Salivette sampling device

Saliva samples were collected with Salivette sampling devices (Salimetrics, Inc., the United States of America) from all adolescents. Samples were collected on three days at home. To capture peak cortisol values in the early morning in response to waking and total cortisol output, saliva samples were collected upon waking, 30 minutes after waking, and at 8 pm. Participants were asked to keep a diary of their waking time, the time of each cortisol sample collection, and any illness symptoms on a given sample collection day. Collection times were verified using a MEMSCap™ device that recorded when each saliva swab was taken from a storage container. Adolescents were instructed to store samples in the freezer before they delivered them to the lab. After their return to the lab, saliva samples were placed in a −80 °C freezer. Experimenters followed standard laboratory safety procedures for handling saliva samples. Salivary cortisol levels were determined by a time-resolved immuno-assay with fluorescence detection (DELFIA). An average coefficient of variation (CV) of duplicate assays of each sample was calculated. Any CV values greater than 12% for cortisol values greater than 5 nmol/L were reanalyzed, as were any values ≥ 100 nmol/L. The average of the two assay values was used for analysis.

Adolescent participants collected saliva samples three times a day (upon waking, 30 minutes after waking, and at 8 pm) over three consecutive days using Salivette sampling devices (Salimetrics, Inc., the United States of America).^{23 (link)} Participants were asked not to eat or drink prior to collecting each sample,^{27 (link)} and to record waking time, each saliva sample collection time, as well as any illness symptoms on a given sampling day in a diary.^{23 (link)} Sample collection times were then verified by an objective time recording device, MEMSCap^™. This device recorded the time when each saliva swab was taken from a storage container.
The saliva samples were stored in the freezer at home until they were delivered to the lab and placed in a −80 °C freezer.^{23 (link)} Experimenters followed standard laboratory safety procedures for handling saliva samples. Saliva samples were assayed in duplicate for cortisol, using a time-resolved immuno-assay with fluorescence detection (DELFIA). An average coefficient of variation (CV) for each sample was calculated. The samples were reanalyzed when CV values were greater than 12% and cortisol values were greater than 5 nmol/L, as well as any cortisol values ≥ 100 nmol/L. The average of the duplicate values was used for the subsequent analyses.