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Our recent work has highlighted that care needs to be taken when interpreting single cell data originating from flow cytometry acquisition or cell sorting: We found that doublets of T cells bound to other immune cells are often present in the live singlet gate of human peripheral blood samples acquired by flow cytometry. This hidden “contamination” generates atypical gene signatures of mixed cell lineage in what is assumed to be single cells, which can lead to data misinterpretation, such as the description of novel immune cell types. Here, based on the example of T cell–monocyte complexes, we identify experimental and data analysis strategies to help distinguishing between singlets and cell–cell complexes in non‐imaging flow cytometry and single‐cell sorting. We found robust molecular signatures in both T cell–monocyte and T cell–B cell complexes that can distinguish them from singlets at both protein and mRNA levels. Imaging flow cytometry with appropriate gating strategy (matching the one used in cell sorting) and direct microscopy imaging after cell sorting were the two methods of choice to detect the presence of cell–cell complexes in suspicious dual‐expressing cells. We finally applied this knowledge to highlight the likely presence of T cell–B cell complexes in a recently published dataset describing a novel cell population with mixed T cell and B cell lineage properties. © 2020 The Authors.  Cytometry Part A  published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

The Challenge of Distinguishing Cell–Cell Complexes from Singlet Cells in Non‐Imaging Flow Cytometry and Single‐Cell Sorting