title: Novel application of a whole blood intracellular cytokine detection assay to quantitate specific T-cell frequency in field studies.
abstract: We optimized a whole blood intracellular cytokine assay to quantitate the frequency of specific CD4+ and CD8+ T cells in small volumes of whole blood from infants from developing countries. The assay is performed in two steps. First, whole blood is stimulated in the presence of specific antigens for 6-18 h, ending with cryopreservation of fixed white cells. These stimulation steps were specifically adapted to be practical and reliable in a rural, developing country field setting. Later, in a more resourceful setting, interferon-gamma producing CD4+ or CD8+ T cells are detected by flow cytometry. The assay proved sensitive and specific for detecting mycobacteria-specific immunity 10 weeks after Bacillus Calmette-Guerin (BCG) vaccination of newborns from a rural field site.
authors.affiliation: Department of Pediatrics, University of Miami School of Medicine, Miami, FL 33101, USA. whanekom@med.miami.edu
authors.forename: Willem A
authors.initials: WA
authors.lastname: Hanekom
authors.forename: Jane
authors.initials: J
authors.lastname: Hughes
authors.forename: Maushumi
authors.initials: M
authors.lastname: Mavinkurve
authors.forename: Megan
authors.initials: M
authors.lastname: Mendillo
authors.forename: Marcia
authors.initials: M
authors.lastname: Watkins
authors.forename: Hoyam
authors.initials: H
authors.lastname: Gamieldien
authors.forename: Sebastian J
authors.initials: SJ
authors.lastname: Gelderbloem
authors.forename: Mzwandile
authors.initials: M
authors.lastname: Sidibana
authors.forename: Nazma
authors.initials: N
authors.lastname: Mansoor
authors.forename: Virginia
authors.initials: V
authors.lastname: Davids
authors.forename: Rose Ann
authors.initials: RA
authors.lastname: Murray
authors.forename: Anthony
authors.initials: A
authors.lastname: Hawkridge
authors.forename: Patrick A J
authors.initials: PA
authors.lastname: Haslett
authors.forename: Stanley
authors.initials: S
authors.lastname: Ress
authors.forename: Gregory D
authors.initials: GD
authors.lastname: Hussey
authors.forename: Gilla
authors.initials: G
authors.lastname: Kaplan
mesh_terms: D001500:BCG Vaccine / Q000276:immunology; D001800:Blood Specimen Collection; D015925:Cryopreservation; D016207:Cytokines / Q000097:blood*; D003906:Developing Countries; D005434:Flow Cytometry; D015658:HIV Infections / Q000097:blood / Q000276:immunology; D006801:Humans; D007156:Immunologic Memory / Q000276:immunology; D007223:Infant; D007371:Interferon-gamma / Q000032:analysis / Q000378:metabolism; D042541:Intracellular Space / Q000737:chemistry*; D009169:Mycobacterium tuberculosis / Q000276:immunology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016176:T-Lymphocyte Subsets / Q000166:cytology* / Q000276:immunology / Q000378:metabolism*; D013997:Time Factors
chemical_list: D001500:BCG Vaccine; D016207:Cytokines; D007371:Interferon-gamma