Antibody reagents are the key components of multiparametric flow cytometry analysis. Their quality performance is an absolute requirement for reproducible flow cytometry experiments. While there is an enormous body of antibody reagents available, there is still a lack of consensus about which criteria should be evaluated to select antibody reagents with the proper performance, how to validate antibody reagents for flow cytometry, and how to interpret the validation results. The achievements of cytometry moved the field to a higher number of measured parameters, large data sets, and computational data analysis approaches. These advancements pose an increased demand for antibody reagent performance quality. This review summarizes the codevelopment of cytometry, antibody development, and validation strategies. It discusses the diverse issues of the specificity, cross-reactivity, epitope, titration, and reproducibility features of antibody reagents, and this review discusses the validation principles and methods that are currently available and those that are emerging. We argue that significant efforts should be invested by antibody users, developers, manufacturers, and publishers to increase the quality and reproducibility of published studies. More validation data should be presented by all stakeholders; however, the data should be presented in sufficient experimental detail to foster reproducibility, and community effort shall lead to the public availability of large data sets that can serve as a benchmark for antibody performance. © 2019 International Society for Advancement of Cytometry.

• Keywords
• flow cytometry, monoclonal antibody, validation,
• MeSH
• Indicators and Reagents MeSH
• Antibodies * MeSH
• Flow Cytometry MeSH
• Reproducibility of Results MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Indicators and Reagents MeSH
• Antibodies * MeSH

Microalgae are photosynthetic microorganisms with a major influence on global ecosystems. Further, owing to the production of various secondary metabolites, microalgae are also intensively studied for their enormous potential in biotechnology and its applications. While flow cytometry (FCM) is a fast and reliable method particularly suitable for genome size estimation in plant and animal studies, its application to microalgae often comes with many methodological challenges due to specific issues (e.g., cell wall composition, and presence of various secondary metabolites). Sample preparation requires considerable amounts of biomass, chemical fixation, and/or extraction of cellular components. In genome size estimation, appropriate methods for isolation of intact nuclei (using lysis buffers, razor-blade chopping, various enzymes, or bead-beating of cells) are essential for successful and high-quality analyses. Nuclear DNA amounts of microalgae diverge greatly, varying by almost 30,000-fold (0.01 to 286 pg). Even though new algal reference standards for genome size are now being introduced, animal red blood cells and nuclei from plant tissues are still predominantly used. Due to our limited knowledge of microalgal life cycles, particular caution should be taken during 1C/2C-value (or ploidy level) assignments.

• Keywords
• algal FCM standards, best practices, flow cytometry, genome size, microalgae, nuclear isolation,
• MeSH
• Biomass MeSH
• Biotechnology MeSH
• Genome Size MeSH
• Ecosystem MeSH
• Microalgae * MeSH
• Flow Cytometry MeSH
• Publication type
• Journal Article MeSH

The Flow Cytometry Standard (FCS) format is a widely accepted norm for storing Flow Cytometry (FCM) data. Its goal as a standard is to allow FCM data sharing and re-analysis. Over more than three decades of its existence FCS has evolved into a well-defined, flexible file format reflecting technical changes in the FCM field. Its flexibility as well as rising numbers of instrument vendors leads to suboptimal implementations of FCS in some cases. Such situations compromise the primary goal of the standard and hinder the ability to reproduce FCM analyses. It is further underlined by rapid rise of advanced FCM analyses, often carried out outside traditional software tools and heavily relying on standard data storage and presentation. We have developed flowIO, an R package which tests FCS file conformance with the standard as defined by International Society for Advancement of Cytometry (ISAC) normative. Along with the package we provide a web based application (also at http://bioinformin.cesnet.cz/flowIO/) allowing user friendly access to the conformance testing as well as FCS file editing and export for further analysis.

