What is the protocol for a cortisol (cortisone) saliva test?

Protocol for Salivary Cortisol Testing

For screening Cushing's syndrome, collect late-night salivary cortisol at the patient's usual bedtime (at least 2-3 samples on different nights), while for assessing diurnal cortisol rhythm, collect samples at standardized time points throughout the day with morning collection between 8:00-9:00 AM. 1, 2

Pre-Collection Patient Instructions

Timing Before Sample Collection

• No food or drink for 30-60 minutes before collection to avoid contamination and pH alterations that affect cortisol measurement 3
• No teeth brushing or oral hygiene products for 1-2 hours prior to collection to minimize blood contamination that falsely elevates biomarker levels 3
• No smoking, vaping, or nicotine products for 4 hours before sampling 3
• No alcohol consumption for 12 hours preceding the test 3
• Abstain from strenuous physical activity for 24-48 hours before collection, as exercise significantly increases salivary cortisol levels 3

Conditions That Invalidate Testing

• Postpone testing if acute infection (especially upper respiratory tract infections) occurred within the past 2 weeks; reschedule 2 weeks after resolution 3
• Avoid cognitive assessments or mental stressors immediately before sampling, as these elevate salivary cortisol and amylase levels 3
• Do not test shift workers or patients with disrupted circadian rhythms using late-night salivary cortisol, as their cortisol peaks occur at different times and yield unreliable results 1, 2

Collection Timing Protocols

For Cushing's Syndrome Screening (Late-Night Salivary Cortisol)

• Collect at patient's usual bedtime rather than strictly at midnight, as cortisol nadir is tightly entrained to sleep onset 2
• Obtain at least 2-3 samples on different nights to account for variability and detect cyclic hypercortisolism 2
• Diagnostic accuracy: sensitivity 92-100%, specificity 93-100% for detecting Cushing's syndrome 2
• Cut-off values vary by age: For adults age 21-60, values >1.9 µg/L at 10:00 PM indicate hypercortisolism with 97.6% sensitivity and 100% specificity 4

For Diurnal Cortisol Rhythm Assessment

• Morning sample: 8:00-9:00 AM to capture the physiologic peak of cortisol secretion 1
• Additional time points if assessing full diurnal pattern: 11:00 AM-12:00 PM, 4:00-6:00 PM, 7:00-8:00 PM, and 10:00 PM 4
• For single time-point collections: standardize to 9:00-11:00 AM window and use the same time for follow-up testing 3
• Serial samplings over 24-48 hours are advised for biomarkers affected by diurnal variation, with statistical analyses using area under the curve 3

Collection Methods

Preferred Collection Techniques

• Passive drooling is the most commonly used method and provides whole "mixed" resting-state saliva from all major salivary glands 3
• Absorbent-pad-based methods (using inert polypropylene or polyethylene materials) are gaining acceptance for ease of use, especially with older individuals and those with cognitive issues 3
• Avoid cotton materials as they retain cortisol and other hormones, impeding correct quantification 3
• Alternative methods include: direct spitting into a tube, passive drooling through a funnel/straw, or oral rinsing with 5 mL of 0.25% citric acid or saline for 15 seconds 3

Volume and Container Requirements

• Collect 2-3 mL of total saliva before centrifugation to provide adequate volume for aliquoting into 500-µL portions after centrifugation 3
• Use high-grade polypropylene tubes or low-affinity plastic collection tubes to prevent binding of cortisol to tube walls 3

Sample Handling and Storage

Immediate Post-Collection

• Collect at room temperature, then immediately place the collection device on ice to minimize proteolytic activity 3
• Steroid hormones (including cortisol) are stable at ambient temperature for extended periods, unlike protein biomarkers 3
• Visually inspect immediately after collection for blood contamination, lipstick, coffee, or other contaminants that falsely elevate biomarker levels 3

Processing and Preservation

• Centrifuge samples (unless using commercial devices with specific manufacturer instructions that don't require centrifugation) 3
• Adding protease inhibitors is recommended for protein biomarkers but less critical for cortisol, which is a steroid hormone 3
• Aliquot into 500-µL portions for storage after centrifugation 3

Assisted vs. Self-Collection

Advantages of Assisted Collection (Recommended)

• Trained collector ensures adherence to pre-analytic instructions and verifies patient health condition 3
• Timing and duration can be controlled, allowing measurement of flow and secretion rates 3
• Immediate inspection for contamination allows rectification and re-collection on the spot 3

Self-Collection Considerations

• Main advantage is convenience (no travel required), but introduces greater variability in pre-analytical variables 3
• Electronic monitoring devices are recommended when self-collection is used, as 26% of subjects fail to collect samples at correct times, significantly altering cortisol profiles 5
• Noncompliant subjects show blunted cortisol awakening response and less steep circadian decline compared to compliant subjects 5

Critical Pitfalls to Avoid

• Topical hydrocortisone preparations must be avoided before testing, as they contaminate samples 2
• Medications affecting cortisol binding globulin (oral estrogens, pregnancy) increase total cortisol levels and cause false interpretation 1
• CYP3A4 inducers affect dexamethasone metabolism and can cause false positives on dexamethasone suppression tests 1
• Sleep disturbances correlate with decreased morning awakening salivary cortisol in patients with insomnia 3
• Compliance monitoring is essential: inform patients about electronic monitoring devices to improve adherence, as uninformed subjects show significantly lower compliance 5