{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,7]],"date-time":"2026-04-07T11:08:52Z","timestamp":1775560132860,"version":"3.50.1"},"reference-count":18,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2026,4,3]],"date-time":"2026-04-03T00:00:00Z","timestamp":1775174400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Fundo de Incentivo \u00e0 Pesquisa e Eventos","award":["FIPE 2022-0410"],"award-info":[{"award-number":["FIPE 2022-0410"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["MPs"],"abstract":"<jats:p>Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy. AML classification is currently based on cytogenetic and molecular alterations as well as immunophenotyping, although risk stratification still relies primarily on cytogenetic findings. However, approximately 45% of AML patients present with a normal karyotype, which makes accurate risk classification and treatment stratification more challenging. Therefore, the identification of molecular prognostic markers described in the literature has become essential in routine diagnostic laboratories, enabling the more precise categorization of patients into risk groups. In this study, we present a simple, rapid, step-by-step multiplex PCR protocol combined with capillary electrophoresis for the detection of two of the most prevalent molecular alterations in AML: nucleophosmin 1 (NPM1) mutations and Fms-like tyrosine kinase 3 internal tandem duplications (FLT3\/ITD). This protocol provides a practical workflow that can assist diagnostic laboratories in implementing and optimizing multiplex mutation detection in routine practice.<\/jats:p>","DOI":"10.3390\/mps9020059","type":"journal-article","created":{"date-parts":[[2026,4,6]],"date-time":"2026-04-06T04:10:27Z","timestamp":1775448627000},"page":"59","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A Simple and Fast Protocol to Detect Nucleophosmin 1 (NPM1) Mutation and Fms-like Tyrosine Kinase 3 Internal Tandem Duplication (FLT3\/ITD): Optimizing Laboratory Routine"],"prefix":"10.3390","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8704-8183","authenticated-orcid":false,"given":"Alessandra Helena da Silva","family":"Hellwig","sequence":"first","affiliation":[{"name":"Resid\u00eancia Multidisciplinar em \u00c1rea Profissional An\u00e1lises Cl\u00ednicas, Hospital de Cl\u00ednicas de Porto Alegre, Porto Alegre 90035-903, Brazil"},{"name":"Programa de P\u00f3s-Gradua\u00e7\u00e3o em Medicina Ci\u00eancias M\u00e9dicas, Universidade Federal do Rio Grande do Sul, Porto Alegre 90035-903, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gisele Menezes","family":"Ewald","sequence":"additional","affiliation":[{"name":"Unidade de Imunologia de Transplantes e Medicina Personalizada, Servi\u00e7o de Diagn\u00f3stico Laboratorial, Hospital de Cl\u00ednicas de Porto Alegre, Porto Alegre 90035-903, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3881-6526","authenticated-orcid":false,"given":"Grazielle Motta","family":"Rodrigues","sequence":"additional","affiliation":[{"name":"Resid\u00eancia Multidisciplinar em \u00c1rea Profissional An\u00e1lises Cl\u00ednicas, Hospital de Cl\u00ednicas de Porto Alegre, Porto Alegre 90035-903, Brazil"},{"name":"Programa de P\u00f3s-Gradua\u00e7\u00e3o em Medicina Ci\u00eancias M\u00e9dicas, Universidade Federal do Rio Grande do Sul, Porto Alegre 90035-903, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5587-208X","authenticated-orcid":false,"given":"William Latosinski","family":"Matos","sequence":"additional","affiliation":[{"name":"Resid\u00eancia Multidisciplinar em \u00c1rea Profissional An\u00e1lises Cl\u00ednicas, Hospital de Cl\u00ednicas de Porto Alegre, Porto Alegre 90035-903, Brazil"},{"name":"Programa de P\u00f3s-Gradua\u00e7\u00e3o em Medicina Ci\u00eancias M\u00e9dicas, Universidade Federal do Rio Grande do Sul, Porto Alegre 90035-903, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Juliana","family":"Bergmann","sequence":"additional","affiliation":[{"name":"Unidade de Microbiologia, Setor de Biologia Molecular, Servi\u00e7o de Diagn\u00f3stico Laboratorial, Hospital de Cl\u00ednicas de Porto Alegre, Porto Alegre 90035-903, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Viviane Horn de","family":"Melo","sequence":"additional","affiliation":[{"name":"Unidade de Microbiologia, Setor de Biologia Molecular, Servi\u00e7o de Diagn\u00f3stico Laboratorial, Hospital de Cl\u00ednicas de Porto Alegre, Porto Alegre 90035-903, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4112-0880","authenticated-orcid":false,"given":"Rodrigo Minuto","family":"Paiva","sequence":"additional","affiliation":[{"name":"Unidade de Microbiologia, Setor de Biologia Molecular, Servi\u00e7o de Diagn\u00f3stico Laboratorial, Hospital de Cl\u00ednicas de Porto Alegre, Porto Alegre 90035-903, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9119-6242","authenticated-orcid":false,"given":"Dariane Castro","family":"Pereira","sequence":"additional","affiliation":[{"name":"Resid\u00eancia Multidisciplinar em \u00c1rea Profissional An\u00e1lises Cl\u00ednicas, Hospital de Cl\u00ednicas de Porto Alegre, Porto Alegre 90035-903, Brazil"},{"name":"Unidade de Microbiologia, Setor de Biologia Molecular, Servi\u00e7o de Diagn\u00f3stico Laboratorial, Hospital de Cl\u00ednicas de Porto Alegre, Porto Alegre 90035-903, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-7968-7509","authenticated-orcid":false,"given":"Eduardo Wandame","family":"Gomez","sequence":"additional","affiliation":[{"name":"Resid\u00eancia Multidisciplinar em \u00c1rea Profissional An\u00e1lises Cl\u00ednicas, Hospital de Cl\u00ednicas de Porto Alegre, Porto Alegre 90035-903, Brazil"},{"name":"Unidade de Microbiologia, Setor de Biologia Molecular, Servi\u00e7o de Diagn\u00f3stico Laboratorial, Hospital de Cl\u00ednicas de Porto Alegre, Porto Alegre 90035-903, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,3]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"489","DOI":"10.1111\/j.1365-2141.2008.07205.x","article-title":"A Rapid, One Step Assay for Simultaneous Detection of FLT3\/ITD and NPM1 Mutations in AML with Normal Cytogenetics","volume":"142","author":"Huang","year":"2008","journal-title":"Br. 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