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

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 £1025, 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 98% of patients with sensitivities at least similar to RQ-PCR (£1025), if sufficient cells (>4 3 106, preferably more) are evaluated

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

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, 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 98% of patients with sensitivities at least similar to RQ-PCR (£10), if sufficient cells (>4 3 10, preferably more) are evaluated.E.F. was supported by the Grant Agency of the Czech Republic (project of Centre of Excellence No. P302/12/G101). L.S., T.S., and P.T. were supported by European Research Area Network (ERA-NET) PrioMedChild, grant 40-41800-98-027. E.M. was supported by Ministry of Health of the Czech Republic, grant 15-28525A. M.K. was supported by the University Hospital Motol, Prague, Czech Republic (00064203). E.S.d.C., Q.L., and A.O. acknowledge the Bilateral Cooperation Program between Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Brasília, Brazil) and Dirección General de Políticas Universitárias–Ministério de Educación, Cultura y Deportes (Madrid, Spain) (311/15). E.S.d.C. acknowledges Research Foundation of the State of Rio de Janeiro, Rio de Janeiro, Brazil (E26/110.105/2014, E26/102.191/2013) and Conselho Nacional de Desenvolvimento Científico e Tecnológico–CNPQ of Brazil (400194/2014-7). G.G., C.B., and P.B. were supported by Fondazione Tettamanti.Peer Reviewe