Required section · Section 3 of 6

What can turn a valid draw into a misleading result

A basal, single time-point cortisol or ACTH answers a screening question only. Deciding whether the adrenal gland has functional reserve, or whether the HPA axis will appropriately suppress, requires a dynamic test: a stimulation test for suspected insufficiency, or a suppression test for suspected excess. The Endocrine Society's adrenal insufficiency guideline frames the cosyntropin (synthetic ACTH1-24) stimulation test as a reserve question: give exogenous ACTH and observe whether the adrenal cortex can respond. A stimulated cortisol that stays below the assay-specific cutoff supports insufficiency. The Endocrine Society's Cushing syndrome guideline frames the overnight low-dose dexamethasone suppression test, late-night salivary cortisol, and 24-hour urine free cortisol as feedback questions: give exogenous glucocorticoid, or sample at the expected nadir, and ask whether the axis suppresses appropriately. Random serum cortisol, random plasma ACTH, and high-dose dexamethasone testing are not recommended as initial Cushing syndrome screening.

Every dose, sampling time, and numeric cutoff in dynamic testing is assay-dependent and governed by the local controlled procedure, not by a portable universal number. Under CLIA (42 CFR 493.1253), a laboratory using an unmodified FDA-cleared test must verify that the manufacturer's reference interval fits its own patient population, and a laboratory using a modified or laboratory-developed test must establish its own reference intervals and performance specifications. A cutoff is therefore always the assay's or the local procedure's, never a portable fixed number.

In a two-site assay, extreme antigen excess can saturate both capture and signal antibodies, causing paradoxically low signal; dilution can restore proportional recovery.

Side by side cards contrasting ACTH plasma, whose handling requirements (including any prechilling, ice, chilled centrifugation, and frozen storage) are set by the cited assay manufacturer, reference laboratory, or local controlled procedure and cannot be copied between systems, with cortisol serum, which tolerates routine ambient handling, with a note that one routine handling path applied to both tubes damages the ACTH result. — Figure 1Same venipuncture, two different handling paths for ACTH plasma and cortisol serum.

Factors that can invalidate or complicate cortisol, ACTH, prolactin, or GH/IGF-1 interpretation.
Factor	Affected analyte	Effect	Resolution
Oral estrogen (raises CBG)	Total cortisol	Raises total cortisol without proportionate free cortisol rise	Note estrogen use; consider free cortisol context
CYP3A4 inducer (e.g., rifampin)	Dexamethasone suppression test	Speeds dexamethasone clearance, falsely non-suppressed	Simultaneous dexamethasone level, per local procedure
Prednisolone cross-reactivity	Immunoassay cortisol	Falsely elevated measured cortisol	Check medication list; confirm assay's cross-reactivity profile
Macroprolactin	Total prolactin	Falsely elevated in an asymptomatic patient	PEG precipitation screen
Hook effect with large mass	Prolactin	Falsely low/near-normal despite very high true value	Serial dilution

Knowledge checks

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Knowledge check 1

What question does a basal, single time-point cortisol or ACTH answer, compared with a dynamic stimulation or suppression test?

Choose one option.

A basal result answers a screening question; a dynamic test asks whether the adrenal gland has reserve or whether the axis appropriately suppresses

A basal result and a dynamic test answer the same question at different costs

A basal result is only useful for suspected excess, and a dynamic test is only useful for suspected insufficiency

Knowledge check 2

Which of the following can make a measured cortisol result misleading without any error in the laboratory's testing process? Select all that apply.

Choose at least 2 options.

Oral estrogen raising cortisol-binding globulin and total cortisol

Prednisolone cross-reacting with the cortisol immunoassay

Acute illness, pain, or venipuncture stress at the time of the draw

A miscalibrated analyzer producing a systematic bias

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