Exhaustive expansion: A novel technique for analyzing complex data generated by higher-order polychromatic flow cytometry experiments

Background: The complex data sets generated by higher-order polychromatic flow cytometry experiments are a challenge to analyze. Here we describe Exhaustive Expansion, a data analysis approach for deriving hundreds to thousands of cell phenotypes from raw data, and for interrogating these phenotypes to identify populations of biological interest given the experimental context.Methods: We apply this approach to two studies, illustrating its broad applicability. The first examines the longitudinal changes in circulating human memory T cell populations within individual patients in response to a melanoma peptide (gp100_209-2M) cancer vaccine, using 5 monoclonal antibodies (mAbs) to delineate subpopulations of viable, gp100-specific, CD8+ T cells. The second study measures the mobilization of stem cells in porcine bone marrow that may be associated with wound healing, and uses 5 different staining panels consisting of 8 mAbs each.Results: In the first study, our analysis suggests that the cell surface markers CD45RA, CD27 and CD28, commonly used in historical lower order (2-4 color) flow cytometry analysis to distinguish memory from naïve and effector T cells, may not be obligate parameters in defining central memory T cells (T_CM). In the second study, we identify novel phenotypes such as CD29+CD31+CD56+CXCR4+CD90+Sca1-CD44+, which may characterize progenitor cells that are significantly increased in wounded animals as compared to controls.Conclusions: Taken together, these results demonstrate that Exhaustive Expansion supports thorough interrogation of complex higher-order flow cytometry data sets and aids in the identification of potentially clinically relevant findings.