Adrenocorticotropic Hormone (Cosyntropin) Stimulation Test

Etiology and Epidemiology

The cosyntropin stimulation test is used to evaluate 3 distinct etiologies of adrenal insufficiency: primary adrenal insufficiency (Addison disease), secondary adrenal insufficiency, and glucocorticoid-induced adrenal insufficiency. Each condition is associated with different underlying pathophysiologic mechanisms and epidemiologic patterns.

Primary Adrenal Insufficiency

Primary adrenal insufficiency is rare, with global prevalence estimates ranging from 4 to 221 cases per 1 million individuals.[1] The most common cause is autoimmune adrenalitis, characterized by autoimmune destruction of the adrenal cortex. Additional etiologies include:

• Congenital adrenal hyperplasia

• Infectious causes such as tuberculosis, HIV, cytomegalovirus, and fungal infections [1][2]

• Pharmacologic inhibition from high-dose azole antifungals or adrenal enzyme inhibitors (eg, mitotaine, ketoconazole, metyrapone, and etomidate) [1][2]

• Surgical removal of adrenal cortical tissue [1]

• Metastatic disease [3]

Primary adrenal insufficiency involves destruction or dysfunction of all zones of the adrenal cortex, resulting in deficiencies of cortisol, aldosterone, and adrenal androgens. Consequently, affected patients often present with manifestations of both glucocorticoid and mineralocorticoid deficiency, including hypotension, hyponatremia, hyperkalemia, and volume depletion.

Secondary Adrenal Insufficiency

Secondary adrenal insufficiency affects approximately 140 to 279 per 1 million individuals worldwide.[1] The condition results from decreased pituitary production of corticotropin (ACTH) due to disorders involving the pituitary gland.[1] Common causes include:

• Pituitary tumors
• Pituitary hemorrhage (apoplexy)
• Inflammatory or infiltrative conditions, including hypophysitis, sarcoidosis, and hemochromatosis
• Pituitary surgery or radiation therapy [1][3]
• Medications that suppress corticotropin production, particularly opioids [1]
• Central nervous system radiation [3]

Unlike primary adrenal insufficiency, aldosterone production remains intact in secondary adrenal insufficiency because regulation of the zona glomerulosa depends primarily on the renin–angiotensin system rather than ACTH. Consequently, hyperkalemia and significant volume depletion are typically absent.

Glucocorticoid-Induced Adrenal Insufficiency

Glucocorticoid-induced adrenal insufficiency is the most common form of adrenal insufficiency worldwide, although its precise incidence remains unknown. The condition occurs in the setting of exogenous glucocorticoid therapy, which is prescribed to approximately 1% to 3% of the adult population.[1] Chronic exposure suppresses hypothalamic corticotropin-releasing hormone (CRH) secretion, thereby reducing pituitary corticotropin (ACTH) release and leading to adrenocortical atrophy.[1][4]

Among patients receiving supraphysiologic glucocorticoid doses, the prevalence of adrenal suppression ranges from approximately 10% to 100%, depending on dose, duration of therapy, and individual susceptibility.[1] Similar to secondary adrenal insufficiency, aldosterone production remains preserved.[4]

Clinical Significance

Most patients with primary and secondary adrenal insufficiency experience months to years of nonspecific symptoms before diagnosis, and up to 50% of patients present with adrenal crisis, a life-threatening emergency.[1] The cosyntropin stimulation test is considered the diagnostic gold standard for primary adrenal insufficiency. However, its sensitivity is lower (approximately 64%) for secondary adrenal insufficiency, particularly in cases of recent onset.[1][2][5]

Pathophysiology

The cosyntropin stimulation test evaluates adrenal cortical function by directly measuring the adrenal gland’s ability to produce cortisol in response to supraphysiologic ACTH stimulation. This assessment provides valuable insight into the integrity of the HPA axis, although important limitations exist when distinguishing between primary and secondary causes of adrenal insufficiency.

