Collecting Saliva and Measuring Salivary Cortisol and Alpha-amylase in Frail Community Residing Older Adults via Family Caregivers

The overall goal of this procedure is to demonstrate the collection and analysis of salivary bio measures in a community residing, medically frail population. This is accomplished by first assembling the necessary saliva collection supplies. The second step is to arrange for the home visit, to provide the supplies and demonstrate the saliva collection procedures to the caregiver.

Next, provide written instructions and answer any questions. The final step is to return the saliva samples to the lab and conduct the assay procedures. Ultimately, the protocol is designed to measure the diurnal profile of salivary alpha amylase and salivary cortisol concentrations in medically frail patients residing in the community.

This method will answer key questions in the fields of biobehavioral research to enhance our understanding of biological sensitivity and susceptibility around stress related processes. Prior to scheduling a home visit to consented, participants gather and assemble the necessary supplies, which includes four psychometrics children’s swabs, four swab storage tubes, four color coded tube caps, five white labels, one plastic Ziploc bag, a saliva collection instruction sheet, five nitro gloves, and a storage box. Using a nitro gloved hand, place four Salome children’s swabs into the four swab storage tubes and cap them with yellow, orange, blue, and purple color caps respectively.

Next place a label on each storage tube to indicate the time of collection. Place the four capped storage tubes, the saliva collection instruction sheet, and four nitro gloves in the Ziploc bag, and label it with the subject ID and date of collection. Following this schedule a short home visit to subjects previously consented and on the day prior to saliva sampling.

After arriving at the subject’s home, provide the plastic bag of supplies and instruction sheet to the saliva collector. Review the instruction sheet and emphasize the need to record the start and stop times of saliva collection by highlighting the written instructions. Record the name, dosage and schedule of all prescription, and over the counter medications taken by the participant within the last 48 hours.

Instruct the saliva collector or SC to apply a nitro glove to the dominant hand. Tell the participant to tilt his or her head forward to allow the saliva to pool on the floor of his or her mouth. Following this, instruct the SC to uncap the swab storage tube and retrieve the oral swab.

Instruct the SC to place the oral swab under the front of the tongue of the older adult to absorb about three drops of the pooled saliva and to hold the oral swab in place for two minutes to ensure that it is saturated, have the SC return the saturated oral swab back into the basket of the swab storage tube, and replace the cap. Next, have the SC immediately place the tube in the storage box and place it in a freezer. After discarding the glove in the trash bin, advise the SC to record the start and stop times on the saliva instruction sheet.

Next, inform the SC that they’ll be collecting saliva samples on four occasions the following day, specifically on awakening 30 minutes post awakening mid-morning and evening, have the SC tell the patient to rinse his or her mouth with water 10 minutes prior to saliva collection. Following saliva collection, discard the samples visibly contaminated with blood. Instruct the SC that the saliva samples should remain in the freezer until retrieved by the study team.

Call the study team to confirm that they will schedule a time and day to retrieve the frozen samples and bring new saliva collection supplies for any future collection date. Once the samples have been removed from the subject’s freezer, keep them frozen during transport to the lab by placing them in an insulated freezer bag. Upon arrival to the lab, replace the white label on each tube with a sequential barcode.

Check that the sequential barcode matches the subject ID date of collection and time of collection. Then return the saliva samples to a minus 20 degrees Celsius freezer to precipitate the mucin. After thawing the saliva samples, centrifuge the samples at 1500 times gravity for 15 minutes.

Once the salivary assay kit reagents are at room temperature for approximately 90 minutes, prepare the wash buffer following the kit instructions pipette 25 microliters of the standards controls and samples into the appropriate wells of a 96 well plate. Following this pipette, 200 microliters of a previously prepared enzyme conjugate solution into the wells. Mix the samples by placing the plate on a rotator for five minutes at 500 RPM.

After incubating the samples at room temperature for 55 minutes, wash and blot the plate with wash buffer four times. Then add 200 microliters of tetraethyl benzine or TMB solution. Once the samples have been mixed for five minutes.

At 500 RPM, cover the plate with aluminum foil and incubate them in the dark for an additional 25 minutes. When finished, add 50 microliters of stop solution and mix the samples for three minutes at 500 RPM. Within 10 minutes of adding the stop solution, wipe off the plate bottom and read the plate with a plate reader at 450 nanometers.

Next, calculate the cortisol concentration using the average optical density. Subtract the average optical density in non-specific binding wells from the average optical density in zero standard control and sample wells. Finally, estimate the concentrations of the controls and unknowns using software capable of logistics.

The procedures described are designed to measure the diurnal profile of salivary, alpha amylase and salivary cortisol concentrations. Since cortisol results have been shown to be positively skewed, it is suggested to use log transformation to the data to yield a normal distribution. Several strategies are available to model differences in diurnal rhythms of cortisol and alpha amylase.

The first strategy is to plot the diurnal change of alpha amylase or cortisol between waking and 30 minutes post waking. The second strategy is to plot total cortisol or alpha amylase concentration over a day. The resulting summary parameters can be grouped into two general categories, the measure of the magnitude of response or the measures of the pattern of response over time.

Growth curve modeling may be useful for studies in which multiple samples are collected in individuals across the day. A growth curve can be fitted to allow for individual differences in the diurnal cycle by adding predictors at the individual or measurement level. After viewing this video, you should have a good understanding of how to collect saliva and how to teach caregivers how to collect saliva from medically for our older adults in the home setting, and you should have a good understanding of how to conduct laboratory analysis of salivary analytes that are feasible, have high compliance and yield quality specimens.