Drop Date: November 2010
SBB – Improving Saliva Study Results
In This Drop: Ensuring Accurate and Reproducible Salivary Bioscience
Ensuring that assay results are accurate and meaningful is a key part of every study. To achieve this goal when saliva is used as a testing fluid, researchers need to be aware that biomarkers enter saliva through a number of different pathways. Analytes that originate in the salivary glands or that enter by active transport will behave differently than those that enter by passive diffusion; consequently, some analytes may be sensitive to mouth location and/or saliva flow rates. Different response and recovery characteristics to stressors may also exist among analytes, and other factors such as diurnal rhythm must be considered as well. It is therefore important that researchers understand these matters so that they can choose a collection method and sampling design appropriate for the focus of their study.
Additionally, once samples have been collected from the subjects, there are still further issues that may be related to the use of saliva; these involve sample handling and storage, immunoassay choice and performance, laboratory equipment, technician proficiency, and finally, data analysis and reporting. Weaknesses in any any of these areas can significantly affect the quality of the data or the way it is interpreted.
Inter- and Intra-Assay Coefficients of Variability
Studies that utilize immunoassays to measure biomarker levels typically report the inter- and intra-assay CVs for their data. Here we offer advice on calculating and interpreting these measures of assay precision.
Collecting Saliva from Infants and Small Children
Saliva is an ideal testing fluid for studies that involve infants or small children because it can be collected in a relatively non-invasive and stress-free fashion. However, even the collection of saliva from these young subjects can at times be a challenge, and sending samples off to be tested only to find out that the samples were “QNS” (Quantity Not Sufficient) can have an adverse impact on a study. In this article we discuss some of the methods and materials that have been used to collect saliva from small children, and we report on a new collection device that is designed to make the process easier.
*Salimetrics provides this information for research use only (RUO). Information is not provided to promote off-label use of medical devices. Consult full text of article.
Assay Variance and Control
Jon Peterson Principal Scientist, Salimetrics
The variance of analyte levels found in any biological fluid should be expected to differ based upon two primary factors: biovariability and measurement error.
Biovariability is beyond the scope of this discussion, but relates to the variance of an analyte level over an individual’s lifetime on daily, monthly or seasonal basis. These factors are typically independent of the measurement method(s).
Measurement error is primarily dependent upon the assay method, precision of pipetting and sample handling. Factors that can influence the performance of an ELISA assay method are:
- Uniformity of the solid phase
- Stability of reagents
- Selection and calibration of the pipettes for various volumes
- Plate washer
- Pre-analytical sample handling
High-quality ELISA kits have stringent quality guidelines to ensure that they meet the exacting accuracy needs of researchers. Assay precision must be critically evaluated, and the same is true of all other performance characteristics such as accuracy and stability of reagents.
We can recommend, but not control, the state of equipment in our customer’s labs. Needless to say, properly calibrated and maintained pipettes and plate washers in the hands of trained professionals will yield a different result than the opposite.
Pipetting proficiency can be evaluated with some simple procedures utilizing pipettes, uncoated microwell plates, a yellow dye solution and a plate reader. Through a series of dye additions, one is able to estimate pipetting proficiency and identify areas for improvement to achieve good data results.
For additional information, please see: (PDF) Salimetrics Pipetting Proficiency Study
REFERENCES & RELATED RESEARCH
Salivary Flow and Alpha-Amylase: Collection Technique, Duration, and Oral Fluid Type.
Beltzer, E.K., Fortunato, C.K., Guaderrama, M.M., et al. (2010). Physiol Behav, 101, 289-96.
Salivary alpha-amylase (sAA) is a digestive and anti-microbial enzyme synthesized locally in the salivary glands. Because its synthesis and release into saliva are controlled by nervous signals to the glands, it can serve as a convenient biomarker of ANS activity. Due to differences in the cellular makeup of each type of salivary gland, concentrations of sAA will vary according to the location in the mouth where the saliva is collected, and levels may also be affected by differences or changes in saliva flow. In this paper, the authors rigorously examine the ways that collection method, duration of collection, and collection location interact to affect estimates of sAA.
The other side of the curve: examining the relationship between pre-stressor physiological responses and stress reactivity.
Balodis, I.M., Wynne-Edwards, K.E., Olmstead, M.C. (2010). Psychoneuroendocrinology, 35, 1363-73.
Many biobehavioral studies now incorporate measurements of salivary cortisol and salivary alpha-amylase to monitor physiological responses to physical or psychological stress, but there is still considerable variation from study to study in the way data on stress reactivity are gathered and analyzed. In this study, the authors examine the strengths and weaknesses of a number of strategies currently used to characterize the physiological and subjective aspects of the stress response. The period of time between the arrival of the subject in the laboratory and the administration of the stressor receives special attention, and the possibility of a relationship between biomarker changes during the initial resting period and an individual’s subsequent response to the stressor is explored.
*Note: Salimetrics provides this information for research use only (RUO). Information is not provided to promote off-label use of medical devices. Please consult the full-text article.