• Keywords
• FCS, bioinformatics, data standard, file format, flow cytometry, reproducibility in science,
• MeSH
• Flow Cytometry standards   MeSH
• Software * MeSH
• Computational Biology * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

There is an agreement in the field that interlaboratory reproducibility of flow cytometry measurements as well as the whole studies might be improved by a consensual use of methodological approach. Typically, a consensus is made on a crucial markers needed in the immunostaining panel, sometimes on the particular fluorochrome conjugates and rarely on a complete set of methods for sample preparation. The term "standardization" is used to describe the complete set of methodical steps, while "harmonization" is used for partial agreement on the method. Standardization can provide a platform for improved reproducibility of cytometry results over prolonged periods of time, across different sites and across different instruments. For the purpose of structured discussion, several desired aims are described: common interpretation of the immunophenotype definition of a target subset, accurate quantification, reproducible pattern of a multicolor immunophenotype, and reproducible intensity of all measured parameters. An overview of how standardization was approached by several large consortia is provided: EuroFlow, The ONE Study, Human Immunology Project Consortium (HIPC), and several other groups. Their particular aims and the tools adopted to reach those aims are noted. How those standardization efforts were adopted in the field and how the resulting outcome was evaluated is reviewed. Multiple challenges in the instrument hardware design, instrument setup tools, reagent design, and quality features need to be addressed to achieve optimal standardization. Furthermore, the aims of different studies vary, and thus, the reasonable requirements for standardization differ. A framework of reference for the reasonable outcomes of different approaches is offered. Finally, it is argued that complete standardization is important not only for the reproducibility of measurements but also for education, for quality assessment and for algorithmic data analysis. The different standardized approaches can and in fact should serve as benchmarking reference tools for the development of future flow cytometry studies. © 2019 International Society for Advancement of Cytometry.

• Keywords
• EuroFlow, data analysis, flow cytometry, standardization,
• MeSH
• Immunophenotyping MeSH
• Indicators and Reagents MeSH
• Humans MeSH
• Flow Cytometry * MeSH
• Reference Standards MeSH
• Reproducibility of Results MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Indicators and Reagents MeSH

• Keywords
• clinical trial, data analysis, flow cytometry, normalization,
• MeSH
• Algorithms * MeSH
• Cytokines metabolism   MeSH
• Clinical Trials as Topic * MeSH
• Humans MeSH
• Flow Cytometry * MeSH
• AIDS Vaccines therapeutic use   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Comment MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cytokines MeSH
• AIDS Vaccines MeSH

Fluorochrome selection is a key step in designing multi-color antibody panels. The list of available fluorochromes is continuously growing, fitting current needs in clinical flow cytometry to simultaneously use more markers to better define multiple leukocyte subpopulations in a single tube. Several criteria guide fluorochrome selection: i) the fluorescence profiles (excitation and emission), ii) relative brightness, iii) fluorescence overlap, iv) fluorochrome stability, and v) reproducible conjugation to antibodies. Here we used 75 samples (45 bone marrow and 30 blood) to illustrate EuroFlow strategies for evaluation of compatible fluorochromes, and how the results obtained guide fluorochrome selection as a critical step in the antibody-panel building process. Our results allowed identification of optimal fluorescence profiles (e.g. higher fluorescence intensity and/or resolution with limited fluorescence overlap into neighbor channels) for brilliant violet (BV)421 and BV510 in the violet laser and allophycocyanin (APC) hilite 7 (H7) or APC C750 in the red laser vs. other candidate fluorochromes generally applied for the same detectors and here evaluated. Moreover, evaluation of the same characteristics for another group of fluorochromes (e.g. BV605, BV650, PE CF594, AF700 or APC AF700) guided selection of the most appropriate fluorochrome conjugates to be combined in a multi-color antibody panel. Albeit this is a demanding approach, it could be successfully applied for selection of fluorochrome combinations for the EuroFlow antibody panels for diagnosis, classification and monitoring of hematological malignancies and primary immunodeficiencies. Consequently, sets of 8-, 10- and 12-color fluorochrome combinations are proposed as frame of reference for initial antibody panel design.

