About Salimetrics

Salimetrics leads the industry in supporting thousands of salivary bioscience researchers around the world with the most accurate, highest-quality, salivary assays, saliva collection devices, and saliva testing services.  Dedicated to the field of salivary bioscience, Salimetrics assays have been used in more saliva-related published papers than any other salivary assay in the field. Salimetrics is headquartered in the United States with locations in State College, Pennsylvania and Carlsbad, California, and also supports a global distribution network.

About SalivaBio

At SalivaBio, we recognize the critical role saliva collection plays in providing consistent, accurate results. SalivaBio is dedicated to the creation of a global standard for collection devices and techniques, as well as the development of new, cutting edge saliva collection methods to increase ease of use and participant compliance. Exclusively from Salimetrics, all SalivaBio products are extensively tested to minimize or eliminate variability from saliva collection and have been optimized for collection with participant-specific groups. Not all saliva collection devices are the same, for example cotton swabs, can interact with analytes in the sample and/or negatively influence assay performance. SalivaBio collection devices have been tested on all Salimetrics assay kits and we have concrete recommendations for which device is compatible for testing the analytes in your study.

What’s In This Guide?

1. Introduction
2. What to Consider Before Collecting Saliva
   • Variability of Saliva Composition
   • Effects of Flow Rate and Mouth Location on Salivary Analytes
   • Saliva Sample Volume and Stimulation
   • Combining DNA Analysis and Salivary Analytes
   • Blood in Saliva
   • Maintaining Analyte Integrity
3. Before Saliva Collection
4. How to Collect Saliva: Collection Methods and Devices
5. Saliva Collection Methods: Adults
   • Passive Drool
   • SalivaBio Oral Swabs
6. Saliva Collection Methods: Infants and Small Children
   • SalivaBio Children’s and Infant’s Swabs
7. Saliva Collection Methods: Animals & Other Non-Human Species
8. How to Collect Saliva for DNA Analysis
   • Collection Methods and Sample Volume
   • Stability of DNA Over Time
   • Storing Saliva for DNA Analysis
   • Avoiding Contamination of Samples
9. After Saliva Collection
10. Long Term Sample Storage
11. How to Prepare Saliva Samples for Testing
12. References

Introduction

Saliva samples can be collected in a convenient, minimally-invasive, and repeated manner. Saliva is a complex, heterogenous biospecimen which can lead to data errors if not collected properly. However, by following proper saliva collection and handling procedures high quality, reproducible data can be obtained from this sample type. To assist investigators in maximizing the integrity of their saliva samples, we offer the following advice.

1. Knowledge about saliva testing is rapidly growing and being revised. The technical team at Salimetrics maintains the latest information and provides Ask an Expert support as part of our mission to support Salivary Bioscience.
2. We advise consulting the literature on the analytes to be measured, and when the available literature appears inadequate, we strongly recommend a pilot study. It is ultimately each investigator’s responsibility to make decisions about the best methods to use for their specific study.
3. For an additional discussion and current updates, we recommend signing up for The Salivary Bioscience Bulletin.

What to Consider Before Collecting Saliva

Variability of Saliva Composition

Levels of many analytes in saliva do not remain static, and concentrations may change in response to a number of influences (1-5). Samples can also vary substantially between individuals. Depending on the analyte of interest and the nature of your study, several factors may be of importance:

• Collection Time – Most hormones display a diurnal rhythm of expression, and this can be true for certain salivary proteins as well. Cortisol based research is especially impacted by the rhythm of cortisol expression through the course of the day – night cycle, and studies specific to diurnal expression of cortisol are central to study design. We suggest looking at the latest recommendations in the literature or reaching out to Ask an Expert for specific recommendations based on the analytes to be tested. For most research, sample collection should be made at specific times or within a window of time for the most meaningful measurements.

• Response and Recovery Characteristics – Sample collections should be timed to properly capture responses if a study involves interventions and stressors. This is true for longer interval studies spread over months, or lab-based stressor studies measuring stress responses.

