Standardization of Hormonal Assays for the 21st Century

We write to reprise and expand the theme of a consensus statement on the subject of standardization of testosterone measurement published in the October issue of the JCEM (1) and encourage your review of that article. The statement from The Endocrine Society and the Centers for Disease Control and Prevention (CDC), with the endorsement of many other groups, addresses the impact of currently available, often inaccurate testosterone assays on clinical care, research, and health care expenditures. The concern is rooted in the fact that there is no recognized reference standard for the assay. Absent such a standard, trustworthy age- and sex-specific reference ranges for testosterone are not attainable. Inaccuracies in plasma testosterone measurement have been noted in our Journal for several years (2–4) and apply to both total and free hormone measurements, particularly at the lower levels. The major problems with sensitivity and specificity of the assays have related to current immunoassay methodology, particularly those that are automated (platform-based), although variability has also been shown with analysis by mass spectrometry (5, 6). There are other problems that are unique to the validity of so-called free testosterone assays. For the most part, free testosterone “measurements” are rarely actually measured but reflect instead derived or calculated values by one or another method that has never been thoroughly validated or standardized. As a result, large errors in this calculation of 20–40% may exist (7–9), typically overestimating values obtained by equilibrium dialysis. The concept of measuring “bioavailable” testosterone is no less conceptually flawed and also provides values of questionable validity (10). Consequently, a calculated free testosterone measurement can in no way substitute for a total testosterone measured in a validated mass spectrometry-based method. An Endocrine Society Position Statement in 2007 warned of the significant limitations of testosterone assays (11). The lack of a “gold standard” test and the implications of inaccurate hormonal assays should be obvious and include mistaken diagnosis with subsequent inappropriate care, misleading research findings, and unnecessary health care costs, to name a few. Indeed, given that we strive to base our clinical practice guidelines (3, 12) upon the results of clinical investigation and clinical trials, such guidelines could be worthless to dangerous when rooted in inaccurate laboratory measurements. A recent study documented adverse cardiovascular events associated with testosterone administration; in that study, both initial participation and subsequent dosage adjustments of testosterone were based upon measurements of total and free testosterone (13). How reliable were those measurements, and how might they have influenced dosage changes directly linked to the observed adverse outcomes? The current consensus statement (1) reflects an ongoing initiative of the CDC (14, 15) in partnership with The Endocrine Society to illuminate this problem. The stated goal of the collaboration is “to improve the quality of research, patient care, and public health through broad implementation of standardized testosterone measurements that are accurate, reliable, and comparable over time.” Although the efforts cited above to standardize testosterone assays are both necessary and laudable, they represent an initiative based on only one hormone. At this time, the best results are achieved by methods based on tandem mass spectrometry. However, the issue is not the specific method, but rather the recognition that an assay’s accuracy be traceable to a scientifically arrived at, universally recognized standard. Do all of our other routinely employed hormonal assays provide truly accurate and precise measurements? The wide variation in plasma thyroglobulin and antithyroglobulin antibody levels in different laboratories comes immediately to mind, but similar variations are commonly seen with assays of the serum estrogens and other steroids (16), vitamin D, free T3 or free T4, GH (17), ACTH, salivary cortisol, etc., the measurements of which form the basis for so many of our clinical decisions. The growing use and availability of tandem mass spectrometry has led to more reliable results for 25-OH-vitamin D (18) and has proven specific and reliable for estradiol and progesterone as well as for testosterone (19). Thus, the recommendations proposed by Rosner and Vesper (1) to those having a stake in the quality of testosterone assays could be applied as well to stakeholders in the measurement of several other hormones. These stakeholders include laboratory directors, regulatory agencies, professional societies, pharmaceutical companies, the National Institutes of Health and other research funding agencies, third party payers for health care, and of course patients and their physicians. If you missed their article in the October issue of JCEM (1), we invite you to read and consider the recommendations and the proposed process for standardization that they suggest, which we will not repeat here. Rather, we will close by mentioning another important stakeholder on the issue of reliable assay results—our medical and scientific journals. Just as journals have collaborated to develop guidelines for clinical trials, standardized nomenclature, aspects of ethical publishing, etc., we can, as a body, insist that research published in our pages must present hormone data measured by assays with the highest standards of accuracy and precision as certified by appropriate bodies. To do so effectively, the JCEM acknowledges that more work would be required by journal staff, manuscript reviewers, and the editors to assure compliance, but the outcome will be well worth the labor. The effective standardization of assays of testosterone and other hormones will be a work in progress, conservatively speaking, over perhaps the next decade. The proximate effort is the standardization of the total testosterone assays, but we should not ignore the inherent conceptual flaws in methods for the estimation of free testosterone as well as the imprecision of these assays. The CDC/Endocrine Society initiative has provided the start, with an emphasis on the importance of mass spectrometry-based testosterone assays. It will take time and dollars for hospital and commercial clinical pathology laboratories to upgrade their technology and methodology. Given the importance of this initiative to both hormone research and clinical care, we hope that the CDC will monitor the quality control of these assays on an ongoing basis as a public service. Once these assays are clearly and optimally standardized, the JCEM pledges to require that all testosterone data submitted by authors will have been measured by laboratories employing the requisite optimal methodology. We will call upon our sister journals to adopt the same policy. Disclosure Summary: The authors have nothing to declare.