title: Semi-automated and standardized cytometric procedures for multi-panel and multi-parametric whole blood immunophenotyping.
abstract: Immunophenotyping by multi-parametric flow cytometry is the cornerstone technology for enumeration and characterization of immune cell populations in health and disease. Standardized procedures are essential to allow for inter-individual comparisons in the context of population based or clinical studies. Herein we report the approach taken by the Milieu Intérieur Consortium, highlighting the standardized and automated procedures used for immunophenotyping of human whole blood samples. We optimized eight-color antibody panels and procedures for staining and lysis of whole blood samples, and implemented pre-analytic steps with a semi-automated workflow using a robotic system. We report on four panels that were designed to enumerate and phenotype major immune cell populations (PMN, T, B, NK cells, monocytes and DC). This work establishes a foundation for defining reference values in healthy donors. Our approach provides robust protocols for affordable, semi-automated eight-color cytometric immunophenotyping that can be used in population-based studies and clinical trial settings.
authors.affiliation: Center for Human Immunology, Institut Pasteur, Paris, France.
authors.forename: Milena
authors.initials: M
authors.lastname: Hasan
authors.affiliation: Center for Human Immunology, Institut Pasteur, Paris, France.
authors.forename: Benoit
authors.initials: B
authors.lastname: Beitz
authors.affiliation: Center for Human Immunology, Institut Pasteur, Paris, France; Center for Bioinformatics, Institut Pasteur, Paris, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Rouilly
authors.affiliation: Center for Human Immunology, Institut Pasteur, Paris, France.
authors.forename: Valentina
authors.initials: V
authors.lastname: Libri
authors.affiliation: Center for Human Immunology, Institut Pasteur, Paris, France; INSERM U818, France; Laboratory of Dendritic Cell Immunobiology, Department of Immunology, Institut Pasteur, Paris, France.
authors.forename: Alejandra
authors.initials: A
authors.lastname: Urrutia
authors.affiliation: Center for Human Immunology, Institut Pasteur, Paris, France; INSERM U818, France; Laboratory of Dendritic Cell Immunobiology, Department of Immunology, Institut Pasteur, Paris, France.
authors.forename: Darragh
authors.initials: D
authors.lastname: Duffy
authors.affiliation: Center for Human Immunology, Institut Pasteur, Paris, France.
authors.forename: Lydie
authors.initials: L
authors.lastname: Cassard
authors.affiliation: Laboratory of Innate Immunity, Department of Immunology, Institut Pasteur, Paris, France.
authors.forename: James P
authors.initials: JP
authors.lastname: Di Santo
authors.affiliation: Center for Human Immunology, Institut Pasteur, Paris, France.
authors.forename: Estelle
authors.initials: E
authors.lastname: Mottez
authors.affiliation: Laboratory of Human Evolutionary Genetics, Department of Genomes & Genetics, Institut Pasteur, Paris, France; CNRS URA3012, France.
authors.forename: Lluis
authors.initials: L
authors.lastname: Quintana-Murci
authors.affiliation: Center for Human Immunology, Institut Pasteur, Paris, France; INSERM U818, France; Laboratory of Dendritic Cell Immunobiology, Department of Immunology, Institut Pasteur, Paris, France; INSERM UMS20, France. Electronic address: albertm@pasteur.fr.
authors.forename: Matthew L
authors.initials: ML
authors.lastname: Albert
authors.affiliation: Center for Human Immunology, Institut Pasteur, Paris, France; Laboratory of Immunoregulation, Department of Immunology, Institut Pasteur, Paris, France. Electronic address: lars.rogge@pasteur.fr.
authors.forename: Lars
authors.initials: L
authors.lastname: Rogge
mesh_terms: D015703:Antigens, CD / Q000276:immunology; D057205:Automation, Laboratory / Q000379:methods*; D001402:B-Lymphocytes; D003713:Dendritic Cells; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D007694:Killer Cells, Natural; D009000:Monocytes; D009504:Neutrophils; D013048:Specimen Handling / Q000379:methods; D013601:T-Lymphocytes
chemical_list: D015703:Antigens, CD
keywords: Automation; Cytometry; Immunophenotyping; Standardization; Whole blood