{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T21:06:57Z","timestamp":1790370417418,"version":"4.1.0"},"reference-count":27,"publisher":"Wiley","license":[{"start":{"date-parts":[[2026,8,6]],"date-time":"2026-08-06T00:00:00Z","timestamp":1785974400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"},{"start":{"date-parts":[[2026,8,6]],"date-time":"2026-08-06T00:00:00Z","timestamp":1785974400000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Cytometry Part B Clinical"],"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>\n                    Flow cytometry can establish T cell clonality by detecting a restricted expression pattern of the T cell receptor (TCR)\n                    <jats:italic>\u03b2<\/jats:italic>\n                    constant region (TRBC), expressed in association with CD3. However, T cell neoplasms frequently lose surface expression of the CD3\/TCR complex, posing a challenge to demonstrating T cell lineage and clonality. To address this challenge, here we present a 12\u2010color flow cytometry panel, called cytoTCR, to characterize cytoplasmic expression of CD3\/TCR complex components. We apply cytoTCR to 38 patient specimens with immunophenotypically abnormal T cell populations, demonstrating this approach can efficiently establish T cell lineage and clonality in challenging T cell neoplasms that have lost surface CD3 expression. While we show that natural killer (NK)\u2010lineage neoplasms can express cytoplasmic CD3 at similar levels to T cells, we show that absent expression of cytoplasmic TCR components by mature lymphocytes can help confirm NK cell lineage. We demonstrate that cytoTCR can detect cytoplasmic TRBC\u2010restriction in challenging cases of null\u2010phenotype anaplastic large cell lymphoma, which lack surface expression of pan\u2010T cell antigens. In cases of T\u2010lymphoblastic leukemia, cytoTCR shows that cytoplasmic TRBC expression matches the expected developmental stage of the leukemia. Finally, we use cytoTCR to characterize atypical cCD3\n                    <jats:sup>\u2212<\/jats:sup>\n                    CD7\n                    <jats:sup>\u2212<\/jats:sup>\n                    T cells in a patient with a history of T\u2010lymphoblastic leukemia as well as recent CAR\u2010T therapy, showing that this atypical population is polytypic and represents CAR\u2010T product rather than residual disease. Our study presents a broadly applicable flow cytometric approach to simultaneously assess T cell lineage and clonality in suspected T lineage populations with absent surface CD3 expression.\n                  <\/jats:p>","DOI":"10.1002\/cyto.b.70061","type":"journal-article","created":{"date-parts":[[2026,8,7]],"date-time":"2026-08-07T06:52:22Z","timestamp":1786085542000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Cytoplasmic staining of T cell receptor components enables efficient assessment of lineage and clonality in surface\n                    <scp>CD3<\/scp>\n                    \u2010negative T cell neoplasms"],"prefix":"10.1002","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1430-5852","authenticated-orcid":false,"given":"Aaron J.","family":"Wilk","sequence":"first","affiliation":[{"name":"Department of Pathology Stanford University School of Medicine  Stanford California USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gary","family":"Gitana","sequence":"additional","affiliation":[{"name":"Department of Pathology Stanford University School of Medicine  Stanford California USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5220-4628","authenticated-orcid":false,"given":"Jean","family":"Oak","sequence":"additional","affiliation":[{"name":"Department of Pathology Stanford University School of Medicine  Stanford California USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,8,6]]},"reference":[{"issue":"7","key":"e_1_2_7_2_1","doi-asserted-by":"crossref","first-page":"1720","DOI":"10.1038\/s41375-022-01620-2","article-title":"The 5th edition of the World Health Organization classification of Haematolymphoid Tumours: Lymphoid neoplasms","volume":"36","author":"Alaggio R.","year":"2022","journal-title":"Leukemia"},{"issue":"6238","key":"e_1_2_7_3_1","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1038\/341159a0","article-title":"CD3\u2010negative natural killer cells express zeta TCR as part of a novel