Reclassifying Assay-Intrinsic HLA Reactivity Expands Donor Availability Without Short-Term Allograft Risk

Reclassifying Assay-Intrinsic HLA Reactivity Expands Donor Availability Without Short-Term Allograft Risk | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Reclassifying Assay-Intrinsic HLA Reactivity Expands Donor Availability Without Short-Term Allograft Risk Anna Mallone, Yuan Tian, Fabian Rössler, Kerstin Huebel, Dusan Harmacek, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9171076/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Accurate interpretation of HLA antibody reactivity is essential for virtual crossmatching (vXM) and organ allocation, particularly for sensitized kidney transplant candidates. Single-antigen bead (SAB) assays based on Luminex technology are widely used for this purpose but are susceptible to assay-intrinsic reactivity that can inflate calculated panel reactive antibody (cPRA) and unnecessarily restrict donor availability. Here, we assess the clinical and immunological impact of orthogonal SAB testing using Immucor Lifecodes in candidates initially evaluated with One Lambda assays. Cross-platform testing revealed reproducible bead-specific intrinsic reactivity affecting distinct HLA Class I and Class II specificities, as identified in a large reference cohort of non-immunized males. Orthogonal testing reclassified a substantial fraction of One Lambda–positive unacceptable antigens as assay intrinsic reactivity, resulting in marked reductions in cPRA and increased donor compatibility across sensitized candidates. Longitudinal post-transplant follow-up demonstrated rapid attenuation or disappearance of antibodies to reclassified antigens, without increased de novo donor-specific antibody formation, impaired allograft function, altered immune cell dynamics, or evidence of subclinical graft injury. Building on these findings, we developed a predictive model integrating assay intrinsic reactivity frequency, cross-platform discordance, and post-transplant antibody kinetics to identify bead specificities prone to assay-driven overcalling, with strong discriminative performance. Together, these results demonstrate that orthogonal SAB testing corrects systematic overestimation of HLA sensitization, reduces cPRA inflation without compromising short-term graft safety, and suggests a quantitative framework for harmonizing HLA antibody interpretation across transplant allocation systems. Health sciences/Medical research/Translational research Health sciences/Nephrology/Kidney diseases Organ Allocation HLA- antibodies Donor-specific antibodies (DSA) Single-antigen bead assay (SAB) Virtual crossmatch Antibody-mediated rejection Transplant immunology Full Text Additional Declarations There is NO Competing Interest. All participants provided informed consent to participate in the study. The study was approved by the Cantonal Ethics Committee of Zurich (BASEC 2018-01182). Supplementary Files SupplementaryMaterial.pdf Supplementary Material Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9171076","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":616660129,"identity":"b059da58-5313-47dc-8863-80c18b48f273","order_by":0,"name":"Anna Mallone","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIiWNgGAWjYBADGT4Q+QHM5iFOCw8bkGCcQbIWZh5itPDP7j34gTHHjoeNvffgY9u2OnsG9rMH8GqRuHMuWYJxWzIPG8+5ZOPctsPMDDx5CfituZFjANTCzMMmkWMmndt2gI1BgscArw75GznGPxi31YO0mP+2bKvjIajF4EaOGdCWw2BbmBnbmCUIajEEarFI3HYc6JczxpI95w4bsPHk4NciB3TYjY/bquX42XsMP/woq7PnZz+DXwsYJCBz2AirHwWjYBSMglFACAAAUCQ10Qehi08AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-7949-9718","institution":"USZ","correspondingAuthor":true,"prefix":"","firstName":"Anna","middleName":"","lastName":"Mallone","suffix":""},{"id":616660130,"identity":"af6f3809-87ca-4464-bc47-c6d0eefe0560","order_by":1,"name":"Yuan Tian","email":"","orcid":"","institution":"University Hospital Zurich","correspondingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Tian","suffix":""},{"id":616660131,"identity":"4f8fda26-b55b-4cf3-a857-135fbe42baaa","order_by":2,"name":"Fabian Rössler","email":"","orcid":"","institution":"University Hospital Zurich","correspondingAuthor":false,"prefix":"","firstName":"Fabian","middleName":"","lastName":"Rössler","suffix":""},{"id":616660132,"identity":"4c1ead97-def8-4ab0-89c2-60db5779a458","order_by":3,"name":"Kerstin