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Three forensic detection tools\u2014Sperm Tracker Lab, Sperm Tracker Spray, and RSID\u2122 tests\u2014were evaluated on multiple substrates (skin, hair, nylon, cotton, and car interiors) and under various contamination conditions, including the presence of blood, dust, soil, and bodily fluids. Detection techniques included contact-pressure methods (Sperm Tracker Lab), application on uneven surfaces (Sperm Tracker Spray), fluorescence-based searches with ALS (alternative light sources), and immunochromatographic testing (RSID\u2122 kits) for sperm-specific proteins. Positive findings were confirmed via microscopic examination and DNA analysis. All the samples were labelled and stored following strict chain-of-custody protocols.\u00a0Sperm Tracker Spray demonstrated consistent effectiveness, successfully detecting minimal volumes (1\u20132 \u00b5L) across a wide range of materials. Conversely, ALS showed reduced sensitivity, especially in the presence of diluted or minimal traces and on textured or dark fabrics. RSID\u2122 kits provided reliable confirmation of the presence of semen, even when environmental or biological contamination was present. Accurate and thorough documentation of the chain of custody proved essential for preserving sample authenticity and reducing the risk of error.\u00a0The findings underscore the importance of a multidisciplinary forensic approach combining specialized reagents, confirmatory immunochromatographic testing, and rigorous adherence to chain-of-custody procedures. This integrated strategy enhances the reliability of seminal trace detection in investigations of sexual assault. Moreover, verifying trace persistence under diverse conditions contributes significantly to the evidentiary value of forensic samples in judicial contexts.<\/jats:p>","DOI":"10.1007\/s12024-025-01149-7","type":"journal-article","created":{"date-parts":[[2025,12,13]],"date-time":"2025-12-13T09:13:06Z","timestamp":1765617186000},"page":"533-540","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Verification of the persistence of sperm traces under different chain-of-custody conditions. 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The study was approved by the Local Bioethics Committee of the General Hospital, University of Palermo.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval"}},{"value":"Informed consent was obtained from all individual participants included in the study.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Consent to participate"}},{"value":"N\/A","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Consent for publication"}},{"value":"All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript.","order":5,"name":"Ethics","group":{"name":"EthicsHeading","label":"Conflict of interest"}},{"value":"The authors declare no conflicts of interest. The biological material used in this study was obtained from healthy human volunteers who were duly informed and provided written informed consent.","order":6,"name":"Ethics","group":{"name":"EthicsHeading","label":"Compliance with ethical standards"}}]}}