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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

1
Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands.
2
Childhood Leukaemia Investigation Prague, Department of Paediatric Haematology and Oncology, Second Faculty of Medicine, Charles University, University Hospital Motol, Prague, Czech Republic.
3
Department of Pediatric Hematology and Oncology, Zabrze, Medical University of Silesia, Katowice, Poland.
4
Dutch Childhood Oncology Group, The Hague, The Netherlands.
5
Centro Ricerca Tettamanti, Clinica Pediatrica Università di Milano Bicocca, Monza, Italy.
6
Department of Hematology, University of Schleswig-Holstein, Campus Kiel, Kiel, Germany.
7
Department of Pediatrics, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
8
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.
9
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.
10
Cytognos SL, Salamanca, Spain.

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.

PMID:
27903527
PMCID:
PMC5291958
DOI:
10.1182/blood-2016-07-726307
[Indexed for MEDLINE]
Free PMC Article

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