New Cutoffs for the Biochemical Diagnosis of Adrenal Insufficiency After ACTH Stimulation Using Specific Cortisol Assays

Bradley R. Javorsky; Hershel Raff; Ty B. Carroll; Alicia Algeciras-Schimnich; Ravinder Jit Singh; Jessica M. Colón-Franco; James W. Findling


J Endo Soc. 2021;5(4) 

In This Article

Abstract and Introduction


Context: The normal cortisol response 30 or 60 minutes after cosyntropin (ACTH[1–24]) is considered to be ≥18 μg/dL (500 nmol/L). This threshold is based on older serum cortisol assays. Specific monoclonal antibody immunoassays or LC-MS/MS may have lower thresholds for a normal response.

Objective: To calculate serum cortisol cutoff values for adrenocorticotropic hormone (ACTH) stimulation testing with newer specific cortisol assays.

Methods: Retrospective analysis of ACTH stimulation tests performed in ambulatory and hospitalized patients suspected of adrenal insufficiency (AI). Serum samples were assayed for cortisol in parallel using Elecsys I and Elecsys II immunoassays, and when volume was available, by Access immunoassay and LC-MS/MS.

Results: A total of 110 patients were evaluated. Using 18 μg/dL as the cortisol cutoff after ACTH stimulation, 14.5%, 29%, 22.4%, and 32% of patients had a biochemical diagnosis of AI using the Elecsys I, Elecsys II, Access, and LC-MS/MS assays, respectively. Deming regressions of serum cortisol were used to calculate new cortisol cutoffs based on the Elecsys I cutoff of 18 μg/dL. For 30-minute values, new cutoffs were 14.6 μg/dL for Elecsys II, 14.8 μg/dL for Access, and 14.5 μg/dL for LC-MS/MS. Baseline cortisol <2 μg/dL was predictive of subnormal stimulated cortisol values.

Conclusion: To reduce false positive ACTH stimulation testing, we recommend a new serum cortisol cutoff of 14 to 15 μg/dL depending on the assay used (instead of the historical value of 18 μg/dL with older polyclonal antibody assays). Clinicians should be aware of the new cutoffs for the assays available to them when evaluating patients for AI.


The accurate and swift diagnosis of adrenal insufficiency (AI) is imperative given the potential for life-threatening consequences if missed.[1,2] Conversely, inappropriate assignment of AI to individuals has the potential for unnecessary glucocorticoid therapy.[3] Therefore, confirmation of the diagnosis of AI mandates precise biochemical testing, often requiring the assessment of adrenocorticotropic hormone (ACTH)-stimulated adrenal function to evaluate the integrity of hypothalamic-pituitary-adrenal (HPA) axis function.

ACTH (synthetic ACTH[1–24]; cosyntropin; synacthen) stimulation testing (CST) is the most commonly performed dynamic test to assess the adequacy of adrenal function in patients with suspected secondary adrenal insufficiency.[4] CST assesses the maximum adrenocortical secretory response to a supraphysiologic dose of ACTH. Patients with primary AI always have an elevated plasma ACTH, so CST is typically not needed for confirmation of the diagnosis.[5,6] Although insulin-induced hypoglycemia has previously been considered the gold standard test for decreased HPA axis function, it is very challenging to perform properly, labor intensive, and risky.[7] Accordingly, it has been abandoned in most clinical settings. The CST cortisol cutoff threshold for the diagnosis of AI 30 or 60 minutes after ACTH administration has evolved over the years, but it has become entrenched at 18 μg/dL (500 nmol/L) despite improved specificity of newer cortisol assays.[5,8–10] Historically, immunoassays using polyclonal antibodies have been used to establish post-cosyntropin cortisol cutoff concentrations as high as 20 μg/dL.[11,12] These assays had cross-reactivity with other serum steroids.[13–15] Newer-generation assays with greater specificity for cortisol have been developed and have already replaced polyclonal antibody assays in many institutions.[13,15,16]

Basal morning serum cortisol concentrations are also used to either increase the suspicion for, or rule-out the diagnosis of AI.[11,12,17–20] Furthermore, basal morning serum cortisol concentrations ranging from 11 to 19 μg/dL have been cited as a criterion to rule-out AI. Conversely, it has been argued that very low cortisol values (ie, <3–6 μg/dL) may establish biochemical AI and thus obviate the need for dynamic CST.

The Elecsys Cortisol generation II (Roche Diagnostics, City, IN) and Beckman Access Cortisol (Beckman Coulter, City, CA) immunoassays utilize monoclonal antibodies to identify cortisol.[16,21] Liquid chromatography–tandem mass spectrometry (LC-MS/MS) is a non-antibody, structural assay highly specific for cortisol.[22–25] Serum cortisol concentrations are approximately 20% lower with the newer assays compared with the older assays in some studies.[8,13,15,26,27] However, other studies have actually suggested that LC-MS/MS yielded a higher peak cortisol cutoff after ACTH stimulation than cortisol measured by immunoassay.[28,29] Considering these discrepancies in the literature, ACTH-stimulated cortisol threshold values using new, more-specific cortisol assays are needed to accurately diagnose AI and minimize overtreatment.

The purpose of the current study was to evaluate basal serum cortisol and the serum cortisol response to synthetic ACTH[1–24] stimulation in patients suspected of having AI and compare more-specific cortisol assays (2 monoclonal antibody assays and LC-MS/MS) with a polyclonal antibody cortisol assay in order to calculate new cutoffs for the cortisol response to CST.