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We hypothesized that a multiplex droplet digital PCR (ddPCR) assay covering key thyroid-cancer driver mutations would increase the pre-operative diagnostic yield.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      A six-gene ddPCR panel was developed to detect\n                      <jats:italic>BRAF<\/jats:italic>\n                      p.V600E,\n                      <jats:italic>TERT<\/jats:italic>\n                      promoter mutations c.-124\u00a0C\u2009&gt;\u2009T (C228T) and c.-146\u00a0C\u2009&gt;\u2009T (C250T),\n                      <jats:italic>PIK3CA<\/jats:italic>\n                      p.H1047R\/L, and all clinically relevant variants in\n                      <jats:italic>NRAS<\/jats:italic>\n                      ,\n                      <jats:italic>HRAS<\/jats:italic>\n                      , and\n                      <jats:italic>KRAS<\/jats:italic>\n                      at codons 12, 13, and 61. Analytical performance was established using plasmid controls. The assays were subsequently applied to 210 prospectively collected fine-needle aspiration (FNA) specimens from 201 patients, with parallel ARMS-PCR testing for\n                      <jats:italic>BRAF<\/jats:italic>\n                      p.V600E and histopathological confirmation available for 49 surgically excised nodules.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      The six-gene ddPCR panel demonstrated 100% analytical specificity and a limit of detection of 0.1% variant allele frequency (VAF). In clinical samples, 103 of 210 nodules (49%) harbored at least one mutation, with\n                      <jats:italic>BRAF<\/jats:italic>\n                      p.V600E being the most prevalent alteration (81\/103, 78.6%). ddPCR identified five low-abundance\n                      <jats:italic>BRAF<\/jats:italic>\n                      p.V600E mutations (VAF 0.37\u20132.96%) that were missed by ARMS-PCR, one of which was confirmed as papillary thyroid carcinoma on postoperative pathology. Mutation prevalence increased progressively across Bethesda categories, ranging from 33% in Bethesda I to 89% in Bethesda VI. Compared with surgical pathology, the combination of the six-gene ddPCR panel and FNAC achieved 100% sensitivity, 71% specificity, and 96% diagnostic accuracy, significantly outperforming FNAC alone or FNAC combined with ARMS-PCR (\n                      <jats:italic>P<\/jats:italic>\n                      \u2009&lt;\u20090.05).\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>This study developed a six-gene mutation detection panel composed of four multiplex droplet digital PCR (ddPCR) assays for thyroid nodule analysis. Integration with cytology substantially reduces indeterminate diagnoses and may guide personalized management, including the avoidance of unnecessary surgery.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s13044-026-00292-9","type":"journal-article","created":{"date-parts":[[2026,3,20]],"date-time":"2026-03-20T13:43:31Z","timestamp":1774014211000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Assessing the clinical diagnostic utility of multiplex ddPCR assays in thyroid nodules"],"prefix":"10.1186","volume":"19","author":[{"given":"Xiubo","family":"Li","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chunhui","family":"Shen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiang","family":"Peng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qianfeng","family":"Xu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jun","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Minghui","family":"He","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Minyi","family":"Kong","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhao","family":"Lin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jingyan","family":"Luo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yan","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,3,20]]},"reference":[{"key":"292_CR1","doi-asserted-by":"publisher","first-page":"04113","DOI":"10.7189\/jogh.15.04113","volume":"15","author":"Q Jin","year":"2025","unstructured":"Jin Q, Wu J, Huang C, et al. 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This study was conducted in accordance with the ethical standards of the Declaration of Helsinki. The study protocol was reviewed and approved by the Medical Ethics Committee of Guangzhou First People\u2019s Hospital (Approval No. K-2025-034-01). As this was a retrospective study based on residual clinical specimens obtained after routine diagnostic procedures and anonymized clinical data, the requirement for written informed consent was waived by the Ethics Committee. All data were de-identified prior to analysis to ensure patient confidentiality.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval and consent to participate"}},{"value":"The authors declare no competing interests.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"11"}}