The test may be performed at any time of day, as results are not significantly influenced by diurnal variation.[2][6] In cases of glucocorticoid-induced adrenal insufficiency, the test helps assess whether chronic exogenous glucocorticoid exposure has led to sufficient adrenocortical atrophy to impair cortisol responsiveness. However, diagnostic reliability may be reduced in patients with shorter therapy durations.[4]

Importantly, the cosyntropin stimulation test should never delay empiric glucocorticoid administration in patients with suspected adrenal crisis. Definitive diagnostic testing can be completed after the patient has been clinically stabilized.[6]

Mechanism of Action

Cosyntropin is a synthetic analog comprising the first 24 amino acids of the native 39–amino acid ACTH molecule.[2] When administered at the standard 250 μg dose, cosyntropin produces supraphysiologic stimulation of the adrenal cortex by binding to melanocortin-2 receptors on adrenocortical cells, thereby triggering cortisol synthesis and secretion.[7] In healthy individuals, doses as low as 5 to 10 μg are sufficient to produce near-maximal adrenal stimulation.[7]

Evaluation of Adrenal Cortical Function

The cosyntropin stimulation test directly measures the functional reserve of the adrenal cortex. Serum cortisol samples are obtained at baseline and at 30 and 60 minutes following cosyntropin administration.[1] A peak cortisol level below 500 nmol/L (18 μg/dL) at either 30 and 60 minutes is consistent with adrenocortical insufficiency, although specific diagnostic thresholds vary by assay. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) assays use lower cutoffs of approximately 412 nmol/L at 30 minutes and 485 nmol/L at 60 minutes.[4][8]

In primary adrenal insufficiency, destruction or dysfunction of the adrenal cortex prevents an appropriate cortisol response to ACTH stimulation, regardless of the dose administered.[2] Under these conditions, the cosyntropin stimulation test functions as the diagnostic gold standard because endogenous ACTH secretion is already maximally elevated and exogenous cosyntropin fails to elicit a significant increase in cortisol secretion.[7]

Assessment of Hypothalamic–Pituitary–Adrenal Axis Integrity

The cosyntropin stimulation test provides an indirect and limited assessment of the entire HPA axis. In secondary adrenal insufficiency (pituitary ACTH deficiency) and tertiary adrenal insufficiency (hypothalamic CRH deficiency), chronic ACTH deficiency leads to progressive adrenocortical atrophy.[7] When atrophy is advanced, the adrenal glands may fail to respond adequately even to supraphysiologic doses of cosyntropin, leading to an abnormal test result.[1]

Important limitations affect the detection of secondary and tertiary adrenal insufficiency. A meta-analysis demonstrated a sensitivity of 64% and a specificity of 93% for secondary adrenal insufficiency.[1] Patients with mild or recent-onset ACTH deficiency may have normal test results because a supraphysiologic cosyntropin dose can partially stimulate atrophied adrenal glands that would not respond appropriately to physiologic ACTH levels.[1][7] In such cases, alternative dynamic testing, such as the insulin tolerance test, overnight metyrapone test, or glucagon stimulation test, may be required when clinical suspicion remains high despite a normal cosyntropin test.[1] 

Specimen Requirements and Procedure

The cosyntropin stimulation test requires the measurement of serum cortisol levels at baseline and at 30 and 60 minutes after cosyntropin administration. The standard adult dose is 0.25 mg (250 μg), administered by intravenous or intramuscular injection.

Pretest Preparation

Glucocorticoids and spironolactone should be withheld on the day of testing. Long-acting glucocorticoids may need to be discontinued for a longer period before testing. Estrogen-containing drugs should be discontinued 4 to 6 weeks before the test, as they increase cortisol-binding globulin (CBG) concentrations and may interfere with the interpretation of results. However, diagnostic testing should not delay emergency glucocorticoid treatment in suspected adrenal crisis; confirmatory testing can be performed after clinical stabilization.[6]

Testing Procedures

The cosyntropin stimulation test can be performed at any time of day, as results are not significantly influenced by diurnal variation.[2][6] As summarized in a recent review, dynamic evaluation involves obtaining a baseline serum cortisol level, followed by a repeat measurement 60 minutes after intravenous or intramuscular administration of 250 µg of cosyntropin (corticotropin). A normal response is defined as a cortisol concentration of at least 18 µg/dL at 60 minutes.[1]

Cosyntropin may be administered intravenously or intramuscularly. Reconstituted solutions should be used immediately and discarded if not administered promptly. Any solution that appears cloudy or contains particulate matter should not be administered.[2][6][7]

Interfering Factors

Several factors can interfere with the accuracy and interpretation of the cosyntropin stimulation test, particularly those that alter measured serum cortisol levels or affect CBG concentrations. Changes in CBG can influence total cortisol values independent of true adrenal function. In addition, conditions that affect cortisol metabolism may produce misleading results if these influences are not carefully considered within the clinical context.

