From Clinical Laboratory News:
Are there times when a test is so unreliable and imprecise that it simply shouldn’t be used with certain patients? That’s the debate swirling around total testosterone assays—direct immunoassays in particular—when used for testing in women and children. Concentrations of this steroid hormone may be as low as 0.17 nmol/L (5 ng/dL) for these populations, compared with 11.1 nmol/L (320 ng/dL), the lower range of normal in men. A growing body of evidence has clearly demonstrated that the performance of some immunoassays at such low concentrations is sub-par at best, prompting experts to question their use in women and children. Earlier this year, a consortium of professional associations and government agencies examined the issue and is now calling for long overdue testosterone assay standardization, an initiative that has major implications for manufacturers, labs, and public health.
William Rosner, MD, who co-chaired the consensus conference, has been a vocal advocate for improving the assays. “This [standardization effort] has implications for individual patients and for researchers. There’s a push on to treat women with testosterone, but we can’t know how much to give them or whether to give them any if we don’t have a way to accurately measure testosterone levels,” he said “At the same time, researchers can’t compare their results to anyone else’s, and they can’t compare their own results to results they obtained 10 years ago if they changed the method they’re using to measure testosterone.” Rosner is a professor of medicine at Columbia University in New York City.
The consensus effort, spearheaded by The Endocrine Society and Centers for Disease Control and Prevention (CDC) and endorsed by AACC, is encouraging the expert scientific and medical communities, third-party payers, manufacturers, funding entities, journals, and other stakeholders to undertake specific efforts to aid in improving the accuracy of testosterone measurements. The group’s consensus statement has been submitted for publication in The Journal of Clinical Endocrinology & Metabolism.
Issues about the performance of testosterone assays have risen to the fore as more potential applications for the analyte have become apparent. Testosterone levels once were primarily used in the work-up of men suspected of hypogonadism and in gender assignment for newborns with ambiguous genitalia.
More recently, research—not all of it concordant—has suggested testosterone may have a protective benefit in osteoporosis, type 2 diabetes, cardiovascular disease, obesity, and depression in men. Androgen replacement therapy in women also has gained favor as a means of boosting libido, bone density, and muscle mass. In addition, there has been growing recognition of and better treatments for polycystic ovary disease, a disease of androgen excess and the most common hormonal disorder in women of reproductive age. Meanwhile, the epidemic of obesity in children has been associated with precocious puberty, thereby increasing the need to determine androgen status in these patients. All of these factors have pumped up the volume of testing, particularly in women and children, who had not been part of the traditional testing population.
Rosner believes a big tent will be needed to achieve standardization of total testosterone assays because a complicated web of factors has contributed to their shortcomings. Medical journals, laboratories, regulators, lab testing and accrediting bodies, diagnostic manufacturers, and even payers all have had a hand in promulgating and perpetuating a lackluster assay, he contends.
Conventional radioimmunoassays (RIA) for measuring testosterone were developed in the early 1970s. These assays, which involve denaturing steroid-binding proteins to release testosterone followed by purification steps that remove numerous potentially interfering metabolites, are highly reliable when properly validated, according to experts. But they are cumbersome, time-consuming, costly, and, like other antibody assays, can have cross-reactivity issues.
As time went on, direct chemiluminescent, enzymatic, and fluorescent immunoassays came on the scene and surpassed conventional RIAs in use. These direct immunoassays provide rapid and inexpensive results, but they have several serious drawbacks as well. Due to lack of specificity of the antibodies, they tend to overestimate testosterone measurements. They also are prone to matrix effects and have inadequate sensitivity to measure low levels of testosterone accurately and reliably.
Perhaps because the direct immunoassays are acceptable in evaluating testosterone levels in most men, it was an easy leap to assume they would work well in women and children, too.
Notable strides towards standardization have been taken by the CDC since 2007. Among other things, the Division of Laboratory Sciences (DLS) has developed an accurate and precise reference method. The method uses a reference material from the Australian National Measurement Institute, the only one that’s currently available for testosterone, according to Hubert Vesper, PhD, chief of the protein biomarkers laboratory in CDC’s DLS. The National Institute of Standards and Technology (NIST) also has developed a serum-based product, which is considered a secondary reference material.
CDC took another important step earlier this year when it launched a standardization program for testosterone. The program is open to manufacturers, commercial and reference labs and involves fresh-frozen, single-donor serum samples with reference values.
CAP also has stepped up to the plate by offering, starting earlier this year, a separate accuracy-based proficiency testing program for steroid hormones, including testosterone. “It’s voluntary and at present is not required for CLIA certification,” said John Eckfeldt, MD, PhD, Ellis Benson professor and vice chair for clinical affairs in the department of laboratory medicine and pathology at the University of Minnesota in Minneapolis. “Labs that are concerned about accuracy and want to pay the extra money and take the extra time to participate will do so.”
As crucial as these efforts are, greater participation by manufacturers will be needed for testosterone immunoassays to leap forward in accuracy and precision at lower concentrations, according to experts.
Major reference labs have invested in and use mass spectrometry to measure testosterone, particularly for women and children.
Until mass spectrometry is a mainstream method, Fitzgerald urged labs relying on immunoassays to consider sending out any samples for measurement on women and children. “All the major reference labs have it available by mass spec, and as far as send-out tests go, it’s not extremely expensive. It’s on par with most send-out tests,” he observed.