Salimetrics

Advancing discovery through salivary assay
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All Things Saliva

03.01.08

Saliva Collection Advice(1)

Saliva is an attractive testing medium because it allows samples to be gathered easily, with relatively little stress, and on a repeated basis. Investigators who intend to use saliva testing need to understand, however, that the methods used to collect and handle saliva samples can have a direct effect on their testing results. The purpose of this bulletin is to offer advice based on our experience with saliva collection and testing, which we hope will help customers gather the highest quality data.

Recommendations for reducing viscosity and particulate matter in saliva.

Whole saliva contains glycoproteins know as mucins, which form mucus when dissolved. This thick and sticky substance can cause problems in transferring small volumes by pipette. Saliva can also contain various other substances (cell debris, food particles, bacteria, etc.). Unless these unwanted materials are removed they can cause problems with the assay procedure.

Salimetrics advises freezing all saliva samples once before performing the assay, followed by vortexing after the sample is thawed. This procedure helps break up the mucus, and it can then be centrifuged into the bottom of the tube. Any other particles that were present are also removed in this step. For the majority of samples this will leave a clear solution that can be used in the assay.

Selection of an appropriate collection device.

Because steroid hormones are non-polar molecules, they have a tendency to be attracted to some types of plastic. Tubes or vials used to collect saliva must be made of a material that will not cause the hormones to stick, since this would cause inaccurate test results. It is also important that the material not release any contaminants that might cause interference in the immunoassay.

All collection tubes and vials sold by Salimetrics are made of high-grade polypropylene, and they are appropriate for the collection of steroid hormones.

Absorbent devices placed in the mouth to collect saliva samples must also not cause any problems with retention of the analyte, or with the immunoassay process. Cotton has been widely used as a collection material, either in the form of braided ropes, simple buds mounted on a shaft, or in cylindrical rolls. Studies have shown, however, that its use is sometimes problematic, for two reasons:

  • When the amount of saliva available for collection is small, cotton may not release enough volume to perform the assay. In the case of cortisol, it also appears that the percent of the compound recovered can be associated with the initial volume available to be absorbed. (2)
  • Cotton causes interference with immunoassays for a number of substances. Erroneously high values have been reported for testosterone, DHEA, progesterone, 17-hydroxyprogesterone, estradiol, and androstenedione; low values have been reported for SIgA. (3,4,5)

 

Salimetrics advises that cotton collection devices should be used only in situations where ample collection volume will be present, and only for cortisol, cotinine, and a-amylase. Furthermore, due to unacceptably large lot-to-lot variations that have been measured for the cotton Salivette® device, Salimetrics no longer approves its use with our kits.(6)

In order to meet the need for an absorbent collection device that is easy to use, and that yields good volume recovery even with small samples, we have introduced the Salimetrics Oral Swab (SOS). It is made of an inert, non-toxic polymer, shaped into a 1 x 4 cm roll. The device is manufactured to rigorous quality control standards to insure that test results will be consistent. We have thus far approved it for use with a-amylase, cortisol, cotinine, c-reactive protein, SIgA, and testosterone, and further tests are underway. It is appropriate for use by adults and children age 6 and older.

Proper handling and storage of saliva can improve assay results.

Bacteria are present in saliva, and unless steps are taken to slow or stop their action, they can cause the degradation of analytes in varying degrees. (7) For example, cortisol is relatively less affected, and it can go for 4 days at room temperature without significant change. Testosterone is more easily affected, and it shows degradation over 4 days even under refrigeration (4°C), when compared to freezing.

We advocate a simple, conservative approach to sample handling and advise that all samples should be refrigerated within 30 minutes, and frozen within 4 hours. (If collection is being carried out in the field, it may not be practical to freeze the samples until the end of the day, but samples should be kept cold until they are returned to the lab). It is important, however, to be aware of the fact that hormones and biomarkers found in saliva have differing levels of stability to the freeze-thaw process. Some, such as cortisol and a-amylase, have good stability through three freeze-thaw cycles. Others, such as DHEA, estradiol, and progesterone have poor stability, and freeze-thaw cycles should be minimized. However, for estradiol, progesterone, and CRP it is better to refreeze than to leave at 4°C for 24 hours. Researchers who archive saliva samples for future use, and who anticipate testing for any of the compounds sensitive to freeze-thawing, should divide their samples into smaller aliquots before freezing.

