Comparison of different preparation techniques of dried blood spot quality controls in newborn screening for congenital adrenal hyperplasia

Abstract

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.

Current Status of Newborn Bloodspot Screening Worldwide 2024: A Comprehensive Review of Recent Activities (2020–2023)

“…A collaboration between universities in Hungary and Germany identified potential interferents in the analysis of methylmalonic acid, which has not previously been reported and represents a pitfall in clinical diagnostics and NBS [ 833 ]. Because no commercial quality control material is available for the second-tier NBS CAH assay, a 2021 report compared 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 bloodspots [ 834 ]. Also in 2021, a report detailed the issues relating to NBS for SMA in Hungary, with particular emphasis on the baby Zente case and the need to have routine NBS for SMA [ 835 ].…”

Advanced Microsamples: Current Applications and Considerations for Mass Spectrometry-Based Metabolic Phenotyping Pipelines

“…Consistent microsample preparation including sub-punches (if using traditional DBSs), extraction, and automation is important for reproducible analyte extraction. Similarly, consistent enrichment is important, should detection limits need to be improved for quantification (preconcentration) and the selective removal of interfering substances (sample clean-up) [110]. These processes are integral in metabolic phenotyping workflows as they determine the molecular concentrations in complex biological matrices (including blood, urine, and body tissues) used to derive clinical and biological conclusions.…”

Advanced Microsamples: Current Applications and Considerations for Mass Spectrometry-Based Metabolic Phenotyping Pipelines

“…Consistent microsample preparation including sub-punches (if using traditional DBSs), extraction, and automation is important for reproducible analyte extraction. Similarly, consistent enrichment is important, should detection limits need to be improved for quantification (preconcentration) and selective removal of interfering substances (sample clean-up) [110]. These processes are integral in metabolic phenotyping workflows as they determine molecular concentrations in complex biological matrices (including blood, urine, and body tissues) used to derive clinical and biological conclusions.…”