We established ELISA protocols and tested over a period of several months 291 CVL samples obtained from two CONRAD-funded microbicide studies. Volunteers for these studies applied either microbicide or control product intravaginally twice a day for 14 intermenstrual days. Lavage samples were obtained in 10 cc saline during three visits: visit 2 before product use; visit 3 and visit 4 at the seventh and 14th day of product use, respectively. Because the following results are presented before data analysis and study personnel unmasking, the treatment group and control group data are pooled.
We established detailed protocols and quality control procedures for the measurement of IL-1α, IL-1β, IL-8, IL-1RA and TNF-α in the CVLs using commercial photometric or luminescence ELISAs (R&D Systems, Minneapolis, MN, USA, and Endogen-Pierce, Rockford, IL, USA), a Multilabel Microplate Counter Victor 2 (Perkin Elmer Life Sciences, Boston, MA, USA) and WorkOut Version 1.5 Wallace Software (DAZDAQ Ltd., Ringmer, East Sussex, UK). The intra-assay and inter-assay coefficients of variation were determined from duplicate measurements in at least three independent assay runs for each assay. For this purpose we used standard spiking in pooled CVLs.
We determined the optimal dilutions of CVLs that avoid matrix interference and allow reproducible measurements within the best-fit detection range. To neutralize the acidic pH of the CVLs, which interferes with some cytokine detection systems, we recommend the dilution of samples at least two times with the assay's sample or calibrator diluent (usually protein/serum base with preservatives which may contain 0.01% Thimerosal). For IL-8 quantitation in 10 cc CVLs we recommend a screening dilution of 10× and for IL-1RA – 100× (). Higher dilutions (40× and, respectively, 400×) are applied to samples showing cytokine values at the top of the assay's dynamic range.
FIGURE 3 Cytokine detection in human cervicovaginal lavages at different sample dilutions. (a) The assay dynamic range and cervicovaginal lavage (CVL) matrix require a 100× screening dilution; (b) CVL matrix interference requires a 10× dilution (more ...)
Using our quality control procedure and taking the dilution factors into consideration we established the following cut-off levels for highly reproducibly quantifiable cytokine levels in human CVLs (10 cc saline): IL-1α 20 pg/ml; IL-1β 10 pg/ml; IL-8 256 pg/ml; IL-1RA 4690 pg/ml; TNF-α 22 pg/ml. These values are close to experimentally established ED50s for these cytokines. Lower concentrations can be measurable in high sensitivity assays; however, the coefficient of variation for duplicate measurements is usually greater than 30%, and the biological significance of such levels is questionable.
To establish the stability of endogenous cytokines detectable in the saline CVLs we pooled CVLs collected before product use and measured cytokine levels by ELISA after different lengths of exposure to various temperature conditions. The results from four independent experiments with different CVL pools showed that IL1α, IL-1β, IL-8 and IL-1RA were stable for 2 h at temperatures as high as 37°C and that most of them were stable at 4°C for up to 24 h, suggesting that CVLs for cytokine monitoring could be collected in field conditions or low resource clinical settings ().
FIGURE 4 The recovery of endogenous cytokines from pooled cervicovaginal lavages collected at baseline. The results represent the mean and SEM from duplicate measurements from two independent experiments for each cytokine. Cervicovaginal lavage pools were divided (more ...)
Our results confirmed the expected considerable variation of cytokine concentrations at baseline, especially for IL-8 and IL-1RA (). The subject stratification based on sexual abstinence, however, showed significantly higher values and baseline ranges in the sexually active cohorts as compared with sexually abstinent cohorts (). The cytokine profiling of subjects before and after product use revealed three major patterns, each of them underscoring some important caveats of interpretation.
Baseline cytokine variability in human cervicovaginal lavages collected from healthy volunteers (n = 90) in CONRAD-sponsored clinical trials. Each dot represents the average of duplicate measurements of individual cervicovaginal lavage samples.
Baseline IL-8 and IL-1RA values are significantly elevated in the cervicovaginal lavages of sexually active versus sexually abstinent women (n = 90).
In the most common pattern, all five cytokines detected in the CVL samples post treatment (visits 3 and 4) were within the range of normal baseline variation for the sexually abstinent or sexually active cohorts, and showed no significant difference from the pre-dosing cytokine background for the individual subject. As interference with the cytokine detection assay was ruled out for the products tested in this study, a conclusion can be made that this pattern is associated with a lack of product effects on the cytokine equilibrium.
The second pattern is illustrated by the case of a sexually active healthy volunteer (). The CVL collected at baseline (pre-treatment visit 2) showed traces of blood. At the same time, the baseline sample showed markedly increased cytokine levels. Whereas the IL-1RA and IL-8 values were consistent with the baseline values observed in the sexually active cohort, the higher IL-1α and IL-1β levels could be explained with the presence of blood in this sample.
FIGURE 7 Case interpretation: concentrations of cytokines in cervicovaginal lavage samples obtained from a sexually active healthy volunteer. Bars represent means ± SD of duplicate cytokine measurements by enzyme-linked immunosorbent assay. The solid line (more ...)
The third cytokine pattern is illustrated by the case of a sexually abstinent woman (). In this case, IL-1α and IL-8 were progressively increased at visits 3 and 4 compared with the baseline visit 2, with the baseline values being within the normal variation for sexually abstinent cohorts (). If no other cytokines had been measured in these CVLs, a product-initiated inflammatory response would have been suspected in this woman. However, the evaluation of the early response inflammatory mediators IL-1β and IL-RA demonstrates a marked increase of both cytokines at baseline (), which is significantly higher than the normal range for the sexually abstinent cohorts. These findings reverse the interpretation pattern, suggesting the presence of an inflammatory background condition before product use (most likely asymptomatic as the subject was enrolled in the study). The cytokine data interpretation in the context of the complete clinical analysis may provide further clues as to whether the dynamics of this cytokine profile have been exacerbated by product use.
FIGURE 8 Case interpretation: dynamic of cytokine concentrations in cervicovaginal lavages obtained from a sexually abstinent woman before microbicide product use (visit 2) and after microbicide product use (visits 2 and 3). Bars represent means ± SD of (more ...)