The challenge of distinguishing cell-cell complexes from singlet cells in non-imaging flow cytometry and single-cell sorting

Abstract

<jats:title>Abstract</jats:title><jats:p>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.</jats:p>

Expression of unique gene signature distinguishes TCRαβ+/BCR+ dual expressers from CD3+CD14+ doublets

“…In contrast, Burel et al did not report any differentially expressed genes by CD3 + CD14 + doublets as compared to T cells or monocytes. This is an important and key difference that distinguishes between transcriptomes of DEs and CD3 + CD14 + doublets, that Burel and colleagues 1 have completely ignored without a clear reason. By doing so, they effectively stripped DEs from their identity and turned them into doublets.…”

“…In their study of CD3 + CD14 + doublets, Burel et al 1 created cell‐type specific scores by summing the TPM (Transcripts Per Million Reads) counts of the 100 top genes exclusively expressed in T cells and monocytes. They did not report any unique signature genes of CD3 + CD14 + doublets.…”

“…Thus, using additional independent methods is necessary to corroborate fluorescence‐activated cell sorting (FACS) results in confirming identity of newly discovered rare cell types. In a recent publication of this journal, Burel et al 1 attempted to assess whether the newly described dual expresser (DEs) lymphocytes by Ahmed et al, 2 which are characterized by the coexpression of lineage markers of conventional B (Bcon) and T (Tcon) cells, are enriched for B‐T cell doublets.…”

Label-free imaging flow cytometry for analysis and sorting of enzymatically dissociated tissues

“…While doublet detection is not trivial in FACS as doublets are defined only indirectly through the ratio of event size and width, in imaging flow cytometers like soRT-FDC, a bright-field image is available for visual inspection 24 , 25 . Because of the large datasets, containing several thousands of images, an automated doublet detection is desired.…”

Distinguishing cell–cell complexes from dual lineage cells using single‐cell transcriptomics is not trivial

“…Finally, we identify multiple transcriptomic features of DE cells that correspond to B cell-T cell complexes, namely the presence of lower average expression of B-and T-cell specific genes, and a higher number of detected genes per cell. Taken together, our results demonstrate that solely based on their scRNAseq profile, it is not possible to ascertain that DE cells are dual expressing cells and not cell-cell complexes.We have recently discovered that cell-cell doublet populations pairing a T cell and an antigen-presenting cell (APC)-such as a monocyte or a B cell-can be detected in the singlet gate of ex vivo human blood samples analyzed by flow cytometry [1,2]. Strikingly, the T cell-…”