BACKGROUND/AIMS: Endothelial progenitor cells (EPCs) and circulating endothelial cells (CECs) have been described as markers of endothelial damage and dysfunction in several diseases, including deep venous thrombosis. Their role in patients with known thrombophilia has not yet been evaluated. Both EPCs and CECs represent extremely rare cell populations. Therefore, it is essential to use standardized methods for their identification and quantification. METHODS: In this study, we used multicolor flow cytometry to analyze the number of EPCs and CECs in patients with thrombophilia with or without a history of thrombosis. Patients with hematological malignancies after high-dose chemotherapy and patients with acute myocardial infarction were used as positive controls. RESULTS: EPC and CEC immunophenotypes were determined as CD45dim/-CD34+CD146+CD133+ and CD45dim/-CD34+CD146+CD133-, respectively. Increased levels of endothelial cells were observed in positive control groups. No significant changes in the number of EPCs or CECs were detected in patients with thrombophilia compared to healthy controls. CONCLUSION: Our optimized multicolor flow cytometry method allows unambiguous identification and quantification of endothelial cells in the peripheral blood. Our results support previous studies showing that elevated levels of CECs could serve as an indicator of endothelial injury or dysfunction. Normal levels of CECs or EPCs were found in patients with thrombophilia.

• Klíčová slova
• Circulating endothelial cells, Endothelial progenitor cells, Flow cytometry, Thrombophilia,
• MeSH
• CD antigeny krev   MeSH
• dospělí MeSH
• endoteliální buňky * metabolismus   patologie   MeSH
• endoteliální progenitorové buňky * metabolismus   patologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• průtoková cytometrie * MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• trombofilie * krev   patologie   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CD antigeny 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

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

Multicolor flow cytometry allows for analysis of tens of cellular parameters in millions of cells at a single-cell resolution within minutes. The lack of technologies that would facilitate feasible and relatively cheap profiling of such a number of cells with an antibody-based approach led us to the development of a high-throughput cytometry-based platform for surface profiling. We coupled the fluorescent cell barcoding with preexisting, commercially available screening tools to analyze cell surface fingerprint at a large scale. This powerful approach will help to identify novel biomarkers and druggable targets and facilitate the discovery of new concepts in immunology, oncology, and developmental biology.

• Klíčová slova
• Cell surface phenotyping, Fluorescent cell barcoding, High-throughput screening, Multicolor flow cytometry,
• MeSH
• antigeny povrchové * MeSH
• biologické markery analýza   MeSH
• fluorescenční barviva MeSH
• průtoková cytometrie MeSH
• výzkum * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny povrchové * MeSH
• biologické markery MeSH
• fluorescenční barviva 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

The combination of color-coded microspheres as carriers and flow cytometry as a detection platform provides new opportunities for multiplexed measurement of biomolecules. Here, we developed a software tool capable of automated gating of color-coded microspheres, automatic extraction of statistics from all subsets and validation, normalization, and cross-sample analysis. The approach presented in this article enabled us to harness the power of high-content cellular proteomics. In size exclusion chromatography-resolved microsphere-based affinity proteomics (Size-MAP), antibody-coupled microspheres are used to measure biotinylated proteins that have been separated by size exclusion chromatography. The captured proteins are labeled with streptavidin phycoerythrin and detected by multicolor flow cytometry. When the results from multiple size exclusion chromatography fractions are combined, binding is detected as discrete reactivity peaks (entities). The information obtained might be approximated to a multiplexed western blot. We used a microsphere set with >1,000 subsets, presenting an approach to extract biologically relevant information. The R-project environment was used to sequentially recognize subsets in two-dimensional space and gate them. The aim was to extract the median streptavidin phycoerythrin fluorescence intensity for all 1,000+ microsphere subsets from a series of 96 measured samples. The resulting text files were subjected to algorithms that identified entities across the 24 fractions. Thus, the original 24 data points for each antibody were compressed to 1-4 integrated values representing the areas of individual antibody reactivity peaks. Finally, we provide experimental data on cellular protein changes induced by treatment of leukemia cells with imatinib mesylate. The approach presented here exemplifies how large-scale flow cytometry data analysis can be efficiently processed to employ flow cytometry as a high-content proteomics method.

