7,807 research outputs found
Frequent issues and lessons learned from EuroFlow QA
No full textEuroFlow Quality Assessment was designed to provide a feedback on the quality of the standardization effort in executing the EuroFlow protocols for sample preparation and instrument setup. It was first beta-tested by the members of the EuroFlow consortium internally (2010−2013) and opened to the external participants from 2015 onwards. The goal of participation in the EuroFlow QA is to evaluate whether the technical quality of the data generated by the laboratory is comparable to the data of the EuroFlow members and thus if a non-EuroFlow member participant can use the EuroFlow reference sample database for his own patient evaluation. Also it assesses whether data are sufficiently standardized for automated population gating and alarm notification. By spring 2018, a total 87 laboratories from 32 countries on five continents have registered for the EuroFlow QA program. We evaluated 163 results of 2015–2016 QA rounds, where we noted clear improvement in the score of first-time participants (median score of 91% correct) when they participated second time or later (median score of 94% correct, p = 0,017), which was comparable to EuroFlow member scores (median score of 97% correct). Among frequent mistakes, we found non-adherence to the EuroFlow protocols (improper reagent used), improper gating and some compensation issues. In summary, we show that EuroFlow QA has a positive impact on improvement of standardized data quality of non-member laboratories adhering to the EuroFlow standard operating procedures and reagent panels.This study was performed within the EuroFlow Consortium, the EU-supported LSHB-CT- 2006-018708 FP6 Specific Targeted Research Project, which obtained sustainability based on income from the development of intellectual property and related patents, which have been licensed to industry. The work of TK and NB was supported by grant 15-28525A from Ministry of Health of the Czech Republic and Instruments and infrastructure were supported by Ministry of Education, Youth and Sports, Czech Republic NPU I no. LO1604.Peer reviewe
Immunophenotypic Analysis of Acute Megakaryoblastic Leukemia: A EuroFlow Study
No full textAcute megakaryoblastic leukemia (AMKL) is a rare and heterogeneous subtype of acute myeloid leukemia (AML). We evaluated the immunophenotypic profile of 72 AMKL and 114 non-AMKL AML patients using the EuroFlow AML panel. Univariate and multivariate/multidimensional analyses were performed to identify most relevant markers contributing to the diagnosis of AMKL. AMKL patients were subdivided into transient abnormal myelopoiesis (TAM), myeloid leukemia associated with Down syndrome (ML-DS), AML—not otherwise specified with megakaryocytic differentiation (NOS-AMKL), and AMKL—other patients (AML patients with other WHO classification but with flowcytometric features of megakaryocytic differentiation). Flowcytometric analysis showed good discrimination between AMKL and non-AMKL patients based on differential expression of, in particular, CD42a.CD61, CD41, CD42b, HLADR, CD15 and CD13. Combining CD42a.CD61 (positive) and CD13 (negative) resulted in a sensitivity of 71% and a specificity of 99%. Within AMKL patients, TAM and ML-DS patients showed higher frequencies of immature CD34+/CD117+ leukemic cells as compared to NOS-AMKL and AMKL-Other patients. In addition, ML-DS patients showed a significantly higher expression of CD33, CD11b, CD38 and CD7 as compared to the other three subgroups, allowing for good distinction of these patients. Overall, our data show that the EuroFlow AML panel allows for straightforward diagnosis of AMKL and that ML-DS is associated with a unique immunophenotypic profile
How to make usage of the standardized EuroFlow 8-color protocols possible for instruments of different manufacturers
No full textInternational audienceA critical component of the EuroFlow standardization of leukemia/lymphoma immunophenotyping is instrument setup. Initially, the EuroFlow consortium developed a step-by-step standard operating protocol for instrument setup of ≥8-color flow cytometers that were available in 2006, when the EuroFlow activities started. Currently, there are 14 instruments from 9 manufacturers capable of 3-laser excitation and ≥8 color measurements. The specific adaptations required in the instrument set-up to enable them to acquire the standardized 8-color EuroFlow protocols are described here. Overall, all 14 instruments can be fitted with similar violet, blue and red lasers for simultaneous measurements of ≥8 fluorescent dyes. Since individual instruments differ both on their dynamic range (scale) and emission filters, it is not accurate to simply recalculate the target values to different scale, but adjustment of PMT voltages to a given emission filter and fluorochrome, is essential. For this purpose, EuroFlow has developed an approach using Type IIB (spectrally matching) particles to set-up standardized and fully comparable fluorescence measurements, in instruments from different manufacturers, as demonstrated here for the FACSCanto II, and Navios and MACSQuant flow cytometers. Data acquired after such adjustment on any of the tested cytometry platforms could be fully superimposed and therefore analyzed together. The proposed approach can be used to derive target values for any combination of spectrally distinct fluorochromes and any distinct emission filter of any new flow cytometry platform, which enables the measurement of the 8-color EuroFlow panels in a standardized way, by creating superimposable datafiles
Comments on EuroFlow standard operating procedures for instrument setup and compensation for BD FACS Canto II, Navios and BD FACS Lyric instruments
