Designing Your Study

The NIDCR recently completed the library for the salivary proteome.  The project identified more than 1,000 anayltes present in oral fluids.  The implications are profound.  This knowledge enables a very wide range of analyses to be conducted now and in the future in salivary specimens.  Plan your project and sample collection, handling, and storage in a way that maximizes your opportunity to take advantage of all that can be learned from this biospecimen.

Consider the frequency, timing, and volume of sample you request from participants.  It’s true that saliva sampling is minimally invasive, but it is easy to add significant participant burden by designing protocols that request participants to donate more sample volume than is really needed, or more often than is really practical, and on schedules that aren’t really appropriate for the flow of their everyday life.

The salivary bioscience literature is spread across a wide range of journals from many different fields.  Gathering this information can be a challenge.   The field is also generating new information at a very rapid pace… faster, than the traditional print publication process can keep up with in many cases.   Its important to reach out to experts in the field to be sure you have the most current information.  Also, studies conducted prior to 1990 were undertaken before the advent of commercially available immunoassay protocols designed specifically for use with saliva and before saliva collection procedures were standardized.  For this reason, the methods of publications that involve salivary bioscience prior to the 1990s should be reviewed carefully.

Many laboratories around the world now enable access to salivary bioscience measurement methods for investigators.  Be certain that the laboratory you choose can provide you with data to support quality assurance and control.  If you are fortunate to have access to a lab in which you or your students can do salivary immunoassays, invest the time to validate the use of the protocols in the hands of those operators and set in place standard operating procedures for equipment maintenance and calibration.

Start your testing off right by choosing an assay that is optimized for the sample matrix in which you are testing. Although convenient to use one assay for multiple sample types, assays designed for a multitude of sample types also plays into the ability to enhance the precision of an immunoassay. Next, make sure your assays are being performed by a highly-trained laboratory technician who follows standardized procedures and the latest industry-accepted protocols. Also realize that while many assay manufactures report the capabilities of offering low CVs, those values often don’t often correlate to real-world results. For research, inter-assay CVs of less than 15% and an intra-assay CVs of less than 10% are generally acceptable. However, with proper saliva handling and experience, Salimetrics assays perform at the lower range of less than 5%. Researchers should carefully consider the maximum %CV acceptable, as variability will reduce data confidence and harm the statistical power of the study when reporting a biological change. Researchers should compare and choose both an assay and lab that is capable of meeting or exceeding your targeted criteria for assay precision.

Recognize and appreciate the degree of unsystematic error that can be introduced by variation in sample collection, handling and storage, as well as in how salivary immunoassays are performed.   In combination with your estimates of “effect size” consider this information carefully when planning your sample size.  Statistical strategies for analyzing salivary bioscience data have advanced in recent years.  Check the literature for updates and contact us for advice.

We recommend all saliva be frozen as soon as possible after collection. Some analytes have been shown to be relatively stable when stored at room temperature, however, sample specific differences have been observed. At a minimum we recommend samples, once collected, be refrigerated at 4°C immediately after collection and transferred to -20°C as soon as possible.

Once frozen in cryovials at –20°C or below, the stability of analytes in saliva is no different than the long term stability of analytes in other traditional biological specimens.  Theoretically, storage at -20°C or below is the best and most economic option for the storage of saliva samples in biorepositories. We do not recommend long term storage, beyond 6 months, of samples collected with swabs unless sample is transferred to cryovials.

We do not recommend the use of cotton for saliva collection due to known interference effects from cotton. We also recommend when collecting saliva samples that you ensure that the swab type used has been validated for the analyte to be tested. Cotton has been shown to interfere with the measurements of several different analytes, and being organic in nature, is prone to variation as well. For more information, you may review the data presented in the Rigor and Reproducibility (Part 2): How Good Are Your Saliva Samples? bulletin. If consistent and true measurements are desired, the best option for swab based collection is using one that is constructed of a synthetic material that has been validated for the analyte to be measured. The only commercially available saliva collection swab that has been validated for the measurement of multiple different analytes is the SalivaBio Oral Swab. Please check to determine if it is compatible for the analytes you wish to measure in your study and note that for some analytes, such as Estradiol, collection by passive drool is the only approved method.

The use of stimulants to increase saliva production is not recommended since it can lead to artificially high or low levels for some analytes. In extreme cases such as a elderly populations, or studies with participants that are unable to generate sufficient saliva due to medication side-effects, it is considered acceptable to use visual aids such as pictures of food to help increase salivation, or the use of olfactory stimulants such as extracts of lemon oil or orange oil inhaled.

Schwartz, E., Granger, D. A., Susman, E. J., Gunnar, M., & Laird, B. (1998).  Assessing salivary cortisol in studies of child development. Child Development, 69, 1503-1513. PMID 9914636

Salimetrics has not validated the use of protease inhibitors with the assay kits. If you determine a need for protease inhibitors in saliva samples, we recommend you validate the use of these inhibitors prior to the study sample collection to ensure that there is no impact on the results of the assay.

Whole saliva contains glycoproteins known as mucins which give saliva its thick and sticky consistency. Using fresh saliva for quantitative estimations leads to inconsistent results due to these mucins. Once frozen, mucins will precipate and fall out of solution. Subsequently, thawed samples, when centrifuged will contain a pellet with the precipated mucins, and other substances such as cell debris and food particles at the bottom, while the supernatant will be a clear, water-like in consistency, and easier to handle. Results obtained with this cleared saliva sample are more accurate and consistent.

