Metabolites of these compounds may additionally cross-react. Interfering molecules can be structurally related endogenous compounds (e.g., 6β-hydroxycortisol for a cortisol assay), drugs (including anabolic steroids and herbal medications), or natural products. One limitation of steroid hormone immunoassays is interference caused by compounds with structural similarity to the target steroid molecule against which the assay antibodies were generated. ![]() Consequently, many clinical laboratories continue to use immunoassays for routine steroid hormone measurement. However, chromatography and mass spectrometry require specialized technical skill along with dedicated and often expensive instrumentation. ![]() LC/MS/MS provides a high degree of specificity for steroid hormone measurement. An increasing number of clinical laboratories, particularly reference laboratories, utilize liquid chromatography-tandem mass spectrometry (LC/MS/MS) for steroid hormone measurement. The most common alternative approach for measurement of steroid hormones is chromatography (especially high-performance liquid chromatography, HPLC), either alone or in combination with mass spectrometry (MS) (HPLC/MS or simply LC/MS). A variety of immunoassay methods are available, ranging from enzyme-linked immunosorbent assays (ELISAs) to homogeneous immunoassays that can be run on high-throughput analyzers commonly found in hospital clinical laboratories. Immunoassays are frequently used in laboratory medicine for quantitation of plasma/serum concentrations of steroid hormones such as cortisol, dehydroepiandrosterone (DHEA) sulfate, estradiol, progesterone, or testosterone. Using two-dimensional similarity calculations, all compounds with high cross-reactivity also showed a high degree of similarity to the target molecule of the immunoassay. Norethindrone therapy may impact immunoassay measurement of testosterone in women. Several anabolic steroids may produce clinically significant false positives on the testosterone assay, although interpretation is limited by sparse pharmacokinetic data for some of these drugs. In addition, 21-deoxycortisol likely produces clinically relevant cross-reactivity for cortisol in patients with 21-hydroxylase deficiency, while 11-deoxycortisol may produce clinically relevant cross-reactivity in 11β-hydroxylase deficiency or following metyrapone challenge. 6-Methylprednisolone and prednisolone showed high cross-reactivity for the cortisol assay, with high likelihood of clinically significant effect for patients administered these drugs. The Roche Elecsys Cortisol and Testosterone II assays showed a wider range of cross-reactivity than the DHEA sulfate, Estradiol II, and Progesterone II assays.
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