{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,8]],"date-time":"2026-04-08T09:45:56Z","timestamp":1775641556210,"version":"3.50.1"},"reference-count":45,"publisher":"Public Library of Science (PLoS)","issue":"6","license":[{"start":{"date-parts":[[2024,6,28]],"date-time":"2024-06-28T00:00:00Z","timestamp":1719532800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:sec id=\"sec001\">\n<jats:title>Background<\/jats:title>\n<jats:p>Many newborn screening programs worldwide have introduced screening for diseases using DNA extracted from dried blood spots (DBS). In Germany, DNA-based assays are currently used to screen for severe combined immunodeficiency (SCID), spinal muscular atrophy (SMA), and sickle cell disease (SCD).<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec002\">\n<jats:title>Methods<\/jats:title>\n<jats:p>This study analysed the impact of pre-analytic DNA carry-over in sample preparation on the outcome of DNA-based newborn screening for SCID and SMA and compared the efficacy of rapid extraction versus automated protocols. Additionally, the distribution of T cell receptor excision circles (TREC) on DBS cards, commonly used for routine newborn screening, was determined.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec003\">\n<jats:title>Results<\/jats:title>\n<jats:p>Contaminations from the punching procedure were detected in the SCID and SMA assays in all experimental setups tested. However, a careful evaluation of a cut-off allowed for a clear separation of true positive polymerase chain reaction (PCR) amplifications. Our rapid in-house extraction protocol produced similar amounts compared to automated commercial systems. Therefore, it can be used for reliable DNA-based screening. Additionally, the amount of extracted DNA significantly differs depending on the location of punching within a DBS.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec004\">\n<jats:title>Conclusions<\/jats:title>\n<jats:p>Newborn screening for SMA and SCID can be performed reliably. It is crucial to ensure that affected newborns are not overlooked. Therefore a carefully consideration of potential contaminating factors and the definition of appropriate cut-offs to minimise the risk of false results are of special concern. It is also important to note that the location of punching plays a pivotal role, and therefore an exact quantification of TREC numbers per \u03bcl may not be reliable and should therefore be avoided.<\/jats:p>\n<\/jats:sec>","DOI":"10.1371\/journal.pone.0306329","type":"journal-article","created":{"date-parts":[[2024,6,28]],"date-time":"2024-06-28T18:33:21Z","timestamp":1719599601000},"page":"e0306329","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":2,"title":["Quality considerations and major pitfalls for high throughput DNA-based newborn screening for severe combined immunodeficiency and spinal muscular 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Sharma","year":"2019","journal-title":"Iran J Parasitol"},{"issue":"6","key":"pone.0306329.ref033","doi-asserted-by":"crossref","first-page":"597","DOI":"10.2217\/14622416.8.6.597","article-title":"High-resolution DNA melting analysis for simple and efficient molecular diagnostics","volume":"8","author":"GH Reed","year":"2007","journal-title":"Pharmacogenomics"},{"issue":"4","key":"pone.0306329.ref034","first-page":"389","article-title":"Preventing PCR amplification carryover contamination in a clinical laboratory","volume":"34","author":"J. Aslanzadeh","year":"2004","journal-title":"Ann Clin Lab Sci"},{"issue":"20","key":"pone.0306329.ref035","first-page":"e135","article-title":"A new method to prevent carry-over contaminations in two-step PCR NGS library preparations","volume":"43","author":"V Seitz","year":"2015","journal-title":"Nucleic Acids Res"},{"issue":"8","key":"pone.0306329.ref036","doi-asserted-by":"crossref","first-page":"e23604","DOI":"10.1371\/journal.pone.0023604","article-title":"Profiling trait anxiety: transcriptome analysis reveals cathepsin B (Ctsb) as a novel candidate gene for emotionality in mice","volume":"6","author":"L Czibere","year":"2011","journal-title":"PLoS One"},{"key":"pone.0306329.ref037","doi-asserted-by":"crossref","first-page":"70","DOI":"10.1016\/j.clinbiochem.2019.10.009","article-title":"Automated analysis of dried urine spot (DUS) samples for toxicology screening","volume":"75","author":"A Pablo","year":"2020","journal-title":"Clin Biochem"},{"issue":"3","key":"pone.0306329.ref038","doi-asserted-by":"crossref","DOI":"10.3390\/v14030646","article-title":"TREC\/KREC Levels and T and B Lymphocyte Subpopulations in COVID-19 Patients at Different Stages of the Disease","volume":"14","author":"AA Savchenko","year":"2022","journal-title":"Viruses"},{"key":"pone.0306329.ref039","doi-asserted-by":"crossref","first-page":"76","DOI":"10.1186\/1824-7288-36-76","article-title":"Genetics of SCID","volume":"36","author":"F. 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