Despite a reliance on first-generation testosterone assays to diagnose conditions such as hyper- and hypogonadism, these assays’ quality is so poor as to make the results little better than a guess in some instances, according to the authors of a study published in The Journal of Applied Laboratory Medicine.

Known weaknesses of these assays are cross-reactivity with other steroids and a high variation in the low concentration range. Now, researchers have identified a third weakness: an ineffective displacement of testosterone sex hormone-binding globulin (SHBG).

The investigators obtained serum from 30 women on oral contraceptives, 30 women not using oral contraceptives, and 30 pregnant women. They compared the results from an isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method to six commercially available testosterone immunoassays, four widely used first-generation assays (Unicel, Centaur, Immulite, and Liaison) and two second-generation tests (Architect and Cobas). They also used the Architect immunoassay to measure SHBG.

The four first-generation tests showed inaccurate testosterone results when compared with the ID-LC-MS/MS method. The Unicel and Liaison assays also demonstrated a very poor standardization. On average, SHBG concentrations were lowest in women not using oral contraceptives and highest in pregnant women, ranging from 18.5 to 633 nmol/L. There was also an inverse relationship between SHBG concentrations and deviations in testosterone from the ID-LC-MS/MS results in the first-generation immunoassays, but not in the second-generation immunoassays.

This, the authors wrote, “strongly suggests that all four first-generation testosterone immunoassays are incapable of completely releasing testosterone from its binding proteins. This may lead to false low results in patients with high SHBG concentrations and to false high results in patients with low SHBG concentrations, respectively.”

In an accompanying editorial, William E. Winter, MD, of the University of Florida in Gainesville, addressed a further question: Should immunoassays be used at all to measure total testosterone in women? A literature search turned up just three relevant articles, including recommendations from the American Association of Clinical Endocrinology and the Centers for Disease Control and Prevention (CDC) that mass spectrometry be used to measure total testosterone.

As Winter writes: “It would be reasonable to state that measuring low concentrations of testosterone by immunoassay is problematic under any circumstance.” His laboratory, as well as three other major reference laboratories contacted, all use mass spectrometry to measure total testosterone in women and children and, in some cases, men, he found.

He concludes by expressing his hope that the CDC will release guidelines calling for the use of mass spectrometry when measuring testosterone in women and children and, possibly, in hypogonadal men.