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  1. Improved graft survival in highly sensitized patients undergoing renal transplantation after the introduction of a clinically validated flow cytometry crossmatch.

    LENUS (Irish Health Repository)

    Limaye, Sandhya

    2009-04-15

    Flow cytometric techniques are increasingly used in pretransplant crossmatching, although there remains debate regarding the clinical significance and predictive value of donor-specific antibodies detected by flow cytometry. At least some of the discrepancies between published studies may arise from differences in cutoffs used and lack of standardization of the test.

  2. Luminex-based virtual crossmatching for renal transplantation in ...

    African Journals Online (AJOL)

    Background. Current practice in the Johannesburg renal transplantation programme is to perform a transplant when the patient's complement-dependent cytotoxicity and flow cytometric crossmatches are negative. However, even in patients with negative crossmatches early graft rejections have occurred. We retrospectively ...

  3. Using real data for a virtual crossmatch.

    Science.gov (United States)

    Zachary, Andrea A; Sholander, Jeffrey T; Houp, Julie A; Leffell, Mary S

    2009-08-01

    Virtual crossmatches have been performed for more than 40 years under the guise of unacceptable antigens. Today, solid-phase assays provide the opportunity for more accurate identification and more precise measurement of the strength of donor-specific antibodies. The process of performing a virtual crossmatch begins with establishing a correlation between the antibody testing assay and the results of actual crossmatches. We provide here data indicating that the identity and strength of DSA defined with solid-phase phenotype panels correlates significantly with the outcome of both cytotoxic (CDC; r = 0.83) and flow cytometric (r = 0.85) crossmatches. Based on the threshold established from these correlations, we were able to correctly predict the results of CDC and flow cytometric crossmatches in 92.8 and 92.4% of cases, respectively. The correlations with single antigen panels were substantially lower (82.6-47.9%) and may be caused by a variety of factors, including variability in the amount and condition of different antigens and extremely high sensitivity, which may make the test less robust. We demonstrate that adding additional information to the solid-phase results can increase the frequency of correct crossmatch prediction. We also present data demonstrating an additional use of the virtual crossmatch in posttransplant monitoring.

  4. A flow cytometric assay for simultaneously measuring the ...

    African Journals Online (AJOL)

    Jane

    2011-10-24

    Oct 24, 2011 ... This research objective was to exploit a novel method for measuring the proliferation, cytotoxicity of cytokine-induced killer (CIK) cells using carboxyfluorescein succinimidyl ester/proliferation index. (CFSE/PI) and flow cytometric assay. As cells divide, CFSE is apportioned equally between the two daughter ...

  5. Flow cytometric immunoassay for sulfonamides in raw milk

    NARCIS (Netherlands)

    Keizer, de W.; Bienenmann-Ploum, M.; Bergwerff, A.A.; Haasnoot, W.

    2008-01-01

    Sulfonamide antibiotics are applied in veterinary medicine for the treatment of microbial infections. For the detection of residues of sulfonamides in milk, a multi-sulfonamide flow cytometric immunoassay (FCI) was developed using the Luminex MultiAnalyte Profiling (xMAP) technology. In this

  6. A flow cytometric assay for simultaneously measuring the ...

    African Journals Online (AJOL)

    This research objective was to exploit a novel method for measuring the proliferation, cytotoxicity of cytokine-induced killer (CIK) cells using carboxyfluorescein succinimidyl ester/proliferation index (CFSE/PI) and flow cytometric assay. As cells divide, CFSE is apportioned equally between the two daughter cells, leading to a ...

  7. Technical discussions II - Flow cytometric analysis

    NARCIS (Netherlands)

    Cunningham, A; Cid, A; Buma, AGJ

    In this paper the potencial of flow cytometry as applied to the aquatic life sciences is discussed. The use of flow cytometry for studying the ecotoxicology of phytoplankton was introduced. On the other hand, the new flow cytometer EUROPA was presented. This is a multilaser machine which has been

  8. Flow cytometric fingerprinting for microbial strain discrimination and physiological characterization.

    Science.gov (United States)

    Buysschaert, Benjamin; Kerckhof, Frederiek-Maarten; Vandamme, Peter; De Baets, Bernard; Boon, Nico

    2018-02-01

    The analysis of microbial populations is fundamental, not only for developing a deeper understanding of microbial communities but also for their engineering in biotechnological applications. Many methods have been developed to study their characteristics and over the last few decades, molecular analysis tools, such as DNA sequencing, have been used with considerable success to identify the composition of microbial populations. Recently, flow cytometric fingerprinting is emerging as a promising and powerful method to analyze bacterial populations. So far, these methods have primarily been used to observe shifts in the composition of microbial communities of natural samples. In this article, we apply a flow cytometric fingerprinting method to discriminate among 29 Lactobacillus strains. Our results indicate that it is possible to discriminate among 27 Lactobacillus strains by staining with SYBR green I and that the discriminatory power can be increased by combined SYBR green I and propidium iodide staining. Furthermore, we illustrate the impact of physiological changes on the fingerprinting method by demonstrating how flow cytometric fingerprinting is able to discriminate the different growth phases of a microbial culture. The sensitivity of the method is assessed by its ability to detect changes in the relative abundance of a mix of polystyrene beads down to 1.2%. When a mix of bacteria was used, the sensitivity was as between 1.2% and 5%. The presented data demonstrate that flow cytometric fingerprinting is a sensitive and reproducible technique with the potential to be applied as a method for the dereplication of bacterial isolates. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

  9. Uncovering Aberrant Mutant PKA Function with Flow Cytometric FRET

    Directory of Open Access Journals (Sweden)

    Shin-Rong Lee

    2016-03-01

    Full Text Available Biology has been revolutionized by tools that allow the detection and characterization of protein-protein interactions (PPIs. Förster resonance energy transfer (FRET-based methods have become particularly attractive as they allow quantitative studies of PPIs within the convenient and relevant context of living cells. We describe here an approach that allows the rapid construction of live-cell FRET-based binding curves using a commercially available flow cytometer. We illustrate a simple method for absolutely calibrating the cytometer, validating our binding assay against the gold standard isothermal calorimetry (ITC, and using flow cytometric FRET to uncover the structural and functional effects of the Cushing-syndrome-causing mutation (L206R on PKA’s catalytic subunit. We discover that this mutation not only differentially affects PKAcat’s binding to its multiple partners but also impacts its rate of catalysis. These findings improve our mechanistic understanding of this disease-causing mutation, while illustrating the simplicity, general applicability, and power of flow cytometric FRET.

  10. Automated High-Dimensional Flow Cytometric Data Analysis

    Science.gov (United States)

    Pyne, Saumyadipta; Hu, Xinli; Wang, Kui; Rossin, Elizabeth; Lin, Tsung-I.; Maier, Lisa; Baecher-Allan, Clare; McLachlan, Geoffrey; Tamayo, Pablo; Hafler, David; de Jager, Philip; Mesirov, Jill

    Flow cytometry is widely used for single cell interrogation of surface and intracellular protein expression by measuring fluorescence intensity of fluorophore-conjugated reagents. We focus on the recently developed procedure of Pyne et al. (2009, Proceedings of the National Academy of Sciences USA 106, 8519-8524) for automated high- dimensional flow cytometric analysis called FLAME (FLow analysis with Automated Multivariate Estimation). It introduced novel finite mixture models of heavy-tailed and asymmetric distributions to identify and model cell populations in a flow cytometric sample. This approach robustly addresses the complexities of flow data without the need for transformation or projection to lower dimensions. It also addresses the critical task of matching cell populations across samples that enables downstream analysis. It thus facilitates application of flow cytometry to new biological and clinical problems. To facilitate pipelining with standard bioinformatic applications such as high-dimensional visualization, subject classification or outcome prediction, FLAME has been incorporated with the GenePattern package of the Broad Institute. Thereby analysis of flow data can be approached similarly as other genomic platforms. We also consider some new work that proposes a rigorous and robust solution to the registration problem by a multi-level approach that allows us to model and register cell populations simultaneously across a cohort of high-dimensional flow samples. This new approach is called JCM (Joint Clustering and Matching). It enables direct and rigorous comparisons across different time points or phenotypes in a complex biological study as well as for classification of new patient samples in a more clinical setting.

  11. A flow cytometric technique for quantification and differentiation of bacteria in bulk tank milk

    DEFF Research Database (Denmark)

    Holm, C.; Mathiasen, T.; Jespersen, Lene

    2004-01-01

    AIMS: The present study describes a flow cytometric technique for quantification and differentiation of bacteria in bulk tank milk according to the main cause of elevated counts. METHODS AND RESULTS: A total of 75 Danish bulk tank milk samples exceeding the grading level of 3.0 x 10(4) CFU ml(-1...... parameters were as follows: staining with Oregon Green conjugated wheat germ agglutinin that binds to the cell wall of bacteria, staining with hexidium iodide that binds to all bacterial DNA, the flow cytometric forward scatter and the flow cytometric side scatter. Three regions in the flow cytometric plot...

  12. Flow Cytometric Bead Sandwich Assay Based on a Split Aptamer.

    Science.gov (United States)

    Shen, Luyao; Bing, Tao; Liu, Xiangjun; Wang, Junyan; Wang, Linlin; Zhang, Nan; Shangguan, Dihua

    2018-01-24

    A few aptamers still bind their targets after being split into two moieties. Split aptamers have shown great potential in the development of aptameric sensors. However, only a few split aptamers have been generated because of lack of knowledge on the binding structure of their parent aptamers. Here, we report the design of a new split aptamer and a flow cytometric bead sandwich assay using a split aptamer instead of double antibodies. Through DMS footprinting and mutation assay, we figured out the target-binding moiety and the structure-stabilizing moiety of the l-selectin aptamer, Sgc-3b. By separating the duplex strand in the structure-stabilizing moiety, we obtained a split aptamer that bound l-selectin. After optimization of one part of the split sequence to eliminate the nonspecific binding of the split sequence pair, we developed a split-aptamer-based cytometric bead assay (SACBA) for the detection of soluble l-selectin. SACBA showed good sensitivity and selectivity to l-selectin and was successfully applied for the detection of spiked l-selectin in the human serum. The strategies for generating split aptamers and designing the split-aptamer-based sandwich assay are simple and efficient and show good practicability in aptamer engineering.

  13. Multivariate analysis of flow cytometric data using decision trees.

    Science.gov (United States)

    Simon, Svenja; Guthke, Reinhard; Kamradt, Thomas; Frey, Oliver

    2012-01-01

    Characterization of the response of the host immune system is important in understanding the bidirectional interactions between the host and microbial pathogens. For research on the host site, flow cytometry has become one of the major tools in immunology. Advances in technology and reagents allow now the simultaneous assessment of multiple markers on a single cell level generating multidimensional data sets that require multivariate statistical analysis. We explored the explanatory power of the supervised machine learning method called "induction of decision trees" in flow cytometric data. In order to examine whether the production of a certain cytokine is depended on other cytokines, datasets from intracellular staining for six cytokines with complex patterns of co-expression were analyzed by induction of decision trees. After weighting the data according to their class probabilities, we created a total of 13,392 different decision trees for each given cytokine with different parameter settings. For a more realistic estimation of the decision trees' quality, we used stratified fivefold cross validation and chose the "best" tree according to a combination of different quality criteria. While some of the decision trees reflected previously known co-expression patterns, we found that the expression of some cytokines was not only dependent on the co-expression of others per se, but was also dependent on the intensity of expression. Thus, for the first time we successfully used induction of decision trees for the analysis of high dimensional flow cytometric data and demonstrated the feasibility of this method to reveal structural patterns in such data sets.

  14. Oxidative product formation in irradiated neutrophils. A flow cytometric analysis

    International Nuclear Information System (INIS)

    Wolber, R.A.; Duque, R.E.; Robinson, J.P.; Oberman, H.A.

    1987-01-01

    The effect of irradiation on neutrophil oxidative function was evaluated using a flow cytometric assay of intracellular hydrogen peroxide (H 2 O 2 ) production. This assay quantitates the H 2 O 2 -dependent conversion of the nonfluorescent compound, 2'-7'-dichlorofluorescein (DCFH), into fluorescent 2'-7'-dichlorofluorescein (DCF) on a single-cell basis. Intracellular H 2 O 2 production in response to stimulation with phorbol myristate acetate was not affected by neutrophil irradiation at doses up to 2500 rad. In addition, irradiation of intracellular DCFH and aqueous 2'-7'-dichlorofluorescein diacetate (DCFH-DA) resulted in DCF production, which suggested that oxidative molecules produced by aqueous radiolysis were detected by this assay. This study indicates that radiation doses of 1500 to 2500 rad, which are sufficient to prevent induction of graft-versus-host disease by transfused blood components, are not deleterious to neutrophil oxidative metabolism

  15. The cytometric future: it ain't necessarily flow!

    Science.gov (United States)

    Shapiro, Howard M

    2011-01-01

    Initial approaches to cytometry for classifying and characterizing cells were based on microscopy; it was necessary to collect relatively high-resolution images of cells because only a few specific reagents usable for cell identification were available. Although flow cytometry, now the dominant cytometric technology, typically utilizes lenses similar to microscope lenses for light collection, improved, more quantitative reagents allow the necessary information to be acquired in the form of whole-cell measurements of the intensities of light transmission, scattering, and/or fluorescence.Much of the cost and complexity of both automated microscopes and flow cytometers arises from the necessity for them to measure one cell at a time. Recent developments in digital camera technology now offer an alternative in which one or more low-magnification, low-resolution images are made of a wide field containing many cells, using inexpensive light-emitting diodes (LEDs) for illumination. Minimalist widefield imaging cytometers can provide a smaller, less complex, and substantially less expensive alternative to flow cytometry, critical in systems intended for in resource-poor areas. Minimalism is, likewise, a good philosophy in developing instrumentation and methodology for both clinical and large-scale research use; it simplifies quality assurance and compliance with regulatory requirements, as well as reduces capital outlays, material costs, and personnel training requirements. Also, importantly, it yields "greener" technology.

  16. Detection of circulating immune complexes by Raji cell assay: comparison of flow cytometric and radiometric methods

    International Nuclear Information System (INIS)

    Kingsmore, S.F.; Crockard, A.D.; Fay, A.C.; McNeill, T.A.; Roberts, S.D.; Thompson, J.M.

    1988-01-01

    Several flow cytometric methods for the measurement of circulating immune complexes (CIC) have recently become available. We report a Raji cell flow cytometric assay (FCMA) that uses aggregated human globulin (AHG) as primary calibrator. Technical advantages of the Raji cell flow cytometric assay are discussed, and its clinical usefulness is evaluated in a method comparison study with the widely used Raji cell immunoradiometric assay. FCMA is more precise and has greater analytic sensitivity for AHG. Diagnostic sensitivity by the flow cytometric method is superior in systemic lupus erythematosus (SLE), rheumatoid arthritis, and vasculitis patients: however, diagnostic specificity is similar for both assays, but the reference interval of FCMA is narrower. Significant correlations were found between CIC levels obtained with both methods in SLE, rheumatoid arthritis, and vasculitis patients and in longitudinal studies of two patients with cerebral SLE. The Raji cell FCMA is recommended for measurement of CIC levels to clinical laboratories with access to a flow cytometer

  17. Detection of circulating immune complexes by Raji cell assay: comparison of flow cytometric and radiometric methods

    Energy Technology Data Exchange (ETDEWEB)

    Kingsmore, S.F.; Crockard, A.D.; Fay, A.C.; McNeill, T.A.; Roberts, S.D.; Thompson, J.M.

    1988-01-01

    Several flow cytometric methods for the measurement of circulating immune complexes (CIC) have recently become available. We report a Raji cell flow cytometric assay (FCMA) that uses aggregated human globulin (AHG) as primary calibrator. Technical advantages of the Raji cell flow cytometric assay are discussed, and its clinical usefulness is evaluated in a method comparison study with the widely used Raji cell immunoradiometric assay. FCMA is more precise and has greater analytic sensitivity for AHG. Diagnostic sensitivity by the flow cytometric method is superior in systemic lupus erythematosus (SLE), rheumatoid arthritis, and vasculitis patients: however, diagnostic specificity is similar for both assays, but the reference interval of FCMA is narrower. Significant correlations were found between CIC levels obtained with both methods in SLE, rheumatoid arthritis, and vasculitis patients and in longitudinal studies of two patients with cerebral SLE. The Raji cell FCMA is recommended for measurement of CIC levels to clinical laboratories with access to a flow cytometer.

  18. Activity of ethanol-stressed Oenococcus oeni cells: a flow cytometric approach

    NARCIS (Netherlands)

    Silveira, da M.G.; Abee, T.

    2009-01-01

    Aims: To study the effect of ethanol on Oenococcus oeni activity at the single cell level. Methods and Results: The active extrusion of the fluorescent probe carboxy fluorescein (cF) was used to assess the metabolic activity of ethanol-stressed O. oeni cells. Subsequent flow cytometric analysis

  19. Double fluorescent flow cytometric assessment of bacterial internalization and binding by epithelial cells

    NARCIS (Netherlands)

    de Boer, E. C.; Bevers, R. F.; Kurth, K. H.; Schamhart, D. H.

    1996-01-01

    This study describes a new flow cytometric method for assessment of phagocytosis of specific bacteria (bacillus Calmette-Guérin (BCG) and Escherichia coli) by bladder epithelial cells. The internalization assay consisted of labeling bacteria chemically with fluorescein isothiocyanate (FITC).

  20. Cluster Analysis of Flow Cytometric List Mode Data on a Personal Computer

    NARCIS (Netherlands)

    Bakker Schut, Tom C.; Bakker schut, T.C.; de Grooth, B.G.; Greve, Jan

    1993-01-01

    A cluster analysis algorithm, dedicated to analysis of flow cytometric data is described. The algorithm is written in Pascal and implemented on an MS-DOS personal computer. It uses k-means, initialized with a large number of seed points, followed by a modified nearest neighbor technique to reduce

  1. Intrinsic properties of so-called dormant probiotic bacteria, determined by flow cytometric viability assays.

    Science.gov (United States)

    Lahtinen, Sampo J; Ouwehand, Arthur C; Reinikainen, Johanna P; Korpela, Jaakko M; Sandholm, Jouko; Salminen, Seppo J

    2006-07-01

    Plate counting and four culture-independent flow cytometric assays were used to determine the viability and intrinsic properties of three probiotic strains during storage. The strains showed reduction in plate counts but were able to maintain esterase activity, intact cytoplasmic membrane, and pH gradient. The apparently uncultivable probiotic cells were active and stress resistant.

  2. Intrinsic Properties of So-Called Dormant Probiotic Bacteria, Determined by Flow Cytometric Viability Assays

    OpenAIRE

    Lahtinen, Sampo J.; Ouwehand, Arthur C.; Reinikainen, Johanna P.; Korpela, Jaakko M.; Sandholm, Jouko; Salminen, Seppo J.

    2006-01-01

    Plate counting and four culture-independent flow cytometric assays were used to determine the viability and intrinsic properties of three probiotic strains during storage. The strains showed reduction in plate counts but were able to maintain esterase activity, intact cytoplasmic membrane, and pH gradient. The apparently uncultivable probiotic cells were active and stress resistant.

  3. Validation of flow cytometric analysis of platelet function in patients with a suspected platelet function defect.

    Science.gov (United States)

    van Asten, I; Schutgens, R E G; Baaij, M; Zandstra, J; Roest, M; Pasterkamp, G; Huisman, A; Korporaal, S J A; Urbanus, R T

    2018-01-16

    Essentials The diagnosis of mild platelet function disorders (PFDs) is challenging. Validation of flow cytometric testing in patients with suspected PFDs is required. Flow cytometry has added value to light transmission aggregometry (LTA) in diagnosis of PFDs. There is fair agreement in diagnosing PFDs between LTA and flow cytometry. Background Light transmission aggregometry (LTA) is the most commonly used test for the diagnosis of platelet function disorders (PFDs), but has moderate sensitivity for mild PFDs. Flow cytometry has been recommended for additional diagnostics of PFDs but is not yet standardized as a diagnostic test. We developed a standardized protocol for flow cytometric analysis of platelet function that measures fibrinogen binding and P-selectin expression as platelet activation markers in response to agonist stimulation. Objectives To determine the additional value of flow cytometric platelet function testing to standard LTA screening in a cross-sectional cohort of patients with a suspected PFD. Methods Platelet function was assessed with flow cytometry and LTA in 107 patients suspected of a PFD in whom von Willebrand disease and coagulation factor deficiencies were excluded. Both tests were compared in terms of agreement and discriminative ability for diagnosing patients with PFDs. Results Out of 107 patients, 51 patients had an elevated bleeding score; 62.7% of the patients had abnormal platelet function measured with flow cytometry and 54.2% of the patients were abnormal based on LTA. There was fair agreement between LTA and flow cytometry (κ = 0.32). The discriminative ability of flow cytometric analysis in patients with an elevated bleeding score was good (AUC 0.82, 0.74-0.90), but moderate for LTA (AUC 0.70, 0.60-0.80). Both tests combined had a better discriminative ability (AUC 0.87, 0.80-0.94). Conclusion Flow cytometric analysis of platelet function has added value in diagnostics of PFDs in patients with unexplained bleeding tendency.

  4. Flow cytometric immunophenotyping of adult T-cell leukemia/lymphoma using CD3 gating.

    Science.gov (United States)

    Yokote, Taiji; Akioka, Toshikazu; Oka, Satoko; Hara, Satoshi; Kobayashi, Kichinosuke; Nakajima, Hideto; Yamano, Takeshi; Ikemoto, Toshiyuki; Shimizu, Akira; Tsuji, Motomu; Hanafusa, Toshiaki

    2005-08-01

    Adult T-cell leukemia/lymphoma (ATLL) is a lymphoproliferative neoplasm of helper T lymphocytes caused by human T-cell leukemia virus type-1 (HTLV-1). The disease was first described in Kyushu, in southwestern Japan, and most frequently occurs in endemic areas, such as Japan, the Caribbean basin, West Africa, Brazil, and northern Iran. ATLL is essentially a disease of adults, characterized clinically by generalized lymphadenopathy, hepatosplenomegaly, skin lesions, and hypercalcemia. The prognosis of most patients is quite poor, with a median survival time of only 13 months, even if multiagent combination chemotherapy is given. In the present study, flow cytometric immunophenotyping with CD3 gating was performed on 30 samples from 26 patients who had been given a diagnosis of ATLL. The records of these patients also were reviewed retrospectively. In 14 of the 30 samples, an abnormal CD3(low) T-cell population was distinguishable from the normal T-cell populations by flow cytometric analysis. Herein we report a novel strategy for flow cytometric immunophenotyping of ATLL facilitated by CD3(low) gating.

  5. Single-Cell Phosphospecific Flow Cytometric Analysis of Canine and Murine Adipose-Derived Stem Cells

    Directory of Open Access Journals (Sweden)

    Harumichi Itoh

    2017-01-01

    Full Text Available This study aimed to demonstrate single-cell phosphospecific flow cytometric analysis of canine and murine adipose-derived stem/stromal cells (ADSCs. ADSCs were obtained from clinically healthy laboratory beagles and C57BL/6 mice. Cell differentiation into adipocytes, osteocytes, and chondrocytes was observed for the cultured canine ADSCs (cADSCs and murine ADSCs (mADSCs to determine their multipotency. We also performed single-cell phosphospecific flow cytometric analysis related to cell differentiation and stemness. Cultured cADSCs and mADSCs exhibited the potential to differentiate into adipocytes, osteocytes, and chondrocytes. In addition, single-cell phosphospecific flow cytometric analysis revealed similar β-catenin and Akt phosphorylation between mADSCs and cADSCs. On the other hand, it showed the phosphorylation of different Stat proteins. It was determined that cADSCs and mADSCs show the potential to differentiate into adipocytes, osteocytes, and chondrocytes. Furthermore, a difference in protein phosphorylation between undifferentiated cADSCs and mADSCs was identified.

  6. Multiparametric flow cytometric analysis of estrogen receptor: a ...

    African Journals Online (AJOL)

    Precise prognostication of breast cancer based on immunohistochemical features is a challenging assay. Thus, there is a need for more sophisticated prognostic determinants. This work aims to investigate the sensitivity of flow cytometry for the accurate evaluation of steroid receptor positive, tumor cells in formalin-fixed ...

  7. Flow cytometric chromosome sorting in plants: The next generation

    Czech Academy of Sciences Publication Activity Database

    Vrána, Jan; Šimková, Hana; Kubaláková, Marie; Čihalíková, Jarmila; Doležel, Jaroslav

    2012-01-01

    Roč. 57, č. 3 (2012), s. 331-337 ISSN 1046-2023 R&D Projects: GA ČR GAP501/10/1740 Grant - others:GA MŠk(CZ) ED0007/01/01 Program:ED Institutional research plan: CEZ:AV0Z50380511 Keywords : Chromosome sorting * Flow cytometry * Fluorescence in situ hybridization Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.641, year: 2012

  8. DNA flow cytometric analysis in variable types of hydropic placentas

    Directory of Open Access Journals (Sweden)

    Fatemeh Atabaki pasdar

    2015-05-01

    Full Text Available Background: Differential diagnosis between complete hydatidiform mole, partial hydatidiform mole and hydropic abortion, known as hydropic placentas is still a challenge for pathologists but it is very important for patient management. Objective: We analyzed the nuclear DNA content of various types of hydropic placentas by flowcytometry. Materials and Methods: DNA ploidy analysis was performed in 20 non-molar (hydropic and non-hydropic spontaneous abortions and 20 molar (complete and partial moles, formalin-fixed, paraffin-embedded tissue samples by flow cytometry. The criteria for selection were based on the histopathologic diagnosis. Results: Of 10 cases histologically diagnosed as complete hydatiform mole, 9 cases yielded diploid histograms, and 1 case was tetraploid. Of 10 partial hydatidiform moles, 8 were triploid and 2 were diploid. All of 20 cases diagnosed as spontaneous abortions (hydropic and non-hydropic yielded diploid histograms. Conclusion: These findings signify the importance of the combined use of conventional histology and ploidy analysis in the differential diagnosis of complete hydatidiform mole, partial hydatidiform mole and hydropic abortion.

  9. Ultrastructural and flow cytometric analyses of lipid accumulation in microalgae

    Energy Technology Data Exchange (ETDEWEB)

    Solomon, J.A.; Hand, R.E. Jr.; Mann, R.C.

    1986-12-01

    Lipid accumulation in three species of microalgae was investigated with flow cytometry (FCM) and transmission electron microscopy (TEM). Previous studies using batch cultures of a algae have led to the assumption that lipid accumulation in microalgae is a gradual process requiring at least several days for completion. However, FCM reveals, through changes in the chlorophyll:lipid ratio, that the time span required for individual cells to change metabolic state is short. Simultaneous FCM measurements of chlorophyll and nile red (neutral lipid) fluorescence in individual cells of nitrogen-deficient Isochrysis populations revealed a bimodal population distribution as one stage in the lipid accumulation process. The fact that two discrete populations exist, with few cells in an intermediate stage, suggests rapid response to a liqid trigger. Interpretations of light and electron microscopic observations are consistent with this hypothesis. The time required for an entire population to achieve maximum lipid content is considerably longer than that required for a single cell, due to the variation in response time among cells. In this study high lipid cultures were sometimes obtained by using FCM to separate high lipid cells from the remainder of the population. FCM holds much promise for strain enhancement but considerable developmental work, directed at providing more consistent results, remains to be done. 8 refs., 35 figs.

  10. Flow cytometric analysis of microbial contamination in food industry technological lines--initial study.

    Science.gov (United States)

    Józwa, Wojciech; Czaczyk, Katarzyna

    2012-04-02

    Flow cytometry constitutes an alternative for traditional methods of microorganisms identification and analysis, including methods requiring cultivation step. It enables the detection of pathogens and other microorganisms contaminants without the need to culture microbial cells meaning that the sample (water, waste or food e.g. milk, wine, beer) may be analysed directly. This leads to a significant reduction of time required for analysis allowing monitoring of production processes and immediate reaction in case of contamination or any disruption occurs. Apart from the analysis of raw materials or products on different stages of manufacturing process, the flow cytometry seems to constitute an ideal tool for the assessment of microbial contamination on the surface of technological lines. In the present work samples comprising smears from 3 different surfaces of technological lines from fruit and vegetable processing company from Greater Poland were analysed directly with flow cytometer. The measured parameters were forward and side scatter of laser light signals allowing the estimation of microbial cell contents in each sample. Flow cytometric analysis of the surface of food industry production lines enable the preliminary evaluation of microbial contamination within few minutes from the moment of sample arrival without the need of sample pretreatment. The presented method of fl ow cytometric initial evaluation of microbial state of food industry technological lines demonstrated its potential for developing a robust, routine method for the rapid and labor-saving detection of microbial contamination in food industry.

  11. Flow cytometric of reticulocytes quantification: radio-induction medullary aplasia application

    International Nuclear Information System (INIS)

    Dubner, D.; Perez, M.; Gisone, P.

    1996-01-01

    Flow cytometric reticulocyte quantification was assayed in ten patients undergoing bone marrow transplantation (BMT) with previous conditioning by chemotherapy and total body irradiation. A reticulocyte maturity index (RMI) was determined taking into account the RNA content. With the aim of testing the utility of RMI as an early predictor of functional recovery in marrow aplasia, other hematological indicators as neutrophils count were comparatively evaluated. Mean time elapsed between BMT and engraftment evidence by RMI was 17,6 days. In six patients the RMI was the earliest indicator of functional recovery. The applicability of this assay in the pursuit of radioinduced bone marrow aplasia is discussed. (authors). 4 refs., 4 figs., 2 tabs

  12. Karyological and flow cytometric evidence of triploid specimens in Bufo viridis (Amphibia Anura

    Directory of Open Access Journals (Sweden)

    D Cavallo

    2010-01-01

    Full Text Available Karyological and flow cytometric (FCM analyses were performed on a group of 14 green toads of the Bufo viridis species from seven Eurasian populations. Both approaches gave concordant results concerning the DNA ploidy level. All the populations examined were represented exclusively by diploid or tetraploid specimens, except one, where triploids were found. Results evidenced an interpopulation variability in DNA content against the same ploidy level, as well as an unusually high number of triploids in a particular reproductive place. The origin of polyploidy and the presence and persistence of a high number of triploids in a particular population are discussed.

  13. Discrimination of bromodeoxyuridine labelled and unlabelled mitotic cells in flow cytometric bromodeoxyuridine/DNA analysis

    DEFF Research Database (Denmark)

    Jensen, P O; Larsen, J K; Christensen, I J

    1994-01-01

    Bromodeoxyuridine (BrdUrd) labelled and unlabelled mitotic cells, respectively, can be discriminated from interphase cells using a new method, based on immunocytochemical staining of BrdUrd and flow cytometric four-parameter analysis of DNA content, BrdUrd incorporation, and forward and orthogonal...... light scatter. The method was optimized using the human leukemia cell lines HL-60 and K-562. Samples of 10(5) ethanol-fixed cells were treated with pepsin/HCl and stained as a nuclear suspension with anti-BrdUrd antibody, FITC-conjugated secondary antibody, and propidium iodide. Labelled mitoses could...

  14. A flow cytometric assay technology based on quantum dots-encoded beads

    International Nuclear Information System (INIS)

    Wang Haiqiao; Liu Tiancai; Cao Yuancheng; Huang Zhenli; Wang Jianhao; Li Xiuqing; Zhao Yuandi

    2006-01-01

    A flow cytometric detecting technology based on quantum dots (QDs)-encoded beads has been described. Using this technology, several QDs-encoded beads with different code were identified effectively, and the target molecule (DNA sequence) in solution was also detected accurately by coupling to its complementary sequence probed on QDs-encoded beads through DNA hybridization assay. The resolution of this technology for encoded beads is resulted from two longer wavelength fluorescence identification signals (yellow and red fluorescent signals of QDs), and the third shorter wavelength fluorescence signal (green reporting signal of fluorescein isothiocyanate (FITC)) for the determination of reaction between probe and target. In experiment, because of QDs' unique optical character, only one excitation light source was needed to excite the QDs and probe dye FITC synchronously comparing with other flow cytometric assay technology. The results show that this technology has present excellent repeatability and good accuracy. It will become a promising multiple assay platform in various application fields after further improvement

  15. Detection of nickel and palladium contact hypersensitivity by a flow cytometric lymphocyte proliferation test.

    Science.gov (United States)

    Spoerri, I; Scherer, K; Michel, S; Link, S; Bircher, A J; Heijnen, I A F M

    2015-03-01

    We established a flow cytometric lymphocyte proliferation test (LPT) for the detection of nickel (Ni) and palladium (Pd) sensitization. Eighty-one consecutive patients with an indication for patch test (PT) were tested by LPT with Ni (NiSO4 ) and Pd (Na2 PdCl4 and PdCl2 ) salts. The imprecision of the LPT was low (coefficient of variation 7.2%). Using PT as a diagnostic reference, the sensitivity and specificity of LPT were 74.4% and 80% for NiSO4 , 74.4% and 78.3% for Na2 PdCl4 , and 57.2% and 85.4% for PdCl2 , respectively. For both Ni and Pd, the likelihood ratio for a positive PT markedly increased with increasing LPT value. With medical history as a reference, the sensitivity and specificity were 40.6% and 82.1% for LPT and 59.4% and 89.7% for PT, respectively. Combination of LPT and PT resulted in a higher specificity of 95%, albeit lower sensitivity of 34.4%. In conclusion, flow cytometric LPT represents a reliable and useful method for the detection of Ni and Pd sensitization. LPT values correlate with PT results and, when used in combination with PT, increase test specificity. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  16. A flow cytometric protocol for titering recombinant adenoviral vectors containing the green fluorescent protein.

    Science.gov (United States)

    Hitt, D C; Booth, J L; Dandapani, V; Pennington, L R; Gimble, J M; Metcalf, J

    2000-03-01

    As the use of adenoviral vectors in gene therapy protocols increases, there is a corresponding need for rapid, accurate, and reproducible titer methods. Multiple methods currently exist for determining titers of recombinant adenoviral vector, including optical absorbance, electron microscopy, fluorescent focus assay, and the "gold standard" plaque assay. This paper introduces a novel flow cytometric method for direct titer determination that relies on the expression of the green fluorescent protein (GFP), a tracking marker incorporated into several adenoviral vectors. This approach was compared to the plaque assay using 10(-4)- to 10(-6)-fold dilutions of a cesium-chloride-purified, GFP expressing adenovirus (AdEasy + GFP + GAL). The two approaches yielded similar titers: 3.25 +/- 1.85 x 10(9) PFU/mL versus 3.46 +/- 0.76 x 10(9) green fluorescent units/(gfu/mL). The flow cytometric method is complete within 24 h in contrast to the 7 x 10 days required by the plaque assay. These results indicate that the GFU/mL is an alternative functional titer method for fluorescent-tagged adenoviral vectors.

  17. Multiplex competitive microbead-based flow cytometric immunoassay using quantum dot fluorescent labels

    International Nuclear Information System (INIS)

    Yu, Hye-Weon; Kim, In S.; Niessner, Reinhard; Knopp, Dietmar

    2012-01-01

    Highlights: ► First time, duplex competitive bead-based flow cytometric immunoassay was developed using ODs. ► Antibody-coated QD detection probes and antigen-immobilized microspheres were synthesized. ► The two model target analytes were low molecular weight compounds of microbial and chemical origin. ► The determination of different water types was possible after simple filtration of samples. - Abstract: In answer to the ever-increasing need to perform the simultaneous analysis of environmental hazards, microcarrier-based multiplex technologies show great promise. Further integration with biofunctionalized quantum dots (QDs) creates new opportunities to extend the capabilities of multicolor flow cytometry with their unique fluorescence properties. Here, we have developed a competitive microbead-based flow cytometric immunoassay using QDs fluorescent labels for simultaneous detection of two analytes, bringing the benefits of sensitive, rapid and easy-of-manipulation analytical tool for environmental contaminants. As model target compounds, the cyanobacterial toxin microcystin-LR and the polycyclic aromatic hydrocarbon compound benzo[a]pyrene were selected. The assay was carried out in two steps: the competitive immunological reaction of multiple targets using their exclusive sensing elements of QD/antibody detection probes and antigen-coated microsphere, and the subsequent flow cytometric analysis. The fluorescence of the QD-encoded microsphere was thus found to be inversely proportional to target analyte concentration. Under optimized conditions, the proposed assay performed well within 30 min for the identification and quantitative analysis of the two environmental contaminants. For microcystin-LR and benzo[a]pyrene, dose–response curves with IC 50 values of 5 μg L −1 and 1.1 μg L −1 and dynamic ranges of 0.52–30 μg L −1 and 0.13–10 μg L −1 were obtained, respectively. Recovery was 92.6–106.5% for 5 types of water samples like bottled

  18. Flow Cytometric Analysis of T, B, and NK Cells Antigens in Patients with Mycosis Fungoides

    Directory of Open Access Journals (Sweden)

    Serkan Yazıcı

    2015-01-01

    Full Text Available We retrospectively analyzed the clinicopathological correlation and prognostic value of cell surface antigens expressed by peripheral blood mononuclear cells in patients with mycosis fungoides (MF. 121 consecutive MF patients were included in this study. All patients had peripheral blood flow cytometry as part of their first visit. TNMB and histopathological staging of the cases were retrospectively performed in accordance with International Society for Cutaneous Lymphomas/European Organization of Research and Treatment of Cancer (ISCL/EORTC criteria at the time of flow cytometry sampling. To determine prognostic value of cell surface antigens, cases were divided into two groups as stable and progressive disease. 17 flow cytometric analyses of 17 parapsoriasis (PP and 11 analyses of 11 benign erythrodermic patients were included as control groups. Fluorescent labeled monoclonal antibodies were used to detect cell surface antigens: T cells (CD3+, CD4+, CD8+, TCRαβ+, TCRγδ+, CD7+, CD4+CD7+, CD4+CD7−, and CD71+, B cells (HLA-DR+, CD19+, and HLA-DR+CD19+, NKT cells (CD3+CD16+CD56+, and NK cells (CD3−CD16+CD56+. The mean value of all cell surface antigens was not statistically significant between parapsoriasis and MF groups. Along with an increase in cases of MF stage statistically significant difference was found between the mean values of cell surface antigens. Flow cytometric analysis of peripheral blood cell surface antigens in patients with mycosis fungoides may contribute to predicting disease stage and progression.

  19. Bivariate flow cytometric analysis and sorting of different types of maize starch grains.

    Science.gov (United States)

    Zhang, Xudong; Feng, Jiaojiao; Wang, Heng; Zhu, Jianchu; Zhong, Yuyue; Liu, Linsan; Xu, Shutu; Zhang, Renhe; Zhang, Xinghua; Xue, Jiquan; Guo, Dongwei

    2018-02-01

    Particle-size distribution, granular structure, and composition significantly affect the physicochemical properties, rheological properties, and nutritional function of starch. Flow cytometry and flow sorting are widely considered convenient and efficient ways of classifying and separating natural biological particles or other substances into subpopulations, respectively, based on the differential response of each component to stimulation by a light beam; the results allow for the correlation analysis of parameters. In this study, different types of starches isolated from waxy maize, sweet maize, high-amylose maize, pop maize, and normal maize were initially classified into various subgroups by flow cytometer and then collected through flow sorting to observe their morphology and particle-size distribution. The results showed that a 0.25% Gelzan solution served as an optimal reagent for keeping individual starch particles homogeneously dispersed in suspension for a relatively long time. The bivariate flow cytometric population distributions indicated that the starches of normal maize, sweet maize, and pop maize were divided into two subgroups, whereas high-amylose maize starch had only one subgroup. Waxy maize starch, conversely, showed three subpopulations. The subgroups sorted by flow cytometer were determined and verified in terms of morphology and granule size by scanning electron microscopy and laser particle distribution analyzer. Results showed that flow cytometry can be regarded as a novel method for classifying and sorting starch granules. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

  20. Improved flow cytometric assessment reveals distinct microvesicle (cell-derived microparticle signatures in joint diseases.

    Directory of Open Access Journals (Sweden)

    Bence György

    Full Text Available INTRODUCTION: Microvesicles (MVs, earlier referred to as microparticles, represent a major type of extracellular vesicles currently considered as novel biomarkers in various clinical settings such as autoimmune disorders. However, the analysis of MVs in body fluids has not been fully standardized yet, and there are numerous pitfalls that hinder the correct assessment of these structures. METHODS: In this study, we analyzed synovial fluid (SF samples of patients with osteoarthritis (OA, rheumatoid arthritis (RA and juvenile idiopathic arthritis (JIA. To assess factors that may confound MV detection in joint diseases, we used electron microscopy (EM, Nanoparticle Tracking Analysis (NTA and mass spectrometry (MS. For flow cytometry, a method commonly used for phenotyping and enumeration of MVs, we combined recent advances in the field, and used a novel approach of differential detergent lysis for the exclusion of MV-mimicking non-vesicular signals. RESULTS: EM and NTA showed that substantial amounts of particles other than MVs were present in SF samples. Beyond known MV-associated proteins, MS analysis also revealed abundant plasma- and immune complex-related proteins in MV preparations. Applying improved flow cytometric analysis, we demonstrate for the first time that CD3(+ and CD8(+ T-cell derived SF MVs are highly elevated in patients with RA compared to OA patients (p=0.027 and p=0.009, respectively, after Bonferroni corrections. In JIA, we identified reduced numbers of B cell-derived MVs (p=0.009, after Bonferroni correction. CONCLUSIONS: Our results suggest that improved flow cytometric assessment of MVs facilitates the detection of previously unrecognized disease-associated vesicular signatures.

  1. Cell cycle flow cytometric analysis in the diagnosis and management of colorectal carcinoma.

    Science.gov (United States)

    Sampedro, A; Salas-Bustamante, A; López-Artimez, M; García-Muñíz, J L; Urdiales, G

    1999-08-01

    To establish prognostic models and protocols for individualized management in colorectal carcinoma patients based on both clinical and DNA flow cytometric parameters. Prospective study of 88 colon carcinoma patients with a minimum follow-up of 12 months, operated on with the intent to cure and not treated with radiotherapy or chemotherapy. All the cases were subjected to a clinical evaluation: age, sex, tumor localization and size, histologic grade, tumor stage, disease-free interval, survival and flow cytometric study (ploidy, DNA index and S-phase fraction [SPF]). From the total of 88 neoplasms studied, 56 (63.6%) were from males and 32 (36.4%) from females; 30 (34%) were located in the right side of the colon, 7 (8%) in the transverse colon and 51 (58%) in the left side of the colon. Eleven (12.5%) were stage I, 52 (59.1%) stage II and 25 (28%) stage III. Forty-two (47.7%) were diploid and 46 (52.3%) aneuploid. The S-phase mean was 14.6% (12% for diploids and 16.9% for aneuploids). During the follow-up period, 26.1% of diploid tumors recurred, whereas aneuploid tumors recurred in 36.9% (P < .05). SPF from diploid and aneuploid tumors was analyzed separately. Regarding relapse-free interval, the behavior of diploid tumors with a high SPF was similar to that of aneuploid ones. Two kinetic profiles were established, favorable (diploid tumors with low S phase) and unfavorable (diploid with high S phase and all aneuploid tumors), that had significant prognostic value for progression and survival and that allowed identification of patients at high risk of recurrence. We formulated a prognostic index according to SPF and tumor stage that has discriminatory capacity for biologic behavior in colorectal tumors.

  2. A new one-platform flow cytometric method for residual cell counting in platelet concentrates.

    Science.gov (United States)

    Schmidt, Michael; Spengler, Hans-Peter; Lambrecht, Bernd; Hourfar, Michael K; Seifried, Erhard; Tonn, Torsten

    2009-12-01

    According to German regulations and guidelines, residual red blood cells (rRBCs) and residual white blood cells (rWBCs) must number fewer than 3 x 10(9) cells/unit and 1 x 10(6) cells/unit in platelet concentrates (PCs), respectively. Due to low levels of residual cells in final products, there is still a need for fast, reliable, and sensitive methods of automated detection of these cell types. In Part A, 21 PCs were spiked with predetermined numbers of red blood cells (RBCs) and white blood cells (WBCs). The linearity, precision, and accuracy of the BD Thrombo Count assay (BD Biosciences Europe) were tested and validated according to international guidelines. Finally in Part B, 100 PCs prepared from pooled buffy coats were tested by the BD Thrombo Count assay and compared with other methods, including Nageotte (rWBCs) and Neubauer (rRBCs) counting chambers and the flow cytometric BD LeucoCOUNT (Becton Dickinson) assay (rWBCs). The unspecific background of blank PC samples was fewer than 0.02 cells/microL for WBCs and fewer than 34 cells/microL for RBCs (mean, 21). Linear regression and precision analyses of spiked PC samples were determined for both WBCs (r(2) = 0.992; range, 0.6-6.0 WBCs/microL) and RBCs (r(2) = 0.999; 800-8000 RBCs/microL). No carryover of cells or drift in results was detected in the automated sample acquisition mode. Analysis according to statistical methods of Bland and Altman demonstrated a high correlation between BD Thrombo Count and the Neubauer manual counting chamber. This novel flow cytometric test is a quick and reliable single-tube assay that has been demonstrated as a potential alternative for the existing manual microscopic counting procedures that are both time-consuming and laborious.

  3. Flow cytometric DNA analysis of ducks accumulating 137Cs on a reactor reservoir

    International Nuclear Information System (INIS)

    George, L.S.; Dallas, C.E.; Brisbin, I.L. Jr.; Evans, D.L.

    1991-01-01

    The objective of this study was to detect red blood cell (rbc) DNA abnormalities in male, game-farm mallard ducks as they ranged freely and accumulated 137Cs (radiocesium) from an abandoned nuclear reactor cooling reservoir. Prior to release, the ducks were tamed to enable recapture at will. Flow cytometric measurements conducted at intervals during the first year of exposure yielded cell cycle percentages of DNA (G0/G1, S, G2 + M phases) of rbc, as well as coefficients of variation (CV) in the G0/G1 phase. DNA histograms of exposed ducks were compared with two sets of controls which were maintained 30 and 150 miles from the study site. 137Cs live wholebody burdens were also measured in these animals in a parallel kinetics study, and an approximate steady-state equilibrium was attained after about 8 months. DNA histograms from 2 of the 14 contaminated ducks revealed DNA aneuploid-like patterns after 9 months exposure. These two ducks were removed from the experiment at this time, and when sampled again 1 month later, one continued to exhibit DNA aneuploidy. None of the control DNA histograms demonstrated DNA aneuploid-like patterns. There were no significant differences in cell cycle percentages at any time point between control and exposed animals. A significant increase in CV was observed at 9 months exposure, but after removal of the two ducks with DNA aneuploidy, no significant difference was detected in the group monitored after 12 months exposure. An increased variation in the DNA and DNA aneuploidy could, therefore, be detected in duck rbc using flow cytometric analysis, with the onset of these effects being related to the attainment of maximal levels of 137Cs body burdens in the exposed animals

  4. Biotinylation of interleukin-2 (IL-2) for flow cytometric analysis of IL-2 receptor expression. Comparison of different methods

    NARCIS (Netherlands)

    M.O. de Jong (Marg); H. Rozemuller (Henk); J.G.J. Bauman (J. G J); J.W.M. Visser (Jan)

    1995-01-01

    textabstractThe main prerequisites for the use of biotinylated ligands to study the expression of growth factor receptors on heterogeneous cell populations, such as peripheral blood or bone marrow, by flow cytometric methods, are that the biotinylated ligand retains its binding ability and that

  5. Clonal evolution demonstrated by flow cytometric DNA analysis of a human colonic carcinoma grown in nude mice

    DEFF Research Database (Denmark)

    Vindeløv, L L; Spang-Thomsen, M; Visfeldt, J

    1982-01-01

    A spontaneous change in DNA content of a human colonic carcinoma grown in nude mice was observed fortuitously. The tumor initially had a G1 cell DNA content of 1.3 times that of normal cells. Flow cytometric DNA analysis showed in transplant generation 56 the appearance of a new subpopulation which...

  6. Role of flow cytometry to define unacceptable HLA antigens in lung transplant recipients with HLA-specific antibodies.

    Science.gov (United States)

    Appel, James Z; Hartwig, Matthew G; Cantu, Edward; Palmer, Scott M; Reinsmoen, Nancy L; Davis, R Duane

    2006-04-15

    Antidonor HLA-specific antibodies have been associated with hyperacute rejection and primary graft failure in lung transplant recipients. Thus, transplant candidates with HLA-specific antibodies generally undergo prospective crossmatching to exclude donors with unacceptable HLA antigens. However, the need to perform a prospective crossmatch limits the donor pool and is associated with increased waiting list times and mortality. A virtual crossmatch strategy using flow cytometry, which enables precise determination of HLA-specific antibody specificity, was compared to prospective crossmatching in sensitized lung transplant candidates. In all, 341 lung transplant recipients were analyzed retrospectively (April 1992 to July 2003). Sixteen patients with HLA-specific antibodies underwent transplantation based on flow cytometric determination of antibody specificity and 10 underwent prospective crossmatching. Freedom from bronchiolitis obliterans syndrome (BOS) at three years was similar in those undergoing a virtual crossmatch, those undergoing prospective crossmatching, and those without HLA-specific antibodies (80.4% +/- 13.4, 85.7% +/- 13.2, and 73.8% +/- 2.8, respectively, P = 0.88). Three-year survival was also comparable (87.5% +/- 8.3, 70.0% +/- 14.5, and 78.5% +/- 2.4, respectively, P = 0.31). Elimination of prospective crossmatching for sensitized patients was associated with a significant decrease in time on the waiting list (P < 0.01) and in waiting list mortality (P < 0.05). All 16 patients undergoing a virtual crossmatch had negative retrospective crossmatches. By carefully determining the specificity of HLA-specific antibodies, flow cytometry methodologies enable the prediction of negative crossmatch results with up to 100% accuracy, enabling the determination of appropriateness of donors. Using this virtual crossmatch strategy, crossmatching can be safely omitted prior to lung transplantation, thereby decreasing waiting list time and mortality rates for

  7. A novel flow cytometric assay for measurement of In Vivo pulmonary neutrophil phagocytosis

    Directory of Open Access Journals (Sweden)

    Gentry-Nielsen Martha J

    2006-07-01

    Full Text Available Abstract Background Phagocytosis assays are traditionally performed in vitro using polymorphonuclear leukocytes (PMNs isolated from peripheral blood or the peritoneum and heat-killed, pre-opsonized organisms. These assays may not adequately mimic the environment within the infected lung. Our laboratory therefore has developed a flow cytometric in vivo phagocytosis assay that enables quantification of PMN phagocytosis of viable bacteria within the lungs of rats. In these studies, rats are injected transtracheally with lipopolysaccharide (LPS to recruit PMNs to their lungs. They are then infected with live 5(-and 6 carboxyfluorescein diacetate succinimidyl ester (CFDA/SE labeled type 3 Streptococcus pneumoniae. Bronchoalveolar lavage is performed and resident alveolar macrophages and recruited PMNs are labeled with monoclonal antibodies specific for surface epitopes on each cell type. Three color flow cytometry is utilized to identify the cell types, quantify recruitment, and determine uptake of the labeled bacteria. Results The viability of the alveolar macrophages and PMNs isolated from the lavage fluid was >95%. The values of the percentage of PMNs in the lavage fluid as well as the percentage of PMNs associated with CFSE-labeled S. pneumoniae as measured through flow cytometry showed a high degree of correlation with the results from manual counting of cytospin slides. Conclusion This assay is suitable for measuring bacterial uptake within the infected lung. It can be adapted for use with other organisms and/or animal model systems.

  8. Flow cytometric analysis of the granulocyte respiratory burst: a comparison study of fluorescent probes.

    Science.gov (United States)

    Vowells, S J; Sekhsaria, S; Malech, H L; Shalit, M; Fleisher, T A

    1995-01-13

    Chronic granulomatous disease (CGD) is a rare recessive disorder caused by defects in the NADPH oxidase enzyme complex of phagocytes (neutrophils, eosinophils and monocytes). CGD phagocytes fail to produce superoxide and other reactive oxygen species following cell activation (Malech, 1993). The products of oxidase activation can be measured in individual cells by flow cytometry using specific fluorescent probes that increase fluorescence upon oxidation (Trinkle et al., 1987). This approach can be used to confirm a diagnosis of CGD, and to detect the normal/abnormal phagocyte mixture that characterizes the X-linked CGD carrier state. Three fluorescent probes have been described as useful for this purpose: 2'7'-dichlorofluorescin diacetate (DCF) (Bass et al., 1983), 5,6-carboxy-2'7'-dichlorofluorescein diacetate, bis(acetoxymethyl) ester (C-DCF) (Hockenbery et al., 1993) and dihydrorhodamine 123 (DHR) (Rothe et al., 1988; Kinsey et al., 1987). A direct comparison between these three probes has not been reported. In this study we performed a direct comparison between these three probes, evaluating their ability in flow cytometric analysis to maximize fluorescent separation between activated CGD patient and normal granulocytes. Using a whole blood technique with phorbol myristate acetate (PMA) as an activator, it was found that DHR loaded normal granulocytes had a fluorescence intensity which, upon activation, was 48-fold higher than that of C-DCF loaded granulocytes and seven-fold higher than DCF loaded granulocytes (P < 0.001). Use of sodium azide to decrease the catabolism of H2O2 enhanced the fluorescence of DCF by 140%, C-DCF by 45% and DHR by 25%, suggesting that DCF is primarily sensitive to H2O2. DCF and DHR were then evaluated for sensitivity in the detection of small percentages of normal cells in a CGD/normal granulocyte mixture. Normal sub-populations as small as 0.1% could clearly be distinguished using DHR, while DCF was insensitive at this level. Based

  9. Novel calibration method for flow cytometric fluorescence resonance energy transfer measurements between visible fluorescent proteins.

    Science.gov (United States)

    Nagy, Peter; Bene, László; Hyun, William C; Vereb, György; Braun, Manuela; Antz, Christof; Paysan, Jacques; Damjanovich, Sándor; Park, John W; Szöllősi, János

    2005-10-01

    The combination of fluorescence resonance energy transfer (FRET) and flow cytometry offers a statistically firm approach to study protein associations. Fusing green fluorescent protein (GFP) to a studied protein usually does not disturb the normal function of a protein, but quantitation of FRET efficiency calculated between GFP derivatives poses a problem in flow cytometry. We generated chimeras in which cyan fluorescent protein (CFP) was separated by amino acid linkers of different sizes from yellow fluorescent protein (YFP) and used them to calibrate the cell-by-cell flow cytometric FRET measurements carried out on two different dual-laser flow cytometers. Then, CFP-Kip1 was coexpressed in yeast cells with YFP and cyclin-dependent kinase-2 (Cdk2) and served as a positive control for FRET measurements, and CFP-Kip1 coexpressed with a random peptide fused to YFP was the negative control. We measured donor, direct, and sensitized acceptor fluorescence intensities and developed a novel way to calculate a factor (alpha) that characterized the fluorescence intensity of acceptor molecules relative to the same number of excited donor molecules, which is essential for quantifying FRET efficiency. This was achieved by calculating FRET efficiency in two different ways and minimizing the squared difference between the two results by changing alpha. Our method reliably detected the association of Cdk2 with its inhibitor, Kip1, whereas the nonspecific FRET efficiency between Cdk2 and a random peptide was negligible. We identified and sorted subpopulations of yeast cells showing interaction between the studied proteins. We have described a straightforward novel calibration method to accurately quantitate FRET efficiency between GFP derivatives in flow cytometry.

  10. A flow cytometric method for characterization of circulating cell-derived microparticles in plasma

    Directory of Open Access Journals (Sweden)

    Morten Hjuler Nielsen

    2014-02-01

    Full Text Available Background and aim: Previous studies on circulating microparticles (MPs indicate that the majority of MPs are of a size below the detection limit of most standard flow cytometers. The objective of the present study was to establish a method to analyze MP subpopulations above the threshold of detection of a new generation BD FACSAria™ III digital flow cytometer. Methods: We analyzed MP subpopulations in plasma from 24 healthy individuals (9 males and 15 females. MPs were identified according to their size (<1.0-µm, by Lactadherin-FITC labelling, and by exposure of cell-specific markers. The sensitivity of the flow cytometer was tested against that of a previous-generation instrument FC500. Reproducibility of the FACSAria and our set-up was investigated, and the percentage of phosphatidylserine (PS exposing MPs binding Lactadherin was determined. Results: By using a flow cytometric approach we identified and quantitated MPs derived from platelets, monocytes, erythrocytes and endothelial cells. In addition, levels of tissue factor-positive MPs were determined. The FACSAria demonstrated improved sensitivity and increased MP detection range compared to the FC500 instrument. The reproducibility of PS+PMP and PS+MP measurements was 11.7 and 23.2%, respectively. When expressed as a percentage of total MPs, the PS-positive MP population represented 15.1±5.5%, and PS-positive MPs were significantly increased in men. Conclusion: We have established a method to measure MPs above the detection limit of a new generation flow cytometer and derived from a number of cell-types in a healthy population of men and women.

  11. Flow cytometric method for measuring chromatin fragmentation in fixed sperm from yellow perch (Perca flavescens)

    Science.gov (United States)

    Jenkins, Jill A.; Draugelis-Dale, Rassa O.; Pinkney, Alfred E.; Iwanowicz, Luke R.; Blazer, Vicki

    2015-01-01

    Declining harvests of yellow perch, Perca flavescens, in urbanized watersheds of Chesapeake Bay have prompted investigations of their reproductive fitness. The purpose of this study was to establish a flow cytometric technique for DNA analysis of fixed samples sent from the field to provide reliable gamete quality measurements. Similar to the sperm chromatin structure assay, measures were made on the susceptibility of nuclear DNA to acid-induced denaturation, but used fixed rather than live or thawed cells. Nuclei were best exposed to the acid treatment for 1 minute at 37 °C followed by the addition of cold (4 °C) propidium iodide staining solution before flow cytometry. The rationale for protocol development is presented graphically through cytograms. Field results collected in 2008 and 2009 revealed DNA fragmentation up to 14.5%. In 2008, DNA fragmentation from the more urbanized watersheds was significantly greater than from reference sites (P = 0.026) and in 2009, higher percentages of haploid testicular cells were noted from the less urbanized watersheds (P = 0.032) indicating better reproductive condition at sites with less urbanization. For both years, total and progressive live sperm motilities by computer-assisted sperm motion analysis ranged from 19.1% to 76.5%, being significantly higher at the less urbanized sites (P chromatin compaction levels from samples translocated over distance and time. The protocol provides an approach that can be modified for other species across taxa.

  12. Flow cytometric analysis using SYBR Green I for genome size estimation in coffee.

    Science.gov (United States)

    Ronildo Clarindo, Wellington; Roberto Carvalho, Carlos

    2011-02-01

    Plant genome size has been measured by flow cytometry using propidium iodide as a dye for nuclear DNA staining. However, some authors have reported the occurrence of genome size estimation errors, especially in plants rich in secondary metabolites, such as the coffee tree. In this context, we tested an alternative cytometric protocol using the SYBR Green I as a fluorochrome for stoichiometrically staining nuclear double-stranded DNA in Coffea canephora (2x) and Coffea arabica (4x). The results showed that the respective mean genome size measured from nuclei stained with SYBR Green I and propidium iodide was statistically identical. However, the G(0)/G(1) peaks of nuclei stained with SYBR Green I exhibited lower coefficient variations (1.57-2.85%) compared to those stained with propidium iodide (2.75-4.80%). Coefficient variation statistical data suggest that SYBR Green I is adequate for stoichiometric nuclei staining using this methodology. Our results provide evidence that SYBR Green I can be used in flow cytometry measurements of plants, with the advantages of minimizing errors in nuclear DNA content quantification, staining relatively quicker, with high affinity, and being less mutagenic than propidium iodide. Copyright © 2009 Elsevier GmbH. All rights reserved.

  13. Dissociation of mono- and co-culture spheroids into single cells for subsequent flow cytometric analysis.

    Science.gov (United States)

    Grässer, Ute; Bubel, Monika; Sossong, Daniela; Oberringer, Martin; Pohlemann, Tim; Metzger, Wolfgang

    2018-03-01

    Spheroids are considered to reflect the natural organization of cells better than 2D cell cultures, but their analysis by flow cytometry requires dissociation into single cells. We established protocols for dissociation of mono- and co-culture spheroids consisting of human fibroblasts and human endothelial cells. Cell recovery rate and viability after dissociation were evaluated with hemocytometer and by flow cytometry. The diameter of cells and the amount of cell aggregates were quantified by Casy ® -technology and the cellular composition was analyzed by flow cytometry. Optimal dissociation conditions with low cell aggregation were determined by size, cultivation time and cellular composition of the spheroids. Smaller spheroids (10,000 cells) could be dissociated with Accutase ® , whereas larger spheroids (50,000 cells) required more stringent dissociation conditions. The size of the cells decreased with increasing cultivation time. Cell recovery rate was dependent upon cellular composition and spheroid size. The highest cell recovery rate was found for co-culture spheroids. The highest cell viability was detected for dissociated fibroblast spheroids. A quantitative analysis of the cellular composition of dissociated co-culture spheroids was possible. Spheroids can be successfully dissociated into singular cells for subsequent flow cytometric analysis. Dissociation conditions as well as cell recovery rate and cell viability depend on size, cultivation time and cellular composition of the spheroids. The observed decrease in cell size in spheroids over time might be responsible for the well-known time-dependent decrease in spheroid size. Copyright © 2017 Elsevier GmbH. All rights reserved.

  14. Early changes in flow cytometric DNA profiles induced by californium-252 neutron brachytherapy in squamocellular carcinomas of the uterine cervix.

    Science.gov (United States)

    Tacev, T; Zaloudík, J; Janáková, L; Vagunda, V

    1998-01-01

    Ninety-five squamocellular carcinomas of the uterine cervix, clinical Stages II and III, were treated by either four schedules combining 252-californium neutron-gamma-radiotherapy with different proportions of a neutron component (9, 6 and 3 Gy) or gamma-irradiation alone. Flow cytometric DNA profiles were obtainable in 72 cases before treatment and 56 cases were monitored for DNA content by flow cytometry (FCM) in weekly intervals by analysis of sequential microbiopsies for one month during and after radiotherapy. DNA aneuploidy was reduced from 40% (25/63) to 19% (9/47) one week within therapy in neutron-treated groups, but not after initial gamma-radiotherapy alone. Extinction of DNA aneuploid subpopulations occurred after neutron therapy in all remaining aneuploid tumors (9/9) during further monitoring, but only in 40% (2/5) of tumors after sole gamma-irradiation. In contrast, proliferation index by more than 50% was more often achieved in groups with a higher gamma-radiation component than after neutrons only. When all therapy-induced DNA flow cytometric events are taken together for evaluation of the effects of various radiotherapy schedules, it appears that the regimen with the maximal neutron dose may not be optimal for all tumors. It is hypothesized that the differences in the early flow cytometric DNA profiles may select the DNA aneuploid squamous cell uterine cervical carcinomas as candidates for combined neutron-brachytherapy, while highly proliferating DNA near-diploid tumors may profit more from treatment with a higher gamma-radiotherapy component. However, these early DNA flow cytometric findings need to be correlated with clinical course of the disease to validate this hypothesis, a process which will be completed at the end of the expected five-year clinical outcome in 2000.

  15. A novel rapid and reproducible flow cytometric method for optimization of transfection efficiency in cells.

    Science.gov (United States)

    Homann, Stefanie; Hofmann, Christian; Gorin, Aleksandr M; Nguyen, Huy Cong Xuan; Huynh, Diana; Hamid, Phillip; Maithel, Neil; Yacoubian, Vahe; Mu, Wenli; Kossyvakis, Athanasios; Sen Roy, Shubhendu; Yang, Otto Orlean; Kelesidis, Theodoros

    2017-01-01

    Transfection is one of the most frequently used techniques in molecular biology that is also applicable for gene therapy studies in humans. One of the biggest challenges to investigate the protein function and interaction in gene therapy studies is to have reliable monospecific detection reagents, particularly antibodies, for all human gene products. Thus, a reliable method that can optimize transfection efficiency based on not only expression of the target protein of interest but also the uptake of the nucleic acid plasmid, can be an important tool in molecular biology. Here, we present a simple, rapid and robust flow cytometric method that can be used as a tool to optimize transfection efficiency at the single cell level while overcoming limitations of prior established methods that quantify transfection efficiency. By using optimized ratios of transfection reagent and a nucleic acid (DNA or RNA) vector directly labeled with a fluorochrome, this method can be used as a tool to simultaneously quantify cellular toxicity of different transfection reagents, the amount of nucleic acid plasmid that cells have taken up during transfection as well as the amount of the encoded expressed protein. Finally, we demonstrate that this method is reproducible, can be standardized and can reliably and rapidly quantify transfection efficiency, reducing assay costs and increasing throughput while increasing data robustness.

  16. Fine needle aspirate flow cytometric phenotyping characterizes immunosuppressive nature of the mesothelioma microenvironment.

    Science.gov (United States)

    Lizotte, Patrick H; Jones, Robert E; Keogh, Lauren; Ivanova, Elena; Liu, Hongye; Awad, Mark M; Hammerman, Peter S; Gill, Ritu R; Richards, William G; Barbie, David A; Bass, Adam J; Bueno, Raphael; English, Jessie M; Bittinger, Mark; Wong, Kwok-Kin

    2016-08-19

    With the emergence of checkpoint blockade and other immunotherapeutic drugs, and the growing adoption of smaller, more flexible adaptive clinical trial designs, there is an unmet need to develop diagnostics that can rapidly immunophenotype patient tumors. The ability to longitudinally profile the tumor immune infiltrate in response to immunotherapy also presents a window of opportunity to illuminate mechanisms of resistance. We have developed a fine needle aspirate biopsy (FNA) platform to perform immune profiling on thoracic malignancies. Matching peripheral blood, bulk resected tumor, and FNA were analyzed from 13 mesothelioma patients. FNA samples yielded greater numbers of viable cells when compared to core needle biopsies. Cell numbers were adequate to perform flow cytometric analyses on T cell lineage, T cell activation and inhibitory receptor expression, and myeloid immunosuppressive checkpoint markers. FNA samples were representative of the tumor as a whole as assessed by head-to-head comparison to single cell suspensions of dissociated whole tumor. Parallel analysis of matched patient blood enabled us to establish quality assurance criteria to determine the accuracy of FNA procedures to sample tumor tissue. FNA biopsies provide a diagnostic to rapidly phenotype the tumor immune microenvironment that may be of great relevance to clinical trials.

  17. Flow cytometric analysis of lymphocyte proliferative responses to food allergens in dogs with food allergy.

    Science.gov (United States)

    Fujimura, Masato; Masuda, Kenichi; Hayashiya, Makio; Okayama, Taro

    2011-10-01

    Two different allergy tests, antigen-specific immunoglobulin E quantification (IgE test) and flow cytometric analysis of antigen-specific proliferation of peripheral lymphocytes (lymphocyte proliferation test), were performed to examine differences in allergic reactions to food allergens in dogs with food allergy (FA). Thirteen dogs were diagnosed as FA based on clinical findings and elimination diet trials. Seven dogs clinically diagnosed with canine atopic dermatitis (CAD) were used as a disease control group, and 5 healthy dogs were used as a negative control group. In the FA group, 19 and 33 allergen reactions were identified using the serum IgE test and the lymphocyte proliferation test, respectively. Likewise, in the CAD group, 12 and 6 allergen reactions and in the healthy dogs 3 and 0 allergen reactions were identified by each test, respectively. A significant difference was found between FA and healthy dogs in terms of positive allergen detection by the lymphocyte proliferation test, suggesting that the test can be useful to differentiate FA from healthy dogs but not from CAD. Both tests were repeated in 6 of the dogs with FA after a 1.5- to 5-month elimination diet trial. The IgE concentrations in 9 of 11 of the positive reactions decreased by 20-80%, whereas all the positive reactions in the lymphocyte proliferation test decreased to nearly zero (Pfood allergens may be involved in the pathogenesis of canine FA.

  18. A Flow Cytometric Analysis of Vitreous Inflammatory Cells in Patients with Proliferative Diabetic Retinopathy

    Directory of Open Access Journals (Sweden)

    Mojca Urbančič

    2013-01-01

    Full Text Available The purpose of this study was to investigate inflammatory cells in vitreous from patients with proliferative diabetic retinopathy (PDR using flow cytometric analysis. Twenty-eight patients with PDR requiring vitrectomy because of macular traction or tractional retinal detachment were enrolled in the study (n=28, and 6 patients with macular hole (MH formed the control group. Samples of vitreous and peripheral venous blood were obtained at the beginning of vitrectomy. T lymphocytes were found in vitreous from patients with PDR, and CD4/CD8 ratio was higher in vitreous (median 4.3 compared to blood (median 1.9; P=0.003. No B lymphocytes were detected in vitreous. The percentage of histiocytes/macrophages was significantly higher in vitreous (median 62.1 in comparison with blood (median 5.5; P<0.0001. No lymphocytes were detected in vitreous of the control group. There were more T lymphocytes in vitreous from patients with active PDR. No association between cells in the vitreous and visual acuity improvement after surgery was found. In conclusion, T lymphocytes are found in vitreous from patients with PDR and reflect the activity of PDR but do not seem to predict visual prognosis. Higher CD4/CD8 ratio in vitreous compared to blood from patients with PDR is consistent with local inflammatory response in PDR.

  19. Flow cytometric probing of mitochondrial function in equine peripheral blood mononuclear cells

    Directory of Open Access Journals (Sweden)

    Coignoul Freddy

    2007-09-01

    Full Text Available Abstract Background The morphopathological picture of a subset of equine myopathies is compatible with a primary mitochondrial disease, but functional confirmation in vivo is still pending. The cationic dye JC-1 exhibits potential-dependent accumulation in mitochondria that is detectable by a fluorescence shift from green to orange. As a consequence, mitochondrial membrane potential can be optically measured by the orange/green fluorescence intensity ratio. A flow cytometric standardized analytic procedure of the mitochondrial function of equine peripheral blood mononuclear cells is proposed along with a critical appraisal of the crucial questions of technical aspects, reproducibility, effect of time elapsed between blood sampling and laboratory processing and reference values. Results The JC-1-associated fluorescence orange and green values and their ratio were proved to be stable over time, independent of age and sex and hypersensitive to intoxication with a mitochondrial potential dissipator. Unless time elapsed between blood sampling and laboratory processing does not exceed 5 hours, the values retrieved remain stable. Reference values for clinically normal horses are given. Conclusion Whenever a quantitative measurement of mitochondrial function in a horse is desired, blood samples should be taken in sodium citrate tubes and kept at room temperature for a maximum of 5 hours before the laboratory procedure detailed here is started. The hope is that this new test may help in confirming, studying and preventing equine myopathies that are currently imputed to mitochondrial dysfunction.

  20. A Flow-Cytometric Gram-Staining Technique for Milk-Associated Bacteria

    Science.gov (United States)

    Holm, Claus; Jespersen, Lene

    2003-01-01

    A Gram-staining technique combining staining with two fluorescent stains, Oregon Green-conjugated wheat germ agglutinin (WGA) and hexidium iodide (HI) followed by flow-cytometric detection is described. WGA stains gram-positive bacteria while HI binds to the DNA of all bacteria after permeabilization by EDTA and incubation at 50°C for 15 min. For WGA to bind to gram-positive bacteria, a 3 M potassium chloride solution was found to give the highest fluorescence intensity. A total of 12 strains representing some of the predominant bacterial species in bulk tank milk and mixtures of these were stained and analyzed by flow cytometry. Overall, the staining method showed a clear differentiation between gram-positive and gram-negative bacterial populations. For stationary-stage cultures of seven gram-positive bacteria and five gram-negative bacteria, an average of 99% of the cells were correctly interpreted. The method was only slightly influenced by the growth phase of the bacteria or conditions such as freezing at −18°C for 24 h. For any of these conditions, an average of at least 95% of the cells were correctly interpreted. When stationary-stage cultures were stored at 5°C for 14 days, an average of 86% of the cells were correctly interpreted. The Gram-staining technique was applied to the flow cytometry analysis of bulk tank milk inoculated with Staphylococcus aureus and Escherichia coli. These results demonstrate that the technique is suitable for analyzing milk samples without precultivation. PMID:12732558

  1. Cholesterol sensitivity of detergent resistance: A rapid flow cytometric test for detecting constitutive or induced raft association of membrane proteins

    OpenAIRE

    Imre Gombos, Zsolt Bacsó, Cynthia Detre, Henrietta Nagy, Katalin Goda, Márton Andrásfalvy, Gábor Szabó, János Matkó; Bacsó Zsolt (1963-) (biofizikus); Goda Katalin (1969-) (biofizikus); Szabó Gábor (1953-) (biofizikus)

    2004-01-01

    Lipid rafts are cholesterol- and glycosphingolipid-rich microdomains in the cellular plasma membranes that play critical roles in compartmentalization (concentration, coupling, and isolation) of receptors and signal molecules. Therefore, detecting constitutive or induced raft associations of such proteins is of central interest in cell biology. This has mostly been done with time- and cell-consuming immunobiochemical techniques affected by several sources of artifacts. A flow cytometric analy...

  2. THE EFFECT OF LABELING INTENSITY, ESTIMATED BY REAL-TIME CONFOCAL LASER SCANNING MICROSCOPY, ON FLOW CYTOMETRIC APPEARANCE AND IDENTIFICATION OF IMMUNOCHEMICALLY LABELED MARINE DINOFLAGELLATES

    NARCIS (Netherlands)

    VRIELING, EG; DRAAIJER, A; VANZEIJL, WJM; PEPERZAK, L; GIESKES, WWC; VEENHUIS, M; Zeijl, Wilhelmus J.M. van

    Two different fluorescein isothiocyanate (FITC) conjugates were used to analyze the effect of labeling intensity on the flow cytometric appearance of marine dinoflagellates labeled with antibodies that specifically recognized the outer cell wall. Location of the labeling was revealed by

  3. Differentiation of HL-60 promyelocytic leukemia cells monitored by flow cytometric measurement of nitro blue tetrazolium (NBT) reduction.

    Science.gov (United States)

    Blair, O C; Carbone, R; Sartorelli, A C

    1985-01-01

    Reduction of nitro blue tetrazolium (NBT) to insoluble blue formazan granules occurs during the stimulus-induced respiratory burst of mature granulocytes and is routinely used as an indicator of the extent of granulocytic differentiation of HL-60 acute promyelocytic leukemia cells. In the present study, the differentiation of HL-60 leukemia cells induced by dimethylsulfoxide (DMSO) or retinoic acid was monitored by flow cytometric (FCM) measurement of forward and 90 degree light scatter of NBT treated cells. Two-parameter correlated analysis permitted a distinction between cells with increased forward and decreased 90 degree light scatter (NBT-), and cells with decreased forward and increased 90 degree light scatter (NBT+). Fixation of NBT treated cells with 1% paraformaldehyde facilitated flow cytometric analysis, and allowed differences in NBT reduction to be quantitated. DMSO-induced cells expressed an all-or-none reduction of NBT to formazan, compared with retinoic acid treated cells that exhibited a graded response. Three parameter flow cytometric analysis of HL-60 leukemia cells stained with propidium iodide in combination with NBT allowed the determination of the cell cycle distribution of NBT-treated cells.

  4. Flow cytometric analysis of viable bacteria in urine samples of febrile patients at the emergency department.

    Science.gov (United States)

    Tavenier, Anne H; de Boer, Foppie J; Moshaver, Bijan; van der Leur, Sjef J C M; Stegeman, Coen A; Groeneveld, Paul H P

    2017-08-16

    Fast and reliable diagnostics are important in febrile patients admitted to the emergency department. Current urine diagnostics are fast but moderately reliable or reliable but time consuming. Flow cytometry (FC) is a new promising technique in the diagnostics of complicated urinary tract infections by counting bacteria in urine samples. The aim of this study is to improve the FC method by counting only viable bacteria. Urine was obtained from 135 consecutive febrile patients at the emergency department. According to protocol regular diagnostic urine tests were performed. In addition, FC counting of viable and non-viable bacteria was executed after staining with thiazole orange and propidium iodide. All test results were compared to the results of urine culture (≥ 10 5 colony forming units/mL). At a cut-off value of 2.01 × 10 5 viable bacteria/mL the sensitivity was 100% and specificity was 78.4% (AUC-value 0.955 on ROC-curve). Spearman correlation test exhibited a higher correlation for flow cytometric counting of only viable bacteria than counting of all bacteria (0.59 vs. 0.37). Using ROC-curves, the AUC-values for FC counting of all bacteria, only viable bacteria and Gram staining were respectively 0.935, 0.955, and 0.968 (P > 0.05). FC counting of only viable bacteria can predict quickly and reliably positive and negative urine cultures in febrile patients admitted to the emergency department. It can help to improve the speed and accuracy of the diagnostic procedure at the emergency department. © 2017 Clinical Cytometry Society. © 2017 International Clinical Cytometry Society.

  5. Flow cytometric analysis of circulating platelet-monocyte aggregates in whole blood: methodological considerations.

    Science.gov (United States)

    Harding, Scott A; Din, Jehangir N; Sarma, Jaydeep; Jessop, Alasdair; Weatherall, Mark; Fox, Keith A A; Newby, David E

    2007-08-01

    Platelet-monocyte aggregates are increasingly being used to quantify platelet activation. The variables that influence platelet-monocyte aggregates have not been well defined. We sought to determine the effect of blood collection, handling and processing techniques on detected levels of platelet-monocyte aggregates using a flow cytometric assay. Whole blood was labelled with anti-CD14-PE and anti-CD42a-FITC. Thereafter, samples were fixed and red cells lysed. Analysis was performed with the flow cytometer initially triggering on light scatter and then on FL-2 to identify CD14-PE positive monocytes. Platelet-monocyte aggregates were defined as monocytes positive for CD42a. The effect of collection, handling and processing techniques on this assay were assessed. Anticoagulation with heparin (20.1 +/- 2.0%), PPACK (16.8 +/- 1.9%), sodium citrate (12.3 +/- 1.6%) and EDTA (9.5 +/- 1.0%) resulted in markedly different levels of platelet-monocyte aggregation (P venepuncture (20.9 +/- 3.9% vs.13.8 +/- 2.4%, P = 0.03). For every 10 minutes of delay prior to processing platelet-monocyte aggregates increased by 2.8% (P = 0.0001) in PPACK anticoagulated blood and 1.7% (P = 0.01) in citrate anticoagulated blood. Erythrocyte lysis together with fixation does not affect platelet-monocyte aggregation. Platelet-monocyte aggregates remained stable over 24 hours when fixed and stored at 4 degrees C. Multiple handling and processing factors may affect platelet-monocyte aggregation. We recommend the measurement of platelet-monocyte aggregates on samples collected by direct venepuncture, using a direct thrombin inhibitor as the anticoagulant and minimising the time delay before sample fixation.

  6. Flow cytometric immunobead assay for quantitative detection of platelet autoantibodies in immune thrombocytopenia patients.

    Science.gov (United States)

    Zhai, Juping; Ding, Mengyuan; Yang, Tianjie; Zuo, Bin; Weng, Zhen; Zhao, Yunxiao; He, Jun; Wu, Qingyu; Ruan, Changgeng; He, Yang

    2017-10-23

    Platelet autoantibody detection is critical for immune thrombocytopenia (ITP) diagnosis and prognosis. Therefore, we aimed to establish a quantitative flow cytometric immunobead assay (FCIA) for ITP platelet autoantibodies evaluation. Capture microbeads coupled with anti-GPIX, -GPIb, -GPIIb, -GPIIIa and P-selectin antibodies were used to bind the platelet-bound autoantibodies complex generated from plasma samples of 250 ITP patients, 163 non-ITP patients and 243 healthy controls, a fluorescein isothiocyanate (FITC)-conjugated secondary antibody was the detector reagent and mean fluorescence intensity (MFI) signals were recorded by flow cytometry. Intra- and inter-assay variations of the quantitative FCIA assay were assessed. Comparisons of the specificity, sensitivity and accuracy between quantitative and qualitative FCIA or monoclonal antibody immobilization of platelet antigen (MAIPA) assay were performed. Finally, treatment process was monitored by our quantitative FCIA in 8 newly diagnosed ITPs. The coefficient of variations (CV) of the quantitative FCIA assay were respectively 9.4, 3.8, 5.4, 5.1 and 5.8% for anti-GPIX, -GPIb, -GPIIIa, -GPIIb and -P-selectin autoantibodies. Elevated levels of autoantibodies against platelet glycoproteins GPIX, GPIb, GPIIIa, GPIIb and P-selectin were detected by our quantitative FCIA in ITP patients compared to non-ITP patients or healthy controls. The sensitivity, specificity and accuracy of our quantitative assay were respectively 73.13, 81.98 and 78.65% when combining all 5 autoantibodies, while the sensitivity, specificity and accuracy of MAIPA assay were respectively 41.46, 90.41 and 72.81%. A quantitative FCIA assay was established. Reduced levels of platelet autoantibodies could be confirmed by our quantitative FCIA in ITP patients after corticosteroid treatment. Our quantitative assay is not only good for ITP diagnosis but also for ITP treatment monitoring.

  7. FlowFP: A Bioconductor Package for Fingerprinting Flow Cytometric Data

    Directory of Open Access Journals (Sweden)

    Wade T. Rogers

    2009-01-01

    Full Text Available A new software package called flowFP for the analysis of flow cytometry data is introduced. The package, which is tightly integrated with other Bioconductor software for analysis of flow cytometry, provides tools to transform raw flow cytometry data into a form suitable for direct input into conventional statistical analysis and empirical modeling software tools. The approach of flowFP is to generate a description of the multivariate probability distribution function of flow cytometry data in the form of a “fingerprint.” As such, it is independent of a presumptive functional form for the distribution, in contrast with model-based methods such as Gaussian Mixture Modeling. FlowFP is computationally efficient and able to handle extremely large flow cytometry data sets of arbitrary dimensionality. Algorithms and software implementation of the package are described. Use of the software is exemplified with applications to data quality control and to the automated classification of Acute Myeloid Leukemia.

  8. A novel flow cytometric HTS assay reveals functional modulators of ATP binding cassette transporter ABCB6.

    Directory of Open Access Journals (Sweden)

    Kishore Polireddy

    Full Text Available ABCB6 is a member of the adenosine triphosphate (ATP-binding cassette family of transporter proteins that is increasingly recognized as a relevant physiological and therapeutic target. Evaluation of modulators of ABCB6 activity would pave the way toward a more complete understanding of the significance of this transport process in tumor cell growth, proliferation and therapy-related drug resistance. In addition, this effort would improve our understanding of the function of ABCB6 in normal physiology with respect to heme biosynthesis, and cellular adaptation to metabolic demand and stress responses. To search for modulators of ABCB6, we developed a novel cell-based approach that, in combination with flow cytometric high-throughput screening (HTS, can be used to identify functional modulators of ABCB6. Accumulation of protoporphyrin, a fluorescent molecule, in wild-type ABCB6 expressing K562 cells, forms the basis of the HTS assay. Screening the Prestwick Chemical Library employing the HTS assay identified four compounds, benzethonium chloride, verteporfin, tomatine hydrochloride and piperlongumine, that reduced ABCB6 mediated cellular porphyrin levels. Validation of the identified compounds employing the hemin-agarose affinity chromatography and mitochondrial transport assays demonstrated that three out of the four compounds were capable of inhibiting ABCB6 mediated hemin transport into isolated mitochondria. However, only verteporfin and tomatine hydrochloride inhibited ABCB6's ability to compete with hemin as an ABCB6 substrate. This assay is therefore sensitive, robust, and suitable for automation in a high-throughput environment as demonstrated by our identification of selective functional modulators of ABCB6. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel modulators of ABCB6 activity.

  9. Air sampling to assess potential generation of aerosolized viable bacteria during flow cytometric analysis of unfixed bacterial suspensions

    Science.gov (United States)

    Carson, Christine F; Inglis, Timothy JJ

    2018-01-01

    This study investigated aerosolized viable bacteria in a university research laboratory during operation of an acoustic-assisted flow cytometer for antimicrobial susceptibility testing by sampling room air before, during and after flow cytometer use. The aim was to assess the risk associated with use of an acoustic-assisted flow cytometer analyzing unfixed bacterial suspensions. Air sampling in a nearby clinical laboratory was conducted during the same period to provide context for the existing background of microorganisms that would be detected in the air. The three species of bacteria undergoing analysis by flow cytometer in the research laboratory were Klebsiella pneumoniae, Burkholderia thailandensis and Streptococcus pneumoniae. None of these was detected from multiple 1000 L air samples acquired in the research laboratory environment. The main cultured bacteria in both locations were skin commensal and environmental bacteria, presumed to have been disturbed or dispersed in laboratory air by personnel movements during routine laboratory activities. The concentrations of bacteria detected in research laboratory air samples were reduced after interventional cleaning measures were introduced and were lower than those in the diagnostic clinical microbiology laboratory. We conclude that our flow cytometric analyses of unfixed suspensions of K. pneumoniae, B. thailandensis and S. pneumoniae do not pose a risk to cytometer operators or other personnel in the laboratory but caution against extrapolation of our results to other bacteria and/or different flow cytometric experimental procedures. PMID:29608197

  10. Fluorescent particles in the antibody solution result in false TF- and CD14-positive microparticles in flow cytometric analysis.

    Science.gov (United States)

    Aass, Hans Christian D; Øvstebø, Reidun; Trøseid, Anne-Marie S; Kierulf, Peter; Berg, Jens Petter; Henriksson, Carola Elisabeth

    2011-12-01

    Tissue factor (TF)-positive microparticles (MPs) are highly procoagulant, and linked to thrombosis in sepsis and cancer. MP-associated TF may be assayed by immunological or functional methods. Several reports have demonstrated discrepancies between TF-protein and TF-activity, which have been explained by antibody binding to "encrypted" or degraded forms of inactive TF-protein. Our goal was to evaluate the possible interference of fluorescent antibody aggregates in solutions containing antibodies against TF and CD14 in flow cytometric analysis. Using monocyte-derived microparticles (MPs) released from human monocytes, incubated with or without lipopolysaccharides (LPS) in vitro, we measured MP-associated TF-protein (flow cytometry) and TF-activity (clot formation assay). MPs released from monocytes exposed to LPS (1 ng mL(-1) ) had ∼14 times higher TF-activity than MPs originated from monocytes exposed to only culture medium. However, using untreated anti-TF antibodies (American Diagnostica and BD) in the flow cytometric analysis, MPs released from unstimulated monocytes had a similar number of TF-positive events as MPs secernated from LPS-stimulated monocytes [∼45,000 events mL(-1) (American Diagnostica); ∼15,000 events mL(-1) (BD)]. These TF-positive events did not exert any TF-activity, and centrifugation (17,000g, 30 min, 4°C) of the antibody solutions prior to use effectively removed the interfering fluorescent events. Removal of fluorescent interference, probably in the form of fluorescent antibody aggregates, from the antibody solutions by centrifugation is essential to prevent the occurrence of false positive flow cytometric events. The events can be mistaken as MP-associated TF-protein, and interpreted as a discrepancy between TF-protein and TF-activity. Copyright © 2011 International Society for Advancement of Cytometry.

  11. Successful Desensitization of T cell Flow Cytometry Crossmatch Positive Renal Transplant Recipients Using Plasmapheresis and Super High-Dose Intravenous Immunoglobulin

    Directory of Open Access Journals (Sweden)

    Yoichi Kakuta, MD, PhD

    2018-01-01

    Full Text Available Background. High-dose IVIG (2 g/kg alone or low-dose IVIG (100 mg/kg in conjunction with plasma exchange is typically administered as a renal transplantation desensitization therapy. Herein, we monitored changes in T cell and B cell flow cytometry crossmatch (FCXM to assess the effects of short-term super high-dose IVIG (4 g/kg administration with plasmapheresis before living-donor renal transplantation. Methods. Seventeen patients, each showing positive T cell FCXM (median ratio, ≥ 1.4 after 2 rounds of double-filtration plasmapheresis, received 4-day regimens of IVIG (1 g/kg per day over 1-week periods. T cell and B cell FCXM determinations were obtained after every IVIG dose and again up to 4 weeks after initiating IVIG to ascertain negative conversion of T cell FCXM (median ratio < 1.4. The primary study endpoint was the percentage of patients achieving T cell FCXM-negative status after the 4-dose IVIG regimen. Results. Upon completion (4 g/kg total or discontinuation of IVIG administration, 8 (47.1% of 17 patients displayed negative T cell FCXM. Based on Kaplan-Meier estimates, the cumulative T cell FCXM-negative conversion rate 4 weeks after IVIG administration initiation was 60.3%. The T cell FCXM-negative conversion rates after cumulative doses of 1, 2, 3, and 4 g/kg IVIG were 29.4%, 35.3%, 56.3%, and 46.7%, respectively. Conclusions. Desensitization of donor-specific antibody-positive renal transplant recipients seems achievable in only a subset of recipients through IVIG dosing (1 g/kg × 4 within 1 week after double-filtration plasmapheresis. The T cell FCXM-negative conversion rate resulting from a cumulative IVIG dose of 3 g/kg or greater surpassed that attained via conventional single-dose IVIG (2 g/kg protocol. This short-term high-dose IVIG desensitization protocol may be an alternative to conventional protocols for recipients with donor-specific antibody.

  12. Comparative analysis of Luminex-based donor-specific antibody mean fluorescence intensity values with complement-dependent cytotoxicity & flow crossmatch results in live donor renal transplantation

    Directory of Open Access Journals (Sweden)

    Ajay Kumar Baranwal

    2017-01-01

    Interpretation & conclusions: A cut-off MFI value of 3000 for Luminex SAB-based assay was found to significantly correlate with the FCXM positivity while a MFI value of 7000 and above predicted a positive CDC crossmatch. MFI cut-off value obtained as a surrogate marker for CDC and FCXM tests will help in resolving the limitations of different cell-based techniques.

  13. A short-term in vitro test for tumour sensitivity to adriamycin based on flow cytometric DNA analysis

    DEFF Research Database (Denmark)

    Engelholm, S A; Spang-Thomsen, M; Vindeløv, L L

    1983-01-01

    A new method to test the sensitivity of tumour cells to chemotherapy is presented. Tumour cells were incubated in vitro on agar, and drug-induced cell cycle perturbation was monitored by flow cytometric DNA analysis. In the present study the method was applied to monitor the effect of adriamycin...... tumours. The dose level causing maximum accumulation in the G2 + M phase is suggested as a parameter for quantifying the sensitivity. The results indicate that the method can be extended to sensitivity testing of human tumours....

  14. Development of a flow cytometric assay to quantify lymphocyte adhesion to cytokine-stimulated human endothelial and biliary epithelial cells.

    Science.gov (United States)

    Korlipara, L V; Leon, M P; Rix, D A; Douglas, M S; Gibbs, P; Bassendine, M F; Kirby, J A

    1996-05-27

    The adhesive interaction between T lymphocytes and parenchymal cells is of importance for many processes of the cellular immune response. This adhesion is regulated by the activation status of the T cell and by cytokines in the microenvironment which can alter adhesion molecule expression by endothelial and epithelial cells. In this study results from an isotopic adhesion assay were compared with those from a flow cytometric assay in order to determine which was most appropriate for the investigation of lymphocyte adhesion to human umbilical vein endothelial cells (HUVEC) and intrahepatic biliary epithelial cells (HIBEC). Treatment of both these cell types with the proinflammatory cytokines interferon-gamma (IFN-gamma) or tumour necrosis factor-alpha (TNF-alpha) significantly upregulated expression of intercellular adhesion molecule-1 (ICAM-1). Treatment with TNF-alpha also induced endothelial cells to express vascular cell adhesion molecule-1 (VCAM-1). The isotopic assay demonstrated increased adhesion of lymphoblasts to HUVEC which had been stimulated with cytokines for 15 h but failed to detect major changes in adhesion following 72 h of cytokine treatment of HUVEC or HIBEC. However, the flow cytometric assay reproducibly demonstrated increased adhesion following cytokine treatment for both these time periods; these increases corresponded with the changes in adhesion molecule expression by cytokine-stimulated HUVEC and HIBEC targets. The differences in apparent adhesion measured by the two assays after cytokine stimulation for 72 h may be explained by cytokine-induced changes in the morphology and confluency of cultured cells. Results of the isotopic assay are proportional to the total number of lymphoid cells bound by the cultured target cells and will be distorted by changes in effective target cell area. The flow cytometric assay measures the mean number of lymphoid cells bound by each target cell and is independent of the total binding area. It is concluded

  15. Flow cytometric analysis of expression of interleukin-2 receptor beta chain (p70-75) on various leukemic cells

    International Nuclear Information System (INIS)

    Hoshino, S.; Oshimi, K.; Tsudo, M.; Miyasaka, M.; Teramura, M.; Masuda, M.; Motoji, T.; Mizoguchi, H.

    1990-01-01

    We analyzed the expression of the interleukin-2 receptor (IL-2R) beta chain (p70-75) on various leukemic cells from 44 patients by flow cytometric analysis using the IL-2R beta chain-specific monoclonal antibody, designated Mik-beta 1. Flow cytometric analysis demonstrated the expression of the IL-2R beta chain on granular lymphocytes (GLs) from all eight patients with granular lymphocyte proliferative disorders (GLPDs), on adult T-cell leukemia (ATL) cells from all three patients with ATL, and on T-cell acute lymphoblastic leukemia (T-ALL) cells from one of three patients with T-ALL. Although GLs from all the GLPD patients expressed the IL-2R beta chain alone and not the IL-2R alpha chain (Tac-antigen: p55), ATL and T-ALL cells expressing the beta chain coexpressed the alpha chain. In two of seven patients with common ALL (cALL) and in both patients with B-cell chronic lymphocytic leukemia, the leukemic cells expressed the alpha chain alone. Neither the alpha chain nor the beta chain was expressed on leukemic cells from the remaining 28 patients, including all 18 patients with acute nonlymphocytic leukemia, five of seven patients with cALL, all three patients with multiple myeloma, and two of three patients with T-ALL. These results indicate that three different forms of IL-2R chain expression exist on leukemic cells: the alpha chain alone; the beta chain alone; and both the alpha and beta chains. To examine whether the results obtained by flow cytometric analysis actually reflect functional aspects of the expressed IL-2Rs, we studied the specific binding of 125I-labeled IL-2 (125I-IL-2) to leukemic cells in 18 of the 44 patients. In addition, we performed 125I-IL-2 crosslinking studies in seven patients. The results of IL-2R expression of both 125I-IL-2 binding assay and crosslinking studies were in agreement with those obtained by flow cytometric analysis

  16. Micronuclei frequency in circulating erythrocytes from rainbow trout (Oncorhynchus mykiss) subjected to radiation, an image analysis and flow cytometric study

    International Nuclear Information System (INIS)

    Schultz, N.; Norrgren, L.; Grawe, J.; Johannisson, A.; Medhage, O.

    1993-01-01

    Rainbow trout (oncorhynchus mykiss) were exposed to a single X-ray dose of 4 Gy. The frequency of micronuclei in the peripheral erythrocytes was investigated at regular intervals up to 58 days after the exposure. A flow cytometric method and a semi-automatic image analysis method were used to estimate the micronuclei frequency. The results show that both methods can detect an increased frequency of micronuclei in peripheral erythrocytes from exposed fish. However, the semi-automatic image analysis method was the most stable and sensitive. (Author)

  17. Clonal evolution demonstrated by flow cytometric DNA analysis of a human colonic carcinoma grown in nude mice

    DEFF Research Database (Denmark)

    Vindeløv, L L; Spang-Thomsen, M; Visfeldt, J

    1982-01-01

    A spontaneous change in DNA content of a human colonic carcinoma grown in nude mice was observed fortuitously. The tumor initially had a G1 cell DNA content of 1.3 times that of normal cells. Flow cytometric DNA analysis showed in transplant generation 56 the appearance of a new subpopulation whi...... evolution of a tumor would be less pronounced if old subpopulations often become extinct as new ones emerge. Heterogeneity of human tumors is of clinical importance because the individual subpopulations may have different sensitivity patterns to antineoplastic drugs.......A spontaneous change in DNA content of a human colonic carcinoma grown in nude mice was observed fortuitously. The tumor initially had a G1 cell DNA content of 1.3 times that of normal cells. Flow cytometric DNA analysis showed in transplant generation 56 the appearance of a new subpopulation which...... in three passages completely overgrew the original population. The DNA content of the new subpopulation was twice that of the original population. The observation supports the hypothesis of clonal evolution of tumor cell populations. The growth rates of the tumor before and after the change showed...

  18. Rapid Detection and Enumeration of Giardia lamblia Cysts in Water Samples by Immunomagnetic Separation and Flow Cytometric Analysis ▿ †

    Science.gov (United States)

    Keserue, Hans-Anton; Füchslin, Hans Peter; Egli, Thomas

    2011-01-01

    Giardia lamblia is an important waterborne pathogen and is among the most common intestinal parasites of humans worldwide. Its fecal-oral transmission leads to the presence of cysts of this pathogen in the environment, and so far, quantitative rapid screening methods are not available for various matrices, such as surface waters, wastewater, or food. Thus, it is necessary to establish methods that enable reliable rapid detection of a single cyst in 10 to 100 liters of drinking water. Conventional detection relies on cyst concentration, isolation, and confirmation by immunofluorescence microscopy (IFM), resulting in low recoveries and high detection limits. Many different immunomagnetic separation (IMS) procedures have been developed for separation and cyst purification, so far with variable but high losses of cysts. A method was developed that requires less than 100 min and consists of filtration, resuspension, IMS, and flow cytometric (FCM) detection. MACS MicroBeads were used for IMS, and a reliable flow cytometric detection approach was established employing 3 different parameters for discrimination from background signals, i.e., green and red fluorescence (resulting from the distinct pattern emitted by the fluorescein dye) and sideward scatter for size discrimination. With spiked samples, recoveries exceeding 90% were obtained, and false-positive results were never encountered for negative samples. Additionally, the method was applicable to naturally occurring cysts in wastewater and has the potential to be automated. PMID:21685159

  19. Crossmatch testing in kidney transplantation: Patterns of practice and associations with rejection and graft survival

    International Nuclear Information System (INIS)

    Salvalaggio, Paolo R; Graff, Ralph J; Pinsky, Brett; Schnitzler, Mark A; Takemoto, Steven K; Burroughs, Thomas E; Santos, Luiz S; Lentine Krista L

    2009-01-01

    Methods of crossmatch testing prior to kidney transplantation are not standardized and there are limited large-scale data on the use and outcomes implications of crossmatch modality. Data describing the most sensitive crossmatch modality for crossmatch-negative kidney transplants were drawn from the Organ Procurement and Transplant Network Registry. Within the cohort transplanted in 1999-2005, we identified patient and transplant characteristics predictive of each testing modality by multivariate logistic regression. We assessed associations of crossmatch modality with rejection risk by logistic regression and with graft survival by Cox's hazards analysis. Among 230,995 transplants, use of flow cytometry with T-and B-lymphocytes (T and B FC) increased progressively in 1987-2005. Among the recent transplants performed in 1999-2005 (n=64,320), negative T and B FC crossmatch was associated with 15% lower relative risk of first-year acute rejection (adjusted HR 0.85, 95% CI 0.80-0.89) compared to negative T-antihuman-globulin and B-National Institutes of Health/Wash (T AHG and B) crossmatch. Five-year graft survival after transplant with negative T and B FC (82.6%) was modestly better than after negative T AHG and B (81.4%, P0.008) or T AHG crossmatch (81.1%, P 60 years. Many subgroups for whom negative T and B FC crossmatch predicted lower rejection risk (Caucasians, deceased donor recipients, re-transplants) were not more likely to be crossmatched by this method. We conclude that current practice patterns have not aligned utilization of T and B FC crossmatch with associated benefits. Prospective evaluation of the relationship of crossmatch modality with outcomes is warranted. (author)

  20. C1Q Assay Results in Complement-Dependent Cytotoxicity Crossmatch Negative Renal Transplant Candidates with Donor-Specific Antibodies: High Specificity but Low Sensitivity When Predicting Flow Crossmatch

    Directory of Open Access Journals (Sweden)

    José M. Arreola-Guerra

    2016-01-01

    Full Text Available The aim of the present study was to describe the association of positive flow cross match (FXM and C1q-SAB. Methods. In this observational, cross-sectional, and comparative study, patients included had negative AHG-CDC-XM and donor specific antibodies (DSA and were tested with FXM. All pretransplant sera were tested with C1q-SAB assay. Results. A total of 50 donor/recipient evaluations were conducted; half of them had at least one C1q+ Ab (n=26, 52%. Ten patients (20.0% had DSA C1q+ Ab. Twenty-five (50% FXMs were positive. Factors associated with a positive FXM were the presence of C1q+ Ab (DSA C1q+ Ab: OR 27, 2.80–259.56, P=0.004, and no DSA C1q+ Ab: OR 5, 1.27–19.68, P=0.021 and the DSA LABScreen-SAB MFI (OR 1.26, 95% CI 1.06–1.49, P=0.007. The cutoff point of immunodominant LABScreen SAB DSA-MFI with the greatest sensitivity and specificity to predict FXM was 2,300 (sensitivity: 72% and specificity: 75%. For FXM prediction, DSA C1q+ Ab was the most specific (95.8%, 85–100 and the combination of DSA-MFI > 2,300 and C1q+ Ab was the most sensitive (92.0%, 79.3–100. Conclusions. C1q+ Ab and LABScreen SAB DSA-MFI were significantly associated with FXM. DSA C1q+ Ab was highly specific but with low sensitivity.

  1. A Protocol for the Comprehensive Flow Cytometric Analysis of Immune Cells in Normal and Inflamed Murine Non-Lymphoid Tissues

    Science.gov (United States)

    Yu, Yen-Rei A.; O’Koren, Emily G.; Hotten, Danielle F.; Kan, Matthew J.; Kopin, David; Nelson, Erik R.; Que, Loretta; Gunn, Michael D.

    2016-01-01

    Flow cytometry is used extensively to examine immune cells in non-lymphoid tissues. However, a method of flow cytometric analysis that is both comprehensive and widely applicable has not been described. We developed a protocol for the flow cytometric analysis of non-lymphoid tissues, including methods of tissue preparation, a 10-fluorochrome panel for cell staining, and a standardized gating strategy, that allows the simultaneous identification and quantification of all major immune cell types in a variety of normal and inflamed non-lymphoid tissues. We demonstrate that our basic protocol minimizes cell loss, reliably distinguishes macrophages from dendritic cells (DC), and identifies all major granulocytic and mononuclear phagocytic cell types. This protocol is able to accurately quantify 11 distinct immune cell types, including T cells, B cells, NK cells, neutrophils, eosinophils, inflammatory monocytes, resident monocytes, alveolar macrophages, resident/interstitial macrophages, CD11b- DC, and CD11b+ DC, in normal lung, heart, liver, kidney, intestine, skin, eyes, and mammary gland. We also characterized the expression patterns of several commonly used myeloid and macrophage markers. This basic protocol can be expanded to identify additional cell types such as mast cells, basophils, and plasmacytoid DC, or perform detailed phenotyping of specific cell types. In examining models of primary and metastatic mammary tumors, this protocol allowed the identification of several distinct tumor associated macrophage phenotypes, the appearance of which was highly specific to individual tumor cell lines. This protocol provides a valuable tool to examine immune cell repertoires and follow immune responses in a wide variety of tissues and experimental conditions. PMID:26938654

  2. A flow-cytometric NK-cytotoxicity assay adapted for use in rat repeated dose toxicity studies

    International Nuclear Information System (INIS)

    Marcusson-Staahl, Maritha; Cederbrant, Karin

    2003-01-01

    A recent regulatory document for immunotoxicity testing of new pharmaceutical drugs includes cytotoxic natural killer (NK)-cell function as a required parameter in repeated dose toxicity studies. The classical 51 Cr-release assay is the conventional test for cytotoxicity testing but several drawbacks with this assay has increased the demand for new reliable test systems. Here, we describe the optimisation of a flow-cytometric cytotoxicity assay especially adapted for regulatory rat studies in drug development. The test principle is based on target cell labelling with 5-(6)-carboxy-fluorescein succinimidyl ester (CFSE) and subsequent DNA-labelling with propidium iodide (PI) for identification of target cells with compromised cell membranes. The results are expressed as percentage of dead targets on a cell-to-cell basis. The final format of the assay includes 0.5 ml peripheral blood, 1.25x10 5 effector cells per sample, and collection of 500 target events by flow-cytometry. When NKR-P1+ cells were removed from the effector cell population by magnetic depletion the relative proportion decreased from 6 to 0.08%. The corresponding cytotoxic activity decreased from 68 to 8%. Also, the cytotoxic activity showed a significant and positive correlation with the proportion of NK-cells present in the effector cell suspension. Thus, the cytotoxicity measured is almost exclusively exerted by NK-cells. The current flow-cytometric test benefits from using peripheral blood as a source for effector cells since it will not conflict with the use of spleen for histopathological investigations in repeated dose toxicity studies. Additionally, since only a minimal number of effector cells are required per sample repeated testing of the same animal is enabled

  3. A Protocol for the Comprehensive Flow Cytometric Analysis of Immune Cells in Normal and Inflamed Murine Non-Lymphoid Tissues.

    Directory of Open Access Journals (Sweden)

    Yen-Rei A Yu

    Full Text Available Flow cytometry is used extensively to examine immune cells in non-lymphoid tissues. However, a method of flow cytometric analysis that is both comprehensive and widely applicable has not been described. We developed a protocol for the flow cytometric analysis of non-lymphoid tissues, including methods of tissue preparation, a 10-fluorochrome panel for cell staining, and a standardized gating strategy, that allows the simultaneous identification and quantification of all major immune cell types in a variety of normal and inflamed non-lymphoid tissues. We demonstrate that our basic protocol minimizes cell loss, reliably distinguishes macrophages from dendritic cells (DC, and identifies all major granulocytic and mononuclear phagocytic cell types. This protocol is able to accurately quantify 11 distinct immune cell types, including T cells, B cells, NK cells, neutrophils, eosinophils, inflammatory monocytes, resident monocytes, alveolar macrophages, resident/interstitial macrophages, CD11b- DC, and CD11b+ DC, in normal lung, heart, liver, kidney, intestine, skin, eyes, and mammary gland. We also characterized the expression patterns of several commonly used myeloid and macrophage markers. This basic protocol can be expanded to identify additional cell types such as mast cells, basophils, and plasmacytoid DC, or perform detailed phenotyping of specific cell types. In examining models of primary and metastatic mammary tumors, this protocol allowed the identification of several distinct tumor associated macrophage phenotypes, the appearance of which was highly specific to individual tumor cell lines. This protocol provides a valuable tool to examine immune cell repertoires and follow immune responses in a wide variety of tissues and experimental conditions.

  4. Flow-cytometric analysis of changes in lymphocyte subsets in the blood of cancer patients during radiation therapy

    International Nuclear Information System (INIS)

    Ogawa, Yasuhiro; Maeda, Tomoho; Ogawa, Yukiko

    1983-01-01

    In this study, we report a clinical application of a rapid flow-cytometric immunofluorescence method for studying lymphocyte subsets in whole blood by using monoclonal antibodies OKT3, OKT4, OKT8, OKIa1, OKT6, OKT10, OKT9, OKT11 and OKM1. Changes in the relative percentages and absolute counts of various lymphocyte subsets during radiotherapy of cancer patients were evaluated by this method. In patients with good response, the elevation of OKT 4 + -lymphocyte level and/or the depression of OKT8 + -lymphocyte level was observed. In contrast, the depression of OKT4 + -lymphocyte level and/or the elevation of OKT8 + -lymphocyte level was observed on patients with poor response. Our data indicate that these monoclonal antibodies can be regarded as invaluable tools for evaluation of immune status of cancer patients during radiotherapy. (author)

  5. Flow cytometric evaluation of physico-chemical impact on Gram-positive and Gram-negative bacteria

    Directory of Open Access Journals (Sweden)

    Antje eFröhling

    2015-09-01

    Full Text Available Since heat sensitivity of fruits and vegetables limits the application of thermal inactivation processes, new emerging inactivation technologies have to be established to fulfil the requirements of food safety without affecting the produce quality. The efficiency of inactivation treatments has to be ensured and monitored. Monitoring of inactivation effects is commonly performed using traditional cultivation methods which have the disadvantage of the time span needed to obtain results.The aim of this study was to compare the inactivation effects of peracetic acid (PAA, ozonated water (O3 and cold atmospheric pressure plasma (CAPP on Gram-positive and Gram-negative bacteria using flow cytometric methods. E. coli cells were completely depolarized after treatment (15 s with 0.25 % PAA at 10 °C, and after treatment (10 s with 3.8 mg l-1 O3 at 12°C. The membrane potential of CAPP treated cells remained almost constant at an operating power of 20 W over a time period of 3 min, and subsequently decreased within 30 s of further treatment. Complete membrane permeabilization was observed after 10 s O3 treatment, but treatment with PAA and CAPP did not completely permeabilize the cells within 2 min and 4 min, respectively. Similar results were obtained for esterase activity. O3 inactivates cellular esterase but esterase activity was detected after 4 min CAPP treatment and 2 min PAA treatment. L. innocua cells and P. carotovorum cells were also permeabilized instantaneously by O3 treatment at concentrations of 3.8 ± 1 mg l-1. However, higher membrane permeabilization of L. innocua and P. carotovorum than of E. coli was observed at CAPP treatment of 20 W. The degree of bacterial damage due to the inactivation processes is highly dependent on treatment parameters as well as on treated bacteria. Important information regarding the inactivation mechanisms can be obtained by flow cytometric measurements and this enables the definition of critical process

  6. Effect of 17 beta-oestradiol on growth curves and flow cytometric DNA distribution of two human breast carcinomas grown in nude mice

    DEFF Research Database (Denmark)

    Brünner, N; Spang-Thomsen, M; Vindeløv, L

    1983-01-01

    The effect of 17 beta-oestradiol on a "receptor positive" and on a "receptor negative" human breast carcinoma grown in nude mice was studied. Experimental growth data were used to determine the effect on tumour growth. Flow cytometric DNA analysis (FCM) performed on tumour tissue obtained...

  7. A heparin-based method for flow cytometric analysis of microparticles directly from platelet-poor plasma in calcium containing buffer

    DEFF Research Database (Denmark)

    Iversen, Line V; Ostergaard, Ole; Nielsen, Christoffer

    2013-01-01

    V negative MPs to clinical parameters in systemic diseases, which emphasize the importance of including characterization of AnxV-binding. An obstacle in flow cytometric analysis of AnxV-binding to MPs is that plasma may clot when adding the Ca(2+)-containing buffers. We here devise a simple method...

  8. Clinical flow cytometric screening of SAP and XIAP expression accurately identifies patients with SH2D1A and XIAP/BIRC4 mutations.

    Science.gov (United States)

    Gifford, Carrie E; Weingartner, Elizabeth; Villanueva, Joyce; Johnson, Judith; Zhang, Kejian; Filipovich, Alexandra H; Bleesing, Jack J; Marsh, Rebecca A

    2014-07-01

    X-linked lymphoproliferative disease is caused by mutations in two genes, SH2D1A and XIAP/BIRC4. Flow cytometric methods have been developed to detect the gene products, SAP and XIAP. However, there is no literature describing the accuracy of flow cytometric screening performed in a clinical lab setting. We reviewed the clinical flow cytometric testing results for 656 SAP and 586 XIAP samples tested during a 3-year period. Genetic testing was clinically performed as directed by the managing physician in 137 SAP (21%) and 115 XIAP (20%) samples. We included these samples for analyses of flow cytometric test accuracy. SH2D1A mutations were detected in 15/137 samples. SAP expression was low in 13/15 (sensitivity 87%, CI 61-97%). Of the 122 samples with normal sequencing, SAP was normal in 109 (specificity 89%, CI 82-94%). The positive predictive values (PPVs) and the negative predictive values (NPVs) were 50% and 98%, respectively. XIAP/BIRC4 mutations were detected in 19/115 samples. XIAP expression was low in 18/19 (sensitivity 95%, CI 73-100%). Of the 96 samples with normal sequencing, 59 had normal XIAP expression (specificity 61%, CI 51-71%). The PPVs and NPVs were 33% and 98%, respectively. Receiver-operating characteristic analysis was able to improve the specificity to 75%. Clinical flow cytometric screening tests for SAP and XIAP deficiencies offer good sensitivity and specificity for detecting genetic mutations, and are characterized by high NPVs. We recommend these tests for patients suspected of having X-linked lymphoproliferative disease type 1 (XLP1) or XLP2. © 2014 Clinical Cytometry Society.

