Handling Multiple Salivary Analytes and Order of Assay

Many bio-behavioral studies now include measurements of multiple salivary biomarkers. Some analytes such as cortisol and alpha-amylase are fairly stable at room temperature, and they present few problems for handling in the laboratory. The sex steroids (estrogens, progesterone, and testosterone) and their precursors (androstenedione, DHEA, DHEA-S, and 17-OH progesterone) are more easily degraded, however, and they need more care in handling. When multiple analytes are to be measured in a study, the samples needs to be handled according to the requirements of the least stable analyte. Since saliva samples are often archived for future assays of undetermined analytes, it is generally a good idea to take a conservative approach to sample handling. We consequently recommend that all samples should be kept cold and frozen as soon as possible after collection. Ordinary -20°C freezers are acceptable for the initial freezing, and ultra-low temperature freezers (-60°C) are recommended for longer-term storage. Sodium azide should not be added to saliva samples to prevent bacterial growth and prevent analyte degradation, since it may cause interference in immunoassays.

Proper handling and storage conditions for salivary biomarkers can vary significantly. Analytes such as estradiol, progesterone, and DHEA are especially sensitive to degradation from storage at room temperature or from repeated freeze/thaw cycles. Testosterone is less fragile, but not as stable as cortisol and alpha-amylase. CRP will experience significant degradation from storage at 2-8°C, but it is minimally affected by freeze/thaw damage. It is therefore important to research analyte stability before beginning to collect samples.

If susceptible analytes are to be included in a study, we recommend the following strategies:

  • Refreezing of samples should be minimized as much as possible. Aliquotting large samples into smaller volumes is advisable.
  • If practical, testing for multiple analytes should be completed on the same day.
  • Assay the most fragile biomarkers first, saving the more stable ones for later. In the Salimetrics Laboratory, we run multiple analyte assays in the following order: estrogens (estrone, estradiol, estriol), progesterone, DHEA, DHEA-S, CRP, testosterone, cotinine, cortisol, alpha-amylase.

One final point: if multiple analytes are to be measured, be sure to collect enough saliva volume. Collection by the passive drool technique into a vial allows the sample volume to be judged visually, but not all subjects are able or willing to donate passive drool. If an absorbent swab such as the Salimetrics SOS, SCS, or SIS is used for collection, the sample can be recovered from the swab immediately afterwards by using a needleless 5 cc syringe to express the sample from the swab into a vial. If too little sample was collected on the first attempt, the procedure can then be repeated as necessary. Taking the time to monitor the saliva collection process in this way will help researchers avoid missing data due to insufficient sample volumes.

March 2012