Effects of Mouth Location (swab method only)

Absorbent devices may collect localized saliva rather than whole saliva, which may affect results for some analytes. Salivary levels of proteins such as alpha-Amylase and Secretory IgA do vary according to mouth location (6-8). We recommend placing absorbent devices in the location where saliva is pooling. For an adult or child sitting upright, the floor of the mouth (under the tongue) is the ideal location. For infants, adults, and children in a reclined position, we recommend turning the head to the side and collecting from the side of the cheek where saliva is “pooling”. When collecting saliva by passive drool, we recommend allowing saliva to pool under the tongue and gently propelling the collected drool into the tube through the straw-like collection aid. This sample will also be representative of saliva collected from all the salivary glands and eliminate any bias of collecting saliva from specific salivary glands.

Effects of Flow Rate

Levels of a few hydrophilic analytes, such as DHEA-S and/or larger proteins, such as SIgA, decrease as saliva flow rates increase, and salivary α-Amylase may also be similarly affected (9, 10). We currently advise that researchers covary the secretion rate (mL/min) for analytes such as α-Amylase, SIgA, and DHEA-S.

Record the total time necessary to collect the desired volume of saliva, so that assay results can be multiplied by the flow rate (mL/min) in order to express the results as a secretion rate (output per unit of time).

Example (SIgA): 205.60 µg/mL x 1.33 mL/min = 273.45 µg/min

To determine flow-rate if an absorbent device is used to collect saliva, weigh the swab and storage tube together before and after collection. (An approximate value of 1.0 g/mL may be assumed for the density of the saliva.) Also record the length of time the swab is in the mouth, so that flow rate can be estimated.

Sample Volume and Salivary Stimulants

Modern immunoassays are designed to use small sample volumes (less than 100 µL), and in most cases, stimulants are not required to collect adequate sample volume. We recommend against the use of oral stimulants when collecting saliva samples due to the possibility of causing assay interference or alteration of levels of some analytes (11); the goal should be to minimize unnecessary sources of variation in saliva test results. For example, even chewing on unflavored paraffin/wax could affect flow-dependent analytes.

If stimulants are absolutely necessary (no saliva can be collected without their use);

• Use sparingly and in a consistent manner throughout the study (12).
• Consider using olfactory or visual stimulation before an oral stimulant is used.
• Try practicing with study participants before actual collection to ensure comfort with the methodology prior to collection for the actual study.
• Conduct a pilot study to ensure that the oral stimulant does not cause assay or analyte interference (11).

Prior to sample collection, view analyte collection protocols or Ask an Expert concerning the sample volume requirement based on the number and type of assays to be performed.

Combining Salivary Analyte and DNA Analysis

If you plan to include DNA analysis in your current study, are archiving for DNA analysis at some future date, or would like to perform DNA analysis on existing samples that have been previously archived, please see “How to Collect Saliva for DNA Analysis”

Blood Contamination in Saliva

Levels of many analytes are higher in the general circulation than in saliva and blood contamination can be a concern under specific conditions (21-24). We recommend the following:

We recommend the following:

• Participants should not brush their teeth within 45 minutes prior to sample collection (23).
• Dental work should not be performed within 24 hours prior to sample collection.
• Research participants should be screened for oral health problems or injuries.
• Saliva samples visibly contaminated with blood should be discarded and recollected (22).
• Samples collected from populations at high risk for oral health problems, may be screened with the Salimetrics Blood Contamination Assay Kit (Salimetrics Item 1-1302; 1-1302-5).

Saliva Collection Device Considerations

It is critical that the collection protocol used is compatible with the analyte to be tested. Some analytes can only be tested with samples collected by passive drool. Using a swab to measure these analytes can result in over- or under-recovery of the analyte being measured.

Not all swabs are equal either. We recommend using a swab that has been validated for the measurement of the analyte of interest. Some manufacturers have swabs that are specific to cortisol and have only been validated for the measurement of cortisol. Using this swab to measure another hormone, such as testosterone, can lead to erroneous results. Use of cotton as a swab material has already been demonstrated in the literature as a source of variable results and is not recommended if consistent results, not confounded by a biological material, are desired. Salimetrics has confirmed that data from cotton collection devices often matches with the well-cited Shirtcliff, EA et al paper (2001). However, cotton-based methods also increase variability and provide data results that significantly differ from “true passive drool” values, as illustrated below;

Maintaining Analyte Integrity

It is critical to research and follow basic analyte stability and storage recommendations prior to sample collection. Use only high-quality POLYPROPYLENE collection tubes and vials to store samples. Polystyrene or other non-validated plastic tubes can adversely affect the measured values of analytes. All SalivaBio collection tubes and vials are made from high quality polypropylene.