Huebel","email":"","orcid":"","institution":"University Hospital Zurich","correspondingAuthor":false,"prefix":"","firstName":"Kerstin","middleName":"","lastName":"Huebel","suffix":""},{"id":616660133,"identity":"c894364d-6c78-42e9-b779-c848cfb1cf13","order_by":4,"name":"Dusan Harmacek","email":"","orcid":"","institution":"University Hospital Zurich","correspondingAuthor":false,"prefix":"","firstName":"Dusan","middleName":"","lastName":"Harmacek","suffix":""},{"id":616660134,"identity":"71a25705-4ab9-4c7e-8dc4-82357cf371d4","order_by":5,"name":"Lukas Frischknecht","email":"","orcid":"","institution":"University Hospital Zurich","correspondingAuthor":false,"prefix":"","firstName":"Lukas","middleName":"","lastName":"Frischknecht","suffix":""},{"id":616660135,"identity":"1a90f34b-d1cf-48be-958f-94c9928251ab","order_by":6,"name":"Jakob Nilsson","email":"","orcid":"https://orcid.org/0000-0001-5091-8133","institution":"University Hospital Zurich","correspondingAuthor":false,"prefix":"","firstName":"Jakob","middleName":"","lastName":"Nilsson","suffix":""}],"badges":[],"createdAt":"2026-03-19 15:17:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9171076/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9171076/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106523337,"identity":"b3edfeec-d534-430f-ab31-3d60baeee9ff","added_by":"auto","created_at":"2026-04-09 13:17:46","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1120868,"visible":true,"origin":"","legend":"Article File","description":"","filename":"Mallone2026.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9171076/v1_covered_59a57037-c1c4-44d4-8f33-c36be7fe5588.pdf"},{"id":106523330,"identity":"b1be54a5-9dcb-41aa-bf22-3d76bc7dcc28","added_by":"auto","created_at":"2026-04-09 13:17:27","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":323523,"visible":true,"origin":"","legend":"Supplementary Material","description":"","filename":"SupplementaryMaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9171076/v1/e87fb77b535a41ace616fe77.pdf"}],"financialInterests":"\u003cp\u003eThere is \u003cstrong\u003eNO\u003c/strong\u003e Competing Interest.\u003c/p\u003e\n\u003cp\u003eAll participants provided informed consent to participate in the study. 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Single-antigen bead (SAB) assays based on Luminex technology are widely used for this purpose but are susceptible to assay-intrinsic reactivity that can inflate calculated panel reactive antibody (cPRA) and unnecessarily restrict donor availability. Here, we assess the clinical and immunological impact of orthogonal SAB testing using Immucor Lifecodes in candidates initially evaluated with One Lambda assays. Cross-platform testing revealed reproducible bead-specific intrinsic reactivity affecting distinct HLA Class I and Class II specificities, as identified in a large reference cohort of non-immunized males. Orthogonal testing reclassified a substantial fraction of One Lambda–positive unacceptable antigens as assay intrinsic reactivity, resulting in marked reductions in cPRA and increased donor compatibility across sensitized candidates. 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Together, these results demonstrate that orthogonal SAB testing corrects systematic overestimation of HLA sensitization, reduces cPRA inflation without compromising short-term graft safety, and suggests a quantitative framework for harmonizing HLA antibody interpretation across transplant allocation systems.","manuscriptTitle":"Reclassifying Assay-Intrinsic HLA Reactivity Expands Donor Availability Without Short-Term Allograft Risk","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-09 13:17:24","doi":"10.21203/rs.3.rs-9171076/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b8103081-2c53-444c-ab70-7e46be361271","owner":[],"postedDate":"April 9th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":65621922,"name":"Health sciences/Medical research/Translational research"},{"id":65621923,"name":"Health sciences/Nephrology/Kidney diseases"}],"tags":[],"updatedAt":"2026-04-21T11:12:15+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-09 13:17:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9171076","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9171076","identity":"rs-9171076","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}