Comparative analysis of two TREC/KREC quantification methods for newborn screening of primary immunodeficiency with T- and B-lymphopenia used in Ukraine

Background. Primary immunodeficiency (PID) compromise the immune system, leaving newborns highly vulnerable to infections. Severe combined immunodeficiency (SCID) is the most severe form, characterized by the absence or dysfunction of T and B cells. Without early treatment, most infants with SCID do not survive their first year. In Ukraine, after a successful pilot project, newborn screening for SCID and other types of PID is now part of Advanced Neonatal Screening, using T-cell receptor excision circles (TREC) and kappa-deleting recombination excision circles (KREC) measurements. Since the pilot project used a different method than the current screening program, the purpose of this study was to compare these two methods, evaluating their benefits and downsides, aiming to optimize screening for early, effective treatment. Materials and Methods. In the pilot project method (Method 1), TREC and KREC quantification was performed using a custom real-time PCR assay with melting curve analysis. Method 1 included standards with known TREC and KREC copy numbers, no-template controls (NTCs), and positive controls to ensure reliable results. The method currently employed in the Advanced Neonatal Screening (Method 2) uses the Biocore® SMA/TKID PLUS Diagnostic Kit, a commercial kit, for TREC, KREC and SMN1 quantification via real-time PCR. Measurements for both methods are reported per one million cells. Results and Discussion. While there are differences in the general parameters of DNA extraction, PCR, and result analysis and interpretation, both Method 1 and Method 2 showed a significant difference in Cq values. Despite these differences, both methods demonstrated the capability of inentifying abnormal TREC/KREC values, enabling the detection of SCID and some PID cases. Conclusion. The pilot project demonstrated the effectiveness of TREC/KREC quantification for SCID screening and led to its implementation in Advanced Neonatal Screening in Ukraine. Over 121,000 newborns were tested, confirming six positive cases. Method 1 provides higher precision and versatility, while Method 2 is faster, simpler, and capable of automation but lacks precise quantification. Adding standards to Method 2 could enhance its utility for widespread SCID screening.