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

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

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.

cell–cell complexes, doublets, flow cytometry, single-cell immune profiling, single-cell RNA sequencing
1552-4922
1127-1135
Burel, Julie G.
d043b967-6296-4f02-b628-e397a82de132
Pomaznoy, Mikhail
c483a11a-a48a-4eb5-9637-007b043afda8
Lindestam Arlehamn, Cecilia S.
972358b3-c46c-4de5-b7e1-afa2cf65ed35
Seumois, Gregory
0be7d3d6-5526-458c-aa5c-cce52410a2ed
Vijayanand, Pandurangan
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Sette, Alessandro
240988b3-3c05-4041-a39b-8be8e145803c
Peters, Bjoern
c49863c8-d2c6-4db4-9d2c-72a7c0cbe117
Burel, Julie G.
d043b967-6296-4f02-b628-e397a82de132
Pomaznoy, Mikhail
c483a11a-a48a-4eb5-9637-007b043afda8
Lindestam Arlehamn, Cecilia S.
972358b3-c46c-4de5-b7e1-afa2cf65ed35
Seumois, Gregory
0be7d3d6-5526-458c-aa5c-cce52410a2ed
Vijayanand, Pandurangan
79514f33-66cf-47cc-a8fa-46bbfc21b7d1
Sette, Alessandro
240988b3-3c05-4041-a39b-8be8e145803c
Peters, Bjoern
c49863c8-d2c6-4db4-9d2c-72a7c0cbe117

Burel, Julie G., Pomaznoy, Mikhail, Lindestam Arlehamn, Cecilia S., Seumois, Gregory, Vijayanand, Pandurangan, Sette, Alessandro and Peters, Bjoern (2020) The challenge of distinguishing cell–cell complexes from singlet cells in non-imaging flow cytometry and single-cell sorting. Cytometry Part A, 97 (11), 1127-1135. (doi:10.1002/cyto.a.24027).

Record type: Article

Abstract

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.

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cyto.a.24027 (1) - Version of Record
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Accepted/In Press date: 18 April 2020
e-pub ahead of print date: 13 May 2020
Published date: November 2020
Additional Information: © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.
Keywords: cell–cell complexes, doublets, flow cytometry, single-cell immune profiling, single-cell RNA sequencing

Identifiers

Local EPrints ID: 440945
URI: http://eprints.soton.ac.uk/id/eprint/440945
ISSN: 1552-4922
PURE UUID: b569e3a6-e166-4bc5-92e2-3249a298505d
ORCID for Pandurangan Vijayanand: ORCID iD orcid.org/0000-0001-7067-9723

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Date deposited: 26 May 2020 16:30
Last modified: 17 Mar 2024 12:40

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Contributors

Author: Julie G. Burel
Author: Mikhail Pomaznoy
Author: Cecilia S. Lindestam Arlehamn
Author: Gregory Seumois
Author: Pandurangan Vijayanand ORCID iD
Author: Alessandro Sette
Author: Bjoern Peters

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