What is the protocol for a cortisol stimulation test using Adrenocorticotropic hormone (ACTH)?

Cortisol Stimulation Test Protocol Using ACTH

The high-dose (250-μg) ACTH stimulation test is the recommended protocol for cortisol stimulation testing due to its ease of administration, safety profile, and comparable accuracy to alternative methods. 1

Standard Protocol for ACTH Stimulation Test

Preparation

• Patients receiving cortisone, hydrocortisone, or spironolactone should omit their pre-test doses on the day of testing
• Women taking estrogen-containing medications may exhibit abnormally high basal cortisol levels
• Testing can be performed at any time of day, though morning testing is traditionally preferred 2

Administration Method

1. Collect baseline sample:

   • Draw 6-7 mL of blood in a heparinized tube for baseline cortisol measurement

2. Administer ACTH:

   • Reconstitute 0.25 mg (250 μg) of cosyntropin with 1 mL of 0.9% Sodium Chloride Injection, USP
   • Administer via one of these routes:
     • Intramuscular injection (most common)
     • Direct intravenous injection over 2 minutes (in 2-5 mL of saline)
     • Intravenous infusion over 4-8 hours (at approximately 40 μg/hour)

3. Collect post-stimulation samples:

   • Draw blood at 30 minutes after ACTH administration
   • Draw blood at 60 minutes after ACTH administration (recommended)
   • Both samples should be refrigerated until laboratory analysis 3

Interpretation of Results

Normal response criteria:

1. Baseline cortisol level should exceed 5 μg/dL
2. 30-minute level should show an increment of at least 7 μg/dL above baseline
3. 30-minute level should exceed 18 μg/dL
4. 60-minute level typically shows an approximate doubling of the baseline value 3

Important Clinical Considerations

Timing of Sample Collection

• While 30-minute samples are commonly used, collecting both 30 and 60-minute samples is strongly recommended
• Studies show that 54% of patients reach peak cortisol levels at 60 minutes 4
• 13.4% of patients may fail to reach adequate cortisol levels at 30 minutes but will demonstrate normal response at 60 minutes 2
• Using only 30-minute values could result in overdiagnosis of adrenal insufficiency in approximately 1 in 7 patients 2

Dose Considerations

• The high-dose (250-μg) ACTH test is preferred over low-dose (1-μg) testing due to:
  • Similar diagnostic accuracy
  • Easier practical administration (no bedside preparation required)
  • Better standardization across institutions 1
• For pediatric patients aged 2 years or less, a reduced dose of 0.125 mg may be sufficient 3

Testing Time of Day

• Research indicates that time of day does not significantly affect 30 and 60-minute cortisol responses to ACTH stimulation
• No significant differences in mean cortisol levels at 30 and 60 minutes when comparing morning vs afternoon testing 2

Patient Factors Affecting Results

• Weight status may influence timing of peak cortisol:
  • Overweight/obese individuals tend to peak at 30 minutes
  • Normal/underweight individuals tend to peak at 60 minutes 4
• Medications that can affect results:
  • Recent glucocorticoid use (within 72-96 hours) may suppress response 5
  • Estrogen-containing medications can elevate baseline cortisol
  • Spironolactone may affect baseline values 3

Pitfalls to Avoid

• Using only 30-minute cortisol values may lead to false positive diagnosis of adrenal insufficiency
• Failing to discontinue interfering medications before testing
• Not collecting both 30 and 60-minute samples
• Improper handling of samples (should be refrigerated if not analyzed within 12 hours)
• Testing within 3 days of initiating high-dose glucocorticoid therapy may yield unreliable results 5

The high-dose ACTH stimulation test remains the most widely used and reliable diagnostic test for adrenal insufficiency, with the 60-minute cortisol value providing the most accurate assessment of adrenal function.