Core Facility Fluorescence Cytometry, Research Center Borstel, Leibniz Lung Center, Borstel, Germany.
Division of Clinical and Molecular Allergology, Priority Area Asthma and Allergy, Airway Research Center North (ARCN), German Center for Lung Research (DZL, Borstel, Germany.
Allergy. 2021 Dec;76(12):3776-3788. doi: 10.1111/all.14900. Epub 2021 May 28.
Allergy is one of the most common chronic diseases in Europe. Therefore, an increased need for specific and sensitive diagnostic tests that truly detect allergy exists. This study aimed at establishing a highly specific high-throughput and automated basophil activation test (BAT) that proves the existence of an allergy with utmost probability.
BAT from 1104 samples was analyzed; a novel gating strategy with three antibodies (FcεRIα, CD203c, CD63) was established and compared with our published protocol (12 antibodies). Based on the novel gating strategy, storage conditions, automated measurement, and analyses using R (1376 samples out of 1389) were optimized to set up a high-throughput BAT.
No differences in sensitivity and specificity were found between the novel three antibody (FcεRIα, CD203c, CD63) and the 12 antibody gating strategy or between automated and manually analyzed samples (saving up to 90% of labor time). The time frame for basophil activation measurement after blood donation has been extended considerably (whole blood storage ≤7 days (RT) and 17 days (4°C) prior to BAT preparation and measurement). Respective storage conditions were optimized for samples after stimulation, staining, and preparation (≤7 days (RT) and 28 days (4°C)). These achievements were confirmed by a nationwide ring trial showing robustness and applicability of our BAT on a variety of flow cytometers.
Our considerable optimizations overcame the hurdles that until now prevented the BAT from being used as high-throughput allergy diagnostic test in routine laboratories and shall allow for collaborative studies between clinics and research centers.
过敏是欧洲最常见的慢性疾病之一。因此，人们对真正能够检测过敏的特异性和敏感性诊断测试的需求日益增加。本研究旨在建立一种高度特异性的高通量和自动化嗜碱性粒细胞活化试验（BAT），以最大概率证明过敏的存在。
分析了 1104 个样本的 BAT；建立了一种新的三抗体（FcεRIα、CD203c、CD63）门控策略，并与我们发表的方案（12 种抗体）进行了比较。基于新的门控策略、存储条件、自动化测量和 R 分析（1389 个样本中的 1376 个），优化了高通量 BAT 的设置。
新型三抗体（FcεRIα、CD203c、CD63）与 12 抗体门控策略之间，以及自动化和手动分析样本之间，在敏感性和特异性方面没有差异（可节省高达 90%的劳动力时间）。从献血到嗜碱性粒细胞活化测量的时间框架已经大大延长（全血在准备和测量前可在 RT 下储存 7 天（室温）和 17 天（4°C），在 4°C 下储存 17 天）。分别优化了刺激、染色和准备后的样本的储存条件（在 RT 下储存≤7 天（室温）和在 4°C 下储存 28 天）。全国性的环试验证实了这些成就，该试验显示了我们的 BAT 在各种流式细胞仪上的稳健性和适用性。
我们的大量优化克服了迄今为止阻碍 BAT 成为常规实验室高通量过敏诊断测试的障碍，这将使临床和研究中心之间能够进行合作研究。
