How is salivary cortisol collected and what does it indicate?

Salivary Cortisol Collection and Clinical Significance

Salivary cortisol is collected through passive drooling or absorbent pad-based methods, with standardized timing to account for diurnal variations, and provides valuable information about stress levels, adrenal function, and cortisol's diurnal rhythm patterns that directly impact patient morbidity and mortality. 1

Collection Methods

Non-Absorbent-Pad-Based Methods

• Passive drooling: Most commonly used method where saliva is drooled through a funnel or straw into a collection tube
• Direct spitting: Expectoration of saliva into a tube
• Oral rinsing: Swishing with citric acid or saline solution and then expectorating into a tube 1

Absorbent-Pad-Based Methods

• Salivette (Sarstedt): Subject chews on an absorbent pad for 60 seconds, then places it in a plastic tube for centrifugation 1
• Pure•SAL (Oasis Diagnostics): Uses compression to collect a cell-free, highly purified specimen 1

Important Collection Considerations

• Use high-grade polypropylene tubes rather than cotton materials which can retain cortisol and other analytes 1
• Collect 2-3 mL of total saliva before processing 1
• Avoid cotton-based collection materials as they can retain hormones and impede accurate quantification 1

Pre-Collection Guidelines

1. Timing considerations:

   • Account for diurnal variations - cortisol levels are highest in the morning and lowest at night 1
   • Collection between 8-10 AM or in the afternoon when levels are more stable 1
   • For screening Cushing's syndrome, late-night (2300h) collection is most valuable 2

2. Dietary restrictions:

   • No eating, drinking, or smoking 1-2 hours before collection 1
   • Avoid alcohol for 12 hours before collection 1

3. Physical activity:

   • Abstain from strenuous exercise for 24-48 hours before collection as physical activity increases salivary cortisol levels 1

4. Mental stressors:

   • Perform collection before cognitive assessments or stressful procedures 1
   • Note any acute stress as it elevates cortisol levels 1

5. Oral hygiene:

   • No teeth brushing or oral hygiene products 1-2 hours before collection to minimize blood contamination 1

Clinical Significance of Salivary Cortisol

Advantages Over Blood Testing

• Non-invasive collection method
• Stress-free sampling (avoids stress-induced cortisol elevation from venipuncture)
• Reflects unbound (free) cortisol, the biologically active fraction 3, 4
• Can be collected at home or any setting
• Stable at room temperature for steroid hormones 1

Clinical Applications

1. Diagnosis of adrenal disorders:

   • Excellent screening test for Cushing's syndrome with 92% sensitivity using late-night samples 2
   • Evaluation of adrenal insufficiency 3
   • Combination of elevated late-night salivary cortisol and elevated UFC identifies 100% of patients with Cushing's syndrome 2

2. Assessment of stress response:

   • Monitors physiological stress levels
   • Evaluates hypothalamic-pituitary-adrenal (HPA) axis function 4

3. Diurnal rhythm evaluation:

   • Normal subjects show significant diurnal variation (morning: 15.5 nmol/L, evening: 3.9 nmol/L) 3
   • Loss of diurnal rhythm suggests pathology 1

4. Dynamic testing:

   • Dexamethasone suppression test (DST) can be performed using salivary samples 3
   • ACTH stimulation testing 3

Post-Collection Processing

1. Immediate handling:

   • Place samples on ice immediately after collection to minimize proteolytic activity 1
   • Add protease inhibitors for protein biomarkers 1

2. Processing:

   • Centrifugation (typically 600 × g for 10 min at 4°C) 1
   • Aliquot into 500-μL portions 1
   • Store at -80°C for long-term storage 1

Potential Pitfalls and Caveats

• Collection material interference: Cotton-based materials can retain cortisol, leading to falsely low results 1
• Blood contamination: Can falsely elevate cortisol levels 1
• Medication effects: Certain medications (thyroid hormones, psychotropic agents) may cause discrepancies between salivary and plasma cortisol 3
• Acute infections: Immune and related salivary markers are elevated due to infections; ideally, postpone collection for 2 weeks after infection 1
• Collection method variability: Salivette collection shows stronger correlation with serum cortisol compared to passive drooling 5

By following standardized collection protocols and understanding the clinical significance of results, salivary cortisol measurement provides valuable insights into adrenal function and stress response that can directly impact patient management and outcomes.