Steroid Assays and Endocrinology: Best Practices for Basic Scientists
Open accessAbstract
Endocrinologists measure hormones. In fact, the development of endocrinology as a discipline parallels the development of hormone assays. Early on, these assays were time consuming and cumbersome, with exhaustive controls and replicates, an entire research project unto itself. Initially, hormone activity was defined and quantified with bioassays, but these assays cannot distinguish among different hormones with overlapping activities, such as the 2 androgens testosterone and dihydrotestosterone. In addition, quantitation with bioassays is relatively imprecise, operator dependent, and vulnerable to misinterpretation. The advent of immunoassays in the 1960s heralded a new era for endocrinology. Small molecules covalently linked to immunogenic proteins elicited antibodies directed to epitopes of the target molecule, such as a steroid. The conjugation process, however, requires at least minimal modification of the steroid structure, and the antibody might be directed against an epitope common to several steroids. Consequently, Abraham's estradiol assay involved extraction and chromatography to prevent cross-reaction with other steroids (1). Quantitation was limited to the linear portion of the standard curve, because error rose sharply at the high and low ends where the curve flattens. Consequently, the assay took over a day to perform, and sensitivity was limited. Over time, efforts were directed to shorten the procedure and to improve sensitivity. More specific antibodies were developed, standard curves were extended (albeit with increased error) using computer-based curve fitting, and for abundant analytes such as cortisol in human serum, assays were run directly on serum or culture medium without extraction and chromatography, a so-called “direct” immunoassay. With these advances, endocrinology has moved on. We no longer spend most of our time measuring hormones. We transfect cells and challenge them with hormones, drugs, and other stimuli; we create genetically engineered animals and perform complex physiology experiments. Our ability to generate complex animal models with tissue-specific gene deletions as well as knockdown of target genes in cell cultures has advanced at an amazing pace over the last decade. In the end, however, the final readout of our experiments is often hormone measurements. Now, here is the rub. We have pushed our immunoassays beyond their limits. Routinely, immunoassays employ small quantities of various sample types from several species, often for which the assay was neither designed nor validated. Rarely do assays incorporate labor-intensive extraction and chromatography. Laboratories purchase kits and perilously use these assays without the traditional controls, such as spiked samples, mixing experiments, and serial dilutions. Assay performance characteristics are reported from the instruction sheet rather than generated for the specific samples used in the experiments. As an example, the University of Virginia Ligand Assay and Analysis Core, one of the “gold standards” of endocrinology research for decades, changed their estradiol assay for mouse serum in 2011 when a direct comparison found the previous assay kit to be unreliable (2). Are all or some of the papers reporting estradiol measurements by the University of Virginia Ligand Core previous to this change thus invalid? The high standards and competitiveness of endocrinology research today divert our attention from the scrupulous attention to detail for hormone assays; however, can we believe these results? Should we impose standards on investigators? Is it time to consider what constitutes best practices for steroid assays in endocrinology research? The limitations of direct testosterone immunoassays for clinical use, particularly for low concentrations found in women and children, have been recognized for some time. This problem prompted a The Endocrine Society position statement recommending either liquid chromatography/tandem mass spectrometry (LC-MS/MS) or immunoassay after extraction and chromatography for measuring testosterone in women and children (3). Subsequently, our sister journal, The Journal of Clinical Endocrinology and Metabolism announced that new submissions starting 2015 must employ LC-MS/MS assays for sex steroids (4). This editorial has sparked a wave of long overdue discussions among journal editors, investigators, leadership of The Endocrine Society, and members of Partnership for Accurate Testing of Hormones (https://www.endocrine.org/advocacy-and-outreach/path) on the broader topic of steroid assay quality in all forms of endocrinology research. As a result, The Endocrine Society has convened a Sex Steroid Assays Reporting Task Force to address this topic in depth. At the time this editorial was written, the Task Force's work is just beginning, and The Endocrine Society has suspended The Journal of Clinical Endocrinology and Metabolism's policy while deliberations are underway (5). This exercise is
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Moderate or severe comedonal acne with 10 or more facial lesions in early puberty, or moderate inflammatory acne through the perimenarcheal years, is uncommon, with a prevalence below 5%.
“While comedonal acne is common in adolescent girls, moderate or severe comedonal acne (i.e., 10 or more facial lesions) in early puberty or moderate inflammatory acne through the perimenarcheal years is uncommon (<5% prevalence) [42,43]”
From The Diagnosis of Polycystic Ovary Syndrome during Adolescence (2015) · cites this paper as [43]
There is a drive within the endocrine community to phase out immunoassays where possible.
“Indeed, there is a drive within the endocrine community to phase out immunoassays where possible [343].”
From Human steroid biosynthesis, metabolism and excretion are differentially reflected by serum and urine steroid metabolomes: a comprehensive review (2019) · cites this paper as [343]
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- Authors
- Auchus, R. J.
- Affiliations
- University of Michigan (US)
- Corresponding author
- Auchus, Richard J.
- ORCID
- Auchus, Richard J.
- Venue
- Endocrinology
- Publisher
- The Endocrine Society
- Published
- 2014-06-01
- Volume
- 155
- Issue
- 6
- Pages
- 2049-2051
- ISSN
- 0013-7227 (ISSN-L)
- 1945-7170
- PMC
- PMC4020931
- OpenAlex
- W2026073784
- Cited by
- 67
- References
- 8
- Citations by year
- 2026: 3 · 2025: 5 · 2024: 14 · 2023: 5 · 2022: 1 · 2021: 6 · 2020: 2 · 2019: 4 · 2018: 4 · 2017: 14 · 2016: 5 · 2015: 3
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Endocrinology (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- University of Michigan (48109)
- College of Pharmacy, University of Michigan
- National Institute of General Medical Sciences (R01GM086596)
- Topics
- Health Sciences › Medicine › Physiology › Clinical Laboratory Practices and Quality Control
- Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Adrenal Hormones and Disorders
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Estrogen and related hormone effects
- Keywords
- steroid hormone assay, immunoassay validation, assay specificity, cross-reactivity, assay sensitivity, assay precision, assay accuracy, direct immunoassay, cortisol, estradiol, testosterone, dihydrotestosterone, immunoassay kit
- MeSH terms
- Endocrinology, Humans, Immunoassay, Mass Spectrometry, Steroids
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