Medications

Glucocorticoids and spironolactone can falsely elevate measured plasma cortisol levels and should be withheld on the day of testing. Long-acting glucocorticoids may require a longer washout period before evaluation. Specifically for suspected glucocorticoid-induced adrenal insufficiency, the Endocrine Society recommends discontinuing exogenous glucocorticoid therapy for at least 24 hours before dynamic testing to minimize assay interference.[4]

Estrogen-containing medications increase CBG concentrations, thereby raising total plasma cortisol levels and potentially masking adrenal insufficiency.[8] Ideally, these agents should be discontinued 4 to 6 weeks before testing to allow CBG levels to normalize. Alternatively, CBG may be measured concurrently; if elevated, total plasma cortisol values may not accurately reflect free cortisol concentrations.

Physiologic and Pathologic Conditions

Conditions that alter CBG levels can significantly affect the interpretation of the cosyntropin stimulation test. States associated with increased CBG, such as pregnancy and oral estrogen therapy, raise total plasma cortisol concentrations and may mask underlying adrenal insufficiency.[4][8] Conversely, conditions that reduce CBG or albumin levels, including advanced cirrhosis, nephrotic syndrome, malnutrition, and prolonged critical illness, lower measured serum cortisol concentrations and may result in overdiagnosis of adrenal insufficiency.[4]

Rare disorders, including congenital CBG deficiency, glucocorticoid resistance, and glucocorticoid hypersensitivity, can also markedly influence test results and complicate clinical interpretation.[2] In such cases, assessing free cortisol levels or consulting an endocrinologist may be necessary to ensure an accurate diagnosis. 

Disruption of the circadian rhythm can lead to falsely low morning cortisol levels in individuals with night-shift work, jet lag, or severe insomnia.[4] Although the cosyntropin stimulation test itself is not significantly influenced by diurnal variation and may be performed at any time of day, baseline cortisol measurements obtained in conjunction with the test may be affected.[2]

Assay Variability

Interpretation of cortisol concentrations varies considerably depending on the assay method. Immunoassays that use polyclonal antibodies are generally less specific and less accurate than those using monoclonal antibodies. In contrast, LC-MS/MS is considered the most accurate method.[4] In a large study, baseline cortisol thresholds used to exclude adrenal insufficiency ranged from 336 nmol/L (12.2 μg/dL) to 506 nmol/L (18.3 μg/dL) across 3 different immunoassays.[4]  When LC-MS/MS is used, lower diagnostic cutoffs are applied, approximately 412 nmol/L at 30 minutes and 485 nmol/L at 60 minutes, compared with the traditional 500 nmol/L threshold commonly used for immunoassays.[8]

Duration of ACTH Deficiency

In secondary adrenal insufficiency, the cosyntropin stimulation test may be less reliable in patients with recent-onset or mild ACTH deficiency (within days to weeks).[4] A supraphysiologic dose of cosyntropin can partially stimulate atrophied adrenal glands that would not respond appropriately to physiologic ACTH concentrations, potentially resulting in false-negative findings.

