Which laboratory tests should be ordered to diagnose adrenal insufficiency and assess its cause?

Laboratory Tests for Diagnosing Adrenal Insufficiency

Order an early morning (8 AM) serum cortisol, plasma ACTH, and serum DHEAS simultaneously as your initial diagnostic workup, followed by a 250 µg cosyntropin (ACTH) stimulation test if the morning cortisol is indeterminate (between 5-15 µg/dL or 140-415 nmol/L). 1

Initial Baseline Laboratory Tests

Start with these three tests drawn simultaneously in the early morning:

• Morning serum cortisol (8 AM): This is your first-line screening test

  • <5 µg/dL (<140 nmol/L): Highly suggestive of adrenal insufficiency 1, 2

  • 15 µg/dL: Effectively rules out adrenal insufficiency 2

  • 5-15 µg/dL: Indeterminate—proceed to dynamic testing 2

• Plasma ACTH: Distinguishes primary from secondary adrenal insufficiency 1

  • 2-fold the upper limit of normal (typically >66 pmol/L or 300 ng/L): Indicates primary adrenal insufficiency 1

  • Low or low-normal: Suggests secondary or glucocorticoid-induced adrenal insufficiency 2
  • Critical point: The combination of cortisol <5 µg/dL with ACTH >2-fold elevated is diagnostic for primary adrenal insufficiency without requiring further testing 1

• Serum DHEAS (age- and sex-adjusted): Particularly useful for primary adrenal insufficiency 1, 3

  • Well below the lower limit of normal suggests primary adrenal insufficiency 1
  • Cannot be used in isolation (may be low in elderly patients without disease) 1
  • Recent evidence supports using DHEAS to help rule out primary adrenal insufficiency in indeterminate cases 3

Confirmatory Dynamic Testing

Cosyntropin (ACTH) Stimulation Test - The Diagnostic Gold Standard

When to perform: If morning cortisol is indeterminate (5-15 µg/dL) or when baseline results are not absolutely unequivocal 1

Protocol 1:

• Administer 250 µg cosyntropin (tetracosactide/Synacthen) IV or IM as bolus
• Measure serum cortisol at baseline, 30 minutes, and 60 minutes
• Can be performed at any time of day (not affected by diurnal variation) 1

Interpretation:

• Peak cortisol <18 µg/dL: Diagnostic of adrenal insufficiency 1, 4
• Peak cortisol ≥18 µg/dL: Normal adrenal function 4
• The test has 92% sensitivity for primary adrenal insufficiency 1
• Important caveat: The test performs better for primary (97% sensitivity at 95% specificity) than secondary adrenal insufficiency (57% sensitivity at 95% specificity) 5

Note on low-dose (1 µg) testing: The 1 µg test provides no better diagnostic accuracy than the 250 µg test for primary adrenal insufficiency, though it may have slightly higher sensitivity for secondary adrenal insufficiency 1, 5. The standard 250 µg dose remains preferred for practical reasons 1.

Additional Tests for Primary Adrenal Insufficiency

Plasma renin and serum aldosterone (measured simultaneously) 1:

• Order these in all patients with confirmed primary adrenal insufficiency to assess mineralocorticoid deficiency
• Elevated plasma renin with inappropriately normal or low aldosterone indicates mineralocorticoid deficiency 1
• Mineralocorticoid deficiency may be the only early sign of evolving primary adrenal insufficiency 1

Critical Assay-Specific Considerations

Be aware that cortisol assay methodology significantly affects diagnostic thresholds 6. The traditional 18 µg/dL cutoff was established using polyclonal antibody assays. Newer monoclonal antibody assays may require different cutoffs—recent data suggests optimal cutoffs ranging from 14.5-17.0 µg/dL depending on the specific assay platform 6. Always interpret results using your laboratory's specific reference ranges and validated cutoffs.

Alternative/Emerging Tests

• Salivary cortisone: Emerging as a practical home-based screening alternative that avoids cortisol-binding globulin variability 3
• Insulin tolerance test: Remains the gold standard specifically for secondary adrenal insufficiency when cosyntropin testing is equivocal 7, 4
• CRH stimulation test: Useful for distinguishing hypothalamic from pituitary causes of secondary adrenal insufficiency 4

Common Pitfalls to Avoid

• Cortisol-binding globulin alterations: Conditions affecting CBG (pregnancy, estrogen therapy, critical illness) can falsely lower or raise total cortisol measurements 1. Consider free cortisol or salivary cortisone in these situations 3.
• ACTH sample handling: ACTH is unstable and must be collected in chilled EDTA tubes and processed immediately 1
• Timing errors: While the cosyntropin test can be done anytime, baseline cortisol and ACTH should be drawn in the early morning (8 AM) for proper interpretation 2
• Assay variability: ACTH assays demonstrate significant analytical bias between platforms 1—interpret based on your laboratory's specific reference ranges