  9. CD33 monoclonal antibody conjugated Au cluster nano-bioprobe for targeted flow-cytometric detection of acute myeloid leukaemia

    Energy Technology Data Exchange (ETDEWEB)

    Retnakumari, Archana; Jayasimhan, Jasusri; Chandran, Parwathy; Menon, Deepthy; Nair, Shantikumar; Mony, Ullas; Koyakutty, Manzoor, E-mail: manzoork@aims.amrita.edu, E-mail: ullasmony@aims.amrita.edu [Amrita Centre for Nanoscience and Molecular Medicine, Amrita Institute of Medical Science, Cochin 682 041 (India)

    2011-07-15

    Protein stabilized gold nanoclusters (Au-NCs) are biocompatible, near-infrared (NIR) emitting nanosystems having a wide range of biomedical applications. Here, we report the development of a Au-NC based targeted fluorescent nano-bioprobe for the flow-cytometric detection of acute myeloid leukaemia (AML) cells. Au-NCs with {approx} 25-28 atoms showing bright red-NIR fluorescence (600-750 nm) and average size of {approx} 0.8 nm were prepared by bovine serum albumin assisted reduction-cum-stabilization in aqueous phase. The protein protected clusters were conjugated with monoclonal antibody against CD33 myeloid antigen, which is overexpressed in {approx} 99.2% of the primitive population of AML cells, as confirmed by immunophenotyping using flow cytometry. Au-NC-CD33 conjugates having average size of {approx} 12 nm retained bright fluorescence over an extended duration of {approx} a year, as the albumin protein protects Au-NCs against degradation. Nanotoxicity studies revealed excellent biocompatibility of Au-NC conjugates, as they showed no adverse effect on the cell viability and inflammatory response. Target specificity of the conjugates for detecting CD33 expressing AML cells (KG1a) in flow cytometry showed specific staining of {approx} 95.4% of leukaemia cells within 1-2 h compared to a non-specific uptake of {approx} 8.2% in human peripheral blood cells (PBMCs) which are CD33{sup low}. The confocal imaging also demonstrated the targeted uptake of CD33 conjugated Au-NCs by leukaemia cells, thus confirming the flow cytometry results. This study demonstrates that novel nano-bioprobes can be developed using protein protected fluorescent nanoclusters of Au for the molecular receptor targeted flow cytometry based detection and imaging of cancer cells.

  10. CD33 monoclonal antibody conjugated Au cluster nano-bioprobe for targeted flow-cytometric detection of acute myeloid leukaemia

    Science.gov (United States)

    Retnakumari, Archana; Jayasimhan, Jasusri; Chandran, Parwathy; Menon, Deepthy; Nair, Shantikumar; Mony, Ullas; Koyakutty, Manzoor

    2011-07-01

    Protein stabilized gold nanoclusters (Au-NCs) are biocompatible, near-infrared (NIR) emitting nanosystems having a wide range of biomedical applications. Here, we report the development of a Au-NC based targeted fluorescent nano-bioprobe for the flow-cytometric detection of acute myeloid leukaemia (AML) cells. Au-NCs with ~ 25-28 atoms showing bright red-NIR fluorescence (600-750 nm) and average size of ~ 0.8 nm were prepared by bovine serum albumin assisted reduction-cum-stabilization in aqueous phase. The protein protected clusters were conjugated with monoclonal antibody against CD33 myeloid antigen, which is overexpressed in ~ 99.2% of the primitive population of AML cells, as confirmed by immunophenotyping using flow cytometry. Au-NC-CD33 conjugates having average size of ~ 12 nm retained bright fluorescence over an extended duration of ~ a year, as the albumin protein protects Au-NCs against degradation. Nanotoxicity studies revealed excellent biocompatibility of Au-NC conjugates, as they showed no adverse effect on the cell viability and inflammatory response. Target specificity of the conjugates for detecting CD33 expressing AML cells (KG1a) in flow cytometry showed specific staining of ~ 95.4% of leukaemia cells within 1-2 h compared to a non-specific uptake of ~ 8.2% in human peripheral blood cells (PBMCs) which are CD33low. The confocal imaging also demonstrated the targeted uptake of CD33 conjugated Au-NCs by leukaemia cells, thus confirming the flow cytometry results. This study demonstrates that novel nano-bioprobes can be developed using protein protected fluorescent nanoclusters of Au for the molecular receptor targeted flow cytometry based detection and imaging of cancer cells.

  11. CD33 monoclonal antibody conjugated Au cluster nano-bioprobe for targeted flow-cytometric detection of acute myeloid leukaemia

    International Nuclear Information System (INIS)

    Retnakumari, Archana; Jayasimhan, Jasusri; Chandran, Parwathy; Menon, Deepthy; Nair, Shantikumar; Mony, Ullas; Koyakutty, Manzoor

    2011-01-01

    Protein stabilized gold nanoclusters (Au-NCs) are biocompatible, near-infrared (NIR) emitting nanosystems having a wide range of biomedical applications. Here, we report the development of a Au-NC based targeted fluorescent nano-bioprobe for the flow-cytometric detection of acute myeloid leukaemia (AML) cells. Au-NCs with ∼ 25-28 atoms showing bright red-NIR fluorescence (600-750 nm) and average size of ∼ 0.8 nm were prepared by bovine serum albumin assisted reduction-cum-stabilization in aqueous phase. The protein protected clusters were conjugated with monoclonal antibody against CD33 myeloid antigen, which is overexpressed in ∼ 99.2% of the primitive population of AML cells, as confirmed by immunophenotyping using flow cytometry. Au-NC-CD33 conjugates having average size of ∼ 12 nm retained bright fluorescence over an extended duration of ∼ a year, as the albumin protein protects Au-NCs against degradation. Nanotoxicity studies revealed excellent biocompatibility of Au-NC conjugates, as they showed no adverse effect on the cell viability and inflammatory response. Target specificity of the conjugates for detecting CD33 expressing AML cells (KG1a) in flow cytometry showed specific staining of ∼ 95.4% of leukaemia cells within 1-2 h compared to a non-specific uptake of ∼ 8.2% in human peripheral blood cells (PBMCs) which are CD33 low . The confocal imaging also demonstrated the targeted uptake of CD33 conjugated Au-NCs by leukaemia cells, thus confirming the flow cytometry results. This study demonstrates that novel nano-bioprobes can be developed using protein protected fluorescent nanoclusters of Au for the molecular receptor targeted flow cytometry based detection and imaging of cancer cells.

  12. Immuno-flow cytometric detection of the ichthyotoxic dinoflagellates Gyrodinium aureolum and Gymnodinium nagasakiense: independence of physiological state

    Science.gov (United States)

    Vrieling, Engel G.; van de Poll, Willem H.; Vriezekolk, Gertie; Gieskes, Winfried W. C.

    1997-05-01

    The ichthyotoxic dinoflagellates Gyrodinium aureolum and Gymnodinium nagasakiense were cultured under different environmental conditions to test possible variability in immunochemical labelling intensity of cell-surface antigens using species-specific monoclonal antibodies. Variation of antigen abundance (which is directly related to labelling intensity) at the cell surface, determined by immuno-flow cytometry of cells labelled with FITC, appeared to be small but significant compared to control cultures. In general, a minor decrease in FIX fluorescence was recorded during exponential growth, followed by an increase during stationary growth. FITC fluorescence was correlated with cell size, shape and structure. This suggests a constant number of antigens per unit of cell surface. In all cultures, immunochemically labelled cells were distinguished clearly from unlabelled cells; immuno-flow cytometric identification is apparently not affected by growth conditions. Only at the end of the stationary growth phase in batch cultures did the FITC fluorescence values drop, which suggests that unhealthy, dying or lysing cells may either alter the composition of the cell surface or just fail to express the antigen.

  13. An improved flow cytometric immunobead array to detect autoantibodies in plasma from patients with immune thrombocytopenic purpura.

    Science.gov (United States)

    Zhao, Yunxiao; Zhu, Mingqing; Jiang, Miao; Zuo, Bin; Wu, Qingyu; Ruan, Changgeng; He, Yang

    2015-01-01

    Autoantibodies against platelet glycoproteins (GPs) play an important role in immune thrombocytopenic purpura (ITP). This study was to develop an improved flow cytometric immunobead array (FCIA) assay to detect platelet autoantibodies in ITP patient plasma. Plasma samples were isolated from 71 ITP patients and 136 non-ITP controls and incubated with platelets from normal individuals. After washing, platelets were lysed and the platelet lysates were incubated with polystyrene microbeads coupled with monoclonal antibodies against human GPs IX (SZ1), Ib (SZ2), IIIa (SZ21), IIb (SZ22), and P-selectin (SZ51). Platelet GP-autoantibody complexes were detected by flow cytometry using a FITC-labeled secondary antibody. Autoantibodies against platelet GPIb, GPIIb, GPIIIa, GPIX and P-selectin were detected in plasma from ITP patients, as indicated by high mean fluorescent intensity values when microbeads with antibodies SZ1, SZ2, SZ21, SZ22, and SZ51 were used. In ROC analysis, values of the area under the curve were 0.74, 0.83, 0.80, 0.79 and 0.87, respectively. Compared with the previously reported assays, this new FCIA eliminated the need of isolating platelets from ITP patients without compromising assay sensitivity and accuracy in predicting ITP. This simplified FICA assay may be more suitable for ITP diagnosis in clinical laboratory settings. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Flow cytometric-membrane potential detection of sodium channel active marine toxins: application to ciguatoxins in fish muscle and feasibility of automating saxitoxin detection.

    Science.gov (United States)

    Manger, Ronald; Woodle, Doug; Berger, Andrew; Dickey, Robert W; Jester, Edward; Yasumoto, Takeshi; Lewis, Richard; Hawryluk, Timothy; Hungerford, James

    2014-01-01

    Ciguatoxins are potent neurotoxins with a significant public health impact. Cytotoxicity assays have allowed the most sensitive means of detection of ciguatoxin-like activity without reliance on mouse bioassays and have been invaluable in studying outbreaks. An improvement of these cell-based assays is presented here in which rapid flow cytometric detection of ciguatoxins and saxitoxins is demonstrated using fluorescent voltage sensitive dyes. A depolarization response can be detected directly due to ciguatoxin alone; however, an approximate 1000-fold increase in sensitivity is observed in the presence of veratridine. These results demonstrate that flow cytometric assessment of ciguatoxins is possible at levels approaching the trace detection limits of our earlier cytotoxicity assays, however, with a significant reduction in analysis time. Preliminary results are also presented for detection of brevetoxins and for automation and throughput improvements to a previously described method for detecting saxitoxins in shellfish extracts.

  15. A flow cytometric method for characterization of circulating cell-derived microparticles in plasma

    DEFF Research Database (Denmark)

    Nielsen, Morten Hjuler; Beck-Nielsen, Henning; Andersen, Morten Nørgaard

    2014-01-01

    BACKGROUND AND AIM: Previous studies on circulating microparticles (MPs) indicate that the majority of MPs are of a size below the detection limit of most standard flow cytometers. The objective of the present study was to establish a method to analyze MP subpopulations above the threshold...... of detection of a new generation BD FACSAria™ III digital flow cytometer. METHODS: We analyzed MP subpopulations in plasma from 24 healthy individuals (9 males and 15 females). MPs were identified according to their size (

  16. Laser-based flow cytometric analysis of genotoxicity of humans exposed to ionizing radiation during the Chernobyl accident

    Science.gov (United States)

    Jensen, Ronald H.; Bigbee, William L.; Langlois, Richard G.; Grant, Stephen G.; Pleshanov, Pavel G.; Chirkov, Andre A.; Pilinskaya, Maria A.

    1991-05-01

    An analytical technique has been developed that allows laser-based flow cytometric measurement of the frequency of red blood cells that have lost allele-specific expression of a cell surface antigen due to genetic toxicity in bone marrow precursor cells. Previous studies demonstrated a correlation of such effects with the exposure of each individual to mutagenic phenomena, such as ionizing radiation, and the effects can persist for the lifetime of each individual. During the emergency response to the nuclear power plant accidert at Chemobyl, Ukraine, USSR, a number of people were exposed to whole body doses of ioniing radiation. Some of these individuals were tested with this laser-based assay and found to express a dose-dependent increase in the frequency of variant red blood cells that appears to be a persistent biological effect. This effect is similar to that which was previously observed in individuals who were exposed to ionizing radiation at Hiroshima in 1945 because of the A-bomb explosion. All data indicate that this assay might well be used as a biodosimeter to estimate radiation dose and also as an element to be used for estimating the risk of each individual to develop cancer due to radiation exposure.

  17. Simple and easy method to evaluate uptake potential of nanoparticles in mammalian cells using a flow cytometric light scatter analysis.

    Science.gov (United States)

    Suzuki, Hiroshi; Toyooka, Tatsushi; Ibuki, Yuko

    2007-04-15

    Many classes of nanoparticles have been synthesized and widely applied, however, there is a serious lack of information concerning their effects on human health and the environment. Considering that their use will increase, accurate and cost-effective measurement techniques for characterizing "nanotoxicity" are required. One major toxicological concern is that nanoparticles are easily taken up in the human body. In this study, we developed a method of evaluating the uptake potential of nanosized particles using flow cytometric light scatter. Suspended titanium dioxide (TiO2) particles (5, 23, or 5000 nm) were added to Chinese hamster ovary cells. Observation by confocal laser scanning microscopy showed that the TiO2 particles easily moved to the cytoplasm of the cultured mammalian cells, not to the nucleus. The intensity of the side-scattered light revealed that the particles were taken up in the cells dose-, time-, and size-dependently. In addition, surface-coating of TiO2 particles changed the uptake into the cells, which was accurately reflected in the intensity of the side-scattered light. The uptake of other nanoparticles such as silver (Ag) and iron oxide (Fe3O4) also could be detected. This method could be used for the initial screening of the uptake potential of nanoparticles as an index of "nanotoxicity".

  18. Flow Cytometric Evaluation of Human Neutrophil Apoptosis During Nitric Oxide Generation In Vitro: The Role of Exogenous Antioxidants

    Directory of Open Access Journals (Sweden)

    Zofia Sulowska

    2005-01-01

    in vitro. The effect of exogenous supply of NO donors such as SNP, SIN-1, and GEA-3162 on the course of human neutrophil apoptosis and the role of extracellular antioxidants in this process was investigated. Isolated from peripheral blood, neutrophils were cultured in the presence or absence of NO donor compounds and antioxidants for 8, 12, and 20 hours. Apoptosis of neutrophils was determined in vitro by flow cytometric analysis of cellular DNA content and Annexin V protein binding to the cell surface. Exposure of human neutrophils to GEA-3162 and SIN-1 significantly accelerates and enhances their apoptosis in vitro in a time-dependent fashion. In the presence of SNP, intensification of apoptosis has not been revealed until 12 hours after the culture. The inhibition of GEA-3162- and SIN-1-mediated neutrophil apoptosis by superoxide dismutase (SOD but not by catalase (CAT was observed. Our results show that SOD and CAT can protect neutrophils against NO-donors-induced apoptosis and suggest that the interaction of NO and oxygen metabolites signals may determine the destructive or protective role of NO donor compounds during apoptotic neutrophil death.

  19. Flow cytometric analysis of platelet cyclooxygenase-1 and -2 and surface glycoproteins in patients with immune thrombocytopenia and healthy individuals.

    Science.gov (United States)

    Rubak, Peter; Kristensen, Steen D; Hvas, Anne-Mette

    2017-06-01

    Immature platelets may contain more platelet enzymes such as cyclooxygenase (COX)-1 and COX-2 than mature platelets. Patients with immune thrombocytopenia (ITP) have a higher fraction of immature platelets and can therefore be utilized as a biological model for investigating COX-1 and COX-2 platelet expression. The aims were to develop flow cytometric assays for platelet COX-1 and COX-2 and to investigate the COX-1 and COX-2 platelet expression, platelet turnover, and platelet glycoproteins in ITP patients (n = 10) compared with healthy individuals (n = 30). Platelet count and platelet turnover parameters (mean platelet volume (MPV), immature platelet fraction (IPF), and immature platelet count (IPC)) were measured by flow cytometry (Sysmex XE-5000). Platelet COX-1, COX-2, and the glycoproteins (GP)IIb, IX, Ib, Ia, and IIIa were all analyzed by flow cytometry (Navios) and expressed as median fluorescence intensity. COX analyses were performed in both whole blood and platelet rich plasma (PRP), whereas platelet glycoproteins were analyzed in whole blood only. ITP patients had significantly lower platelet count (55 × 10 9 /L) than healthy individuals (240 × 10 9 /L, p platelet count and IPC (both p-values Platelet COX-1 expression was higher in ITP patients than healthy individuals using whole blood (p COX-1 platelet turnover and COX-1 expression (all p-values platelet turnover and COX-1 and COX-2 expressions (all p-values platelet turnover in ITP patients (all p-values 0.14, rho = 0.11-0.28). In conclusion, ITP patients expressed higher COX-1 and platelet glycoprotein levels than healthy individuals. COX-1 and platelet glycoproteins demonstrated positive correlations with platelet turnover in ITP patients. In healthy individuals, COX-1 and COX-2 expression correlated positively with platelet turnover. PRP was more sensitive compared with whole blood as regards determination of COX. Therefore, PRP is the recommended matrix for investigating COX-1 and COX-2 in

  20. A flow cytometric assay to quantify invasion of red blood cells by rodent Plasmodium parasites in vivo.

    Science.gov (United States)

    Lelliott, Patrick M; Lampkin, Shelley; McMorran, Brendan J; Foote, Simon J; Burgio, Gaetan

    2014-03-17

    Malaria treatments are becoming less effective due to the rapid spread of drug resistant parasites. Increased understanding of the host/parasite interaction is crucial in order to develop treatments that will be less prone to resistance. Parasite invasion of the red blood cell (RBC) is a critical aspect of the parasite life cycle and is, therefore, a promising target for the development of malaria treatments. Assays for analysing parasite invasion in vitro have been developed, but no equivalent assays exist for in vivo studies. This article describes a novel flow cytometric in vivo parasite invasion assay. Experiments were conducted with mice infected with erythrocytic stages of Plasmodium chabaudi adami strain DS. Exogenously labelled blood cells were transfused into infected mice at schizogony, and collected blood samples stained and analysed using flow cytometry to specifically detect and measure proportions of labelled RBC containing newly invaded parasites. A combination of antibodies (CD45 and CD71) and fluorescent dyes, Hoechst (DNA) and JC-1 (mitochondrial membrane potential), were used to differentiate parasitized RBCs from uninfected cells, RBCs containing Howell-Jolly bodies, leukocytes and RBC progenitors. Blood cells were treated ex vivo with proteases to examine the effects on in vivo parasite invasion. The staining and flow cytometry analysis method was accurate in determining the parasitaemia down to 0.013% with the limit of detection at 0.007%. Transfused labelled blood supported normal rates of parasite invasion. Protease-treated red cells resulted in 35% decrease in the rate of parasite invasion within 30 minutes of introduction into the bloodstream of infected mice. The invasion assay presented here is a versatile method for the study of in vivo red cell invasion efficiency of Plasmodium parasites in mice, and allows direct comparison of invasion in red cells derived from two different populations. The method also serves as an accurate

  1. Microbial Eco-Physiology of the human intestinal tract: a flow cytometric approach

    NARCIS (Netherlands)

    Amor, Ben K.

    2004-01-01

    This thesis describes a multifaceted approach to further enhance our view of the complex human intestinal microbial ecosystem. This approach combines me advantages of flow cyrometry (FCM), a single cell and high-throughput technology, and molecular techniques that have proven themselves to be

  2. Flow cytometric characterization of peripheral blood leukocyte populations of 3 neotropical snake species: Boa constrictor, Bothrops jararaca, and Crotalus durissus.

    Science.gov (United States)

    Carvalho, Marcelo P N; Queiroz-Hazarbassanov, Nicolle G T; Massoco, Cristina O; Rossi, Silmara; Sant'Anna, Sávio S; Catão-Dias, José L; Grego, Kathleen F

    2016-06-01

    The reptilian immune system is represented by innate, humoral, and cell-mediated mechanisms, involving different types of blood leukocytes. The development of optimized methods for the advanced study of origin and function of reptilian blood leukocytes is needed. The purpose of the study was to optimize leukocyte density gradient isolation protocols from snake peripheral blood samples, and characterize recovered cells by flow cytometry based on size and internal complexity for a qualitative and semi-quantitative assessment of leukocyte populations in one boa (Boa constrictor), and 2 viper species (Bothrops jararaca, Crotalus durissus). Blood samples from 30 snakes (10 from each species, 5 males and 5 females) were collected in tubes with sodium heparin. Fresh blood was centrifuged with either ficoll-paque PLUS or percoll density gradients for leukocyte isolation. Flow cytometric leukocyte gates were defined based on size (forward scatter [FSC]) and internal complexity (side scatter [SSC]). Relative leukocyte differential counts after sorting the cells in these gates in one snake for each species were compared to conventional light microscopic differential counts on unsorted isolated leukocytes. There was no statistical difference in the relative leukocyte populations, including heterophils, azurophils, and small and large lymphocytes between samples isolated by ficoll or percoll. Four leukocyte gates were identified based on their location in FSC/SSC cytograms. The relative leukocyte differential counts after sorting in single animals showed some agreement with the light microscopy differential count on unsorted cells. Based on FSC and SSC, 4 distinct leukocyte populations were found in ficoll or percoll density gradient isolated leukocytes from peripheral blood from boa and viper species. Further optimization of the technique should allow the performance of functional assays. © 2016 American Society for Veterinary Clinical Pathology.

  3. Flow cytometric gating for spleen monocyte and DC subsets: differences in autoimmune NOD mice and with acute inflammation.

    Science.gov (United States)

    Dong, Matthew B; Rahman, M Jubayer; Tarbell, Kristin V

    2016-05-01

    The role of antigen presenting cells (APCs) in the pathogenesis of autoimmune and other inflammatory diseases is now better understood due to advances in multicolor flow cytometry, gene expression analysis of APC populations, and functional correlation of mouse to human APC populations. A simple but informative nomenclature of conventional and plasmacytoid dendritic cell subsets (cDC1, cDC2, pDC) and monocyte-derived populations incorporates these advances, but accurate subset identification is critical. Ambiguous gating schemes and alterations of cell surface markers in inflammatory condition can make comparing results between studies difficult. Both acute inflammation, such as TLR-ligand stimulation, and chronic inflammation as found in mouse models of autoimmunity can alter DC subset gating. Here, we address these issues using in vivo CpG stimulation as an example of acute inflammation and the non-obese diabetic (NOD) mouse as a model of chronic inflammation.We provide a flow cytometric antibody panel and gating scheme that differentiate 2 monocytic and 3DC subsets in the spleen both at steady state and after CpG stimulation. Using this method, we observed differences in the composition of NOD DCs that have been previously reported, and newly identified increases in the number of NOD monocyte-derived DCs. Finally, we established a protocol for DC phosphoflow to measure the phosphorylation state of intracellular proteins, and use it to confirm functional differences in the identified subsets. Therefore, we present optimized methods for distinguishing monocytic and DC populations with and without inflammation and/or autoimmunity associated with NOD mice. Published by Elsevier B.V.

  4. Single-laboratory validation of a multiplex flow cytometric immunoassay for the simultaneous detection of coccidiostats in eggs and feed.

    Science.gov (United States)

    Bienenmann-Ploum, Monique E; Vincent, Ursula; Campbell, Katrina; Huet, Anne-Catherine; Haasnoot, Willem; Delahaut, Philippe; Stolker, Linda A A M; Elliott, Christopher T; Nielen, Michel W F

    2013-11-01

    Coccidiostats are authorized in the European Union (EU) to be used as poultry feed additives. Maximum (residue) levels (M(R)Ls) have been set within the EU for consumer and animal protection against unintended carry-over, and monitoring is compulsory. This paper describes the single-laboratory validation of a previously developed multiplex flow cytometric immunoassay (FCIA) as screening method for coccidiostats in eggs and feed and provides and compares different approaches for the calculation of the cut-off levels which are not described in detail within Commission Decision 2002/657/EC. Comparable results were obtained between the statistical (reference) approach and the rapid approaches. With the most rapid approach, the cut-off levels for narasin/salinomycin, lasalocid, diclazuril, nicarbazin (DNC) and monensin in egg, calculated as percentages of inhibition (%B/B0), were 60, 32, 76, 80 and 84, respectively. In feed, the cut-off levels for narasin/salinomycin, lasalocid, nicarbazin (DNC) and monensin were 70, 64, 72 and 78, respectively, and could not be determined for diclazuril. For all analytes, except for diclazuril in feed, the rate of false positives (false non-compliant) in blank samples was lower than 1 %, and the rate of false negatives (false compliant) at the M(R)Ls was below 5 %. Additionally, very good correlations (r ranging from 0.994 to 0.9994) were observed between two different analysers, a sophisticated flow cytometer (FlexMAP 3D(®)) and a more cost-efficient and transportable planar imaging detector (MAGPIX(®)), hence demonstrating adequate transferability.

  5. Flow cytometric evaluation of sperm parameters in relation to fertility potential.

    Science.gov (United States)

    Gillan, Lindsay; Evans, Gareth; Maxwell, W M C

    2005-01-15

    Most laboratory methods used to evaluate semen quality have not correlated highly with fertilizing capacity. The discovery of a variety of fluorochromes and compounds conjugated to fluorescent probes has enabled a more widespread analysis of sperm attributes, and in conjunction with the flow cytometer, permit the evaluation of a large number of spermatozoa. A number of characteristics of sperm integrity, viability and function can be assessed by flow cytometry. The DNA status of spermatozoa has been determined using the metachromatic properties of acridine orange (AO). AO staining, when used in the sperm chromatin structure assay (SCSA), correlates with fertility in a number of species. DNA fragmentation can also be assessed using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, which identifies DNA strand breaks by labeling free 3'-OH termini with modified nucleotides. The status of the sperm acrosome can be determined using fluorescently labeled lectins and LysoTracker Green DND-26, a fluorescent acidotropic probe. Capacitation status has been observed through calcium-mediated changes using chlortetracycline (CTC) or by changes in membrane fluidity monitored by the binding of the fluorescent amphiphilic probe, Merocyanine 540. Fluorescently labeled annexin-V, C6NBD and Ro-09-0198 can also be used to detect changes in membrane phospholipid distribution. Cell viability can be determined using the propensity of propidium iodide (PI), ethidium homodimer-1 (EthD-1) or Yo-Pro-1 to permeate damaged membranes. These are generally more adaptable to clinical flow cytometry than the bisbenzimide membrane impermeable stain, Hoechst 33258, which excites in the ultraviolet range and requires UV laser equipment. Mitochondrial function can be determined using rhodamine 123 (R123) and MitoTracker Green FM (MITO) and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1). Flow cytometry is a tool that may be used

  6. Flow cytometric determination of osmotic behaviour of animal erythrocytes toward their engineering for drug delivery

    Directory of Open Access Journals (Sweden)

    Kostić Ivana T.

    2015-01-01

    Full Text Available Despite the fact that the methods based on the osmotic properties of the cells are the most widely used for loading of drugs in human and animal erythrocytes, data related to the osmotic properties of erythrocytes derived from animal blood are scarce. This work was performed with an aim to investigate the possibility of use the flow cytometry as a tool for determination the osmotic behaviour of porcine and bovine erythrocytes, and thus facilitate the engineering of erythrocytes from animal blood to be drug carriers. The method of flow cytometry successfully provided the information about bovine and porcine erythrocyte osmotic fragility, and made the initial steps in assessment of erythrocyte shape in a large number of erythrocytes. Although this method is not able to confirm the swelling of pig erythrocytes, it indicated to the differences in pig erythrocytes that had basic hematological parameters inside and outside the reference values. In order to apply/use the porcine and bovine erythrocytes as drug carriers, the method of flow cytometry, confirming the presence of osmotically different fractions of red blood cells, indicated that various amounts of the encapsulated drug in porcine and bovine erythrocytes can be expected.

  7. Multiparameter Flow Cytometric Assays to Quantify Effector and Regulatory T-Cell Function in Multiple Sclerosis.

    Science.gov (United States)

    Sinha, Sushmita; Crawford, Michael P; Ortega, Sterling B; Karandikar, Nitin J

    2015-01-01

    The immune system plays a major pathological and regulatory role in multiple sclerosis (MS) and, therefore, is a focus of extensive research. Animal models of MS have been crucial in understanding the pathological processes in MS and developing certain treatments, however, all crucial aspects of the human disease may not be appropriately modeled. With the exception of detecting oligoclonal bands and IgG synthesis in cerebrospinal fluids of MS patients, there has not been major progress in the development of immunologic tests that can be used for diagnosis of MS. Further, due to the lack of validated immune assays, routine monitoring of the immune system following therapy initiation is not a part of standard patient care in MS. This is critical since immunomodulatory therapies used for MS treatment are not benign and, more importantly, there is a considerable variation in clinical responses in MS patients initiating such therapies. Flow cytometry is a powerful tool that can be used for studying both the phenotype and function of immune cells. The studies described here will demonstrate how flow cytometry can be used to apply current knowledge about the MS immune system to develop a diagnostic laboratory test for the immunologic monitoring of this disease. Importantly, we will also show that the multiparameter flow cytometry based assay developed by us can also be implemented for the immunologic evaluation of therapeutic success in MS patients.

  8. A flow cytometric method for characterization of circulating cell-derived microparticles in plasma.

    Science.gov (United States)

    Nielsen, Morten Hjuler; Beck-Nielsen, Henning; Andersen, Morten Nørgaard; Handberg, Aase

    2014-01-01

    Previous studies on circulating microparticles (MPs) indicate that the majority of MPs are of a size below the detection limit of most standard flow cytometers. The objective of the present study was to establish a method to analyze MP subpopulations above the threshold of detection of a new generation BD FACSAria™ III digital flow cytometer. We analyzed MP subpopulations in plasma from 24 healthy individuals (9 males and 15 females). MPs were identified according to their size (derived from platelets, monocytes, erythrocytes and endothelial cells. In addition, levels of tissue factor-positive MPs were determined. The FACSAria demonstrated improved sensitivity and increased MP detection range compared to the FC500 instrument. The reproducibility of PS+PMP and PS+MP measurements was 11.7 and 23.2%, respectively. When expressed as a percentage of total MPs, the PS-positive MP population represented 15.1±5.5%, and PS-positive MPs were significantly increased in men. We have established a method to measure MPs above the detection limit of a new generation flow cytometer and derived from a number of cell-types in a healthy population of men and women.

  9. Simple flow cytometric detection of haemozoin containing leukocytes and erythrocytes for research on diagnosis, immunology and drug sensitivity testing

    Directory of Open Access Journals (Sweden)

    Grobusch Martin P

    2011-03-01

    Full Text Available Abstract Background Malaria pigment (haemozoin, Hz has been the focus of diverse research efforts. However, identification of Hz-containing leukocytes or parasitized erythrocytes is usually based on microscopy, with inherent limitations. Flow cytometric detection of depolarized Side-Scatter is more accurate and its adaptation to common bench top flow cytometers might allow several applications. These can range from the ex-vivo and in-vitro detection and functional analysis of Hz-containing leukocytes to the detection of parasitized Red-Blood-Cells (pRBCs to assess antimalarial activity. Methods A standard benchtop flow cytometer was adapted to detect depolarized Side-Scatter. Synthetic and Plasmodium falciparum Hz were incubated with whole blood and PBMCs to detect Hz-containing leukocytes and CD16 expression on monocytes. C5BL/6 mice were infected with Plasmodium berghei ANKA or P. berghei NK65 and Hz-containing leukocytes were analysed using CD11b and Gr1 expression. Parasitized RBC from infected mice were identified using anti-Ter119 and SYBR green I and were analysed for depolarized Side Scatter. A highly depolarizing RBC population was monitored in an in-vitro culture incubated with chloroquine or quinine. Results A flow cytometer can be easily adapted to detect depolarized Side-Scatter and thus, intracellular Hz. The detection and counting of Hz containing leukocytes in fresh human or mouse blood, as well as in leukocytes from in-vitro experiments was rapid and easy. Analysis of CD14/CD16 and CD11b/Gr1 monocyte expression in human or mouse blood, in a mixed populations of Hz-containing and non-containing monocytes, appears to show distinct patterns in both types of cells. Hz-containing pRBC and different maturation stages could be detected in blood from infected mice. The analysis of a highly depolarizing population that contained mature pRBC allowed to assess the effect of chloroquine and quinine after only 2 and 4 hours, respectively

  10. Flow Cytometric Detection of PrPScin Neurons and Glial Cells from Prion-Infected Mouse Brains.

    Science.gov (United States)

    Yamasaki, Takeshi; Suzuki, Akio; Hasebe, Rie; Horiuchi, Motohiro

    2018-01-01

    In prion diseases, an abnormal isoform of prion protein (PrP Sc ) accumulates in neurons, astrocytes, and microglia in the brains of animals affected by prions. Detailed analyses of PrP Sc -positive neurons and glial cells are required to clarify their pathophysiological roles in the disease. Here, we report a novel method for the detection of PrP Sc in neurons and glial cells from the brains of prion-infected mice by flow cytometry using PrP Sc -specific staining with monoclonal antibody (MAb) 132. The combination of PrP Sc staining and immunolabeling of neural cell markers clearly distinguished neurons, astrocytes, and microglia that were positive for PrP Sc from those that were PrP Sc negative. The flow cytometric analysis of PrP Sc revealed the appearance of PrP Sc -positive neurons, astrocytes, and microglia at 60 days after intracerebral prion inoculation, suggesting the presence of PrP Sc in the glial cells, as well as in neurons, from an early stage of infection. Moreover, the kinetic analysis of PrP Sc revealed a continuous increase in the proportion of PrP Sc -positive cells for all cell types with disease progression. Finally, we applied this method to isolate neurons, astrocytes, and microglia positive for PrP Sc from a prion-infected mouse brain by florescence-activated cell sorting. The method described here enables comprehensive analyses specific to PrP Sc -positive neurons, astrocytes, and microglia that will contribute to the understanding of the pathophysiological roles of neurons and glial cells in PrP Sc -associated pathogenesis. IMPORTANCE Although formation of PrP Sc in neurons is associated closely with neurodegeneration in prion diseases, the mechanism of neurodegeneration is not understood completely. On the other hand, recent studies proposed the important roles of glial cells in PrP Sc -associated pathogenesis, such as the intracerebral spread of PrP Sc and clearance of PrP Sc from the brain. Despite the great need for detailed analyses

  11. Flow cytometric analysis of apoptosis in cryoconserved chicken primordial germ cells.

    Science.gov (United States)

    Sawicka, Dorota; Chojnacka-Puchta, Luiza; Zielinski, Marcin; Plucienniczak, Grazyna; Plucienniczak, Andrzej; Bednarczyk, Marek

    2015-03-01

    Our research aimed to compare the effects of four cryoprotectants and four slow freezing programs on the viability and apoptosis of primordial germ cells (PGCs) in vitro. PGCs were collected from chicken embryonic blood at Hamburger and Hamilton (HH) stages 14-16 and purified by Percoll density gradient centrifugation and then subjected to cryopreservation. We applied microscopy to determine the survival of PGCs after trypan blue staining and flow cytometry to examine apoptosis and viability after annexin V kit staining. We also examined the functionality of cryopreserved PGCs in vivo. Significant differences in viability of PGCs determined via microscopy and flow cytometry were observed. The most unfavorable combination for slow freezing PGCs was program 3 and MIX H (10% DMSO and 5% glycerol in Hank's solution supplemented with 10% FBS) as the cryoprotectant (48.43 and 15.37% live and early apoptotic PGCs, respectively). The highest average percentage of live PGCs (93.1%) and the lowest percentage of early apoptotic PGCs (6.5%) were achieved by slow freezing PGCs in the presence of DMSO F (10% DMSO in FBS) via program 1. Therefore, this method was chosen for the in vivo test. Cryopreserved (group 1) and freshly isolated (group 2) PGCs were transfectedwith a pEGFP-N1 plasmid, cultured under antibiotic selection, and then injected into 3-day-old embryos. After 5 days of incubation, we identified the EGFP marker gene in the gonads of 40 and 45% of recipients in groups 1 and 2, respectively. This is the first study to apply flow cytometry to examine the apoptosis and viability of cryopreserved PGCs. The in vitro and in vivo findings showed that the developed PGC cryoconservation method, depending on slow freezing at the rate of 2°C/min (program 1) in the presence of 10% DMSO F, is an improvement over previous cryoconservation methods and may be a useful tool for the ex situ strategy of poultry biodiversity preservation.

  12. Quick cytogenetic screening of breeding bulls using flow cytometric sperm DNA histogram analysis.

    Science.gov (United States)

    Nagy, Szabolcs; Polgár, Péter J; Andersson, Magnus; Kovács, András

    2016-09-01

    The aim of the present study was to test the FXCycle PI/RNase kit for routine DNA analyses in order to detect breeding bulls and/or insemination doses carrying cytogenetic aberrations. In a series of experiments we first established basic DNA histogram parameters of cytogenetically healthy breeding bulls by measuring the intraspecific genome size variation of three animals, then we compared the histogram profiles of bulls carrying cytogenetic defects to the baseline values. With the exception of one case the test was able to identify bulls with cytogenetic defects. Therefore, we conclude that the assay could be incorporated into the laboratory routine where flow cytometry is applied for semen quality control.

  13. APPLICATION OF SIX-COLOR FLOW CYTOMETRIC ANALYSIS FOR IMMUNE PROFILE MONITORING

    Directory of Open Access Journals (Sweden)

    I. V. Kudryavtsev

    2015-01-01

    Full Text Available The article deals with applications of six-color flow analysis for studying the immune state parameters by means of flow cytometry. Whole blood from healthy donors was stained with combination of monoclonal antibodies, i.e., HLA-DR-FITC, CD16+56-PE, CD4-ECD, CD19-ECD, CD8-PC5.5, CD3-PC7, CD45-APC (Beckman Coulter, USA using a “no-wash” technology. To adjust the photomultiplier (PMT voltage, we used the tubes with each of the tested monoclonal antibodies, PMT voltage was considered optimal when the negative population was located in the middle of the first decade at the logarithmic scale. The compensatory adjustment was performed in automatic mode, using Navios software (Beckman Coulter, USA. We discuss an optimal gating strategy in order to assess the populations of interest: total T cells (CD3+CD19-, T helper cells (CD3+CD4+, cytotoxic T lymphocytes (CD3+CD8+, B cells (CD3-CD19+, NK cells (CD3-CD16+CD56+, NKT cells (CD3+CD16+CD56+, activated T lymphocytes (CD3+HLA-DR+. 

  14. Optimization of flow cytometric detection and cell sorting of transgenic Plasmodium parasites using interchangeable optical filters.

    Science.gov (United States)

    Vorobjev, Ivan A; Buchholz, Kathrin; Prabhat, Prashant; Ketman, Kenneth; Egan, Elizabeth S; Marti, Matthias; Duraisingh, Manoj T; Barteneva, Natasha S

    2012-09-05

    Malaria remains a major cause of morbidity and mortality worldwide. Flow cytometry-based assays that take advantage of fluorescent protein (FP)-expressing malaria parasites have proven to be valuable tools for quantification and sorting of specific subpopulations of parasite-infected red blood cells. However, identification of rare subpopulations of parasites using green fluorescent protein (GFP) labelling is complicated by autofluorescence (AF) of red blood cells and low signal from transgenic parasites. It has been suggested that cell sorting yield could be improved by using filters that precisely match the emission spectrum of GFP. Detection of transgenic Plasmodium falciparum parasites expressing either tdTomato or GFP was performed using a flow cytometer with interchangeable optical filters. Parasitaemia was evaluated using different optical filters and, after optimization of optics, the GFP-expressing parasites were sorted and analysed by microscopy after cytospin preparation and by imaging cytometry. A new approach to evaluate filter performance in flow cytometry using two-dimensional dot blot was developed. By selecting optical filters with narrow bandpass (BP) and maximum position of filter emission close to GFP maximum emission in the FL1 channel (510/20, 512/20 and 517/20; dichroics 502LP and 466LP), AF was markedly decreased and signal-background improve dramatically. Sorting of GFP-expressing parasite populations in infected red blood cells at 90 or 95% purity with these filters resulted in 50-150% increased yield when compared to the standard filter set-up. The purity of the sorted population was confirmed using imaging cytometry and microscopy of cytospin preparations of sorted red blood cells infected with transgenic malaria parasites. Filter optimization is particularly important for applications where the FP signal and percentage of positive events are relatively low, such as analysis of parasite-infected samples with in the intention of gene

  15. Optimization of flow cytometric detection and cell sorting of transgenic Plasmodium parasites using interchangeable optical filters

    Directory of Open Access Journals (Sweden)

    Vorobjev Ivan A

    2012-09-01

    Full Text Available Abstract Background Malaria remains a major cause of morbidity and mortality worldwide. Flow cytometry-based assays that take advantage of fluorescent protein (FP-expressing malaria parasites have proven to be valuable tools for quantification and sorting of specific subpopulations of parasite-infected red blood cells. However, identification of rare subpopulations of parasites using green fluorescent protein (GFP labelling is complicated by autofluorescence (AF of red blood cells and low signal from transgenic parasites. It has been suggested that cell sorting yield could be improved by using filters that precisely match the emission spectrum of GFP. Methods Detection of transgenic Plasmodium falciparum parasites expressing either tdTomato or GFP was performed using a flow cytometer with interchangeable optical filters. Parasitaemia was evaluated using different optical filters and, after optimization of optics, the GFP-expressing parasites were sorted and analysed by microscopy after cytospin preparation and by imaging cytometry. Results A new approach to evaluate filter performance in flow cytometry using two-dimensional dot blot was developed. By selecting optical filters with narrow bandpass (BP and maximum position of filter emission close to GFP maximum emission in the FL1 channel (510/20, 512/20 and 517/20; dichroics 502LP and 466LP, AF was markedly decreased and signal-background improve dramatically. Sorting of GFP-expressing parasite populations in infected red blood cells at 90 or 95% purity with these filters resulted in 50-150% increased yield when compared to the standard filter set-up. The purity of the sorted population was confirmed using imaging cytometry and microscopy of cytospin preparations of sorted red blood cells infected with transgenic malaria parasites. Discussion Filter optimization is particularly important for applications where the FP signal and percentage of positive events are relatively low, such as analysis

  16. Flow cytometric measurement of RNA synthesis using bromouridine labelling and bromodeoxyuridine antibodies

    DEFF Research Database (Denmark)

    Jensen, P O; Larsen, J; Christiansen, J

    1993-01-01

    Nuclear RNA synthesis can be analysed by flow cytometry of cells labelled with 5-bromouridine (BrUrd) and stained with anti-bromodeoxyuridine (BrdUrd) antibody and FITC-conjugated secondary antibody. A panel of 5 different commercially available anti-BrdUrd antibodies was tested on cells of a HL-60...... human leukemia cell line, stained as a methanol-fixed nuclear suspension. The BrUrd-induced fluorescence signals were highest with the antibody ABDM (Partec), moderate but reproducible with B-44 (Becton Dickinson), variable or low with BR-3 and IU-4 (Caltag), and not detectable with Bu20a (DAKO...... the variation of RNA synthesis during the cell cycle. The BrUrd incorporation was high in the S and G2 phase, variable in G1, and negligible in mitosis. Similar results were obtained using other cell types....

  17. Flow cytometric measurement of the metabolism of benzo [a] pyrene by mouse liver cells in culture

    International Nuclear Information System (INIS)

    Bartholomew, J.C.; Wade, C.G.; Dougherty, K.

    1984-01-01

    The metabolism of benzo[a]pyrene in individual cells was monitored by flow cytometry. The measurements are based on the alterations that occur in the fluorescence emission spectrum of benzo[a]pyrene when it is converted to various metabolities. Using present instrumentation the technique could easily detect 1 x 10/sup 6/ molecules per cells of benzo [a]pyrene and 1 x 10/sup 7/ molecules per cell of the diol epoxide. The analysis of C3H IOT 1/2 mouse fibroblasts growing in culture indicated that there was heterogeneity in the conversion of the parent compound into diol epoxide derivative suggesting that some variation in sensitivity to transformation by benzo[a]pyrene may be due to differences in cellular metabolism

  18. DEA 1 expression on dog erythrocytes analyzed by immunochromatographic and flow cytometric techniques.

    Science.gov (United States)

    Acierno, M M; Raj, K; Giger, U

    2014-01-01

    The Dog erythrocyte antigen (DEA) 1 blood group system was thought to contain types DEA 1.1 and 1.2 (and possibly 1.3 [A3]). However, DEA 1.2+ dogs are very rare and newer typing methods reveal varying degrees of DEA 1 positivity. To assess if variation in DEA 1 positivity is because of quantitative differences in surface antigen expression. To determine expression patterns in dogs over time and effects of blood storage (4°C). To evaluate DEA 1.2+ samples by DEA 1 typing methods. Anticoagulated blood samples from 66 dogs in a research colony and from a hospital, and 9 previously typed DEA 1.2+ dogs from an animal blood bank. Research study: Samples were analyzed by flow cytometry and immunochromatographic strip using a monoclonal anti-DEA 1 antibody. Twenty dogs were DEA 1-, whereas 46 dogs were weakly to strongly DEA 1+. Antigen quantification revealed excellent correlation between strip and flow cytometry (r = 0.929). Both methods reclassified DEA 1.2+ samples as weakly to moderately DEA 1+, but they were not retyped with the polyclonal anti-DEA 1.1/1.X antibodies. Dogs and blood samples retained their relative DEA 1 antigen densities over time. The blood group system DEA 1 is a continuum from negative to strongly positive antigen expression. Previously typed DEA 1.2+ appears to be DEA 1+. These findings further the understanding of the DEA 1 system and suggest that all alleles within the DEA 1 system have a similarly based epitope recognized by the monoclonal antibody. Copyright © 2014 by the American College of Veterinary Internal Medicine.

  19. Flow cytometric analysis of lymphocytes and lymphocyte subpopulations in induced sputum from patients with asthma

    Directory of Open Access Journals (Sweden)

    Yutaro Shiota

    2000-01-01

    Full Text Available Study objectives were to compare the numbers of lymphocytes and lymphocyte subpopulations in induced sputum from asthmatic patients and from healthy subjects, and to determine the effect of inhaled anti-asthmatic steroid therapy on these cell numbers. Hypertonic saline inhalation was used to non-invasively induce sputum samples in 34 patients with bronchial asthma and 21 healthy subjects. The sputum samples were reduced with dithioerythritol and absolute numbers of lymphocytes and lymphocyte subpopulations were assessed by direct immunofluorescence and flow cytometry. To assess the effect of beclomethasone dipropionate (BDP on induced sputum, numbers of lymphocytes and lymphocyte subpopulations in sputum also were evaluated after 4 weeks of BDP inhalation treatment in seven asthmatic patients. An adequate sample was obtained in 85.3% of patients with asthma and in 79.2% of the healthy subjects. Induced sputum from patients with asthma had increased numbers of lymphocytes (P = 0.009; CD4+ cells (P = 0.044; CD4+ cells-bearing interleukin-2 receptor (CD25; P = 0.016; and CD4+ cells bearing human histocompatibility leukocyte antigen (HLA-DR (P = 0.033. CD8+ cells were not increased in asthmatic patients. In patients treated with inhaled steroids, numbers of lymphocytes, CD4+ cells, CD25-bearing CD4+ cells and HLA-DR-bearing CD4+ cells in sputum decreased from pretreatment numbers (P = 0.016, 0.002, 0.003 and 0.002, respectively. Analysis of lymphocytes in induced sputum by flow cytometry is useful in assessing bronchial inflammation, and activated CD4+ lymphocytes may play a key role in the pathogenesis of airway inflammation in bronchial asthma.

  20. Flow cytometric assessment of microbial abundance in the near-field area of seawater reverse osmosis concentrate discharge

    KAUST Repository

    Van Der Merwe, Riaan

    2014-06-01

    The discharge of concentrate and other process waters from seawater reverse osmosis (SWRO) plant operations into the marine environment may adversely affect water quality in the near-field area surrounding the outfall. The main concerns are the increase in salt concentration in receiving waters, which results in a density increase and potential water stratification near the outfall, and possible increases in turbidity, e.g., due to the discharge of filter backwash waters. Changes in ambient water quality may affect microbial abundance in the area, for example by hindering the photosynthesis process or disrupting biogenesis. It is widely accepted that marine biodiversity is lower in more extreme conditions, such as high salinity environments. As aquatic microbial communities respond very rapidly to changes in their environment, they can be used as indicators for monitoring ambient water quality. The objective of this study was to assess possible changes in microbial abundance as a result of concentrate discharge into the near-field area (<. 25. m) surrounding the outfall of the King Abdullah University of Science and Technology (KAUST) SWRO plant. Flow cytometric (FCM) analysis was conducted in order to rapidly determine microbial abundance on a single-cell level in 107 samples, taken by diving, from the discharge area, the intake area and two control sites. FCM analysis combined the measurement of distinct scatter of cells and particles, autofluorescence of cyanobacteria and algae, and fluorescence after staining of nucleic acids with SYBR® Green for a total bacterial count. The results indicate that changes in microbial abundance in the near-field area of the KAUST SWRO outfall are minor and appear to be the result of a dilution effect rather than a direct impact of the concentrate discharge. © 2014 Elsevier B.V.

  1. Flow cytometric bacterial cell counts challenge conventional heterotrophic plate counts for routine microbiological drinking water monitoring.

    Science.gov (United States)

    Van Nevel, S; Koetzsch, S; Proctor, C R; Besmer, M D; Prest, E I; Vrouwenvelder, J S; Knezev, A; Boon, N; Hammes, F

    2017-04-15

    Drinking water utilities and researchers continue to rely on the century-old heterotrophic plate counts (HPC) method for routine assessment of general microbiological water quality. Bacterial cell counting with flow cytometry (FCM) is one of a number of alternative methods that challenge this status quo and provide an opportunity for improved water quality monitoring. After more than a decade of application in drinking water research, FCM methodology is optimised and established for routine application, supported by a considerable amount of data from multiple full-scale studies. Bacterial cell concentrations obtained by FCM enable quantification of the entire bacterial community instead of the minute fraction of cultivable bacteria detected with HPC (typically water samples per day, depending on the laboratory and selected staining procedure(s). Moreover, many studies have shown FCM total (TCC) and intact (ICC) cell concentrations to be reliable and robust process variables, responsive to changes in the bacterial abundance and relevant for characterising and monitoring drinking water treatment and distribution systems. The purpose of this critical review is to initiate a constructive discussion on whether FCM could replace HPC in routine water quality monitoring. We argue that FCM provides a faster, more descriptive and more representative quantification of bacterial abundance in drinking water. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Evaluation of chromatin condensation in human spermatozoa: a flow cytometric assay using acridine orange staining.

    Science.gov (United States)

    Golan, R; Shochat, L; Weissenberg, R; Soffer, Y; Marcus, Z; Oschry, Y; Lewin, L M

    1997-01-01

    The quality of sperm chromatin is an important factor in fertilization and is especially critical where one spermatozoon is artificially selected for fertilizing an egg (as in intracytoplasmic sperm injection). In this study, flow cytometry after staining of human spermatozoa with Acridine Orange was used to study chromatin structure. A method is described for estimating the percentage of cells in a human sperm sample that have completed epididymal maturation in regard to chromatin condensation. Of the 121 samples of the semen that were examined, nine contained a higher percentage of hypocondensed spermatozoa and six samples contained elevated amounts of hypercondensed spermatozoa. In addition to aberrancies in chromatin condensation other defects showed up as satellite populations of spermatozoa with higher than normal ratios of red/green fluorescence after Acridine Orange staining. Such defects were found in 15 semen samples. The use of swim-up and Percoll gradient centrifugation methods was shown to improve the percentage of spermatozoa with normal chromatin structure in some samples with poor initial quality.

  3. Comparison of quantitative flow cytometric data provided by panels with lower and increased color number

    Science.gov (United States)

    Bocsi, József; Mittag, Anja; Pierzchalski, Arkadiusz; Baumgartner, Adolf; Dähnert, Ingo; Tárnok, Attila

    2012-03-01

    To date the flow cytometry (FCM) industry is booming with new generations of commercial clinical instruments. Long-term clinical studies have the dilemma that moving to new instruments being capable of more complex cell-analysis makes it difficult to compare new data with those obtained on older instruments with less complex analysis panels. Since 15 years we conduct follow-up studies on children with congenital heart diseases. In this period we moved from 2- to 3- and now to 10-color FCM immunophenotyping panels. Questions arise how to compare and transfer data from lower to higher level of complexity. Two comparable antibody panels for leukocyte immunophenotyping (12-tube 2-colors, and 9-tube 4-colors) were measured on a BD FACScalibur FCM (calibration: Spherotech beads) in 19 blood samples from children with congenital heart disease. This increase of colors was accompanied by moving antibodies that were in the 2-color panel either FITC or PE labeled to red dyes such as PerCP or APC. Algorithms were developed for bridging data for quantitative characterization of antigen expression (mean fluorescence intensity) and frequency of different cell subpopulations in combination with rainbow bead standard data. This approach worked for the most relevant antibodies (CD3, CD4, CD8 etc.) well, but rendered substantial uncertainty for activation markers (CD69 etc.). Our techniques are particularly well suited to the analysis in long-term studies and have the potential to compare older and recent results in a standardized way.

  4. Flow cytometric analysis of X-ray sensitivity in ataxia telangiectasia

    International Nuclear Information System (INIS)

    Rudolph, N.S.; Latt, S.A; Harvard Medical School, Boston

    1989-01-01

    Flow cytrometric analysis of 5-bromodeoxyuridine (BrdU) incorporation during DNA synthesis was used to characterize the effects of X-rays on cell-cycle kinetics in the DNA-repair deficiency disease ataxia telangiectasia (AT). Cultured fibroblasts from homozygotes (at/at), heterozygotes (at/+) and normal controls (+/+) were either: (1) irradiated, cultured, then pulsed with BrdU and harvested, or (2) pulsed with BrdU, irradiated, cultured and then harvested. Cells were then fixed and stained with both a fluoresceinated monoclonal antibody against BrdU to identify S-phase cells and with propidium diiodide to measure total DNA content. Irradiation of +/+ and at/+ cells induced a similar, transient G 2 /M arrest detectable within 8 h, which subsequently delayed by 6-8 h the passage of cells into G 1 and depleted early S phase. In contrast, at/at cells failed to arrest in G 2 /M phase and entered the next cell cycle withou pausing to repair radiation-induced damage. X-Rays also blocked entry of +/+ G 1 cells into S phase, subsequently reducing the total S-phase population. This effect was not observed in at/at cells. These cell-cycle responses to radiation may be of diagnostic use and ultimately may help explain the basic defect in AT. (author). 38 refs.; 7 figs.; 2 tabs

  5. Development of a novel flow cytometric approach to evaluate fish sperm chromatin using fixed samples

    Science.gov (United States)

    Jenkins, Jill A.

    2013-01-01

    The integrity of the paternal DNA is essential for the accurate transmission of genetic information, yet fertilization is not inhibited by chromatin breakage. Some methods are available for the sensitive detection of DNA damage and can be applied in studies of environmental toxicology, carcinogenesis, aging, and assisted reproduction techniques in both clinical and experimental settings. Because semen samples obtained from remote locations undergo chromatin damage prior to laboratory assessment, the present study was undertaken to evaluate treatments for effective chromatin staining in the development of a DNA fragmentation assay using fixed milt from yellow perch (Perca flavescens). Similar to the sperm chromatin structure assay (SCSA), susceptibility of nuclear DNA to acid-induced denaturation was measured by flow cytometry (FCM). Use of 10% buffered formalin for milt fixation allowed easier peak discrimination than 4% paraformaldehyde. The effects of time and temperature of incubation in 0.08 N HCl were evaluated in order to determine the ideal conditions for promoting DNA decondensation and making strand breaks more available for staining and detection by FCM. The best results were obtained with incubation at 37°C for 1 minute, followed by cold propidium iodide staining for 30 minutes.

  6. Flow cytometric investigation of immune-response-related surface molecules on human colorectal cancers

    DEFF Research Database (Denmark)

    Diederichsen, Axel Cosmus Pyndt; Stenholm, A C; Kronborg, O

    1998-01-01

    different colorectal cancers were monitored by multiparameter flow cytometry. Gating on EP4+ cells, the expression of the surface molecules HLA class I, HLA class II, CD80 (B7-1), CD54 (ICAM-I) and CD58 (LFA-3) was evaluated. In 60 of 70 tumours, all tumour cells expressed HLA class I, in 10 tumours 15......Our purpose was to clarify whether human colorectal cancer cells are equipped to present tumour-associated-antigens to the immune system, and whether this ability correlates with lymphoid infiltration, the Dukes' stage and Jass classification. Enzymatically dissociated tumour cells from 70......-96% of the tumour cells expressed HLA class I. In 1 tumour, all tumour cells expressed HLA class II, in 67 tumours some expressed HLA class II, in 2 tumours none expressed HLA class II. Expression of CD58 was heterogeneous, and there was no or only sparse expression of CD80 and CD54. Expression of the HLA class I...

  7. Flow cytometric analysis of microparticle phenotype and their role in thrombin generation.

    Science.gov (United States)

    Macey, M G; Enniks, N; Bevan, S

    2011-01-01

    Microparticles may be generated from a number of cell types and are known to play a role in haemostasis by a variety of mechanisms. We investigated the role of platelet, red cell, and leucocyte-derived microparticles in the measurement of thrombin generation. Four parameters of thrombin generation (the endogenous thrombin potential (ETP), lag time, time to peak, peak height) and microparticle content was determined in 35 plasma samples from normal individuals pre and post filtration to remove microparticles. Immunofluorescent flow cytometry was used to identify and enumerate platelet, leucocyte, monocyte and red cell derived microparticles in plasma samples based on the expression of CD42b, CD45, CD15, and Glycophorin A respectively. Expression of phosphatidylserine and tissue factor by microparticles was determined by Annexin V and anti CD142 binding. The pre and post filtration results were compared. There was a significant decrease in ETP and Peak Height, and an increase in the time to peak post filtration (P microparticles was also observed. The change in CD42b+ microparticles correlated highly with the change in Annexin V+ microparticles (r = 0.68). Whilst the change in ETP correlated best with the change in CD15+ microparticles (r = 0.45) and the change in time to peak correlated with the change in Annexin V binding (r = 0.52) (P < 0.01). The presence of micropartcles in plasma significantly affects thrombin generation. Copyright © 2010 International Clinical Cytometry Society.

  8. Flow cytometric studies of the survival of cytochalasin-induced polyploidy in Chinese hamster ovary cells.

    Science.gov (United States)

    Court, J B; Burn, C; Moore, J L

    1990-05-01

    A method has been developed to estimate the post-irradiation survival of cytochalasin B-induced polyploidization of adherent Chinese hamster ovary cell using the flow cytometer. After exposure to radiation, surviving cells are allowed to become polyploid in the presence of cytochalasin, and are detached using trypsin, fixed by the addition of glutaraldehyde and stained using mithramycin. DNA content distributions are polymodal, and the absolute number of cells per culture in any given ploidy class is estimated by reference to a non-fluorescent bead internal standard, detected using forward scatter. Post-irradiation survival is defined as the ability to reach a given DNA content, and is reduced exponentially with dose. A bioassay to determine optimum cytochalasin concentrations can be derived from the relative size of the 2C (G0/G1) peak in the DNA content distribution. At culture densities greater than about 8 x 10(4) cell/cm2 the relative number of cells reaching at least 16C is reduced, but this inhibition is partially reversible by an increase in the medium glucose concentration, but not by the use of cytochalasin D or dihydro B.

  9. Proposed criterion for distinguishing ABO mosaics from ABO chimeras using flow cytometric analysis.

    Science.gov (United States)

    Oda, Akira; Matsuyama, Nobuki; Hirashima, Mizuko; Ishii, Hiroyuki; Kimura, Keiko; Matsukura, Harumichi; Hirayama, Fumiya; Kawa, Keisei; Fukumori, Yasuo

    2015-01-01

    Differentiation of ABO mosaics from chimeras is performed using flow cytometry (FCM) analysis. Although mosaics and chimeras have been distinguished by presence or absence of clear resolution using FCM analysis, the lack of quantitative metrics and definitive criteria for this differentiation has made some cases difficult to differentiate. In this study, therefore, we attempted to establish a definitive and quantitative criterion for this differentiation. When FCM histogram gates for group "A" or "B" antigen-negative and -positive red blood cells (RBCs) were set such that group O RBCs were classified as 99 percent negative and group A or B RBCs as 99 percent positive, the percentages of RBCs in the middle region of six chimeras and 23 mosaics (12 A mosaics and 11 B mosaics) were 0.1-0.6 percent and 7.0-19.0 percent, respectively. This results suggested that ABO mosaics and chimeras can be unambiguously differentiated when the cutoff point of the intermediate region is set to 1 percent.

  10. Chemosensitivity of human small cell carcinoma of the lung detected by flow cytometric DNA analysis of drug-induced cell cycle perturbations in vitro

    DEFF Research Database (Denmark)

    Engelholm, S A; Spang-Thomsen, M; Vindeløv, L L

    1986-01-01

    A method based on detection of drug-induced cell cycle perturbation by flow cytometric DNA analysis has previously been described in Ehrlich ascites tumors as a way to estimate chemosensitivity. The method is extended to test human small-cell carcinoma of the lung. Three tumors with different...... sensitivities to melphalan in nude mice were used. Tumors were disaggregated by a combined mechanical and enzymatic method and thereafter have incubated with different doses of melphalan. After incubation the cells were plated in vitro on agar, and drug induced cell cycle changes were monitored by flow...

  11. Monitoring microbiological changes in drinking water systems using a fast and reproducible flow cytometric method

    KAUST Repository

    Prest, Emmanuelle I E C

    2013-12-01

    Flow cytometry (FCM) is a rapid, cultivation-independent tool to assess and evaluate bacteriological quality and biological stability of water. Here we demonstrate that a stringent, reproducible staining protocol combined with fixed FCM operational and gating settings is essential for reliable quantification of bacteria and detection of changes in aquatic bacterial communities. Triplicate measurements of diverse water samples with this protocol typically showed relative standard deviation values and 95% confidence interval values below 2.5% on all the main FCM parameters. We propose a straightforward and instrument-independent method for the characterization of water samples based on the combination of bacterial cell concentration and fluorescence distribution. Analysis of the fluorescence distribution (or so-called fluorescence fingerprint) was accomplished firstly through a direct comparison of the raw FCM data and subsequently simplified by quantifying the percentage of large and brightly fluorescent high nucleic acid (HNA) content bacteria in each sample. Our approach enables fast differentiation of dissimilar bacterial communities (less than 15min from sampling to final result), and allows accurate detection of even small changes in aquatic environments (detection above 3% change). Demonstrative studies on (a) indigenous bacterial growth in water, (b) contamination of drinking water with wastewater, (c) household drinking water stagnation and (d) mixing of two drinking water types, univocally showed that this FCM approach enables detection and quantification of relevant bacterial water quality changes with high sensitivity. This approach has the potential to be used as a new tool for application in the drinking water field, e.g. for rapid screening of the microbial water quality and stability during water treatment and distribution in networks and premise plumbing. © 2013 Elsevier Ltd.

  12. Flow-cytometric study of vital cellular functions in Escherichia coli during solar disinfection (SODIS).

    Science.gov (United States)

    Berney, Michael; Weilenmann, Hans-Ulrich; Egli, Thomas

    2006-06-01

    The effectiveness of solar disinfection (SODIS), a low-cost household water treatment method for developing countries, was investigated with flow cytometry and viability stains for the enteric bacterium Escherichia coli. A better understanding of the process of injury or death of E. coli during SODIS could be gained by investigating six different cellular functions, namely: efflux pump activity (Syto 9 plus ethidium bromide), membrane potential [bis-(1,3-dibutylbarbituric acid)trimethine oxonol; DiBAC4(3)], membrane integrity (LIVE/DEAD BacLight), glucose uptake activity (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose; 2-NBDG), total ATP concentration (BacTiter-Glo) and culturability (pour-plate method). These variables were measured in E. coli K-12 MG1655 cells that were exposed to either sunlight or artificial UVA light. The inactivation pattern of cellular functions was very similar for both light sources. A UVA light dose (fluence) of 80 % of the cells was observed at a fluence of approximately 1500 kJ m(-2), and the cytoplasmic membrane of bacterial cells became permeable at a fluence of >2500 kJ m(-2). Culturable counts of stressed bacteria after anaerobic incubation on sodium pyruvate-supplemented tryptic soy agar closely correlated with the loss of membrane potential. The results strongly suggest that cells exposed to >1500 kJ m(-2) solar UVA (corresponding to 530 W m(-2) global sunlight intensity for 6 h) were no longer able to repair the damage and recover. Our study confirms the lethal effect of SODIS with cultivation-independent methods and gives a detailed picture of the 'agony' of E. coli when it is stressed with sunlight.

  13. Flow cytometric assay for analysis of cytotoxic effects of potential drugs on human peripheral blood leukocytes

    Science.gov (United States)

    Nieschke, Kathleen; Mittag, Anja; Golab, Karolina; Bocsi, Jozsef; Pierzchalski, Arkadiusz; Kamysz, Wojciech; Tarnok, Attila

    2014-03-01

    Toxicity test of new chemicals belongs to the first steps in the drug screening, using different cultured cell lines. However, primary human cells represent the human organism better than cultured tumor derived cell lines. We developed a very gentle toxicity assay for isolation and incubation of human peripheral blood leukocytes (PBL) and tested it using different bioactive oligopeptides (OP). Effects of different PBL isolation methods (red blood cell lysis; Histopaque isolation among others), different incubation tubes (e.g. FACS tubes), anticoagulants and blood sources on PBL viability were tested using propidium iodide-exclusion as viability measure (incubation time: 60 min, 36°C) and flow cytometry. Toxicity concentration and time-depended effects (10-60 min, 36 °C, 0-100 μg /ml of OP) on human PBL were analyzed. Erythrocyte lysis by hypotonic shock (dH2O) was the fastest PBL isolation method with highest viability (>85%) compared to NH4Cl-Lysis (49%). Density gradient centrifugation led to neutrophil granulocyte cell loss. Heparin anticoagulation resulted in higher viability than EDTA. Conical 1.5 mL and 2 mL micro-reaction tubes (both polypropylene (PP)) had the highest viability (99% and 97%) compared to other tubes, i.e. three types of 5.0 mL round-bottom tubes PP (opaque-60%), PP (blue-62%), Polystyrene (PS-64%). Viability of PBL did not differ between venous and capillary blood. A gentle reproducible preparation and analytical toxicity-assay for human PBL was developed and evaluated. Using our assay toxicity, time-course, dose-dependence and aggregate formation by OP could be clearly differentiated and quantified. This novel assay enables for rapid and cost effective multiparametric toxicological screening and pharmacological testing on primary human PBL and can be adapted to high-throughput-screening.°z

  14. Flow cytometric comparison of the effects of trialkyltins on the murine erythroleukemic cell.

    Science.gov (United States)

    Zucker, R M; Elstein, K H; Easterling, R E; Massaro, E J

    1989-10-02

    Cellular effects of exposure to tributyltin (TBT), triethyltin (TET), or trimethyltin (TMT) were investigated by flow cytometry employing the murine erythroleukemic cell (MELC) as a model cellular system. Cell viability was investigated by the carboxyfluorescein diacetate (CFDA) uptake/propidium iodide (PI) exclusion method: above a critical concentration (exposure for 4 h), which was specific for each of the trialkyltin compounds, the cell becomes permeable to PI, indicating loss of viability. Cellular CF fluorescence (derived from intracellular hydrolysis of CFDA) increased as a function of alkyltin concentration below the critical concentration and decreased as viability decreased above the critical concentration. Relative membrane potential, monitored with a cyanine dye (DiOC6), correlated with viability (PI exclusion), remaining essentially unaltered below the critical concentration and decreasing above it. At/above 1 microM TBT, 5 microM TET, or 100 microM TMT, the cell cycle was blocked in the G2/M phase. The 90 degrees light scatter (a measure of refractive index), axial light loss (a measure of volume), and fluorescein isothiocyanate (FITC) fluorescence (a measure of protein content) of nuclei isolated from trialkyltin-treated MELC by detergent treatment, increased as a function of organotin dose. Fluorescence and interference microscopy revealed increased quantities of residual cytoplasmic tags adherent to the nuclei as a function of organotin dose, apparently resulting from increased cytoplasmic resistance to detergent-mediated solubilization. The effects of the trialkyltins correlated with their lipophilicity (octanol/water coefficient). These data support the hypothesis that fixation (protein denaturation, cross-linking, etc.) is an important mode of organotin cytotoxicity.

  15. Flow Cytometric Analysis of Leishmania Reactive CD4+/CD8+ Lymphocyte Proliferation in Cutaneous Leishmaniasis

    Directory of Open Access Journals (Sweden)

    H Keshavarz

    2008-12-01

    Full Text Available Background: Determination of the division history of T cells in vitro is helpful in the study of effector mechanisms against infections. Technique described here uses the intracellular fluorescent label carboxyfluorescein diacetate succinimidyl ester (CFSE to monitor the proliferation. Methods: In a cross sectional study, blood samples were collected from 7 volunteers with history of cutaneous leishmania­sis (CL and one healthy control from endemic areas in Isfahan province who referred to the Center for Research and Training in Skin Diseases and Leprosy (CRTSDL, then CD4+/CD8+ lymphocytes and CD14+ monocytes were isolated from peri­pheral blood mononuclear cells (PBMC using mAbs and magnetic nanoparticles. CFSE labeled CD4+ or CD8+ lympho­cytes cultured with autologous monocytes in the presence of PHA, SLA, live Leishmania major or as control with­out sti­mulation. Cells were harvested after 7 days and were analyzed using flow cytometry. Results: Five consecutive divisions were monitored separately. Stimulation of CD4+ or CD8+ lymphocytes from CL sub­jects with SLA showed a significant difference in proliferation comparing with unstimulated cells (P< 0.05. The signifi­cant difference in the percentages of CD4+ cells stimulated with SLA was revealed at different divisions for each subject. In CD8+ lymphocyte, significant stronger stimulation of SLA was evident later in the proliferation process. The mean number of divisions in both CD4+/CD8+ lymphocytes stimulated with SLA was significantly greater than when stimulated with live L. major (P=0.007 / P=0.012, respectively Conclusion: The percentage of divided cells might be calculated separately in each division. The cells remained active following CFSE staining and there is possibility of functional analysis simultaneously.

  16. Flow-cytometric assessment of cellular poly(ADP-ribosylation capacity in peripheral blood lymphocytes

    Directory of Open Access Journals (Sweden)

    Hyland Paul

    2006-07-01

    Full Text Available Abstract Background Poly(ADP-ribosylation is a posttranslational modification of nuclear proteins catalysed by poly(ADP-ribose polymerases (PARPs, using NAD+ as a substrate. Activation of PARP-1 is in immediate response to DNA damage generated by endogenous and exogenous damaging agents. It has been implicated in several crucial cellular processes including DNA repair and maintenance of genomic stability, which are both intimately linked with the ageing process. The measurement of cellular poly(ADP-ribosylation capacity, defined as the amount of poly(ADP-ribose produced under maximal stimulation, is therefore relevant for research on ageing, as well as for a variety of other scientific questions. Results This paper reports a new, robust protocol for the measurement of cellular poly(ADP-ribosylation capacity in PBMC or Jurkat T-cells using flow cytometry, based on a previously established immuno-dot-blot assay. In order to validate the new assay, we determined the dose-response curve of 3-aminobenzamide, a well-known competitive PARP inhibitor, and we derived an IC50 that is very close to the published value. When testing a set of PBMC samples taken from fifteen healthy young human donors, we could confirm the presence of a substantial interindividual variation, as previously observed using a radiometric assay. Conclusion The methodology described in this paper should be generally useful for the determination of cellular poly(ADP-ribosylation capacity in a wide variety of settings, especially for the comparison of large sets of samples, such as population studies. In contrast to previously published radiometric or immuno-dot-blot assays, the new FACS-based method allows (i selective analysis of mononuclear cells by gating and (ii detection of a possible heterogeneity in poly(ADP-ribosylation capacity between cells of the same type.

  17. Dysregulated angiogenesis in B-chronic lymphocytic leukemia: Morphologic, immunohistochemical, and flow cytometric evidence

    Directory of Open Access Journals (Sweden)

    Crawford Susan E

    2008-04-01

    Full Text Available Abstract Background The extent of enhanced bone marrow angiogenesis in chronic lymphocytic leukemia (CLL and relationship to proangiogenic factors and prognostic indicators is largely unexplored. Methods To further investigate the role of angiogenesis in CLL by evaluating the topography and extent of angiogenesis in a group of CLL bone marrow biopsies, to study the expression of pro and antiangiogenic vascular factors in CLL cells to more precisely document the cell types producing these factors, and to evaluate the role, if any, of localized hypoxia in upregulation of angiogenesis in CLL We used immunohistochemistry (IHC (n = 21 pts with antibodies to CD3 and CD20, proangiogenic (VEGF, HIF-1a and antiangiogenic (TSP-1 factors, and VEGF receptors -1 and -2 to examine pattern/extent of CLL marrow involvement, microvessel density (MVD, and angiogenic characteristics; flow cytometry (FC was performed on 21 additional cases for VEGF and TSP-1. Results CLL patients had higher MVD (23.8 vs 14.6, p~0.0002 compared to controls (n = 10. MVD was highest at the periphery of focal infiltrates, was not enhanced in proliferation centers, and was increased irrespective of the presence or absence of cytogenetic/immunophenotypic markers of aggressivity. By IHC, CLL cells were VEGF(+, HIF-1a (+, TSP-1(-, VEGFR-1(+, and VEGFR-2(+. By FC, CLL cells were 1.4–2.0-fold brighter for VEGF than T cells and were TSP-1(-. Conclusion CLL demonstrates enhanced angiogenesis, with increased MVD, upregulated VEGF and downregulated TSP-1. Upregulation of HIF-1a in all CLL cases suggests localized tissue hypoxia as an important stimulant of microvessel proliferation. The presence of VEGF receptors on CLL cells implies an autocrine effect for VEGF. Differences in MVD did not correlate with traditional genetic/immunophenotypic markers of aggressiveness.

  18. Flow cytometric bacterial cell counts challenge conventional heterotrophic plate counts for routine microbiological drinking water monitoring

    KAUST Repository

    Van Nevel, S.

    2017-02-08

    Drinking water utilities and researchers continue to rely on the century-old heterotrophic plate counts (HPC) method for routine assessment of general microbiological water quality. Bacterial cell counting with flow cytometry (FCM) is one of a number of alternative methods that challenge this status quo and provide an opportunity for improved water quality monitoring. After more than a decade of application in drinking water research, FCM methodology is optimised and established for routine application, supported by a considerable amount of data from multiple full-scale studies. Bacterial cell concentrations obtained by FCM enable quantification of the entire bacterial community instead of the minute fraction of cultivable bacteria detected with HPC (typically < 1% of all bacteria). FCM measurements are reproducible with relative standard deviations below 3% and can be available within 15 min of samples arriving in the laboratory. High throughput sample processing and complete automation are feasible and FCM analysis is arguably less expensive than HPC when measuring more than 15 water samples per day, depending on the laboratory and selected staining procedure(s). Moreover, many studies have shown FCM total (TCC) and intact (ICC) cell concentrations to be reliable and robust process variables, responsive to changes in the bacterial abundance and relevant for characterising and monitoring drinking water treatment and distribution systems. The purpose of this critical review is to initiate a constructive discussion on whether FCM could replace HPC in routine water quality monitoring. We argue that FCM provides a faster, more descriptive and more representative quantification of bacterial abundance in drinking water.

  19. Color encoded microbeads-based flow cytometric immunoassay for polycyclic aromatic hydrocarbons in food

    International Nuclear Information System (INIS)

    Meimaridou, Anastasia; Haasnoot, Willem; Noteboom, Linda; Mintzas, Dimitrios; Pulkrabova, Jana; Hajslova, Jana; Nielen, Michel W.F.

