title: Standardizing Flow Cytometry Immunophenotyping Analysis from the Human ImmunoPhenotyping Consortium.
abstract: Standardization of immunophenotyping requires careful attention to reagents, sample handling, instrument setup, and data analysis, and is essential for successful cross-study and cross-center comparison of data. Experts developed five standardized, eight-color panels for identification of major immune cell subsets in peripheral blood. These were produced as pre-configured, lyophilized, reagents in 96-well plates. We present the results of a coordinated analysis of samples across nine laboratories using these panels with standardized operating procedures (SOPs). Manual gating was performed by each site and by a central site. Automated gating algorithms were developed and tested by the FlowCAP consortium. Centralized manual gating can reduce cross-center variability, and we sought to determine whether automated methods could streamline and standardize the analysis. Within-site variability was low in all experiments, but cross-site variability was lower when central analysis was performed in comparison with site-specific analysis. It was also lower for clearly defined cell subsets than those based on dim markers and for rare populations. Automated gating was able to match the performance of central manual analysis for all tested panels, exhibiting little to no bias and comparable variability. Standardized staining, data collection, and automated gating can increase power, reduce variability, and streamline analysis for immunophenotyping.
authors: Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, 98109, WA.
authors: Greg
authors: G
authors: Finak
authors: Hematology Branch, National Institutes of Health, Bethesda, Maryland, USA.
authors: Marc
authors: M
authors: Langweiler
authors: BD Biosciences, San Jose, CA, USA.
authors: Maria
authors: M
authors: Jaimes
authors: Terry Fox Laboratory , British Columbia Cancer Agency, V3J 4W6, Canada.
authors: Mehrnoush
authors: M
authors: Malek
authors: Terry Fox Laboratory , British Columbia Cancer Agency, V3J 4W6, Canada.
authors: Jafar
authors: J
authors: Taghiyar
authors: UCLA Pathology and Laboratory Medicine, Los Angeles, CA.
authors: Yael
authors: Y
authors: Korin
authors: Dept of Neurology, Yale School of Medicine, New Haven, CT.
authors: Khadir
authors: K
authors: Raddassi
authors: Dept of Neurology, Yale School of Medicine, New Haven, CT.
authors: Lesley
authors: L
authors: Devine
authors: Baylor Institute for Immunology Research, Dallas, TX.
authors: Gerlinde
authors: G
authors: Obermoser
authors: University of Cambridge, JDRF/Wellcome Trust Diabetes and Inflammation Laboratory, Cambridge Institute for Medical Research, Cambridge, UK.
authors: Marcin L
authors: ML
authors: Pekalski
authors: University of Cambridge, JDRF/Wellcome Trust Diabetes and Inflammation Laboratory, Cambridge Institute for Medical Research, Cambridge, UK.
authors: Nikolas
authors: N
authors: Pontikos
authors: Dept Microbiology &Immunology, University of Miami Miller School of Medicine, Miami, FL.
authors: Alain
authors: A
authors: Diaz
authors: Guys and St Thomas Hospital, Guy's Hospital, London, UK.
authors: Susanne
authors: S
authors: Heck
authors: Guys and St Thomas Hospital, Guy's Hospital, London, UK.
authors: Federica
authors: F
authors: Villanova
authors: School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton, BN2 4GJ, United Kingdom.
authors: Nadia
authors: N
authors: Terrazzini
authors: Brighton and Sussex Medical School, Division of Medicine, Brighton, BN1 9PS, United Kingdom.
authors: Florian
authors: F
authors: Kern
authors: Department of Informatics, J. Craig Venter Institute, La Jolla, 92037, CA.
authors: Yu
authors: Y
authors: Qian
authors: Department of Informatics, J. Craig Venter Institute, La Jolla, 92037, CA.
authors: Rick
authors: R
authors: Stanton
authors: School of Mathematics and Physics, University of Queensland, Brisbane, Australia.
authors: Kui
authors: K
authors: Wang
authors: The Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
authors: Aaron
authors: A
authors: Brandes
authors: Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, 98109, WA.
authors: John
authors: J
authors: Ramey
authors: Terry Fox Laboratory , British Columbia Cancer Agency, V3J 4W6, Canada.|Baxter Laboratory in Stem Cell Biology, Stanford University, Stanford, California, 94305, USA.
authors: Nima
authors: N
authors: Aghaeepour
authors: Hematology Branch, National Institutes of Health, Bethesda, Maryland, USA.|University of Rochester Medical Center, School of Medicine and Dentistry, Rochester, 14642, NY.
authors: Tim
authors: T
authors: Mosmann
authors: Department of Informatics, J. Craig Venter Institute, La Jolla, 92037, CA.
authors: Richard H
authors: RH
authors: Scheuermann
authors: UCLA Pathology and Laboratory Medicine, Los Angeles, CA.
authors: Elaine
authors: E
authors: Reed
authors: Baylor Institute for Immunology Research, Dallas, TX.
authors: Karolina
authors: K
authors: Palucka
authors: Baylor Institute for Immunology Research, Dallas, TX.
authors: Virginia
authors: V
authors: Pascual
authors: Dept Microbiology &Immunology, University of Miami Miller School of Medicine, Miami, FL.
authors: Bonnie B
authors: BB
authors: Blomberg
authors: Guys and St Thomas Hospital, Guy's Hospital, London, UK.
authors: Frank
authors: F
authors: Nestle
authors: Laboratory of Immunology, National Eye Institute, National Institutes of Health, Bethesda, Maryland, USA.
authors: Robert B
authors: RB
authors: Nussenblatt
authors: Terry Fox Laboratory , British Columbia Cancer Agency, V3J 4W6, Canada.|Department of Medical Genetics, University of British Columbia, Canada.
authors: Ryan Remy
authors: RR
authors: Brinkman
authors: Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, 98109, WA.
authors: Raphael
authors: R
authors: Gottardo
authors: Institute for Immunity, Transplantation, and Infection, Stanford University School of Medicine, Stanford, 94305, CA.
authors: Holden
authors: H
authors: Maecker
authors: NHLBI Flow Cytometry Core, NIH, Bethesda, 20892, MD.
authors: J Philip
authors: JP
authors: McCoy
mesh_terms: D000465:Algorithms; D001331:Automation; D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods / Q000592:standards*; D006801:Humans; D016130:Immunophenotyping / Q000379:methods / Q000592:standards*; D007753:Laboratories / Q000592:standards*; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism