Lab Tests to Rule Out Cushing's Syndrome
To rule out Cushing's syndrome, perform at least two of three first-line screening tests: 24-hour urinary free cortisol (UFC), late-night salivary cortisol (LNSC), or overnight 1-mg dexamethasone suppression test (DST), with UFC and LNSC being preferred for their superior sensitivity (>90%) and specificity (approaching 100%). 1
Recommended Screening Tests
First-Line Options (Choose at Least 2)
24-hour urinary free cortisol (UFC): Collect for 3 consecutive days with diagnostic cutoff >193 nmol/24h (>70 μg/m²), demonstrating 89% sensitivity and 100% specificity 1. Values >100 μg/24h (1.6 μmol/24h) are typically diagnostic in symptomatic patients 1. When measured by LC-MS/MS, UFC achieves the best accuracy among screening tests with positive likelihood ratio of 10.7 and negative likelihood ratio of 0.03 2.
Late-night salivary cortisol (LNSC): Obtain samples on 2-3 separate nights with abnormal threshold >3.6 nmol/L, achieving 95% sensitivity and 100% specificity 1. This test has the highest specificity among screening modalities 3 and is particularly useful for outpatients since cortisol is stable at room temperature 4.
Overnight 1-mg dexamethasone suppression test (DST): Administer 1 mg dexamethasone at midnight with serum cortisol measured at 8 AM; values >1.8 μg/dL (50 nmol/L) indicate Cushing's syndrome 1. Cortisol >5 μg/dL (138 nmol/L) indicates overt disease 3. This test has >90% sensitivity and is particularly effective for ruling out the condition when negative 5.
Critical Testing Principles
Never rely on a single test: Perform at least 2-3 abnormal screening tests before confirming the diagnosis, as no single test achieves 100% diagnostic accuracy 1. This is especially important because 24-hour UFC can vary by up to 50% between collections 1.
Exclude exogenous glucocorticoid use before any biochemical testing, including oral prednisolone, dexamethasone, and fluticasone inhalers, as these confuse interpretation 1, 3.
Diagnostic Algorithm
Initial screening: Order at least two of the three first-line tests (UFC, LNSC, DST) simultaneously 1
If results are discordant: Obtain 2-3 additional collections of each test to account for variability and cyclic disease 1, 3
If ≥2 screening tests are abnormal: Measure morning (09:00h) plasma ACTH to determine if Cushing's syndrome is ACTH-dependent (ACTH >5 ng/L) or ACTH-independent (low/undetectable ACTH) 1
If ACTH is normal/elevated: Proceed to pituitary MRI 1
Common Pitfalls to Avoid
Factors Causing False-Positive Results
Oral contraceptives and estrogen therapy: Increase cortisol-binding globulin, falsely elevating total cortisol 3. Consider alternative tests or discontinue estrogen before testing 5.
CYP3A4 inducers: Accelerate dexamethasone metabolism, causing false-positive DST results 3. Consider measuring dexamethasone levels concomitantly with cortisol to confirm adequate drug absorption 1, 5.
Pseudo-Cushing's states: Depression, alcoholism, severe obesity, and polycystic ovary syndrome can cause mild hypercortisolism mimicking true disease 3. Consider Dex-CRH test to distinguish if results remain equivocal 1.
Night-shift workers: Should not undergo LNSC testing due to disrupted circadian rhythm 3. DST may be preferred in this population 5.
Factors Causing False-Negative Results
CYP3A4 inhibitors: Increase dexamethasone levels, causing false-negative DST results 3
Renal impairment or polyuria: Invalidate UFC results; use LNSC instead 3
Cyclic Cushing's syndrome: Can produce weeks to months of normal cortisol secretion interspersed with excess, requiring extended monitoring during symptomatic periods 1, 3
Collection Issues
Incomplete 24-hour urine collection: Measure volume and total creatinine excretion to assess completeness 1. Avoid copper contamination of collection apparatus 1.
Salivary cortisol contamination: Avoid dental work, teeth brushing, or oral trauma within 1-2 hours of collection 3. Topical hydrocortisone preparations can contaminate samples 3.
Special Populations
Children and Adolescents
Use the same three screening tests with age-appropriate cutoffs: UFC >193 nmol/24h (>70 μg/m²), midnight sleeping cortisol ≥50 nmol/L (≥1.8 μg/dL), or LNSC based on local assay cutoff 1
Low-dose dexamethasone suppression test (LDDST): 0.5 mg 6-hourly for 48 hours, with serum cortisol >50 nmol/L (>1.8 μg/dL) indicating Cushing's syndrome 1
Adrenal Incidentalomas
- DST is particularly useful as first-line test, with cortisol values >5 μg/dL identifying patients with dysregulated cortisol secretion 5