    2010-01-01

    Food contamination caused by chemical hazards such as persistent organic pollutants (POPs) is a worldwide public health concern and requires continuous monitoring. The chromatography-based analysis methods for POPs are accurate and quite sensitive but they are time-consuming, laborious and expensive. Thus, there is a need for validated simplified screening tools, which are inexpensive, rapid, have automation potential and can detect multiple POPs simultaneously. In this study we developed a flow cytometry-based immunoassay (FCIA) using a color-encoded microbeads technology to detect benzo[a]pyrene (BaP) and other polycyclic aromatic hydrocarbons (PAHs) in buffer and food extracts as a starting point for the future development of rapid multiplex assays including other POPs in food, such as polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs). A highly sensitive assay for BaP was obtained with an IC 50 of 0.3 μg L -1 using a monoclonal antibody (Mab22F12) against BaP, similar to the IC 50 of a previously described enzyme-linked immunosorbent assay (ELISA) using the same Mab. Moreover, the FCIA was 8 times more sensitive for BaP compared to a surface plasmon resonance (SPR)-based biosensor immunoassay (BIA) using the same reagents. The selectivity of the FCIAs was tested, with two Mabs against BaP for 25 other PAHs, including two hydroxyl PAH metabolites. Apart from BaP, the FCIAs can detect PAHs such as indenol[1,2,3-cd]pyrene (IP), benz[a]anthracene (BaA), and chrysene (CHR) which are also appointed by the European Food Safety Authority (EFSA) as suitable indicators of PAH contamination in food. The FCIAs results were in agreement with those obtained with gas chromatography-mass spectrometry (GC-MS) for the detection of PAHs in real food samples of smoked carp and wheat flour and has great potential for the future routine application of this assay in a simplex or multiplex format in combination with simplified extraction procedure which are

  20. Development of gammabeta thymocyte subsets during prenatal and postnatal ontogeny: a flow cytometric study in miniature pigs

    Czech Academy of Sciences Publication Activity Database

    Šinkora, Marek; Šinkorová, J.

    2003-01-01

    Roč. 87, č. 1 (2003), s. 101 ISSN 0165-2478 R&D Projects: GA ČR GA524/01/0919 Institutional research plan: CEZ:AV0Z5020903 Keywords : thymocyte subsets * cytometric Subject RIV: EE - Microbiology, Virology Impact factor: 1.710, year: 2003

  1. Flow Cytometric Quantification of Peripheral Blood Cell β-Adrenergic Receptor Density and Urinary Endothelial Cell-Derived Microparticles in Pulmonary Arterial Hypertension.

    Directory of Open Access Journals (Sweden)

    Jonathan A Rose

    Full Text Available Pulmonary arterial hypertension (PAH is a heterogeneous disease characterized by severe angiogenic remodeling of the pulmonary artery wall and right ventricular hypertrophy. Thus, there is an increasing need for novel biomarkers to dissect disease heterogeneity, and predict treatment response. Although β-adrenergic receptor (βAR dysfunction is well documented in left heart disease while endothelial cell-derived microparticles (Ec-MPs are established biomarkers of angiogenic remodeling, methods for easy large clinical cohort analysis of these biomarkers are currently absent. Here we describe flow cytometric methods for quantification of βAR density on circulating white blood cells (WBC and Ec-MPs in urine samples that can be used as potential biomarkers of right heart failure in PAH. Biotinylated β-blocker alprenolol was synthesized and validated as a βAR specific probe that was combined with immunophenotyping to quantify βAR density in circulating WBC subsets. Ec-MPs obtained from urine samples were stained for annexin-V and CD144, and analyzed by a micro flow cytometer. Flow cytometric detection of alprenolol showed that βAR density was decreased in most WBC subsets in PAH samples compared to healthy controls. Ec-MPs in urine was increased in PAH compared to controls. Furthermore, there was a direct correlation between Ec-MPs and Tricuspid annular plane systolic excursion (TAPSE in PAH patients. Therefore, flow cytometric quantification of peripheral blood cell βAR density and urinary Ec-MPs may be useful as potential biomarkers of right ventricular function in PAH.

  2. Flow cytometric cell sorting and in vitro pre-osteoinduction are not requirements for in vivo bone formation by human adipose-derived stromal cells.

    Science.gov (United States)

    Liu, Yunsong; Zhao, Yan; Zhang, Xiao; Chen, Tong; Zhao, Xianghui; Ma, Gui-e; Zhou, Yongsheng

    2013-01-01

    Human adipose-derived stromal cells (hASCs) are a promising cell source for bone tissue engineering. However, before the clinical application of hASCs for the treatment of bone defects, key questions require answers, including whether pre-osteoinduction (OI) and flow cytometric cell purification are indispensible steps for in vivo bone formation by hASCs. In this study, hASCs were purified by flow cytometric cell sorting (FCCS). The osteogenic capabilities of hASCs and purified hASCs with or without pre-osteoinduction were examined through in vitro and in vivo experiments. We found that pre-OI enhanced the in vitro osteogenic capacity of hASCs. However, 8 weeks after in vivo implantation, there were no significant differences between hASCs and hASCs that had undergone OI (hASCs+OI) or between purified hASCs and purified hASCs+OI (P>0.05). Interestingly, we also found that purified hASCs had an osteogenic potential similar to that of unpurified hASCs in vitro and in vivo. These results suggest that FCCS and in vitro pre-OI are not requirements for in vivo bone formation by hASCs.

  3. Flow cytometric cell sorting and in vitro pre-osteoinduction are not requirements for in vivo bone formation by human adipose-derived stromal cells.

    Directory of Open Access Journals (Sweden)

    Yunsong Liu

    Full Text Available Human adipose-derived stromal cells (hASCs are a promising cell source for bone tissue engineering. However, before the clinical application of hASCs for the treatment of bone defects, key questions require answers, including whether pre-osteoinduction (OI and flow cytometric cell purification are indispensible steps for in vivo bone formation by hASCs. In this study, hASCs were purified by flow cytometric cell sorting (FCCS. The osteogenic capabilities of hASCs and purified hASCs with or without pre-osteoinduction were examined through in vitro and in vivo experiments. We found that pre-OI enhanced the in vitro osteogenic capacity of hASCs. However, 8 weeks after in vivo implantation, there were no significant differences between hASCs and hASCs that had undergone OI (hASCs+OI or between purified hASCs and purified hASCs+OI (P>0.05. Interestingly, we also found that purified hASCs had an osteogenic potential similar to that of unpurified hASCs in vitro and in vivo. These results suggest that FCCS and in vitro pre-OI are not requirements for in vivo bone formation by hASCs.

  4. The evaluation of histo-blood group ABO typing by flow cytometric and PCR-amplification of specific alleles analyses and their application in clinical laboratories.

    Science.gov (United States)

    Aki, Kensaku; Izumi, Azusa; Hosoi, Eiji

    2012-01-01

    ABO antigens are oligosaccharide antigens, and are widely distributed on red blood and tissue cells as well as in saliva and body fluid. Therefore, these antigens are important not only for blood transfusion, but also for tissue cell and organ transplantations. Also, blood, hair, and seminal fluid are important sources of evidence at crime scenes, and these antigens are some of the most important markers for personal identification in forensic investigations. Here, we describe the development and use of quantitative analysis of A, B, and H antigens on red blood cells by employing flow cytometric analysis and the ABO genotyping method based on PCR-amplification of specific alleles (PASA) within DNA, especially from blood and saliva. In this study, flow cytometric analysis could be used to compare the differences between the expression of A and/or B and H antigens on red blood cells with various phenotypes, and the PASA method was able to determine the genotype of the type cisA(2)B(3) pedigree using only DNA extracted from saliva. These analysis methods are simple and useful for judging the ABO blood group system and genotyping, and are used widely throughout research and clinical laboratories and forensic fields.

  5. Development of a flow cytometric bead immunoassay and its assessment as a possible aid to potency evaluation of enterotoxaemia vaccines

    Directory of Open Access Journals (Sweden)

    Angela Buys

    2014-03-01

    Full Text Available Enterotoxaemia, an economically important disease of sheep, goats and calves, is caused by systemic effects of the epsilon toxin produced by the anaerobic bacterium Clostridium perfringens type D. The only practical means of controlling the occurrence of enterotoxaemia is to immunise animals by vaccination. The vaccine is prepared by deriving a toxoid from the bacterial culture filtrate and the potency of the vaccine is tested with the in vivo mouse neutralisation test (MNT. Due to ethical, economic and technical reasons, alternative in vitro assays are needed. In this study an indirect cytometric bead immunoassay (I-CBA was developed for use in vaccine potency testing and the results were compared with those obtained using an indirect enzyme-linked immunosorbent assay (I-ELISA and the MNT. Sera were collected from guinea pigs immunised with three different production batches of enterotoxaemia vaccine and the levels of anti-epsilon toxin antibodies were determined. Although the intra- and inter-assay variability was satisfactory, epsilon antitoxin levels determined by both the I-ELISA and indirect cytometric bead immunoassay (I-CBA tests were higher than those of the MNT assay. In contrast to the MNT, all of the serum samples were identified as having antitoxin levels above the required minimum (not less than 5 U/mL. These results indicate that the respective in vitro tests in their current formats are not yet suitable alternatives to the in vivo MNT. The growing demand for a more humane, cost-effective and efficient method for testing the potency of enterotoxaemia vaccines, however, provides a strong impetus for further optimisation and standardisation of the I-CBA assay but further analytical research is required.

  6. Effect of 17 beta-oestradiol on growth curves and flow cytometric DNA distribution of two human breast carcinomas grown in nude mice

    DEFF Research Database (Denmark)

    Brünner, N; Spang-Thomsen, M; Vindeløv, L

    1983-01-01

    The effect of 17 beta-oestradiol on a "receptor positive" and on a "receptor negative" human breast carcinoma grown in nude mice was studied. Experimental growth data were used to determine the effect on tumour growth. Flow cytometric DNA analysis (FCM) performed on tumour tissue obtained...... by sequential fine-needle aspirations was used to estimate the effect on the cell cycle. In the receptor-positive breast carcinoma, oestradiol induced complete tumour regression and characteristic cell cycle changes. In the receptor-negative breast carcinoma, no changes in tumour growth and cell cycle...... prior to any reduction in the tumour size, the results suggest that FCM may prove a valuable method in the early detection of tumour response to hormone treatment in human breast cancer....

  7. Japanese Society for Laboratory Hematology flow cytometric reference method of determining the differential leukocyte count: external quality assurance using fresh blood samples.

    Science.gov (United States)

    Kawai, Y; Nagai, Y; Ogawa, E; Kondo, H

    2017-04-01

    To provide target values for the manufacturers' survey of the Japanese Society for Laboratory Hematology (JSLH), accurate standard data from healthy volunteers were needed for the five-part differential leukocyte count. To obtain such data, JSLH required an antibody panel that achieved high specificity (particularly for mononuclear cells) using simple gating procedures. We developed a flow cytometric method for determining the differential leukocyte count (JSLH-Diff) and validated it by comparison with the flow cytometric differential leukocyte count of the International Council for Standardization in Haematology (ICSH-Diff) and the manual differential count obtained by microscopy (Manual-Diff). First, the reference laboratory performed an imprecision study of JSLH-Diff and ICSH-Diff, as well as performing comparison among JSLH-Diff, Manual-Diff, and ICSH-Diff. Then two reference laboratories and seven participating laboratories performed imprecision and accuracy studies of JSLH-Diff, Manual-Diff, and ICSH-Diff. Simultaneously, six manufacturers' laboratories provided their own representative values by using automated hematology analyzers. The precision of both JSLH-Diff and ICSH-Diff methods was adequate. Comparison by the reference laboratory showed that all correlation coefficients, slopes and intercepts obtained by the JSLH-Diff, ICSH-Diff, and Manual-Diff methods conformed to the criteria. When the imprecision and accuracy of JSLH-Diff were assessed at seven laboratories, the CV% for lymphocytes, neutrophils, monocytes, eosinophils, and basophils was 0.5~0.9%, 0.3~0.7%, 1.7~2.6%, 3.0~7.9%, and 3.8~10.4%, respectively. More than 99% of CD45 positive leukocytes were identified as normal leukocytes by JSLH-Diff. When JSLH-Diff method were validated by comparison with Manual-Diff and ICSH-Diff, JSLH-Diff showed good performance as a reference method. © 2016 John Wiley & Sons Ltd.

  8. Flow cytometric method for enumeration and characterization of newly released polymorphonuclear leukocytes from the bone marrow using 5'-bromo-2'-deoxyuridine.

    Science.gov (United States)

    Shih, Chih-Horng; Whalen, Beth A; Goto, Yukinobu; Hogg, James C; van Eeden, Stephan F

    2005-09-01

    Inflammation accelerates polymorphonuclear leukocyte (PMN) release from the bone marrow, and these PMNs are implicated in inappropriate tissue injury. We have previously developed a method using 5'-bromo-2'-deoxyuridine (BrdU) to study PMN kinetics using an immunocytochemical grading system of PMN on cytospin slides. The aim of this study was to develop a flow cytometric method to quantify the number of positively stained PMN and grade the intensity of staining for the transit time calculation of PMN through the marrow. Dividing myeloid progenitors in the marrow of rabbits were labeled with a pulse dosage of intravenous BrdU. BrdU-labeled PMN (PMN(BrdU)) were detected in the circulation using a FITC-conjugated anti-BrdU monoclonal antibody. The PMN(BrdU) were assigned to five groups according to their FITC intensity, and the transit times of PMN at different stages of development in the marrow were calculated. Results were compared using parallel immunocytochemical analysis of the same samples. In control animals, PMN(BrdU) in the circulation peaked at 72 h after BrdU labeling with 36.0% of PMN labeled. In normal rabbits, the transit times of PMN through the mitotic pool (49.5 +/- 4.2 h) and maturation pool (65.5 +/- 3.1 h) correlated well with immunocytochemical analysis and previously published values. Using this method, we demonstrated that exposure to air pollution particles accelerates the release of PMN(BrdU) from the marrow. We conclude that a flow cytometric approach for identifying BrdU-labeled leukocytes provides an objective and accurate method for studying leukocyte kinetics and behavior.

  9. Fluorescence in situ hybridization and sequential catalysed reporter deposition (2C-FISH for the flow cytometric sorting of freshwater ultramicrobacteria

    Directory of Open Access Journals (Sweden)

    Stefan M Neuenschwander

    2015-03-01

    Full Text Available Flow cytometric sorting is a powerful tool to physically separate cells within mixed microbial communities. If combined with phylogenetic staining (fluorescence in situ hybridization, FISH it allows to specifically sort defined genotypic microbial populations from complex natural samples. However, the targeted enrichment of freshwater ultramicrobacteria, such as members of the LD12 clade of Alphaproteobacteria (SAR11-IIIb, is still challenging. Current FISH protocols, even in combination with signal amplification by catalysed reporter deposition (CARD, are not sufficiently sensitive for the distinction of these bacteria from background noise by flow cytometry, presumably due to their low ribosome content and small cell sizes. We, therefore, modified a CARD based flow sorting protocol with the aim of increasing its sensitivity to a level sufficient for ultramicrobacteria. This was achieved by a second signal amplification step mediated by horseradish peroxidase labelled antibodies targeted to the fluorophores that were previously deposited by CARD-FISH staining. The protocol was tested on samples from an oligo-mesotrophic lake. Ultramicrobacteria affiliated with LD12 Alphaproteobacteria could be successfully sorted to high purity by flow cytometry. The ratios of median fluorescence signal to background ranged around 20, and hybridization rates determined by flow cytometry were comparable to those obtained by fluorescence microscopy. Potential downstream applications of our modified cell staining approach range from the analysis of microdiversity within 16S rRNA-defined populations to that of functional properties, such as the taxon-specific incorporation rates of organic substrates.

  10. Effect of acidic pH on flow cytometric detection of bacteria stained with SYBR Green I and their distinction from background

    International Nuclear Information System (INIS)

    Baldock, Daniel; Nocker, Andreas; Nebe-von-Caron, Gerhard; Bongaerts, Roy

    2013-01-01

    Unspecific background caused by biotic or abiotic particles, cellular debris, or autofluorescence is a well-known interfering parameter when applying flow cytometry to the detection of microorganisms in combination with fluorescent dyes. We present here an attempt to suppress the background signal intensity and thus to improve the detection of microorganisms using the nucleic acid stain SYBR ® Green I. It has been observed that the fluorescent signals from SYBR Green I are greatly reduced at acidic pH. When lowering the pH of pre-stained samples directly prior to flow cytometric analysis, we hypothesized that the signals from particles and cells with membrane damage might therefore be reduced. Signals from intact cells, temporarily maintaining a neutral cytosolic pH, should not be affected. We show here that this principle holds true for lowering background interference, whereas the signals of membrane-compromised dead cells are only affected weakly. Signals from intact live cells at low pH were mostly comparable to signals without acidification. Although this study was solely performed with SYBR ® Green I, the principle of low pH flow cytometry (low pH-FCM) might hold promise when analyzing complex matrices with an abundance of non-cellular matter, especially when expanded to non-DNA binding dyes with a stronger pH dependence of fluorescence than SYBR Green I and a higher pK a value. (paper)

  11. Flow cytometric analysis of p21 protein expression on irradiated human lymphocytes; Analise por citometria de fluxo da expressao da proteina p21 em linfocitos humanos irradiados

    Energy Technology Data Exchange (ETDEWEB)

    Santos, N.F.G.; Amaral, A., E-mail: neyliane@gmail.com [Universidade Federal de Pernambuco (UFPE), Recife, PE (Brazil). Departamento de Energia Nuclear. Laboratorio de Modelagem e Biodosimetria Aplicada; Freitas-Silva, R. [Universidade Federal de Pernambuco (UFPE), Garanhuns, PE (Brazil). Departamento de Ciencias Naturais e Exatas; Pereira, V.R.A. [Fundacao Oswaldo Cruz (FIOCRUZ), Recife, PE (Brazil). Centro de Pesquisas Aggeu Magalhaes. Departamento de Imunologia. Lab. de Imunoparasitologia; Tasat, D.R. [Universidad Nacional de General San Martin, Buenos Aires (Argentina). Escuela de Ciencia y Tecnologia. Laboratorio de Biologia Celular del Pulmon

    2013-08-15

    Cell cycle blockage in G1 is a mechanism p21 protein-regulated and coupled to DNA damage response to permit genetic content analysis, damage repair and cell death. Analysis of proteins that participates of this response has progressed with new analytic tools, and data contributes to comprehension of radioinduced molecular events as well as to new approaches on practices that employ ionizing radiation. On this perspective, the aim of this research was to evaluate, by flow cytometry, p21 expression on irradiated human lymphocytes, maintained under different experimental conditions. Peripheral blood samples from 10 healthy subjects were irradiated with doses of 0 (non-irradiated), 1, 2 and 4 Gy. Lymphocytes were processed to analysis on ex vivo (no cultured) condition and after 24; 48 and 72 hours culture, with and without phytohemagglutinin stimulation. p21 protein expression levels were measured by flow cytometry, as percentage values. Results indicate that flow cytometric assay allows detection of changes on p21 expression, since it was detected significant increase on phytohemagglutinin-stimulated samples, for all times, against basal expression (ex vivo). However, it was not observed significant alterations on p21 protein radioinduced levels, for all doses, times and culture conditions analyzed. These results not indicate so p21 protein as bioindicator of ionizing radiation exposure. Nevertheless, data confirmation may to require analysis of a more numerous population. (author)

  12. Importance of the autocontrol crossmatch in human renal transplantation.

    Science.gov (United States)

    Cross, D E; Greiner, R; Whittier, F C

    1976-04-01

    The killing of donor cells in the standard lymphocyte crossmatch is considered strong evidence for preformed antibodies in the recipients's serum. Moreover, it is generally accepted that presensitization has occurred if any of the stored sera kill the donor cells. In our hands, if either the current or the stored sera kill the donor cells, it precludes transplantation. In nine cases we discovered that the recipient's sera also killed the recipient's own lymphocytes, a positive autocontrol test, indicating that factors other than conventional preformed cytotoxic antibodies were responsible for the positive standard crossmatch. The nine patients who demonstrated a positive standard crossmatch and a positive autocontrol for those sera received cadaver allografts. None of the kidneys were rejected hyperacutely and all are functioning adequately. We conclude that the autocontrol crossmatch is an important adjunct for uncovering false positive reactions in the standard lymphocyte crossmatch test.

  13. Simple flow cytometric protocol of CD4+/CD8+ lymphocyte ratio assessment in bronchoalveolar lavage fluids from patients with interstitial lung diseases.

    Science.gov (United States)

    Szpechcinski, Adam; Kopinski, Piotr; Giedronowicz, Dorota; Rozy, Adriana; Jagus, Paulina; Szolkowska, Malgorzata; Chorostowska-Wynimko, Joanna

    2011-10-01

    To validate the fast and accurate flow cytometric (FCM) protocol using blood-standardized antibodies for alveolar lymphocyte subtyping with respect to standard immunocytochemistry (IC). FCM and IC were applied to immunophenotype T cell subsets in bronchoalveolar lavage (BAL) fluids from patients with interstitial lung diseases. Diagnostic BAL specimens from 50 patients with suspected sarcoidosis, idiopathic pulmonary fibrosis, and hypersensitivity pneumonitis were evaluated by both IC and FCM. In FCM, CD4+ and CD8+ T cells were identified by light scatter gating with CD3 selection using basic tricolor cytometer. Relative amounts of CD4+, CD8+ T cells, and CD4+/CD8+ ratios demonstrated by the FCM showed excellent, significant correlations with IC results. FCM values did not differ significantly from IC results. However, the sensitivity and specificity of conventional IC staining were not sufficient to assess CD4+/ CD8+ ratio in most idiopathic pulmonary fibrosis cases. Additionally, performing IC immunophenotyping in BAL samples with low lymphocyte content introduced a remarkable error into CD4+/CD8+ ratio assessment. FCM allowed reliable, precise, and fast T-cell subset measurement in all BAL samples, overcoming the IC disadvantages. Our validated FCM protocol provides diagnostically relevant CD4+/CD8+ ratio determination by simple light scatter gating strategy with CD3 selection.

  14. Nuclear chromatin variations in human spermatozoa undergoing swim-up and cryopreservation evaluated by the flow cytometric sperm chromatin structure assay.

    Science.gov (United States)

    Spanò, M; Cordelli, E; Leter, G; Lombardo, F; Lenzi, A; Gandini, L

    1999-01-01

    The sperm chromatin structure assay (SCSA) is a flow cytometric (FCM) technique which exploits the metachromatic properties of Acridine Orange to monitor the susceptibility of sperm chromatin DNA to in-situ acid denaturation. SCSA was used to study the chromatin structure variations of human spermatozoa in semen, both before and after swim-up and after cryopreservation. Semen samples were provided by 19 healthy normozoospermic subjects attending pre-marriage checks. Each sample was divided into three aliquots: the first aliquot was evaluated without further treatment, the second underwent swim-up, and the third was stored according to standard cryopreservation techniques in liquid nitrogen at -196 degrees C. Samples were also analysed by light and fluorescence microscopy (after Acridine Orange staining to evaluate the number of green fluorescent sperm heads), and by computer-assisted semen analysis. The results showed that post-rise spermatozoa represent a subpopulation characterized by a general improvement of the morphological (reduction of the percentage of abnormal forms and heads, increase of the green head sperm percentage) and kinetic parameters. This subpopulation also exhibited improved chromatin structure properties, confirming that these cells have the best structural and functional characteristics, indicative of optimal fertilizing ability. On the other hand, overall sperm quality deteriorates after cryopreservation. When thawed spermatozoa underwent an additional swim-up round, a general improvement of nuclear maturity was seen in the post-rise spermatozoa.

  15. Molecular pathways of early CD105-positive erythroid cells as compared with CD34-positive common precursor cells by flow cytometric cell-sorting and gene expression profiling

    International Nuclear Information System (INIS)

    Machherndl-Spandl, S; Suessner, S; Danzer, M; Proell, J; Gabriel, C; Lauf, J; Sylie, R; Klein, H-U; Béné, M C; Weltermann, A; Bettelheim, P

    2013-01-01

    Special attention has recently been drawn to the molecular network of different genes that are responsible for the development of erythroid cells. The aim of the present study was to establish in detail the immunophenotype of early erythroid cells and to compare the gene expression profile of freshly isolated early erythroid precursors with that of the CD34-positive (CD34 + ) compartment. Multiparameter flow cytometric analyses of human bone marrow mononuclear cell fractions (n=20) defined three distinct early erythroid stages. The gene expression profile of sorted early erythroid cells was analyzed by Affymetrix array technology. For 4524 genes, a differential regulation was found in CD105-positive erythroid cells as compared with the CD34 + progenitor compartment (2362 upregulated genes). A highly significant difference was observed in the expression level of genes involved in transcription, heme synthesis, iron and mitochondrial metabolism and transforming growth factor-β signaling. A comparison with recently published data showed over 1000 genes that as yet have not been reported to be upregulated in the early erythroid lineage. The gene expression level within distinct pathways could be illustrated directly by applying the Ingenuity software program. The results of gene expression analyses can be seen at the Gene Expression Omnibus repository

  16. Commercially Available Antibodies to Human Tumour Necrosis Factor-α Tested for Cross-Reactivity with Ovine and Bovine Tumour Necrosis Factor-α using Flow Cytometric Assays

    Directory of Open Access Journals (Sweden)

    Waller K Persson

    2004-06-01

    Full Text Available A thorough understanding of the immune system, including the role of different cytokines, during inflammatory diseases in ruminants could lead to the development of new diagnostic methods and treatments. Tumour necrosis factor-α (TNF-α is an important cytokine in the onset of the inflammatory responses. Unfortunately, the number of studies on cytokines, like TNF-α, in ruminants is limited due to a lack of species-specific reagents. As cytokines have remained rather conserved during evolution, cross-reactivity between animal species may occur. Therefore, the aim of the present study was to investigate 5 commercially available antibodies against human TNF-α for their ability to cross-react with ovine and/or bovine TNF-α, using a bead-based flow cytometric method. Two of the antibody clones (Mab 11 and 6401.1111 showed cross reactivity with ovine recombinant TNF-α in concentrations above 2.5 ng/ml. However, none of the antibodies detected TNF-α in bovine milk, or serum containing known concentrations of bovine TNF-α, as earlier determined with ELISA. The results could be due to inability of the antibodies to cross-react between species, but quenching of the signal by matrix proteins might also have lowered the response.

  17. Response of Syngonium podophyllum L. ‘White Butterfly’ shoot cultures to alternative media additives and gelling agents, and flow cytometric analysis of regenerants

    Directory of Open Access Journals (Sweden)

    JAIME A. TEIXEIRA DA SILVA

    2015-05-01

    Full Text Available Abstract. Teixeira da Silva JA. 2015. Response of Syngonium podophyllum L. ‘White Butterfly’ shoot cultures to alternative media additives and gelling agents, and flow cytometric analysis of regenerants. Nusantara Bioscience 7: 26-32. Syngonium podophyllum L. (arrowhead vine is a popular leafy indoor pot plant whose tissue culture has been established, primarily through in vitro shoot culture, but several interesting aspects have not yet been explored. In this study, cv. ‘White Butterfly’ was used to investigate the response of shoot formation to alternative gelling agents and media additives. Gellan gum (Gelrite® at 2 g/L resulted in greater leaf production, plantlet fresh weight and higher chlorophyll content (SPAD value than all other gelling agents tested, including agar, Bacto agar, phytagel, oatmeal agar, potato dextrose agar, barley starch and corn starch, when on a basal Hyponex® (NPK = 6.5: 6: 19; 3 g/L medium. Several alternative liquid medium additives tested (low and full fat milk, Coca-Cola®, coffee, Japanese green, Oolong and Darjeeling teas negatively impacted plant growth, stunted roots and decreased chlorophyll content (SPAD value of leaves. Plant growth on medium with refined sucrose or table sugar responded similarly. Poor growth was observed when crude extract from a high rebaudioside-containing stevia (Stevia rebaudiana Bertoni line - an artificial sweetener - was used. Leaf tissue from the control did not show any endopolyploidy but low levels of endopolyploidy (8C were detected in some treatments.

  18. Effect of chronic pesticide exposure on murine cornea: a histopathological, cytological and flow cytometric approach to study ocular damage by xenobiotics.

    Science.gov (United States)

    Sanyal, Shalini; Das, Prosun; Law, Sujata

    2016-02-01

    Pesticide exposure can occur directly or indirectly in an occupational setting or otherwise. The health hazards of pesticides have long been studied; however, little is known about the ocular insult of these potent chemicals. In this study, we examined the consequences of long-term pesticide exposure on the ocular tissue in animal model with special focus on the cornea. Swiss Albino mice were sacrificed to obtain the eye globes and various cytological, cytotoxic and histological evaluations, in vitro growth kinetic studies and flow cytometric analyses of select cytokeratins were performed to determine the structural and functional damage due to pesticide exposure. Our study revealed the detrimental impact of this xenobiotic insult by cataloguing the damage to each layer of the cornea wherein it was discovered that all the functional layers as well as the membranes were compromised. We hope that our investigation will pave the way for future studies in this oft overlooked area of affront caused by pesticide exposure to the ocular surface.

  19. Lymphocyte subset enumeration in HIV seronegative and HIV-1 seropositive adults in Dar es Salaam, Tanzania: determination of reference values in males and females and comparison of two flow cytometric methods.

    Science.gov (United States)

    Urassa, W K; Mbena, E M; Swai, A B; Gaines, H; Mhalu, F S; Biberfeld, G

    2003-06-01

    The level of CD4(+) T-lymphocytes represents a useful marker with which to monitor the progression of HIV infection. Sex and geographical differences in the reference values of lymphocyte subsets have been reported. We have compared two flow cytometric methods (MultiSET and SimulSET) for the quantification of lymphocyte subsets using whole blood from 92 HIV seropositive and 241 seronegative adults, and determined the reference values of lymphocyte subsets in HIV seronegative Tanzanian subjects. In seronegative Tanzanian subjects, the percentages of CD3(+) and CD4(+) T-lymphocytes and the CD4(+):CD8(+) T-lymphocyte ratios were lower while the percentage of natural killer cells was higher compared to the levels of the corresponding parameters reported for Europeans. Seronegative Tanzanian females had significantly higher levels of CD3(+) and CD4(+) T-lymphocytes and CD4(+):CD8(+) T-lymphocyte ratios compared to seronegative males. The correlation coefficients of CD3(+), CD4(+) and CD8(+) T lymphocyte counts and percentages obtained by the two flow cytometric methods were high. The median values of the number of CD4(+) T-lymphocytes obtained by the two methods were not significantly different. In conclusion, determination of the reference values of lymphocyte subsets in HIV seronegative Tanzanian adults showed significant sex differences and differences in percentage values compared to those reported in certain other geographical areas. There was acceptable agreement in the levels of CD4(+) T-lymphocyte values obtained by the two flow cytometric methods.

  20. Flow cytometric analysis of variation in the level of nuclear DNA endoreduplication in the cotyledons amongst Vigna radiata cultivars

    Czech Academy of Sciences Publication Activity Database

    Pal, A.; Vrána, Jan; Doležel, Jaroslav

    2004-01-01

    Roč. 57, č. 3 (2004), s. 262-266 ISSN 0008-7114 R&D Projects: GA AV ČR IBS5038104 Grant - others:Indian National Science Academy(IN) INSA Institutional research plan: CEZ:AV0Z5038910 Keywords : Cotyledon * endoreduplication * flow cytometry Subject RIV: GE - Plant Breeding Impact factor: 0.366, year: 2004

  1. Flow cytometric and microscopic analysis of the effect of tannic acid on plant nuclei and estimation of DNA content

    Czech Academy of Sciences Publication Activity Database

    Loureiro, J.; Rodriguez, E.; Doležel, Jaroslav; Santos, C.

    2006-01-01

    Roč. 98, - (2006), s. 515-527 ISSN 0305-7364 R&D Projects: GA MŠk(CZ) LC06004 Institutional research plan: CEZ:AV0Z50380511 Keywords : genome size * flow cytometry * nuclear DNA content Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.448, year: 2006

  2. Stereologic, histopathologic, flow cytometric, and clinical parameters in the prognostic evaluation of 74 patients with intraoral squamous cell carcinomas

    DEFF Research Database (Denmark)

    Bundgaard, T; Sørensen, Flemming Brandt; Gaihede, M

    1992-01-01

    BACKGROUND AND METHODS: A consecutive series of all 78 incident cases of intraoral squamous cell carcinoma occurring during a 2-year period in a population of 1.4 million inhabitants were evaluated by histologic score (the modified classification of Jacobsson et al.), flow cytometry, stereology, ...

  3. Development of a flow cytometric method to analyze subpopulations of bacteria in probiotic products and dairy starters

    NARCIS (Netherlands)

    Bunthof, C.J.; Abee, T.

    2002-01-01

    Flow cytometry (FCM) is a rapid and sensitive technique that can determine cell numbers and measure various physiological characteristics of individual cells by using appropriate fluorescent probes. Previously, we developed an FCM assay with the viability probes carboxyfluorescein diacetate (cFDA)

  4. [Standardization of the quantitative flow cytometric test with anti-D antibodies for fetomaternal hemorrhage in RhD negative women].

    Science.gov (United States)

    Spychalska, Justyna; Uhrynowska, Małgorzata; Pyl, Hanna; Klimczak-Jajor, Edyta; Kopeć, Izabella; Peciakowska, Małgorzata; Gutowska, Renata; Gawlak, Maciej; Słomska, Sylwia; Dąbkowska, Syiwia; Szczecina, Roman; Dębska, Marzena; Brojer, Ewa

    2015-07-01

    In order to determine the appropriate dose of anti-D immunoglobulin to be administered as a preventive measure against hemolytic disease of the fetus/newborn in the subsequent pregnancy it is necessary to assess the number of fetal red blood cells that infiltrate/penetrate into the maternal circulation as a result of fetomaternal hemorrhage (FMH). One of the quantitative methods of FMH analysis is based on flow cytometry (FACS) which makes use of monoclonal antibodies to RhD antigen (anti-D test). The aim of the study was to further develop the method, evaluate its sensitivity and reproducibility and to compare it with the test based on the detection of fetal hemoglobin (HbF). The FACS study involved 20 RhD negative pregnant women and 80 RhD negative women after delivery. The following monoclonal antibodies were used: BRAD 3 FITC (anti-RhD antigen), CD45 PerCP (anti leukocyte antigen CD45), and anti-HbF PE. The fluorescence intensity of cells incubated with BRAD 3 FITC was demonstrated to depend on the RhD antigen expression, though the anti-D test also detects the weak D variant. The CD45 PerCP antibodies increased the sensitivity of anti-D test since they eliminated the leukocytes which non-specifically bind anti-D from the analysis. The presence of anti-D antibodies in maternal plasma does not affect the quantitative assessment of the fetal RhD positive fetal cells with BRAD 3 FITC. In case of FMH, the results of the anti-D test were similar to those with anti-HbF antibodies. The flow cytometric test with anti-D and anti-CD45 is useful in the assessment of the fetomaternal hemorrhage in RhD negative women. The sensitivity of the test is estimated at 0.05%.

  5. Simultaneous flow cytometric quantification of plant nuclear DNA contents over the full range of described angiosperm 2C values.

    Science.gov (United States)

    Galbraith, David W

    2009-08-01

    Flow cytometry provides a rapid, accurate, and simple means to determine nuclear DNA contents (C-value) within plant homogenates. This parameter is extremely useful in a number of applications in basic and applied plant biology; for example, it provides an important starting point for projects involving whole genome sequencing, it facilitates characterization of plant species within natural and agricultural settings, it allows facile identification of engineered plants that are euploid or that represent desired ploidy classes, it points toward studies concerning the role of C-value in plant growth and development and in response to the environment and in terms of evolutionary fitness, and, in uncovering new and unexpected phenomena (for example endoreduplication), it uncovers new avenues of scientific enquiry. Despite the ease of the method, C-values have been determined for only around 2% of the described angiosperm (flowering plant) species. Within this small subset, one of the most remarkable observations is the range of 2C values, which spans at least two orders of magnitude. In determining C-values for new species, technical issues are encountered which relate both to requirement for a method that can provide accurate measurements across this extended dynamic range, and that can accommodate the large amounts of debris which accompanies flow measurements of plant homogenates. In this study, the use of the Accuri C6 flow cytometer for the analysis of plant C-values is described. This work indicates that the unusually large dynamic range of the C6, a design feature, coupled to the linearity of fluorescence emission conferred by staining of nuclei using propidium iodide, allows simultaneous analysis of species whose C-values span that of almost the entire described angiosperms. Copyright 2009 International Society for Advancement of Cytometry.

  6. Flow cytometric assessment of chicken T cell-mediated immune responses after Newcastle disease virus vaccination and challenge

    DEFF Research Database (Denmark)

    Dalgaard, T. S.; Norup, L. R.; Pedersen, A.R.

    2010-01-01

    . Despite a delayed NDV-specific antibody response to vaccination, L133 appeared to be better protected than L130 in the subsequent infection challenge as determined by the presence of viral genomes. Peripheral blood was analyzed by flow cytometry and responses in vaccinated/challenged birds were studied...... by 5-color immunophenotyping as well as by measuring the proliferative capacity of NDV-specific T cells after recall stimulation. Immunophenotyping identified L133 as having a significantly lower CD4/CD8 ratio and a lower frequency of γδ T cells than L130 in the peripheral T cell compartment...

  7. Flow cytometric sexing of spider sperm reveals an equal sperm production ratio in a female-biased species

    DEFF Research Database (Denmark)

    Vanthournout, Bram; Deswarte, K; Hammad, H

    2014-01-01

    that other factors influence sex ratio variation. In this paper, we investigate whether this additional variation can be explained by the unequal production of male- and female-determining sperm cells during sperm production. Using flow cytometry, we show that males produce equal amounts of male- and female......-determining sperm cells; thus bias in sperm production does not contribute to the sex ratio bias observed in this species. This demonstrates that other factors such as parental genes suppressing endosymbiont effects and cryptic female choice might play a role in sex allocation in this species....

  8. Improved flow cytometric identification of myelopoiesis by the simultaneous labelling with CD13, CD14 and CD66 monoclonal antibodies

    DEFF Research Database (Denmark)

    Bonde, J; Meyer, K; Broe, M K

    1996-01-01

    in the fast determination of remission state. In MDS, the immature myeloid component could be distinguished in patients defined according to the FAB classification with the possibility of identifying aberrant phenotypes, the assay should also be of interest in other myeloproliferative disorders. Moreover......The aim of the present study was to increase our knowledge of myelopoiesis evaluated by flow cytometry. We therefore designed a triple-marker assay employing monoclonal antibodies against the CD13 (immature), the CD14 (monocytic), and the CD66 (mature myeloid) antigens using three...