molecular complex","volume":"341","author":"Anderson P.","year":"1989","journal-title":"Nature"},{"issue":"3","key":"e_1_2_7_4_1","doi-asserted-by":"crossref","first-page":"361","DOI":"10.1002\/cyto.b.21881","article-title":"Flow cytometric evaluation of TRBC1 expression in tissue specimens and body fluids is a novel and specific method for assessment of T\u2010cell clonality and diagnosis of T\u2010cell neoplasms","volume":"100","author":"Berg H.","year":"2021","journal-title":"Cytometry Part B, Clinical Cytometry"},{"issue":"2","key":"e_1_2_7_5_1","doi-asserted-by":"crossref","first-page":"152","DOI":"10.1056\/NEJMoa2505478","article-title":"Universal base\u2010edited CAR7 T cells for T\u2010cell acute lymphoblastic leukemia","volume":"394","author":"Chiesa R.","year":"2026","journal-title":"The New England Journal of Medicine"},{"issue":"3","key":"e_1_2_7_6_1","doi-asserted-by":"crossref","first-page":"192","DOI":"10.1002\/cyto.b.22175","article-title":"TRBC1 in flow cytometry: Assay development, validation, and reporting considerations","volume":"106","author":"Devitt K. A.","year":"2024","journal-title":"Cytometry Part B, Clinical Cytometry"},{"issue":"3","key":"e_1_2_7_7_1","doi-asserted-by":"crossref","first-page":"285","DOI":"10.1182\/blood-2017-01-761320","article-title":"CD7\u2010edited T cells expressing a CD7\u2010specific CAR for the therapy of T\u2010cell malignancies","volume":"130","author":"Gomes\u2010Silva D.","year":"2017","journal-title":"Blood"},{"key":"e_1_2_7_8_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2021.04.048"},{"issue":"4","key":"e_1_2_7_9_1","doi-asserted-by":"crossref","DOI":"10.3390\/ijms22041817","article-title":"Emerging role of T\u2010cell receptor constant \u03b2 chain\u20101 (TRBC1) expression in the flow cytometric diagnosis of T\u2010cell malignancies","volume":"22","author":"Horna P.","year":"2021","journal-title":"International Journal of Molecular Sciences"},{"issue":"1","key":"e_1_2_7_10_1","doi-asserted-by":"crossref","DOI":"10.1038\/s41408-024-01002-0","article-title":"Dual T\u2010cell constant \u03b2 chain (TRBC)1 and TRBC2 staining for the identification of T\u2010cell neoplasms by flow cytometry","volume":"14","author":"Horna P.","year":"2024","journal-title":"Blood Cancer Journal"},{"key":"e_1_2_7_11_1","volume-title":"Defining, establishing, and verifying reference intervals in the clinical laboratory; Approved guideline","author":"Horowitz G. L.","year":"2008"},{"issue":"3","key":"e_1_2_7_12_1","doi-asserted-by":"crossref","first-page":"285","DOI":"10.6004\/jnccn.2022.0015","article-title":"T\u2010cell lymphomas, version 2.2022, NCCN clinical practice guidelines in oncology","volume":"20","author":"Horwitz S. M.","year":"2022","journal-title":"Journal of the National Comprehensive Cancer Network"},{"issue":"3","key":"e_1_2_7_13_1","doi-asserted-by":"crossref","first-page":"352","DOI":"10.1002\/cyto.b.21874","article-title":"Lymphocytic variant of hypereosinophilic syndrome: A report of seven cases from a single institution","volume":"100","author":"Hu Z.","year":"2021","journal-title":"Cytometry Part B, Clinical Cytometry"},{"issue":"2","key":"e_1_2_7_14_1","doi-asserted-by":"crossref","first-page":"99","DOI":"10.1002\/cyto.b.21768","article-title":"Flow cytometry applications in the diagnosis of T\/NK\u2010cell lymphoproliferative disorders","volume":"96","author":"Jevremovic D.","year":"2019","journal-title":"Cytometry Part B, Clinical Cytometry"},{"issue":"7","key":"e_1_2_7_15_1","doi-asserted-by":"crossref","first-page":"969","DOI":"10.5858\/arpa.2013-0074-CR","article-title":"De novo CD3 negative hepatosplenic T\u2010cell lymphoma: Diagnostic challenges and pitfalls","volume":"138","author":"Kapur L. H.","year":"2014","journal-title":"Archives of Pathology & Laboratory Medicine"},{"issue":"2","key":"e_1_2_7_16_1","doi-asserted-by":"crossref","first-page":"314","DOI":"10.1309\/GUHKGAJEJ72CEAL7","article-title":"Anaplastic large cell lymphoma: A flow cytometric analysis of 29 cases: A flow cytometric analysis of 29 cases","volume":"128","author":"Kesler M. V.","year":"2007","journal-title":"American Journal of Clinical Pathology"},{"issue":"5","key":"e_1_2_7_17_1","doi-asserted-by":"crossref","first-page":"555","DOI":"10.1182\/blood.2022018200","article-title":"Hepatosplenic T\u2010cell lymphoma with STAT5B and SETD2 mutations recurring as cells