{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T20:51:35Z","timestamp":1792788695616,"version":"4.5.0"},"reference-count":45,"publisher":"Ovid Technologies (Wolters Kluwer Health)","issue":"10","funder":[{"name":"European Society for Organ Transplantation","id":[{"id":"https:\/\/ror.org\/05gsxqf59","id-type":"ROR","asserted-by":"crossref"}]},{"name":"OrganX"}],"content-domain":{"domain":["lww.com","ovid.com"],"crossmark-restriction":true},"short-container-title":["JASN"],"published-print":{"date-parts":[[2026,10]]},"abstract":"<jats:sec>\n                    <jats:title>Key Points<\/jats:title>\n                    <jats:p>\n                      <jats:list list-type=\"bullet\">\n                        <jats:list-item>\n                          <jats:p>Normalization methods affected the performance of gene-based diagnostic classifiers for kidney transplant rejection across a large multicenter cohort.<\/jats:p>\n                        <\/jats:list-item>\n                        <jats:list-item>\n                          <jats:p>Complex normalization reduced classifier performance by overcorrecting signal, while simpler methods preserved rejection signature and key robustness.<\/jats:p>\n                        <\/jats:list-item>\n                        <jats:list-item>\n                          <jats:p>By identifying optimal normalization methods, this work advances a standardized preprocessing framework for molecular diagnostics in transplantation.<\/jats:p>\n                        <\/jats:list-item>\n                      <\/jats:list>\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>The Banff 2022 classification endorses intragraft gene expression profiling using the Banff Human Organ Transplant (B-HOT) consensus gene panel for rejection diagnosis. However, lack of standardized analytical pipelines, including data normalization, limits clinical implementation, with its effect on diagnostic performance yet to be determined.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      We evaluated ten normalization methods in 868 kidney allograft biopsies from nine European and North American centers, all Banff-graded and B-HOT\u2013profiled on nCounter, comprising derivation (\n                      <jats:italic toggle=\"yes\">n<\/jats:italic>\n                      =441), internal (\n                      <jats:italic toggle=\"yes\">n<\/jats:italic>\n                      =186), and external (\n                      <jats:italic toggle=\"yes\">n<\/jats:italic>\n                      =241) validation cohorts. Each method was assessed through its downstream impact on (\n                      <jats:italic toggle=\"yes\">1<\/jats:italic>\n                      ) gene count stability, (\n                      <jats:italic toggle=\"yes\">2<\/jats:italic>\n                      ) differential expression and cross-platform concordance with RNA sequencing (RNA-seq) data, and (\n                      <jats:italic toggle=\"yes\">3<\/jats:italic>\n                      ) discrimination and calibration of predictive models for antibody-mediated rejection (AMR) and\n                      <jats:italic toggle=\"yes\">T<\/jats:italic>\n                      -cell\u2013mediated rejection (TCMR).\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      Most methods improved count stability and showed high concordance with RNA-seq for overall gene expression. They also produced robust differential expression signatures consistent with those detected by RNA-seq, except for\n                      <jats:italic toggle=\"yes\">RUVSeq<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">RCRNorm<\/jats:italic>\n                      , which identified fewer differentially expressed genes and showed lower concordance. In the overall validation cohort (\n                      <jats:italic toggle=\"yes\">n<\/jats:italic>\n                      =427), diagnostic performance was consistently high across\n                      <jats:italic toggle=\"yes\">nSolver<\/jats:italic>\n                      -based approaches,\n                      <jats:italic toggle=\"yes\">nanostringr<\/jats:italic>\n                      ,\n                      <jats:italic toggle=\"yes\">NanoStringDiff<\/jats:italic>\n                      ,\n                      <jats:italic toggle=\"yes\">MetaNorm<\/jats:italic>\n                      , and\n                      <jats:italic toggle=\"yes\">RCRNormFast<\/jats:italic>\n                      (AMR, area under the ROC curve [AUROC], 0.88\u20130.91; area under precision-recall curves [AUPRC], 0.86\u20130.89; TCMR AUROC, 0.90\u20130.92; AUPRC, 0.78\u20130.83). Performance declined with\n                      <jats:italic toggle=\"yes\">RCRNorm<\/jats:italic>\n                      (AMR AUROC\/AUPRC, 0.55\/0.41; TCMR, 0.53\/0.18) and, for TCMR, with\n                      <jats:italic toggle=\"yes\">RUVSeq<\/jats:italic>\n                      (AUROC, 0.84\u20130.85; AUPRC, 0.64\u20130.65). Calibration was satisfactory for most methods, except for\n                      <jats:italic toggle=\"yes\">RCRNorm<\/jats:italic>\n                      and for TCMR models after\n                      <jats:italic toggle=\"yes\">RUVSeq<\/jats:italic>\n                      .\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>Normalization choice significantly impacted gene expression profiles and diagnostic classifier performance. Most methods, including nSolver-based pipelines, achieved robust discrimination for both AMR and TCMR. Complex methods, including RCRNorm, and RUVSeq for TCMR, reduced performance, with simpler approaches consistently outperforming them for B-HOT\u2013based molecular diagnostics.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1681\/asn.0000001107","type":"journal-article","created":{"date-parts":[[2026,4,23]],"date-time":"2026-04-23T16:01:03Z","timestamp":1776960063000},"page":"2176-2193","update-policy":"https:\/\/doi.org\/10.1097\/lww.0000000000001000","source":"Crossref","is-referenced-by-count":2,"title":["Normalization of nCounter Gene Expression Data Alters Molecular Diagnostics in Kidney Transplantation"],"prefix":"10.1681","volume":"37","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5955-6770","authenticated-orcid":false,"given":"Alexis","family":"Piedrafita","sequence":"first","affiliation":[{"name":"Universit\u00e9 Paris Cit\u00e9, INSERM U970, Paris Institute for Transplantation and Organ Regeneration, Paris, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-4662-4010","authenticated-orcid":false,"given":"Marta","family":"Sablik","sequence":"additional","affiliation":[{"name":"Universit\u00e9 Paris Cit\u00e9, INSERM U970, Paris Institute for Transplantation and Organ Regeneration, Paris, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7130-0439","authenticated-orcid":false,"given":"Evgenia","family":"Preka","sequence":"additional","affiliation":[{"name":"Universit\u00e9 Paris Cit\u00e9, INSERM U970, Paris Institute for Transplantation and Organ Regeneration, Paris, France"},{"name":"Department of Pediatric Nephrology, Necker-Enfants Malades Hospital, Assistance Publique - H\u00f4pitaux de Paris, Paris, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6191-6889","authenticated-orcid":false,"given":"Valentin","family":"Goutaudier","sequence":"additional","affiliation":[{"name":"Universit\u00e9 Paris Cit\u00e9, INSERM U970, Paris Institute for Transplantation and Organ Regeneration, Paris, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zeynep","family":"Demir","sequence":"additional","affiliation":[{"name":"Universit\u00e9 Paris Cit\u00e9, INSERM U970, Paris Institute for Transplantation and Organ Regeneration, Paris, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fariza","family":"Mezine","sequence":"additional","affiliation":[{"name":"Universit\u00e9 Paris Cit\u00e9, INSERM U970, Paris Institute for Transplantation and Organ Regeneration, Paris, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3450-0033","authenticated-orcid":false,"given":"Gillian","family":"Divard","sequence":"additional","affiliation":[{"name":"Universit\u00e9 Paris Cit\u00e9, INSERM U970, Paris Institute for Transplantation and Organ Regeneration, Paris, France"},{"name":"Kidney Transplant Department, Saint-Louis Hospital, Assistance Publique-H\u00f4pitaux de