This chapter explains normal adrenocortical function and provides an overview of related disorders, including: hypercortisolemia, Cushing’s syndrome, hypocortisolemia, Addison’s disease, congenital adrenal hyperplasia, Nelson’s syndrome and adrenocortical insufficiency. For each immunoassay analyte commonly used in the adrenocortical field the biological function is explained, with the clinical applications of the test and its limitations, including test protocols. Typical assay technology is described. The type of sample and frequency of use are included, with an example reference interval (for background information only). The analytes included are cortisol and adrenocorticotropic hormone. In the final section, management of patients with Cushing’s syndrome is described.

An original article in Clinical Endocrinology (Thynne et al., Clin. Endo. 80, 328−332 (2014)) entitled Factitious Cushing’s Syndrome Masquerading as Cushing’s Disease was followed with a Commentary (Clin. Endo. 80, 333−334 (2014)) on the Pitfalls in the Diagnosis and Differential Diagnosis of Cushing’s Disease by one of the world’s leading endocrinologists on Cushing’s syndrome.

The first paper demonstrated the problems of non-specificity. The patient had a grossly elevated urine free cortisol concentration, mildly raised saliva cortisol and normal serum cortisol concentrations while simultaneously had low but not totally suppressed plasma ACTH concentration (13 ng/L). The patient underwent extensive investigations and initial treatment with ketoconazole for suspected Cushing’s disease.  It was the surgeon who decided to investigate the discordant cortisol and ACTH results and so prevented the patient undergoing pituitary surgery.  Liquid-chromatography tandem mass spectrometry (LCMSMS) analysis revealed a very low urine free cortisol excretion of 20 nmol/24h, but prednisolone excretion was 16,200 nmol/24h.

The Commentary article nicely illustrated a number of important points. Firstly it is essential that laboratory scientists and clinical endocrinologists understand the vagaries of each immunoassay that we use, in particular the cross-reactivities with similar structured compounds. Secondly, a robust plasma ACTH assay is vital to differentiate ACTH-dependent from ACTH-independent Cushing’s syndrome. Traditionally, a suppressed ACTH is associated with ACTH-independent Cushing’s syndrome; in this case the low ACTH was discordant with a diagnosis of Cushing’s disease.

Most ACTH assays used in clinical service laboratories are now performed on automated immunoassay platforms or done manually using commercial analytical kits. Pecori Giraldi questioned the performance of plasma ACTH assays, particularly at low concentrations (Eur. J. Endocrinol. 164, 505−512 (2011)).  The concerns related to the precision of these assays at low concentrations. At high concentrations imprecision errors had minimal impact on diagnosis but this was very different at low concentrations where as much as 40% of results were classified incorrectly. Some of the methods in that paper have since been withdrawn and automation has improved imprecision.

Another factor to consider in modern ACTH assays with regards variability is the calibration. Not all ACTH (1–39) formulations are equally recognized by assay antibodies. The different potency of ACTH standards had already been observed in the past (ref. 1). Since there is no International Standard for human ACTH this will be difficult to rectify in the near future.

The problems of immunoassay specificity as indicated in the paper by Thynne have generated significant discussions with regards to the role of LCMSMS. In their paper, LCMSMS did resolve the discordance between ACTH and cortisol results. The prestigious endocrine journal  J. Clin. Endocrinol. Metab. has gone as far as stating that from Jan 2014, any steroid assay results have to be backed using LCMSMS evidence (Ref. 2). Others have suggested that LCMSMS still has its own problems and have offered an alternative view for the future.

The analysis of adrenal hormones still has a major role in the differential diagnosis of cortisol excess and the adrenal insufficiency syndromes (Ref. 4)

1. Dobson, S.H., Gibson, S., White, A. Assessment of the potency of different standards in the immunoradiometric assay of ACTH. Ann. Clin. Biochem. 26, 96−101 (1989).
2. Handelsman, D.J., Wartofsky, L. Requirement for mass spectrometry sex steroid assays in the Journal of Clinical Endocrinology and Metabolism. J. Clin. Endocrinol. Metab. 98, 3971–3973 (2013).
3. Auchus, R.J. Steroid Assays and Endocrinology: Best Practices for Basic Scientists. Endocrinology, 155(6), 2049–2051 (2014).
4. Charmandari et al. Adrenal insufficiency. The Lancet 383  2152–2167 (2014).

Dr. Les Perry is a Consultant Clinical Scientist in the Department of Clinical Biochemistry at the Croydon University Hospital NHS Trust, London. He took up this post in May 2012. Previously, he was at Bart’s Hospital, London and provided expert advice on the appropriate use and the interpretation of routine and specialized endocrine test results to facilitate the diagnosis, treatment and monitoring of rare, complex diseases. He has a particular interest in pediatric and adult endocrinology. He led the introduction of LCMSMS technology into the Department 5 years ago as a new analytical tool for service provision. He obtained his BSc in 1976, MSc (Leeds) in Steroid Endocrinology in 1977, PhD (London) in 1987 and the FRC Path 2008. He has written over 120 peer reviewed publications which are a mixt of methodological and clinical papers. He is the current Chair of the UKNEQAS Specialist Advisory Group for Immunoassay & Endocrinology.

This chapter also contains material contributed by Dr. Sami Medbak to the first edition of The Immunoassay Handbook.

Adrenal, circadian rhythm, stress, hypercortisolemia, Cushing’s syndrome, hypocortisolemia, Addison’s disease, congenital adrenal hyperplasia, Nelson’s syndrome, adrenocortical insufficiency, cortisol, adrenocorticotropic hormone, corticotropin, corticotropin-releasing hormone, corticosteroid, aldosterone, dehydroepiandrostenedione, dehydroepiandrostenedione sulfate, androstenedione, testosterone, cortisol-binding globulin.