Numerous studies have shown evidence supporting the benefits of universal newborn screening for primary immunodeficiencies (PID) and for Spinal Muscular Atrophy (SMA). We have developed a four-plex, real-time PCR assay to screen for Severe Combined Immune Deficiencies (SCID), X-linked agammaglobulinemia (XLA), and SMA in DNA extracted from a single 3.2 mm punch of a dried blood spot (DBS). A simple, high-throughput, semi-automated DNA extraction method was developed for a Janus liquid handler that can process 384 DBS punches in four 96-well plates in just over one hour with sample tracking capability. The PCR assay identifies the absence of exon 7 in the SMN1 gene, while simultaneously evaluating the copy number of T-cell receptor excision circles (TREC) and Kappa-deleting recombination excision circles (KREC) molecules. Additionally, the amplification of a reference gene, RPP30, was included in the assay as a quality/quantity indicator of DNA isolated from the DBS. The assay performance was demonstrated on over 3000 DNA samples isolated from punches of putative normal newborn DBS. The reliability and analytical accuracy were further evaluated using DBS controls, and contrived and confirmed positive samples. The results from this study demonstrate the potential of future molecular DBS assays, and highlight how a multiplex assay could benefit newborn screening programs.

Galactosaemia has been included in various newborn screening programs since 1963. Several methods are used for screening; however, the predominant methods used today are based on the determination of either galactose-1-phosphate uridyltransferase (GALT) activity or the concentration of total galactose. These methods cannot be multiplexed and therefore require one full punch per sample. Since the introduction of mass spectrometry in newborn screening, many diseases have been included in newborn screening programs. Here, we present a method for including classical galactosaemia in an expanded newborn screening panel based on the specific determination of galactose-1-phosphate by tandem mass spectrometry. The existing workflow only needs minor adjustments, and it can be run on the tandem mass spectrometers in routine use. Furthermore, compared to the currently used methods, this novel method has a superior screening performance, producing significantly fewer false positive results. We present data from 5500 routine newborn screening samples from the Danish Neonatal Screening Biobank. The cohort was enriched by including 14 confirmed galactosaemia positive samples and 10 samples positive for other metabolic disorders diagnosed through the Danish newborn screening program. All galactosaemia positive samples were identified by the method with no false positives. Furthermore, the screening performance for other metabolic disorders was unaffected.