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Interpretation is particularly challenging in HTLV-1 infection, in which persistent antigenic stimulation may produce clonal or oligoclonal T-cell expansions overlapping with non-neoplastic patterns. This study aimed to assess the diagnostic performance and interpretative contribution of two EuroClonality\/BIOMED-2 assays (TCR\u03b3-A and TCR\u03b3-B), considered complementary components of the same analytical panel, and a single-tube multiplex PCR assay (TCR\u03b3-O), and to develop an integrated workflow for TCR\u03b3 clonality interpretation. Methods: A total of 107 peripheral blood samples were analyzed, including 19 healthy donors, 75 HTLV-1 carriers, and 13 patients with adult T-cell leukemia\/lymphoma (ATLL). TCR\u03b3 clonality was evaluated using three multiplex PCR assays followed by capillary electrophoresis. Profiles were classified as polyclonal, oligoclonal, or monoclonal. Diagnostic performance analysis was performed using healthy donors as the reference negative group and ATLL patients as the reference positive group. Results: The combined EuroClonality approach (TCR\u03b3-A+B) showed the highest sensitivity (100%), whereas TCR\u03b3-O showed the highest specificity (94.7%). TCR\u03b3-A and TCR\u03b3-B each showed a sensitivity of 76.9%, with specificities of 84.2% and 78.9%, respectively. Classification was highly concordant in healthy donors and ATLL patients employing both assays. However, among HTLV-1 carriers, classification varied across the assays. The integrated workflow was associated with a significant change in the distribution of molecular classification patterns in the HTLV-1 group compared with EuroClonality-based interpretation alone. (Stuart\u2013Maxwell, p &lt; 0.001). Conclusions: The EuroClonality\/BIOMED-2 strategy provided greater sensitivity, whereas TCR\u03b3-O showed greater specificity. Their integration supported a complementary and hierarchical approach to TCR\u03b3 clonality assessment, particularly in HTLV-1 infection. These findings support an exploratory integrated framework for TCR\u03b3 clonality interpretation in HTLV-1 infection and ATLL, requiring validation in larger independent cohorts.<\/jats:p>","DOI":"10.3390\/diagnostics16142288","type":"journal-article","created":{"date-parts":[[2026,7,22]],"date-time":"2026-07-22T09:20:39Z","timestamp":1784712039000},"page":"2288","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Integrated TCR\u03b3 Clonality Workflow for Molecular Diagnosis in HTLV-1 Carriers and Adult T-Cell Leukemia\/Lymphoma"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0009-0000-3585-2105","authenticated-orcid":false,"given":"Davi Ferreira","family":"Jofre","sequence":"first","affiliation":[{"name":"Laboratory of Pathogenesis and Directed Therapy in Onco-Immuno-Hematology (LIM-31), Department of Hematology, Hemotherapy, and Cell Therapy, Faculty of Medicine, University of Sao Paulo (FM-USP), Sao Paulo 05419-000, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Giulia Sales","family":"Guimar\u00e3es","sequence":"additional","affiliation":[{"name":"Laboratory of Pathogenesis and Directed Therapy in Onco-Immuno-Hematology (LIM-31), Department of Hematology, Hemotherapy, and Cell Therapy, Faculty of Medicine, University of Sao Paulo (FM-USP), Sao Paulo 05419-000, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-5105-7992","authenticated-orcid":false,"given":"Ana Beatriz","family":"Rocha","sequence":"additional","affiliation":[{"name":"Laboratory of Pathogenesis and Directed Therapy in Onco-Immuno-Hematology (LIM-31), Department of Hematology, Hemotherapy, and Cell Therapy, Faculty of Medicine, University of Sao Paulo (FM-USP), Sao Paulo 05419-000, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-2364-5598","authenticated-orcid":false,"given":"J\u00e9ssica Bilar","family":"Cavalcanti","sequence":"additional","affiliation":[{"name":"Laboratory of Pathogenesis and Directed Therapy in Onco-Immuno-Hematology (LIM-31), Department of Hematology, Hemotherapy, and Cell Therapy, Faculty of Medicine, University of Sao Paulo (FM-USP), Sao Paulo 05419-000, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-3222-9990","authenticated-orcid":false,"given":"Priscila de Lima","family":"Barros","sequence":"additional","affiliation":[{"name":"Laboratory of Pathogenesis and Directed Therapy in Onco-Immuno-Hematology (LIM-31), Department of