A-060 Comparative Evaluation of three Immunoassays and LC-MS/MS for the Measurement of Urinary Free Cortisol in Cushing’s Syndrome Diagnosis

Abstract Background Measurement of urinary free cortisol (UFC) is crucial for diagnosing Cushing’s syndrome (CS), as it offers a non-invasive approach to assess hypercortisolism. CS, a rare but serious endocrine disorder, is characterized by prolonged exposure to elevated cortisol levels and is associated with increased mortality and diminished quality of life due to comorbidities. In this study, we compared UFC determination using three immunoassays: Autobio A6200 (Autobio, Zhengzhou, China), Mindray CL-1200i (Mindray, Shenzhen, China), and Snibe MAGLUMI X8 (Shenzhen New Industries Biomedical Engineering, Shenzhen, China) against liquid chromatography-tandem mass spectrometry (LC-MS/MS). Additionally, we assessed the diagnostic value of 24-hour UFC measurements obtained from these three immunoassays in identifying CS. Methods Residual 24-hour urine samples from 94 patients diagnosed with CS and 243 non-CS patients collected from our previous cohort were utilized. A laboratory-developed LC-MS/MS method was used as the reference. UFC was measured through direct assays on Autobio, Mindray, and Snibe platforms. Method comparisons were conducted using Passing–Bablok regression and Bland–Altman plot analyses. Cut-off values for each assay along with their corresponding sensitivities and specificities were determined via ROC analysis. Results The immunoassays from Autobio, Mindray, and Snibe exhibited strong correlations with LC-MS/MS [Correlation coefficient r=0.961 (95% CI, 0.951–0.968), 0.989 (95% CI, 0.987–0.992), and 0.963 (95% CI, 0.954–0.970), respectively; p<0.0001]. All immunoassays demonstrated a proportionally positive bias when compared to LC-MS/MS; the regression equations were y=0.13+1.60 x for Autobio; y=-0.111+1.126x for Mindray; and y=0.35+1.22x for Snibe respectively. The areas under the curve (AUC) were calculated as follows: AUC = 0.962 (95% CI, 0.935–0.980) for Autobio, AUC =0.969 (95% CI, 0.944–0.985) for Mindray, and AUC = 0.963 (95% CI, 0.937–0.981) for Snibe. Comparing these results to that of LC-MS/MS (AUC = 0.972, 95% CI, 0.948–0.987), no significant differences among the three immunoassays were observed regarding their performance. The cut-off values of immunoassays were found to be higher than that obtained from LC-MS/MS, with ranges of 64.9–98.6 µg/24 hr compared to 56.1 µg/24 hr for LC-MS/MS. The sensitivity and specificity of the assays ranged from 89.8% to 93.2% and from 93.3% to 96.2%, respectively. Conclusion Recently, there has been a notable increase in the availability of highly specific antibodies and innovative detection principles, alongside new reagents introduced by manufacturers worldwide that are gradually being integrated into clinical laboratories. In this study, we have demonstrated that three UFC reagents from Autobio, Mindray, and Snibe exhibit reliable consistency with LC-MS/MS results. Furthermore, these reagents demonstrate high diagnostic accuracy for the identification of CS.