EuroFlow standardization of flow cytometer instrument settings and immunophenotyping protocols

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Abstract

The EU-supported EuroFlow Consortium aimed at innovation and standardization of immunophenotyping for diagnosis and classification of hematological malignancies by introducing 8-color flow cytometry with fully standardized laboratory procedures and antibody panels in order to achieve maximally comparable results among different laboratories. This required the selection of optimal combinations of compatible fluorochromes and the design and evaluation of adequate standard operating procedures (SOPs) for instrument setup, fluorescence compensation and sample preparation. Additionally, we developed software tools for the evaluation of individual antibody reagents and antibody panels. Each section describes what has been evaluated experimentally versus adopted based on existing data and experience. Multicentric evaluation demonstrated high levels of reproducibility based on strict implementation of the EuroFlow SOPs and antibody panels. Overall, the 6 years of extensive collaborative experiments and the analysis of hundreds of cell samples of patients and healthy controls in the EuroFlow centers have provided for the first time laboratory protocols and software tools for fully standardized 8-color flow cytometric immunophenotyping of normal and malignant leukocytes in bone marrow and blood; this has yielded highly comparable data sets, which can be integrated in a single database.

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Spectral compensation for flow cytometry: Visualization artifacts, limitations, and caveats

Mario Roederer (2001)

In multicolor flow cytometric analysis, compensation for spectral overlap is nearly always necessary. For the most part, such compensation has been relatively simple, producing the desired rectilinear distributions. However, in the realm of multicolor analysis, visualization of compensated often results in unexpected distributions, principally the appearance of a large number of events on the axis, and even more disconcerting, an inability to bring the extent of compensated data down to "autofluorescence" levels. A mathematical model of detector measurements with variable photon intensities, spillover parameters, measurement errors, and data storage characteristics was used to illustrate sources of apparent error in compensated data. Immunofluorescently stained cells were collected under conditions of limiting light collection and high spillover between detectors to confirm aspects of the model. Photon-counting statistics contribute a nonlinear error to compensated parameters. Measurement errors and log-scale binning error contribute linear errors to compensated parameters. These errors are most apparent with the use of red or far-red fluorochromes (where the emitted light is at low intensity) and with large spillover between detectors. Such errors can lead to data visualization artifacts that can easily lead to incorrect conclusions about data, and account for the apparent "undercompensation" previously described for multicolor staining. There are inescapable errors arising from imperfect measurements, photon-counting statistics, and even data storage methods that contribute both linearly and nonlinearly to a "spreading" of a properly compensated autofluorescence distribution. This phenomenon precludes the use of "quadrant" statistics or gates to analyze affected data; it also precludes visual adjustment of compensation. Most importantly, it is impossible to properly compensate data using standard visual graphical interfaces (histograms or dot plots). Computer-assisted compensation is required, as well as careful gating and experimental design to determine the distinction between positive and negative events. Finally, the use of special staining controls that employ all reagents except for the one of interest (termed fluorescence minus one, or "FMO" controls) becomes necessary to accurately identify expressing cells in the fully stained sample. Copyright 2001 Wiley-Liss, Inc.

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Flow cytometric immunophenotyping for hematologic neoplasms.

Fiona F Craig, Kenneth A. Foon (2008)

Flow cytometric immunophenotyping remains an indispensable tool for the diagnosis, classification, staging, and monitoring of hematologic neoplasms. The last 10 years have seen advances in flow cytometry instrumentation and availability of an expanded range of antibodies and fluorochromes that have improved our ability to identify different normal cell populations and recognize phenotypic aberrancies, even when present in a small proportion of the cells analyzed. Phenotypically abnormal populations have been documented in many hematologic neoplasms, including lymphoma, chronic lymphoid leukemias, plasma cell neoplasms, acute leukemia, paroxysmal nocturnal hemoglobinuria, mast cell disease, myelodysplastic syndromes, and myeloproliferative disorders. The past decade has also seen refinement of the criteria used to identify distinct disease entities with widespread adoption of the 2001 World Health Organization (WHO) classification. This classification endorses a multiparametric approach to diagnosis and outlines the morphologic, immunophenotypic, and genotypic features characteristic of each disease entity. When should flow cytometric immunophenotyping be applied? The recent Bethesda International Consensus Conference on flow cytometric immunophenotypic analysis of hematolymphoid neoplasms made recommendations on the medical indications for flow cytometric testing. This review discusses how flow cytometric testing is currently applied in these clinical situations and how the information obtained can be used to direct other testing.

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Flow cytometry controls, instrument setup, and the determination of positivity.

Holden T. Maecker, Martin Trotter (2006)

A frequent goal of flow cytometric analysis is to classify cells as positive or negative for a given marker, or to determine the precise ratio of positive to negative cells. This requires good and reproducible instrument setup, and careful use of controls for analyzing and interpreting the data. The type of controls to include in various kinds of flow cytometry experiments is a matter of some debate and discussion. In this tutorial, we classify controls in various categories, describe the options within each category, and discuss the merits of each option. (c) 2006 International Society for Analytical Cytology.

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Author and article information

Journal

Journal ID (nlm-ta): Leukemia

Journal ID (iso-abbrev): Leukemia

Title: Leukemia

Publisher: Nature Publishing Group

ISSN (Print): 0887-6924

ISSN (Electronic): 1476-5551

Publication date (Print): September 2012

Publication date (Electronic): 05 September 2012

Volume: 26

Issue: 9

Pages: 1986-2010

Affiliations

[1 ]simpleDepartment of Pediatric Hematology and Oncology, 2nd Faculty of Medicine, Charles University (DPH/O) , Prague, Czech Republic

[2 ]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

[3 ]simpleDepartment of Immunology, Erasmus MC, University Medical Center Rotterdam , Rotterdam, The Netherlands

[4 ]simpleCytognos SL , Salamanca, Spain

[5 ]simpleSecond Department of Medicine, University Hospital of Schleswig Holstein, Campus Kiel (UNIKIEL) , Kiel, Germany

[6 ]simpleDepartment of Hematology, Hôpital Necker and UMR CNRS 8147, University of Paris Descartes (AP-HP) , Paris, France

[7 ]simpleDepartment of Hematology, Portuguese Institute of Oncology (IPOLFG) , Lisbon, Portugal

[8 ]simpleHaematological Malignancy Diagnostic Service (HMDS), University of Leeds (UNIVLEEDS) , Leeds, UK

[9 ]simpleDepartment of Pediatric Hematology and Oncology, Medical University of Silesia (SUM) , Zabrze, Poland

[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 or b.vanbodegom@ 123456erasmusmc.nl

[11]

Shared first authorship, because TK and JFM have equally contributed to this manuscript.

Article

Publisher Item ID: leu2012122

DOI: 10.1038/leu.2012.122

PMC ID: 3437409

PubMed ID: 22948490

SO-VID: f64f4936-14a2-441c-93d7-72ca6e0ee109

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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/

EuroFlow standardization of flow cytometer instrument settings and immunophenotyping protocols

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Abstract

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Karger: Oncology

Most cited references 45

Spectral compensation for flow cytometry: Visualization artifacts, limitations, and caveats

Flow cytometric immunophenotyping for hematologic neoplasms.

Flow cytometry controls, instrument setup, and the determination of positivity.

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