Department of Surgery, Kennemer Gasthuis, Haarlem, The Netherlands.
J Immunol Methods. 2012 Jan 31;375(1-2):189-95. doi: 10.1016/j.jim.2011.10.011. Epub 2011 Oct 26.
The sentinel lymph node (SLN) is an emerging focus for immunological research in breast cancer. Cryopreservation of SLN single-cell suspensions allows for simultaneous phenotypic multi-parameter analyses and minimizes operator dependent variability. This is of particular importance for immunomonitoring of large multicenter trials. However, little data are available regarding the influence of cryopreservation on phenotypic characteristics of lymph node dendritic cells and T cells. In this study we assessed the feasibility of cryopreservation of viable SLN cell samples for flowcytometric analysis, by comparing quantitative analyses of SLN cell samples after freeze-thawing with direct analysis of fresh SLN cell samples. SLN were collected from nine breast cancer patients. From each SLN cell sample, half was used for immediate analysis and half was analyzed after cryopreservation and thawing. Conventional dendritic cell (cDC) and T cell subsets were quantified and phenotypically characterized by flow cytometry. The observed frequencies of both CD1a(+) and CD1a(-)CD11c(+)CD14(-) cDC subsets showed significant correlation between the fresh and frozen-thawed samples. Similar high correlations were found for CD83 and CD86 expression markers on the more frequent (>0.2%) CD1a(+) and CD1a(-)CD11c(+)CD14(-) cDC subset, but not on the low-frequency (<0.2%) CD1a(+)CD11c(+)CD14(+) cDC subset. CD4/CD8 T cell ratios were comparable and were significantly correlated pre- and post-freezing. Regulatory CD4(+)CD25(hi) T cell frequencies and their FoxP3 expression levels were significantly higher after freezing-thawing than in the freshly analyzed samples. Nevertheless, a highly significant correlation was found for both parameters pre- and post-freezing. Cryopreservation and thawing seems a valid and practical alternative to direct analysis of fresh viable lymph node cells, without introducing cryo-dependent variance between SLN samples. However, enumeration of low-frequency cell populations and assessment of their marker expression levels are less reliable after cryopreservation and should be assessed and considered in the design of each clinical trial.
前哨淋巴结 (SLN) 是乳腺癌免疫研究的新兴焦点。SLN 单细胞悬液的冷冻保存允许同时进行表型多参数分析，并最大程度地减少操作人员依赖性变异。这对于大型多中心试验的免疫监测尤其重要。然而，关于冷冻保存对淋巴结树突状细胞和 T 细胞表型特征的影响的数据很少。在这项研究中，我们通过比较冷冻解冻后 SLN 细胞样品的定量分析与直接分析新鲜 SLN 细胞样品，评估了冷冻保存活 SLN 细胞样品进行流式细胞分析的可行性。从 9 名乳腺癌患者中收集 SLN。从每个 SLN 细胞样品中，一半用于立即分析，另一半用于冷冻保存和解冻后分析。通过流式细胞术定量和表型分析常规树突状细胞 (cDC) 和 T 细胞亚群。新鲜和冷冻解冻样品之间观察到的 CD1a(+) 和 CD1a(-)CD11c(+)CD14(-) cDC 亚群的频率均呈显著相关性。在更常见 (>0.2%) CD1a(+) 和 CD1a(-)CD11c(+)CD14(-) cDC 亚群的 CD83 和 CD86 表达标志物上也发现了类似的高相关性，但在低频 (<0.2%) CD1a(+)CD11c(+)CD14(+) cDC 亚群上则没有。CD4/CD8 T 细胞比值在冷冻前和冷冻后相似且呈显著相关性。冷冻解冻后调节性 CD4(+)CD25(hi) T 细胞频率及其 FoxP3 表达水平显著高于新鲜分析的样品。尽管如此，在冷冻前和冷冻后，这两个参数均具有高度显著的相关性。冷冻保存和解冻似乎是一种有效的替代方法，可替代直接分析新鲜活淋巴结细胞，而不会在 SLN 样本之间引入与冷冻相关的变异性。然而，冷冻保存后，低频细胞群的计数及其标志物表达水平的评估不太可靠，在设计每个临床试验时应进行评估并加以考虑。
