Impact of blood storage and sample handling on quality of high dimensional flow cytometric data in multicenter clinical research
Impact of blood storage and sample handling on quality of high dimensional flow cytometric data in multicenter clinical research
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DOI：
10.1016/j.jim.2019.06.007
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发表时间：
2019-12-01
影响因子：
2.2
通讯作者：
Berkowska, M. A.
中科院分区：
文献类型：
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作者：
Diks, A. M.;Bonroy, C.;Berkowska, M. A.
在多中心临床研究环境中获得可靠和可重复的高质量数据需要设计最佳的标准操作程序。虽然人们普遍认识到需要在样品处理和数据分析方面实现标准化，但分析前阶段样品处理的影响仍然被低估。我们评估了样本储存时间（近似于运输时间）和温度、抗凝剂类型以及有限血量对流式细胞术研究再现性的影响。采用EuroFlow批量裂解方案处理的EDTA和肝素钠样本，染色并在4 ℃下储存，显示细胞表面标志物的表达和主要白细胞群的分布相当稳定，长达72 h。额外的样品固定（1% PFA，Fix & Perm）没有任何有益效果。在处理和染色之前在室温下储存< 24小时的血液样品似乎适合可靠的免疫表型分析，尽管观察到绝对细胞数的损失。在髓系细胞和单核细胞中观察到主要损失，而淋巴细胞似乎受影响较小。细胞表面标志物的表达和群体分布在肝素钠血液中比EDTA血液中更稳定。然而，肝素钠样本的储存与白细胞计数随时间的快速下降相关。全血固定策略（Cyto-Chex，TransFix）改善了长期群体分布，但不利于细胞标志物的表达。在处理前不同时间延迟的EDTA临床（患者）血液样本中，成功证实了本研究对健康供体血液样本的主要结论。最后，我们认识到，在血容量不足的情况下，需要调整批量裂解。尽管总体结论明确，但单个标记物和细胞群有不同的首选条件。因此，应始终根据临床应用和主要目标白细胞群体调整样本处理的具体指南。
Obtaining reliable and reproducible high quality data in multicenter clinical research settings requires design of optimal standard operating procedures. While the need for standardization in sample processing and data analysis is well-recognized, the impact of sample handling in the pre-analytical phase remains underestimated. We evaluated the impact of sample storage time (approximate to transport time) and temperature, type of anticoagulant, and limited blood volume on reproducibility of flow cytometric studies.EDTA and Na-Heparin samples processed with the EuroFlow bulk lysis protocol, stained and stored at 4 degrees C showed fairly stable expression of cell surface markers and distribution of the major leukocyte populations for up to 72 h. Additional sample fixation (1% PFA, Fix & Perm) did not have any beneficial effects. Blood samples stored for < 24 h at room temperature before processing and staining seemed suitable for reliable immunophenotyping, although losses in absolute cell numbers were observed. The major losses were observed in myeloid cells and monocytes, while lymphocytes seemed less affected. Expression of cell surface markers and population distribution were more stable in Na-Heparin blood than in EDTA blood. However, storage of Na-Heparin samples was associated with faster decrease in leukocyte counts over time. Whole blood fixation strategies (Cyto-Chex, TransFix) improved long-term population distribution, but were detrimental for expression of cellular markers. The main conclusions from this study on healthy donor blood samples were successfully confirmed in EDTA clinical (patient) blood samples with different time delays until processing. Finally, we recognized the need for adjustments in bulk lysis in case of insufficient blood volumes.Despite clear overall conclusions, individual markers and cell populations had different preferred conditions. Therefore, specific guidelines for sample handling should always be adjusted to the clinical application and the main target leukocyte population.