What laboratory tests are recommended for a patient suspected of having Cushing syndrome?

Laboratory Testing for Cushing Syndrome

For patients with suspected Cushing syndrome, begin screening with 2-3 first-line tests: late-night salivary cortisol (LNSC), 24-hour urinary free cortisol (UFC), and/or overnight 1-mg dexamethasone suppression test (DST), followed by plasma ACTH measurement if screening confirms hypercortisolism. 1, 2

Initial Screening Tests

The diagnostic approach requires multiple tests due to variability in cortisol secretion and the possibility of cyclic disease. 1, 2

Late-Night Salivary Cortisol (LNSC)

• Collect at least 2-3 samples on consecutive days at the patient's usual bedtime (typically 11 PM-midnight) when cortisol should be at its nadir 1, 2
• Offers the highest specificity (93-100%) among first-line screening tests 2
• Sensitivity is 95% for detecting Cushing syndrome 2
• Critical contraindication: Do NOT use in night-shift workers or anyone with disrupted sleep-wake cycles, as the test relies on intact circadian rhythm 2
• Avoid topical hydrocortisone preparations before collection, as they can contaminate samples 2

24-Hour Urinary Free Cortisol (UFC)

• Collect 2-3 separate 24-hour urine collections to account for day-to-day variability 1, 3
• Sensitivity exceeds 90% for detecting hypercortisolism 3
• Diagnostic threshold: values >100 μg/24h (1.6 μmol/24h) are typically diagnostic in symptomatic patients 3
• Less reliable in patients with renal impairment (CrCl <60 mL/min) or polyuria (>5 L/24h) 3
• Ensure complete urine collections with appropriate total volumes to avoid false results 4

Overnight 1-mg Dexamethasone Suppression Test (DST)

• Administer 1 mg dexamethasone at 11 PM, measure serum cortisol at 8 AM 1, 2
• Normal suppression: cortisol <1.8 μg/dL (50 nmol/L) 1, 5
• Preferred for shift workers and patients with disrupted circadian rhythm 1
• Less reliable in women taking oral estrogen-containing contraceptives, as estrogen increases corticosteroid-binding globulin 1
• Consider measuring dexamethasone levels alongside cortisol to rule out abnormal metabolism or malabsorption 1, 5

Confirming the Diagnosis

When Screening Tests Are Abnormal

• Repeat 1-2 screening tests to confirm before proceeding with further evaluation 1, 2
• If all tests remain normal but clinical suspicion is high, refer to an endocrinologist for extended monitoring 2
• Consider cyclic Cushing syndrome if results are inconsistent—perform multiple sequential LNSC measurements over weeks to months 2

Ruling Out Pseudo-Cushing States

Pseudo-Cushing states (severe obesity, uncontrolled diabetes, depression, alcoholism) can cause false-positive screening results. 1, 2

• Monitor for 3-6 months to see if symptoms resolve with treatment of underlying conditions 1
• Consider dexamethasone-CRH test (Dex-CRH) at an expert center, with cortisol rise >38 nmol/L at 15 minutes after CRH indicating true Cushing syndrome 5
• Measure dexamethasone levels to ensure adequate suppression and rule out false positives 1, 5

Determining the Etiology

Plasma ACTH Measurement

Once hypercortisolism is confirmed, measure morning (8-9 AM) plasma ACTH to differentiate ACTH-dependent from ACTH-independent causes. 2, 5

• ACTH >5 ng/L (>1.1 pmol/L): Indicates ACTH-dependent Cushing syndrome (pituitary adenoma or ectopic ACTH source) 2, 5
• ACTH >29 ng/L: 70% sensitivity and 100% specificity for Cushing disease (pituitary source) 5
• Low or undetectable ACTH: Indicates ACTH-independent Cushing syndrome (adrenal source) 2, 5

For ACTH-Dependent Cushing Syndrome

Proceed with pituitary MRI (preferably 3T with thin slices) to detect pituitary adenoma. 1, 5

• If adenoma ≥10 mm: Presume Cushing disease and proceed to surgery 5
• If adenoma 6-9 mm: Consider CRH stimulation test for additional confirmation 5
• If no adenoma or <6 mm lesion: Perform bilateral inferior petrosal sinus sampling (BIPSS) to definitively distinguish pituitary from ectopic ACTH sources 1, 5

BIPSS Diagnostic Criteria

• Central-to-peripheral ACTH ratio ≥2:1 at baseline or ≥3:1 after CRH/desmopressin stimulation confirms pituitary source 5
• Sensitivity 96-100%, specificity approaching 100% when performed correctly 5
• Must be performed at specialized centers by experienced interventional radiologists 5
• Measure prolactin levels simultaneously to confirm adequate venous sampling 5

For ACTH-Independent Cushing Syndrome

Perform adrenal CT or MRI to identify adrenal lesion(s). 5

Common Pitfalls to Avoid

• Never use ACTH stimulation testing to diagnose Cushing syndrome—it is not a screening test for hypercortisolism 3
• Do not rely on a single UFC collection—always obtain 2-3 samples 1, 3
• Always exclude exogenous glucocorticoid use (including topical, inhaled, and injected preparations) before any biochemical testing 2
• Do not perform BIPSS to diagnose hypercortisolism—it is only for localization after confirming ACTH-dependent disease 1
• Ensure patients are in the active phase before performing BIPSS in cyclic Cushing disease by confirming hypercortisolism immediately prior with LNSC, DST, or UFC 5
• Stop medical therapy for Cushing disease (steroidogenesis inhibitors) before BIPSS to enable accurate interpretation 5