Chemiluminescent immunoassay overestimates hormone concentrations and obscures testosterone sex differences relative to LC-MS/MS in a field study of diverse adolescents

BACKGROUND: Methodological comparisons of hormone quantification techniques have repeatedly demonstrated that, in adults, enzyme immunoassay (EIA) inflates steroid hormone concentrations relative to mass spectrometry. However, methodological comparisons in adolescent samples remain rare, and few studies have examined how chemiluminescent immunoassay (CLIA), another popular immunoassay, compares to mass spectrometry. Additionally, no studies have examined how differences in analytical techniques may be affecting relationships between steroid hormone levels and outcomes of interest, such as psychopathology. This pre-registered analysis of an existing dataset measured salivary cortisol and testosterone using both CLIA and liquid chromatography dual mass spectrometry (LC-MS/MS) in a repeated measures (516 samples) sample of 207 9th graders. METHODS: In aim 1, this study sought to expand on past findings by 1) measuring inflation of testosterone and cortisol by CLIA in a relatively large adolescent sample, and 2) showing that CLIA (like EIA) testosterone inflation was especially true in groups with low ‘true’ testosterone levels. In aim 2, this study sought to examine the impact of hormone quantification method on relationships between hormone levels and psychopathological measures (the Children's Depression Inventory, the Perceived Social Stress Scale, the UCLA Loneliness Scale, and the Anxious Avoidant and Negative Self Evaluation subscales of the Social Anxiety Scale for Adolescents). RESULTS: We found that CLIA, like EIA, inflated testosterone and cortisol levels and overestimated female testosterone resulting in suppressed sex differences in testosterone. We did not observe these same patterns when examining testosterone in individuals with differing levels of pubertal development. Results of psychopathology analyses demonstrated no significant method differences in hormone-psychopathology relationships. CONCLUSIONS: Our findings show that CLIA introduces proportional bias in cortisol and testosterone in a manner that suppresses sex differences in testosterone. Steroid measurement method did not significantly moderate the relationship between hormones and psychopathology in our sample, though more work is needed to investigate this question in larger, clinical samples.