  9. Enumeration of extracellular vesicles by a new improved flow cytometric method is comparable to fluorescence mode nanoparticle tracking analysis.

    Science.gov (United States)

    Pasalic, Leonardo; Williams, Rebekka; Siupa, Agnieszka; Campbell, Heather; Henderson, Michelle J; Chen, Vivien M Y

    2016-05-01

    Extracellular vesicles (EVs) play a role in a variety of physiological and pathological processes. However, use of EVs as biomarkers has been hampered by limitations of current detection and enumeration methods. We compared fluorescence-threshold flow cytometry (FT-FC) to nanoparticle tracking analysis (NTA) for enumeration of cell culture-derived EVs. FT-FC and NTA utilising fluorescence mode (F-NTA) enumerated similar numbers of EVs stained with a membrane dye PKH67. Both methods were sufficiently sensitive to detect cell-derived EVs above the background of culture medium. Light scatter NTA (LS-NTA) detected 10-100× more particles than either fluorescence-based method but demonstrated poor specificity. F-NTA appeared to have better sensitivity for vesicles, however, the FT-FC method combined direct enumeration of EVs with high sensitivity and specificity in the >100nm range. Due to wider availability and higher degree of automation and standardisation, FT-FC is a reasonable surrogate to F-NTA for quantification of EVs. Extracellular vesicles are small particles, which can act as tools for intercellular communication. One recent area of interest in EVs is their potentials as biomarkers. In this article, the authors investigated and compared fluorescence-threshold flow cytometry (FT-FC) to nanoparticle tracking analysis (NTA) for the detection of EVs and showed that FT- FC method could be more advantageous. This technique should provide a new alternative for the future. Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.

  10. Flow cytometric minimal residual disease monitoring in children with acute lymphoblastic leukemia treated by regimens with reduced intensity

    Directory of Open Access Journals (Sweden)

    A. M. Popov

    2015-01-01

    Full Text Available 191 consecutive unselected children with acute lymphoblastic leukemia aged from 1 to 16 years were enrolled in the study. Bone marrow samples were obtained at the time of initial diagnostics as well as at days 15 (n = 188, 36 (n = 191, and 85 (n = 187 of remission induction. Minimal residual disease (MRD was assessed by 6–10-color flow cytometry. Flow cytometry data at day 15 allowed distinguishing three patients groups with significantly different outcome (p ˂ 0.0001: 35.64 % patients with MRD < 0.1 % represented 5-year event-free survival (EFS of 100 %; 48.40 % cases with 0.1 % ≤ MRD< 10 % had EFS 84.6 ± 4.2 %; 15.96 % patients with very high MRD (≥ 10 % belonged to group with poor outcome (EFS 56.7 ± 9.0 %. At the end of remission induction (day 36 36 children (18.85 % with MRD higher than 0.1 % had significantly worse outcome compared to remaining ones (EFS 49.4 ± 9.0 and 93.5 ± 2.1 % respectively; p ˂ 0.0001. From a clinical standpoint it is relevant to evaluate both low-risk and high-risk criteria. Multivariate analysis showed that day 15 MRD data is better for low-risk patients definition while end-induction MRD is the strongest unfavorable prognostic factor.

  11. Influence of a radioprotector WR-638 on the lymphoid compartment of the irradiated rat thymus: a flow cytometric analysis

    International Nuclear Information System (INIS)

    Dragojevic-Simic, V.; Colic, M.; Gasic, S.

    1994-01-01

    The T cell composition of the thymus of X-ray irradiated (3.5 Gy) Wistar rat protected with WR-638 was analyzed by flow cytometry using monoclonal antibodies directed to the Thy 1.1, CD43, CD2, CD5, CD4, CD8 and class I and II MHC antigens. It was shown that this dose of X-rays caused cyclic changes in thymic cellularity manifested as: primary involution (until day 2), primary regeneration (from days 2 to 14), secondary involution (from days 14 to 21) and secondary regeneration (from days 21 to 30). WR-638 reduced the magnitude of thymocyte depletion in the primary involutive phase of the irradiated thymi. (author)

  12. Genetic stock assessment of spawning arctic cisco (Coregonus autumnalis) populations by flow cytometric determination of DNA content.

    Science.gov (United States)

    Lockwood, S F; Bickham, J W

    1991-01-01

    Intraspecific variation in cellular DNA content was measured in five Coregonus autumnalis spawning populations from the Mackenzie River drainage, Canada, using flow cytometry. The rivers assayed were the Peel, Arctic Red, Mountain, Carcajou, and Liard rivers. DNA content was determined from whole blood preparations of fish from all rivers except the Carcajou, for which kidney tissue was used. DNA content measurements of kidney and blood preparations of the same fish from the Mountain River revealed statistically indistinguishable results. Mosaicism was found in blood preparations from the Peel, Arctic Red, Mountain, and Liard rivers, but was not observed in kidney tissue preparations from the Mountain or Carcajou rivers. The Liard River sample had significantly elevated mean DNA content relative to the other four samples; all other samples were statistically indistinguishable. Significant differences in mean DNA content among spawning stocks of a single species reinforces the need for adequate sample sizes of both individuals and populations when reporting "C" values for a particular species.

  13. Flow cytometric measurement of the metabolism of benzo[a]pyrene by mouse liver cells in culture

    International Nuclear Information System (INIS)

    Bartholomew, J.C.; Wade, C.G.; Dougherty, K.K.

    1984-01-01

    The metabolism of benzo[a]pyrene in individual cells was monitored by flow cytometry. The measurements are based on the alterations that occur in the fluorescence emission spectrum of benzo[a]pyrene when it is converted to various metabolites. Using present instrumentation the technique could easily detect 1x10 6 molecules per cells of benzo[a]pyrene and 1x10 7 molecules per cell of the diol epoxide. The analysis of C3H IOT 1/2 mouse fibroblasts growing in culture indicated that there was heterogeneity in the conversion of the parent compound into diol epoxide derivatives suggesting that some variation in sensitivity to transformation by benzo[a]pyrene may be due to differences in cellular metabolism. The technique allows sensitive detection of metabolites in viable cells, and provides a new approach to the study of factors that influence both metabolism and transformation. (orig.)

  14. Flow cytometric quantification of all phases of the cell cycle and apoptosis in a two-color fluorescence plot.

    Directory of Open Access Journals (Sweden)

    Christine Vignon

    Full Text Available An optimal technology for cell cycle analysis would allow the concomitant measurement of apoptosis, G0, G1, S, G2 and M phases in combination with cell surface phenotyping. We have developed an easy method in flow cytometry allowing this discrimination in an only two-color fluorescent plot. It is based on the concomitant use of 7-amino-actinomycin D and the antibodies anti-Ki67 and anti-phospho(Ser10-histone H3, both conjugated to Alexa Fluor®488 to discriminate G0 and M phases, respectively. The method is particularly valuable in a clinical setting as verified in our laboratory by analyzing human leukemic cells from marrow samples or after exposure to cell cycle modifiers.

  15. Evaluation of flow cytometric immunophenotyping and DNA analysis for detection of malignant cells in serosal cavity fluids.

    Science.gov (United States)

    Sayed, Douaa M; el-Attar, Madiha M; Hussein, Aliaa A R Mohamed

    2009-07-01

    The serosal cavities are frequent sites of tumor metastasis. The distinction between carcinoma cells, inflammatory cells, and reactive or malignant mesothelial cells can be difficult in cytology. Multicolor flow cytometry (FCM) provides the opportunity to evaluate multiple antigens simultaneously, making it possible to characterize various cell populations. In this study, we aimed to assess the diagnostic accuracy of FCM immunophenotyping and DNA in comparison with serum tumor markers and classic cytology for detection of malignant cells in pleural and ascitic fluids. One hundred and nineteen samples of body cavity fluids were analyzed. Immunophenotyping was performed by four-color immunofluorescent staining using monoclonal antibodies against Ber-EP4, cytokeratin, CD3, and CD45. The DNA analysis by FCM was also performed. In addition, serum CA19-9, CEA, AFP, and CA125 were analyzed. Ber-EP4 marker had the highest sensitivity (73%) and specificity (95.5%) in the detection of carcinoma cells in serous fluid and correlated with cytology in most of cases (73%). The mean of DI differed statistically in patients with malignant effusions than in benign one. DI showed no difference in fluids due to infiltration of malignant epithelial cells or hematopoietic malignancy or due to hepatocellular carcinoma developing in cirrhotic liver. Thus, flow cytometry appears to aid not only in the detection of malignant cells but also in the characterization of cell type. On the other hand, although DNA ploidy examination had better sensitivity; it had no advantage over conventional cytopathological examination in identification of malignant cells. 2009 Wiley-Liss, Inc.

  16. Optimized multiparametric flow cytometric analysis of circulating endothelial cells and their subpopulations in peripheral blood of patients with solid tumors: a technical analysis.

    Science.gov (United States)

    Zhou, Fangbin; Zhou, Yaying; Yang, Ming; Wen, Jinli; Dong, Jun; Tan, Wenyong

    2018-01-01

    Circulating endothelial cells (CECs) and their subpopulations could be potential novel biomarkers for various malignancies. However, reliable enumerable methods are warranted to further improve their clinical utility. This study aimed to optimize a flow cytometric method (FCM) assay for CECs and subpopulations in peripheral blood for patients with solid cancers. An FCM assay was used to detect and identify CECs. A panel of 60 blood samples, including 44 metastatic cancer patients and 16 healthy controls, were used in this study. Some key issues of CEC enumeration, including sample material and anticoagulant selection, optimal titration of antibodies, lysis/wash procedures of blood sample preparation, conditions of sample storage, sufficient cell events to enhance the signal, fluorescence-minus-one controls instead of isotype controls to reduce background noise, optimal selection of cell surface markers, and evaluating the reproducibility of our method, were integrated and investigated. Wilcoxon and Mann-Whitney U tests were used to determine statistically significant differences. In this validation study, we refined a five-color FCM method to detect CECs and their subpopulations in peripheral blood of patients with solid tumors. Several key technical issues regarding preanalytical elements, FCM data acquisition, and analysis were addressed. Furthermore, we clinically validated the utility of our method. The baseline levels of mature CECs, endothelial progenitor cells, and activated CECs were higher in cancer patients than healthy subjects ( P technical issues found in previously published assays and validated the reproducibility and sensitivity of our proposed method. Future work is required to explore the potential of our optimized method in clinical oncologic applications.

  17. Challenges of flow-cytometric estimation of nuclear genome size in orchids, a plant group with both whole-genome and progressively partial endoreplication.

    Science.gov (United States)

    Trávníček, Pavel; Ponert, Jan; Urfus, Tomáš; Jersáková, Jana; Vrána, Jan; Hřibová, Eva; Doležel, Jaroslav; Suda, Jan

    2015-10-01

    Nuclear genome size is an inherited quantitative trait of eukaryotic organisms with both practical and biological consequences. A detailed analysis of major families is a promising approach to fully understand the biological meaning of the extensive variation in genome size in plants. Although Orchidaceae accounts for ∼10% of the angiosperm diversity, the knowledge of patterns and dynamics of their genome size is limited, in part due to difficulties in flow cytometric analyses. Cells in various somatic tissues of orchids undergo extensive endoreplication, either whole-genome or partial, and the G1-phase nuclei with 2C DNA amounts may be lacking, resulting in overestimated genome size values. Interpretation of DNA content histograms is particularly challenging in species with progressively partial endoreplication, in which the ratios between the positions of two neighboring DNA peaks are lower than two. In order to assess distributions of nuclear DNA amounts and identify tissue suitable for reliable estimation of nuclear DNA content, we analyzed six different tissue types in 48 orchid species belonging to all recognized subfamilies. Although traditionally used leaves may provide incorrect C-values, particularly in species with progressively partial endoreplication, young ovaries and pollinaria consistently yield 2C and 1C peaks of their G1-phase nuclei, respectively, and are, therefore, the most suitable parts for genome size studies in orchids. We also provide new DNA C-values for 22 orchid genera and 42 species. Adhering to the proposed methodology would allow for reliable genome size estimates in this largest plant family. Although our research was limited to orchids, the need to find a suitable tissue with dominant 2C peak of G1-phase nuclei applies to all endopolyploid species. © 2015 International Society for Advancement of Cytometry.

  18. Flow cytometric monitoring of bacterioplankton phenotypic diversity predicts high population-specific feeding rates by invasive dreissenid mussels.

    Science.gov (United States)

    Props, Ruben; Schmidt, Marian L; Heyse, Jasmine; Vanderploeg, Henry A; Boon, Nico; Denef, Vincent J

    2018-02-01

    Species invasion is an important disturbance to ecosystems worldwide, yet knowledge about the impacts of invasive species on bacterial communities remains sparse. Using a novel approach, we simultaneously detected phenotypic and derived taxonomic change in a natural bacterioplankton community when subjected to feeding pressure by quagga mussels, a widespread aquatic invasive species. We detected a significant decrease in diversity within 1 h of feeding and a total diversity loss of 11.6 ± 4.1% after 3 h. This loss of microbial diversity was caused by the selective removal of high nucleic acid populations (29 ± 5% after 3 h). We were able to track the community diversity at high temporal resolution by calculating phenotypic diversity estimates from flow cytometry (FCM) data of minute amounts of sample. Through parallel FCM and 16S rRNA gene amplicon sequencing analysis of environments spanning a broad diversity range, we showed that the two approaches resulted in highly correlated diversity measures and captured the same seasonal and lake-specific patterns in community composition. Based on our results, we predict that selective feeding by invasive dreissenid mussels directly impacts the microbial component of the carbon cycle, as it may drive bacterioplankton communities toward less diverse and potentially less productive states. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  19. Transmission electron microscopic morphological study and flow cytometric viability assessment of Acinetobacter baumannii susceptible to Musca domestica cecropin.

    Science.gov (United States)

    Gui, Shuiqing; Li, Rongjiang; Feng, Yongwen; Wang, Sanming

    2014-01-01

    Multidrug-resistant (MDR) Acinetobacter baumannii infections are difficult to treat owing to the extremely limited armamentarium. Expectations about antimicrobial peptides' use as new powerful antibacterial agents have been raised on the basis of their unique mechanism of action. Musca domestica cecropin (Mdc), a novel antimicrobial peptide from the larvae of Housefly (Musca domestica), has potently active against Gram-positive and Gram-negative bacteria standard strain. Here we evaluated the antibacterial activity of Mdc against clinical isolates of MDR-A. baumannii and elucidate the related antibacterial mechanisms. The minimal inhibitory concentration (MIC) of Mdc was 4 μg/mL. Bactericidal kinetics of Mdc revealed rapid killing of A. baumannii (30 min). Flow cytometry using viability stain demonstrated that Mdc causes A. baumannii membrane permeabilization in a concentration- and time-dependent process, which correlates with the bactericidal action. Moreover, transmission electron microscopic (TEM) examination showed that Mdc is capable of disrupting the membrane of bacterial cells, resulting in efflux of essential cytoplasmic components. Overall, Mdc could be a promising antibacterial agent for MDR-A. baumannii infections.

  20. Transmission Electron Microscopic Morphological Study and Flow Cytometric Viability Assessment of Acinetobacter baumannii Susceptible to Musca domestica cecropin

    Directory of Open Access Journals (Sweden)

    Shuiqing Gui

    2014-01-01

    Full Text Available Multidrug-resistant (MDR Acinetobacter baumannii infections are difficult to treat owing to the extremely limited armamentarium. Expectations about antimicrobial peptides' use as new powerful antibacterial agents have been raised on the basis of their unique mechanism of action. Musca domestica cecropin (Mdc, a novel antimicrobial peptide from the larvae of Housefly (Musca domestica, has potently active against Gram-positive and Gram-negative bacteria standard strain. Here we evaluated the antibacterial activity of Mdc against clinical isolates of MDR-A. baumannii and elucidate the related antibacterial mechanisms. The minimal inhibitory concentration (MIC of Mdc was 4 μg/mL. Bactericidal kinetics of Mdc revealed rapid killing of A. baumannii (30 min. Flow cytometry using viability stain demonstrated that Mdc causes A. baumannii membrane permeabilization in a concentration- and time-dependent process, which correlates with the bactericidal action. Moreover, transmission electron microscopic (TEM examination showed that Mdc is capable of disrupting the membrane of bacterial cells, resulting in efflux of essential cytoplasmic components. Overall, Mdc could be a promising antibacterial agent for MDR-A. baumannii infections.

  1. The combination of kinetic and flow cytometric semen parameters as a tool to predict fertility in cryopreserved bull semen.

    Science.gov (United States)

    Gliozzi, T M; Turri, F; Manes, S; Cassinelli, C; Pizzi, F

    2017-11-01

    Within recent years, there has been growing interest in the prediction of bull fertility through in vitro assessment of semen quality. A model for fertility prediction based on early evaluation of semen quality parameters, to exclude sires with potentially low fertility from breeding programs, would therefore be useful. The aim of the present study was to identify the most suitable parameters that would provide reliable prediction of fertility. Frozen semen from 18 Italian Holstein-Friesian proven bulls was analyzed using computer-assisted semen analysis (CASA) (motility and kinetic parameters) and flow cytometry (FCM) (viability, acrosomal integrity, mitochondrial function, lipid peroxidation, plasma membrane stability and DNA integrity). Bulls were divided into two groups (low and high fertility) based on the estimated relative conception rate (ERCR). Significant differences were found between fertility groups for total motility, active cells, straightness, linearity, viability and percentage of DNA fragmented sperm. Correlations were observed between ERCR and some kinetic parameters, and membrane instability and some DNA integrity indicators. In order to define a model with high relation between semen quality parameters and ERCR, backward stepwise multiple regression analysis was applied. Thus, we obtained a prediction model that explained almost half (R 2=0.47, Pfertility. Once the accuracy of fertility prediction has been confirmed, the model developed in the present study could be used by artificial insemination centers for bull selection or for elimination of poor fertility ejaculates.

  2. Flow cytometric analysis of FSHR, BMRR1B, LHR and apoptosis in granulosa cells and ovulation rate in merino sheep.

    Science.gov (United States)

    Regan, Sheena L P; McFarlane, James R; O'Shea, Tim; Andronicos, Nicholas; Arfuso, Frank; Dharmarajan, Arun; Almahbobi, Ghanim

    2015-08-01

    The aim of the present study was to determine the direct cause of the mutation-induced, increased ovulation rate in Booroola Merino (BB) sheep. Granulosa cells were removed from antral follicles before ovulation and post-ovulation from BB (n=5) and WT (n=12) Merino ewes. Direct immunofluorescence measurement of mature cell surface receptors using flow cytometry demonstrated a significant up-regulation of FSH receptor (FSHR), transforming growth factor beta type 1, bone morphogenetic protein receptor (BMPR1B), and LH receptor (LHR) in BB sheep. The increased density of FSHR and LHR provide novel evidence of a mechanism for increasing the number of follicles that are recruited during dominant follicle selection. The compounding increase in receptors with increasing follicle size maintained the multiple follicles and reduced the apoptosis, which contributed to a high ovulation rate in BB sheep. In addition, we report a mutation-independent mechanism of down-regulation to reduce receptor density of the leading dominant follicle in sheep. The suppression of receptor density coincides with the cessation of mitogenic growth and steroidogenic differentiation as part of the luteinization of the follicle. The BB mutation-induced attenuation of BMPR1B signaling led to an increased density of the FSHR and LHR and a concurrent reduction in apoptosis to increase the ovulation rate. The role of BMPs in receptor modulation is implicated in the development of multiple ovulations. © 2015 Society for Reproduction and Fertility.

  3. Multiparameter flow cytometric analysis of CD4 and CD8 T cell subsets in young and old people

    Directory of Open Access Journals (Sweden)

    Özcelik Dennis

    2008-07-01

    Full Text Available Abstract Background T cell-mediated immunity in elderly people is compromised in ways reflected in the composition of the peripheral T cell pool. The advent of polychromatic flow cytometry has made analysis of cell subsets feasible in unprecedented detail. Results Here we document shifts in subset distribution within naïve (N, central memory (CM and effector memory (EM cells defined by CD45RA and CCR7 expression in the elderly, additionally using the costimulatory receptors CD27 and CD28, as well as the coinhibitory receptors CD57 and KLRG-1, to further dissect these. Although differences between young and old were more marked in CD8 than in CD4 cells, a similar overall pattern prevailed in both. Thus, the use of all these markers together, and inclusion of assays of proliferation and cytokine secretion, may enable the construction of a differentiation scheme applicable to CD4 as well as CD8 cells, with the model (based on Romero et al. suggesting the progression N→CM→EM1→EM2→pE1→pE2→EM4→EM3→E end-stage non-proliferative effector cells. Conclusion Overall, the results suggest that both differences in subset distribution and differences between subsets are responsible for age-related changes in CD8 cells but that differences within rather than between subsets are more prominent for CD4 cells.

  4. Flow cytometric investigations of diploid and tetraploid plants and in vitro cultures of Datura stramonium and Hyoscyamus niger.

    Science.gov (United States)

    Weber, Jost; Georgiev, Vasil; Pavlov, Atanas; Bley, Thomas

    2008-10-01

    Plant in vitro systems are valuable sources for the production of biological active substances. However, changed profiles of secondary metabolites, and low, variable yields possibly caused by genetic instabilities complicate their industrial implementation. DNA profiling of plant in vitro cultures may provide data for the selection of highly producing in vitro cultures. Diploid and tetraploid Datura stramonium and Hyoscyamus niger plant as well as calli, and hairy root lines derived from them were analyzed by flow cytometry. Plant in vitro cultures undergo several cycles of endoreduplication more than the explants from which they were obtained. The highest cycle values were observed in calli (e.g. 1.19 for diploid H. niger) possibly induced by the growth factors. However, hairy roots cultivated without growth factor exhibited significant degrees of endoreduplication (cycle value 0.88 for diploid H. niger). Sets of five hairy root lines from each plant and ploidy level showed consistent within-set ploidy patterns. The ploidy profiles of investigated plant in vitro and in vivo differ. For the first time we report that hairy roots of two Solanaceae species undergo endoreduplication. Theploidy profiles of in vitro cultures (hairy roots and calli) seem to be influenced by the genome size, the growth factors applied, and the type of in vitro culture. The transformation of several hairy root lines showed no differences in the ploidy patterns. Copyright 2008 International Society for Advancement of Cytometry.

  5. Flow Cytometric Clinical Immunomonitoring Using Peptide–MHC Class II Tetramers: Optimization of Methods and Protocol Development

    Directory of Open Access Journals (Sweden)

    Diahann T. S. L. Jansen

    2018-01-01

    Full Text Available With the advent of novel strategies to induce tolerance in autoimmune and autoimmune-like conditions, clinical trials of antigen-specific tolerizing immunotherapy have become a reality. Besides safety, it will be essential to gather mechanistic data on responding CD4+ T cells to assess the effects of various immunomodulatory approaches in early-phase trials. Peptide–MHC class II (pMHCII multimers are an ideal tool for monitoring antigen-specific CD4+ T cell responses in unmanipulated cells directly ex vivo. Various protocols have been published but there are reagent and assay limitations across laboratories that could hinder their global application to immune monitoring. In this methodological analysis, we compare protocols and test available reagents to identify sources of variability and to determine the limitations of the tetramer binding assay. We describe a robust pMHCII flow cytometry-based assay to quantify and phenotype antigen-specific CD4+ T cells directly ex vivo from frozen peripheral blood mononuclear cell samples, which we suggest should be tested across various laboratories to standardize immune-monitoring results.

  6. Flow cytometric analysis of peripheral blood and tumor-infiltrating regulatory T cells in dogs with oral malignant melanoma.

    Science.gov (United States)

    Tominaga, Makiko; Horiuchi, Yutaka; Ichikawa, Mika; Yamashita, Masao; Okano, Kumiko; Jikumaru, Yuri; Nariai, Yoko; Kadosawa, Tsuyoshi

    2010-05-01

    It is well known that tumor-infiltrating lymphocytes (TILs) and peripheral blood lymphocytes (PBLs) from patients with advanced-stage cancer have a poor immune response. Regulatory T cells (Tregs), characterized by the expression of a cluster of differentiation 4 and intracellular FoxP3 markers, can inhibit antitumor immunoresponse. In the present study, the prevalence of Tregs in peripheral blood and tumor tissue from dogs with oral malignant melanoma was evaluated by triple-color flow cytometry. The percentage of Tregs in the peripheral blood of the dogs with malignancy was significantly increased compared with healthy control dogs, and the percentage of Tregs within tumors was significantly increased compared with Tregs in peripheral blood of dogs with oral malignant melanoma. This finding suggests that the presence of tumor cells induced either local proliferation or selective migration of Tregs to tumor-infiltrated sites. A better understanding of the underlying mechanisms of Treg regulation in patients with cancer may lead to an effective anticancer immunotherapy against canine malignant melanoma and possibly other tumors.

  7. Immunophenotypic Characterization of Human Bone Marrow Mast Cells. A Flow Cytometric Study of Normal and Pathological Bone Marrow Samples

    Directory of Open Access Journals (Sweden)

    Luis Escribano

    1998-01-01

    Full Text Available The goal of the present paper was to define the immunophenotype of bone marrow mast cells (BMMC from healthy controls and patients with hematologic malignancies (HM based on the use of multiple stainings with monoclonal antibodies analyzed by flow cytometry. Our results show that BMMC from both groups of individuals display a similar but heterogenous immunophenotype. The overall numbers of BMMC are higher in the HM group of individuals (p = 0.08. Three patterns of antigen expression were detected: (1 markers constantly positive in all cases analyzed (CD9, CD29, CD33, CD43, CD44, CD49d, CD49e, CD51, CD71, CD117, and FcεRI, (2 antigens that were constantly negative (CD1a, CD2, CD3, CD5, CD6, CD11a, CD14, CD15, CD16, CD19, CD20, CD21, CD23, CD25, CD30, CD34, CD38, CD41a, CD42b, CD65, CD66b, HLA-DR, and CD138, and (3 markers that were positive in a variable proportion of cases – CD11b (50%, CD11c (77%, CD13 (40%, CD18 (20%, CD22 (68%, CD35 (27%, CD40 (67%, CD54 (88% and CD61 (40%. In addition, BMMC from all cases explored were CD45+, and this antigen was expressed at an intensity similar to that of mature granulocytes.

  8. Flow cytometric analysis of pollen grains collected from individual bees provides information about pollen load composition and foraging behaviour.

    Science.gov (United States)

    Kron, Paul; Kwok, Allison; Husband, Brian C

    2014-01-01

    Understanding the species composition of pollen on pollinators has applications in agriculture, conservation and evolutionary biology. Current identification methods, including morphological analysis, cannot always discriminate taxa at the species level. Recent advances in flow cytometry techniques for pollen grains allow rapid testing of large numbers of pollen grains for DNA content, potentially providing improved species resolution. A test was made as to whether pollen loads from single bees (honey-bees and bumble-bees) could be classified into types based on DNA content, and whether good estimates of proportions of different types could be made. An examination was also made of how readily DNA content can be used to identify specific pollen species. The method allowed DNA contents to be quickly found for between 250 and 9391 pollen grains (750-28 173 nuclei) from individual honey-bees and between 81 and 11 512 pollen grains (243-34 537 nuclei) for bumble-bees. It was possible to identify a minimum number of pollen species on each bee and to assign proportions of each pollen type (based on DNA content) present. The information provided by this technique is promising but is affected by the complexity of the pollination environment (i.e. number of flowering species present and extent of overlap in DNA content). Nevertheless, it provides a new tool for examining pollinator behaviour and between-species or cytotype pollen transfer, particularly when used in combination with other morphological, chemical or genetic techniques.

  9. Optimized multiparametric flow cytometric analysis of circulating endothelial cells and their subpopulations in peripheral blood of patients with solid tumors: a technical analysis

    Directory of Open Access Journals (Sweden)

    Zhou F

    2018-03-01

    Full Text Available Fangbin Zhou,1,2 Yaying Zhou,3 Ming Yang,1 Jinli Wen,3 Jun Dong,4 Wenyong Tan1 1Department of Oncology, The Second Clinical Medical College, Shenzhen People’s Hospital, Jinan University, Shenzhen, People’s Republic of China; 2Integrated Chinese and Western Medicine Postdoctoral Research Station, Jinan University, Guangzhou, People’s Republic of China; 3Clinical Medical Research Center, The Second Clinical Medical College, Shenzhen People’s Hospital, Jinan University, Shenzhen, People’s Republic of China; 4Department of Pathophysiology, Key Laboratory of the State Administration of Traditional Chinese Medicine, Medical College of Jinan University, Guangzhou, People’s Republic of China Background: Circulating endothelial cells (CECs and their subpopulations could be potential novel biomarkers for various malignancies. However, reliable enumerable methods are warranted to further improve their clinical utility. This study aimed to optimize a flow cytometric method (FCM assay for CECs and subpopulations in peripheral blood for patients with solid cancers.Patients and methods: An FCM assay was used to detect and identify CECs. A panel of 60 blood samples, including 44 metastatic cancer patients and 16 healthy controls, were used in this study. Some key issues of CEC enumeration, including sample material and anticoagulant selection, optimal titration of antibodies, lysis/wash procedures of blood sample preparation, conditions of sample storage, sufficient cell events to enhance the signal, fluorescence-minus-one controls instead of isotype controls to reduce background noise, optimal selection of cell surface markers, and evaluating the reproducibility of our method, were integrated and investigated. Wilcoxon and Mann–Whitney U tests were used to determine statistically significant differences.Results: In this validation study, we refined a five-color FCM method to detect CECs and their subpopulations in peripheral blood of patients

  10. Flow cytometric evaluation of the contribution of ionic silver to genotoxic potential of nanosilver in human liver HepG2 and colon Caco2 cells.

    Science.gov (United States)

    Sahu, Saura C; Njoroge, Joyce; Bryce, Steven M; Zheng, Jiwen; Ihrie, John

    2016-04-01

    Exposure to nanosilver found in food- and cosmetics-related consumer products is of public concern because of the lack of information about its safety. In this study, two widely used in vitro cell culture models, human liver HepG2 and colon Caco2 cells, and the flow cytometric micronucleus (FCMN) assay were evaluated as tools for rapid predictive screening of the potential genotoxicity of nanosilver. Recently, we reported the genotoxicity of 20 nm nanosilver using these systems. In the current study presented here, we tested the hypothesis that the nanoparticle size and cell types were critical determinants of its genotoxicity. To test this hypothesis, we used the FCMN assay to evaluate the genotoxic potential of 50 nm nanosilver of the same shape, composition, surface charge and obtained from the same commercial source using the same experimental conditions and in vitro models (HepG2 and Caco2) as previously tested for the 20 nm silver. Results of our study show that up to the concentrations tested in these cultured cell test systems, the smaller (20 nm) nanoparticle is genotoxic to both the cell types by inducing micronucleus (MN). However, the larger (50 nm) nanosilver induces MN only in HepG2 cells, but not in Caco2 cells. Also in this study, we evaluated the contribution of ionic silver to the genotoxic potential of nanosilver using silver acetate as the representative ionic silver. The MN frequencies in HepG2 and Caco2 cells exposed to the ionic silver in the concentration range tested are not statistically significant from the control values except at the top concentrations for both the cell types. Therefore, our results indicate that the ionic silver may not contribute to the MN-forming ability of nanosilver in HepG2 and Caco2 cells. Also our results suggest that the HepG2 and Caco2 cell cultures and the FCMN assay are useful tools for rapid predictive screening of a genotoxic potential of food- and cosmetics-related chemicals including nanosilver

  11. A novel and easy FxCycle™ violet based flow cytometric method for simultaneous assessment of DNA ploidy and six-color immunophenotyping.

    Science.gov (United States)

    Tembhare, Prashant; Badrinath, Yajamanam; Ghogale, Sitaram; Patkar, Nikhil; Dhole, Nilesh; Dalavi, Pooja; Kunder, Nikesh; Kumar, Ashok; Gujral, Sumeet; Subramanian, P G

    2016-03-01

    Abnormal DNA ploidy is a valuable prognostic factor in many neoplasms, especially in hematological neoplasms like B-cell acute lymphoblastic leukemia (B-ALL) and multiple myeloma (MM). Current methods of flow-cytometric (FC) DNA-ploidy evaluation are either technically difficult or limited to three- to four-color immunophenotyping and hence, challenging to evaluate DNA-ploidy in minute tumor population with background rich of its normal counterpart cells and other hematopoietic cells. We standardized a novel sensitive and easy method of simultaneous evaluation of six- to seven-color immunophenotyping and DNA-ploidy using a dye-FxCycle Violet (FCV). Linearity, resolution, and coefficient of variation (CV) for FCV were studied using chicken erythrocyte nuclei. Ploidy results of FCV were compared with Propidium iodide (PI) in 20 samples and intra-assay variation for FCV was studied. Using this six-color immunophenotyping & FCV-protocol DNA-ploidy was determined in bone-marrow samples from 124 B-ALL & 50 MM patients. Dilution experiment was also conducted to determine the sensitivity in detection of aneuploidy in minute tumor population. FCV revealed high linearity and resolution in 450/50 channel. On comparison with PI, CV of Go/G1-peak with FCV (mean-CV 4.1%) was slightly higher than PI (mean-CV 2.9%) but had complete agreement in ploidy results. Dilution experiment showed that aneuploidy could be accurately detected up to the limit of 0.01% tumor cells. Intra-assay variation was very low with CV of 0.005%. In B-ALL, hypodiploidy was noted in 4%, hyperdiploidy in 24%, near-hyperdiploidy in 13% and remaining 59% were diploid. In MM, hypodiploidy was in 2%, hyperdiploidy in 58%, near-hyperdiploidy in 8% and remaining 30% were diploid. FCV-based DNA-ploidy method is a sensitive and easy method for simultaneous evaluation of six-color immunophenotyping and DNA analysis. It is useful in DNA-ploidy evaluation of minute tumor population in cases like minimal residual

  12. Flow cytometric immunophenotyping of regulatory T cells in chronic lymphocytic leukemia: comparative assessment of various markers and use of novel antibody panel with CD127 as alternative to transcription factor FoxP3.

    Science.gov (United States)

    Dasgupta, Alakananda; Mahapatra, Manoranjan; Saxena, Renu

    2013-04-01

    This study analyzed the frequency of regulatory T cells (Tregs) in chronic lymphocytic leukemia (CLL) by multiparameter flow cytometric immunophenotyping. Patients showed significantly increased frequencies of Tregs as compared to controls, a significantly higher percentage than that identified by previous studies, possibly indicating a different prognosis of CLL in different parts of the world and, more precisely, a worse prognosis of CLL in the Indian population. A higher frequency of Tregs was also seen in advanced stage of disease with significantly reduced frequencies of Tregs in patients with CLL after chemotherapy. A significant proportion of CD127low/-FoxP3+ Tregs expressed only low levels of CD25. Thus, CD127 appears to be a better marker than CD25 for the identification of CD4+FoxP3+ T cells as potential Tregs. Our results suggest that the specificity and sensitivity of CD4+CD127low/- cells are comparable to those of CD4+FoxP3+, which is the gold standard, and can be used as an alternative. This novel flow cytometric antibody panel with fewer number of antibodies is cost-effective and can be used to enumerate Tregs in resource-limited settings.

  13. Cytometric analysis of mammalian sperm for induced morphologic and DNA content errors

    Energy Technology Data Exchange (ETDEWEB)

    Pinkel, D.

    1983-06-27

    Some flow-cytometric and image analysis procedures under development for quantitative analysis of sperm morphology are reviewed. The results of flow-cytometric DNA-content measurements on sperm from radiation exposed mice are also summarized, the results related to the available cytological information, and their potential dosimetric sensitivity discussed. (ACR)

  14. Cytometric analysis of mammalian sperm for induced morphologic and DNA content errors

    International Nuclear Information System (INIS)

    Pinkel, D.

    1983-01-01

    Some flow-cytometric and image analysis procedures under development for quantitative analysis of sperm morphology are reviewed. The results of flow-cytometric DNA-content measurements on sperm from radiation exposed mice are also summarized, the results related to the available cytological information, and their potential dosimetric sensitivity discussed

  15. An extended leukocyte differential count (16 types of circulating leukocytes) using the CytoDiff flow cytometric system can provide information for the discrimination of sepsis severity and prediction of outcome in sepsis patients.

    Science.gov (United States)

    Park, Sang Hyuk; Park, Borae G; Park, Chan-Jeoung; Kim, Sue; Kim, Duck-Hee; Jang, Seongsoo; Hong, Suk-Kyung; Chi, Hyun-Sook

    2014-07-01

    The Beckman Coulter CytoDiff flow cytometric system (Beckman Coulter, Miami, FL) was recently developed for performing leukocyte differential counts in up to 16 leukocyte subpopulations. We compared these leukocyte subpopulation levels among patients with three stages of sepsis (uncomplicated sepsis, severe sepsis, septic shock), especially focused on the discrimination of complicated sepsis from uncomplicated sepsis. We examined a total of 181 samples with sepsis who were admitted to the surgical intensive care unit. In addition, we examined samples obtained from 60 normal healthy volunteers. Both the proportions and absolute numbers of each cell type in the four groups were obtained using the CytoDiff flow cytometric system and compared. Mature neutrophils and immature granulocytes failed to discriminate patients with complicated sepsis from those with uncomplicated sepsis although their absolute numbers were increased compared with normal controls. In contrast, almost all lymphocyte subpopulations and CD16(-) monocytes decreased significantly in patients with complicated sepsis compared with uncomplicated sepsis. Among them, only B lymphocytes showed independent ability to discriminate two groups. Both B lymphocytes and CD16(-) monocytes possessed a significant adverse prognostic impact on overall survival when their absolute numbers decreased. Almost all lymphocyte subpopulations and CD16(-) monocytes decrease in size with increasing sepsis severity. Among them, only B lymphocytes showed independent ability to discriminate patients with complicated sepsis from those with uncomplicated sepsis. Both B lymphocytes and CD16 (-) monocytes show a significant adverse prognostic impact on overall survival outcomes in sepsis patients when their absolute numbers are decreased. Copyright © 2013 Clinical Cytometry Society.

