Centro Internacional de Entrenamiento e Investigaciones Médicas-CIDEIM, Cali, Colombia.
Universidad Icesi, Cali, Colombia.
BMC Immunol. 2019 Jan 18;20(1):5. doi: 10.1186/s12865-019-0286-z.
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.
人类外周血单核细胞（PBMC）广泛用于研究免疫细胞功能、鉴定生物标志物以及开发人类疾病的诊断和治疗方法等。在科学界，人们一直认为“陈旧的血样”不适合用于分离 PBMC 进行功能检测，这一观点已成定论。然而，关于静脉穿刺后时间对人 PBMC 制备物质量和稳定性影响的部分数据，会影响到那些时间控制采血有难度的研究设计，例如涉及多个采样中心/地点的现场研究。在这项研究中，我们评估了静脉穿刺后 24 小时时间过程中时间对用于免疫分析的人类血液白细胞稳定性的影响。从 8 名健康成年志愿者中采集血液样本并分成 4 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。在每个时间点，所有样本都独立进行单核细胞亚群定量、细胞活力、基因表达和细胞因子分泌分析。
血液样本处理时间延迟 24 小时不会影响 PBMC 的活力。然而，在静脉穿刺后 12 小时，CD3+T 细胞的频率显著降低（p<0.05），并且 LPS 诱导的 CXCL10 分泌增加。早在采血后 7 小时，就发现 TNFα、CCL8、CCR2 和 CXCL10 基因表达发生改变。
这些数据揭示了之前未被认识到的样本处理控制的早期时间点，并为涉及人类血液样本免疫分析的研究设计提供了特定于检测的时间参考。
