Performance of spectral flow cytometry and mass cytometry for the study of innate myeloid cell populations

Kyra van der Pan; Indu Khatri; Anniek L de Jager; Alesha Louis; Sara Kassem; Brigitta A E Naber; Inge F de Laat; Marjolijn Hameetman; Suzanne E T Comans; Alberto Orfao; Jacques J M van Dongen; Paula Díez; Cristina Teodosio

doi:10.3389/fimmu.2023.1191992

Performance of spectral flow cytometry and mass cytometry for the study of innate myeloid cell populations

Front Immunol. 2023 May 19:14:1191992. doi: 10.3389/fimmu.2023.1191992. eCollection 2023.

Authors

Affiliations

¹ Department of Immunology, Leiden University Medical Center (LUMC), Leiden, Netherlands.
² Flow Cytometry Core Facility, Leiden University Medical Center (LUMC), Leiden, Netherlands.
³ Translational and Clinical Research Program, Cancer Research Center (IBMCC; University of Salamanca - CSIC), Cytometry Service, NUCLEUS, Department of Medicine, University of Salamanca and Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain.
⁴ Sarcomas and Experimental Therapeutics Laboratory, Health Research Institute of Asturias (ISPA) and Asturias Central University Hospital (HUCA), Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, Oviedo, Asturias, Spain.

Abstract

Introduction: Monitoring of innate myeloid cells (IMC) is broadly applied in basic and translational research, as well as in diagnostic patient care. Due to their immunophenotypic heterogeneity and biological plasticity, analysis of IMC populations typically requires large panels of markers. Currently, two cytometry-based techniques allow for the simultaneous detection of ≥40 markers: spectral flow cytometry (SFC) and mass cytometry (MC). However, little is known about the comparability of SFC and MC in studying IMC populations.

Methods: We evaluated the performance of two SFC and MC panels, which contained 21 common markers, for the identification and subsetting of blood IMC populations. Based on unsupervised clustering analysis, we systematically identified 24 leukocyte populations, including 21 IMC subsets, regardless of the cytometry technique.

Results: Overall, comparable results were observed between the two technologies regarding the relative distribution of these cell populations and the staining resolution of individual markers (Pearson's ρ=0.99 and 0.55, respectively). However, minor differences were observed between the two techniques regarding intra-measurement variability (median coefficient of variation of 42.5% vs. 68.0% in SFC and MC, respectively; p<0.0001) and reproducibility, which were most likely due to the significantly longer acquisition times (median 16 min vs. 159 min) and lower recovery rates (median 53.1% vs. 26.8%) associated with SFC vs. MC.

Discussion: Altogether, our results show a good correlation between SFC and MC for the identification, enumeration and characterization of IMC in blood, based on large panels (>20) of antibody reagents.

Keywords: cyTOF; immunophenotyping; mass cytometry; myeloid cells; spectral flow cytometry.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Flow Cytometry* / methods
Humans
Myeloid Cells*
Reproducibility of Results

Grants and funding

The work here presented was funded by the European Research Council under the European Union’s Horizon 2020 Research and Innovation Programme with an ERC Advanced Grant (ERC-2015-AdG 695655, TiMaScan). CT is the recipient of an USAL4EXCELLENCE fellowship, funded by the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 101034371. PD is supported by a Sara Borrell postdoctoral fellowship (Institute of Health Carlos III, Spanish Government, CD22/00009).