The number of tests that can be conducted for each assay is noted in individual kit inserts and on the assay kits specifications page. We recommend running all samples in duplicate and to add sufficient plates to your order to account for repeat testing. Between 5-10% of samples may require repeat testing depending on the lab’s level of experience and sample specific handling issues.

Choosing the right sample collection device is critical to high quality results. There are multiple variables that need to be considered prior to choosing the collection device appropriate to your study design and for your population. A list of collection methods for certain participant groups and validated by analytes is available at Saliva Collection Methods & Devices.  However, we strongly suggest that you use our on-line service or Ask An Expert to get detailed advice.  Factors to consider include analyte to be tested, study participant age, whether samples will be collected in the field, and how much sample is needed from each participant.

Salimetrics QC team monitors kit and collection device lot-to-lot variation and does not approve lots that do not meet these specifications to ensure minimal lot to lot variability.  However, to minimize variables from any source and when feasible, we recommend using the same lot of kits and collection supplies whenever possible.

With modern online search tools and the vast number of salivary bioscience articles published each year, we no longer maintain records of this type.  We suggest Pubmed or Psychlit for searching the scientific literature. We know that the quality of the literature is higher, with respect to assay technology, starting around 1998. Publications before that time, should be reviewed carefully. You can also  “Ask an Expert” if you are having trouble finding an answer. Salimetrics scientists are actively participating in reviewing the scientific literature and have a wealth of information they are happy to share.

We recommend that you (a) use Pubmed and Psychlit to conduct a literature search. We know that the quality of the literature is higher, with respect to assay technology, starting around 1998. Publications before that time, should be reviewed carefully and (b) contact us via “Ask an Expert”. Salimetrics scientists are actively participating in reviewing the scientific literature and have a wealth of information they are happy to share.

If the analyte you are interested in is not mentioned in our website, please “Ask an Expert” to provide you with information on this analyte, we are always interested in developing assays for new salivary analytes.

Internal testing suggests a higher degree of variability between analyte concentrations when sample is collected by passive drool or the SalivaBio swab and compared to Salivettes – both the cortisol specific Salivette (blue top) or the cotton Salivette (white top). We believe that any swab used to collect a saliva sample must have results consistent with passive drool, the gold standard of the saliva collection, and our data indicates that Salivette derived data is significantly different from that obtained using the SalivaBio Swabs. For more information, you may review the data presented in the Rigor and Reproducibility (Part 2): How Good Are Your Saliva Samples? bulletin.

To ensure precision and consistency in saliva based research, we recommend the use of SalivaBio Swabs for saliva collection when swab collection is required.

Disposal of saliva samples is Institute specific. Please check with the EHS department at your organization to determine the regulations associated with saliva disposal. Saliva is considered a Class II biohazard and disposal will be consistent with policy determining Biohazardous Waste at your organization.

A single saliva sample can be used for the measurement of multiple analytes, and can also be a source of DNA for SNP analysis. The number of analytes tested depends on the type of analyte to be tested. Please call us for sample volume requirements specific to your study.

Please call us to speak with an expert to help guide you with your study design and provide you with relevant references specific for the analytes to be tested. With most hormones, the time of collection can play a critical part in the level detected; and the time of collection should be standardized for all study participants.  Most steroid hormone levels are higher in the am.  These papers present a very good overview.

Granger, D. A., et al. (2012).  Salivary Bioscience and research on adolescence: A integrated perspective.  Journal of Adolescence. 32, 1081-1095 PMID 22401843

Skoluda, N., et al. (2015) Intra-individual psychological and physiological responses to acute laboratory stressors of different intensity. Psychoneuroendocrinology. 51:227-36. PMID 25462896

Please refer to the following articles for medication and its impact on cortisol levels:

Granger DA, Hibel LC, Fortunato CK, Kapelewski CH. (2009) Medication effects on salivary cortisol: tactics and strategy to minimize impact in behavioral and developmental science. Psychoneuroendocrinology. 34(10):1437-48. PMID: 19632788

Hibel LC, Granger DA, Kivlighan KT, Blair C. (2006) Individual differences in salivary cortisol: associations with common over-the-counter and prescription medication status in infants and their mothers. Hormones and Behavior. 50(2):293-300 PMID: 16682032

In addition, follow the recommendations in the Salimetrics Saliva Collection Handbook for additional advice

Although salivary alpha amylase (sAA) can sometimes be detected at birth the linkage between the ANS and salivary gland is not mature, thus sAA may not reflect the activity of the ANS in newborns.  Salivary alpha amylase can be detected in the saliva of infants as young as 3 months old.  Over the first several months the amount of sAA produced in infants increases until it reaches the levels seen in adults at  the toddler period.

Davis, E. P., Granger, D. A. (2009) Developmental differences in infant salivary alpha-amylase and cortisol responses to stress. Psychoneuroendocrinology. 34(6): 795–804. PMCID: PMC2693709

Salimetrics believes that the best information about cortisol timing are based on studies of the Trier Social Stress Test (TSST) although various other lab based stressors have also been used successfully in the literature. Many studies have been published and that information is very widely available. The reference below is useful for timing of saliva samples after TSST and other stressors.

Skoluda, N. et al. (2015) Intra-individual psychological and physiological responses to acute laboratory stressors of different intensity. Psychoneuroendocrinology. 51:227-36. PMID: 25462896