Division of Blood Disorders, National Center on Birth Defects and Developmental Disabilities, Centers for Disease Control and Prevention, Atlanta, GA, USA.
Indiana Hemophilia and Thrombosis Center, Indianapolis, IN, USA.
J Thromb Haemost. 2017 Oct;15(10):1971-1976. doi: 10.1111/jth.13795. Epub 2017 Sep 14.
Essentials Immunologic methods detect factor VIII (FVIII) antibodies in some inhibitor-negative specimens. Specimens were tested by modified Nijmegen-Bethesda assay (NBA) and fluorescence immunoassay. The NBA with preanalytical heat inactivation detects FVIII inhibitors down to 0.2 NBU. IgG frequency validates the established threshold for positivity of ≥ 0.5 NBU for this NBA.
Background The Bethesda assay for measurement of factor VIII inhibitors called for quantification of positive inhibitors by using dilutions producing 25-75% residual activity (RA), corresponding to 0.4-2.0 Bethesda units, with the use of 'more sensitive methods' for samples with RA closer to 100% being recommended. The Nijmegen modification (Nijmegen-Bethesda assay [NBA]) changed the reagents used but not these calculations. Some specimens negative by the NBA have been shown to have FVIII antibodies detectable with sensitive immunologic methods. Objective To examine the performance at very low inhibitor titers of the Centers for Disease Control and Prevention (CDC)-modified NBA (CDC-NBA), which includes preanalytic heat inactivation to liberate bound anti-FVIII antibodies. Methods Specimens with known inhibitors were tested with the CDC-NBA. IgG anti-FVIII antibodies were measured by fluorescence immunoassay (FLI). Results Diluted inhibitors showed linearity below 0.4 Nijmegen-Bethesda units (NBU). With four statistical methods, the limit of detection of the CDC-NBA was determined to be 0.2 NBU. IgG anti-FVIII antibodies, which correlate most strongly with functional inhibitors, were present at rates above the background rate of healthy controls in specimens with titers ≥ 0.2 NBU and showed an increase in frequency from 14.3% at 0.4 NBU to 67% at the established threshold for positivity of 0.5 NBU. Conclusions The CDC-NBA can detect inhibitors down to 0.2 NBU. The FLI, which is more sensitive, demonstrates anti-FVIII IgG in some patients with negative (< 0.5) NBU. The sharp increase in IgG frequency between 0.4 and 0.5 NBU validates the established threshold for positivity of ≥ 0.5 NBU for the CDC-NBA, supporting the need for method-specific thresholds.
用于测量因子 VIII 抑制剂的贝塞斯达测定法要求通过使用产生 25-75%残余活性 (RA) 的稀释液来定量阳性抑制剂，这对应于 0.4-2.0 贝塞斯达单位，建议对于 RA 更接近 100%的样本使用“更敏感的方法”。尼姆egen 修正（Nijmegen-Bethesda 测定法 [NBA]）改变了所用试剂，但未改变这些计算。一些通过 NBA 呈阴性的标本已被证明具有可通过敏感免疫方法检测到的因子 VIII 抗体。目的：检查包括预分析热灭活以释放结合的抗因子 VIII 抗体在内的疾病预防控制中心（CDC）改良 NBA（CDC-NBA）在极低抑制剂滴度下的性能，该 NBA 包括预分析热灭活以释放结合的抗因子 VIII 抗体。方法：用 CDC-NBA 检测已知抑制剂的标本。荧光免疫测定法 (FLI) 测定 IgG 抗因子 VIII 抗体。结果：稀释抑制剂在低于 0.4 尼姆egen-Bethesda 单位（NBU）时呈线性。使用四种统计方法，确定 CDC-NBA 的检测限为 0.2 NBU。与功能抑制剂相关性最强的 IgG 抗因子 VIII 抗体在滴度≥0.2 NBU 的标本中以高于健康对照的背景率存在，并从 0.4 NBU 时的 14.3%频率增加到建立的阳性阈值 0.5 NBU 时的 67%。结论：CDC-NBA 可检测低至 0.2 NBU 的抑制剂。更敏感的 FLI 在一些 NBA 呈阴性（<0.5）的患者中显示抗因子 VIII IgG。在 0.4 和 0.5 NBU 之间 IgG 频率的急剧增加验证了为 CDC-NBA 建立的阳性阈值≥0.5 NBU 的合理性，支持针对特定方法的阈值的必要性。
