{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,10]],"date-time":"2026-04-10T05:09:14Z","timestamp":1775797754398,"version":"3.50.1"},"reference-count":45,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,4,10]],"date-time":"2026-04-10T00:00:00Z","timestamp":1775779200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Oncol."],"abstract":"<jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>\n                      The best way to monitor Chimeric Antigen Receptor (CAR)-T cells persistence and expansion\n                      <jats:italic>in vivo<\/jats:italic>\n                      after infusion, and the significance of observed fluctuations over time remains controversial. As living drugs, these therapies do not follow classical pharmacokinetic patterns. Therefore, reliable quantification of circulating CAR-T cells is essential to explore the relations between\n                      <jats:italic>in vivo<\/jats:italic>\n                      expansion and persistence on one hand, tumor response and occurrence of side-effects on the other hand; this work will prepare integration of this information in harmonized post-CAR-T Cells intervention algorithms. Conventional flow cytometry protocols rely on multi-step wash procedures that increase processing time and may reduce sensitivity. We here developed and validated a one-step no-wash flow cytometry assay for routine CAR-T monitoring in patients treated with approved autologous CAR-T Cells.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>CAR-T cell monitoring was implemented between 2021 and 2024 in patients treated with autologous CD19-directed CAR-T therapies at our institution with a classical two steps and wash flow cytometry method. Analytical validation included determination of detection and quantification limits, linearity, precision, and inter-laboratory reproducibility. In 2024, the classical method was considerably optimized to a one-step no-wash format to reduce manual handling and improve sensitivity. Clinical relevance was assessed in a cohort of 29 patients treated with axicabtagene ciloleucel, correlating CAR-T expansion metrics with clinical endpoints.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>The optimized one-step no-wash assay markedly improved analytical sensitivity, achieving a limit of detection of 0.3 cells\/\u00b5L and a lower limit of quantification of 1.0 cells\/\u00b5L, versus 2.0 and 5.0 cells\/\u00b5L, respectively, when using the previous two steps and wash protocol, while demonstrating a strong concordance (r\u00b2 = 0.984). Inter-assay coefficients of variation remained below 9%, confirming maintained precision despite workflow simplification. In 29 patients treated with axicabtagene ciloleucel (axicel), peak CAR-T expansion significantly correlated with objective responses (p&amp;lt; 0.01) and occurrences of immune effector cell\u2013associated neurotoxicity syndrome (ICANS) (p = 0.02), supporting the clinical relevance of flow cytometry\u2013based CAR-T monitoring in routine practice.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>This one-step no-wash flow cytometry assay combines enhanced sensitivity with improved operational efficiency and provides clinically informative CAR-T cell monitoring in standard-of-care settings.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.3389\/fonc.2026.1774431","type":"journal-article","created":{"date-parts":[[2026,4,10]],"date-time":"2026-04-10T04:27:24Z","timestamp":1775795244000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Clinical implementation of a one-step no-wash flow cytometry method allows for real-time monitoring of patients treated with autologous CAR-T cells"],"prefix":"10.3389","volume":"16","author":[{"given":"Nassim","family":"Salem","sequence":"first","affiliation":[{"name":"Centre de Th\u00e9rapie Cellulaire, D\u00e9partement de Biologie du Cancer, Institut Paoli-Calmettes","place":["Marseille, France"]},{"name":"Module Bioth\u00e9rapies du Centre d\u2019Investigation Clinique de Marseille, AP-HM, Aix-Marseille Universit\u00e9, Institut Paoli-Calmettes, CBT-1409, INSERM","place":["Marseille, 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