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Early diagnosis and timely treatment can prevent infections and secondary complications, emphasizing a role for newborn screening (NBS). NBS for XLA is based on quantification of kappa-deleting recombination excision circles (KRECs). KREC-based screening could result in a large number of false-positive referrals associated with high impact for parents and health care systems, indicating the need for a second tier test.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      KRECs were measured in NBS cards (\n                      <jats:italic>N<\/jats:italic>\n                      \u2009=\u2009110,491) with a multiplex TREC\/KREC qPCR assay. As second tier test options, an alternative qPCR multiplex assay, epigenetic immune cell counting for relative B-cell quantification and targeted next-generation sequencing with B-cell deficiency gene panel including 73 genes were performed on NBS cards of newborns with low KRECs.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>In total, 136\/110,491 newborns had KRECs below cut-off. With the alternative qPCR multiplex assay, 16\/110 of these newborns (14.5%) had KRECs above cut-off and would not have been referred. With epigenetic immune cell counting, 16.5% (17\/103) had relative B-cell counts in the range of healthy controls. Targeted NGS showed promising results as 87 out of 103 (84%) newborns with low KRECs did not show any pathogenic\/likely pathogenic variants and would not have been referred for follow-up diagnostics.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>Several second tier tests can potentially reduce the number of false-positive referrals in NBS for XLA. NGS seems to be the most effective technique in NBS for XLA and other forms of agammaglobulinemia. Our results show promising first steps towards the implementation of NBS for XLA.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1007\/s10875-025-01927-6","type":"journal-article","created":{"date-parts":[[2025,11,8]],"date-time":"2025-11-08T02:52:45Z","timestamp":1762570365000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Next-generation Sequencing and Other Second Tier Tests in Newborn Screening for (X-linked) Agammaglobulinemia"],"prefix":"10.1007","volume":"45","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2993-7847","authenticated-orcid":false,"given":"Maartje","family":"Blom","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Annelotte J.","family":"Duintjer","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ingrid","family":"Pico-Knijnenburg","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sandra","family":"Imholz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sahila","family":"Balkassmi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hermine A.","family":"van Duyvenvoorde","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1510-3104","authenticated-orcid":false,"given":"Mirjam","family":"van der Burg","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,11,8]]},"reference":[{"issue":"2","key":"1927_CR1","doi-asserted-by":"publisher","first-page":"279","DOI":"10.1016\/0092-8674(93)90667-F","volume":"72","author":"S Tsukada","year":"1993","unstructured":"Tsukada S, Saffran DC, Rawlings DJ, Parolini O, Allen RC, Klisak I, et al. 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