title: Biological and technical variables affecting immunoassay recovery of cytokines from human serum and simulated vaginal fluid: a multicenter study.
abstract: The increase of proinflammatory cytokines in vaginal secretions may serve as a surrogate marker of unwanted inflammatory reaction to microbicide products topically applied for the prevention of sexually transmitted diseases, including HIV-1. Interleukin (IL)-1beta and IL-6 have been proposed as indicators of inflammation and increased risk of HIV-1 transmission; however, the lack of information regarding detection platforms optimal for vaginal fluids and interlaboratory variation limit their use for microbicide evaluation and other clinical applications. This study examines fluid matrix variants relevant to vaginal sampling techniques and proposes a model for interlaboratory comparisons across current cytokine detection technologies. IL-1beta and IL-6 standards were measured by 12 laboratories in four countries, using 14 immunoassays and four detection platforms based on absorbance, chemiluminescence, electrochemiluminescence, and fluorescence. International reference preparations of cytokines with defined biological activity were spiked into (1) a defined medium simulating the composition of human vaginal fluid at pH 4.5 and 7.2, (2) physiologic salt solutions (phosphate-buffered saline and saline) commonly used for vaginal lavage sampling in clinical studies of cytokines, and (3) human blood serum. Assays were assessed for reproducibility, linearity, accuracy, and significantly detectable fold difference in cytokine level. Factors with significant impact on cytokine recovery were determined by Kruskal-Wallis analysis of variance with Dunn's multiple comparison test and multiple regression models. All assays showed acceptable intra-assay reproducibility; however, most were associated with significant interlaboratory variation. The smallest reliably detectable cytokine differences ( P < 0.05) derived from pooled interlaboratory data varied from 1.5- to 26-fold depending on assay, cytokine, and matrix type. IL-6 but not IL-1beta determinations were lower in both saline and phosphate-buffered saline as compared to vaginal fluid matrix, with no significant effect of pH. The (electro)chemiluminescence-based assays were most discriminative and consistently detected <2-fold differences within each matrix type. The Luminex-based assays were less discriminative with lower reproducibility between laboratories. These results suggest the need for uniform vaginal sampling techniques and a better understanding of immunoassay platform differences and cross-validation before the biological significance of cytokine variations can be validated in clinical trials. This investigation provides the first standardized analytic approach for assessing differences in mucosal cytokine levels and may improve strategies for monitoring immune responses at the vaginal mucosal interface.
authors.affiliation: Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA. rfichorova@rics.bwh.harvard.edu
authors.forename: Raina N
authors.initials: RN
authors.lastname: Fichorova
authors.forename: Nicola
authors.initials: N
authors.lastname: Richardson-Harman
authors.forename: Massimo
authors.initials: M
authors.lastname: Alfano
authors.forename: Laurent
authors.initials: L
authors.lastname: Belec
authors.forename: Cedric
authors.initials: C
authors.lastname: Carbonneil
authors.forename: Silvia
authors.initials: S
authors.lastname: Chen
authors.forename: Lisa
authors.initials: L
authors.lastname: Cosentino
authors.forename: Kelly
authors.initials: K
authors.lastname: Curtis
authors.forename: Charlene S
authors.initials: CS
authors.lastname: Dezzutti
authors.forename: Betty
authors.initials: B
authors.lastname: Donoval
authors.forename: Gustavo F
authors.initials: GF
authors.lastname: Doncel
authors.forename: Melissa
authors.initials: M
authors.lastname: Donaghay
authors.forename: Jean-Charles
authors.initials: JC
authors.lastname: Grivel
authors.forename: Esmeralda
authors.initials: E
authors.lastname: Guzman
authors.forename: Madeleine
authors.initials: M
authors.lastname: Hayes
authors.forename: Betsy
authors.initials: B
authors.lastname: Herold
authors.forename: Sharon
authors.initials: S
authors.lastname: Hillier
authors.forename: Carol
authors.initials: C
authors.lastname: Lackman-Smith
authors.forename: Alan
authors.initials: A
authors.lastname: Landay
authors.forename: Leonid
authors.initials: L
authors.lastname: Margolis
authors.forename: Kenneth H
authors.initials: KH
authors.lastname: Mayer
authors.forename: Jenna-Malia
authors.initials: JM
authors.lastname: Pasicznyk
authors.forename: Melanie
authors.initials: M
authors.lastname: Pallansch-Cokonis
authors.forename: Guido
authors.initials: G
authors.lastname: Poli
authors.forename: Patricia
authors.initials: P
authors.lastname: Reichelderfer
authors.forename: Paula
authors.initials: P
authors.lastname: Roberts
authors.forename: Irma
authors.initials: I
authors.lastname: Rodriguez
authors.forename: Hela
authors.initials: H
authors.lastname: Saidi
authors.forename: Rosaria Rita
authors.initials: RR
authors.lastname: Sassi
authors.forename: Robin
authors.initials: R
authors.lastname: Shattock
authors.forename: James E
authors.initials: JE
authors.lastname: Cummins
mesh_terms: D001826:Body Fluids / Q000737:chemistry*; D005260:Female; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D053583:Interleukin-1beta / Q000032:analysis* / Q000097:blood*; D015850:Interleukin-6 / Q000032:analysis* / Q000097:blood*; D012015:Reference Standards; D015203:Reproducibility of Results; D014621:Vagina / Q000378:metabolism*
chemical_list: D053583:Interleukin-1beta; D015850:Interleukin-6