title: Influence of specimen age and use of different negative controls in determination of intracytoplasmic levels of cytokines after whole-blood culture assay.
abstract: Intracytoplasmic detection of cytokines by flow cytometry has become a powerful tool in the characterization of cytokine-producing cells. However, it is not known to what extent specimen age and the use of various negative controls may influence the amount of cytokine-positive cells. We therefore compared different times of storage and the use of several negative controls in the determination of intracytoplasmic levels of cytokines. There was a substantial decline of interleukin-2- and gamma interferon-positive lymphocytes after 20 h and especially after 48 h of storage. The precision of intracytoplasmic interleukin-6 determination decreases after long-term storage compared to 2 h of storage, whereas the amount of interleukin-8-positive monocytes remained rather stable. Therefore, we recommend performing the analysis as fast as possible after the blood sample is drawn. Under consideration of isotype-matched antibodies and nonstimulated cells as negative controls instead of the purified antibody-blocking control, strikingly higher amounts of interleukin-2-, gamma interferon, interleukin-6-, and interleukin-8-positive cells were found. For a meaningful interpretation of data these differences have to be kept in mind. Further studies should evaluate the exact specificity of these controls.
authors.affiliation: Department of Pediatrics, Medical University of Lübeck, Lübeck, Germany. ChrSchultz@aol.com
authors.forename: Christian
authors.initials: C
authors.lastname: Schultz
authors.forename: Christina
authors.initials: C
authors.lastname: Rott
authors.forename: Petra
authors.initials: P
authors.lastname: Temming
authors.forename: Julia
authors.initials: J
authors.lastname: von Puttkammer
authors.forename: Peter
authors.initials: P
authors.lastname: Bucsky
mesh_terms: D000328:Adult; D019138:Antibodies, Blocking / Q000494:pharmacology; D000918:Antibody Specificity; D016207:Cytokines / Q000032:analysis*; D003593:Cytoplasm / Q000737:chemistry / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods* / Q000592:standards*; D006801:Humans; D007371:Interferon-gamma / Q000032:analysis; D015850:Interleukin-6 / Q000032:analysis; D016209:Interleukin-8 / Q000032:analysis; D008214:Lymphocytes / Q000737:chemistry; D008828:Microbiological Techniques; D009000:Monocytes / Q000737:chemistry; D013048:Specimen Handling
chemical_list: D019138:Antibodies, Blocking; D016207:Cytokines; D015850:Interleukin-6; D016209:Interleukin-8; D007371:Interferon-gamma