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Effects of storage time and temperature on highly multiparametric flow analysis of peripheral blood samples; implications for clinical trial samples.
Bioscience Reports ( IF 3.8 ) Pub Date : 2021-02-15 , DOI: 10.1042/bsr20203827
Amelia Jerram 1 , Thomas V Guy 2 , Lucinda Beutler 1 , Bavani Gunasegaran 1 , Ronald Sluyter 2, 3 , Barbara Fazekas de St Groth 1, 4 , Helen M McGuire 1, 4
Affiliation  

We sought to determine the effect of time and temperature of blood sample storage before preparation of human PBMCs by Ficoll-hypaque density gradient centrifugation. Blood samples from healthy donors were stored at room temperature (RT) or refrigerated at 4oC before preparation of PBMCs. Cell yield and viability, and proportions of major cell populations within PBMCs, as determined by fluorescence flow cytometry, were assessed for both fresh and cryopreserved samples. Highly multiparametric mass cytometry was performed on cryopreserved PBMCs. We found that refrigeration had marked negative effects on subsequent PBMC yield. Storage at RT led to co-purification of low density neutrophils with PBMCs, but had no detectable effects on the proportions of multiple cell subsets including, but not limited to, monocytes, NK cells, B cells, Treg cells, and naïve, central memory and effector memory CD4+ and CD8+ T cells and CD45RA-positive terminal effector CD8+ T cells. Expression of a number of cell surface receptors, including CXCR5, CCR6, CXCR3 and TIGIT, but not CD247 was reduced after RT storage before PBMC preparation, and this effect correlated with the degree of low density neutrophil contamination. As such, when PBMC preparation cannot be undertaken immediately after blood draw, storage at RT is far superior to refrigeration. RT storage leads to neutrophil activation, but does not compromise measurement of PBMC subset distribution. However caution must be applied to interpretation of cytometric measurements of surface molecules such as chemokine receptors.

中文翻译:

保存时间和温度对外周血样品高多参数流量分析的影响 对临床试验样本的影响。

我们试图在通过 Ficoll-hypaque 密度梯度离心制备人 PBMC 之前确定血样储存时间和温度的影响。在制备 PBMC 之前,来自健康供体的血液样本在室温 (RT) 下储存或在 4oC 下冷藏。通过荧光流式细胞术测定的细胞产量和活力,以及 PBMC 内主要细胞群的比例,对新鲜和冷冻保存的样品进行了评估。对冷冻保存的 PBMC 进行了高度多参数质谱流式细胞术。我们发现冷藏对随后的 PBMC 产量有明显的负面影响。RT 储存导致低密度中性粒细胞与 PBMC 共同纯化,但对多个细胞亚群的比例没有可检测到的影响,包括但不限于单核细胞、NK 细胞、B 细胞、Treg 细胞、和幼稚的中枢记忆和效应记忆 CD4+ 和 CD8+ T 细胞以及 CD45RA 阳性终末效应 CD8+ T 细胞。在 PBMC 制备前 RT 储存后,许多细胞表面受体(包括 CXCR5、CCR6、CXCR3 和 TIGIT,但不是 CD247)的表达降低,这种影响与低密度中性粒细胞污染的程度相关。因此,当抽血后不能立即进行 PBMC 制备时,RT 储存远优于冷藏。RT 储存会导致中性粒细胞激活,但不会影响 PBMC 子集分布的测量。然而,必须谨慎解释表面分子(如趋化因子受体)的细胞计量学测量值。在 PBMC 制备前 RT 储存后,许多细胞表面受体(包括 CXCR5、CCR6、CXCR3 和 TIGIT,但不是 CD247)的表达降低,这种影响与低密度中性粒细胞污染的程度相关。因此,当抽血后不能立即进行 PBMC 制备时,RT 储存远优于冷藏。RT 储存会导致中性粒细胞激活,但不会影响 PBMC 子集分布的测量。然而,必须谨慎解释表面分子(如趋化因子受体)的细胞计量学测量值。在 PBMC 制备前 RT 储存后,许多细胞表面受体(包括 CXCR5、CCR6、CXCR3 和 TIGIT,但不是 CD247)的表达降低,这种影响与低密度中性粒细胞污染的程度相关。因此,当抽血后不能立即进行 PBMC 制备时,RT 储存远优于冷藏。RT 储存会导致中性粒细胞激活,但不会影响 PBMC 子集分布的测量。然而,必须谨慎解释表面分子(如趋化因子受体)的细胞计量学测量值。当抽血后不能立即进行 PBMC 制备时,RT 储存远优于冷藏。RT 储存会导致中性粒细胞激活,但不会影响 PBMC 子集分布的测量。然而,必须谨慎解释表面分子(如趋化因子受体)的细胞计量学测量值。当抽血后不能立即进行 PBMC 制备时,RT 储存远优于冷藏。RT 储存会导致中性粒细胞激活,但不会影响 PBMC 子集分布的测量。然而,必须谨慎解释表面分子(如趋化因子受体)的细胞计量学测量值。
更新日期:2021-02-22
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