with NK\u2010cell immunophenotype","volume":"141","author":"Khodadoust M.","year":"2023","journal-title":"Blood"},{"issue":"6","key":"e_1_2_7_18_1","doi-asserted-by":"crossref","first-page":"1876","DOI":"10.4049\/jimmunol.149.6.1876","article-title":"Expression of cytoplasmic CD3 epsilon proteins in activated human adult natural killer (NK) cells and CD3 gamma, delta, epsilon complexes in fetal NK cells. Implications for the relationship of NK and T lymphocytes","volume":"149","author":"Lanier L. L.","year":"1992","journal-title":"Journal of Immunology"},{"issue":"6251","key":"e_1_2_7_19_1","doi-asserted-by":"crossref","first-page":"803","DOI":"10.1038\/342803a0","article-title":"Co\u2010association of CD3 zeta with a receptor (CD16) for IgG fc on human natural killer cells","volume":"342","author":"Lanier L. L.","year":"1989","journal-title":"Nature"},{"issue":"9","key":"e_1_2_7_20_1","doi-asserted-by":"crossref","first-page":"1115","DOI":"10.1002\/ajh.25775","article-title":"Loss of surface CD3 expression in allogeneic CAR T\u2010cells","volume":"95","author":"Margolskee E.","year":"2020","journal-title":"American Journal of Hematology"},{"key":"e_1_2_7_21_1","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.b.22258"},{"issue":"4","key":"e_1_2_7_22_1","doi-asserted-by":"crossref","first-page":"1055","DOI":"10.1084\/jem.175.4.1055","article-title":"Ontogeny of human natural killer (NK) cells: Fetal NK cells mediate cytolytic function and express cytoplasmic CD3 epsilon,delta proteins","volume":"175","author":"Phillips J. H.","year":"1992","journal-title":"Journal of Experimental Medicine"},{"issue":"5","key":"e_1_2_7_23_1","doi-asserted-by":"crossref","first-page":"683","DOI":"10.1093\/ajcp\/aqaa086","article-title":"Cytoplasmic expression of CD3\u03b5 heterodimers by flow cytometry rapidly distinguishes between mature T\u2010cell and natural killer\u2010cell neoplasms","volume":"154","author":"Shi M.","year":"2020","journal-title":"American Journal of Clinical Pathology"},{"issue":"2","key":"e_1_2_7_24_1","doi-asserted-by":"crossref","first-page":"172","DOI":"10.1002\/cyto.b.22204","article-title":"Appropriate interpretation of TRBC1\u2010dim positive subsets in T\u2010cell immunophenotyping by flow cytometry","volume":"108","author":"Shi M.","year":"2025","journal-title":"Cytometry Part B, Clinical Cytometry"},{"issue":"6","key":"e_1_2_7_25_1","doi-asserted-by":"crossref","first-page":"701","DOI":"10.1089\/hyb.1992.11.701","article-title":"Generation of monoclonal antibodies against a human T cell receptor beta chain expressed in transgenic mice","volume":"11","author":"Viney J. L.","year":"1992","journal-title":"Hybridoma"},{"key":"e_1_2_7_26_1","volume-title":"Haematolymphoid tumours","author":"WHO Classification of Tumours Editorial Board","year":"2024"},{"key":"e_1_2_7_27_1","doi-asserted-by":"publisher","DOI":"10.1084\/jem.20210582"},{"issue":"10","key":"e_1_2_7_28_1","doi-asserted-by":"crossref","first-page":"2534","DOI":"10.4049\/jimmunol.2001319","article-title":"Human cytomegalovirus infection promotes expansion of a functionally superior cytoplasmic CD3+ NK cell subset with a Bcl11b\u2010regulated T cell signature","volume":"207","author":"Wu Z.","year":"2021","journal-title":"The Journal of Immunology"}],"container-title":["Cytometry Part B: Clinical Cytometry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cyto.b.70061","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/full-xml\/10.1002\/cyto.b.70061","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cyto.b.70061","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,7]],"date-time":"2026-08-07T06:52:26Z","timestamp":1786085546000},"score":1,"resource":{"primary":{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/cyto.b.70061"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,6]]},"references-count":27,"alternative-id":["10.1002\/cyto.b.70061"],"URL":"https:\/\/doi.org\/10.1002\/cyto.b.70061","archive":["Portico"],"relation":{},"ISSN":["1552-4949","1552-4957"],"issn-type":[{"value":"1552-4949","type":"print"},{"value":"1552-4957","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,6]]},"assertion":[{"value":"2026-06-03","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-07-30","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-08-06","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"cyto.b.70061"}}