No full textThis commentary discusses particularities of application of the EuroFlow standardization of flow cytometric analyses on three different flow cytometers. The EuroFlow consortium developed a fully standardized approach for flow cytometric immunophenotyping of hematological malignancies and primary immunodeficiencies. Standardized instrument setup is an essential part of EuroFlow standardization. Initially, the EuroFlow Consortium developed and optimized a step-by-step standard operating procedure (SOP) to setup 8-color BD FACSCanto II flow cytometer (Canto), with the later inclusion of Navios (Beckman Coulter) and BD FACSLyric (Lyric). Those SOPs were developed to enable standardized and fully comparable fluorescence measurements in the three flow cytometers. In Canto and Navios, mean fluorescence intensity (MFI) of a reference peak of Rainbow beads calibration particles is used to set up photomultiplier (PMT) voltages for each detector channel in individual instruments to reach the same MFI across distinct instruments. In turn, a new feature of Lyric instruments allows to share collection of attributes that are used to place the positive population at the same position among instruments in the form of assays, as one of its components integrated in the Cytometer Setup and Tracking (CS&T) module. The EuroFlow Lyric assays thus allow for standardized acquisition of 8-color EuroFlow panels on Lyric without the need to setup the PMT voltages on the individual instruments manually. In summary, the standardized instrument setup developed by EuroFlow enables cross-platform inter- and intra-laboratory standardization of flow cytometric measurements. This commentary provides a perspective on the modifications of the standardized EuroFlow instrument setup of Canto, Navios and Lyric instruments that are described in detail in individual instrument-specfic SOPs available at the EuroFlow website.TK and MN were supported by Ministry of Health of the Czech Republic, grant nr. NV18-03-00343, Ministry of Education, Youth and Sports of the Czech Republic, NPU I project nr.LO1604 and EU-Prague project CZ.2.16/3.1.00/24505. JFM and AO were supported by CB16/12/00400 grant (CIBER-ONC, Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Madrid, Spain and FONDOS FEDER).Peer reviewe
EuroFlow standardization of flow cytometer instrument settings and immunophenotyping protocols
No full textThe 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
The EuroFlow PID Orientation Tube for Flow Cytometric Diagnostic Screening of Primary Immunodeficiencies of the Lymphoid System
No full textIn the rapidly evolving field of primary immunodeficiencies (PID), the EuroFlow
consortium decided to develop a PID orientation and screening tube that facilitates
fast, standardized, and validated immunophenotypic diagnosis of lymphoid PID, and
allows full exchange of data between centers. Our aim was to develop a tool that would
be universal for all lymphoid PIDs and offer high sensitivity to identify a lymphoid PID
(without a need for specificity to diagnose particular PID) and to guide and prioritize
further diagnostic modalities and clinical management. The tube composition has been
defined in a stepwise manner through several cycles of design-testing-evaluationredesign in a multicenter setting. Equally important appeared to be the standardized preanalytical procedures (sample preparation and instrument setup), analytical procedures
(immunostaining and data acquisition), the software analysis (a multidimensional view
based on a reference database in Infinicyt software), and data interpretation. This
standardized EuroFlow concept has been tested on 250 healthy controls and 99 PID
patients with defined genetic defects. In addition, an application of new EuroFlow
software tools with multidimensional pattern recognition was designed with inclusion of
maturation pathways in multidimensional patterns (APS plots). The major advantage of
the EuroFlow approach is that data can be fully exchanged between different laboratories
in any country of the world, which is especially of interest for the PID field, with generally
low numbers of cases per center
EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes
No full textEuroFlow Consortium (EU-FP6, LSHB-CT-2006-018708): et al.This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivative Works 3.0 Unported License.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.We are grateful to Dr Jean-Luc Sanne of the European Commission for his support
and monitoring of the EuroFlow project. The research activities of the EuroFlow Consortium were supported by the European Commission (grant STREP
EU-FP6, LSHB-CT-2006-018708, entitled ‘Flow cytometry for fast and sensitive diagnosis and follow-up of hematological malignancies’) and the following national grants: Spanish Network of Cancer Research Centers (ISCIII RTICC-RD06/0020/0035-FEDER), FIS 08/90881 and FIS PS09/02430 from the ‘Fondo de Investigación Sanitaria’, Ministerio de Ciencia e Innovación (Madrid, Spain); GR37 EDU/894/2009 and SA016-A-09 from the Consejería de Educación, Junta de Castilla y León (Valladolid, Spain); PIB2010BZ-00565 from the Dirección General de Cooperación Internacional y Relaciones Institucionales, Secretaría de Estado de Investigación, Ministerio de Ciencia e Innovación (Madrid, Spain); Ministry of Health Czech Republic (NT/12425-4 and NS/10480-3); and Charles University grant No. 23010 and UNCE No. 204012; Tomas Kalina is supported as an ‘ISAC Scholar’ by the International Society for Advancement of Cytometry (ISAC); Andy Rawstron was supported by Leukemia and
Lymphoma Research (LLR).Peer Reviewe
EuroFlow-based flowcytometric diagnostic screening and classification of primary immunodeficiencies of the lymphoid system
