A fully-standardized EuroFlow 8-color antibody panel and laboratory procedure was stepwise designed to measure minimal residual disease (MRD) in B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) patients with a sensitivity of ≤10-5, comparable to real-time quantitative polymerase chain reaction (RQ-PCR)-based MRD detection via antigen-receptor rearrangements. Leukocyte markers and the corresponding antibodies and fluorochromes were selected based on their contribution in separating BCP-ALL cells from normal/regenerating BCP cells in multidimensional principal component analyses. After 5 multicenter design-test-evaluate-redesign phases with a total of 319 BCP-ALL patients at diagnosis, two 8-color antibody tubes were selected, which allowed separation between normal and malignant BCP cells in 99% of studied patients. These 2 tubes were tested with a new erythrocyte bulk-lysis protocol allowing acquisition of high cell numbers in 377 bone marrow follow-up samples of 178 BCP-ALL patients. Comparison with RQ-PCR-based MRD data showed a clear positive relation between the percentage concordant cases and the number of cells acquired. For those samples with >4 million cells acquired, concordant results were obtained in 93% of samples. Most discordances were clarified upon high-throughput sequencing of antigen-receptor rearrangements and blind multicenter reanalysis of flow cytometric data, resulting in an unprecedented concordance of 98% (97% for samples with MRD < 0.01%). In conclusion, the fully standardized EuroFlow BCP-ALL MRD strategy is applicable in >98% of patients with sensitivities at least similar to RQ-PCR (≤10-5), if sufficient cells (>4 × 106, preferably more) are evaluated.

Cancer Research Center Department of Medicine and Cytometry Service University of Salamanca Institute of Biomedical Research of Salamanca Salamanca Spain; and

Centro Ricerca Tettamanti Clinica Pediatrica Università di Milano Bicocca Monza Italy

Childhood Leukaemia Investigation Prague Department of Paediatric Haematology and Oncology 2nd Faculty of Medicine Charles University University Hospital Motol Prague Czech Republic

Cytognos SL Salamanca Spain

Department of Hematology Hôpital Necker Enfants Malades and Unité Mixte de Recherche Centre National de la Recherche Scientifique 8147 University of Paris Descartes Paris France

Department of Hematology University of Schleswig Holstein Campus Kiel Kiel Germany

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

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

Department of Pediatrics Federal University of Rio de Janeiro Rio de Janeiro Brazil

Dutch Childhood Oncology Group The Hague The Netherlands

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Cytometry B Clin Cytom. 2019 Jan;96(1):12-15. doi: 10.1002/cyto.b.21720. PubMed

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