Steroid Assays and Endocrinology: Best Practices for Basic Scientists

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 analogous to the development of clinical practice guidelines or “best practice” policies for the care of patients with specific diseases, which are widely embraced in endocrinology practice. Mass spectrometry methods for measuring steroids have existed for decades, yet its widespread use is a recent phenomenon. Gas chromatography coupled to mass spectrometry features high chromatographic resolution and moderate instrument costs but requires derivatization, which reduces throughput (6). LC-MS/MS assays became popular when electrospray sources, which introduce the LC effluent into the MS, became efficient and reliable. Modern triple quadrupole instruments achieve required sensitivities to measure physiologic concentrations of several low-abundance steroids from small samples. LC-MS/MS assays can be automated or semiautomated without derivatization for many steroids, particularly the major Δ4-steroids, but instrumentation cost is high (∼$500 000), and expert technical proficiency is necessary to develop and to implement high-quality assays. In proper hands, LC-MS/MS offers the potential for sensitive, accurate assay of multiple steroids in small samples regardless of origin (7). Furthermore, LC-MS/MS assays do not require antibodies, which can be proprietary and/or exist in limited supplies. Most clinical reference laboratories (Quest Diagnostics, Mayo Medical Laboratories, ARUP) have converted most if not all of their steroid and vitamin D assays to LC-MS/MS with no looking back. So what prevents all endocrinology investigators from embracing a wholesale transition to LC-MS/MS assays for all steroids? Well, the first reason is access. This instrumentation is too complex, costly, and vulnerable to malfunction for even large research groups or centers to purchase and to maintain enough instruments to satisfy the need. Core facilities with capabilities for steroid profiling assays by LC-MS/MS are only in their infancy, and even the local demand would quickly outstrip the capacity. Our own efforts at Michigan have been partially successful yet hampered by setbacks that have delayed our goals to meet the demands of assay volume and performance. The second reason is cost. Although a flat fee might be economical if 12 steroids are assayed simultaneously, most experiments only require 1–4, and when manpower costs dominate the fee, investigators who require only a few analytes will not get much of a discount. The third issue, the elephant in the room if you will, is uncertainty about the advantage of changing assays. When research is based on previous studies using one assay, the interpretation of subsequent studies often rests on comparison with previous results, which frequently is not possible. Even if the new assay is more “correct,” the wrong conclusion might result if the assays are not directly compared. Furthermore, the endpoints of many experiments are the relative amounts, differences, or changes in steroid analytes rather than the absolute values. Some assays might reliably yield this comparative information and support the correct conclusions yet not meet the strictest standards of accuracy. Finally, MS assays are not intrinsically infallible. Every step, from the preanalytical sample preparation to making and diluting standards to the calculation of results from raw data, is a part of the method. Any assay is only as good as its most vulnerable component. You might have a million-dollar instrument with precision to 4 decimal places, but you still have to pipette the serum or culture medium with a $200 pipette that might be accurate to 5%. Nevertheless, the time has come to pause, to deliberate, and to make hard decisions about what level of quality and evidence of performance we will accept as the standard for steroid assays in all forms of endocrinology research. We must focus not on the type of assay but on the performance of the assay for the specific use in every study. We must engage investigators, journals, scientific societies, and funding agencies in the awkward discussions required to determine how to proceed into the future. Striking a balance between cost, convenience, and accuracy will be difficult and painful. These points need to be considered carefully by researchers and journal reviewers, also keeping in mind the survival of laboratories with young investigators or those in developing nations. Facilitating access to high-quality assays should be a priority worldwide. Only 1 fact remains clear: the endocrinology community must clean up its act and assure our readership that we can measure our steroid endpoints reliably. The alternative of remaining on our current trajectory equates to speeding down the proverbial road blindfolded. It is time to find a balanced, equitable, and scientifically sound solution to the problem.