Optimization of processing female genital tissue samples for lymphocyte analysis by flow cytometry.

Abstract

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. 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. 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 ≥0.25mg/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 30seconds had significant impacts on viability or quantities of genital immune cells of interest. 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 1mg/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.