AHA 患者的早期抑制剂检测和治疗调整中具有附加价值。
