Human peripheral blood mononuclear cells (PBMCs) are extensively used for research of immune cell functions, identification of biomarkers and development of diagnostics and therapeutics for human diseases, among others. The assumption that “old blood samples” are not appropriate for isolation of PBMCs for functional assays has been a dogma in the scientific community. However, partial data on the impact of time after phlebotomy on the quality and stability of human PBMCs preparations impairs the design of studies in which time-controlled blood sampling is challenging such as field studies involving multiple sampling centers/sites. In this study, we evaluated the effect of time after phlebotomy over a 24 h time course, on the stability of human blood leukocytes used for immunological analyses. Blood samples from eight healthy adult volunteers were obtained and divided into four aliquots, each of which was left in gentle agitation at room temperature (24 °C) for 2 h (control), 7 h, 12 h and 24 h post phlebotomy. All samples at each time point were independently processed for quantification of mononuclear cell subpopulations, cellular viability, gene expression and cytokine secretion. A 24 h time delay in blood sample processing did not affect the viability of PBMCs. However, a significantly lower frequency of CD3+ T cells (p < 0.05) and increased LPS-induced CXCL10 secretion were observed at 12 h post-phlebotomy. Alterations in TNFα, CCL8, CCR2 and CXCL10 gene expression were found as early as 7 h after blood sample procurement. These data reveal previously unrecognized early time-points for sample processing control, and provide an assay-specific time reference for the design of studies that involve immunological analyses of human blood samples.

中文翻译：

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人外周血样本中白细胞的表型和功能稳定性：免疫学研究设计的考虑因素

人类外周血单个核细胞 (PBMC) 广泛用于免疫细胞功能的研究、生物标志物的鉴定以及人类疾病诊断和治疗的开发等。“旧血样”不适合用于功能测定的 PBMC 分离的假设一直是科学界的教条。然而，关于采血后时间对人类 PBMC 制剂质量和稳定性影响的部分数据损害了时间控制采血具有挑战性的研究设计，例如涉及多个采样中心/站点的现场研究。在这项研究中，我们评估了 24 小时内放血后时间对用于免疫学分析的人类血液白细胞稳定性的影响。获得来自八名健康成年志愿者的血液样本并将其分成四份，每份在室温（24°C）下轻轻搅拌2小时（对照）、7小时、12小时和24小时后放血。每个时间点的所有样品都经过独立处理，以量化单核细胞亚群、细胞活力、基因表达和细胞因子分泌。血液样本处理中的 24 小时延迟不会影响 PBMC 的生存能力。然而，在放血后 12 小时观察到显着较低的 CD3+ T 细胞频率（p < 0.05）和增加的 LPS 诱导的 CXCL10 分泌。早在采集血样后 7 小时就发现 TNFα、CCL8、CCR2 和 CXCL10 基因表达发生变化。这些数据揭示了以前无法识别的样品处理控制的早期时间点，