A user's guide to multicolor flow cytometry panels for comprehensive immune profiling

Anal Biochem. 2021 Aug 15:627:114210. doi: 10.1016/j.ab.2021.114210.

Staffan Holmberg-Thyden ¹, Kirsten Grønbæk ², Anne Ortved Gang ³, Daniel El Fassi ³, Sine Reker Hadrup ⁴

Affiliations

1 Dept. of Hematology, Copenhagen University Hospital, Rigshospitalet, Denmark; T-cells and Cancer, Experimental & Translational Immunology (XTI), Health Technology, Technical University of Denmark, Denmark.
2 Dept. of Hematology, Copenhagen University Hospital, Rigshospitalet, Denmark; Dept. of Clinical Medicine, University of Copenhagen, Denmark; Biotech Research and Innovation Centre, BRIC, University of Copenhagen, Denmark.
3 Dept. of Hematology, Copenhagen University Hospital, Rigshospitalet, Denmark; Dept. of Clinical Medicine, University of Copenhagen, Denmark.
4 T-cells and Cancer, Experimental & Translational Immunology (XTI), Health Technology, Technical University of Denmark, Denmark. Electronic address: [email protected].

PMID: 34033799 DOI: 10.1016/j.ab.2021.114210

Abstract

Multicolor flow cytometry is an essential tool for studying the immune system in health and disease, allowing users to extract longitudinal multiparametric data from patient samples. The process is complicated by substantial variation in performance between each flow cytometry instrument, and analytical errors are therefore common. Here, we present an approach to overcome such limitations by applying a systematic workflow for pairing colors to markers optimized for the equipment intended to run the experiments. The workflow is exemplified by the design of four comprehensive flow cytometry panels for patients with hematological Cancer. Methods for quality control, titration of antibodies, compensation, and staining of cells for obtaining optimal results are also addressed. Finally, to handle the large amounts of data generated by multicolor flow cytometry, unsupervised clustering techniques are used to identify significant subpopulations not detected by conventional sequential gating.

Keywords

Flow cytometry; Immune monitoring; Immunology; Myelodysplastic syndrome; Unsupervised clustering.

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