The High Relevance of 21-Deoxycortisol, (Androstenedione+17 alpha-Hydroxyprogesterone)/Cortisol, and 11-Deoxycortisol/17 alpha-Hydroxyprogesterone for Newborn Screening of 21-Hydroxylase Deficiency

Context: There are limited reports on the detailed examination of steroid profiles for setting algorithms for 21-hydroxylase deficiency (21OHD) screening by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Objective: We aimed to define an algorithm for newborn screening of 21OHD by LC-MS/MS, measuring a total of 2077 dried blood spot samples in Tokyo. Methods: Five steroids (17 alpha-hydroxyprogesterone [17 alpha OHP], 21-deoxycortisol [21 DOF], 11-deoxycortisol [11DOF], androstenedione [4AD], and cortisol [F1) were included in the panel of LC-MS/MS. Samples from 2 cohorts were assayed: Cohort A, 63 "screening positive" neonates who were referred to an endocrinologist (n = 26 with 21OHD; n = 37 false-positive; obtained from 2015 to 2020); and Cohort B, samples (n = 2014) with 17 alpha OHP values in the 97th percentile or above, in the first-tier test with 17 alpha OHP ELISA from 2020 to 2021. Results: Analysis of Cohort A revealed that the 3 indexes 21DOF, 11DOF/17 alpha OHP, and (4AD + 17 alpha OHP)/F had higher area under the curve (AUC) values (0.999, 0.997, 0.989, respectively), while the 17 alpha OHP AUC was lower (0.970). Accordingly, in addition to 17 alpha OHP, the 3 markers were included for defining the screening algorithm. The assay of Cohort B revealed that the new algorithm gave 92% of predicted positive predictive value without false-negative cases. We also determined the reference values for the 5 steroids at 4 to 7 days after birth, according to sex and gestational age (GA), revealing extremely low levels of 21DOF at any GA irrespective of sex differences. Conclusion: Our study demonstrated the high relevance of 21DOF, (4AD + 17 alpha OHP)/F, and 11DOF/17 alpha OHP, rather than 17 alpha OHP, for 21OHD screening.