title: Standardized flow cytometry for highly sensitive MRD measurements in B-cell acute lymphoblastic leukemia.
abstract: 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.
authors.affiliation: Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands.
authors.forename: Prisca
authors.initials: P
authors.lastname: Theunissen
authors.affiliation: Childhood Leukaemia Investigation Prague, Department of Paediatric Haematology and Oncology, Second Faculty of Medicine, Charles University, University Hospital Motol, Prague, Czech Republic.
authors.forename: Ester
authors.initials: E
authors.lastname: Mejstrikova
authors.affiliation: Department of Pediatric Hematology and Oncology, Zabrze, Medical University of Silesia, Katowice, Poland.
authors.forename: Lukasz
authors.initials: L
authors.lastname: Sedek
authors.affiliation: Dutch Childhood Oncology Group, The Hague, The Netherlands.
authors.forename: Alita J
authors.initials: AJ
authors.lastname: van der Sluijs-Gelling
authors.affiliation: Centro Ricerca Tettamanti, Clinica Pediatrica Università di Milano Bicocca, Monza, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Gaipa
authors.affiliation: Department of Hematology, University of Schleswig-Holstein, Campus Kiel, Kiel, Germany.
authors.forename: Marius
authors.initials: M
authors.lastname: Bartels
authors.affiliation: Department of Pediatrics, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Elaine
authors.initials: E
authors.lastname: Sobral da Costa
authors.affiliation: Childhood Leukaemia Investigation Prague, Department of Paediatric Haematology and Oncology, Second Faculty of Medicine, Charles University, University Hospital Motol, Prague, Czech Republic.
authors.forename: Michaela
authors.initials: M
authors.lastname: Kotrová
authors.affiliation: Childhood Leukaemia Investigation Prague, Department of Paediatric Haematology and Oncology, Second Faculty of Medicine, Charles University, University Hospital Motol, Prague, Czech Republic.|Department of Hematology, University of Schleswig-Holstein, Campus Kiel, Kiel, Germany.
authors.forename: Michaela
authors.initials: M
authors.lastname: Novakova
authors.affiliation: Dutch Childhood Oncology Group, The Hague, The Netherlands.
authors.forename: Edwin
authors.initials: E
authors.lastname: Sonneveld
authors.affiliation: Centro Ricerca Tettamanti, Clinica Pediatrica Università di Milano Bicocca, Monza, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Buracchi
authors.affiliation: Centro Ricerca Tettamanti, Clinica Pediatrica Università di Milano Bicocca, Monza, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Bonaccorso
authors.affiliation: Department of Pediatrics, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Elen
authors.initials: E
authors.lastname: Oliveira
authors.affiliation: Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands.
authors.forename: Jeroen G
authors.initials: JG
authors.lastname: Te Marvelde
authors.affiliation: Department of Pediatric Hematology and Oncology, Zabrze, Medical University of Silesia, Katowice, Poland.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Szczepanski
authors.affiliation: 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.
authors.forename: Ludovic
authors.initials: L
authors.lastname: Lhermitte
authors.affiliation: Childhood Leukaemia Investigation Prague, Department of Paediatric Haematology and Oncology, Second Faculty of Medicine, Charles University, University Hospital Motol, Prague, Czech Republic.
authors.forename: Ondrej
authors.initials: O
authors.lastname: Hrusak
authors.affiliation: 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.
authors.forename: Quentin
authors.initials: Q
authors.lastname: Lecrevisse
authors.affiliation: Cytognos SL, Salamanca, Spain.
authors.forename: Georgiana Emilia
authors.initials: GE
authors.lastname: Grigore
authors.affiliation: Childhood Leukaemia Investigation Prague, Department of Paediatric Haematology and Oncology, Second Faculty of Medicine, Charles University, University Hospital Motol, Prague, Czech Republic.
authors.forename: Eva
authors.initials: E
authors.lastname: Froňková
authors.affiliation: Childhood Leukaemia Investigation Prague, Department of Paediatric Haematology and Oncology, Second Faculty of Medicine, Charles University, University Hospital Motol, Prague, Czech Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Trka
authors.affiliation: Department of Hematology, University of Schleswig-Holstein, Campus Kiel, Kiel, Germany.
authors.forename: Monika
authors.initials: M
authors.lastname: Brüggemann
authors.affiliation: 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.
authors.forename: Alberto
authors.initials: A
authors.lastname: Orfao
authors.affiliation: Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands.
authors.forename: Jacques J M
authors.initials: JJ
authors.lastname: van Dongen
authors.affiliation: Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands.
authors.forename: Vincent H J
authors.initials: VH
authors.lastname: van der Velden
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D002648:Child; D002675:Child, Preschool; D005260:Female; D005434:Flow Cytometry / Q000379:methods* / Q000592:standards; D015321:Gene Rearrangement; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D008875:Middle Aged; D018365:Neoplasm, Residual / Q000175:diagnosis*; D015452:Precursor B-Cell Lymphoblastic Leukemia-Lymphoma / Q000175:diagnosis*; D060888:Real-Time Polymerase Chain Reaction; D011947:Receptors, Antigen, B-Cell / Q000235:genetics; D012680:Sensitivity and Specificity; D055815:Young Adult
chemical_list: D011947:Receptors, Antigen, B-Cell