Sodium azide should not be added to saliva samples as a preservative, as was done in the past when radio immunoassays were more common, because it inhibits the action of the horseradish peroxidase commonly used in enzyme immunoassay methods.(8)

Commercially available absorbent devices are not appropriate for all analytes.

Some steroid hormones cannot be accurately measured if the saliva is collected with either the SOS or cotton swabs. Another factor that limits the use of absorbent devices is that the sample volume collected may not be adequate for some types of studies, especially when more than one analyte is being assayed.

In the case of analytes not approved for collection with the SOS or cotton materials, and if subjects are six years of age or greater, Salimetrics advises the collection of whole, unstimulated saliva by the following method: donors should tilt their heads forward and allow the saliva to pool on the floor of their mouth. The saliva is then passed through a short plastic straw into a cryogenic storage vial.

The advantages of this method are:

  • It is safe to use with almost all analytes.
  • It generates a relatively large sample (1-5 mL) within a few minutes, which can be used to test for numerous compounds, or frozen for future assays.

The disadvantages of the method are:

  • It requires a conscious subject who is willing and able to follow the instructions.
  • It does not work well for small children.

Alternative saliva collection devices may be helpful for children, or with the extreme elderly.

Infants under the age of 3 months often have only a small volume of saliva available, and a number of factors, including irregular sleep patterns, crankiness, or fear of strangers, can make it difficult to collect adequate samples from older infants (3-18 months).(5) Also, any collection device used must not present a choking or swallowing hazard.

At the other end of the spectrum are the frail elderly, who frequently take numerous medications, many of which can lead to dry mouth syndrome (xerostomia). Attempts to collect adequate samples from these subjects can also be problematic.(9)

Cotton in the form of braided ropes has frequently been used for collection with children, since a parent or technician can hold one end while the child sucks on the other end. The wet portion of the rope can then be cut off, and the saliva recovered by compression in a needle-less syringe, or by centrifugation. Unfortunately, the taste of the cotton may be unappealing to children, making it hard to secure their cooperation. The restrictions mentioned earlier for cotton devices apply to the rope as well, and we therefore advise limiting its use to assays for cortisol, cotinine, and a-amylase. If the volume of saliva available to be collected is small, the cotton may not release enough sample to perform the assay. If the percent volume recovery is below 95% the cortisol recovery may be lower than expected.(2)

Because of the need to be able to collect usable samples when the volume of saliva is likely to be small, attempts have been made to find additional absorbent materials that can be used for collection. Researchers have reported the successful use of hydrocellulose microsponges (Sorbettes) for the collection of saliva. (2,10,11)

These devices, which consist of small, arrowhead-shaped sponges attached to a shaft, are particularly useful for collecting samples from children due to their small size, and the presence of the shaft, which can be held so that the device does not pose a choking hazard. (Still, parents or researchers using the device should not allow children to chew on the sponge, since it could potentially come off.) In our experience, children are more tolerant of this type of collection device. The Sorbette should be left in the mouth for a full minute, and not removed when it starts to puff up. Under ideal circumstances, this will allow the sponge to absorb 200-300 µL of saliva. If more sample is desired two sponges may be used simultaneously. After collection, saliva is recovered from the device by centrifugation. (More detailed collection instructions are available on request.) When sample volumes are below 100 µL the volume recovery from the microsponge is much better than from the cotton swab.(5)

Unfortunately, the microsponge is not suitable for all analytes. Salimetrics has approved it only for cortisol, cotinine, a-amylase, and SIgA.

