The effects of saliva collection, handling and storage on salivary testosterone measurement

• Salivary testosterone concentrations were influenced neither by repeated sampling nor by stimulation of salivary flow.
• Centrifugation decreased the measured concentration of salivary testosterone.
• Blood contaminated samples interfere with Testosterone ELISA.
• There are several factors that should be assessed before planning the analysis.

Several endocrine parameters commonly measured in plasma, such as steroid hormones, can be measured in the oral fluid. However, there are several technical aspects of saliva sampling and processing that can potentially bias the validity of salivary testosterone measurement. The aim of this study was to evaluate the effects caused by repeated sampling; 5 min centrifugation (at 2000, 6000 or 10,000g); the stimulation of saliva flow by a cotton swab soaked in 2% citric acid touching the tongue; different storage times and conditions as well as the impact of blood contamination on salivary testosterone concentration measured using a commercially available ELISA kit. Fresh, unprocessed, unstimulated saliva samples served as a control. Salivary testosterone concentrations were influenced neither by repeated sampling nor by stimulation of salivary flow. Testosterone levels determined in samples stored in various laboratory conditions for time periods up to 1 month did not differ in comparison with controls. For both genders, salivary testosterone levels were substantially reduced after centrifugation (men F = 29.1; women F = 56.17, p < 0.0001). Blood contamination decreased salivary testosterone levels in a dose-dependent manner (men F = 6.54, p < 0.01, F = 5.01, p < 0.05). Salivary testosterone can be considered A robust and stable marker. However, saliva processing and blood leakage can introduce bias into measurements of salivary testoterone using ELISA. Our observations should be considered in studies focusing on salivary testosterone.

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