Detection of minimal/measurable residual disease (MRD) is a critical prognostic marker in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The EuroFlow Consortium previously developed an 8-color flow cytometric MRD protocol, effective for >98% of BCP-ALL patients treated with chemotherapy. This study aimed to enhance MRD detection, particularly for patients treated with CD19-targeted therapies, by expanding the EuroFlow protocol to a 12-color panel. This new panel incorporates additional B-cell markers and exclusion T/NK-cell markers (CD3 and CD7). Through an evaluation of 237 diagnostic BCP-ALL samples, CD22, CD24, and HLA-DR were selected as additional B-cell gating markers. Two 12-color tubes were technically optimized and clinically validated across 101 patient follow-up samples, demonstrating excellent concordance with molecular MRD levels (R 2 = 0.88). The 12-color BCP-ALL MRD tubes were compatible with the previously developed 8-color automated gating and identification (AGI) tool and demonstrated good reproducibility. Our findings indicate that the 12-color panel performs comparably to the 8-color BCP-ALL MRD panel, including both CD19-positive and CD19-negative cases. However, it offers an improved definition of the B-cell lineage, particularly for expert-guided manual data analysis, and provides additional information on the expression of the targetable marker CD22.

Biomedical Research Networking Centre Consortium of Oncology Instituto de Salud Carlos 3 Madrid Spain

Clinic 3 Special Hematology Laboratory Rostock University Medical Center Rostock Germany

CLIP Department of Pediatric Hematology and Oncology 2nd Faculty of Medicine Charles University and University Hospital Motol Prague Czech Republic

Department of Diagnostic Sciences Ghent University Ghent Belgium

Department of Hematology University of Schleswig Holstein Campus Kiel Kiel Germany

Department of Immunology Laboratory for Medical Immunology Erasmus MC University Medical Center Rotterdam Rotterdam The Netherlands

Department of Immunology LUMC Leiden The Netherlands

Department of Laboratory Medicine Ghent University Hospital Ghent Belgium

Department of Microbiology and Immunology Medical University of Silesia Katowice Poland

Department of Pediatric Hematology and Oncology Medical University of Silesia Katowice Poland

Faculty of Medicine Pediatrics Institute IPPMG Federal University of Rio de Janeiro Rio de Janeiro Brazil

Institute of Biomedical Research of Salamanca Salamanca Spain

Instituto Português de Oncologia de Lisboa Francisco Gentil Rua Prof Lima Basto Lisboa Portugal

Pediatrics Fondazione IRCCS San Gerardo dei Tintori Monza Italy

Princess Máxima Center for Pediatric Oncology Utrecht The Netherlands

Translational and Clinical Research Program Department of Medicine Cancer Research Centre Salamanca Spain

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