Braudeau Cecile, Salabert-Le Guen Nina, Chevreuil Justine, Rimbert Marie, Martin Jerome C, Josien Regis
Laboratoire d'Immunologie, CIMNA, LabEx IGO "Immunotherapy, Graft, Oncology", Nantes, France.
CHU Nantes, Nantes Université, Inserm, Centre de Recherche en Transplantation et Immunologie, UMR 1064, ITUN, Nantes, France.
Cytometry B Clin Cytom. 2021 Nov;100(6):652-665. doi: 10.1002/cyto.b.21994. Epub 2021 Feb 5.
Immune profiling by flow cytometry is not always possible on fresh blood samples due to time and/or transport constraints. Furthermore, the cryopreservation of peripheral blood mononuclear cells (PBMC) requires on-site specialized lab facilities, thus severely restricting the extent to which blood immune monitoring can be applied to multicenter clinical studies. These major limitations can be addressed through the development of simplified whole blood freezing methods.
In this report, we describe an optimized easy protocol for rapid whole blood freezing with the CryoStor® CS10 solution. Using flow cytometry, we compared cellular viability and composition on cryopreserved whole blood samples to matched fresh blood, as well as fresh and frozen PBMC.
Though partial loss of neutrophils was observed, leucocyte viability was routinely >75% and we verified the preservation of viable T cells, NK cells, monocytes, dendritic cells, and eosinophils in frequencies similar to those observed in fresh samples. A moderate decrease in B cell frequencies was observed. Importantly, we validated the possibility to analyze major intracellular markers, such as FOXP3 and Helios in regulatory T cells. Finally, we demonstrated good functional preservation of CS10-cryopreserved cells through the analysis of intracellular cytokine production in ex vivo stimulated T cells (IFNg, IL-4, IL-17A,) and monocytes (IL-1b, IL-6, TNFa).
In conclusion, our protocol provides a robust method to apply reliable immune monitoring studies to cryopreserved whole blood samples, hence offering new important opportunities for the design of future multicenter clinical trials.
由于时间和/或运输限制，通过流式细胞术进行免疫分析在新鲜血液样本上并不总是可行的。此外，外周血单个核细胞（PBMC）的冷冻保存需要现场专门的实验室设施，因此严重限制了血液免疫监测应用于多中心临床研究的程度。这些主要限制可以通过开发简化的全血冷冻方法来解决。
在本报告中，我们描述了一种使用CryoStor® CS10溶液快速冷冻全血的优化简便方案。我们使用流式细胞术比较了冷冻保存的全血样本与匹配的新鲜血液以及新鲜和冷冻的PBMC的细胞活力和组成。
虽然观察到中性粒细胞有部分损失，但白细胞活力通常>75%，并且我们验证了存活的T细胞、NK细胞、单核细胞、树突状细胞和嗜酸性粒细胞的保存频率与新鲜样本中观察到的频率相似。观察到B细胞频率有适度下降。重要的是，我们验证了分析主要细胞内标志物的可能性，例如调节性T细胞中的FOXP3和Helios。最后，我们通过分析体外刺激的T细胞（IFNg、IL-4、IL-17A）和单核细胞（IL-1b、IL-6、TNFa）中的细胞内细胞因子产生，证明了CS10冷冻保存细胞具有良好的功能保存。
总之，我们的方案提供了一种可靠的方法，可将可靠的免疫监测研究应用于冷冻保存的全血样本，从而为未来多中心临床试验的设计提供了新的重要机会。
