Assessment of the hypothalamic pituitary adrenal (HPA) axis remains a challenge and requires a methodological investigative approach, which is heavily reliant on hormonal assays. Clinically, plasma ACTH quantification is used to determine if the source of hyper- or hypocortisolemia is adrenal or extra-adrenal disease (ACTH- independent vs ACTH-dependent respectively). (1) Unfortunately, no test is infallible and all are subject to potential preanalytical and analytical sources of error or interference (2), which to the unsuspecting user may lead to additional invasive and inappropriate investigations.

In our recent series, we discuss 12 cases that in the process of investigation for possible hypothalamic pituitary axis abnormalities were found to have falsely elevated ACTH measurements due to interference in the assay. (3) This discrepancy in clinical and biochemical findings posed a significant diagnostic conundrum with clinical consequences. Additional unnecessary investigations were requested, which for some included invasive assessment such as inferior sinus petrosal sampling. With the assistance of laboratory medicine, appropriate trouble shooting revealed an interference resulting in erroneously elevated ACTH concentration. Repeat analysis even with the use heterophile blocking tubes revealed similar results. Rising serial dilutions and markedly different measurements on an alternate platform confirmed the presence of an interfering substance.

Immunometric assays have advanced significantly to overcome known interfering substances, but not all interferences are accounted for or are yet known. More recently, biotin was found to interfere with biotinylated assays and as use of over the counter substances increases, we may note additional potential interfering substances. (4) Unfortunately, establishing the presence of pathology in a patient versus abnormal assay perturbations remains a challenge and therefore a healthy dose of skepticism is recommended when a clinical/biochemical mismatch is noted. Communication with laboratory medicine remains fundamental to the practice of endocrinology and should be considered early in the diagnostic algorithm as highlighted in our recent case series.


  1. Nieman LK, Biller BM, Findling JW, et al. The diagnosis of Cushing's syndrome: an Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab. 2008;93(5):1526-1540.
  2. Levinson SS, Miller JJ. Towards a better understanding of heterophile (and the like) antibody interference with modern immunoassays. Clin Chim Acta. 2002;325(1-2):1-15.
  3. Donegan DM, Algeciras-Schimnich A, Hamidi O, et al. Corticotropin hormone assay interference: A case series. Clin Biochem. 2019;63:143-147.
  4. Samarasinghe S, Meah F, Singh V, et al. Biotin Interference with Routine Clinical Immunoassays: Understand the Causes and Mitigate the Risks. Endocr Pract. 2017;23(8):989-998.