Another collection method described in a recent publication involves the use of strips of filter paper to absorb saliva from preterm infants. (12) The investigators report that the infants readily sucked on the paper strips, and that the time required for collection was shorter than with other methods. Another benefit of this method is that saliva samples collected on paper can be dried and sent through the regular mail without a need for refrigeration. Dried samples and controls were extracted in a controlled fashion and assayed for cortisol, and the results showed acceptable recovery. This collection method therefore appears to have promise, and it seems worthy of further validation. In particular, the applicability of the procedure to analytes other than cortisol needs to be determined.

Controlling for blood contamination in saliva can improve test results.

Small injuries in the mouth, dental work, or even vigorous tooth brushing can sometimes cause blood to appear in the saliva. Because the levels of hormones found in blood are much higher than in saliva, even the presence of minute quantities of blood in saliva can give falsely high test results.(13) The degree of contamination necessary to affect the assay, and the length of time the effect lasts, vary according to the particular hormone being measured. (14,15,16)

Subjects should be interviewed to inquire about injuries in the mouth, or poor oral health. Dental surgery should not be performed within 48 hours of collection, and subjects should be instructed not to brush their teeth within 45 minutes prior to collection. Any samples that show a visible reddish, yellowish, or brownish tinge from blood contamination should be excluded and recollected.

Visual examination alone is not always a reliable means of identifying blood contamination, however. A similar discoloration can also be present from tobacco use, or even from coffee. The use of dipstick methods (Hemastix) to test for the presence of blood is not reliable in saliva either, due to the presence of peroxidases that can give false positive results.(16) It has also been shown that blood contamination can be present even when visible color is absent. (15)

In order to be able to able to check accurately for the presence of blood in saliva samples, Salimetrics has developed an immunoassay method to screen for blood contamination.(16) This test checks for the presence of transferrin, a large protein found in high levels in blood, but not normally in saliva. If detected by the screening kit, it suggests that the sample should be discarded. Use of the kit may be especially appropriate for populations who have little or no dental care, or who have known oral health problems (smokers, HIV infection).

Controlling for food and drink in subjects can improve assay results.

Breast milk or formula can contain various hormones, or hormone-like substances, which can cause interference in immunoassays.(17) Caffeine can affect a-amylase levels in saliva.(18) Alcohol stimulates saliva flow,(19) and when present in saliva may interfere with antibody binding in immunoassays.(20) Food or drink high in sugar content or acidity can also stimulate the flow of saliva,(21) and the acidity can cause a drop in the pH of the mouth. Changes in saliva flow are potentially a concern because they can affect concentrations of some substances in saliva, such as SIgA.(22) Lowering the pH in the mouth is also undesirable because it can compromise antibody-antigen binding and enzyme activity, leading to invalid immunoassay results. (23)

Salimetrics recommends the following instructions for subjects donating saliva:

  • Avoid alcohol for 24 hours before sample collection
  • Do not eat a major meal within 60 minutes of sample collection
  • Avoid dairy products for 20 minutes before sample collection
  • Avoid foods with high sugar or acidity, or high caffeine content, immediately before sample collection
  • Rinse mouth with water to remove food residue before sample collection, and swallow to increase hydration
  • Wait at least 10 minutes after rinsing before collecting saliva to avoid sample dilution.

 

Because of the undesirable effects noted above for acidic substances, we also advise against the use of drink mix crystals, or ascorbic acid, to increase saliva flow for collection purposes. If saliva stimulants are absolutely necessary, they must used sparingly, and in a consistent manner throughout the study.(24)

Salimetrics’ assay kits are designed to avoid pH problems by being properly buffered, and they also include a pH indicator to identify any samples that are outside of the acceptable range.

Concentrations of certain analytes can be related to the location in the mouth where the saliva is collected.

