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)-1β 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-1β 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-1β 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.

Methodological Issues in Sampling the Local Immune System of the Female Genital Tract in the Context of HIV Prevention Trials

“…22 The multiplex kits can be custom made to fit the panel of cytokines selected for any study design. In preparation for the EMPRO Flora Study, a study investigating vaginal flora and mucosal characteristics in 28 young healthy Belgian women not using hormonal contraceptives, we performed a 27 BioPlex multiplex test run on samples from this same population to make a final selection for a multiplex panel (unpublished observation).…”

Investigation of melamine presence in canned tuna fish by capillary zone electrophoresis method

“…Recovery more than 100% indicates that the measured values for a matrix were higher than the nominal value of the spike (Fichorova et al 2008). On the other hand, low level impurities which caused by the sample structure or presence of melamine under the detection limit, increase the recovery of methods.…”

Serum leptin levels and their proportional relationship with pro- and antiinflammatory mediators in aggressive periodontitis: preliminary report

“…multiplex bead assays or ELISA kits) used and possible interlaboratory variations. It should also be kept in mind that it would be logical not to compare the results that belong to one assay to another assay used in different studies (18).…”