Salimetrics recommends that all samples be frozen immediately after collection. However, if this is not possible, researchers should consider that;

• Some unstable analytes can change rapidly at room temperature (i.e. peptides and proteins)
• Freeze-thaw cycles should be minimized for some analytes such as (DHEA, Progesterone, Estradiol)

Before Saliva Collection

Good saliva collection requires documenting items which may affect results, as well as following procedures which avoid the possibility of contaminating saliva with substances that could interfere with the immunoassay, such as;

1. Avoiding foods with high sugar or acidity, or high caffeine content, immediately before sample collection, since they may compromise the assay by lowering saliva pH and increasing bacterial growth (13, 14).
2. Documenting consumption of alcohol, caffeine, nicotine, and prescription/over-the-counter medications within the prior 12 hours (15-18).
3. Documenting vigorous physical activity and the presence of oral diseases or injury (19, 20).
4. Not eating a major meal within 60 minutes of sample collection.
5. Rinsing mouth with water to remove food residue and waiting at least 10 minutes after rinsing to avoid sample dilution before collecting saliva.

How to Collect Saliva: Collection Methods and Devices

Passive Drool Method: Most researchers prefer to use unstimulated, whole saliva collected by the passive drool technique in order to maintain consistency in the type of sample collected.

Absorbent Device Method: Swabs can be placed in the mouth to collect saliva for increased ease of use. Studies with small children or other individuals that have difficulty with the passive drool technique also may require participant-specific swab-based collection methods. However, swabs are limited to certain analytes. All SalivaBio swabs are made from a similar non-toxic, inert polymer which is guaranteed for consistency across all lots, making it ideal for longitudinal and multi-participant group studies.

Consider the following criteria before selecting an appropriate saliva collection method:

• Participant age & species
• Validated for analytes of interest
• Number of samples required
• Sample volume required
• Single vs. multi-analyte and/or DNA analysis
• Self vs. assisted collection
• Biobanking vs. discarding samples

COLLECTION METHODS AND DEVICES:
ADULTS & CHILDREN 6 + YEARS OF AGE

Passive Drool

Passive drool is highly recommended because it is both cost effective and approved for use with almost all analytes and maintains sample integrity (see Passive Drool instructions). If research participants are not willing or able to drool saliva into a vial, the SalivaBio Oral Swab may be used as an alternative collection method, but only for certain analytes.

To avoid problems with analyte retention or the introduction of contaminants, use validated polypropylene vials for collection, such as SalivaBio 2 ml cryovials, Salimetrics Item No. 5004.01.  Vials must seal tightly, be able to withstand temperatures down to -80ºC, and must be externally threaded to allow for use of the Saliva Collection Aid (SCA – Salimetrics Item No. 5016.02) to effectively guide drool directly into the cryovial.

Materials Needed:

1. Passive Drool Method
2. Bar-Coded Labels
3. Cryostorage box (2″)

The SalivaBio Oral Swab (SOS)

For certain analytes (see SOS instructions), the SalivaBio Oral Swab (SOS) (Item No. 5001.02) can be an excellent alternative to passive drool because of its ease of use.  The SOS also helps filter large macro molecules and other particulate matter from the sample, which may help improve assay results.

Materials Needed:

1. SalivaBio Oral Swab Method
2. Bar-Coded Labels
3. Cryostorage box (4″)

Child Saliva Collection Methods and Devices

Due to the potential for choking when collection devices are placed in the mouth, collecting saliva from infants and children under the age of six requires special considerations.

The Children’s Swab and Infant’s Swab – For Children & Infants

We recommend the SalivaBio Children’s Swab (SCS) (Item No. 5001.06), for children under the age of 6, and the SalivaBio Infant’s Swab (SIS) (Item No. 5001.08), for infants under 6 months of age.

The SCS and SIS devices are manufactured in longer lengths and narrower widths to allow one end of the swab to be held by a parent or technician while the other end is placed in the child’s mouth.  The diameters are appropriate for the size of children’s mouths.  The polymer material is durable and can withstand chewing, and its taste and texture are also acceptable to children.  The volume of sample recovered from the SCS and SIS is typically in the range of 200-1000 µL.  Like the adult SOS, samples collected with either the SCS or the SIS may be tested for various analytes (see SCS instructions, see SIS instructions). All SalivaBio swabs are made from a similar, non-toxic, inert polymer which is guaranteed for consistency across all lots, making it ideal for longitudinal and multi-participant group studies.

