Concurrently, the qPCR assay registered HbS in 431 samples which were submitted to 2 nd-tier SCD screening, resulting in 17 HbS/S, five HbS/C, and two HbS/β thalassemia patients. The screening revealed two positive SCID cases, while 14 newborns with SMA were detected. Between July 2021 and March 2022, 96,015 samples were screened by applying the newly implemented assay. Subsequently, the 2 nd tier MS/MS assay is used to distinguish heterozygous HbS/A carriers from samples of patients with homozygous or compound heterozygous SCD. In our two-tier SCD screening strategy, our multiplex qPCR identifies samples carrying the HBB: c.20A>T allele that is coding for sickle cell hemoglobin (HbS). DNA is extracted from a 3.2-mm dried blood spot from which we simultaneously quantify T-cell receptor excision circles for SCID screening, identify the homozygous SMN1 exon 7 deletion for SMA screening, and determine the integrity of the DNA extraction through the quantification of a housekeeping gene. Thus, we developed a combined approach applying a multiplexed quantitative real-time PCR (qPCR) assay for simultaneous SCID, SMA, and 1 st-tier SCD screening, followed by a tandem mass spectrometry (MS/MS) assay for 2 nd-tier SCD screening. Screening for SCD has been included in Germany’s NBS Program since Fall 2021 and typically requires high-throughput NBS laboratories to adopt analytical platforms that are demanding in terms of instrumentation and personnel. A high-throughput nucleic acid-based method in newborn screening (NBS) has been shown to be fast and cost-effective in the early detection of these diseases. Early diagnosis of severe combined immunodeficiency (SCID), spinal muscular atrophy (SMA), and sickle cell disease (SCD) improves health outcomes by providing a specific treatment before the onset of symptoms.
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