What is the proper procedure for drawing an ACTH (adrenocorticotropic hormone) laboratory sample?

ACTH Lab Draw Procedure

Draw ACTH in a pre-chilled EDTA (purple-top) tube, transport on ice, and centrifuge within 2 hours at room temperature or within 8 hours if kept at 4°C, then freeze the plasma immediately. 1

Sample Collection Timing and Pairing

Obtain the plasma ACTH sample either paired with a morning cortisol sample or at the same time as the baseline sample during a corticotropin stimulation test. 1 The Endocrine Society guidelines specifically recommend this approach for establishing primary adrenal insufficiency (PAI), where an ACTH level ≥2-fold the upper limit of normal combined with confirmed cortisol deficiency indicates PAI.

Key Technical Requirements

• Tube type: EDTA plasma (not serum)
• Pre-analytical handling: While traditional protocols demanded immediate chilled processing, recent evidence shows more flexibility:
  • ACTH remains stable at room temperature for up to 6 hours in EDTA plasma 2
  • If samples cannot reach the lab within 2 hours at room temperature, store immediately at 4°C and process within 8 hours 3
  • The Roche Elecsys immunoassay specifically shows stability up to 12 hours at ambient temperature with <10% degradation 4

Critical Pitfalls to Avoid

Time to centrifugation matters more than temperature alone. Samples left uncentrifuged at room temperature show significant degradation (8-12% change) by 4 hours, but remain stable up to 8 hours at 4°C even when uncentrifuged 3. This means:

• If immediate centrifugation isn't possible, refrigerate the whole blood sample immediately
• Don't assume refrigeration alone preserves ACTH after plasma separation—it doesn't prevent degradation beyond 12 hours 4

Assay-specific considerations: The stability data showing relaxed handling requirements applies primarily to modern immunoassays like the Roche Elecsys 4. Older assays may require stricter protocols, so verify with your laboratory.

Interpretation Context

When ACTH is measured to diagnose PAI, interpret it alongside cortisol:

• ACTH >66 pmol/L (300 ng/L) represents maximum adrenal stimulation 1
• ACTH ≥2× upper reference limit with cortisol <140 nmol/L (5 μg/dL) is highly predictive of PAI 1
• An elevated ACTH with normal cortisol may indicate early-stage PAI 1

Important caveat: ACTH assays demonstrate significant analytical bias between platforms 1, so always interpret results using your laboratory's specific reference ranges rather than absolute cutoffs from the literature.

Practical Algorithm

1. Collect blood in EDTA tube (chilled tube preferred but not essential for modern assays)
2. If processing within 2 hours: Transport at room temperature, centrifuge, freeze plasma
3. If processing delayed 2-8 hours: Refrigerate whole blood at 4°C immediately, then centrifuge and freeze
4. After centrifugation: Freeze separated plasma immediately—don't store at 4°C expecting preservation
5. Pair with cortisol measurement from the same draw for diagnostic interpretation

The evidence shows that previously mandated strict pre-chilled collection and immediate processing are unnecessarily restrictive for modern immunoassays 4, 2, but time remains critical—don't exceed 8 hours even with refrigeration 3.