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Blood. 2017 Jan 19;129(3):347-357. doi: 10.1182/blood-2016-07-726307. Epub 2016 Nov 30.

Standardized flow cytometry for highly sensitive MRD measurements in B-cell acute lymphoblastic leukemia.

Author information

Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands.
Childhood Leukaemia Investigation Prague, Department of Paediatric Haematology and Oncology, Second Faculty of Medicine, Charles University, University Hospital Motol, Prague, Czech Republic.
Department of Pediatric Hematology and Oncology, Zabrze, Medical University of Silesia, Katowice, Poland.
Dutch Childhood Oncology Group, The Hague, The Netherlands.
Centro Ricerca Tettamanti, Clinica Pediatrica Università di Milano Bicocca, Monza, Italy.
Department of Hematology, University of Schleswig-Holstein, Campus Kiel, Kiel, Germany.
Department of Pediatrics, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
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.
Cancer Research Center (Instituto de Biología Molecular y Celular del Cancer-Consejo Superior de Investigaciones Científicas), Department of Medicine and Cytometry Service, University of Salamanca, Institute of Biomedical Research of Salamanca, Salamanca, Spain; and.
Cytognos SL, Salamanca, Spain.


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.

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