Batty P, Hart D P, Platton S
The Royal London Hospital Haemophilia Centre, Barts and The London School of Medicine and Dentistry, QMUL, London, UK.
The Royal London Hospital Haemophilia Centre, Barts Health, The Royal London Hospital, London, UK.
Int J Lab Hematol. 2018 Oct;40(5):561-568. doi: 10.1111/ijlh.12862. Epub 2018 May 18.
Factor VIII (FVIII) antibody formation is the greatest clinical and laboratory challenge within the haemophilia centre. The Nijmegen-Bethesda assay (NBA) is the gold standard for inhibitor quantification, but affected by pre-analytical variables including a patient's FVIII activity (FVIII:C). Pre-analytical heat treatment (PHT) provides a methodology for inhibitor testing when measurable FVIII:C is present.
We evaluated the effect of different PHT conditions (time/temperature) on FVIII:C as well as on potency of inhibitory activity in samples containing FVIII:C (endogenous pooled plasma and exogenous recombinant FVIII (rFL-FVIII) concentrate) or FVIII inhibitor.
PHT of endogenous FVIII at 37°C, 47°C and 52°C resulted in declining measurable FVIII:C at 120 minutes (69%, 57% and 13% of the original FVIII:C, respectively). Incubation at 56°C resulted in FVIII:C ≤ 1IU/dL after 60 minutes for endogenous FVIII and 120 minutes for rFL-FVIII. Incubation at 58°C resulted in FVIII:C < 1IU/dL at 15-30 minutes for endogenous FVIII and at 30-60 minutes for rFL-FVIII. No difference was seen for inhibitor detection following PHT (56°C or 58°C) by NBA or anti-FVIII IgG ELISA.
PHT at 58°C for 30 minutes demonstrated consistent reduction in FVIII:C < 1IU/dL without appearing to affect inhibitor detection. Laboratory awareness of differences in thermostability of different sources of FVIII is important when choosing PHT conditions.
凝血因子 VIII（FVIII）抗体的形成是血友病治疗中心面临的最大临床和实验室挑战。奈梅亨 - 贝塞斯达检测法（NBA）是抑制剂定量的金标准，但受包括患者 FVIII 活性（FVIII:C）在内的分析前变量影响。分析前热处理（PHT）为存在可测量 FVIII:C 时的抑制剂检测提供了一种方法。
我们评估了不同 PHT 条件（时间/温度）对 FVIII:C 以及对含有 FVIII:C 的样本（内源性混合血浆和外源性重组 FVIII（rFL - FVIII）浓缩物）或 FVIII 抑制剂中抑制活性效力的影响。
内源性 FVIII 在 37°C、47°C 和 52°C 进行 PHT，120 分钟时可测量的 FVIII:C 下降（分别为原始 FVIII:C 的 69%、57%和 13%）。56°C 孵育时，内源性 FVIII 60 分钟后以及 rFL - FVIII 120 分钟后 FVIII:C≤1IU/dL。58°C 孵育时，内源性 FVIII 15 - 30 分钟以及 rFL - FVIII 30 - 60 分钟后 FVIII:C＜1IU/dL。通过 NBA 或抗 FVIII IgG ELISA 检测 PHT（56°C 或 58°C）后的抑制剂时未发现差异。
58°C 下 30 分钟的 PHT 可使 FVIII:C 持续降至＜1IU/dL，且似乎不影响抑制剂检测。选择 PHT 条件时，实验室了解不同来源 FVIII 热稳定性的差异很重要。
