How is a late-night salivary cortisol sample collected in a patient suspected of having Cushing's disease?

How to Collect a Late-Night Salivary Cortisol Sample

Late-night salivary cortisol should be collected at the patient's normal bedtime (between 2025-2400 hours) using a simple saliva collection device, with the patient at home in a relaxed, non-stressful environment. 1

Timing of Collection

• Patients can collect saliva at their normal bedtime rather than being required to stay awake until 2300-2400 hours, as bedtime sampling has been validated and may actually provide a lower (more specific) upper limit of normal. 1
• The standard recommended collection time is between 2300-2400 hours (11 PM to midnight), which captures the physiologic nadir of cortisol secretion. 2, 3
• Sampling should occur when the patient is in their usual sleep environment, as this reflects the loss of normal circadian rhythm that characterizes Cushing's syndrome. 4

Collection Method

• Use a commercially available saliva collection device (such as a Salivette or similar FDA-cleared collection system) that allows passive drool or gentle absorption of saliva. 3, 5
• Only 50 µL of saliva is required for modern LC-MS/MS assays, though standard immunoassays may require slightly more volume. 1
• The sample should be collected at home in a non-stressful environment, as stress can artificially elevate cortisol levels. 4

Critical Pre-Collection Instructions

• Avoid eating, drinking (except water), or brushing teeth for at least 30-60 minutes before collection to prevent blood contamination from oral trauma or food interference. 6
• Do not use any topical hydrocortisone preparations (creams, ointments) on the day of collection, as contamination can cause falsely elevated results. 6, 5
• Avoid strenuous physical exercise for 24-48 hours before sampling, as this elevates cortisol levels. 6
• Patients should be in their usual sleep-wake cycle; night-shift workers should not undergo late-night salivary cortisol testing due to disrupted circadian rhythms. 6, 7

Number of Samples Required

• Collect at least 2-3 separate late-night salivary cortisol samples on different nights due to test-to-test variability and the possibility of cyclic Cushing's syndrome. 6, 5
• Multiple samples improve diagnostic accuracy, as patients with cyclic disease may have normal cortisol during quiescent periods. 2, 6

Storage and Handling

• Samples can be stored at room temperature for short periods or refrigerated until transport to the laboratory. 4
• The sample should be clearly labeled with collection date and time. 1

Diagnostic Threshold

• The abnormal threshold is >3.6 nmol/L (>0.1 µg/dL) when measured by immunoassay, with sensitivity of 92-100% and specificity of 93-100%. 6, 3
• Local laboratory reference ranges should be verified, as different assays may have slightly different cutoffs. 2, 8

Common Pitfalls to Avoid

• Contamination with topical corticosteroids is the most common cause of false-positive results—specifically ask patients about hydrocortisone creams, ointments, or hemorrhoid preparations. 6, 4
• Blood contamination from recent dental work, gum disease, or aggressive tooth brushing within 1-2 hours can falsely elevate results. 6
• Acute illness, particularly upper respiratory infections, can activate the HPA axis and cause false-positive results. 6, 4
• Severe obesity, psychiatric disorders, and alcohol use disorder can cause physiologic hypercortisolism (pseudo-Cushing's states) that may elevate late-night salivary cortisol. 6

Special Populations

• Late-night salivary cortisol is particularly useful in pregnant women or those on oral contraceptives, as it measures free cortisol and is not affected by elevated corticosteroid-binding globulin. 5
• In children and adolescents with suspected Cushing's disease, late-night salivary cortisol has 95% sensitivity and 100% specificity. 2