Novel application of a whole blood intracellular cytokine detection assay to quantitate specific T-cell frequency in field studies.
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.
Hanekom
Willem A
WA
Department of Pediatrics, University of Miami School of Medicine, Miami, FL 33101, USA. whanekom@med.miami.edu
Hughes
Jane
J
Mavinkurve
Maushumi
M
Mendillo
Megan
M
Watkins
Marcia
M
Gamieldien
Hoyam
H
Gelderbloem
Sebastian J
SJ
Sidibana
Mzwandile
M
Mansoor
Nazma
N
Davids
Virginia
V
Murray
Rose Ann
RA
Hawkridge
Anthony
A
Haslett
Patrick A J
PA
Ress
Stanley
S
Hussey
Gregory D
GD
Kaplan
Gilla
G
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
D001500:BCG Vaccine; D016207:Cytokines; D007371:Interferon-gamma