Laboratory Testing for Adrenal Insufficiency
Start with 8 AM plasma cortisol and ACTH measurement simultaneously—this is your initial diagnostic approach for suspected adrenal insufficiency. 1
Initial Laboratory Workup
First-Line Tests (Perform Together)
8 AM plasma cortisol: Interpret using these thresholds 1:
- <5 µg/dL (138 nmol/L): Adrenal insufficiency very likely—proceed to confirm diagnosis
- 5-18 µg/dL (138-500 nmol/L): Indeterminate zone—requires stimulation testing
- >18 µg/dL (500 nmol/L): Adrenal insufficiency unlikely
Plasma ACTH (measured simultaneously with cortisol) 1:
- Elevated ACTH (>2-fold above upper limit) + low cortisol = Primary adrenal insufficiency
- Normal or low ACTH + low cortisol = Secondary (central) adrenal insufficiency
Important caveat: These cortisol thresholds vary between assay methods—verify your laboratory's specific cutoffs, as different immunoassays can produce significantly different values 1, 2.
Additional Initial Labs for Primary Adrenal Insufficiency
Once primary adrenal insufficiency is diagnosed, immediately check 1:
- Plasma renin (elevated in primary AI)
- Serum aldosterone (low in primary AI)
- These confirm mineralocorticoid deficiency
DHEAS levels can be helpful as an adjunctive marker—values well below the lower limit for age and sex suggest primary adrenal insufficiency, though this cannot be used in isolation 3, 4.
Confirmatory Dynamic Testing
When Baseline Tests Are Indeterminate
Cosyntropin (Synacthen) Stimulation Test 1:
- Standard protocol: 250 µg cosyntropin IV or IM (125 µg in children <2 years)
- Measure cortisol at baseline, 30 minutes, and 60 minutes
- Diagnostic threshold: Peak cortisol <18 µg/dL (500 nmol/L) confirms adrenal insufficiency
- Can be performed as first-line testing or after indeterminate baseline cortisol
- Not time-dependent—can be done at any time of day
Critical limitation: The Synacthen test can give false-normal results in recent secondary adrenal insufficiency (<4 weeks post-pituitary surgery or trauma) or partial ACTH deficiency 1.
For Suspected Secondary Adrenal Insufficiency with Normal Synacthen Test
If clinical suspicion remains high despite normal Synacthen test, proceed to 1:
Insulin Tolerance Test (gold standard for secondary AI):
- Reference test for adults, adolescents, and children >2 years
- Peak cortisol >18 µg/dL (500 nmol/L) after achieving blood glucose <40 mg/dL (2.2 mmol/L) excludes ACTH insufficiency
- Contraindications: Seizure disorder, coronary artery disease, stroke history, advanced age, infants <2 years
Alternative tests when insulin tolerance test is contraindicated 1:
- Metyrapone test (short protocol)
- CRH stimulation test
Special Populations
Infants and Young Children
- <4 months old: No circadian cortisol rhythm—repeat baseline measurements and start treatment based on probability if cortisol is low; confirm later with Synacthen test 1
- Look for hypoglycemia, dehydration, and convulsions as presenting features 5
Acute/Emergency Situations
In critically ill patients with suspected adrenal crisis 1:
- Cortisol <18 µg/dL (500 nmol/L) in acute illness = start hydrocortisone immediately
- Draw cortisol and ACTH before treatment, but never delay treatment for test results 6
Tests to Avoid
Do NOT use urinary free cortisol for diagnosing adrenal insufficiency—it is not validated for this purpose 1.
Common Pitfalls
Assay variability: Different cortisol assays produce different values—the traditional 18 µg/dL cutoff may need adjustment based on your laboratory's specific assay 1, 2
Confounding conditions that elevate cortisol 1:
- Estrogen therapy or pregnancy
- Acute intercurrent illness
- These require higher diagnostic thresholds
Medication interference: Exogenous steroids (oral prednisolone, dexamethasone, inhaled fluticasone) confound cortisol interpretation 6
False reassurance from Synacthen test: Can miss early or partial secondary adrenal insufficiency—maintain clinical suspicion and proceed to insulin tolerance test if needed 1