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Flow Cytometric Immunophenotyping for Hematologic Neoplasms From www.bloodjournal.org by on October 28, 2010. For personal use only. 2008 111: 3941-3967 Prepublished online Jan 15, 2008; doi:10.1182/blood-2007-11-120535 Flow cytometric immunophenotyping for hematologic neoplasms Fiona E. Craig and Kenneth A. Foon Updated information and services can be found at: http://bloodjournal.hematologylibrary.org/cgi/content/full/111/8/3941 Articles on similar topics may be found in the following Blood collections: Immunobiology (4229 articles) Neoplasia (4217 articles) Free Research Articles (1001 articles) Review Articles (294 articles) Information about reproducing this article in parts or in its entirety may be found online at: http://bloodjournal.hematologylibrary.org/misc/rights.dtl#repub_requests Information about ordering reprints may be found online at: http://bloodjournal.hematologylibrary.org/misc/rights.dtl#reprints Information about subscriptions and ASH membership may be found online at: http://bloodjournal.hematologylibrary.org/subscriptions/index.dtl Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published semimonthly by the American Society of Hematology, 1900 M St, NW, Suite 200, Washington DC 20036. Copyright 2007 by The American Society of Hematology; all rights reserved. From www.bloodjournal.org by on October 28, 2010. For personal use only. Review article Flow cytometric immunophenotyping for hematologic neoplasms Fiona E. Craig1 and Kenneth A. Foon2 1Division of Hematopathology, Department of Pathology, and 2Division of Hematology/Oncology, Department of Medicine, University of Pittsburgh School of Medicine, PA Flow cytometric immunophenotyping re- plasms, including lymphoma, chronic lym- should flow cytometric immunophenotyp- mains an indispensable tool for the diag- phoid leukemias, plasma cell neoplasms, ing be applied? The recent Bethesda Interna- nosis, classification, staging, and moni- acute leukemia, paroxysmal nocturnal he- tional Consensus Conference on flow cyto- toring of hematologic neoplasms. The moglobinuria, mast cell disease, myelodys- metric immunophenotypic analysis of last 10 years have seen advances in flow plastic syndromes, and myeloproliferative hematolymphoid neoplasms made recom- cytometry instrumentation and availabil- disorders. The past decade has also seen mendations on the medical indications for ity of an expanded range of antibodies refinement of the criteria used to identify flow cytometric testing. This review dis- and fluorochromes that have improved distinct disease entities with widespread cusses how flow cytometric testing is cur- our ability to identify different normal cell adoption of the 2001 World Health Organiza- rently applied in these clinical situations populations and recognize phenotypic ab- tion (WHO) classification. This classifica- and how the information obtained can be errancies, even when present in a small tion endorses a multiparametric approach used to direct other testing. (Blood. 2008; proportion of the cells analyzed. Pheno- to diagnosis and outlines the morphologic, 111:3941-3967) typically abnormal populations have been immunophenotypic, and genotypic features documented in many hematologic neo- characteristic of each disease entity. When © 2008 by The American Society of Hematology Introduction A decade has passed since the review “Recent advances in flow monoclonal gammopathy; and organomegaly and tissue masses.5 cytometry: application to the diagnosis of hematologic malig- In these clinical situations, flow cytometric immunophenotyping nancy” was published in Blood.1 In the past 10 years, flow can provide a sensitive screen for the presence of hematologic cytometric immunophenotyping has maintained its position as malignancy and assist in demonstrating the absence of disease. In an indispensable diagnostic tool. Improvements in flow cytom- contrast, the Bethesda group agreed that flow cytometry was etry instrumentation and availability of an expanded range of generally not indicated in the following situations: mature neutro- antibodies and fluorochromes has led to more accurate phenotyp- philia, polyclonal hypergammaglobulinemia, polycythemia, throm- ing of cells, leading to enhanced identification of abnormal bocytosis, and basophilia.5 In addition, the consensus group agreed populations.2 The last 10 years have also seen refinement of the that flow cytometry is a useful tool for staging a previously criteria used to identify distinct disease entities among the diagnosed hematolymphoid neoplasm, monitoring response to hematologic malignancies. The World Health Organization treatment including detection of minimal residual disease (MRD), (WHO) classification for tumors of the hematopoietic and documenting relapse or progression, and diagnosing an intercurrent lymphoid tissues