What is the proper timing for the cosyntropin (ACTH) injection and subsequent cortisol draws in an ACTH stimulation test?

ACTH Stimulation Test Protocol: Timing of Injection and Cortisol Draws

Administer 0.25 mg cosyntropin (250 mcg) intravenously or intramuscularly, obtain a baseline cortisol immediately before injection, then draw cortisol samples at exactly 60 minutes post-injection—the 30-minute sample adds no diagnostic value and can be safely omitted. 1, 2, 3

Standard Protocol

Dose and Administration

• Adults: 0.25 mg (250 mcg) cosyntropin IV or IM 1
• Pediatric patients (2-17 years): 0.25 mg (250 mcg) 1
• Pediatric patients (<2 years): 0.125 mg (125 mcg) 1
• Either intravenous or intramuscular route is acceptable, with similar diagnostic accuracy 4

Timing of Blood Draws

• Baseline (time 0): Draw cortisol immediately before cosyntropin administration 1
• 60 minutes post-injection: Draw cortisol at exactly 60 minutes after administration 1, 2, 5, 3
• The 30-minute sample is unnecessary: Multiple studies demonstrate that a single 60-minute cortisol measurement is 99.7% concordant with traditional testing that includes both 30- and 60-minute samples 5, 3

Why the 60-Minute Sample Alone Is Sufficient

The evidence strongly supports omitting the 30-minute draw:

• In a study of 393 tests, approximately 5% of patients showed insufficient cortisol at 30 minutes but adequate response (≥420 nmol/L or ≥15.2 mcg/dL) at 60 minutes—these patients would be misdiagnosed with adrenal insufficiency if only the 30-minute sample were used 2
• Critically, no patient in any study had adequate cortisol at 30 minutes that then dropped below the diagnostic threshold at 60 minutes 2, 3
• A single 60-minute cortisol concentration showed 99.7% concordance with the full traditional test in hospitalized patients, with 98% positive percent agreement 5
• The 30-minute sample adds no incremental diagnostic value compared to the 60-minute measurement alone 3

Diagnostic Interpretation

Cortisol Thresholds

• Peak cortisol <500 nmol/L (<18 mcg/dL) at 60 minutes: Diagnostic of adrenal insufficiency 6, 1
• Peak cortisol >550 nmol/L (>20 mcg/dL) at 60 minutes: Normal response, excludes adrenal insufficiency 6, 7
• Peak cortisol 500-550 nmol/L (18-20 mcg/dL): Borderline zone; clinical context and assay-specific cutoffs guide interpretation 1

Important Caveats

• Cutoff values may vary according to the specific cortisol assay used—verify your laboratory's reference range 1
• In healthy men, the lowest 60-minute cortisol peak observed was 16.3 mcg/dL (450 nmol/L), supporting the <18 mcg/dL cutoff for insufficiency 4
• Most subjects reach maximal cortisol response at 60 minutes, not 30 minutes 4, 8

Pre-Test Medication Management

Medications to Stop Before Testing

• Glucocorticoids and spironolactone: Stop on the day of testing 1
• Long-acting glucocorticoids: May require longer washout period before testing 1
• Estrogen-containing drugs: Stop 4-6 weeks before testing to allow cortisol-binding globulin levels to normalize 1
• Exogenous steroids (including inhaled fluticasone, prednisone, prednisolone): Can suppress the HPA axis and confound results 6, 7

Special Considerations

• If you must treat suspected adrenal crisis but still need diagnostic testing later, use dexamethasone 4 mg IV instead of hydrocortisone, as dexamethasone does not interfere with cortisol assays 6
• Conditions affecting cortisol-binding globulin (pregnancy, hepatitis, cirrhosis, nephrotic syndrome) can alter total cortisol levels—consider measuring cortisol-binding globulin concurrently if these conditions are present 1

Critical Clinical Pitfalls to Avoid

• Never delay treatment of suspected acute adrenal crisis for diagnostic testing—if the patient is clinically unstable with hypotension, collapse, or severe GI symptoms, immediately give 100 mg IV hydrocortisone and 0.9% saline infusion at 1 L/hour 6
• Do not rely on electrolyte abnormalities alone: Hyponatremia is present in 90% of adrenal insufficiency cases, but hyperkalemia occurs in only ~50% of cases 6
• Avoid testing patients currently on corticosteroids: Morning cortisol and ACTH stimulation tests are unreliable in patients taking exogenous steroids due to iatrogenic HPA axis suppression—wait until adequate washout time has elapsed 6
• Using only the 30-minute sample risks overdiagnosing adrenal insufficiency: Approximately 5% of patients with adequate adrenal reserve will have insufficient 30-minute cortisol that rises to normal by 60 minutes 2, 8

Practical Advantages of Single 60-Minute Sampling

• Simplifies the test protocol and reduces patient discomfort from multiple venipunctures 2
• Saves laboratory costs and resources by eliminating unnecessary cortisol measurements 2
• Reduces false-positive screens: Significantly fewer false-positive results occur with a single 60-minute measurement compared to 30-minute sampling (P < .001) 5
• 100% concordance in ICU patients and 99.6% concordance in floor patients when comparing single 60-minute sampling to traditional protocol 5