PROBLEM A variety of methods have been used to process cervical cytobrush and genital tissue for flow cytometric evaluation of immune cell populations. We sought to optimize genital tract specimen processing and to determine if blood could be used as a model for assessment of tissue processing methods. METHOD OF STUDY Cervical cytobrushes, PBMCs, and genital tissue samples (cervical and endometrial biopsies) were subjected to varying processing conditions to characterize the effects on cell yields, lymphocyte viability, and surface receptors. We exposed PBMC and tissue specimens to varied collagenase types, concentrations, and exposure durations and cytobrushes to immediate vs delayed processing with/without vortexing. RESULTS PBMCs and tissues exposed to varying enzymatic digestion conditions demonstrated stability of some cell surface receptors, including CD3+ , CD4+ , and CD8+ , while others, including CCR6+ , were cleaved when exposed to any concentration of collagenase B, or ≥0.25 mg/mL of collagenase D. We observed increased CD69 expression (marker of cell activation) after exposure to collagenase B. Neither a 2-hour delay in cytobrush processing nor vortexing at a setting of 50% for 30 seconds had significant impacts on viability or quantities of genital immune cells of interest. CONCLUSION Although tissue digestion with collagenase D was sufficient to recover and analyze cells from endometrial biopsy specimens, cervical biopsy specimens required a limited exposure to collagenase B at 1 mg/mL to optimize cell yield and viability for cytometric analysis. PBMCs can be used as a model to assess the impact of tissue processing on co-receptor expression and to optimize methods prior to study implementation.

中文翻译：

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优化处理女性生殖器组织样本以通过流式细胞仪进行淋巴细胞分析。

问题已使用多种方法处理宫颈细胞刷和生殖器组织，以进行免疫细胞群的流式细胞术评估。我们试图优化生殖道标本的处理，并确定是否可以将血液用作评估组织处理方法的模型。研究方法宫颈细胞刷，PBMC和生殖器组织样品（宫颈和子宫内膜活检）经受不同的处理条件，以表征对细胞产量，淋巴细胞活力和表面受体的影响。我们将PBMC和组织标本暴露于各种胶原酶类型，浓度，暴露持续时间和细胞刷中，无论有无涡旋，即时处理还是延迟处理。结果暴露于不同酶消化条件下的PBMC和组织表现出某些细胞表面受体（包括CD3 +，CD4 +和CD8 +）的稳定性，而另一些细胞（包括CCR6 +）暴露于任何浓度的胶原酶B或≥0.25mg / mL时均被裂解。胶原酶D。我们观察到暴露于胶原酶B后CD69表达增加（细胞活化的标志）。在2个小时的时间里，细胞刷处理或在50％的环境中涡旋30秒都不会对生殖器免疫力或活力产生显着影响感兴趣的细胞。结论尽管用胶原酶D进行组织消化足以恢复和分析子宫内膜活检标本中的细胞，但宫颈活检标本需要有限地暴露于1 mg / mL的胶原酶B中，以优化细胞产量和细胞计数分析的活力。