What lab tests are used to diagnose Cushing's disease?

Laboratory Tests for Diagnosing Cushing's Disease

The diagnosis of Cushing's disease requires a two-step approach: first, confirm hypercortisolism using 2-3 screening tests (late-night salivary cortisol, 24-hour urinary free cortisol, and overnight dexamethasone suppression test), then differentiate pituitary from ectopic ACTH sources using morning plasma ACTH levels and dynamic testing. 1, 2

Initial Screening Tests to Confirm Hypercortisolism

The Endocrine Society recommends performing 2-3 first-line screening tests rather than relying on a single test, as no single test achieves perfect accuracy 1, 2, 3. The three recommended screening tests are:

Late-Night Salivary Cortisol (LNSC)

• Highest specificity (93-100%) among all first-line screening tests 2
• Sensitivity of 95% for detecting Cushing's syndrome 1
• Collect at least 2-3 samples on consecutive days at the patient's usual bedtime (typically 11 PM-midnight) to account for variability and detect cyclic disease 2
• Critical contraindication: Do NOT use in night-shift workers or anyone with disrupted sleep-wake cycles, as the test relies on normal circadian rhythm 2
• Avoid topical hydrocortisone contamination, which can cause false positives, particularly with mass spectrometry 2

24-Hour Urinary Free Cortisol (UFC)

• Sensitivity of 89% and specificity of 100% 1
• Measures overall cortisol production over 24 hours 2
• Collect at least 2-3 samples to account for day-to-day variability 2
• When measured by liquid chromatography tandem-mass spectrometry, UFC achieves the best combined positive and negative likelihood ratios (10.7 and 0.03 respectively) among first-line tests 4

Overnight Dexamethasone Suppression Test (DST)

• Give 1 mg dexamethasone at midnight, measure serum cortisol at 8 AM 2
• Normal response: cortisol <1.8 μg/dL (50 nmol/L) 2
• Sensitivity of 95% and specificity of 80% 1
• Measuring dexamethasone levels along with cortisol improves test interpretability and helps identify patients with abnormal dexamethasone metabolism 2, 3
• Less useful in women taking estrogen-containing oral contraceptives 2

Interpretation of Screening Results

• If any test is abnormal, repeat 1-2 screening tests to confirm the diagnosis 2, 3
• If all tests are normal, Cushing's syndrome is unlikely 2
• Consider false positives in severe obesity, uncontrolled diabetes, depression, alcoholism, and pregnancy 2, 3
• For inconsistent results, consider cyclic Cushing's syndrome and perform extended monitoring with multiple periodic LNSC measurements 2

Determining the Source: Pituitary vs. Ectopic ACTH

Once hypercortisolism is confirmed, the next step is determining whether the source is ACTH-dependent (pituitary or ectopic) or ACTH-independent (adrenal).

Morning Plasma ACTH Level

• Essential first test to differentiate ACTH-dependent from ACTH-independent causes 1, 2, 3
• Normal or elevated ACTH (>5 ng/L or >1.1 pmol/L): suggests ACTH-dependent Cushing's syndrome (pituitary or ectopic source) 2
• Low or undetectable ACTH: indicates ACTH-independent Cushing's syndrome (adrenal source) 1, 2

Dynamic Testing to Distinguish Pituitary from Ectopic Sources

No single laboratory test can absolutely differentiate between pituitary and ectopic ACTH-secreting tumors, so clinical context and multiple test results must guide management 5.

CRH Stimulation Test

• A ≥20% increase in cortisol from baseline during CRH testing supports pituitary origin (Cushing's disease) 2
• Increased plasma ACTH and cortisol following CRH administration usually indicates Cushing's disease 5

Desmopressin Stimulation Test

• Increased plasma ACTH and cortisol following desmopressin administration usually indicates Cushing's disease 5
• Most pituitary corticotroph adenomas express vasopressin V3 receptors, while most ectopic ACTH-secreting tumors do not 5
• Using more than one dynamic test (CRH plus desmopressin) might further improve accuracy 5

High-Dose Dexamethasone Suppression Test

• Although it has low accuracy overall, it is still used in some countries 5
• A noninvasive alternative using high-dose DST and CRH stimulation test predicts Cushing's disease if both tests are positive, but if tests are discordant, IPSS is necessary 5

Inferior Petrosal Sinus Sampling (IPSS)

IPSS is the gold standard to reliably exclude ectopic ACTH production and should be performed in specialized centers due to potential patient risk 5.

When IPSS is Necessary

• All patients with lesions <6 mm on pituitary MRI should have IPSS 5
• Patients with lesions 6-9 mm: majority of experts recommend IPSS to confirm diagnosis 5
• Patients with lesions ≥10 mm and dynamic testing consistent with Cushing's disease: IPSS is not necessary 5, 3
• If pituitary MRI is negative or equivocal in ACTH-dependent cases 1, 2

IPSS Diagnostic Criteria

• Central-to-peripheral ACTH gradient <2 before or <3 after stimulation suggests an ectopic tumor 5
• Central-to-peripheral ACTH ratio ≥2:1 before stimulation and ≥3:1 after CRH stimulation indicates pituitary source with 100% sensitivity 1, 3
• Prolactin measurement may improve diagnostic accuracy, and it is essential that the patient is hypercortisolemic at the time of IPSS 5
• IPSS is not sufficiently reliable for tumor lateralization to the right or left side of the pituitary gland 5

Emerging Non-Invasive Approaches

A non-invasive approach using CRH and desmopressin stimulation plus pituitary MRI, followed by whole-body CT if diagnosis is equivocal, correctly diagnosed Cushing's disease in approximately half of patients in one series, potentially eliminating the need for IPSS 5. However, this combination is currently limited to specialized centers 5.

Common Pitfalls to Avoid

• Never perform LNSC in night-shift workers or those with disrupted circadian rhythms 2
• Always exclude exogenous glucocorticoid use before any biochemical testing, as this is the most common cause of Cushing's syndrome 3
• Beware of pseudo-Cushing's states (severe obesity, uncontrolled diabetes, depression, alcoholism) that can cause false-positive screening results 2, 3
• Do not rely on a single screening test—perform 2-3 tests to improve diagnostic accuracy 1, 2
• Consider cyclic Cushing's syndrome when results are inconsistent, requiring extended monitoring 2
• Clinical presentation should always be considered when interpreting test results, as a pituitary lesion on MRI could be an incidental nonfunctioning adenoma with an ectopic ACTH source 5