{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,25]],"date-time":"2026-04-25T04:57:00Z","timestamp":1777093020636,"version":"3.51.4"},"reference-count":43,"publisher":"MDPI AG","issue":"10","license":[{"start":{"date-parts":[[2025,5,8]],"date-time":"2025-05-08T00:00:00Z","timestamp":1746662400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003196","name":"Italian Ministry of Health","doi-asserted-by":"publisher","award":["RC-2025-2797277"],"award-info":[{"award-number":["RC-2025-2797277"]}],"id":[{"id":"10.13039\/501100003196","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>The standardization of multiparameter flow cytometry-based measurable residual disease (MFC-MRD) assessment in acute myeloid leukemia (AML) lacks clear criteria to define leukemia-associated immunophenotypes (LAIPs). In addition, the most specific\/sensitive aberrations used to define LAIPs are often partially expressed by the leukemic clone at diagnosis, raising questions about their reliability for accurate MRD quantification. To address this, we investigated whether the quantification of LAIP+ cells reflects residual disease in cases of partial LAIP expression. The following two MFC-MRD approaches were evaluated by comparing their results to RT-qPCR for NPM1 mutations: (1) the LAIP-method, wherein all cells within the patient-specific template created at diagnosis are counted without further gating; (2) the LAIP-based different-from-normal (DfN)-method, wherein cells+ for LAIP-specific aberrant markers are further selected. A total of 125 bone marrow samples from 25 NPM1-mutated AML patients were studied. Our data demonstrate that the LAIP-based DfN-method improves the MFC-MRD accuracy and comparability with molecular MRD. ROC analysis identified cut-offs of 0.034% and 0.095% to discriminate positive\/negative results in patients receiving intensive chemotherapy and hypomethylating agents, respectively. We also found distinct accuracy degrees based on the LAIP-specific aberrant markers used for MRD assessment. These results refine the MFC-MRD method and highlight the importance of therapy-specific MRD cut-offs and LAIP classification based on specificity and sensitivity.<\/jats:p>","DOI":"10.3390\/ijms26104506","type":"journal-article","created":{"date-parts":[[2025,5,8]],"date-time":"2025-05-08T11:37:02Z","timestamp":1746704222000},"page":"4506","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Validation Study of Analytical Methods for Multiparameter Flow Cytometry-Based Measurable Residual Disease Assessment in Acute Myeloid Leukemia"],"prefix":"10.3390","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3356-9861","authenticated-orcid":false,"given":"Martina","family":"Barone","sequence":"first","affiliation":[{"name":"IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia \u201cSer\u00e0gnoli\u201d, 40138 Bologna, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Agnese","family":"Patuelli","sequence":"additional","affiliation":[{"name":"IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia \u201cSer\u00e0gnoli\u201d, 40138 Bologna, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0385-9040","authenticated-orcid":false,"given":"Michele","family":"Dicataldo","sequence":"additional","affiliation":[{"name":"Department of Medical and Surgical Sciences, University of Bologna, 40138 Bologna, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Maria","family":"Irno Consalvo","sequence":"additional","affiliation":[{"name":"Department of Biomedicine and Prevention, University of Rome Tor Vergata, 00133 Rome, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gabriella","family":"Chirumbolo","sequence":"additional","affiliation":[{"name":"Department of Medical and Surgical Sciences, University of Bologna, 40138 Bologna, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lorenza","family":"Bandini","sequence":"additional","affiliation":[{"name":"Department of Medical and Surgical Sciences, University of Bologna, 40138 Bologna, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-4169-8174","authenticated-orcid":false,"given":"Giulia","family":"Atzeni","sequence":"additional","affiliation":[{"name":"IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia \u201cSer\u00e0gnoli\u201d, 40138 Bologna, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8443-605X","authenticated-orcid":false,"given":"Dorian","family":"Forte","sequence":"additional","affiliation":[{"name":"Department of Medical and Surgical Sciences, University of Bologna, 40138 Bologna, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gianluca","family":"Cristiano","sequence":"additional","affiliation":[{"name":"Department of Medical and Surgical Sciences, University of Bologna, 40138 Bologna, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Emanuela","family":"Ottaviani","sequence":"additional","affiliation":[{"name":"IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia \u201cSer\u00e0gnoli\u201d, 40138 Bologna, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Antonio","family":"Curti","sequence":"additional","affiliation":[{"name":"IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia \u201cSer\u00e0gnoli\u201d, 40138 Bologna, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4320-9253","authenticated-orcid":false,"given":"Francesco","family":"Buccisano","sequence":"additional","affiliation":[{"name":"Department of Biomedicine and Prevention, University of Rome Tor Vergata, 00133 Rome, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4650-201X","authenticated-orcid":false,"given":"Lucia","family":"Catani","sequence":"additional","affiliation":[{"name":"IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia \u201cSer\u00e0gnoli\u201d, 40138 Bologna, Italy"},{"name":"Department of Medical and Surgical Sciences, University of Bologna, 40138 Bologna, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2892-1912","authenticated-orcid":false,"given":"Mario","family":"Arpinati","sequence":"additional","affiliation":[{"name":"IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia \u201cSer\u00e0gnoli\u201d, 40138 Bologna, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,5,8]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1275","DOI":"10.1182\/blood-2017-09-801498","article-title":"Minimal\/measurable residual disease in AML: A consensus document from the European LeukemiaNet MRD Working Party","volume":"131","author":"Schuurhuis","year":"2018","journal-title":"Blood"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"2753","DOI":"10.1182\/blood.2021013626","article-title":"2021 Update on MRD in acute myeloid leukemia: A consensus document from the European LeukemiaNet MRD Working Party","volume":"138","author":"Heuser","year":"2021","journal-title":"Blood"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Chea, M., Rigolot, L., Canali, A., and Vergez, F. 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