What labs check cortisol levels?

Laboratory Tests to Check Cortisol Levels

To evaluate cortisol levels, order one or more of the following tests depending on clinical context: serum cortisol (drawn at 8:00-9:00 AM), 24-hour urinary free cortisol (UFC), late-night salivary cortisol (LNSC), or the 1 mg overnight dexamethasone suppression test (DST). 1, 2

Serum Cortisol Testing

Morning Serum Cortisol

• Draw serum cortisol at 8:00-9:00 AM to capture the physiologic peak of cortisol secretion, which provides the most reliable assessment of adrenocortical function 1
• The reference range for morning (0900 hours) plasma cortisol in adults is 140–700 nmol/L (5.1–25.4 μg/dL) 1
• Always measure ACTH simultaneously with cortisol from the same blood draw at 8:00-9:00 AM when distinguishing primary from secondary adrenal insufficiency 1
• ACTH is extremely labile and requires immediate processing on ice, so coordinate with the laboratory before drawing 1

Critical Timing Considerations

• Never order morning cortisol testing in shift workers or patients with disrupted circadian rhythms, as their cortisol peaks occur at different times and yield unreliable results 1
• Never interpret a single cortisol value without knowing the time of collection, as the same value may be normal at 0900 hours but pathologically elevated at midnight 1

Confounding Factors

• Document medications affecting cortisol binding globulin (CBG), including oral estrogens, pregnancy status, and chronic active hepatitis, as these increase total cortisol levels and can lead to false interpretation 1
• Patients should avoid strenuous exercise for 24-48 hours before testing, and should not undergo cognitive assessments immediately before blood draw, as recent physical or mental stress significantly elevates cortisol 1

Screening Tests for Hypercortisolism (Cushing's Syndrome)

First-Line Options

• Start with the 1 mg overnight dexamethasone suppression test (DST) as the preferred screening test for identifying autonomous cortisol secretion 1, 2
• Administer 1 mg dexamethasone at 11 PM and measure serum cortisol at 8 AM the following morning 1, 2
• Interpretation: <50 nmol/L (<1.8 μg/dL) excludes cortisol hypersecretion, 51-138 nmol/L suggests possible autonomous cortisol secretion, and >138 nmol/L indicates evidence of cortisol hypersecretion 1
• DST has sensitivity >90% for detecting hypercortisolism 1

Late-Night Salivary Cortisol (LNSC)

• LNSC offers 95% sensitivity and 100% specificity for diagnosing Cushing's syndrome 2, 3
• Collect at least 2-3 samples at the patient's usual bedtime (typically 11 PM-midnight) on consecutive days 2, 3
• The diagnostic utility relies on the principle that patients with Cushing's syndrome lose their normal circadian nadir of cortisol secretion 3
• Absolute contraindication: Do NOT perform LNSC in night-shift workers or anyone with disrupted day/night cycles 3
• Topical steroid preparations can contaminate samples, particularly when mass spectrometry is used 3

24-Hour Urinary Free Cortisol (UFC)

• UFC measures increased bioavailable cortisol with 89% sensitivity and 100% specificity when values exceed 193 nmol/24h (70 μg/m²) 2
• Collect 2-3 samples to account for variability 4, 2
• UFC has lower sensitivity compared to DST and LNSC, and is useful as an ancillary test when DST or LNSC results are equivocal 1
• For patients with renal impairment (CrCl <60 mL/min) or significant polyuria, LNSC may be preferred over UFC 2

Diagnostic Algorithm for Suspected Hypercortisolism

Initial Testing Strategy

• For suspected hypercortisolism, start with overnight 1 mg DST as the preferred screening test, and consider adding LNSC and/or 24-hour UFC as complementary tests 4, 1, 2
• There is no single preferred diagnostic test for Cushing's syndrome; clinical judgment and index of suspicion are very important 4
• DST may be the preferred test for shift workers and patients with disrupted circadian rhythm due to uneven sleep schedules 4

Confirmatory Testing

• If any screening test is abnormal, repeat 1-2 screening tests to confirm the diagnosis 2, 3
• For cyclic Cushing's syndrome, multiple sequential LNSC tests are particularly useful, as patients exhibit weeks to months of normal cortisol secretion interspersed with episodes of excess 3
• In classical cyclic Cushing's disease or in patients with unpredictable fluctuating cortisol levels, dynamic testing should be preceded by a confirmatory LNSC, DST, or UFC to document that the patients are in the active phase 4

Determining the Source

• Measure morning plasma ACTH at 08:00-09:00 hours simultaneously with cortisol to differentiate ACTH-dependent from ACTH-independent causes 1, 2, 3
• Normal/elevated ACTH (>1.1 pmol/L or >5 ng/L) suggests ACTH-dependent Cushing's syndrome (pituitary or ectopic source), with 68% sensitivity and 100% specificity 1, 2
• Low/undetectable ACTH indicates ACTH-independent Cushing's syndrome (adrenal source) 2, 3

Common Clinical Scenarios

Evaluating Adrenal Insufficiency

• Order morning cortisol when patients present with nonspecific symptoms including fatigue, nausea/vomiting, anorexia, weight loss, orthostatic hypotension, or unexplained hyponatremia with hyperkalemia 1
• In primary adrenal insufficiency, ACTH will be elevated (>1.1 pmol/L) with low cortisol, while in secondary (central) adrenal insufficiency, both ACTH and cortisol will be low 1

Screening Adrenal Incidentalomas

• All patients with adrenal incidentalomas must be screened for autonomous cortisol secretion, with the 1 mg dexamethasone suppression test as the preferred initial test 1

Laboratory Methodology Considerations

• Automated immunoassays are widely used to measure cortisol but lack specificity and show significant inter-assay differences 5, 6
• Liquid chromatography-tandem mass spectrometry (LC-MS/MS) offers improved specificity and sensitivity for measuring cortisol in serum, urine, and saliva 5, 6, 7
• Currently, there is no preference for mass spectrometry over immunoassay in measuring cortisol level for diagnosis to ensure that patients with mild hypercortisolism are not excluded 4

Critical Pitfalls to Avoid

• Always exclude iatrogenic Cushing's syndrome by thoroughly reviewing all glucocorticoid use before any biochemical testing, as failure to do so leads to unnecessary testing 3
• A single normal test does not exclude Cushing's syndrome, especially in mild or cyclic cases, and multiple tests are often needed 2
• Measuring dexamethasone levels along with cortisol may be useful if a false-positive DST is suspected due to the clinical scenario 4, 2
• Oral estrogen therapy may affect DST results, and DST may not be reliable in women treated with oral estrogen 4, 2
• Pseudo-Cushing's states (psychiatric disorders, alcoholism, obesity, polycystic ovary syndrome) can activate the HPA axis and cause mildly elevated cortisol levels 2, 3