Department of Biomarker Analysis and Development, Fraunhofer Institute of Toxicology and Experimental Medicine, Hannover, Germany.
Department of Translational Medicine and Clinical Pharmacology, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany.
Cytometry A. 2022 Jan;101(1):86-94. doi: 10.1002/cyto.a.24338. Epub 2021 Apr 1.
Application of basophil activation test (BAT) in clinical trials requires assay validity. Whether assay variability differs between healthy and asthmatic subjects is mostly unknown. This study compares basophil stimulation using blood from healthy and asthmatic subjects with or without inhibition of spleen tyrosine kinase (SYK).
Whole blood of healthy and mild asthmatic subjects was stimulated with anti-dinitrophenyl (DNP) IgE/DNP bovine serum albumin and anti-IgE. Basophil activation was detected by CD63 and CD203c expression. CD63 expression levels were compared with serum IgE levels. Three operators repeated experiments with three subjects each from both groups at 3 days to observe assay precision. The effect of the SYK inhibitor BI 1002494 was assessed in BAT for both healthy and asthmatic subjects.
BAT was reproducible in both groups. Acceptance criteria of <25% CV were mostly fulfilled. Stimulation with anti-DNP (p < 0.001, r = -0.80) but not anti-IgE (p = 0.74, r = 0.05) was related to serum IgE with levels > 200 IU/ml limiting anti-DNP stimulation. BI 1002494 IC values were 497 nM and 1080 nM in healthy and 287 nM and 683 nM in asthmatics for anti-DNP and anti-IgE stimulation, respectively.
BAT, performed with blood from healthy or asthmatic subjects, is a robust test for the measurement of a physiological response in clinical trials. Blood from asthmatic donors with serum IgE > 200 IU/ml is less feasible when using anti-DNP stimulation. SYK inhibition was not affected by disease status.
嗜碱性粒细胞活化试验（BAT）在临床试验中的应用需要保证检测的有效性。目前尚不清楚健康人群和哮喘患者的检测结果是否存在差异。本研究对比了健康人群和哮喘患者的血液在加入或不加入脾酪氨酸激酶（SYK）抑制剂 BI 1002494 前后对二硝基苯（DNP）免疫球蛋白 E/DNP 牛血清白蛋白和抗免疫球蛋白 E 的刺激反应，以此来评估检测的变异性。
采集健康人群和轻度哮喘患者的全血，分别用抗 DNP 免疫球蛋白 E/DNP 牛血清白蛋白和抗免疫球蛋白 E 进行刺激，通过 CD63 和 CD203c 的表达来检测嗜碱性粒细胞的活化情况。比较 CD63 的表达水平与血清 IgE 水平的相关性。三位操作者在 3 天内重复三次实验，每组各检测 3 名健康人群和 3 名哮喘患者，以评估检测的精密度。
两组人群的 BAT 结果均具有可重复性，大部分符合<25%CV 的接受标准。与血清 IgE 水平>200 IU/ml 限制抗 DNP 刺激的情况类似，DNP（p<0.001，r=-0.80）而非免疫球蛋白 E（p=0.74，r=0.05）刺激与血清 IgE 水平显著相关。健康人群和哮喘患者抗 DNP 和抗免疫球蛋白 E 刺激的 BI 1002494 的 IC50 值分别为 497nM 和 1080nM、287nM 和 683nM。
使用健康人群或哮喘患者的血液进行 BAT 是临床试验中测量生理反应的一种稳健方法。当使用抗 DNP 刺激时，来自血清 IgE>200 IU/ml 的哮喘患者的血液可行性较差。疾病状态并不影响 SYK 的抑制作用。
