Three flowcharts compare current and second-tier NGS approaches for newborn screening with low TREC levels. Panel A shows the current NBS process where 68 cases with TREC levels below the cut-off are divided into two groups: 22 cases with 2 copies and 46 cases with more than 2 and up to 10 copies. All 68 cases are referred for further evaluation, with outcomes including SCID, non-SCID genetic causes, reversible conditions, low birth weight, ITCL, inconclusive results, and normal T-cell subsets. Panel B illustrates the second-tier NGS approach without a safety net. The 68 cases are similarly divided, but only 16 cases are referred after second-tier NGS, while 52 cases are not referred. Panel C depicts the second-tier NGS approach with a safety net. Here, 22 cases with 2 copies are directly referred, 7 cases out of 46 with more than 2 and up to 10 copies undergo second-tier NGS, resulting in 29 referrals and 39 non-referrals. The outcomes for referred cases are similar to those in Panel A.
Effect of second-tier NGS approaches on referrals. (A) Diagnostic outcomes of the analyzed low-TREC cases (n = 68, ≤10 copies/3.2 mm punch, ImmunoIVD) following the current screening algorithm with first-tier TREC analysis only. A more detailed overview of the clinical characteristics is provided in Table S5. (B and C) The number of referred cases if second-tier NGS would be performed without a safety net (B) and with a safety net (C) to maintain high sensitivity for detecting SCID. With the safety net, second-tier NGS would be performed for all newborns with TRECs ≤10, but newborns with TRECs ≤2 would be referred directly after the first-tier result, with NGS results available after the initial clinical evaluation. Newborns with TRECs >2 and ≤10 would be referred only if NGS results are positive. aFive newborns with TRECs >2 and ≤10 were indirectly referred after repeat analysis on a second NBS card according to the adjusted referral scheme after national implementation (see Materials and methods).