Immunophenotyping by flow cytometry (FCM) is a worldwide mainstay in leukemia diagnostics. For concordant multicentric application, however, a gap exists between available classification systems, technologic standardization, and clinical needs. The AIEOP-BFM consortium induced an extensive standardization and validation effort between its nine national reference laboratories collaborating in immunophenotyping of pediatric acute lymphoblastic leukemia (ALL). We elaborated common guidelines which take advantage of the possibilities of multi-color FCM: marker panel requirements, immunological blast gating, in-sample controls, tri-partite antigen expression rating (negative vs. weak or strong positive) with capturing of blast cell heterogeneities and subclone formation, refined ALL subclassification, and a dominant lineage assignment algorithm able to distinguish "simple" from bilineal/"complex" mixed phenotype acute leukemia (MPAL) cases, which is essential for choice of treatment. These guidelines are a first step toward necessary inter-laboratory standardization of pediatric leukemia immunophenotyping for a concordant multicentric application. © 2017 International Clinical Cytometry Society.

Cancer Research Center and the Hematology Laboratory Jeffrey Modell Foundation Center Edmond and Lily Safra Children's Hospital Sheba Medical Center Tel Hashomer Israel

Children's Cancer Research Institute and St Anna Children's Hospital Department of Pediatrics Medical University of Vienna Vienna Austria

Clinic for Oncology and Tumor Immunology HELIOS Klinikum Berlin Buch Berlin Germany

Department of Pediatric Hematology and Oncology 2nd Faculty of Medicine Charles University and University Hospital Motol Prague Czech Republic

Department of Pediatric Hematology and Oncology Schneider's Children's Medical Center Petach Tikva Israel

Department of Pediatric Oncology Hematology Charité Universitätsmedizin Berlin Germany

Department of Pediatrics University Medical Center Schleswig Holstein Kiel Germany

Division of Oncology and Children's Research Center University Children's Hospital University of Zurich Zurich Switzerland

Flow Cytometry Unit Institute of Clinical Pathology and Medical Research Westmead Hospital Sydney Australia

Laboratory of Pediatric Onco Hematology Women and Child Department University of Padova Padova Italy

Tettamanti Research Center and Department of Pediatrics Ospedale San Gerardo University of Milano Bicocca Monza Italy

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