Sorbonne Université, UPMC Univ Paris 06, INSERM, UMR_S 959, Immunology-Immunopathology-Immunotherapy (I3), Paris F-75005, France.
Biotherapy (CIC-BTi) and Inflammation-Immunopathology-Biotherapy Department (I2B), AP-HP, Hôpital Pitié-Salpêtrière, Paris, F-75651, France.
Cytometry B Clin Cytom. 2018 Sep;94(5):621-626. doi: 10.1002/cyto.b.21622. Epub 2018 Jan 23.
Quantification of regulatory T cells (Tregs) is crucial in immunomonitoring in clinical trials as this cell population has been shown to be involved in a wide range of diseases, including cancers, autoimmune diseases, infections, and allergies. Human Tregs are defined as CD4 CD25 CD127 FoxP3 cells, and the standardization of their staining by flow cytometry is a challenge, especially in multicenter clinical trials, notably because of the intracellular location of FoxP3.
A flow cytometry staining procedure was settled and standardized to measure human Tregs in peripheral whole blood using precoated dried antibodies in ready-to-use tubes. It was compared with reference methods and implemented and validated to be suitable with different cytometer platforms.
The standardized protocol developed with dried antibodies and reduced volumes of whole blood allows an optimal identification of Tregs. Compared with classical staining procedure, it reduces the number of steps required, in a very fast and simple technique. The accuracy of the method was confirmed by a multicenter comparison with different cytometer brands.
Our results highlight the reliability of this high-standard protocol that could become a reference method for the monitoring of Tregs in clinical trials. © 2018 International Clinical Cytometry Society.
在临床试验的免疫监测中，定量检测调节性 T 细胞（Tregs）至关重要，因为该细胞群已被证明与多种疾病有关，包括癌症、自身免疫性疾病、感染和过敏。人类 Tregs 被定义为 CD4 CD25 CD127 FoxP3 细胞，流式细胞术对其染色的标准化是一项挑战，特别是在多中心临床试验中，这主要是因为 FoxP3 的细胞内位置。
我们确定并标准化了一种使用预包被的即用型干抗体在全血中测量人类 Tregs 的流式细胞术染色程序。它与参考方法进行了比较，并被证明适用于不同的流式细胞仪平台。
使用干抗体和减少的全血体积开发的标准化方案允许最佳识别 Tregs。与经典染色程序相比，它减少了所需的步骤数量，是一种非常快速和简单的技术。该方法的准确性通过与不同流式细胞仪品牌的多中心比较得到了确认。
我们的结果强调了这种高标准方案的可靠性，它可能成为临床试验中 Tregs 监测的参考方法。© 2018 国际临床细胞化学学会。
