What tests are needed to check cortisol levels?

Testing to Check Cortisol Levels

The 1 mg dexamethasone suppression test (DST) with serum cortisol measured at 8 AM is the preferred initial screening test for autonomous cortisol secretion, and should be performed in all patients with adrenal incidentalomas. 1

Initial Screening Tests for Cortisol Excess

For screening suspected Cushing's syndrome or autonomous cortisol secretion, three primary tests are available 1:

1 mg Overnight Dexamethasone Suppression Test (DST)

• Administration: 1 mg dexamethasone taken between 11 PM and midnight 1
• Measurement: Serum cortisol measured at 8 AM the following morning 1
• Interpretation 1:
  • <50 nmol/L (<1.8 μg/dL): Excludes cortisol hypersecretion
  • 51-138 nmol/L (1.8-5 μg/dL): Possible autonomous cortisol secretion

  • 138 nmol/L (>5 μg/dL): Evidence of cortisol hypersecretion

• Advantages: Highest sensitivity (>90%) among screening tests, strongly predicts absence of Cushing's syndrome when negative 1

Late Night Salivary Cortisol (LNSC)

• Collection: At least two or three samples collected at usual bedtime (not necessarily midnight) 1
• Advantages: Most specific screening test, useful for cyclic Cushing's syndrome with periodic sequential measurements 1
• Limitations: Should not be performed in night-shift workers with disrupted circadian rhythm 1

24-Hour Urinary Free Cortisol (UFC)

• Collection: Complete 24-hour urine collection 1
• Advantages: Measures total daily cortisol production, not affected by CBG levels 1, 2
• Limitations: Lowest sensitivity among the three screening tests 1

Confirmatory Testing After Positive Screening

Plasma ACTH Measurement

• Timing: Morning (8-9 AM) measurement is optimal 3
• Purpose: Determines if Cushing's syndrome is ACTH-dependent or ACTH-independent 1, 3
• Interpretation 3:
  • ACTH >5 ng/L: Detectable, suggests ACTH-dependent Cushing's syndrome
  • ACTH >29 ng/L: 70% sensitivity and 100% specificity for Cushing's disease
  • Low or undetectable ACTH: Indicates ACTH-independent (adrenal) source

Additional Confirmatory Tests

When initial screening is positive, the following should be measured 1:

• 24-hour urinary free cortisol: If not already done as initial screening
• Midnight salivary cortisol: To confirm loss of circadian rhythm
• DHEAS: To assess for adrenal androgen excess

Testing for Adrenal Insufficiency

Single Serum Cortisol Measurement

• Morning samples (8 AM-12 PM): Cortisol <275 nmol/L identifies subnormal adrenal function with 96.2% sensitivity 4
• Afternoon samples (12 PM-6 PM): Cortisol <250 nmol/L achieves 96.1% sensitivity in outpatients 4
• Interpretation: Values below these thresholds require dynamic testing (short Synacthen test) 4

Short Synacthen (ACTH Stimulation) Test

• Dose: 250 μg (standard) or 1 μg (low-dose, more sensitive) of 1-24 ACTH 5
• Timing: Can be performed at any time of day, though morning is standard 5
• Measurement: Serum cortisol at baseline, 30 minutes, and optionally 60 minutes 5
• Interpretation: Peak cortisol <500 nmol/L is diagnostic of adrenal insufficiency 1

Important Caveats and Pitfalls

False Positive DST Results

Several conditions can cause falsely elevated cortisol after dexamethasone 1:

• Rapid dexamethasone absorption/malabsorption (chronic diarrhea, celiac disease)
• CYP3A4 inducers (phenobarbital, carbamazepine, St. John's wort, rifampin) 1, 6
• Increased CBG from oral estrogens, pregnancy, or chronic active hepatitis 1
• Solution: Measure dexamethasone levels concomitantly with cortisol to confirm adequate absorption 1

Pseudo-Cushing's States

The following can mimic Cushing's syndrome on screening tests 1, 3:

• Severe obesity
• Uncontrolled diabetes mellitus
• Depression
• Chronic alcoholism

Assay-Specific Considerations

• Immunoassays: Lack specificity and show significant inter-assay differences 7, 8
• LC-MS/MS: Offers improved specificity and sensitivity, particularly for salivary cortisol and UFC 7, 8, 2
• Important: Cortisol cut-offs for DST have not been fully validated for LC-MS/MS assays 7

Special Populations

• Patients on medications: Indomethacin can cause false-negative DST results 6
• Cyclic Cushing's: Requires multiple periodic measurements; hypercortisolemia must be confirmed immediately before diagnostic procedures like BIPSS 3
• Critically ill patients: Cortisol interpretation is difficult due to stress response and altered protein binding 7