Results, Reporting, and Critical Findings

Stimulated plasma cortisol levels below 18 μg/dL (500 nmol/L) at 30 or 60 minutes are suggestive of adrenocortical insufficiency, although diagnostic cutoffs vary by assay.[1][2][1][8] When LC-MS/MS  is used, lower thresholds of approximately 412 nmol/L at 30 minutes and 485 nmol/L at 60 minutes are recommended.[8] Because some patients who fail to meet the 30-minute threshold achieve adequate cortisol levels by 60 minutes, measuring at both time points is important to reduce the risk of overdiagnosis.[8] 

Measurement at both 30 and 60 minutes is important because some patients who fail to meet the diagnostic threshold at 30 minutes achieve adequate cortisol concentrations by 60 minutes, reducing the risk of overdiagnosis of adrenal insufficiency.[8] The highest plasma cortisol concentration, whether obtained at baseline or after cosyntropin administration, is generally used to determine normality rather than the absolute increase from baseline.[7]

Results should be reported with the following information:

• Baseline, 30-minute, and 60-minute serum cortisol values with units (μg/dL or nmol/L)
• Peak cortisol concentration achieved
• Assay method used, such as immunoassay or LC-MS/MS
• Assay-specific reference ranges and diagnostic cutoffs
• Interpretation relative to the applicable diagnostic threshold

The report should clearly state whether the findings are consistent with normal adrenal function or are suggestive of adrenocortical insufficiency, and explicitly acknowledge that cutoff values used to exclude adrenal insufficiency vary by assay. When appropriate, interpretation should incorporate relevant clinical context and baseline ACTH concentrations to guide further diagnostic evaluation and management.

Critical Findings

Peak cortisol levels below the diagnostic threshold, typically less than 18 μg/dL or 500 nmol/L, constitute critical findings that require prompt clinical correlation and management.[1][2] Such results are suggestive of adrenal insufficiency and warrant immediate evaluation for compatible symptoms and signs, including fatigue, nausea, hypotension, hyponatremia, and hyperkalemia.

Further assessment should determine the type of adrenal insufficiency by measuring plasma ACTH, dehydroepiandrosterone sulfate (DHEAS), aldosterone, and renin levels. In primary adrenal insufficiency, laboratory findings typically show low cortisol, markedly elevated ACTH (often more than double above the upper limit of normal), low DHEAS, low aldosterone, and elevated renin. In secondary or tertiary adrenal insufficiency, cortisol levels are low, with low or inappropriately normal ACTH and low or low-normal DHEAS concentrations.

Patients with confirmed adrenal insufficiency are at risk for life-threatening adrenal crisis, particularly during periods of physiological stress. Diagnostic evaluation should never delay immediate initiation of empiric glucocorticoid therapy when adrenal crisis is suspected, as formal confirmation can be completed after clinical stabilization.

In secondary adrenal insufficiency, the cosyntropin stimulation test has limited sensitivity (approximately 64%) and may yield false-negative results, particularly in patients with recent-onset or mild ACTH deficiency.[1] A normal test result does not exclude secondary adrenal insufficiency when clinical suspicion remains high, and additional dynamic testing, such as the overnight metyrapone test, insulin tolerance test, or glucagon stimulation test, may be required for further evaluation.[1]

Clinical Significance

The cosyntropin stimulation test has high clinical significance as the diagnostic gold standard for primary adrenal insufficiency, but has limited sensitivity for secondary adrenal insufficiency.[1][2] The test plays a critical role in confirming adrenal cortical dysfunction and guiding appropriate glucocorticoid replacement therapy to prevent adrenal crisis.

Diagnostic Utility

The cosyntropin stimulation test is most useful when early-morning cortisol levels are indeterminate, typically between 5 and 10 μg/dL, and clinical suspicion for adrenal insufficiency remains high.[1] In primary adrenal insufficiency, the cosyntropin stimulation test serves as the diagnostic gold standard and has been validated against the insulin tolerance test. Elevated endogenous ACTH levels already maximally stimulate the adrenal cortex, preventing an adequate cortisol response even to supraphysiologic doses of cosyntropin.[2][7]

In secondary adrenal insufficiency, the cosyntropin stimulation test has limited diagnostic performance, with a sensitivity of approximately 64% and specificity of 93%.[8] False-negative results may occur in mild or recent-onset ACTH deficiency, as supraphysiologic cosyntropin doses can stimulate partially atrophied adrenal glands.[1][7][1] When clinical suspicion persists despite normal or equivocal results, alternative dynamic testing, including the overnight metyrapone, insulin tolerance, or glucagon stimulation tests, should be considered.[1]

Differentiating Types of Adrenal Insufficiency

When combined with baseline ACTH and DHEAS measurements, the cosyntropin stimulation test can help differentiate subtypes of adrenal insufficiency, as mentioned below.[6]

