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.