High Throughput Newborn Screening for Sickle Cell Disease - Application of Two-Tiered Testing with a qPCR-Based Primary screen.

ABSTRACT

BACKGROUND: Sickle cell disease (SCD) is a group of hemoglobinopathies with a common point mutation causing the production of sickle cell hemoglobin (HbS). In high-throughput newborn screening (NBS) for SCD, a two-step procedure is suitable, in which qPCR first pre-selects relevant samples that are differentiated by a second method. METHODS: Three NBS centers using qPCR-based primary screening for SCD performed a laboratory comparison. Methods using tandem MS or HPLC were used for differentiation. RESULTS: In a benchmarking test, 450 dried blood samples were analyzed. Samples containing HbS were detected as reliably by qPCR as by methods established for hemoglobinopathy testing. In a two-step screening approach, the 2nd-tier-analyses have to distinguish the carrier status from pathological variants. In nine months of regular screening, a total of 353,219 samples were analyzed using two-stage NBS procedures. The 1st-tier screening by qPCR reduced the number of samples for subsequent differentiation by>99.5%. Cases with carrier status or other variants were identified as inconspicuous while 78 cases with SCD were revealed. The derived incidence of 14,773, is in good agreement with previously published incidences. CONCLUSION: In high-throughput NBS for SCD, qPCR is suitable to focus 2nd-tier analyses on samples containing HbS, while being unaffected by factors such as prematurity or transfusions. The substantial reduction of samples numbers positively impacts resource conservation, sustainability, and cost-effectiveness. No false negative cases came to attention.