What diagnostic tests should be performed in a patient with elevated cortisol to confirm hypercortisolism and identify its cause?

Diagnostic Testing for Elevated Cortisol Levels

When a patient presents with elevated cortisol, you must first confirm true hypercortisolism using at least two screening tests, then measure plasma ACTH to determine whether the condition is ACTH-dependent or ACTH-independent, followed by targeted imaging and potentially bilateral inferior petrosal sinus sampling (BIPSS) to identify the specific cause. 1

Initial Confirmation of Hypercortisolism

Before pursuing extensive workup, exclude all exogenous glucocorticoid sources including oral steroids, inhaled fluticasone, topical preparations, intra-articular injections, and undeclared supplements, as these suppress endogenous cortisol and invalidate testing 2. Failure to exclude exogenous sources is the most common pitfall leading to unnecessary investigations 2.

Required Screening Tests (Obtain 2-3 Measurements of Each)

Perform at least two of the following three tests, with multiple measurements to account for biological variability and cyclic disease 1, 2:

• 24-hour urinary free cortisol (UFC): Values >100 μg/24h on multiple collections are diagnostic, with sensitivity >90% 1, 2. Ensure complete collection by measuring volume and creatinine; incomplete collections are the most common cause of false results 2.

• Late-night salivary cortisol (LNSC): Collect between 23:00-24:00 hours; levels >3.6 nmol/L indicate loss of circadian rhythm with sensitivity 92-100% and specificity 93-100% 2. In pediatric patients, LNSC demonstrates 95% sensitivity and 100% specificity 2.

• 1-mg overnight dexamethasone suppression test (DST): Administer 1 mg at 23:00-24:00, measure cortisol at 08:00; failure to suppress to <1.8 μg/dL (50 nmol/L) is abnormal 1, 2. Consider measuring dexamethasone levels concomitantly to exclude false-positives from abnormal drug metabolism 2.

Critical pitfall: Cyclic Cushing's syndrome produces weeks to months of normal cortisol interspersed with hypercortisolism, requiring extended monitoring during symptomatic periods 2. Obtain 2-3 measurements of each test on different days 2.

Determine ACTH Dependency

Once hypercortisolism is confirmed, measure morning (08:00-09:00h) plasma ACTH to classify the etiology 1, 3:

ACTH-Dependent Cushing's Syndrome (75-80% of cases)

• ACTH >5 ng/L (>1.1 pmol/L): Detectable ACTH indicates ACTH-dependent disease with high certainty 4, 1, 3
• ACTH >29 ng/L (>6.4 pmol/L): Provides 70% sensitivity and 100% specificity for pituitary Cushing's disease specifically 4, 1, 3

ACTH-Independent Cushing's Syndrome

• ACTH <5 ng/L or undetectable: Indicates adrenal source (adenoma, carcinoma, or bilateral hyperplasia) 4, 1

Important: Morning timing is essential because ACTH follows circadian rhythm; afternoon measurements are unreliable and do not correspond to established diagnostic thresholds 1. Patient does not need to be fasting 1.

Diagnostic Algorithm Based on ACTH Results

For ACTH-Dependent Disease (ACTH >5 ng/L)

Step 1: High-resolution pituitary MRI 1

Obtain 3-Tesla MRI with 1-mm thin slices and gadolinium contrast to detect microadenomas as small as 2-3 mm 1. MRI has only 63% sensitivity for ACTH-secreting adenomas, missing approximately one-third of cases 1.

Interpretation based on MRI findings:

• Adenoma ≥10 mm: Proceed directly to transsphenoidal surgery without further testing 1

• Adenoma 6-9 mm: Perform CRH or desmopressin stimulation test; cortisol rise >38 nmol/L at 15 minutes with corresponding ACTH rise supports pituitary source with >70% sensitivity 4, 1

• No adenoma or lesion <6 mm: BIPSS is mandatory 4, 1

Step 2: Bilateral Inferior Petrosal Sinus Sampling (BIPSS) 4, 1

BIPSS is the gold standard for distinguishing pituitary from ectopic ACTH sources, with 96-100% sensitivity and near-100% specificity 4, 1. Perform only in specialized centers by experienced interventional radiologists 4, 1.

Diagnostic criteria:

• Central-to-peripheral ACTH ratio ≥2:1 at baseline OR ≥3:1 after CRH/desmopressin stimulation confirms pituitary source 4, 1
• Inter-petrosal gradient ≥1.4 after stimulation suggests lateralization (58-87.5% concordance with surgical findings) 4, 1

Prerequisites for BIPSS:

• Confirm active hypercortisolism on the morning of the procedure (critical for cyclic disease) 4, 1
• Discontinue all steroidogenesis inhibitors with appropriate washout based on drug half-life 4, 1
• Measure prolactin simultaneously from petrosal sinuses to confirm adequate catheter placement 1

Step 3: If BIPSS indicates peripheral source (ratio <2:1 baseline or <3:1 after stimulation)

Search for ectopic ACTH secretion 1:

• Neck-to-pelvis thin-slice CT scan 1
• If CT negative, obtain ⁶⁸Ga-DOTATATE PET imaging (identifies ~65% of occult neuroendocrine tumors) 1
• Pulmonary carcinoid tumors account for up to 40% of ectopic ACTH cases 1

For ACTH-Independent Disease (ACTH <5 ng/L or undetectable)

Perform adrenal CT or MRI to identify adrenal lesion(s) 1:

• Adrenal adenoma: Laparoscopic adrenalectomy 1
• Adrenal carcinoma: Open adrenalectomy with possible adjuvant therapy 1
• Bilateral hyperplasia: Medical management or unilateral adrenalectomy 1

Common Pitfalls to Avoid

False-positive screening results occur with: 2

• Oral estrogens/contraceptives (increase cortisol-binding globulin, elevating total cortisol without true hypercortisolism) 2
• CYP3A4 inducers (accelerate dexamethasone metabolism, causing false-positive DST) 2
• Pseudo-Cushing's states: severe obesity, depression, alcoholism, polycystic ovary syndrome 2
• Night-shift workers (disrupted circadian rhythm invalidates LNSC) 2

False-negative results occur with: 2

• CYP3A4 inhibitors (increase dexamethasone levels, causing false-negative DST) 2
• Renal impairment or polyuria (invalidate UFC; use LNSC instead) 2

For discordant results: Repeat testing with 2-3 additional collections, measure dexamethasone levels during DST, consider Dex-CRH test to distinguish true Cushing's from pseudo-Cushing's states, or monitor for 3-6 months to capture cyclic disease 2.

Special Considerations

Midnight cortisol testing: A sleeping midnight serum cortisol >207 nmol/L (>7.5 μg/dL) provides 96% sensitivity and 100% specificity, outperforming 24-hour UFC 2. Late-night salivary cortisol is the preferred outpatient alternative 2.

Subclinical hypercortisolism: Found in 5-30% of adrenal incidentalomas; diagnosed by cortisol >1.8 μg/dL after 1-mg DST with low ACTH and altered circadian rhythm 5. Associated with metabolic complications, cardiovascular disease, and osteoporosis despite absence of classic Cushingoid features 5.