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.

• Klíčová slova
• flow cytometry, monoclonal antibody, validation,
• MeSH
• indikátory a reagencie MeSH
• protilátky * MeSH
• průtoková cytometrie MeSH
• reprodukovatelnost výsledků MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• indikátory a reagencie MeSH
• protilátky * 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.

• Klíčová slova
• Antibody panel combination, Fluorochromes, Multicolor flow cytometry,
• MeSH
• fluorescenční barviva * MeSH
• imunofenotypizace metody   MeSH
• lidé MeSH
• průtoková cytometrie metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fluorescenční barviva * 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.

• Klíčová slova
• algal FCM standards, best practices, flow cytometry, genome size, microalgae, nuclear isolation,
• MeSH
• biomasa MeSH
• biotechnologie MeSH
• délka genomu MeSH
• ekosystém MeSH
• mikrořasy * MeSH
• průtoková cytometrie MeSH
• Publikační typ
• časopisecké články 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.

• Klíčová slova
• FCS, bioinformatics, data standard, file format, flow cytometry, reproducibility in science,
• MeSH
• průtoková cytometrie normy   MeSH
• software * MeSH
• výpočetní biologie * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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.

• Klíčová slova
• EuroFlow, data analysis, flow cytometry, standardization,
• MeSH
• imunofenotypizace MeSH
• indikátory a reagencie MeSH
• lidé MeSH
• průtoková cytometrie * MeSH
• referenční standardy MeSH
• reprodukovatelnost výsledků MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• indikátory a reagencie MeSH

• Klíčová slova
• clinical trial, data analysis, flow cytometry, normalization,
• MeSH
• algoritmy * MeSH
• cytokiny metabolismus   MeSH
• klinické zkoušky jako téma * MeSH
• lidé MeSH
• průtoková cytometrie * MeSH
• vakcíny proti AIDS terapeutické užití   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• komentáře MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytokiny MeSH
• vakcíny proti AIDS 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
• CD antigeny analýza   MeSH
• imunofenotypizace MeSH
• lidé MeSH
• paraproteinemie imunologie   patologie   MeSH
• plazmatické buňky klasifikace   imunologie   MeSH
• průtoková cytometrie * metody   normy   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• CD antigeny MeSH

Despite remarkable recent progress in the analysis of plant genome organization and chromosome structure, there is a need for methods enabling DNA sequences to be mapped by fluorescence in situ hybridization (FISH) at high spatial resolution. We sorted mitotic metaphase chromosomes of wheat by flow cytometry and observed the occurrence of hyperexpanded chromosomes among them. However, this phenomenon was not reproducible in subsequent experiments. An investigation into the procedures of flow cytometry revealed that the hyperexpansion of chromosomes became reproducible when the concentration of formaldehyde used in sample fixation was reduced. We conducted FISH analysis with 45S rDNA, 5S rDNA and wheat centromeric repeat sequences as probes on flow-sorted chromosomes and also on chromosomes from squash preparations. We measured the length of chromosomes 1B and 6B, identified by FISH. On average, the hyperexpanded 1B and 6B chromosomes were 7.26 and 7.53 times longer, respectively, than the same chromosomes from the squash preparations. The most stretched 1B and 6B chromosomes both exceeded 100 micrometers.

• MeSH
• chromozomy rostlin genetika   ultrastruktura   MeSH
• hybridizace in situ fluorescenční MeSH
• průtoková cytometrie MeSH
• pšenice genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Chromosome analysis and sorting using flow cytometry (flow cytogenetics) is an attractive tool for fractionating plant genomes to small parts. The reduction of complexity greatly simplifies genetics and genomics in plant species with large genomes. However, as flow cytometry requires liquid suspensions of particles, the lack of suitable protocols for preparation of solutions of intact chromosomes delayed the application of flow cytogenetics in plants. This chapter outlines a high-yielding procedure for preparation of solutions of intact mitotic chromosomes from root tips of young seedlings and for their analysis using flow cytometry and sorting. Root tips accumulated at metaphase are mildly fixed with formaldehyde, and solutions of intact chromosomes are prepared by mechanical homogenization. The advantages of the present approach include the use of seedlings, which are easy to handle, and the karyological stability of root meristems, which can be induced to high degree of metaphase synchrony. Chromosomes isolated according to this protocol have well-preserved morphology, withstand shearing forces during sorting, and their DNA is intact and suitable for a range of applications.

• MeSH
• buněčný cyklus MeSH
• chromozomy rostlin * MeSH
• cytogenetika MeSH
• DNA rostlinná genetika   MeSH
• hybridizace in situ fluorescenční metody   MeSH
• karyotypizace MeSH
• meristém cytologie   MeSH
• průtoková cytometrie metody   MeSH
• rostlinné buňky MeSH
• rostliny genetika   MeSH
• semena rostlinná růst a vývoj   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA rostlinná MeSH

The fluorescence detected using fluorochrome-labelled monoclonal antibodies depends not only on the abundance of the target antigen, but amongst many other factors also on the effective fluorochrome-to-antibody ratio. The diagnostic approach of the EuroFlow consortium relies on reproducible fluorescence intensities over time. A capture bead system for mouse immunoglobulin light chains was utilized to compare the mean fluorescence intensity of 1323 consecutive antibody lots to the currently used lot of the same monoclonal antibody. In total, 157 different monoclonal antibodies were assessed over seven years. Median relative difference between consecutive lots was 3.8% (range: 0.01% to 164.7%, interquartile range: 1.3% to 10.1%). The relative difference exceeded 20% in 8.8% of all comparisons. FITC labelled monoclonal antibodies (median relative difference: 2.1%) showed a significantly smaller variation between lots than antibodies conjugated to PE (3.5%), PECy7 (3.9%), PerCPCy5.5 (5.8%), APC (5.8%), APCH7 (7.4%), and APCC750 (14.5%). Reagents labelled with Pacific Blue (1.4%), Pacific Orange (2.4%), HV450 (0.7%), and HV500 (1.7%) demonstrated more consistent results compared to conjugates of BV421 (4.1%) and BV510 (16.2%). Additionally, significant differences in lot-to-lot fluorescence stability amongst antibodies labelled with the same fluorochrome were observed between manufacturers. These observations might guide future quality recommendations for the production and application of fluorescence-labelled monoclonal antibodies in multicolor flow cytometry.

• Klíčová slova
• Flow cytometry, Fluorescence intensity, Fluorochrome-to-antibody ratio, Variability,
• MeSH
• fluorescenční barviva * normy   MeSH
• monoklonální protilátky * MeSH
• myši MeSH
• průtoková cytometrie metody   normy   MeSH
• reprodukovatelnost výsledků MeSH
• stabilita proteinů MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fluorescenční barviva * MeSH
• monoklonální protilátky * MeSH