Materials Needed:

1. SalivaBio Children’s Swab Method or SalivaBio Infant’s Swab Method
2. Bar-Coded Labels
3. Cryostorage box (4″)

Collecting Saliva for DNA Analysis

Collection Methods and Sample Volume

SalivaBio Oral Swab samples are preferred for salivary DNA analysis if Salimetrics is to perform DNA extraction. However, passive drool is also acceptable, and may be preferred if DNA extraction is to be performed by the researcher if high yields are critical for studies such as microarray genomic analysis. The Salimetrics’ in-house proprietary method for DNA extraction from swabs provides a yield of 2-5 µg/mL of high purity DNA, with a A260:A280 ratio of 1.8 or higher.

DNA can also be obtained from samples that have already been collected for other analyte/biomarker analysis and does not normally require collection of new samples specifically for DNA. For salivary DNA testing, the pellet formed by centrifugation of samples contains cells that provide DNA.  Do not discard the pellet or swab if genetic testing is desired.  We do, however, recommend that the sample be frozen immediately and the pellet or the swab not be subjected to multiple freeze thaws to minimize degradation of DNA.

Additional collection devices such as buccal swabs may be acceptable; Ask an Expert for the latest advice.  When collecting buccal cells, be sure to rub the inside of cheeks for 30-60 seconds with firm pressure.

Stability of Salivary DNA

The DNA sequence of every individual is constant throughout life.  Samples can be pulled from different “waves” of your project and submitted for DNA testing.

Storing Salivary DNA Samples

Freeze saliva samples as soon as possible. However, DNA can tolerate storage at room temperature for up to 5 days without compromising the quality of the DNA for some genetic testing (38). DNA also withstands multiple freeze-thaw cycles without significantly effecting DNA quality.

If samples are to be tested for analytes in addition to DNA analysis, only use Salimetrics-validated collection methods and devices. Do not use a device which dilutes the saliva by an unknown amount or has the potential to interfere with the assays. In addition, follow storage instructions for the more sensitive analyte (i.e., the hormone or biomarker) of interest first.

If room-temperature storage is necessary, the Oragene•DNA® self-collection kit (OG-500) may be used.  Detailed collection and mailing instructions can be found in the Oragene•DNA kit package or at dnagenotek.com.  For young children or people who are unable to drool freely, use the Oragene•DNA assisted collection format (OG-575) to collect whole saliva.

® Oragene is a registered trademark of DNA Genotek Inc.

Avoiding Contamination of Samples

To prevent contamination of saliva samples for DNA analysis, we recommend the following in addition to our recommendations for immunoassay testing collection:

1. Employ only single-use materials (disposable forceps, etc.) for sample collection/transfer to prevent possible contamination between research subjects.
2. Those researchers assisting with collections should wear gloves and avoid touching collection device materials and samples.
3. Do NOT touch the swab or brushes with your fingers.

After Saliva Collection

Freeze samples at or below -20ºC (temperature of a household freezer) immediately after collection.

SalivaBio Swab Collection: Samples can be frozen in the swab for up to 6 months with no decline in levels. However, if it is known ahead of time that samples may need to be stored for longer than 6 months, we recommend expressing the saliva out of the swab either by centrifugation or squeezing through a syringe immediately after collection, and storing the expressed saliva in cryovials, ideally at -80 C.

Long Term Sample Storage

Samples can be stored at -80ºC for several years; the exact time has not yet been determined and may vary by analyte.  However, many samples that have been stored properly for over four years have shown little or no degradation.  We recommend you consult the literature or Ask an Expert for details.

Prior to Sample Testing

On the day samples are to be assayed, bring samples to room temperature, vortex, and then centrifuge at 1500 x g for 15 minutes.  If the samples appear viscous, centrifuge at a higher speed such as 2500 x g or break up the clot with a pipette tip and re-centrifuge.  Assays should be performed using only clear saliva, avoiding the pellet formed at the bottom of the tube. When pipetting viscous solutions such as saliva, greater accuracy is obtained by aspirating slowly to avoid the formation of bubbles.  Vortex and re-centrifuge tubes following each freeze-thaw cycle since additional precipitates may develop upon refreezing.