Singapore Immunology Network, Agency for Science, Technology and Research, 8A Biomedical Grove, Immunos Building, Level 3, Biopolis 138648, Singapore.
J Immunol Methods. 2012 Nov 30;385(1-2):79-89. doi: 10.1016/j.jim.2012.08.010. Epub 2012 Aug 24.
High dimensional flow cytometry is best served by centralized facilities. However, the difficulties around sample processing, storage and shipment make large scale international studies impractical. We therefore sought to identify optimized fixation procedures which fully leverage the analytical capability of high dimensional flow cytometry without the need for complex cell processing or a sustained cold chain. Whole blood staining procedure was employed to investigate the applicability of fixatives including Cyto-Chex® Blood Collection tube (Streck), Transfix® (Cytomark), 1% and 4% paraformaldehyde to centralized analysis of field trial samples. Samples were subjected to environmental conditions which mimic field studies, without refrigerated shipment and analyzed across 10 days, based on cell count and marker expression. This study showed that Cyto-Chex® demonstrated the least variability in absolute cell count relative to samples analyzed directly from donors in the absence of fixation. Transfix® was better at preserving the marker expression among all fixatives. However, Transfix® caused marked increased cell membrane permeabilization and was detrimental to intracellular marker identification. Paraformaldehyde fixation, at either 1% or 4% concentrations, was unfavorable for cell preservation under the conditions tested and thus not recommended. Using these data, we have created an online interactive tool which enables researchers to evaluate the impact of different fixatives on their panel of interest. In this study, we have identified Cyto-Chex® as the optimal cellular preservative for high dimensional flow cytometry in large scale studies for shipped whole blood samples, even in the absence of a sustained cold chain.
高维流式细胞术最好由集中式设施提供服务。然而，围绕样本处理、存储和运输的困难使得大规模的国际研究变得不切实际。因此，我们试图确定优化的固定程序，这些程序可以充分利用高维流式细胞术的分析能力，而无需复杂的细胞处理或持续的冷链。采用全血染色程序来研究固定剂的适用性，包括 Cyto-Chex® Blood Collection tube（Streck）、Transfix®（Cytomark）、1%和 4%多聚甲醛，以对现场试验样本进行集中分析。根据细胞计数和标记物表达，将样本置于模拟现场研究的环境条件下，无需冷藏运输，并在 10 天内进行分析。该研究表明，与未固定直接从供体分析的样本相比，Cyto-Chex®在绝对细胞计数方面表现出最小的变异性。在所有固定剂中，Transfix®更能保持标记物的表达。然而，Transfix®导致细胞膜通透性显著增加，对细胞内标记物的识别有害。在测试条件下，1%或 4%浓度的多聚甲醛固定不利于细胞保存，因此不推荐使用。利用这些数据，我们创建了一个在线交互式工具，使研究人员能够评估不同固定剂对其感兴趣的标志物面板的影响。在这项研究中，我们确定 Cyto-Chex®是大规模研究中用于运输全血样本的高维流式细胞术的最佳细胞保存剂，即使在没有持续冷链的情况下也是如此。
