Concurrent Confirmation and Differential Diagnosis of Congenital Adrenal Hyperplasia from Dried Blood Spots: Application of a Second-Tier LC-MS/MS Assay in a Cross-Border Cooperation for Newborn Screening

Abstract Background: Newborn screening for maple syrup urine disease (MSUD) relies on finding increased concentrations of the branched-chain amino acids (BCAAs) leucine, isoleucine, and valine by tandem mass spectrometry (MS/MS). d-Alloisoleucine (allo-Ile) is the only pathognomonic marker of MSUD, but it cannot be identified by existing screening methods because it is not differentiated from isobaric amino acids. Furthermore, newborns receiving total parenteral nutrition often have increased concentrations of BCAAs. To improve the specificity of newborn screening for MSUD and to reduce the number of diet-related false-positive results, we developed a LC-MS/MS method for quantifying allo-Ile. Methods: Allo-Ile and other BCAAs were extracted from a 3/16-inch dried blood spot punch with methanol/H2O, dried under nitrogen, and reconstituted into mobile phase. Quantitative LC-MS/MS analysis of allo-Ile, its isomers, and isotopically labeled internal standards was achieved within 15 min. To determine a reference interval for BCAAs including allo-Ile, we analyzed 541 dried blood spots. We also measured allo-Ile in blinded samples from 16 MSUD patients and 21 controls and compared results to an HPLC method. Results: Intra- and interassay imprecision (mean CVs) for allo-Ile, leucine, isoleucine, and valine ranged from 1.8% to 7.4%, and recovery ranged from 91% to 129%. All 16 MSUD patients were correctly identified. Conclusions: The LC-MS/MS method can reliably measure allo-Ile in dried blood spots for the diagnosis of MSUD. Applied to newborn screening as a second-tier test, it will reduce false-positive results, which produce family anxiety and increase follow-up costs. The assay also appears suitable for use in monitoring treatment of MSUD patients.

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Cortisol assay in dried blood spots to reduce false positive rate in congenital adrenal hyperplasia screening

Clinica Chimica Acta ◽

10.1016/j.cca.2012.04.022 ◽

2012 ◽

Vol 413 (15-16) ◽

pp. 1306-1307

Author(s):

Julie Brossaud ◽

Pascal Barat ◽

Laurence Fagour ◽

Jean-Benoît Corcuff

Keyword(s):

Congenital Adrenal Hyperplasia ◽

False Positive ◽

False Positive Rate ◽

Dried Blood Spots ◽

Adrenal Hyperplasia ◽

Blood Spots ◽

Positive Rate

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Monitoring of Congenital Adrenal Hyperplasia by Microbore HPLC–Electrospray Ionization Tandem Mass Spectrometry of Dried Blood Spots

Clinical Chemistry ◽

10.1093/clinchem/48.2.354 ◽

2002 ◽

Vol 48 (2) ◽

pp. 354-356 ◽

Cited By ~ 10

Author(s):

Chien-Chen Lai ◽

Chang-Hai Tsai ◽

Fuu-Jen Tsai ◽

Jer-Yuarn Wu ◽

Wei-De Lin ◽

...

Keyword(s):

Mass Spectrometry ◽

Tandem Mass Spectrometry ◽

Congenital Adrenal Hyperplasia ◽

Electrospray Ionization ◽

Dried Blood Spots ◽

Tandem Mass ◽

Adrenal Hyperplasia ◽

Blood Spots ◽

Ionization Tandem Mass Spectrometry

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Diagnosis of congenital adrenal hyperplasia by rapid determination of 17α-hydroxyprogesterone in dried blood spots by gas chromatography/mass spectrometry following microwave-assisted silylation

Rapid Communications in Mass Spectrometry ◽

10.1002/rcm.2163 ◽

2005 ◽

Vol 19 (20) ◽

pp. 2974-2978 ◽

Cited By ~ 22

Author(s):

Chunhui Deng ◽

Jie Ji ◽

Lijuan Zhang ◽

Xiangmin Zhang

Keyword(s):

Mass Spectrometry ◽

Gas Chromatography ◽

Congenital Adrenal Hyperplasia ◽

Rapid Determination ◽

Dried Blood Spots ◽

Gas Chromatography Mass Spectrometry ◽

Adrenal Hyperplasia ◽

Blood Spots ◽

Microwave Assisted

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

PLoS ONE ◽

10.1371/journal.pone.0252091 ◽

2021 ◽

Vol 16 (5) ◽

pp. e0252091

Author(s):

Nóra Grecsó ◽

Anita Zádori ◽

Ákos Baráth ◽

Zsolt Galla ◽

Gábor Rácz ◽

...

Keyword(s):

Congenital Adrenal Hyperplasia ◽

Newborn Screening ◽

Dried Blood Spots ◽

Dried Blood Spot ◽

Adrenal Hyperplasia ◽

Freeze Thaw ◽

Quality Controls ◽

17 Hydroxyprogesterone ◽

Preparation Techniques ◽

Blood Spot

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.

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Next-generation sequencing as a second-tier diagnostic test for newborn screening

Journal of Pediatric Endocrinology and Metabolism ◽

10.1515/jpem-2018-0088 ◽

2018 ◽

Vol 31 (8) ◽

pp. 927-931 ◽

Cited By ~ 2

Author(s):

Xiaomei Luo ◽

Ruifang Wang ◽

Yanjie Fan ◽

Xuefan Gu ◽

Yongguo Yu

Keyword(s):

Next Generation Sequencing ◽

Newborn Screening ◽

Diagnostic Test ◽

Genomic Dna ◽

Sanger Sequencing ◽

Dried Blood Spots ◽

Next Generation ◽

Blood Spots ◽

Second Tier ◽

Generation Sequencing

Abstract Background Tandem mass spectrometry (MS/MS) has been used for newborn screening (NBS) of inherited metabolic diseases (IMDs) for decades. However, the traditional approach can yield false-positive or false-negative results and is affected by biochemical substrate-level fluctuations. To overcome the current limitations, we explored the possibility of using next-generation sequencing (NGS) as a second-tier diagnostic test to detect gene mutations in samples with abnormal MS/MS results. Methods Genomic DNA was extracted from dried blood spots and we designed a multigene panel, comprising 77 genes related to over 40 IMDs, for NBS. The prepared libraries were sequenced on the Ion Personal Genome Machine (PGM) platform. Thirty-eight samples identified as abnormal by MS/MS were tested for the diagnostic accuracy of NGS compared with Sanger sequencing. Results The concentration of DNA extracted from the 38 dried blood spots was sufficient for library preparation. The coverage and depth of the sequencing data were sufficient for the analysis. For all samples, the NGS results were consistent with the Sanger sequencing results. Conclusions The genomic DNA extracted from dried blood spots could be used for NGS, generating reliable sequencing results, and NGS may function as a second-tier diagnostic test for NBS. Ion PGM could facilitate the molecular diagnosis of IMDs with appropriate primers designed for candidate genes.

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