What is the late night salivary cortisol (cortisol) collection test?

Late Night Salivary Cortisol Collection

Late-night salivary cortisol (LNSC) is a highly sensitive and specific screening test for Cushing's syndrome that requires collecting 2-3 saliva samples at bedtime (around 11 PM-midnight) on consecutive days, with values >3.6 nmol/L considered abnormal. 1

Physiologic Basis

The diagnostic utility of LNSC relies on the principle that patients with Cushing's syndrome lose their normal circadian nadir of cortisol secretion that occurs at night. 2 In healthy individuals, cortisol reaches its lowest point during sleep, but this pattern is disrupted in hypercortisolism. 3

• Salivary cortisol concentrations directly reflect the active free (unbound) cortisol in plasma, making it an accurate measure of bioavailable hormone. 3
• The cortisol nadir is tightly entrained to sleep onset, so sampling at the patient's usual bedtime rather than strictly at midnight may decrease false positive results. 2

Collection Technique

• Timing: Collect saliva samples at the patient's usual bedtime (typically 11 PM-midnight) when cortisol should be at its nadir. 2, 1
• Number of samples: At least 2-3 samples collected on consecutive days are required due to test variability and to detect cyclic Cushing's syndrome. 2, 1
• Collection device: Use a commercially available saliva collection device that allows simple, non-stressful sampling at home. 3, 4
• Patient instructions: Samples can be collected at home in a non-stressful environment, which is a major advantage over blood draws. 3

Diagnostic Performance

• Sensitivity: 92-100% across multiple studies, making it highly effective at detecting Cushing's syndrome. 1, 3, 4
• Specificity: 93-100%, the highest among all first-line screening tests for Cushing's syndrome. 2, 1, 3
• The test performs excellently even in high-risk populations such as patients with diabetes and obesity. 5

Interpretation of Results

• Normal threshold: Values <3.6 nmol/L are considered normal. 1, 4
• Abnormal threshold: Values >3.6 nmol/L suggest Cushing's syndrome and warrant further evaluation. 1
• Mild disease: Patients with mild Cushing's syndrome may have LNSC values just above the upper limit of normal. 2
• Cyclic disease: Multiple sequential LNSC measurements are particularly useful for detecting cyclic Cushing's syndrome, where patients exhibit weeks to months of normal cortisol secretion interspersed with episodes of excess. 2

Critical Contraindications and Pitfalls

Absolute contraindication: LNSC should NOT be performed in night-shift workers or anyone with disrupted day/night cycles, as the normal circadian rhythm is lost in these individuals. 2, 6

Factors Causing False Positive Results:

• Topical hydrocortisone contamination: Topical steroid preparations can contaminate samples, particularly when mass spectrometry is used. 2, 7
• Pseudo-Cushing's states: Depression, alcoholism, severe obesity, uncontrolled diabetes, and polycystic ovary syndrome can cause mild hypercortisolism mimicking true Cushing's syndrome. 1, 7
• Acute stress: Physical or psychological stress can transiently elevate cortisol levels. 7
• Sample contamination: Blood contamination from recent dental work, teeth brushing, or oral trauma within 1-2 hours of collection invalidates results. 7

Assay Considerations:

• Mass spectrometry vs. immunoassay: Mass spectrometry can detect both cortisol and cortisone, avoiding contamination from topical hydrocortisone, and offers better sensitivity but reduced specificity compared to immunoassay. 2
• Second-generation assays: Newer electrochemiluminescence immunoassay (ECLIA) kits may have different cut-offs (≥6.73 nmol/L) with excellent diagnostic performance. 8
• LC-MS/MS method: Some studies using liquid chromatography/tandem mass spectrometry report lower sensitivity (74.5-83%) with cut-offs around 2.1-2.8 nmol/L. 9

Clinical Application Algorithm

1. Screen appropriate patients: Those with clinical features of Cushing's syndrome (weight gain with decreased linear growth in children, central obesity, wide purple striae, proximal myopathy). 1

2. Exclude contraindications: Verify patient does not work night shifts and has regular sleep-wake cycles. 2, 6

3. Collect 2-3 samples: Obtain samples on consecutive nights at patient's usual bedtime. 2, 1

4. If any test is abnormal: Repeat 1-2 additional screening tests (including 24-hour urinary free cortisol or dexamethasone suppression test) to confirm diagnosis. 1

5. If results are inconsistent: Consider cyclic Cushing's syndrome and perform extended monitoring with multiple periodic sequential LNSC measurements. 2, 1