A Two-Step LC-MS/MS method to differentiate 21OHD and PORD in Screen-Positive newborns.

BACKGROUND: The multiplex steroid assay of liquid chromatography-tandem mass spectrometry (LC-MS/MS) has the potential to diagnose 21-hydroxylase deficiency (21OHD) directly from screening results. AIM: To establish an endocrinological diagnostic algorithm for 21OHD from screening positive cases based on LC-MS/MS screening. METHODS: For determining the cut-off criteria of 21-deoxycortisol (21DOF), the Tokyo screening cohort (21OHD, n = 26; false positives(FPs), n = 37) was examined. For identifying the biomarker to differentiate 21OHD from P450 oxidoreductase (POR) deficiency (PORD), another condition to cause 21DOF elevation in addition to 17α-OHP, we employed two cohorts from different origins, namely, 21OHD patients from the Tokyo screening cohort and the endocrinological data of previously reported PORD cases (n = 42 of 17 reports). RESULTS: The 21DOF levels between 21OHD patients and FPs yielded 0.999 AUC. The combination of 17α-OHP > 15.1 nmol/L (5.0 ng/mL) and 21DOF > 2.88 nmol/L (1.0 ng/mL) yielded 1.000 and 0.949 of the positive and negative predictive values, respectively. Using both data, 21OHD and PORD can be narrowed down from other conditions in which 17α-OHP is elevated. To estimate 17,20-lyase activity, which is compromised only in PORD, we examined the androstenedione/cortisol (4AD/F) molar ratio, revealing a significant, non-overlapping difference (AUC, 1.000). Postnatal and gestational age dependency of the 4AD/F ratio was not apparent. CONCLUSION: We propose a two-step method to endocrinologically diagnose 21OHD in screening positive newborns. After, narrowing down to 21OHD and PORD using a cut-off of 21DOF > 2.88 nmol/L (1.0 ng/mL), a 4AD/F ratio > 0.06 would be indicative of 21OHD.