delineates many of these entities and has been hematologic malignancy, such as a therapy-related myelodysplastic widely adopted.3 This classification endorses a multiparametric syndrome (MDS). approach to diagnosis with identification of morphologic, pheno- Taking a similar practical approach, this review discusses how typic, and genotypic features that are characteristic of each flow cytometric immunophenotyping is currently applied in these disease entity. However, it is neither necessary nor cost- clinical settings to establish the diagnosis of a hematologic effective to perform multiple studies on every specimen. When malignancy, including how the information obtained can be used to should flow cytometric testing be ordered? direct other ancillary testing. In 2006, a group of international experts met in Bethesda, Maryland, to formulate consensus recommendations for flow cytometric testing.4 In contrast to previous consensus meetings that had considered the reagents required to evaluate a specific disease Flow cytometric immunophenotyping for the entity, the Bethesda group took a more practical approach and diagnosis and monitoring of hematologic addressed the flow cytometric evaluation of specimens based on neoplasms the clinical presentation.5 Consensus was reached that flow cytomet- ric immunophenotyping is indicated in the following clinical Flow cytometric immunophenotyping evaluates individual cells in situations: cytopenias, especially bicytopenia and pancytopenia; suspension for the presence and absence of specific antigens elevated leukocyte count, including lymphocytosis, monocytosis, (phenotype). In the assessment for hematologic malignancies, and eosinophilia; the presence of atypical cells or blasts in the several steps are taken in the application and interpretation of this peripheral blood, bone marrow, or body fluids; plasmacytosis or immunophenotypic information: (1) identification of cells from Submitted October 30, 2007; accepted December 20, 2007. Prepublished online as © 2008 by The American Society of Hematology Blood First Edition paper, January 15, 2008; DOI 10.1182/blood-2007-11-120535. BLOOD, 15 APRIL 2008 ⅐ VOLUME 111, NUMBER 8 3941 From www.bloodjournal.org by on October 28, 2010. For personal use only. 3942 CRAIG and FOON BLOOD, 15 APRIL 2008 ⅐ VOLUME 111, NUMBER 8 different lineages and determination of whether they are mature or separate disease entities. In addition, flow cytometry can be used to immature, such as detection of mature B-lymphoid cells and identify expression of targets for potential antibody-directed therapy myeloblasts; (2) detection of abnormal cells through identification and provide some additional prognostic information such as CD38 of antigen expression that differs significantly from normal; (3) and ZAP-70 expression in chronic lymphocytic leukemia/small detailed documentation of the phenotype of abnormal cell popula- lymphocytic lymphoma (CLL/SLL).8 Following therapy, flow tions (ie, the presence or absence of antigens) and, in comparison to cytometry is becoming an established method for the evaluation of their normal cell counterpart, documentation of increased or minimal residual disease.9,10 Table 1 outlines the normal pattern of decreased intensity of staining by fluorochrome labeled antibodies; staining and clinical utility of reagents recommended by the 2006 (4) evaluation of whether the information available is diagnostic of Bethesda consensus group for the evaluation of the B-lineage. a distinct disease entity and, if not, development of a list of possible Plasma cell neoplasms (PCNs) will be considered separately entities with suggestion of additional studies that might be of because their clinical presentation, morphologic appearance, and diagnostic value such as immunohistochemistry, conventional phenotype are usually distinct. cytogenetic, fluorescence in situ hybridization (FISH), and molecu- lar diagnostic studies; and (5) provision of immunophenotypic Identification of abnormal mature B-lymphoid cells information that might be of additional prognostic value, including Neoplastic mature B-lymphoid cells can be distinguished from the identification of targets for potential directed therapy. normal cells by the identification of 2 main types of phenotypic When a specimen is received for flow cytometric testing, a abnormality: immunoglobulin light chain class restriction and decision is made regarding the cell lineages and antigens to be aberrant antigen expression. evaluated that is based on the type of specimen and other available Immunoglobulin light chain class restriction In contrast to information, such as the medical indication for testing listed on the most normal and reactive populations, neoplasms of mature B cells requisition, clinical history, morphologic findings, history
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