IN THIS ISSUE
- June 2010
- Cortisol Awakening Response
- April 2010
- Reproductive and Sexual Health
- January 2010
- Exercise, Sports Science and Medicine, Measuring SIgA in Saliva
- November 2009
- Salivary Biomarkers and Periodontal Disease, Links Between Periodontal Disease and Systemic Health
- August 2009
- Polymorphisms Affecting HPA Activity, Collecting and Handling Saliva for DNA Analysis
- May 2009
- Salivary Alpha-Amylase, Importance of Mouth Location During Saliva Collection
- March 2009
- Salivary Cortisol, Saliva Collection Techniques
- November 2008
- DHEA, CRP, Cortisol, Entry of Substances into Saliva
IMPROVING SALIVA STUDY RESULTS
Ensuring that assay results are accurate and meaningful is a key part of every study. To acheive 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.
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:
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.
Saliva Handling and Storage Advice
Following proper sample handling procedures is important in order to avoid retention or degradation of salivary analytes.