At what times should a serum cortisol be collected and how should the specimen be processed and stored?

Timing and Handling of Cortisol Specimens

Morning serum cortisol should be drawn between 0800-0900h (within 1 hour of awakening) to capture the physiologic peak, and the specimen can be stored in either serum or plasma at 4°C for up to 40 hours without degradation, though samples with high cortisol concentrations (>15 ng/mL) should be refrigerated if not processed within 5-9 days. 1, 2, 3

Optimal Collection Timing

Morning Cortisol (Primary Screening)

• Draw between 0800-0900h to capture the circadian peak that occurs just minutes before and after awakening 1
• Morning cortisol is the most practical and cost-effective initial assessment for adrenal function 1
• Timing is critical because cortisol secretion follows a sleep-wake cycle with peak levels upon awakening 1

Important Timing Caveats

• Avoid testing in patients with disrupted circadian rhythms (night shift workers, jet lag, severe insomnia) as morning cortisol can appear falsely low 1
• For glucocorticoid-induced adrenal insufficiency assessment, draw at least 24 hours after the last glucocorticoid dose (excluding dexamethasone) 1
• The baseline sample for ACTH can be obtained simultaneously with the morning cortisol sample 4

Midnight Cortisol (Cushing's Syndrome Evaluation)

• When evaluating for Cushing's syndrome, midnight serum cortisol >7.5 μg/dL (207 nmol/L) has 96% sensitivity at 100% specificity for distinguishing Cushing's syndrome from pseudo-Cushing states 5
• Multiple samples between 2300-0100h can be collected, though a single midnight value is highly diagnostic 5

Specimen Handling and Storage

Serum vs. Plasma

• Either serum or plasma is acceptable for cortisol measurement—no significant differences exist between EDTA-treated plasma, heparinized plasma, or serum 2, 3
• Samples can remain uncentrifuged at 4°C for up to 40 hours without cortisol degradation 2

Storage Conditions

• Refrigeration at 4°C is preferred for short-term storage 2, 3
• Samples with initial cortisol concentrations <15 ng/mL remain stable at room temperature for up to 15 days 2
• High cortisol samples (>55-80 ng/mL) degrade significantly after 5-9 days at room temperature 2
• For serum cortisol, ship samples cold to prevent degradation, particularly for elevated values 3
• Long-term storage at -20°C is stable for at least 5 days 3

Container Type

• Glass or plastic tubes are equally acceptable—no effect of tube type on cortisol concentrations has been demonstrated 2, 3

Interpretation Considerations

Assay Variability

• Substantial variability exists between different cortisol assays, affecting interpretation of cutoff values 1
• Liquid chromatography-tandem mass spectrometry is the most accurate method, followed by monoclonal antibody immunoassays, then polyclonal antibody immunoassays 1
• Consider assay-specific cutoffs when available for your laboratory platform 6

Factors Affecting Results

• Elevated cortisol-binding globulin (pregnancy, oral estrogens) falsely elevates total serum cortisol 1
• Low albumin/cortisol-binding globulin (cirrhosis, nephrotic syndrome, malnutrition, critical illness) falsely lowers total serum cortisol 1
• These conditions affect total cortisol but not free cortisol levels 1

Clinical Cutoffs for Adrenal Function

• Morning cortisol <150 nmol/L (5 μg/dL) indicates very likely persistent adrenal insufficiency 1
• Morning cortisol >300 nmol/L (10 μg/dL) suggests adequate HPA axis recovery 1
• Morning cortisol ≥354 nmol/L (12.8 μg/dL) predicted normal adrenal function with 100% sensitivity in one study 1
• Values between 150-300 nmol/L represent a grey zone where dynamic testing may be considered 1, 6