Paris, Paris, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jessy","family":"Dagobert","sequence":"additional","affiliation":[{"name":"Universit\u00e9 Paris Cit\u00e9, INSERM U970, Paris Institute for Transplantation and Organ Regeneration, Paris, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7769-1418","authenticated-orcid":false,"given":"Blaise","family":"Robin","sequence":"additional","affiliation":[{"name":"Universit\u00e9 Paris Cit\u00e9, INSERM U970, Paris Institute for Transplantation and Organ Regeneration, Paris, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Agathe","family":"Truchot","sequence":"additional","affiliation":[{"name":"Universit\u00e9 Paris Cit\u00e9, INSERM U970, Paris Institute for Transplantation and Organ Regeneration, Paris, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-0079-0697","authenticated-orcid":false,"given":"Thibaut","family":"Thalamas","sequence":"additional","affiliation":[{"name":"Universit\u00e9 Paris Cit\u00e9, INSERM U970, Paris Institute for Transplantation and Organ Regeneration, Paris, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aur\u00e9lie","family":"Sannier","sequence":"additional","affiliation":[{"name":"Universit\u00e9 Paris Cit\u00e9, INSERM U970, Paris Institute for Transplantation and Organ Regeneration, Paris, France"},{"name":"Service d'Anatomie et Cytologie Pathologiques, H\u00f4pital Bichat, Assistance Publique - H\u00f4pitaux de Paris, Paris, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5696-6478","authenticated-orcid":false,"given":"Marion","family":"Rabant","sequence":"additional","affiliation":[{"name":"Laboratoire d'Anatomie et Cytologie Pathologiques, Assistance Publique-H\u00f4pitaux de Paris, H\u00f4pital Necker-Enfants malades, Universit\u00e9 Paris Cit\u00e9, Paris, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6284-3757","authenticated-orcid":false,"given":"Olivier","family":"Aubert","sequence":"additional","affiliation":[{"name":"Department of Kidney Transplantation, Necker Hospital, Assistance Publique-H\u00f4pitaux de Paris, Paris, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mehdi","family":"Maanaoui","sequence":"additional","affiliation":[{"name":"Department of Nephrology, Service de N\u00e9phrologie, CHU de Lille, H\u00f4pital Huriez, Lille, France"},{"name":"Inserm, CHU Lille, Institut Pasteur Lille, University in Lille, Lille, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5857-1207","authenticated-orcid":false,"given":"Moglie","family":"Le Quintrec","sequence":"additional","affiliation":[{"name":"Department of Nephrology, Dialysis and Transplantation, Montpellier University Hospital, Montpellier, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9213-6196","authenticated-orcid":false,"given":"Lionel","family":"Couzi","sequence":"additional","affiliation":[{"name":"Department of Nephrology and Transplantation, Bordeaux University Hospital, Bordeaux, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3250-4604","authenticated-orcid":false,"given":"Bertrand","family":"Chauveau","sequence":"additional","affiliation":[{"name":"Department of Pathology, Bordeaux University Hospital, Bordeaux, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9468-7920","authenticated-orcid":false,"given":"Oriol","family":"Bestard","sequence":"additional","affiliation":[{"name":"Department of Nephrology and Kidney Transplantation, Vall d'Hebr\u00f3n University Hospital, Barcelona, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michelle","family":"Elias","sequence":"additional","affiliation":[{"name":"Kidney Transplant Department, Saint-Louis Hospital, Assistance Publique-H\u00f4pitaux de Paris, Paris, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1766-2548","authenticated-orcid":false,"given":"Kevin","family":"Louis","sequence":"additional","affiliation":[{"name":"Kidney Transplant Department, Saint-Louis Hospital, Assistance Publique-H\u00f4pitaux de Paris, Paris, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0621-3202","authenticated-orcid":false,"given":"Ivy A.","family":"Rosales","sequence":"additional","affiliation":[{"name":"Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"R. 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