Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide toward the relevant classification panel and final diagnosis. In this study, we designed and validated an algorithm for automated (database-supported) gating and identification (AGI tool) of cell subsets within samples stained with ALOT. A reference database of normal peripheral blood (PB, n = 41) and bone marrow (BM; n = 45) samples analyzed with the ALOT was constructed, and served as a reference for the AGI tool to automatically identify normal cells. Populations not unequivocally identified as normal cells were labeled as checks and were classified by an expert. Additional normal BM (n = 25) and PB (n = 43) and leukemic samples (n = 109), analyzed in parallel by experts and the AGI tool, were used to evaluate the AGI tool. Analysis of normal PB and BM samples showed low percentages of checks (<3% in PB, <10% in BM), with variations between different laboratories. Manual analysis and AGI analysis of normal and leukemic samples showed high levels of correlation between cell numbers (r2 > 0.95 for all cell types in PB and r2 > 0.75 in BM) and resulted in highly concordant classification of leukemic cells by our previously published automated database-guided expert-supervised orientation tool for immunophenotypic diagnosis and classification of acute leukemia (Compass tool). Similar data were obtained using alternative, commercially available tubes, confirming the robustness of the developed tools. The AGI tool represents an innovative step in minimizing human intervention and requirements in expertise, toward a "sample-in and result-out" approach which may result in more objective and reproducible data analysis and diagnostics. The AGI tool may improve quality of immunophenotyping in individual laboratories, since high percentages of checks in normal samples are an alert on the quality of the internal procedures.

Applied Medical Research Center Pamplona Spain

Biomedical Research Networking Centre Consortium of Oncology Instituto de Salud Carlos 3 Madrid Spain

Cytognos SL Salamanca Spain

Department of Hematology University of Schleswig Holstein Campus Kiel Kiel Germany

Department of Immunohematology and Blood Transfusion Leiden The Netherlands

Department of Immunology Laboratory for Medical Immunology Erasmus MC University Medical Center Rotterdam Rotterdam The Netherlands

Department of Medicine University of Salamanca Salamanca Spain

Department of Microbiology and Immunology Medical University of Silesia in Katowice Zabrze Poland

Department of Pediatric Hematology and Oncology Medical University of Silesia in Katowice Zabrze Poland

Department of Pediatric Hematology and Oncology University Hospital Motol Charles University Prague Czechia

FACS Stem Cell Laboratory Kantonsspital Aarau Aarau Switzerland

Hemato Oncology Laboratory Portuguese Institute of Oncology Lisbon Portugal

Institut Necker Enfants Malades Institut National de Recherche Médicale U1151 Laboratory of Onco Hematology Assistance Publique Hôpitaux de Paris Hôpital Necker Enfants Malades Université de Paris Paris France

Institute of Biomedical Research of Salamanca Salamanca Spain

Laboratory of Hematology University Hospital of Saint Etienne Saint Etienne France

Pediatrics Institute IPPMG Faculty of Medicine Federal University of Rio de Janeiro Av Horacio Macedo Predio do CT CEP Rio de Janeiro 21941 914 Brazil

Princess Máxima Center for Pediatric Oncology Utrecht The Netherlands

Systems and Computing Department Rio de Janeiro Brazil

Tettamanti Research Center Pediatric Clinic University of Milano Bicocca Monza MB Italy

Translational and Clinical Research Program Cancer Research Centre Cytometry Service NUCLEUS Salamanca Spain

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Phenotypic profiling of CD34+ cells by advanced flow cytometry improves diagnosis of juvenile myelomonocytic leukemia