• Keywords
• Antibody panel combination, Fluorochromes, Multicolor flow cytometry,
• MeSH
• Fluorescent Dyes * MeSH
• Immunophenotyping methods   MeSH
• Humans MeSH
• Flow Cytometry methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Fluorescent Dyes * MeSH

Flow cytometry (FCM) is now the most widely used method to determine ploidy levels and genome size of plants. To get reliable estimates and allow reproducibility of measurements, the methodology should be standardized and follow the best practices in the field. In this article, we discuss instrument calibration and quality control and various instrument and acquisition settings (parameters, flow rate, number of events, scales, use of discriminators, peak positions). These settings must be decided before measurements because they determine the amount and quality of the data and thus influence all downstream analyses. We describe the two main approaches to raw data analysis (gating and histogram modeling), and we discuss their advantages and disadvantages. Finally, we provide a summary of best practice recommendations for data acquisition and raw data analysis in plant FCM.

• Keywords
• best practices, calibration, flow cytometry, gating, histogram modeling, instrument settings, plant sciences,
• MeSH
• Genome Size MeSH
• Calibration MeSH
• Ploidies * MeSH
• Flow Cytometry methods   MeSH
• Reproducibility of Results MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

Genome size has played an important role in the evolution of plants and animals because changes in genome size seem to accompany if not facilitate evolutionary adaptation to environmental conditions. Flow cytometry (FCM) is a widespread method for determining genome size thanks to its high accuracy and speed of measurements. Nevertheless, only a few comparative studies of FCM methods exist in the field of mycology, and reviews are absent. In this study, we compared the suitability of several concentrations and RNAse A incubation times, fixatives and buffers for estimating genome size in fungi. We chose the genus Geosmithia as a model filamentous fungus. We also introduced a new standard, Aspergillus fumigatus CEA10, to determine absolute genome size. We found FCM to be an appropriate method for measuring genome size in fungi, but optimization steps showed that incorrect propidium iodide staining of nuclei can overestimate genome size due to cytoplasmic staining. We identified fixation with methanol:glacial acetic acid (3:1 v/v), 10% DMSO, 0.1% Triton-X 100, and 5 mM EDTA in combination with Tris-MgCl2 buffer as the best treatment.

• Keywords
• RNAse, buffer, confocal microscopy, fixation, flow cytometry, fungi, genome size, standard,
• MeSH
• Cell Nucleus genetics   MeSH
• Genome Size genetics   MeSH
• Eurotiales genetics   MeSH
• Flow Cytometry methods   MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH

Flow cytometry (FCM) is currently the most widely-used method to establish nuclear DNA content in plants. Since simple, 1-3-parameter, flow cytometers, which are sufficient for most plant applications, are commercially available at a reasonable price, the number of laboratories equipped with these instruments, and consequently new FCM users, has greatly increased over the last decade. This paper meets an urgent need for comprehensive recommendations for best practices in FCM for different plant science applications. We discuss advantages and limitations of establishing plant ploidy, genome size, DNA base composition, cell cycle activity, and level of endoreduplication. Applications of such measurements in plant systematics, ecology, molecular biology research, reproduction biology, tissue cultures, plant breeding, and seed sciences are described. Advice is included on how to obtain accurate and reliable results, as well as how to manage troubleshooting that may occur during sample preparation, cytometric measurements, and data handling. Each section is followed by best practice recommendations; tips as to what specific information should be provided in FCM papers are also provided.

• Keywords
• DNA base composition, DNA content, cell cycle, endoreduplication, flow cytometric seed screening, genome size, in vitro cultures, intraspecific variation, ploidy,
• MeSH
• Genome Size MeSH
• DNA, Plant genetics   MeSH
• Genome, Plant MeSH
• Ploidies * MeSH
• Flow Cytometry methods   MeSH
• Plants * genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• DNA, Plant MeSH

The technological development of flow cytometry (FC) together with new findings reveal the need for immunophenotyping in research of monoclonal gammopathy (MG) because of its diagnostic, prognostic and predictive significance. The aim of the European Myeloma Network (EMN) is to standardize this analytical method and implement it into routine clinical examination. Since the overall significance and application of FC are still analysed, standardisation could help obtain more clinical relevant information in terms of MG pathophysiology.

• MeSH
• Antigens, CD analysis   MeSH
• Immunophenotyping MeSH
• Humans MeSH
• Paraproteinemias immunology   pathology   MeSH
• Plasma Cells classification   immunology   MeSH
• Flow Cytometry * methods   standards   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Antigens, CD MeSH