• MeSH
• algoritmy MeSH
• automatizace MeSH
• barva MeSH
• bcr-abl fúzní proteiny metabolismus   MeSH
• benzamidy MeSH
• časové faktory MeSH
• databáze proteinů * MeSH
• gelová chromatografie MeSH
• imatinib mesylát MeSH
• lidé MeSH
• mikrosféry MeSH
• nádorové buněčné linie MeSH
• nádorové proteiny metabolismus   MeSH
• piperaziny farmakologie   MeSH
• proteom metabolismus   MeSH
• proteomika metody   MeSH
• průtoková cytometrie metody   MeSH
• pyrimidiny farmakologie   MeSH
• referenční standardy MeSH
• řízení kvality MeSH
• software MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bcr-abl fúzní proteiny MeSH
• benzamidy MeSH
• imatinib mesylát MeSH
• nádorové proteiny MeSH
• piperaziny MeSH
• proteom MeSH
• pyrimidiny MeSH

The advent of new therapeutic approaches to multiple myeloma made necessary the introduction of novel methods for detection of minimal residual disease. Among others approaches residual disease can be detected by the immunofluorescence using flow cytometry. We have examined the co-expression of CD19, CD38, CD45, CD54, CD56, and CD138 molecules in cells of peripheral blood and bone marrow aspirates in patients with multiple myeloma by 3-color flow cytometry. For the detection and characterization of multiple myeloma cells, combinations of following antibodies were used: anti-CD19 FITC, anti-CD38 FITC, anti-CD38 PE, anti-CD54 FITC, anti-CD56 PE-Cy5, anti-CD45 PE, anti-CD45 PE-Cy5 (Immunotech) and anti CD138 PE (Serotec). The samples were analyzed using EPICS XL (Coulter) flow cytometer, and the analysis was based on at least 10,000 events. Samples from 17 patients were analyzed. The percentage of multiple myeloma cells ranged between 0.3% and 54.2% in bone marrow aspirates and between 0.0 and 11.8% in periferal blood. The expression of CD138, CD38, CD54 and CD56 molecules was found in 100%, 100%, 85% and 68% of examined cases, respectively. In our opinion, multiple myeloma cells are best characterized by following combinations of antibodies: CD38 FITC/CD138 PE/CD45 PE-Cy5, CD54 FITC/CD138 PE/CD56 PE-Cy5 or CD54 FITC/CD38 PE/CD 56 PE-Cy5. The identification of a malignant clone is the first and the most important step in the characterization of the disease, determination of its prognosis and the detection of residual disease after treatment. Three-color flow cytometry represents a method which can meet these goals.

• MeSH
• CD antigeny analýza   MeSH
• fluorescenční protilátková technika MeSH
• lidé MeSH
• mnohočetný myelom diagnóza   patologie   terapie   MeSH
• nádorové biomarkery analýza   MeSH
• průtoková cytometrie MeSH
• reziduální nádor MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• anglický abstrakt MeSH
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CD antigeny MeSH
• nádorové biomarkery MeSH

Complex analysis of cellular responses after experimental treatment is important for screening, mechanistic understanding of treatment effects, and the identification of sensitive and resistant cell phenotypes. Modern multicolor flow cytometry has demonstrated its power for such analyses. Here, we introduce a multiparametric protocol for complex analysis of cytokinetics by the simultaneous detection of seven fluorescence parameters. This analysis includes the detection of two surface markers for immunophenotyping, analysis of proliferation based on the cell cycle and the measurement of incorporated nucleoside analogue 5-ethynyl-2'-deoxyuridine (EdU) in newly synthesized DNA, analysis of DNA damage using an anti-phospho-histone H2A.X (Ser139) antibody, and determination of cell death using a fixable viability probe and intracellular detection of caspase-3 activation. To demonstrate the applicability of this protocol for the analysis of heterogeneous and complex cell responses, we used different treatments and model cell lines. We demonstrated that this protocol has the potential to provide complex and simultaneous analysis of cytokinetics and analyze the heterogeneity of the response at the single-cell level. © 2017 International Society for Advancement of Cytometry.