Hematology, Hemotherapy, and Cell Therapy, Faculty of Medicine, University of Sao Paulo (FM-USP), Sao Paulo 05419-000, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Karine Sobral","family":"Marques","sequence":"additional","affiliation":[{"name":"Laboratory of Pathogenesis and Directed Therapy in Onco-Immuno-Hematology (LIM-31), Department of Hematology, Hemotherapy, and Cell Therapy, Faculty of Medicine, University of Sao Paulo (FM-USP), Sao Paulo 05419-000, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3769-933X","authenticated-orcid":false,"given":"N\u00e9lio C\u00e9zar de","family":"Aquino","sequence":"additional","affiliation":[{"name":"Laboratory of Pathogenesis and Directed Therapy in Onco-Immuno-Hematology (LIM-31), Department of Hematology, Hemotherapy, and Cell Therapy, Faculty of Medicine, University of Sao Paulo (FM-USP), Sao Paulo 05419-000, Brazil"},{"name":"Real-World Evidence & Precision Public Health Team, Laboratory of Pathogenesis and Directed Therapy in Onco-Immuno-Hematology (LIM-31), Department of Hematology, Hemotherapy, and Cell Therapy, Faculty of Medicine, University of Sao Paulo, Sao Paulo 05419-000, Brazil"},{"name":"Brazilian Health Regulatory Agency (Ag\u00eancia Nacional de Vigil\u00e2ncia Sanit\u00e1ria\u2014ANVISA), Brasilia 71205-050, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-6584-6413","authenticated-orcid":false,"given":"Vin\u00edcius de Camargo","family":"Callefi","sequence":"additional","affiliation":[{"name":"Laboratory of Pathogenesis and Directed Therapy in Onco-Immuno-Hematology (LIM-31), Department of Hematology, Hemotherapy, and Cell Therapy, Faculty of Medicine, University of Sao Paulo (FM-USP), Sao Paulo 05419-000, Brazil"},{"name":"Real-World Evidence & Precision Public Health Team, Laboratory of Pathogenesis and Directed Therapy in Onco-Immuno-Hematology (LIM-31), Department of Hematology, Hemotherapy, and Cell Therapy, Faculty of Medicine, University of Sao Paulo, Sao Paulo 05419-000, 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Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vanderson","family":"Rocha","sequence":"additional","affiliation":[{"name":"Laboratory of Pathogenesis and Directed Therapy in Onco-Immuno-Hematology (LIM-31), Department of Hematology, Hemotherapy, and Cell Therapy, Faculty of Medicine, University of Sao Paulo (FM-USP), Sao Paulo 05419-000, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9228-3276","authenticated-orcid":false,"given":"Lu\u00eds Alberto de P\u00e1dua Covas","family":"Lage","sequence":"additional","affiliation":[{"name":"Laboratory of Pathogenesis and Directed Therapy in Onco-Immuno-Hematology (LIM-31), Department of Hematology, Hemotherapy, and Cell Therapy, Faculty of Medicine, University of Sao Paulo (FM-USP), Sao Paulo 05419-000, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cadiele Oliana","family":"Reichert","sequence":"additional","affiliation":[{"name":"Laboratory of Pathogenesis and Directed Therapy in Onco-Immuno-Hematology (LIM-31), Department of Hematology, Hemotherapy, and Cell Therapy, Faculty of Medicine, University of Sao Paulo (FM-USP), Sao Paulo 05419-000, Brazil"},{"name":"Real-World Evidence & Precision Public Health Team, Laboratory of Pathogenesis and Directed Therapy in Onco-Immuno-Hematology (LIM-31), Department of Hematology, Hemotherapy, and Cell Therapy, Faculty of Medicine, University of Sao Paulo, Sao Paulo 05419-000, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0655-2821","authenticated-orcid":false,"given":"Juliana","family":"Pereira","sequence":"additional","affiliation":[{"name":"Laboratory of Pathogenesis and Directed Therapy in Onco-Immuno-Hematology (LIM-31), Department of Hematology, Hemotherapy, and Cell Therapy, Faculty of Medicine, University of Sao Paulo (FM-USP), Sao Paulo 05419-000, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6850-8247","authenticated-orcid":false,"given":"Hebert Fabricio","family":"Culler","sequence":"additional","affiliation":[{"name":"Laboratory of Pathogenesis and Directed Therapy in Onco-Immuno-Hematology (LIM-31), Department of Hematology, Hemotherapy, and Cell Therapy, Faculty of Medicine, University of Sao Paulo (FM-USP), Sao Paulo 05419-000, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,7,22]]},"reference":[{"key":"ref_1","unstructured":"WHO Classification of Tumours Editorial Board (2024). 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