What laboratory test is used to assess cortisol levels?

Laboratory Tests for Assessing Cortisol Levels

The recommended initial laboratory tests for assessing cortisol levels include 24-hour urinary free cortisol (UFC), late-night salivary cortisol (LNSC), and/or the overnight 1-mg dexamethasone suppression test (DST). 1

Primary Screening Tests

The Endocrine Society recommends three main tests for cortisol assessment:

1. Late-night salivary cortisol (LNSC)

   • Sensitivity >90% and specificity ~100% when properly collected
   • Reflects free (biologically active) cortisol
   • Particularly useful for initial screening and monitoring cyclic Cushing's syndrome
   • Simple and convenient collection method 1, 2

2. 24-hour urinary free cortisol (UFC)

   • Sensitivity of 97% and specificity of 91% when measured by LC-MS/MS
   • Reflects overall cortisol production over 24 hours
   • Useful for supporting diagnosis 1

3. Overnight 1-mg dexamethasone suppression test (DST)

   • Normal response: cortisol suppression to <50 nmol/L (1.8 μg/dL)
   • Alternative cutoff: <80 nmol/L (3 μg/dL) (may increase false positives)
   • Particularly useful for adrenal incidentalomas and patients with disrupted circadian rhythms 1

Additional Diagnostic Tests

• Plasma ACTH measurement

  • Used to determine if cortisol excess is ACTH-dependent or independent
  • Critical for differentiating between pituitary, ectopic, or adrenal sources of hypercortisolism 1

• Morning serum cortisol

  • Less specific than other tests due to significant overlap between normal subjects and patients with cortisol disorders
  • Measures total cortisol (bound and unbound) 3, 4

Laboratory Methods for Cortisol Measurement

• Immunoassays (IA) and Enzyme Immunoassays (EIA)

  • Widely used in routine clinical laboratories
  • Available in commercial kits and on automated platforms
  • Less specific than chromatographic methods 5

• Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)

  • Higher specificity and sensitivity
  • Preferred method for measuring cortisol in serum, urine, and saliva
  • Particularly valuable for salivary cortisol measurement 5, 6

Special Considerations and Potential Pitfalls

False Positives

• Can occur in patients with:
  • Severe obesity
  • Uncontrolled diabetes
  • Pregnancy
  • Polycystic ovary syndrome (PCOS)
  • Psychiatric disorders
  • Alcohol use disorder 1

False Negatives

• Can occur in patients with:
  • Cyclic Cushing's syndrome (periodic hypercortisolism)
  • Mild Cushing's syndrome 1

Medication Interference

• Women on estrogen-containing oral contraceptives may have false positive results in the 1-mg DST
• Medications that affect CYP3A4 (dexamethasone metabolism) can interfere with test results 1

Choosing the Appropriate Test

1. For initial screening: LNSC or 1-mg DST
2. For confirmation: 24-hour UFC
3. For determining source: Plasma ACTH followed by appropriate imaging (adrenal CT/MRI for low ACTH; pituitary MRI for normal/high ACTH)

The combination of elevated LNSC and/or elevated UFC has been shown to identify 100% of patients with Cushing's syndrome, demonstrating excellent sensitivity when used together 2.

Pediatric Considerations

• Growth failure with weight gain is a key distinguishing feature of Cushing's disease in children
• The same laboratory tests are used, but interpretation may differ 1

Remember that cortisol testing should be interpreted in the clinical context, as various conditions and medications can affect results. When results are equivocal, repeating tests or using multiple testing modalities is often necessary for accurate diagnosis.