No full textGuidelines for screening for primary immunodeficiencies (PID) are well-defined and several consensus diagnostic strategies have been proposed. These consensus proposals have only partially been implemented due to lack of standardization in laboratory procedures, particularly in flow cytometry. The main objectives of the EuroFlow Consortium were to innovate and thoroughly standardize the flowcytometric techniques and strategies for reliable and reproducible diagnosis and classification of PID of the lymphoid system. The proposed EuroFlow antibody panels comprise one orientation tube and seven classification tubes and corresponding databases of normal and PID samples. The 8-color 12-antibody PID Orientation tube (PIDOT) aims at identification and enumeration of the main lymphocyte and leukocyte subsets; this includes naïve pre-germinal center (GC) and antigen-experienced post-GC memory B-cells and plasmablasts. The seven additional 8(-12)-color tubes can be used according to the EuroFlow PID algorithm in parallel or subsequently to the PIDOT for more detailed analysis of B-cell and T-cell subsets to further classify PID of the lymphoid system. The Pre-GC, Post-GC, and immunoglobulin heavy chain (IgH)-isotype B-cell tubes aim at identification and enumeration of B-cell subsets for evaluation of B-cell maturation blocks and specific defects in IgH-subclass production. The severe combined immunodeficiency (SCID) tube and T-cell memory/effector subset tube aim at identification and enumeration of T-cell subsets for assessment of T-cell defects, such as SCID. In case of suspicion of antibody deficiency, PIDOT is preferably directly combined with the IgH isotype tube(s) and in case of SCID suspicion (e.g., in newborn screening programs) the PIDOT is preferably directly combined with the SCID T-cell tube. The proposed ≥8-color antibody panels and corresponding reference databases combined with the EuroFlow PID algorithm are designed to provide fast, sensitive and cost-effective flowcytometric diagnosis of PID of the lymphoid system, easily applicable in multicenter diagnostic settings world-wide
Table_1_The EuroFlow PID Orientation Tube for Flow Cytometric Diagnostic Screening of Primary Immunodeficiencies of the Lymphoid System.pdf
No full textIn the rapidly evolving field of primary immunodeficiencies (PID), the EuroFlow consortium decided to develop a PID orientation and screening tube that facilitates fast, standardized, and validated immunophenotypic diagnosis of lymphoid PID, and allows full exchange of data between centers. Our aim was to develop a tool that would be universal for all lymphoid PIDs and offer high sensitivity to identify a lymphoid PID (without a need for specificity to diagnose particular PID) and to guide and prioritize further diagnostic modalities and clinical management. The tube composition has been defined in a stepwise manner through several cycles of design-testing-evaluation-redesign in a multicenter setting. Equally important appeared to be the standardized pre-analytical procedures (sample preparation and instrument setup), analytical procedures (immunostaining and data acquisition), the software analysis (a multidimensional view based on a reference database in Infinicyt software), and data interpretation. This standardized EuroFlow concept has been tested on 250 healthy controls and 99 PID patients with defined genetic defects. In addition, an application of new EuroFlow software tools with multidimensional pattern recognition was designed with inclusion of maturation pathways in multidimensional patterns (APS plots). The major advantage of the EuroFlow approach is that data can be fully exchanged between different laboratories in any country of the world, which is especially of interest for the PID field, with generally low numbers of cases per center.</p
From big flow cytometry datasets to smart diagnostic strategies: The EuroFlow approach
No full textOn behalf of EuroFlow.The rise in the analytical speed of mutiparameter flow cytometers made possible by the introduction of digital instruments, has brought up the possibility to manage progressively higher number of parameters simultaneously on significantly greater numbers of individual cells. This has led to an exponential increase in the complexity and volume of flow cytometry data generated about cells present in individual samples evaluated in a single measurement. This increase demands for new developments in flow cytometry data analysis, graphical representation, and visualization and interpretation tools to address the new big data challenges, i.e. processing data files of ≥10–25 parameters per cell in samples with >5–10 million cells (= up to 250 million data points per cell sample) obtained in a few minutes. Here, we present a comprehensive review of some of the tools developed by the EuroFlow consortium for processing flow cytometric big data files in diagnostic laboratories, particularly focused on automated EuroFlow approaches for: i) identification of all cell populations coexisting in a sample (automated gating); ii) smart classification of aberrant cell populations in routine diagnostics; iii) automated reporting; together with iv) new tools developed to visualize n-dimensional data in 2-dimensional plots to support expert-guided automated data analysis. The concept of using reference data bases implemented into software programs, in combination with multivariate statistical analysis pioneered by EuroFlow, provides an innovative, highly efficient and fast approach for diagnostic screening, classification and monitoring of patients with distinct hematological and immune disorders, as well as other diseases.E.S.d.C. and C.E.P. acknowledge Research Foundation of the State of Rio de Janeiro, Rio de Janeiro, Brazil (FAPERJ - E26/010.001659/2016, E26/110.105/2014, E26/102.191/2013, E26/202.838/2017-CNE), CAPES - Coordination of Improvement of Higher Education Personnel (PROEX - 1201036), Programa de Oncobiologia and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico– CNPQ of Brazil (409440/2016-7, 304243/2015, PVE4001942014-7).Peer reviewe
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