• Document: Standardized flow cytometry for highly sensitive MRD measurements in B-cell acute lymphoblastic leukemia
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Supplemental Methods & Figures Standardized flow cytometry for highly sensitive MRD measurements in B-cell acute lymphoblastic leukemia 1# 2# 3 4 Prisca Theunissen , Ester Mejstrikova , Lukasz Sedek , Alita J. van der Sluijs-Gelling , Giuseppe 5 6 7 2 2,6 Gaipa , Marius Bartels , Elaine Sobral da Costa , Michaela Kotrová , Michaela Novakova , Edwin 4 5 5 7 1 Sonneveld , Chiara Buracchi , Paola Bonaccorso , Elen Oliveira , Jeroen G. te Marvelde , Tomasz 3 8 2 9 10 Szczepanski , Ludovic Lhermitte , Ondrej Hrusak , Quentin Lecrevisse , Georgiana Emilia Grigore , Eva Froňková , Jan Trka , Monika Brüggemann , Alberto Orfao , Jacques J.M. van Dongen , and 2 2 6 9 1 1 Vincent H.J. van der Velden , on behalf of the EuroFlow Consortium 1 Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, 2 Netherlands; CLIP - Childhood Leukaemia Investigation Prague, Department of Paediatric Haematology and Oncology, Second Faculty of Medicine, Charles University, University Hospital 3 Motol, Prague, Czech Republic; Department of Pediatric Hematology and Oncology, Zabrze, Medical 4 University of Silesia (SUM), Katowice, Poland; Dutch Childhood Oncology Group, The Hague, 5 Netherlands; Centro Ricerca Tettamanti, Clinica Pediatrica Università di Milano Bicocca, Monza 6 (MB),Italy; Department of Hematology, University of Schleswig-Holstein, Campus Kiel, Kiel, Germany; 7 8 Department of Pediatrics, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil; Department of Hematology, Hôpital Necker-Enfants-Malades (AP-HP) and UMR CNRS 8147, University of Paris 9 Descartes, Paris, France; Cancer Research Center (IBMCC-CSIC), Department of Medicine and Cytometry Service, University of Salamanca (USAL) and Institute of Biomedical Research of 10 Salamanca (IBSAL), Salamanca, Spain; Cytognos SL, Salamanca, Spain. # PT and EM equally contributed Page 1 of 14 Supplemental methods Flowcytometric analysis. Flowcytometric (FCM) data were acquired using BD Biosciences FACS Diva software (BD Biosciences, Erembodegem, BE) and further analyzed using Infinicyt software (Cytognos, Salamanca, ES). In addition to the classical 2-dimensional dot plots, the Infinicyt sofware allows visualization of the data using the Automatic Population Separator (APS) graphical representation. Such APS is based on principal component analysis (PCA) with bi-dimensional graphical representations of Principal Component (PC) X versus PC Y. On the basis of such APS representation, information about the separation between two populations is obtained through definition of median and/or mean ± standard deviations (SD) borders together with information about the most informative (versus redundant) 1-4 parameters. To prevent the number of normal cells influencing the PCA, we opted to use a 2 previously reported balanced PCA , implying a fixed ratio between normal and pathological events. Briefly, data on normal or regenerating B-cell precursor (BCP) cells were obtained from a merge of 7 to 14 different data-files, each corresponding to a normal or regenerating BM sample. From this merged file, a pool of 28.000 to 1.124.900 normal/regenerating BCP events was obtained. Next, each BCP-ALL case (generally 5000 events/case) was merged with this normal/regenerating BCP cells pool in a balanced ratio of 1/1 of normal/leukemic events. Finally, a Principal Component Analysis (PCA) transformation was calculated to achieve the maximum variance (‘distance’) between the normal/regenerating BCP cells and the leukemic cells for each individual BCP-ALL patient. Therefore, by using the balanced APS the composition of the sample (number of events) will not have an impact 1-4 on the final results. Of note, the above described strategy was only used for optimizing the antibody panel for the BCP-ALL MRD tubes, and not for actual MRD analyses. Bulk lysis evaluations 5 To allow acquisition of sufficient numbers of cells (up to 5 mil

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