The saliva glands in the mouth are made up principally of two different kinds of secretory cells, which secrete different components of saliva. The various saliva glands located in the mouth each have different ratios of the secretory cells, and consequently the saliva produced by each can be made up of a different blend of the components. (See the bulletin on Saliva and Its Diagnostic Uses in this series.) This is not a concern for the steroid hormones measured with Salimetics’ kits, but certain other analytes may be affected. In particular, a-amylase concentrations are usually higher in saliva collected from the parotid glands in the cheeks than in the whole saliva from under the tongue,(25) and SIgA concentrations in labial saliva are much higher than in parotid saliva.(26)

For most analytes Salimetrics advises the collection of the whole saliva that pools on the floor of the mouth under the tongue. The same is true if a-amylase is measured in combination with other analytes. However, if a-amylase is being measured by itself we recommend placing an SOS collection device between the gum and cheek next to the second upper molar, where the duct from the parotid gland is located. Studies that investigate other differences in saliva function and composition may also require the collection of distinct samples from separate glands.

The flow and composition of saliva changes with stimulation.

Saliva includes many components produced directly in the saliva glands, including glycoproteins known as mucins, and enzymes like a-amylase. Nervous stimulation of saliva glands results in increases in both saliva flow and concentrations of these locally produced components.(27,28,29) In the case of a-amylase, at least one study suggests that increases in the enzyme concentrations are independent of changes in the flow rate. (30)

Some compounds of interest in saliva, such as steroid hormones, originate in blood, and enter saliva mainly by diffusion or filtration.(31) Active transport through cells is also possible for some substances, such as secretory immunoglobulin A (SIgA).(32) As saliva production is stimulated and flow rates increase some of these substances produced outside of the glands may not be able to travel into saliva fast enough to maintain constant concentrations.(33,34,35) Fortunately, this is not a problem for most of the steroid hormones, whose concentrations in saliva have been shown to be independent of flow rate.(31) It can be a concern for other substances, however, including conjugated hormones such as dehydroepiandrosterone sulfate (DHEA-S), SIgA, and peptides (Neuropeptide Y and Vasoactive Intestinal Peptide).(31,36)

In order to maintain a consistent basis for comparison, we recommend the collection of unstimulated saliva whenever possible. Investigators need to know if the concentrations of substances they intend to measure in saliva are affected by saliva flow rates. For substances that are affected, such as SIgA, we advise measuring the length of time needed to collect a specified volume of saliva, in order to express the results as a function of time (e.g., µg/minute).

Levels of salivary hormones and other biomarkers can fluctuate over time.

Levels of some analytes fluctuate markedly during the 24 hour period according to a regular cycle, known as the diurnal rhythm. Some analytes are released periodically in short bursts, a pattern known as pulsatile release.(37) In addition, the timing of patterns of response to stress followed by recovery can be significantly different for certain analytes (e.g. cortisol and a-amylase).(1) Samples collected in a non-standardized fashion and without regard for the characteristic secretion patterns of the analytes involved may produce data that is difficult to interpret.

Researchers must have a thorough understanding of the underlying secretory pattern for each analyte to be measured, and of any differences in response to stress and recovery. The timing of sample collection should then be planned accordingly. For example, in the case of analytes that have pulsatile release, we recommend taking a minimum of 3 samples over a 2-hour period and pooling the samples before testing. We strongly urge consulting the published literature on the analytes in question, and asking us for advice on these issues. When the available knowledge is slim, we recommend carrying out pilot studies.

The effect of drugs on levels of hormones and other biomarkers needs to be considered.

Recent research has shown that medications prescribed to control children’s problem behavior can have an effect on salivary hormone levels.(38) Other work has demonstrated that other over-the-counter and prescription medicines can also have an effect on salivary cortisol levels.(39) Exposure to tobacco smoke has also been shown to have an effect on salivary cortisol and a-amylase levels.(40)

Due to the prevalence of drugs in modern society, and the potential effects that they can have on assay results, Salimetrics advises documentation of the use of prescription and over-the-counter medications taken by subjects, including consumption of nicotine products.

