title: Effect of sample handling on analysis of cytokine responses to Mycobacterium tuberculosis in clinical samples using ELISA, ELISPOT and quantitative PCR.
abstract: Measuring cytokine responses to infection has proven to be invaluable for the understanding of immunity to tuberculosis in the laboratory. However, far less data are available from studies in humans and these have often produced conflicting results. Here we describe a comprehensive multi-center comparison of the most commonly used protocols for cytokine analysis: ELISA, ELISPOT and RT-PCR, in cohorts of TB patients, their household contacts and community controls. In particular, we have studied the effect on these protocols of conditions that commonly prevail in field studies, such as delays between sample collection and analysis, or different source material, such as whole blood or frozen PBMC. The results clearly show that while there is good correlation between the methods under optimal conditions, each method has strengths and weaknesses that render them more or less suitable for particular types of analyses. Researchers should carefully consider these factors when planning human field studies.
authors.affiliation: Department of Tuberculosis Immunology, Statens Serum Institute, Copenhagen, Denmark. TMD@ssi.dk
authors.forename: T Mark
authors.initials: TM
authors.lastname: Doherty
authors.forename: Abebech
authors.initials: A
authors.lastname: Demissie
authors.forename: Dick
authors.initials: D
authors.lastname: Menzies
authors.forename: Peter
authors.initials: P
authors.lastname: Andersen
authors.forename: Graham
authors.initials: G
authors.lastname: Rook
authors.forename: Alimuddin
authors.initials: A
authors.lastname: Zumla
mesh_terms: D000293:Adolescent; D000328:Adult; D000818:Animals; D016207:Cytokines / Q000097:blood* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007118:Immunoassay; D008297:Male; D009169:Mycobacterium tuberculosis / Q000276:immunology*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013048:Specimen Handling
chemical_list: D016207:Cytokines