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Xenium is widely adopted for its reliability, accessibility and data quality, yet the properties and limitations of Xenium-derived data remain poorly characterized. Here we present one of the most comprehensive Xenium datasets so far, encompassing over 40 breast and lung tumor sections profiled using diverse gene panels. Leveraging this resource, we systematically dissect technical noise\u2014including transcript spillover\u2014along with assay specificity, panel performance and segmentation strategies. We demonstrate that single-nucleus RNA sequencing enables precise quantification of transcript contamination. Building on these insights, we introduce SPLIT (Spatial Purification of Layered Intracellular Transcripts), a method that improves signal purity by resolving mixed transcriptomic signals. SPLIT enhances background correction and cell-type resolution and enables the revelation of T-cell exhaustion signatures associated with malignant cell colocalization\u2014signals that would otherwise remain obscured. Together, our findings provide a critical benchmark for Xenium performance and introduce a scalable strategy for signal refinement.<\/jats:p>","DOI":"10.1038\/s41592-026-03089-8","type":"journal-article","created":{"date-parts":[[2026,4,30]],"date-time":"2026-04-30T09:02:05Z","timestamp":1777539725000},"page":"1152-1162","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":25,"title":["Resolving sensitivity, specificity and signal contamination in Xenium spatial transcriptomics"],"prefix":"10.1038","volume":"23","author":[{"given":"Mariia","family":"Bilous","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-9874-9791","authenticated-orcid":false,"given":"Daria","family":"Buszta","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jonathan","family":"Bac","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6696-3983","authenticated-orcid":false,"given":"Senbai","family":"Kang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yixing","family":"Dong","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stephanie","family":"Tissot","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sylvie","family":"Andre","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marina","family":"Alexandre Gaveta","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-9349-1465","authenticated-orcid":false,"given":"Christel","family":"Voize","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Solange","family":"Peters","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0912-6198","authenticated-orcid":false,"given":"Krisztian","family":"Homicsko","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3867-0232","authenticated-orcid":false,"given":"Raphael","family":"Gottardo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,4,30]]},"reference":[{"key":"3089_CR1","doi-asserted-by":"publisher","first-page":"9","DOI":"10.1038\/s41592-020-01033-y","volume":"18","author":"V Marx","year":"2021","unstructured":"Marx, V. 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K.H. and R.G. have received research funding from Owkin and 10x Genomics. K.H received research funding from Bristol Myers Squibb, ROCHE\/Genentech, Merck Sharp & Dohme, Tolremo AG and Boehringer Ingelheim. S.P. has received educational grants, provided consultation, served on advisory boards and\/or delivered lectures for which honoraria were paid to her institution from AbbVie, Amgen, Arcus, AstraZeneca, Bayer, BeiGene, BioNTech, BerGenBio, Bicycle Therapeutics, Bicara, BioInvent, Blueprint Medicines, Boehringer Ingelheim, Bristol Myers Squibb, Clovis, Daiichi Sankyo, Debiopharm, Eli Lilly, F-Star, Foundation Medicine, Genmab, Genzyme, Gilead, GSK, Hutchmed, Illumina, Incyte, Ipsen, iTeos, Janssen, Merck Sharp & Dohme, Merck Serono, Novartis, Nuvalent, Nykode Therapeutics, Novocure, PharmaMar, Promontory Therapeutics, Pfizer, Regeneron, Roche\/Genentech, Sanofi, Takeda and Zymeworks. She has also participated in company-organized public events for AstraZeneca, Boehringer Ingelheim, Bristol Myers Squibb, Eli Lilly, Foundation Medicine, GSK, Illumina, Ipsen, Merck Sharp & Dohme, Novartis, Pfizer, Roche\/Genentech, Sanofi and Takeda. She has received research support as principal investigator for institutionally sponsored clinical trials from Amgen, Arcus, AstraZeneca, BeiGene, Boehringer Ingelheim, Bristol Myers Squibb, Eli Lilly, GSK, iTeos, Merck Sharp & Dohme, Mirati, PharmaMar, Pfizer, Promontory Therapeutics and Roche\/Genentech. The other authors declare no competing interests.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}]}}