Institute of Applied Molecular Medicine (IMMA) Nemesio Díez, Department of Basic Medical Sciences, School of Medicine, San Pablo-CEU University, CEU Universities, Boadilla del Monte, Spain.
Centre for Metabolomics and Bioanalysis (CEMBIO), Department of Chemistry and Biochemistry, School of Pharmacy, San Pablo-CEU University, CEU Universities, Boadilla del Monte, Spain.
Clin Exp Allergy. 2022 Oct;52(10):1157-1168. doi: 10.1111/cea.14192. Epub 2022 Jul 16.
In previous studies with peripheral blood cells, platelet factors were found to be associated with severe allergic phenotypes. A reliable method yielding highly concentrated and pure platelet samples is usually not available for immunological studies. Plateletpheresis is widely used in the clinics for donation purposes. In this study, we designed a protocol based on plateletpheresis to obtain Platelet-Rich Plasma (PRP), Platelet-Poor Plasma (PPP) as well as CD3 and CD14 cells matched samples from a waste plateletpheresis product for immunological studies.
Twenty-seven subjects were voluntarily subjected to plateletpheresis. PRP, PPP and blood cell concentrate contained in a leukocyte reduction system chamber (LRSC) were obtained in this process. CD3 and CD14 cells were isolated from the LRSC by density-gradient centrifugation and positive magnetic bead isolation. RNA was isolated from PRP, CD3 and CD14 cell samples and used for transcriptomic studies by Affymetrix. PRP and PPP samples were used for platelet protein quantification by multiplex assays.
A reliable high yield method to obtain matched samples of PRP, PPP, CD3 and CD14 from a single donor for RNA and protein analyses has been designed. The RNA quality indicators (RQI) routinely used for other cell types were not suitable for platelet RNA characterization. Despite this, the platelet RNA was valid for transcriptomic studies by Affymetrix, as platelet transcripts obtained in our previous studies were confirmed in PRP samples. Platelet samples were enriched in platelet factors as determined in protein multiplex analysis.
We have developed a method that yields not only high content and pure platelet samples from a single donor but also CD3 and CD14 matched samples that can be used for RNA and protein analyses in immunological studies.
在之前的外周血细胞研究中，发现血小板因子与严重过敏表型有关。通常，没有可靠的方法可以获得高度浓缩和纯血小板样本，用于免疫学研究。血小板分离术广泛用于临床捐献。在这项研究中，我们设计了一种基于血小板分离术的方案，从废弃的血小板分离术产品中获得富含血小板的血浆（PRP）、血小板贫浆（PPP）以及与 CD3 和 CD14 细胞匹配的样本，用于免疫学研究。
27 名志愿者自愿接受血小板分离术。在此过程中，从白细胞减少系统室（LRSC）中获得 PRP、PPP 和血液细胞浓缩物。通过密度梯度离心和阳性磁珠分离从 LRSC 中分离 CD3 和 CD14 细胞。从 PRP、CD3 和 CD14 细胞样本中分离 RNA，并通过 Affymetrix 进行转录组学研究。通过多重分析测定 PRP 和 PPP 样本中的血小板蛋白含量。
设计了一种可靠的高产方法，从单个供体中获得 PRP、PPP、CD3 和 CD14 的匹配样本，用于 RNA 和蛋白质分析。常规用于其他细胞类型的 RNA 质量指标（RQI）不适用于血小板 RNA 特征描述。尽管如此，血小板 RNA 仍可用于 Affymetrix 的转录组学研究，因为我们之前的研究中获得的血小板转录本在 PRP 样本中得到了证实。通过蛋白质多重分析确定血小板样本富含血小板因子。
我们已经开发出一种方法，不仅可以从单个供体中获得高含量和纯血小板样本，还可以获得与 CD3 和 CD14 匹配的样本，用于免疫研究中的 RNA 和蛋白质分析。
