1,721,220 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
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
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
EuroFlow: Resetting leukemia and lymphoma immunophenotyping. Basis for companion diagnostics and personalized medicine
No full textThis work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivative Works 3.0 Unported License.-- Editorial.We are grateful to Dr Jean-Luc Sanne of the European Commission for his support and monitoring of the EuroFlow project.Peer Reviewe
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-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
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
MRD detection in multiple myeloma: comparison between MSKCC 10-color single-tube and EuroFlow 8-color 2-tube methods
No full text[EN] In patients with multiple myeloma, obtaining posttreatment minimal residual disease (MRD) negativity is associated with longer progression-free survival and overall survival. Here, we compared the diagnostic performance of a single 10-color tube with that of a EuroFlow 8-color 2-tube panel for MRD testing. Bone marrow samples from 41 multiple myeloma patients were tested in parallel using the 2 approaches. Compared with the sum of the cells from the EuroFlow two 8-color tubes, the Memorial Sloan Kettering Cancer Center (MSKCC) single 10-color tube had a slight reduction in total cell number with a mean ratio of 0.85 (range, 0.57-1.46; P < .05), likely attributable to permeabilization of the cells. Percent of plasma cells showed a high degree of concordance (r2 = 0.97) as did normal plasma cells (r2 = 0.96), consistent with no selective plasma cell loss. Importantly, concordant measurement of residual disease burden was seen with abnormal plasma cells (r2 = 0.97). The overall concordance between the 2 tests was 98%. In 1 case, there was a discrepancy near the limit of detection of both tests in favor of the slightly greater theoretical sensitivity of the EuroFlow 8-color 2-tube panel (analytical sensitivity limit of MSKCC single 10-color tube: 6 cells in 1 million with at least 3 million cell acquisitions; EuroFlow 8-color 2-tube panel: 2 cells in 1 million with the recommended 10 million cell acquisitions)
- …
