High sensitivity salivary cortisol enzyme immunoassay kit

The procedures used for collecting and assaying cortisol followed established protocols (for description, see^{5 (link)}). Parents collected saliva samples from children twice per day (within 30 minutes of wake-up, and right before bedtime) over a 3-day period. Table 2 shows descriptive statistics of sampling times.
The saliva samples were stored in a freezer at −20°C prior to assay procedures. Samples were assayed using a high-sensitivity salivary cortisol enzyme immunoassay kit (Salimetrics, LLC, State College, Pennsylvania). All samples from a child were assayed in duplicate on the same plate to minimize variability. The intra-assay and interassay coefficients of variation fell below 3.7% and 6.4%, respectively.

The saliva samples were stored in a freezer at the laboratory at −20 °C prior to being assayed. Samples were assayed using the Salimetrics, Inc. High Sensitivity Salivary Cortisol Enzyme Immunoassay Kit (catalog No. 1-1102/1-1112). All samples from an individual for one day were assayed in duplicate on the same assay plate. Pairs of samples were rerun in a latter assay if coefficients of variation were greater than 10%. Inter- and intra-assay coefficients of variation fell below 7% and 14%, respectively.
Of a possible 2,016 child care samples (2 per day over 6 days), 1219 (60%) were included in final analyses. Child care cortisol data were missing because samples were not collected (e.g., due to absence or child being unavailable at the time of sampling) (36%), there was not sufficient saliva collected (2%), or because cortisol values were over 3 standard deviations above the mean and excluded as outliers (2%), following common procedures (Dettling, Gunnar, & Donzella, 1999 (link)). Of a possible 516 home samples (2 per day over 2 days), 371 (72%) were included in final analyses. Home cortisol data were missing either because samples were not collected (12%), there was insufficient saliva collected (12%), or because cortisol values were outliers (3%). As is standard, log 10 transformation was used to normalize the cortisol distributions due to a positive skew.

To assess cortisol response, unstimulated salivary samples were collected from participants in polypropylene vials and stored on ice at seven different time points (see ‘Laboratory Procedures’ below). Saliva samples were divided into 1.8 nunc tubes and frozen at −70°C until assayed. Using a high sensitivity salivary cortisol enzyme immunoassay kit (intra-assay precision of 3.35%–3.65%, lower sensitivity limit of <0.003 μg/dL; Salimetrics, LLC), saliva samples were assay twice. Samples were then analyzed simultaneously with a PowerWave HT Microplate Spectrophotometer and a Precision Series Automated Liquid Handling System (BioTek Instruments, Inc.).

Two baseline samples were collected ~30 minutes after arrival to the study to allow children to adapt to the study location. These samples were averaged to get a baseline cortisol level. A “cognitive stressor” response sample was taken 20 minutes after the end of computerized executive functions tasks that required inhibitory control, working memory and visual spatial memory skills, that preterm children find challenging (Kulseng et al., 2006 (link); Shum et al., 2008 (link); Mulder et al., 2009 (link); Aarnoudse-Moens et al., 2012 (link)). Saliva samples were stored at −20°C until assayed using the Salimetrics High Sensitivity Salivary Cortisol Enzyme Immunoassay Kit for quantitative determination of salivary cortisol (Salimetrics LLC, State College, PA). Cortisol values were examined for outliers, then winsorized (Tukey, 1977 ) and log transformed prior to analysis (Gunnar et al., 2009 (link)). Reactivity salivary cortisol was calculated using the following formula: [log (post-cognitive stressor cortisol) – log(baseline cortisol)]/[log (baseline cortisol)].