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Endocrinology, Diabetes, and Metabolism

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Henry Ford Hospital


Introduction: Immunoassays, specifically chemiluminescent assays are commonly used in clinical laboratories for analyzing protein antigens and steroid hormones such as testosterone due to their availability as commercial kits, low cost and simplicity. We present a case of falsely elevated serum testosterone. Clinical Case: A 17-year-old female with facial acne was referred to us for elevated testosterone levels. She was of Greek descent and reported having had coarse dark hair growth on upper lip, chin, chest for which she previously underwent laser therapy. Her menstrual cycles were regular and predictable. She was not on any medications. On physical examination, she was normotensive with BMI of 22kg/m2. She had coarse dark hair on upper abdomen, lower abdomen and medial thighs with Ferriman-Gallwey score of 7, genital exam did not reveal clitoromegaly. On initial testing, serum total testosterone was 417ng/dL (range: <75ng/dL). The referring facility advised pelvic and renal ultrasound which revealed normal appearing ovaries, kidneys and adrenal glands, MRI was also requested but not done prior to her evaluation with us. On repeat testing, using chemiluminescent assay, her serum testosterone levels were again elevated to 422ng/dL, with elevated free androgen index and elevated bioavailable testosterone. Since she had regular, predictable menstrual cycles we suspected falsely elevated testosterone levels. On dilution, results were non-linear indicating interference. Total testosterone levels after addition of heterophilic antibody blocking reagent resulted as 114ng/dL (from 422nl/dL) which indicated heterophile antibody interference. Since this did not reflect true testosterone levels, we repeated the test using liquid chromatography tandem-mass spectrometry (LC-MS/MS) which revealed total testosterone level as 19ng/dL, which correlated with her clinical presentation and the pending MRI was canceled. Immunoassays, although widely used, are limited by cross-reactivity, matrix effects and heterophilic antibodies. LC-MS/MS has superior performance and sensitivity for measurements at low concentrations including steroid hormones such as testosterone, estradiol, cortisol, 17-hydroxyprogesterone. Conclusion: Immunoassays are widely used due to their commercial availability and lower cost. However, discordance between clinical presentation and laboratory tests measuring steroid hormones should prompt endocrinologists to consider laboratory errors, including interfering substances. Repeat testing using LC-MS/MS for steroid hormones, such as testosterone, should be considered to prevent unnecessary repeat testing and imaging.

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Falsely Elevated Testosterone Levels: Role of LC-MS/MS