{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,30]],"date-time":"2026-08-30T00:40:43Z","timestamp":1788050443112,"version":"build-2784847793"},"reference-count":36,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2025,10,19]],"date-time":"2025-10-19T00:00:00Z","timestamp":1760832000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJNS"],"abstract":"<jats:p>Newborn screening (NBS) for spinal muscular atrophy (SMA) and severe combined immunodeficiency (SCID) faces challenges. Accurate and precise SMN1 and SMN2 copy number determination, confirmed by two orthogonal methods, are vital for SMA prognostication and treatment. Single SMN1 copy detection also enables the further feasibility to screen for compound heterozygotes. In SCID, low-level T-cell receptor excision circle (TREC) quantification by quantitative PCR is imprecise, necessitating replicates for reliable results. An assay with enhanced accuracy, precision, and high throughput is warranted for NBS SMA and SCID. False positive of SMN1 deletions due to allele dropout are also a potential pitfall in PCR-based methods. We evaluated a first-tier fully automated quadruplex droplet digital PCR (ddPCR) assay detecting SMN1, SMN2, TREC, and RPP30 using dried blood spots together with a second-tier Sanger sequencing to exclude SMN1 allele dropout. Five proficiency test samples and six patient samples with known SMN1 and SMN2 copy numbers confirmed by multiplex ligation-dependent probe amplification were used for accuracy evaluation with full concordance. The ddPCR assay showed high precision for SMN1 and SMN2 (&lt;7% coefficient of variation (CV) for \u22650 copy) and TREC (14.6% CV at 37 copies\/\u00b5L blood). Second-tier Sanger sequencing identified all SMA cases with homozygous deletions. Accuracy for TREC classification was concordant with 10 proficiency samples. The reference interval of TREC concentration was established for newborns \u2265 34 weeks (n = 1812) and the 2.5th percentile was 57 copies\/\u00b5L blood. A two-tiered approach with fully automated quadruplex ddPCR and Sanger sequencing delivers accurate and precise quantitation for NBS SMA and SCID, enabling early treatment and counseling.<\/jats:p>","DOI":"10.3390\/ijns11040097","type":"journal-article","created":{"date-parts":[[2025,10,20]],"date-time":"2025-10-20T13:54:41Z","timestamp":1760968481000},"page":"97","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Validation on the First-Tier Fully Automated High-Throughput SMN1, SMN2, TREC, and RPP30 Quantification by Quadruplex Droplet Digital PCR for Newborn Screening for Spinal Muscular Atrophy and Severe Combined Immunodeficiency"],"prefix":"10.3390","volume":"11","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1419-489X","authenticated-orcid":false,"given":"Chloe Miu","family":"Mak","sequence":"first","affiliation":[{"name":"Newborn Screening Laboratory, Department of Pathology, Hong Kong Children\u2019s Hospital, Hong Kong SAR, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-1025-4761","authenticated-orcid":false,"given":"Timothy Yiu Cheong","family":"Ho","sequence":"additional","affiliation":[{"name":"Newborn Screening Laboratory, Department of Pathology, Hong Kong Children\u2019s Hospital, Hong Kong SAR, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-3914-691X","authenticated-orcid":false,"given":"Man Kwan","family":"Yip","sequence":"additional","affiliation":[{"name":"Newborn Screening Laboratory, Department of Pathology, Hong Kong Children\u2019s Hospital, Hong Kong SAR, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Felicite Enyu","family":"Song","sequence":"additional","affiliation":[{"name":"Newborn Screening Laboratory, Department of Pathology, Hong Kong Children\u2019s Hospital, Hong Kong SAR, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Raymond Chiu Mo","family":"Tam","sequence":"additional","affiliation":[{"name":"Newborn Screening Laboratory, Department of Pathology, Hong Kong Children\u2019s Hospital, Hong Kong SAR, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Leanne Wing Ying","family":"Yu","sequence":"additional","affiliation":[{"name":"Newborn Screening Laboratory, Department of Pathology, Hong Kong Children\u2019s Hospital, Hong Kong SAR, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ann Anhong","family":"Ke","sequence":"additional","affiliation":[{"name":"Newborn Screening Laboratory, Department of Pathology, Hong Kong Children\u2019s Hospital, Hong Kong SAR, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eric Chun Yiu","family":"Law","sequence":"additional","affiliation":[{"name":"Newborn Screening Laboratory, Department of Pathology, Hong Kong Children\u2019s Hospital, Hong Kong SAR, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9099-4720","authenticated-orcid":false,"given":"Toby Chun Hei","family":"Chan","sequence":"additional","affiliation":[{"name":"Newborn Screening Laboratory, Department of Pathology, Hong Kong Children\u2019s Hospital, Hong Kong SAR, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3861-7033","authenticated-orcid":false,"given":"Matthew Chun Wing","family":"Yeung","sequence":"additional","affiliation":[{"name":"Newborn Screening Laboratory, Department of Pathology, Hong Kong Children\u2019s Hospital, Hong Kong SAR, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,10,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"412","DOI":"10.1373\/clinchem.2014.231019","article-title":"Newborn blood spot screening test using multiplexed real-time PCR to simultaneously screen for spinal muscular atrophy and severe combined immunodeficiency","volume":"61","author":"Taylor","year":"2015","journal-title":"Clin. 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