September 6, 2023

The Two Sides of Cell Doublets in Flow Cytometry Data

Flow cytometry and cell sorting provide unprecedented levels of detail about blood samples for both research and clinical applications, including the phenotypic heterogeneity of human immune cells. However, a recent talk at CYTO 2023 by Julie Burel, La Jolla Institute for Immunology, highlighted that blood-derived cell doublets can be present in single-cell data derived by these techniques.

Methods like exclusion based on Area/Width/Height can remove cell doublets, but some complexes remain undetected, leading to data misinterpretation. While cell doublets often need removing, interestingly, Burel reported that some doublets form naturally — which could provide greater disease understanding.
Here we look at what cell doublets are, their risk and promise, and how analysts can ensure accurate results.

Seeing double

While dual expressor cells exist in human peripheral blood samples acquired by flow cytometry, Burel’s team recently unveiled through imaging that some of these events are actually cell–cell complexes. In the live singlet gate of blood samples, T cells can bind to other immune cells, such as antigen-presenting cells (APC), forming a cell doublet. But without deeper analysis, it can be easy to analyze cell doublets as single cells. The cell doublets can subsequently generate atypical gene signatures of mixed cell lineage, meaning that data misinterpretation can occur, including:

• Specific cell population percentage deflation,

• Novel immune cell type misidentification, and

• Incorrect functional marker expression attribution.

Distinguishing cell-cell complexes for more accurate results

Experimental and data analysis strategies can distinguish between singlets and cell doublets for more accurate data. To detect the presence of cell-cell complexes within potential dual-expressing cells, Burel’s team used two different approaches: cell sorting followed by direct microscopy imaging to confirm the expression of markers on distinct cells, and imaging flow cytometry to generate metrics from pictures of the detected events, enabling doublet exclusion using brightfield area and aspect ratio parameter calculation.

Notably, Burel’s team found that cell-cell complexes have molecular signatures that clearly distinguish them from singlets at both protein and mRNA levels, providing analysts with a way to avoid data misinterpretation. When looking at cell–cell complex signatures with SSC/FSC and SSC/CD45, the team discovered that the complexes had higher levels of CD45, SSC and FSC expression compared to single cells.

But cell doublets aren’t just contaminants. The team found that T cell-APC complexes are formed in vivo as a natural immunological process, and quantities vary depending on disease state. As such, we believe they could provide deeper insights in research and diagnosis.

Leverage expertise for maximum data confidence

Flow cytometry and cell sorting are fundamental techniques to provide deeper insights from blood samples, but careful set-ups and accurate analysis are essential to ensure that cell doublets aren’t compromising results integrity. Our analytical team has the expertise to tailor gating strategies based on the presence of cell-cell complex doublets. CellCarta offers a broad range of advanced services using robust techniques to get greater insights from your studies — contact the team to learn more.


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Roxanne Collin is a Research Scientist within the Development group of the Data Analysis Unit at CellCarta. She holds a PhD in immunology with a focus on the immunogenetics of rare cell populations. At CellCarta, she is participating in the optimisation of flow cytometry panels and gating strategies for a broad variety of immune cell types.