 

References

  1. A major part of the information in this bulletin is abstracted from Granger, D.A. and Fortunato, C.K. Integration of salivary biomarkers and analytes into developmental science: A critical review of theory, tactics, and strategies (In press, 2008).
  2. Harmon, A.G., Hibel, L.C., Rumyantseva, O., & Granger, D.A. (2007). Measuring salivary cortisol in studies of child development: Watch out–what goes in may not come out of saliva collection devices. Dev Psychobiol 49(5), 495-500.
  3. Shirtcliff, E.A., Granger, D.A., Schwartz, E., & Curran, M. J. (2001). Use of salivary biomarkers in biobehavioral research: Cotton-based sample collection methods can interfere with salivary immunoassay results. Psychoneuroendocrinology 26, 165-173.
  4. Gröschl, M., Rauh, M., (2006). Influence of commercial collection devices for saliva on the reliability of salivary steroids analysis. Steroids 71, 1097-1100.
  5. Granger, D.A., Kivlighan, K.T., Fortunato, C., et al. (2007). Integration of salivary biomarkers into developmental and behaviorally-oriented research: Problems and solution for collecting specimens. Physiol Behav 92(4), 583-90.
  6. Salimetrics Technical Bulletin 1001-06.
  7. Whembolua, G.L., Granger, D.A., Singer, S., et al. (2006). Bacteria in the oral mucosa and its effects on the measurement of cortisol, dehydroepiandrosterone, and testosterone in saliva. Horm Behav 49(4), 478-83.
  8. Richardson, T.C., Chapman, D.V., Heyderman, E. (1983). Immunoperoxidase techniques: the deleterious effect of sodium azide on the activity of peroxidase conjugates. J Clin Pathol 36(4), 411-14.
  9. Hodgson, N., Freedman, V.A., Granger, D.A., & Erro, B.(2004). Biobehavioral correlates of relocation in the frail elderly: salivary cortisol, affect, and cognitive function. J Am Geriatr Soc 52, 1856-62.
  10. Strazdins, L., Meyerkort, S., Brent, V., et al. (2005). Impact of saliva collection methods on sIgA and cortisol assays and acceptability to participants. J Immunol Methods 307(1-2), 167-71.
  11. de Weerth, C., Jansen, J, Vos, M., et al. (2007). A new device for collecting saliva for cortisol determination. Psychoneuroendocrinology 32, 1144-48.
  12. Neu, M., Goldstein, M., Gao, D., & Laudenslager, M. (2007). Salivary cortisol in preterm infants: Validation of a simple method for collecting saliva for cortisol determination. Early Human Development 83, 47-54.
  13. Malamud,D., Tabak, L. (1993). Saliva as a diagnostic fluid. Annals N Y Academy Sciences 694,
  14. Granger, D.A., Cicchetti, D., Rogosch, F.A., et al. (2007). Blood contamination in children’s saliva: Prevalence, stability, and impact on the measurement of salivary cortisol, testosterone, and dehydroepiandrosterone. Psychoneuroendocrinonology 32(6), 724-33.
  15. Kivlighan, K.T., Granger, D.A., Schwartz, E.B., et al.(2004). Quantifying blood leakage into the oral mucosa and its effects on the measurement of cortisol, dehydroepiandrosterone, and testosterone in saliva. Horm Behav 46(1), 39-46.
  16. Schwartz, E.B., Granger, D.A. (2004). Transferrin enzyme immunoassay for quantitative monitoring of blood contamination in saliva. Clin Chem 50(3), 654-6.
  17. Magnano, C.L., Diamond, E.J., Gardner, J.M. (1989). Use of salivary cortisol measurements in young infants: A note of caution. Child Dev 60(5), 1099-110.
  18. Klein,L.C., Whetzel, C.A., Ritter, F.E., & Granger, D.A. (2006). Effects of caffeine and stress on salivary alpha-amylase in young men: A salivary biomarker of sympathetic activity. Presented at the annual meeting of the American Psychosomatic Society, Denver, CO.
  19. Martin, S., Pangborn, R.M. (1971). Human parotid secretion in response to ethyl alcohol. J Dent Res 50(2), 485-90.
