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In newborn screening, samples suspected for congenital adrenal hyperplasia (CAH), a potentially lethal inborn error of steroid biosynthesis, need to be confirmed using liquid chromatography-tandem mass spectrometry. Daily quality controls (QCs) for the 2nd-tier CAH assay are not commercially available and are therefore generally prepared within the laboratory. For the first time, we aimed to compare five different QC preparation approaches used in routine diagnostics for CAH on the concentrations of cortisol, 21-deoxycortisol, 11-deoxycortisol, 4-androstenedione and 17-hydroxyprogesterone in dried blood spots. The techniques from Prep1 to Prep5 were tested at two analyte concentrations by spiking aliquots of a steroid-depleted blood, derived from washed erythrocyte suspension and steroid-depleted serum. The preparation processes differed in the sequence of the preparation steps and whether freeze-thaw cycles were used to facilitate blood homogeneity. The five types of dried blood spot QCs were assayed and quantitated in duplicate on five different days using a single calibration row per day. Inter-assay variations less than 15% and concentrations within ±15% of the nominal values were considered acceptable. Results obtained by means of the four dried blood spot QC preparation techniques (Prep1, Prep2, Prep4 and Prep5) were statistically similar and remained within the ±15% ranges in terms of both reproducibility and nominal values. However, concentration results for Prep3 (spiking prior to three freeze-thaw cycles) were significantly lower than the nominal values in this setting, with differences exceeding the ±15% range in many cases despite acceptable inter-assay variations. These findings have implications for the in-house preparation of QC samples in laboratory developed tests for CAH, including 2nd-tier assays in newborn screening.

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

Increased propionylcarnitine levels in newborn screening are indicative for a group of potentially severe disorders including propionic acidemia (PA), methylmalonic acidemias and combined remethylation disorders (MMACBL). This alteration is relatively non-specific, resulting in the necessity of confirmation and differential diagnosis in subsequent tests. Thus, we aimed to develop a multiplex approach for concurrent determination of 3-hydroxypropionic acid, methylmalonic acid and methylcitric acid from the same dried blood spot (DBS) as in primary screening (second-tier test). We also set out to validate the method using newborn and follow-up samples of patients with confirmed PA or MMACBL.The assay was developed using liquid chromatography-tandem mass spectrometry and clinically validated with retrospective analysis of DBS samples from PA or MMACBL patients.Reliable determination of all three analytes in DBSs was achieved following simple and fast (