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 (<7% coefficient of variation (CV) for ≥0 copy) and TREC (14.6% CV at 37 copies/µL 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 ≥ 34 weeks (n = 1812) and the 2.5th percentile was 57 copies/µL 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.

X-linked adrenoleukodystrophy (X-ALD) is the most common peroxisomal disorder and is associated with serious clinical consequences. While newborn screening (NBS) could facilitate early identification and timely intervention, its implementation for X-ALD remains controversial due to ethical concerns arising from its X-linked recessive inheritance pattern, particularly regarding female newborns. This study aims to explore the perspectives of healthcare professionals and the general public on NBS for X-ALD in Hong Kong. An online survey with 20 quantitative questions on ethical considerations was conducted from May to August 2024. Among a total of 259 responses, most respondents (99.2%) supported NBS for their male newborns, primarily because it facilitates early diagnosis and effective management. The majority of the respondents were in favor of offering NBS to female newborns, citing potential benefits for the management of adult-onset disease, enhanced family planning and support, and opportunities for extended family screening. However, some respondents expressed concerns regarding (1) psychological stress and anxiety from uncertain disease onset and frequent monitoring; (2) potential genetic discrimination and adverse impact on insurance premiums/coverage; (3) affordability and accessibility of expensive treatments, such as gene therapy; and (4) ethical issues regarding children’s “right to an open future”, particularly for late-onset female X-ALD. To address these concerns while respecting family autonomy, we propose an opt-in system with clear, balanced information for parents, combined with a three-tier screening algorithm. In summary, while inclusion of X-ALD in Hong Kong’s NBS program receives strong community support, targeted measures are needed to mitigate the identified ethical and practical barriers.