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      EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes


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          Most consensus leukemia & lymphoma antibody panels consist of lists of markers based on expert opinions, but they have not been validated. Here we present the validated EuroFlow 8-color antibody panels for immunophenotyping of hematological malignancies. The single-tube screening panels and multi-tube classification panels fit into the EuroFlow diagnostic algorithm with entries defined by clinical and laboratory parameters. The panels were constructed in 2–7 sequential design–evaluation–redesign rounds, using novel Infinicyt software tools for multivariate data analysis. Two groups of markers are combined in each 8-color tube: (i) backbone markers to identify distinct cell populations in a sample, and (ii) markers for characterization of specific cell populations. In multi-tube panels, the backbone markers were optimally placed at the same fluorochrome position in every tube, to provide identical multidimensional localization of the target cell population(s). The characterization markers were positioned according to the diagnostic utility of the combined markers. Each proposed antibody combination was tested against reference databases of normal and malignant cells from healthy subjects and WHO-based disease entities, respectively. The EuroFlow studies resulted in validated and flexible 8-color antibody panels for multidimensional identification and characterization of normal and aberrant cells, optimally suited for immunophenotypic screening and classification of hematological malignancies.

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          Prognostically useful gene-expression profiles in acute myeloid leukemia.

          In patients with acute myeloid leukemia (AML) a combination of methods must be used to classify the disease, make therapeutic decisions, and determine the prognosis. However, this combined approach provides correct therapeutic and prognostic information in only 50 percent of cases. We determined the gene-expression profiles in samples of peripheral blood or bone marrow from 285 patients with AML using Affymetrix U133A GeneChips containing approximately 13,000 unique genes or expression-signature tags. Data analyses were carried out with Omniviz, significance analysis of microarrays, and prediction analysis of microarrays software. Statistical analyses were performed to determine the prognostic significance of cases of AML with specific molecular signatures. Unsupervised cluster analyses identified 16 groups of patients with AML on the basis of molecular signatures. We identified the genes that defined these clusters and determined the minimal numbers of genes needed to identify prognostically important clusters with a high degree of accuracy. The clustering was driven by the presence of chromosomal lesions (e.g., t(8;21), t(15;17), and inv(16)), particular genetic mutations (CEBPA), and abnormal oncogene expression (EVI1). We identified several novel clusters, some consisting of specimens with normal karyotypes. A unique cluster with a distinctive gene-expression signature included cases of AML with a poor treatment outcome. Gene-expression profiling allows a comprehensive classification of AML that includes previously identified genetically defined subgroups and a novel cluster with an adverse prognosis. Copyright 2004 Massachusetts Medical Society
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            A clinical evaluation of the International Lymphoma Study Group classification of non-Hodgkin's lymphoma. The Non-Hodgkin's Lymphoma Classification Project.

            The recognition of several new types of non-Hodgkin's lymphoma (NHL) in recent years has led to proposals for changing lymphoma classifications, including a new proposal put forth by the International Lymphoma Study Group (ILSG). However, the clinical significance of the new entities and the practical utility of this new proposal have not been studied. Therefore, we performed a clinical evaluation of the ILSG classification. A cohort of 1,403 cases of NHL was organized at nine study sites around the world and consisted of consecutive patients seen between 1988 and 1990 who were previously untreated. A detailed protocol for histologic and clinical analysis was followed at each site, and immunologic characterization as to T- or B-cell phenotype was required. Five expert hematopathologists visited the sites and each classified each case using the ILSG classification. A consensus diagnosis was also reached in each case, and each expert rereviewed a 20% random sample of the cases. Clinical correlations and survival analyses were then performed. A diagnosis of NHL was confirmed in 1,378 (98.2%) of the cases. The most common lymphoma types were diffuse large B-cell lymphoma (31%) and follicular lymphoma (22%), whereas the new entities comprised 21% of the cases. Diagnostic accuracy was at least 85% for most of the major lymphoma types, and reproducibility of the diagnosis was 85%. Immunophenotyping improved the diagnostic accuracy by 10% to 45% for a number of the major types. The clinical features of the new entities were distinctive. Both the histologic types and the patient characteristics as defined by the International Prognostic Index predicted for patient survival. In conclusion we found that the ILSG classification can be readily applied and identifies clinically distinctive types of NHL. However, for clinical application, prognostic factors as defined by the International Prognostic Index must be combined with the histologic diagnosis for appropriate clinical decisions.
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              Mechanisms of B-cell lymphoma pathogenesis.

              Chromosomal translocations involving the immunoglobulin loci are a hallmark of many types of B-cell lymphoma. Other factors, however, also have important roles in the pathogenesis of B-cell malignancies. Most B-cell lymphomas depend on the expression of a B-cell receptor (BCR) for survival, and in several B-cell malignancies antigen activation of lymphoma cells through BCR signalling seems to be an important factor for lymphoma pathogenesis. Recent insights into the lymphomagenic role of factors supplied by the microenvironment also offer new therapeutic strategies.

                Author and article information

                Nature Publishing Group
                September 2012
                03 May 2012
                05 September 2012
                : 26
                : 9
                : 1908-1975
                [1 ]simpleDepartment of Immunology, Erasmus MC, University Medical Center Rotterdam (Erasmus MC) , Rotterdam, The Netherlands
                [2 ]simpleDepartment of Hematology, Hôpital Necker-Enfants-Malades (AP-HP) and UMR CNRS 8147, University of Paris Descartes , Paris, France
                [3 ]simpleSecond Department of Medicine, University Hospital of Schleswig Holstein, Campus Kiel (UNIKIEL) , Kiel, Germany
                [4 ]simpleCancer Research Center (IBMCC-CSIC), Department of Medicine and Cytometry Service, University of Salamanca (USAL) and Institute of Biomedical Research of Salamanca (IBSAL) , Salamanca, Spain
                [5 ]simpleHaematological Malignancy Diagnostic Service (HMDS), University of Leeds (UNIVLEEDS) , Leeds, UK
                [6 ]simpleDepartment of Hematology, Portuguese Institute of Oncology (IPOLFG) , Lisbon, Portugal
                [7 ]simpleDepartment of Pediatric Hematology and Oncology, 2nd Faculty of Medicine, Charles University (DPH/O) , Prague, Czech Republic
                [8 ]simpleDepartment of Pediatric Hematology and Oncology, Medical University of Silesia (SUM) , Zabrze, Poland
                [9 ]simpleCytognos SL , Salamanca, Spain
                [10 ]simpleDepartment of Hematology, University Hospital Salamanca (HUS) and IBSAL , Salamanca, Spain
                Author notes
                [* ]simpleDepartment of Immunology, Erasmus MC, University Medical Center Rotterdam , Dr Molewaterplein 50, 3015 GE Rotterdam, The Netherlands. E-mail: j.j.m.vandongen@ 123456erasmusmc.nl
                Copyright © 2012 Macmillan Publishers Limited

                This work is licensed under the Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/

                : 21 March 2011
                : 14 February 2012
                : 19 April 2012
                Original Article

                Oncology & Radiotherapy
                lymphoma,flow cytometry,euroflow,8-color immunostaining,antibody panel,standardization,hematological malignancies


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