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

Abstract

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-hydroxyprogesterone (17OHP) 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˚C, -20˚C or -70˚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 17OHP were not significantly changed for 1 year even at room temperature at either analyte levels. In contrast, Cort required storage at 4˚C, -20˚C or -70˚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˚C, -20˚C or -70 C and at least for 4 weeks at room temperature. These findings have implications for analyses of stored DBS samples in 2 nd -tier assays in newborn screening and for retrospective CAH studies.

Bobcat Hair Cortisol Correlates with Land Use and Climate

“…Second, most of the carcasses we sampled had been exposed to ambient conditions for weeks or months prior to being sampled, and samples were stored in paper envelopes for months between sampling and assaying. Although cortisol bound in hair is likely stable on the scale of years to decades (Webb et al 2010, cortisol exogenous to hair would significantly degrade over weeks to months (Grecsó et al 2020). We concluded the high specificity of the cortisol assay and the unlikelihood of exogenous cortisol were sufficient to produce quality data without risking loss of endogenous cortisol through washing.…”

Implementing steroid profiling by liquid chromatography‐tandem mass spectrometry improves newborn screening for congenital adrenal hyperplasia in New Zealand

“…Recently, a study has shown that that retrospective LCMSMS analysis of newborn screening samples may not be appropriate as cortisol and 11-deoxycortisol are not stable in specimens undergoing long term storage at room temperature. 19 The prospective nature of this study was a strength, as it ensured the reliability of measured steroid parameters with no risk to screening sensitivity during the period of the study. However, this approach also limited the number of available samples from newborns with CAH to a relatively small number.…”

“…Further evaluation, over a much longer period, of this parameter is warranted if it is to be included in newborn screening protocols as retrospective studies are not possible because of the instability of 11DF in stored samples. 19 The incorporation of 21-deoxycortisol into newborn screening is gaining increasing attention. 22 21DF concentrations in this study however were not elevated in all specimens from newborns with confirmed CAH and 21DF was also detected in a small number of samples with a negative screening result.…”

Quantitative microsampling for bioanalytical applications related to the SARS-CoV-2 pandemic: Usefulness, benefits and pitfalls

“… LC-UV [ 78 ] (continued) Microsampling technique Matrix Analyte(s) Sample volume (μL) Analytical technique Reference DBS Blood Endogenous glucocorticoids 60 LC-MS/MS [ 79 ] VAMS Blood Endogenous glucocorticoids 10 LC-MS/MS [ 79 ] Noviplex (DPS) Plasma Endogenous glucocorticoids 40 LC-MS/MS [ 79 ] DBS Blood Endogenous glucocorticoids n.s. LC-MS/MS [ 80 ] DBS Blood Endogenous glucocorticoids n.s. LC-MS/MS [ 81 ] DBS Blood Endogenous glucocorticoids n.s.…”

“…In general, microsampling applications have been reported for endogenous corticosteroid determination (thus including cortisol and/or cortisone) in DBS [ [80] , [81] , [82] ] and in urine pretreated by SPME [ 83 , 84 ]. Regarding specifically the exogenous corticosteroids most often involved in SARS-CoV-2 therapy, budesonide has been analysed in DBS [ 85 ].…”