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Sentinel lymph node (SLN) mapping may improve staging and reduce morbidity. This study evaluated a dual-tracer technique (superparamagnetic iron oxide: SPIO and methylene blue) for ex vivo detection; assessed one-step nucleic acid amplification (OSNA) versus conventional histology; and applied OSNA-pooling analysis in non-SLN. Prospective study of patients undergoing curative gastrectomy with lymphadenectomy (2017\u20132022). SLNs were identified in fresh specimens by Sentimag\n                    <jats:sup>\u00ae<\/jats:sup>\n                    and\/or macroscopic staining. Each lymph node was bisected, with one half analyzed by hematoxylin\u2013eosin (H&amp;E) and the other by OSNA. Non-SLN were assessed using pooled OSNA to enhance diagnostic performance. Diagnostic performance metrics and agreement (Cohen\u2019s \u03ba) were calculated. Survival was estimated by Kaplan\u2013Meier. Thirty-eight patients were included (mean age 64.3 years; 58% male); 71.1% received neoadjuvant therapy. SLN detection rate was 84.2% (32\/38), with 80 SLNs identified. The dual-tracer approach demonstrated high sensitivity (93.1%) and moderate specificity (44.4%), resulting in an accuracy of 81.6%. Tracer concordance was moderate (\u03ba\u2009=\u20090.42;\n                    <jats:italic>p<\/jats:italic>\n                    \u2009&lt;\u20090.007). OSNA showed good agreement with H&amp;E (sensitivity 80%, specificity 87%), and better performance for micrometastases. The pooling analysis evaluated 1155 non-SLN simultaneously; this novel strategy showed good concordance with conventional histology. After a median follow-up of 53 months, OSNA and H&amp;E status were associated with overall survival in exploratory analyses; however, these findings should be interpreted with caution given the limited sample size and ex vivo proof-of-concept design. Combining SPIO and methylene blue is technically feasible for ex vivo SLN mapping. OSNA complements histology, and its pooled use represents an innovative methodological approach. These results are preliminary and hypothesis-generating; larger prospective in vivo studies are required before clinical applicability can be evaluated. Dual-tracer sentinel node mapping in gastric cancer proved feasible and accurate. OSNA complemented histology, pooled analysis improved detection efficiency, and neoadjuvant therapy did not affect diagnostic performance.\n                  <\/jats:p>","DOI":"10.1038\/s41598-026-43345-7","type":"journal-article","created":{"date-parts":[[2026,3,19]],"date-time":"2026-03-19T09:57:08Z","timestamp":1773914228000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Sentinel lymph node detection in gastric cancer using a dual tracer (Superparamagnetic iron oxide and methylene blue): a prospective study with histological and OSNA validation"],"prefix":"10.1038","volume":"16","author":[{"given":"Raquel","family":"Escalera-P\u00e9rez","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carlos","family":"Medina-Achirica","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francisco J.","family":"Garc\u00eda-Molina","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juan J.","family":"Del Rio-Ignacio","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carolina","family":"Lagares-Franco","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Angel","family":"Estella","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francisco A.","family":"Mateo-Vallejo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,3,19]]},"reference":[{"key":"43345_CR1","unstructured":"7-stomach-fact. -sheet.pdf [Internet]. 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