Division of Blood Disorders, National Center on Birth Defects and Developmental Disabilities, Centers for Disease Control and Prevention, Atlanta, GA, USA.
Haemophilia. 2018 May;24(3):487-491. doi: 10.1111/hae.13435. Epub 2018 Feb 20.
The use of pre-analytical heat treatment (PHT) with the Nijmegen-Bethesda assay (NBA) for inhibitors to factor VIII (FVIII) can remove/destroy infused or endogenous FVIII from patient plasma samples, allowing testing of recently infused patients with haemophilia. Two PHT methods have been described as follows: heating to 56°C for 30 minutes and heating to 58°C for 90 minutes. Data examining the effects of PHT on anti-FVIII IgG , the antibodies known to correlate most closely with the presence of FVIII inhibitors, are limited.
To assess the effect of PHT on the levels of detectable anti-FVIII IgG .
Nijmegen-Bethesda assay-positive specimens were incubated at 56, 58 or 60°C for 90 minutes, and anti-FVIII IgG was measured by fluorescence immunoassay (FLI) at 30-minute intervals. The effects of PHT on the ability of recombinant FVIII (rFVIII) to inhibit detection of patient antibodies by FLI was also examined to assess the stability of rFVIII under the various PHT conditions tested.
Levels of anti-FVIII IgG showed little change following incubations at 56°C (mean 101% of original value at 30 minutes and 100% at 60 minutes) but decreased upon exposure to 58°C (mean 85% at 30 minutes and 66% at 60 minutes). In addition, heating to 56°C effectively decreased the ability of rFVIII to block antibody binding compared to unheated rFVIII.
The optimal temperature for PHT in the FVIII NBA is 56°C. Higher temperatures may lead to loss of inhibitory antibodies.
使用 Nijmegen-Bethesda assay（NBA）对因子 VIII（FVIII）抑制剂进行预分析热处理（PHT），可以从患者血浆样本中去除/破坏输注或内源性 FVIII，从而可以对最近输注过的血友病患者进行检测。有两种 PHT 方法如下：加热至 56°C 30 分钟和加热至 58°C 90 分钟。关于 PHT 对抗 FVIII IgG 的影响的数据，目前研究有限，而抗 FVIII IgG 是与 FVIII 抑制剂存在最密切相关的抗体。
评估 PHT 对可检测的抗 FVIII IgG 水平的影响。
将 Nijmegen-Bethesda assay 阳性标本在 56°C、58°C 或 60°C 下孵育 90 分钟，并用荧光免疫测定法（FLI）在 30 分钟间隔测量抗 FVIII IgG。还检查了 PHT 对重组 FVIII（rFVIII）抑制 FLI 检测患者抗体能力的影响，以评估在测试的各种 PHT 条件下 rFVIII 的稳定性。
在 56°C 孵育后，抗 FVIII IgG 水平变化不大（30 分钟时的原始值的平均为 101%，60 分钟时为 100%），但在暴露于 58°C 时下降（30 分钟时的平均为 85%，60 分钟时为 66%）。此外，与未加热的 rFVIII 相比，加热至 56°C 可有效降低 rFVIII 阻断抗体结合的能力。
在 FVIII NBA 中，PHT 的最佳温度为 56°C。更高的温度可能导致抑制性抗体的丢失。