• Primary adrenal insufficiency:[2]
  • Low cortisol with ACTH levels elevated to more than 2-fold above the upper limit of normal
  • Low DHEAS concentrations
  • Inadequate cortisol response (<18 μg/dL at 30 or 60 minutes)

• Secondary and tertiary adrenal insufficiency:[8]
  • Low cortisol with low or low-normal ACTH and DHEAS concentrations
  • Variable cortisol response depending on duration and severity

• Glucocorticoid-induced adrenal insufficiency:[4]
  • Low cortisol with low or low-normal ACTH concentrations
  • Reduced test reliability in patients receiving glucocorticoids for shorter durations

Quality Control and Lab Safety

Quality control and laboratory safety for the cosyntropin stimulation test emphasize proper specimen handling, precise timing of blood draws, and appropriate assay selection. Patients should also be monitored for rare hypersensitivity reactions following cosyntropin administration.

Specimen Collection and Handling

Blood samples must be collected at precisely defined intervals, including baseline and exactly 30 and 60 minutes after cosyntropin administration. Accurate timing is essential because some patients who fail to meet diagnostic thresholds at 30 minutes achieve adequate cortisol levels at 60 minutes; deviations in sampling time can result in overdiagnosis of adrenal insufficiency.[8]

Serum cortisol concentrations should be measured using validated assays with established reference ranges, as assay selection significantly influences the interpretation of results. Laboratory reports should clearly specify the assay method and provide assay-specific reference ranges with results.

Drug Preparation and Administration Safety

Cosyntropin solutions should be visually inspected before administration, and any solution that appears cloudy or contains particulate matter should not be used. If the reconstituted solution is not administered immediately, it must be discarded to maintain sterility and drug potency.

Hypersensitivity reactions, including anaphylaxis, have been reported with cosyntropin administration. Patients should be monitored during and after testing, with appropriate emergency treatment readily available, as this important safety consideration requires ongoing clinical vigilance.

Preanalytical Quality Control

To ensure diagnostic accuracy, medications that interfere with cortisol measurement or adrenal responsiveness must be appropriately managed. Glucocorticoids and spironolactone should be withheld on the day of testing, with longer discontinuation periods required for long-acting glucocorticoids. Estrogen-containing medications should ideally be discontinued 4 to 6 weeks before testing, or CBG levels should be measured concurrently to aid interpretation. Thorough documentation of the patient’s medication history, including the timing of drug discontinuation, is essential for quality assurance and accurate interpretation of test results.

Assay Standardization and Validation

Laboratories should use validated cortisol assays with established diagnostic cutoffs, recognizing that thresholds for excluding adrenocortical insufficiency vary by assay. Robust internal quality control and participation in external quality assurance programs are essential to ensure consistent and accurate cortisol measurement.

Enhancing Healthcare Team Outcomes

Optimal management of patients undergoing cosyntropin stimulation testing, as well as those diagnosed with adrenal insufficiency, depends on coordinated interprofessional collaboration, patient education, and systematic strategies to prevent adrenal crisis. Healthcare teams must balance diagnostic accuracy while prioritizing patient safety and supporting patients to manage their conditions effectively.

Nurses play a critical role in monitoring for hypersensitivity reactions during and after cosyntropin administration, as anaphylaxis has been reported. Nursing responsibilities also include ensuring the precise timing of blood draws at baseline and at 30 and 60 minutes, as timing accuracy directly influences diagnostic reliability.

Pharmacists should review medication histories to identify agents that may interfere with test interpretation. Glucocorticoids and spironolactone should be withheld on the day of testing, and estrogen-containing medications should be discontinued 4 to 6 weeks in advance. In suspected glucocorticoid-induced adrenal insufficiency, glucocorticoid therapy should generally be withheld for at least 24 hours before dynamic testing, when clinically appropriate.

Early-morning cortisol measurement offers a practical initial approach to evaluating adrenal insufficiency in the appropriate clinical setting. Clinicians must nonetheless balance diagnostic evaluation with the imperative to avoid delaying life-saving treatment when an adrenal crisis is suspected.