Inaccurate First-Generation Testosterone Assays Are Influenced by Sex Hormone–Binding Globulin Concentrations

Background: The quality of testosterone assays has been amatter of debate for several years. Known limitations of testosterone immunoassays are the cross-reactivity with other steroids and a high variation in the low concentration range. We hypothesized that one of the additional limitations of testosterone immunoassays is an ineffective displacement of testosterone from its binding protein. Methods: Thirty samples from women not using oral contraceptives (OAC), 30 samples from women using OAC, and 30 samples from pregnant women were used to measure testosterone by an isotope dilution (ID)-LC-MS/MSmethod and by 6 commercially available testosterone immunoassays (UniCel®, ARCHITECT®, Centaur®, Cobas®, Immulite®, and Liaison®). In addition, sex hormone–binding globulin (SHBG)4 was measured by immunoassay (ARCHITECT). Results: The first-generation immunoassays (UniCel, Centaur, Immulite, and Liaison) showed inaccurate testosterone results in the method comparisons with the ID-LC-MS/MS method (R between 0.61 and 0.86) and for some assays (UniCel and Liaison) also a very poor standardization (slopes of 0.59 and 0.67, respectively). On average, SHBG concentrationswere lowest inwomennot usingOAC and highest in pregnantwomen, andoverall ranged from18.5 to 633nmol/L. In thefirst-generation immunoassays, but not in the second-generation immunoassays, we observed an inverse relationship between SHBG concentrations and deviations in testosterone from the ID-LC-MS/MS results. Conclusions: Widely used first-generation testosterone immunoassays are influenced by SHBG concentrations, which lead to inaccurate results in samples from patients with high or low SHBG concentrations, respectively. Laboratory specialists, clinicians, and researchers should be aware of this limitation in testosterone assays.