• Klíčová slova
• DNA damage, apoptosis, flow cytometry, immunophenotyping, multiparametric analysis, proliferation,
• MeSH
• apoptóza fyziologie   MeSH
• imunofenotypizace metody   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• poškození DNA fyziologie   MeSH
• proliferace buněk fyziologie   MeSH
• průtoková cytometrie metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

OBJECTIVE: The aim of this study was to evaluate changes in the relative counts of different leukocyte subsets in peripheral and umbilical cord blood in pregnancies complicated by preterm prelabor rupture of membranes (PPROM) with respect to the presence of intraamniotic inflammation (IAI) and fetal inflammatory response syndrome (FIRS). METHODS: Fifty-two women with singleton pregnancies complicated by PPROM were included in this study. From samples of peripheral and umbilical cord blood, relative counts of these leukocyte subpopulations were determined using multicolor flow cytometry: granulocytes, monocytes, lymphocytes, T cells and their subpopulations, B cells and their subpopulations, and NK cells and their subpopulations. IAI was defined as increased concentrations of interleukin 6 in the amniotic fluid. Amniotic fluid samples were obtained by transabdominal amniocentesis. RESULTS: Women with IAI had higher relative counts of monocytes (p = 0.04) in peripheral blood. There was an increased relative number of granulocytes (p = 0.003) and a decreased number of lymphocytes (p = 0.0048), helper CD4+ T cells (p = 0.019), NK cells (p = 0.0001) within leukocytes, NK cells within lymphocytes (p = 0.003) and CD16+ NK cells within NK cells (p = 0.005) in umbilical cord blood samples of women with FIRS. However, after adjusting the results for gestational age at sampling, all differences disappeared. CONCLUSIONS: The presence of IAI or FIRS is not accompanied by significant changes in the relative counts of immune cells in peripheral blood or umbilical cord blood in pregnancies complicated by PPROM.

• Klíčová slova
• FIRS, PPROM, flow cytometry, intraamniotic inflammation, leukocyte subpopulations,
• MeSH
• chorioamnionitida imunologie   krev   MeSH
• dospělí MeSH
• fetální krev * imunologie   cytologie   MeSH
• interleukin-6 krev   metabolismus   MeSH
• leukocyty imunologie   MeSH
• lidé MeSH
• plodová voda imunologie   metabolismus   MeSH
• počet leukocytů MeSH
• předčasný odtok plodové vody * imunologie   krev   MeSH
• průtoková cytometrie MeSH
• syndrom systémové zánětlivé reakce imunologie   krev   MeSH
• těhotenství MeSH
• zánět imunologie   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• interleukin-6 MeSH

We have characterized a panel of commercially available anti-human monoclonal antibodies (mAbs) suitable for B-cell identification in pigs and dogs. The specificities of the mAbs were against CD20, CD21, CD22, and CD86. In addition to HM57, originally raised against human CD79alpha the broad cross-reactivity of which was documented more than 10 years ago, we recommend here a panel of several other mAbs as a useful tool for immunophenotyping and multicolor flow cytometry of canine and porcine B-lymphocytes. All six investigated antibodies did bind weakly to either canine or porcine lymphocytes (or both), but considerable weaker than for the human control cells. Four of them did bind to canine or porcine spleen section in immunohistochemistry. Monoclonal antibody against CD22 (clone RFB-4) was the only antibody in the tested panel the cross-reactivity of which was confirmed by Western blot. The advantages and limits of cross-reactive mAbs in studies on animal B-cells are discussed.

• MeSH
• B-lymfocyty imunologie   MeSH
• CD antigeny imunologie   MeSH
• imunofenotypizace MeSH
• lidé MeSH
• monoklonální protilátky imunologie   MeSH
• prasata imunologie   MeSH
• průtoková cytometrie MeSH
• psi imunologie   MeSH
• zkřížené reakce MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• psi imunologie   MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CD antigeny MeSH
• monoklonální protilátky MeSH