  20. Unpublished data collected by Salimetrics.
  21. Newbrun, E. (1962). Observations on the amylase content and flow rate of human saliva following gustatory stimulation. J Dent Res 41(2), 459-65.
  22. Kugler, J., Hess, M., Haake, D. (1992). Secretion of salivary immunoglobulin A in relation to age, saliva flow, mood states, secretion of albumin, cortisol, and catecholamines in saliva. J Clin Immun 12, 45-49.
  23. Schwartz, E.B., Granger, D.A., Susman, E.J., et al. (1998). Assessing salivary cortisol in studies of child development. Child Development 69, 1503-13.
  24. Talge,N.M., Conzella, B., Kryzer, E.M., et al. (2005). It’s not that bad: error introduced by oral stimulants in salivary cortisol research. Dev Psychobiol 47(4), 369-76.
  25. Veerman, E.C., van den Keybus, P.A., Vissink, A. Nieuw Amerongen, A.V. Human glandular salivas: Their separate collection and analysis (1996). Eur J Oral Sci 104(4), 346-52.
  26. Crawford JM, Taubman MA, Smith DJ (1975). Minor salivary glands as a major source of secretory immunoglobin A in the human oral cavity. Science 190 (4220), 1206-9.
  27. Proctor, G.B., Carpenter, G.H. (2007). Regulation of salivary gland function by autonomic nerves. Auton Neurosci 133(1), 3-18.
  28. Proctor, G.B., Carpenter, G.H., Anderson, L.C., & Garrett, J.R. (2000). Nerve-evoked secretion of immunoglobulin A in relation to other proteins by parotid glands in anaesthetized rat. Exper Physiol 85(5), 511-18.
  29. Garrett, J.R. (1987). The proper role of nerves in salivary secretion: a review. J Dent Res 66(2), 387-97.
  30. Rohleder, N., Wolf, J.M. Maldonado, E.F., and Kirschbaum, C. (2006). The psychosocial stress-induced increase in salivary alpha-amylase is independent of saliva flow rate. Psychophysiology 43(6), 645-52.
  31. Vining, R.F., McGinley, R.A. and Symons, R.G. (1983). Hormones in saliva: Mode of entry and consequent implications for clinical interpretation. Clin Chem 29(10), 1752-56.
  32. Mostov, K.E. (1994). Transepithelial transport of immunoglobulins. Annu Rev Immunol 12, 63-84.
  33. Kugler, J., Hess, M., and Haake, D. (1992). Secretion of salivary immunoglobulin A in relation to age, saliva flow, mood states, secretion of albumin, cortisol, and catecholamines in saliva. J Clin Immunol, 12(1), 45-9.
  34. Proctor, G.B., Carpenter, G.H. (2001). Chewing stimulates secretion of human salivary secretory immunoglobulin A. J Dent Res 80(3), 909-13.
  35. Rudney, J.D. (1995). Does variability in salivary protein concentrations influence oral microbial ecology and oral health? Crit Rev Oral Biol Med 6(4), 343-67.
  36. Dawidson, I., Blom, M., Lundeberg, T., Theodorsson, E., and Angmar-Månsson, B (1997). Neropeptides in the saliva of healthy subjects. Life Sciences 60(4-5), 269-78.
  37. West, C.D., Mahajan, D.K., Chavre, V.J., Nabors, C.J. (1973). Simultaneous measurement of multiple plasma steroids by radioimmunoassay demonstrating episodic secretions. J Clin Endocinol Metab 36, 1230-36.
  38. Hibel, L.C., Granger, D.A., Cicchetti, D. & Rogosch, F. (2007). Salivary biomarker levels and diurnal variation: Associations with medications prescribed to control children’s problem behavior. Child Development 78, 927-937.
  39. Hibel, L.C., Granger, D.A., Kivlighan, K.T., Blair, C. & the Family Life Project Investigators (2006). Individual differences in salivary cortisol: Effects of common over the counter and prescription medications in infants and their mothers. Hormones and Behavior 50, 293-300.
  40. Granger, D.A., Blair, C., Willoughby, M., et al. (2007). Individual differences in salivary cortisol and alpha-amylase in mothers and infants: Relation to tobacco smoke exposure. Dev Psychobiol 49(7), 692-701.