Storage stability of five steroids and in dried blood spots for newborn screening and retrospective diagnosis of congenital adrenal hyperplasia

Congenital adrenal hyperplasia (CAH) is a severe inherited disorder of cortisol biosynthesis that is potentially lethal or can seriously affect quality of life. For the first time, we aimed to assess the stability of 21-deoxycortisol (21Deox), 11-deoxycortisol (11Deox), 4-androstenedione (4AD), 17-hydroxprogesterone (170HP) and cortisol (Cort), diagnostic for CAH, in dried blood spots (DBSs) during a 1 year storage at different temperatures. Spiked DBS samples were stored at room temperature, 4 degrees C, -20 degrees C or -70 degrees C, respectively and analyzed in triplicates using liquid chromatography tandem mass spectrometry at Weeks 0, 1, 2, 3 and 4, Month 6 and Year 1. Analyte levels within 15% vs the baseline were considered stable. Our observations show that 21Deox, 4AD and 170HP were not significantly changed for 1 year even at room temperature at either analyte levels. In contrast, Cort required storage at 4 degrees C, -20 degrees C or -70 degrees C for long-term stability, being significantly decreased at room temperature from Month 6 (p<0.01) in both the 30(60) nM and the 90(180) nM samples. 11Deox was significantly decreased at room temperature at Year 1 (p<0.01) and only in the 30(60) nM samples. Thus, all biomarkers were stable for up to 1 year at 4 degrees C, -20 degrees C or -70 degrees C and at least for 4 weeks at room temperature. These findings have implications for analyses of stored DBS samples in 2nd-tier assays in newborn screening and for retrospective CAH studies.