title: An Integrated Workflow To Assess Technical and Biological Variability of Cell Population Frequencies in Human Peripheral Blood by Flow Cytometry.
abstract: In the context of large-scale human system immunology studies, controlling for technical and biological variability is crucial to ensure that experimental data support research conclusions. In this study, we report on a universal workflow to evaluate both technical and biological variation in multiparameter flow cytometry, applied to the development of a 10-color panel to identify all major cell populations and T cell subsets in cryopreserved PBMC. Replicate runs from a control donation and comparison of different gating strategies assessed the technical variability associated with each cell population and permitted the calculation of a quality control score. Applying our panel to a large collection of PBMC samples, we found that most cell populations showed low intraindividual variability over time. In contrast, certain subpopulations such as CD56 T cells and Temra CD4 T cells were associated with high interindividual variability. Age but not gender had a significant effect on the frequency of several populations, with a drastic decrease in naive T cells observed in older donors. Ethnicity also influenced a significant proportion of immune cell population frequencies, emphasizing the need to account for these covariates in immune profiling studies. We also exemplify the usefulness of our workflow by identifying a novel cell-subset signature of latent tuberculosis infection. Thus, our study provides a universal workflow to establish and evaluate any flow cytometry panel in systems immunology studies.
authors.affiliation: La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037; jburel@lji.org.
authors.forename: Julie G
authors.identifier: 0000-0003-1692-2758
authors.initials: JG
authors.lastname: Burel
authors.affiliation: J. Craig Venter Institute, La Jolla, CA 92037.
authors.forename: Yu
authors.initials: Y
authors.lastname: Qian
authors.affiliation: La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Lindestam Arlehamn
authors.affiliation: La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037.
authors.forename: Daniela
authors.initials: D
authors.lastname: Weiskopf
authors.affiliation: La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037.
authors.forename: Jose
authors.identifier: 0000-0002-9098-1652
authors.initials: J
authors.lastname: Zapardiel-Gonzalo
authors.affiliation: Division of Infectious Diseases, University of California, San Diego, La Jolla, CA 92093.
authors.forename: Randy
authors.initials: R
authors.lastname: Taplitz
authors.affiliation: Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD 21205.|Universidad Peruana Caytano Hereida, Lima 15102, Peru.
authors.forename: Robert H
authors.initials: RH
authors.lastname: Gilman
authors.affiliation: Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD 21205.|Universidad Peruana Caytano Hereida, Lima 15102, Peru.|Department of Virology, Tohoku University, Sendai 9808575, Japan.
authors.forename: Mayuko
authors.initials: M
authors.lastname: Saito
authors.affiliation: Genetech Research Center, Colombo 00800, Sri Lanka; and.
authors.forename: Aruna D
authors.initials: AD
authors.lastname: de Silva
authors.affiliation: La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037.
authors.forename: Pandurangan
authors.initials: P
authors.lastname: Vijayanand
authors.affiliation: La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037.|J. Craig Venter Institute, La Jolla, CA 92037.|Department of Pathology, University of California, San Diego, La Jolla, CA 92093.
authors.forename: Richard H
authors.identifier: 0000-0003-1355-892X
authors.initials: RH
authors.lastname: Scheuermann
authors.affiliation: La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Sette
authors.affiliation: La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037.
authors.forename: Bjoern
authors.identifier: 0000-0002-8457-6693
authors.initials: B
authors.lastname: Peters
mesh_terms: D000328:Adult; D000367:Age Factors; D015496:CD4-Positive T-Lymphocytes; D019002:CD56 Antigen / Q000235:genetics / Q000276:immunology; D002452:Cell Count / Q000379:methods; D005260:Female; D005434:Flow Cytometry / Q000379:methods* / Q000592:standards* / Q000706:statistics & numerical data; D006801:Humans; D016130:Immunophenotyping / Q000379:methods; D055985:Latent Tuberculosis / Q000175:diagnosis / Q000276:immunology; D007963:Leukocytes, Mononuclear; D008297:Male; D012737:Sex Factors; D016176:T-Lymphocyte Subsets / Q000276:immunology*; D057188:Workflow
chemical_list: D019002:CD56 Antigen; C522918:NCAM1 protein, human