Feline testosterone assay: analytical and clinical validation in intact and castrated tom cats

Introduction- Testosterone measurement plays a crucial role in differentiating intact from neutered male cats. However, reliable cut-off values and validated analytical methodologies for feline testosterone quantification remain limited. This study aimed to validate a fluorescence enzyme immunoassay (FEIA) for testosterone measurement in tomcats and establish clinically relevant reference intervals to differentiate intact from neutered individuals with high specificity and sensitivity. Material and methods- Both frozen and fresh serum samples were collected from 80 male cats (40 intact, 40 neutered) presented at the Ospedale Veterinario Universitario Didattico (OVUD), University of Padova between January 1, 2022, and October 30, 2024, and used for testosterone analysis. Testosterone concentrations were measured using the AIA-360 Automated Immunoassay Analyzer, employing a fluorescence enzyme immunoassay (FEIA) methodology. Analytical validation included intra-assay and inter-assay Coefficient of Variations and Linearity Under Dilution (LUD). For clinical validation, a receiver operating characteristic (ROC) curve analysis was conducted to establish optimal testosterone cut-off values for distinguishing intact from castrated cats. Additionally, the upper reference limit of testosterone in castrated cats was estimated with a 95% confidence interval (CI). Results- Analytical validation confirmed good intra-assay and inter-assay precision at high testosterone concentrations, while variability increased at lower levels. LUD analysis demonstrated proportionality between expected and observed values, with minimal systematic proportional error. Clinical validation identified > 0.12 ng/mL as the optimal cut-off for balancing sensitivity (60%) and specificity (90%). However, a more stringent threshold of > 0.27 ng/mL ensured 100% specificity, preventing misclassification of neutered cats as intact. The 95% CI for the upper reference limit of testosterone in neutered cats was 0.16 ng/mL, with a 90% CI ranging from 0.12. to 0.20 ng/mL. Conclusions- This study provides a validated analytical approach for testosterone measurement in male cats and establishes clinically relevant cut-off. A testosterone level above 0.27 ng/mL guarantees intact status with 100% specificity. However, values below this threshold do not definitively confirm castrated status, as 52.5% intact cats may have lower testosterone levels. These findings offer a reliable diagnostic framework for assessing feline reproductive status in both clinical and research settings.