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

AuthorsTheunissen, Prisca; Mejstrikova, Ester; Sedek, Lukasz; Sluijs-Gelling, Alita J. van der; Gaipa, Giuseppe; Bartels, Marius; Sobral da Costa, E.; Kotrová, M.; Novákova, Michaela; Sonneveld, Edwin; Buracchi, Chiara; Bonaccorso, Paola; Oliveira, Elen; Marvelde, Jeroen G. te; Szczepanski, Tomasz; Lhermitte, Ludovic; Hrusak, Ondrej; Lécrevisse, Quentin; Grigore, Georgiana Emilia; Froňková, Eva; Trka, Jan; Brüggemann, Monika; Orfao, Alberto ; Dongen, J. J. M. van; Velden, Vincent H. J. van der
Issue Date2017
PublisherAmerican Society of Hematology
CitationBlood 129(3): 347-357 (2017)
AbstractA 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, 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), if sufficient cells (>4 3 10, preferably more) are evaluated.
Identifiersdoi: 10.1182/blood-2016-07-726307
e-issn: 1528-0020
issn: 0006-4971
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