  16. An extended leukocyte differential count (16 types of circulating leukocytes) using the cytodiff flow cytometric system can provide informations for the discrimination of sepsis severity and prediction of outcome in sepsis patients.

    Science.gov (United States)

    Park, Sang Hyuk; Park, Borae G; Park, Chan-Jeoung; Kim, Sue; Kim, Duck-Hee; Jang, Seongsoo; Hong, Suk-Kyung; Chi, Hyun-Sook

    2013-08-20

    Background: The Beckman Coulter CytoDiff flow cytometric system (Beckman Coulter, Miami, FL, USA) was recently developed for performing leukocyte differential counts in up to 16 leukocyte subpopulations. We compared these leukocyte subpopulation levels among patients with three stages of sepsis (uncomplicated sepsis, severe sepsis, septic shock), especially focused on the discrimination of complicated sepsis from uncomplicated sepsis. Methods: We examined a total of 181 samples with sepsis who were admitted to the surgical intensive care unit. In addition, we examined samples obtained from 60 normal healthy volunteers. Both the proportions and absolute numbers of each cell type in the four groups were obtained using the CytoDiff flow cytometric system and compared. Results: Mature neutrophils and immature granulocytes failed to discriminate patients with complicated sepsis from those with uncomplicated sepsis although their absolute numbers were increased compared with normal controls. In contrast, almost all lymphocyte subpopulations and CD16(-) monocytes decreased significantly in patients with complicated sepsis compared with uncomplicated sepsis. Among them, only B lymphocytes showed independent ability to discriminate two groups. Both B lymphocytes and CD16(-) monocytes possessed a significant adverse prognostic impact on overall survival when their absolute numbers decreased. Conclusions: Almost all lymphocyte subpopulations and CD16(-) monocytes decrease in size with increasing sepsis severity. Among them, only B lymphocytes showed independent ability to discriminate patients with complicated sepsis from those with uncomplicated sepsis. Both B lymphocytes and CD16(-) monocytes show a significant adverse prognostic impact on overall survival outcomes in sepsis patients when their absolute numbers are decreased. © 2013 Clinical Cytometry Society. Copyright © 2013 Clinical Cytometry Society.

  17. Immunoadsorption using protein A columns complicates interpretation of cytotoxic crossmatch

    DEFF Research Database (Denmark)

    Koefoed-Nielsen, Pernille Bundgaard; Bistrup, Claus; Christiansen, Mette

    2014-01-01

    Transplanting immunized patients requires immunological monitoring in the pretransplant phase, in order to follow reduction of donor specific HLA antibodies (DSA) after immunoadsorption (IA)/ plasmapheresis and concurrent IvIg and Rituximab administration. Our Tissue Typing Lab performs the immun......Transplanting immunized patients requires immunological monitoring in the pretransplant phase, in order to follow reduction of donor specific HLA antibodies (DSA) after immunoadsorption (IA)/ plasmapheresis and concurrent IvIg and Rituximab administration. Our Tissue Typing Lab performs...... material mixed and incubated with either negative patient serum or AB serum reproduced a positive CDC T-cell crossmatch, supporting a hypothesis of possible leached material as the main cause. To substantiate these observations we aim at detecting leached material in patient samples using ELISA....... In conclusion, the results emphasize the importance of carefully considering crossmatch results subsequent to IA, before a planned transplantation is either postponed or refused....

  18. A radiolabeled antiglobulin test for crossmatching platelet transfusions

    International Nuclear Information System (INIS)

    Kickler, T.S.; Braine, H.G.; Ness, P.M.; Koester, A.; Bias, W.

    1983-01-01

    Despite the use of HLA-matched platelets for alloimmunized recipients, transfusion failures occur. In order to reduce these failures, researchers investigated the use of a radiolabeled antiglobulin technique for platelet crossmatching. The principle of the test is that of an indirect Coombs test using 125 I labeled goat anti-human IgG. Incompatibility is determined by calculating a radioactivity antiglobulin test (RAGT) index. Using this technique, researchers performed 89 crossmatches on 19 leukemic or aplastic patients who were refractory to random donor platelets and receiving varying degrees of HLA-matched platelets. Effectiveness of the transfusion was assessed from the posttransfusion corrected platelet count increment (CCI) determined at 1 and 20 hr. When the RAGT index was 1.9 or less, the mean CCI at 1 lhr was 17,570 +/- 7003/cu mm, n . 55. When the RAGT index was 2.0 or greater, the mean CCI was 4237 +/- 4100/cu mm, n . 34. At 20 hr when the RAGT index was 1.9 or less, the mean CCI was 8722 +/- 3143/cu mm, n . 33, and when the index was 2.0 or greater, the mean CCI was 571 +/- 1286/cu mm, n . 23. Using this technique, one false negative resulted. Nine positive crossmatches with good increments at 1 hr were found; at 20 hr, however, the survival of these units was zero. These data suggest that this method is a useful adjunct in the selection of platelets in the refractory patient

  19. Acute Antibody-Mediated Rejection in Presence of MICA-DSA and Successful Renal Re-Transplant with Negative-MICA Virtual Crossmatch.

    Directory of Open Access Journals (Sweden)

    Yingzi Ming

    Full Text Available The presence of donor-specific alloantibodies (DSAs against the MICA antigen results in high risk for antibody-mediated rejection (AMR of a transplanted kidney, especially in patients receiving a re-transplant. We describe the incidence of acute C4d+ AMR in a patient who had received a first kidney transplant with a zero HLA antigen mismatch. Retrospective analysis of post-transplant T and B cell crossmatches were negative, but a high level of MICA alloantibody was detected in sera collected both before and after transplant. The DSA against the first allograft mismatched MICA*018 was in the recipient. Flow cytometry and cytotoxicity tests with five samples of freshly isolated human umbilical vein endothelial cells demonstrated the alloantibody nature of patient's MICA-DSA. Prior to the second transplant, a MICA virtual crossmatch and T and B cell crossmatches were used to identify a suitable donor. The patient received a second kidney transplant, and allograft was functioning well at one-year follow-up. Our study indicates that MICA virtual crossmatch is important in selection of a kidney donor if the recipient has been sensitized with MICA antigens.

  20. The Crossmatch/Issue Ratio:  Use of a Novel Quality Indicator and Results of an International Survey on RBC Crossmatching and Issuing Practices

    DEFF Research Database (Denmark)

    Yazer, Mark H; Alcantara, Ramir; Beizai, Pouneh

    2016-01-01

    , and the mean ± SD was 1.30 ± 0.34. There was no difference in C/I ratios between services that use the electronic or serologic crossmatch techniques (P = .49). The ratio was the same at the four sites that crossmatch RBCs at the time of issue compared with the time of order receipt (mean ± SD, 1.11 ± 0.09 vs......OBJECTIVES: To understand the worldwide scope of RBC crossmatching and issuing practices and measure efficiency using a novel quality indicator, the crossmatch/issue (C/I) ratio. METHODS: An electronic survey was disseminated to hospital transfusion services collecting details about RBC...

  1. Flow cytometric sensitivity and characteristics of plasma cells in patients with multiple myeloma or its precursor disease: influence of biopsy site and anticoagulation method.

    Science.gov (United States)

    Manasanch, Elisabet E; Salem, Dalia A; Yuan, Constance M; Tageja, Nishant; Bhutani, Manisha; Kwok, Mary; Kazandjian, Dickran; Carter, George; Steinberg, Seth M; Zuchlinski, Diamond; Mulquin, Marcia; Calvo, Katherine; Maric, Irina; Roschewski, Mark; Korde, Neha; Braylan, Raul; Landgren, Ola; Stetler-Stevenson, Maryalice

    2015-05-01

    Flow cytometry has increasing relevance for prognosis in myeloma and precursor disease (monoclonal gammopathy of unknown significance/smoldering myeloma), yet it has been reported that plasma cell enumeration by flow varies depending on the quality of marrow aspirate and field biopsied in patchy disease. We demonstrated increased sensitivity of flow over immunohistochemistry in abnormal-plasma cell detection in monoclonal gammopathy (n = 59)/smoldering myeloma (n = 87). We prospectively evaluated treatment-na ve smoldering myeloma (n = 9)/myeloma (n = 11) patients for the percentage of abnormal plasma cells/total plasma cell compartment, plasma cell viability/infiltration and flow immunophenotype depending on anticoagulant use, biopsy site and pull sequence in uni-and-bilateral bone marrow biopsies and aspirates. We found no statistical difference regarding the percentage of abnormal plasma cells, their immunophenotype or number/distribution in marrow samples even when obtained by different sequence in aspirates, or anticoagulants (p > 0.05). Our results show that plasma cell enumeration and immunophenotyping by flow cytometry is consistent under different conditions in these populations.

  2. Flow cytometric readout based on Mitotracker Red CMXRos staining of live asexual blood stage malarial parasites reliably assesses antibody dependent cellular inhibition

    DEFF Research Database (Denmark)

    Jogdand, Prajakta S; Singh, Susheel K; Christiansen, Michael

    2012-01-01

    asynchronous and tightly synchronized asexual blood stage cultures of Plasmodium falciparum were stained with CMXRos and subjected to detection by flow cytometry and fluorescence microscopy. The parasite counts obtained by flow cytometry were compared to standard microscopic counts obtained through examination......ABSTRACT: BACKGROUND: Functional in vitro assays could provide insights into the efficacy of malaria vaccine candidates. For estimating the anti-parasite effect induced by a vaccine candidate, an accurate determination of live parasite count is an essential component of most in vitro bioassays....... Although traditionally parasites are counted microscopically, a faster, more accurate and less subjective method for counting parasites is desirable. In this study mitochondrial dye (Mitotracker Red CMXRos) was used for obtaining reliable live parasite counts through flow cytometry. METHODS: Both...

  3. Flow Cytometric Analysis of the Expression Pattern of Peroxisomal Proteins, Abcd1, Abcd2, and Abcd3 in BV-2 Murine Microglial Cells.

    Science.gov (United States)

    Debbabi, Meryam; Nury, Thomas; Helali, Imen; Karym, El Mostafa; Geillon, Flore; Gondcaille, Catherine; Trompier, Doriane; Najid, Amina; Terreau, Sébastien; Bezine, Maryem; Zarrouk, Amira; Vejux, Anne; Andreoletti, Pierre; Cherkaoui-Malki, Mustapha; Savary, Stéphane; Lizard, Gérard

    2017-01-01

    Microglial cells play important roles in neurodegenerative diseases including peroxisomal leukodystrophies. The BV-2 murine immortalized cells are widely used in the context of neurodegenerative researches. It is therefore important to establish the expression pattern of peroxisomal proteins by flow cytometry in these cells. So, the expression pattern of various peroxisomal transporters (Abcd1, Abcd2, Abcd3) contributing to peroxisomal β-oxidation was evaluated on BV-2 cells by flow cytometry and complementary methods (fluorescence microscopy, and RT-qPCR). By flow cytometry a strong expression of peroxisomal proteins (Abcd1, Abcd2, Abcd3) was observed. These data were in agreement with those obtained by fluorescence microscopy (presence of numerous fluorescent dots in the cytoplasm characteristic of a peroxisomal staining pattern) and RT-qPCR (high levels of Abcd1, Abcd2, and Abcd3 mRNAs). Thus, the peroxisomal proteins (Abcd1, Abcd2, Abcd3) are expressed in BV-2 cells, and can be analyzed by flow cytometry.

  4. Challenges of flow-cytometric estimation of nuclear genome size in orchids, a plant group with both whole-genome and progressively partial endoreplication

    Czech Academy of Sciences Publication Activity Database

    Trávníček, Pavel; Ponert, J.; Urfus, Tomáš; Jersáková, Jana; Vrána, Jan; Hřibová, Eva; Doležel, Jaroslav; Suda, Jan

    2015-01-01

    Roč. 87, č. 10 (2015), s. 958-966 ISSN 1552-4922 R&D Projects: GA ČR GAP506/12/1320 Institutional support: RVO:67985939 ; RVO:67179843 ; RVO:61389030 Keywords : flow cytometry * genome size * hyporeduplication Subject RIV: EF - Botanics; EF - Botanics (UEB-Q); EH - Ecology, Behaviour (UEK-B) Impact factor: 3.181, year: 2015

  5. Improving the resolution of cryopreserved X- and Y-sperm during DNA flow cytometric analysis with the addition of Percoll to quench the fluorescence of dead sperm

    NARCIS (Netherlands)

    Stap, J.; Hoebe, R. A.; Merton, J. S.; Haring, R. M.; Bakker, P. J.; Aten, J. A.

    1998-01-01

    The most effective method to control the sex of offspring is by separating X- from Y-bearing sperm on the basis of their DNA content. Sperm can be stained with Hoechst 33342 and efficiently sexed using a flow cytometer/cell sorter. However, applying this established assay to cryopreserved bovine

  6. Colour-encoded paramagnetic microbead-based direct inhibition triplex flow cytometric immunoassay for ochratoxin A, fumonisins and zearalenone in cereals and cereal-based feed

    NARCIS (Netherlands)

    Peters, J.; Thomas, D.; Boers, E.A.M.; Rijk, de T.C.; Berthiller, F.; Haasnoot, W.; Nielen, M.W.F.

    2013-01-01

    A combined (triplex) immunoassay for the simultaneous detection of three mycotoxins in grains was developed with superparamagnetic colour-encoded microbeads, in combination with two bead-dedicated flow cytometers. Monoclonal antibodies were coupled to the beads, and the amounts of bound mycotoxins

  7. High-Throughput Flow Cytometric Method for the Simultaneous Measurement of CAR-T Cell Characterization and Cytotoxicity against Solid Tumor Cell Lines.

    Science.gov (United States)

    Martinez, Emily M; Klebanoff, Samuel D; Secrest, Stephanie; Romain, Gabrielle; Haile, Samuel T; Emtage, Peter C R; Gilbert, Amy E

    2018-04-01

    High-throughput flow cytometry is an attractive platform for the analysis of adoptive cellular therapies such as chimeric antigen receptor T cell therapy (CAR-T) because it allows for the concurrent measurement of T cell-dependent cellular cytotoxicity (TDCC) and the functional characterization of engineered T cells with respect to percentage of CAR transduction, T cell phenotype, and measurement of T cell function such as activation in a single assay. The use of adherent tumor cell lines can be challenging in these flow-based assays. Here, we present the development of a high-throughput flow-based assay to measure TDCC for a CAR-T construct co-cultured with multiple adherent tumor cell lines. We describe optimal assay conditions (such as adherent cell dissociation techniques to minimize impact on cell viability) that result in robust cytotoxicity assays. In addition, we report on the concurrent use of T cell transduction and activation antibody panels (CD25) that provide further dissection of engineered T cell function. In conclusion, we present the development of a high-throughput flow cytometry method allowing for in vitro interrogation of solid tumor, targeting CAR-T cell-mediated cytotoxicity, CAR transduction, and engineered T cell characterization in a single assay.

  8. Flow cytometric readout based on Mitotracker Red CMXRos staining of live asexual blood stage malarial parasites reliably assesses antibody dependent cellular inhibition

    Directory of Open Access Journals (Sweden)

    Jogdand Prajakta S

    2012-07-01

    Full Text Available Abstract Background Functional in vitro assays could provide insights into the efficacy of malaria vaccine candidates. For estimating the anti-parasite effect induced by a vaccine candidate, an accurate determination of live parasite count is an essential component of most in vitro bioassays. Although traditionally parasites are counted microscopically, a faster, more accurate and less subjective method for counting parasites is desirable. In this study mitochondrial dye (Mitotracker Red CMXRos was used for obtaining reliable live parasite counts through flow cytometry. Methods Both asynchronous and tightly synchronized asexual blood stage cultures of Plasmodium falciparum were stained with CMXRos and subjected to detection by flow cytometry and fluorescence microscopy. The parasite counts obtained by flow cytometry were compared to standard microscopic counts obtained through examination of Giemsa-stained thin smears. A comparison of the ability of CMXRos to stain live and compromised parasites (induced by either medium starvation or by anti-malarial drug treatment was carried out. Finally, parasite counts obtained by CMXRos staining through flow cytometry were used to determine specific growth inhibition index (SGI in an antibody-dependent cellular inhibition (ADCI assay. Results Mitotracker Red CMXRos can reliably detect live intra-erythrocytic stages of P. falciparum. Comparison between staining of live with compromised parasites shows that CMXRos predominantly stains live parasites with functional mitochondria. Parasite counts obtained by CMXRos staining and flow cytometry were highly reproducible and can reliably determine the ability of IgG from hyper-immune individuals to inhibit parasite growth in presence of monocytes in ADCI assay. Further, a dose-dependent parasite growth inhibitory effect could be detected for both total IgG purified from hyper-immune sera and affinity purified IgGs against the N-terminal non-repeat region of GLURP

  9. Flow cytometric readout based on Mitotracker Red CMXRos staining of live asexual blood stage malarial parasites reliably assesses antibody dependent cellular inhibition.

    Science.gov (United States)

    Jogdand, Prajakta S; Singh, Susheel K; Christiansen, Michael; Dziegiel, Morten H; Singh, Subhash; Theisen, Michael

    2012-07-20

    Functional in vitro assays could provide insights into the efficacy of malaria vaccine candidates. For estimating the anti-parasite effect induced by a vaccine candidate, an accurate determination of live parasite count is an essential component of most in vitro bioassays. Although traditionally parasites are counted microscopically, a faster, more accurate and less subjective method for counting parasites is desirable. In this study mitochondrial dye (Mitotracker Red CMXRos) was used for obtaining reliable live parasite counts through flow cytometry. Both asynchronous and tightly synchronized asexual blood stage cultures of Plasmodium falciparum were stained with CMXRos and subjected to detection by flow cytometry and fluorescence microscopy. The parasite counts obtained by flow cytometry were compared to standard microscopic counts obtained through examination of Giemsa-stained thin smears. A comparison of the ability of CMXRos to stain live and compromised parasites (induced by either medium starvation or by anti-malarial drug treatment) was carried out. Finally, parasite counts obtained by CMXRos staining through flow cytometry were used to determine specific growth inhibition index (SGI) in an antibody-dependent cellular inhibition (ADCI) assay. Mitotracker Red CMXRos can reliably detect live intra-erythrocytic stages of P. falciparum. Comparison between staining of live with compromised parasites shows that CMXRos predominantly stains live parasites with functional mitochondria. Parasite counts obtained by CMXRos staining and flow cytometry were highly reproducible and can reliably determine the ability of IgG from hyper-immune individuals to inhibit parasite growth in presence of monocytes in ADCI assay. Further, a dose-dependent parasite growth inhibitory effect could be detected for both total IgG purified from hyper-immune sera and affinity purified IgGs against the N-terminal non-repeat region of GLURP in ADCI assays coupled with determination of

  10. Exploring the feasibility of multi-site flow cytometric processing of gut associated lymphoid tissue with centralized data analysis for multi-site clinical trials.

    Science.gov (United States)

    McGowan, Ian; Anton, Peter A; Elliott, Julie; Cranston, Ross D; Duffill, Kathryn; Althouse, Andrew D; Hawkins, Kevin L; De Rosa, Stephen C

    2015-01-01

    The purpose of this study was to determine whether the development of a standardized approach to the collection of intestinal tissue from healthy volunteers, isolation of gut associated lymphoid tissue mucosal mononuclear cells (MMC), and characterization of mucosal T cell phenotypes by flow cytometry was sufficient to minimize differences in the normative ranges of flow parameters generated at two trial sites. Forty healthy male study participants were enrolled in Pittsburgh and Los Angeles. MMC were isolated from rectal biopsies using the same biopsy acquisition and enzymatic digestion protocols. As an additional comparator, peripheral blood mononuclear cells (PBMC) were collected from the study participants. For quality control, cryopreserved PBMC from a single donor were supplied to both sites from a central repository (qPBMC). Using a jointly optimized standard operating procedure, cells were isolated from tissue and blood and stained with monoclonal antibodies targeted to T cell phenotypic markers. Site-specific flow data were analyzed by an independent center which analyzed all data from both sites. Ranges for frequencies for overall CD4+ and CD8+ T cells, derived from the qPBMC samples, were equivalent at both UCLA and MWRI. However, there were significant differences across sites for the majority of T cell activation and memory subsets in qPBMC as well as PBMC and MMC. Standardized protocols to collect, stain, and analyze MMC and PBMC, including centralized analysis, can reduce but not exclude variability in reporting flow data within multi-site studies. Based on these data, centralized processing, flow cytometry, and analysis of samples may provide more robust data across multi-site studies. Centralized processing requires either shipping of fresh samples or cryopreservation and the decision to perform centralized versus site processing needs to take into account the drawbacks and restrictions associated with each method.

  11. Exploring the feasibility of multi-site flow cytometric processing of gut associated lymphoid tissue with centralized data analysis for multi-site clinical trials.

    Directory of Open Access Journals (Sweden)

    Ian McGowan

    Full Text Available The purpose of this study was to determine whether the development of a standardized approach to the collection of intestinal tissue from healthy volunteers, isolation of gut associated lymphoid tissue mucosal mononuclear cells (MMC, and characterization of mucosal T cell phenotypes by flow cytometry was sufficient to minimize differences in the normative ranges of flow parameters generated at two trial sites. Forty healthy male study participants were enrolled in Pittsburgh and Los Angeles. MMC were isolated from rectal biopsies using the same biopsy acquisition and enzymatic digestion protocols. As an additional comparator, peripheral blood mononuclear cells (PBMC were collected from the study participants. For quality control, cryopreserved PBMC from a single donor were supplied to both sites from a central repository (qPBMC. Using a jointly optimized standard operating procedure, cells were isolated from tissue and blood and stained with monoclonal antibodies targeted to T cell phenotypic markers. Site-specific flow data were analyzed by an independent center which analyzed all data from both sites. Ranges for frequencies for overall CD4+ and CD8+ T cells, derived from the qPBMC samples, were equivalent at both UCLA and MWRI. However, there were significant differences across sites for the majority of T cell activation and memory subsets in qPBMC as well as PBMC and MMC. Standardized protocols to collect, stain, and analyze MMC and PBMC, including centralized analysis, can reduce but not exclude variability in reporting flow data within multi-site studies. Based on these data, centralized processing, flow cytometry, and analysis of samples may provide more robust data across multi-site studies. Centralized processing requires either shipping of fresh samples or cryopreservation and the decision to perform centralized versus site processing needs to take into account the drawbacks and restrictions associated with each method.

  12. Corrigendum to "Flow cytometric quantitation of platelet phagocytosis by monocytes using a pH-sensitive dye, pHrodo-SE" [Journal of Immunological Methods 447 (2017) 57-64].

    Science.gov (United States)

    Takahashi, Daisuke; Fujihara, Mitsuhiro; Miyazaki, Toru; Matsubayashi, Keiji; Sato, Shinichiro; Azuma, Hiroshi; Kato, Toshiaki; Kino, Shuichi; Ikeda, Hisami; Takamoto, Shigeru; Sato, Noriyuki; Torigoe, Toshihiko

    2018-03-01

    Antibody-mediated phagocytosis of platelets using a flow cytometric monocyte-based phagocytosis assay (FMPA) has been shown to predict the outcome of platelet transfusion. The easy adherence between platelets and monocytes even in the absence of an antibody is regarded as one of limitations of the FMPA. To improve the FMPA for prediction of transfusion outcome, we used the pH-sensitive dye pHrodo succinimidyl ester (pHrodo-SE), which has weak fluorescence at neutral pH and has increased fluorescence intensity in low pH conditions such as in lysomes. Platelets stained with pHrodo-SE were sensitized with an HLA class I monoclonal antibody (w6/32 clone) or anti-HLA class I containing antisera. The platelets were incubated with monocyte-enriched mononuclear cells. Phagocytic activity was assessed by the percentage of monocytes that phagocytosed platelets. Sensitization of platelets with w6/32 significantly increased platelet phagocytosis by monocytes in dose- and time-dependent manners. Anti-HLA class I antibody-containing sera caused platelet phagocytosis in a cognate antigen-antibody-dependent manner. There was a significant correlation (r=0.69, p<0.01) between phagocytic index and titer of HLA class I antibody measured by lymphocyte immunofluorescence test-flow cytometry. In addition, the phagocytic index obtained by FMPA with pHrodo-SE was significantly higher than that obtained by FMPA with the previously used dye, carboxyfluorescein diacetate succinimidyl ester, when platelets were sensitized by w6/32 and anti-HLA class I antibody-containing sera. Because of the higher resolution and higher sensitivity than those of the previous method, the pHrodo-SE-based FMPA may be suitable for more precise quantitation of phagocytosis activity, which would enable qualitative evaluation of transfusion effectiveness. Copyright © 2018.

  13. Flow Cytometric DNA index, G-band Karyotyping, and Comparative Genomic Hybridization in Detection of High Hyperdiploidy in Childhood Acute Lymphoblastic Leukemia

    DEFF Research Database (Denmark)

    Nygaard, Ulrikka; Larsen, Jacob; Kristensen, Tim D

    2006-01-01

    High hyperdiploid acute lymphoblastic leukemia in children is related to a good outcome. Because these patients may be stratified to a low-intensity treatment, we have investigated the sensitivity of flow cytometry (FCM), G-band karyotyping (GBK), and high-resolution comparative genomic hybridiza......High hyperdiploid acute lymphoblastic leukemia in children is related to a good outcome. Because these patients may be stratified to a low-intensity treatment, we have investigated the sensitivity of flow cytometry (FCM), G-band karyotyping (GBK), and high-resolution comparative genomic...... hybridization (HR-CGH) in detecting high hyperdiploid leukemic clones. Twenty-six girls and 34 boys with acute lymphoblastic leukemia diagnosed in 1998 to 1999 were analyzed by FCM, GBK, and HR-CGH. The correlations between DNA indices obtained by FCM, GBK, and HR-CGH were significant (rs=0.61 to 0.77; P

  14. Flow cytometric applications of tumor biology: prospects and pitfalls. [Applications in study of spontaneous dog tumors and in drug and radiation effects on cultured V79 cells

    Energy Technology Data Exchange (ETDEWEB)

    Raju, M.R.; Johnson, T.S.; Tokita, N.; Gillette, E.L.

    1979-01-01

    A brief review of cytometry instrumentation and its potential applications in tumor biology is presented using our recent data. Age-distribution measurements of cells from spontaneous dog tumors and cultured cells after exposure to x rays, alpha particles, or adriamycin are shown. The data show that DNA fluorescence measurements have application in the study of cell kinetics after either radiation or drug treatment. Extensive and careful experimentation is needed to utilize the sophisticated developments in flow cytometry instrumentation.

  15. In-depth validation of acridine orange staining for flow cytometric parasite and reticulocyte enumeration in an experimental model using Plasmodium berghei

    DEFF Research Database (Denmark)

    Hein-Kristensen, L; Wiese, L; Kurtzhals, J A L

    2009-01-01

    Flow cytometry is potentially an effective method for counting malaria parasites, but inconsistent results have hampered its routine use in rodent models. A published two-channel method using acridine orange offers clear discrimination between the infected and uninfected erythrocytes. However......, preliminary studies showed concerns when dealing with Plasmodium berghei-infected blood samples with high numbers of reticulocytes. In hyperparasitemic or chronic P. berghei infection, enhanced erythropoietic activity results in high numbers of circulating immature reticulocytes. We show that even though...

  16. Super-resolved calibration-free flow cytometric characterization of platelets and cell-derived microparticles in platelet-rich plasma.

    Science.gov (United States)

    Konokhova, Anastasiya I; Chernova, Darya N; Moskalensky, Alexander E; Strokotov, Dmitry I; Yurkin, Maxim A; Chernyshev, Andrei V; Maltsev, Valeri P

    2016-02-01

    Importance of microparticles (MPs), also regarded as extracellular vesicles, in many physiological processes and clinical conditions motivates one to use the most informative and precise methods for their characterization. Methods based on individual particle analysis provide statistically reliable distributions of MP population over characteristics. Although flow cytometry is one of the most powerful technologies of this type, the standard forward-versus-side-scattering plots of MPs and platelets (PLTs) overlap considerably because of similarity of their morphological characteristics. Moreover, ordinary flow cytometry is not capable of measurement of size and refractive index (RI) of MPs. In this study, we 1) employed the potential of the scanning flow cytometer (SFC) for identification and characterization of MPs from light scattering; 2) suggested the reference method to characterize MP morphology (size and RI) with high precision; and 3) determined the lowest size of a MP that can be characterized from light scattering with the SFC. We equipped the SFC with 405 and 488 nm lasers to measure the light-scattering profiles and side scattering from MPs, respectively. The developed two-stage method allowed accurate separation of PLTs and MPs in platelet-rich plasma. We used two optical models for MPs, a sphere and a bisphere, in the solution of the inverse light-scattering problem. This solution provides unprecedented precision in determination of size and RI of individual spherical MPs-median uncertainties (standard deviations) were 6 nm and 0.003, respectively. The developed method provides instrument-independent quantitative information on MPs, which can be used in studies of various factors affecting MP population. © 2015 International Society for Advancement of Cytometry.

  17. Flow Cytometric DNA index, G-band Karyotyping, and Comparative Genomic Hybridization in Detection of High Hyperdiploidy in Childhood Acute Lymphoblastic Leukemia

    DEFF Research Database (Denmark)

    Nygaard, Ulrikka; Larsen, Jacob; Kristensen, Tim D

    2006-01-01

    High hyperdiploid acute lymphoblastic leukemia in children is related to a good outcome. Because these patients may be stratified to a low-intensity treatment, we have investigated the sensitivity of flow cytometry (FCM), G-band karyotyping (GBK), and high-resolution comparative genomic hybridiza......High hyperdiploid acute lymphoblastic leukemia in children is related to a good outcome. Because these patients may be stratified to a low-intensity treatment, we have investigated the sensitivity of flow cytometry (FCM), G-band karyotyping (GBK), and high-resolution comparative genomic.......001 for all comparisons). However, in 4 of 18 patients, high hyperdiploidy was overlooked by GBK or HR-CGH, and even when FCM was applied, 2 of 18 patients with high hyperdiploidy by GBK and/or HR-CGH were classified as nonhigh hyperdiploid. If high hyperdiploid subclones were included, FCM could detect all...... high hyperdiploid patients found by either GBK or HR-CGH, but would then in addition classify 15% to 20% of the remaining patients as high hyperdiploid. Thus, both GBK and HR-CGH overlook patients with high hyperdiploidy, and FCM only detects all high hyperdiploid patients if small high hyperdiploid...

  18. Identification of microbes from the surfaces of food-processing lines based on the flow cytometric evaluation of cellular metabolic activity combined with cell sorting.

    Science.gov (United States)

    Juzwa, W; Duber, A; Myszka, K; Białas, W; Czaczyk, K

    2016-09-01

    In this study the design of a flow cytometry-based procedure to facilitate the detection of adherent bacteria from food-processing surfaces was evaluated. The measurement of the cellular redox potential (CRP) of microbial cells was combined with cell sorting for the identification of microorganisms. The procedure enhanced live/dead cell discrimination owing to the measurement of the cell physiology. The microbial contamination of the surface of a stainless steel conveyor used to process button mushrooms was evaluated in three independent experiments. The flow cytometry procedure provided a step towards monitoring of contamination and enabled the assessment of microbial food safety hazards by the discrimination of active, mid-active and non-active bacterial sub-populations based on determination of their cellular vitality and subsequently single cell sorting to isolate microbial strains from discriminated sub-populations. There was a significant correlation (r = 0.97; p vitality and the identification of species from defined sub-populations, although the identified microbes were limited to culturable cells.

  19. Assessment of the viability and fertilizing potential of cryopreserved bovine spermatozoa using dual fluorescent staining and two-flow cytometric systems.

    Science.gov (United States)

    Ericsson, S A; Garner, D L; Redelman, D; Ahmad, K

    1989-04-01

    A dual fluorescent staining system utilizing 5 (and-6)-carboxy-4',5'-dimethyl fluorescein diacetate (CDMFDA) and Hydroethidine (HED) was developed to provide quantifiable information reflective of spermatozoal viability and fertilizing potential. Cryopreserved spermatozoa from ten bulls on which there was fertilizing capacity information were incubated for 1.5, and 3 hr at 39 degrees C prior to fluorogenic staining. Spermatozoa were analyzed using both a FACS Analyzer and an EPICS V flow cytometer to determine if a particular fluorescence pattern was due to an instrumental artifact or cellular processes. Five fluorescent cellular populations were identified by the FACS Analyzer and three populations by the EPICS V. Spermatozoa were quantified after each incubation time for red (HED) and green (CDMFDA) fluorescence. Viable spermatozoa retained the greatest amount of both green and red fluorescence. Dead or moribund spermatozoa had a decrease in over-all fluorescence. The number of viable cells at 0 hr plus the number of dead or morbid cells at any time period were identified by the FACS Analyzer as important in estimating the potential fertility of a bull. The EPICS V identified the number of dead or moribund cells as being related to nonreturn rates. Incubation of samples decreased cellular viability, which resulted in reduced levels of both green and red fluorescence. Similarities between data obtained with both flow cytometers illustrated that cellular processes, not instrumental artifacts, were responsible for the decrease in over-all fluorescence when viability declined, the relationship between the number of cells with specific fluorescence levels and nonreturn rates, and the incubative-induced changes in fluorescence patterns.

  20. Flow-cytometric measurement of CD4-8- T cells bearing T-cell receptor αβ chains, 1

    International Nuclear Information System (INIS)

    Kusunoki, Yoichiro; Hirai, Yuko; Kyoizumi, Seishi; Akiyama, Mitoshi.

    1992-09-01

    In this study we detected rare, possibly abnormal, T cells bearing CD3 surface antigen and T-cell receptor (TCR) αβ chains but lacking both CD4 and CD8 antigens (viz., TCRαβ + CD4 - 8 - cells, as determined by flow cytometry). The TCRαβ + CD4 - 8 - T cells were detected at a mean frequency of 0.63 ± 0.35 % (mean ± standard deviation) in peripheral blood TCRαβ + cells of 119 normal persons. Two unusual cases besides the 119 normal persons showed extremely elevated frequencies of TCRαβ + CD4 - 8 - T cells, viz., approximately 5 % to 10 % and 14 % to 19 % in whole TCRαβ + cells. Both individuals were males who were otherwise physiologically quite normal with no history of severe illness, and these high frequencies were also observed in blood samples collected 2 or 8 years prior to the current measurements. The TCRαβ + CD4 - 8 - T cells of the two individuals were found to express mature T-cell markers such as CD2,3, and 5 antigens, as well as natural killer (NK) cell markers, viz., CD11b, 16, 56, and 57 antigens, when peripheral blood lymphocytes were subjected to three-color flow cytometry. Lectin-dependent or redirected antibody-dependent cell-mediated cytotoxicities were observed for both freshly sorted TCRαβ + CD4 - 8 - cells and in vitro established clones. Nevertheless, NK-like activity was not detected. Further, Southern blot analysis of TCRβ and γ genes revealed identical rearrangement patterns for all the TCRαβ + CD4 - 8 - clones established in vitro. These results suggest that the TCRαβ + CD4 - 8 - T cells from these two mean exhibit unique characteristics and proliferate clonally in vivo. (author)

  1. Lymphocyte subsets show different response patterns to in vivo bound natalizumab--a flow cytometric study on patients with multiple sclerosis.

    Directory of Open Access Journals (Sweden)

    Andrea Harrer

    Full Text Available Natalizumab is an effective monoclonal antibody therapy for the treatment of relapsing-remitting multiple sclerosis (RRMS and interferes with immune cell migration into the central nervous system by blocking the α(4 subunit of very-late activation antigen-4 (VLA-4. Although well tolerated and very effective, some patients still suffer from relapses in spite of natalizumab therapy or from unwanted side effects like progressive multifocal leukoencephalopathy (PML. In search of a routine-qualified biomarker on the effectiveness of natalizumab therapy we applied flow cytometry and analyzed natalizumab binding to α(4 and α(4 integrin surface levels on T-cells, B-cells, natural killer (NK cells, and NKT cells from 26 RRMS patients under up to 72 weeks of therapy. Four-weekly infusions of natalizumab resulted in a significant and sustained increase of lymphocyte-bound natalizumab (p<0.001 which was paralleled by a significant decrease in detectability of the α(4 integrin subunit on all lymphocyte subsets (p<0.001. We observed pronounced natalizumab accumulations on T and B cells at single measurements in all patients who reported clinical disease activity (n = 4. The natalizumab binding capacity of in vitro saturated lymphocytes collected during therapy was strongly diminished compared to treatment-naive cells indicating a therapy-induced reduction of α(4. Summing up, this pilot study shows that flow cytometry is a useful method to monitor natalizumab binding to lymphocytes from RRMS patients under therapy. Investigating natalizumab binding provides an opportunity to evaluate the molecular level of effectiveness of natalizumab therapy in individual patients. In combination with natalizumab saturation experiments, it possibly even provides a means of studying the feasability of patient-tailored infusion intervals. A routine-qualified biomarker on the basis of individual natalizumab saturation on lymphocyte subsets might be an effective tool to

  2. Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting: Phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive CD4{sup +} T cells compared

    Energy Technology Data Exchange (ETDEWEB)

    Ford, A.L.; Goodsall, A.L.; Sedgwick, J.D. [Centenary Institute of Cancer Medicine and Cell Biology, Sydney (Australia)] [and others

    1995-05-01

    Ramified microglia in the adult central nervous system (CNS) are the principal glial element up-regulating MHC class I and II expression in response to inflammatory events or neuronal damage. A proportion of these cells also express MHC class II constitutively in the normal CNS. The role of microglia as APCs for CD4{sup +} cells extravasating into the CNS remains undefined. In this study, using irradiation bone marrow chimeras in CD45-congenic rats, the phenotype CD45{sup low}CD11b/c{sup +} is shown to identify microglial cells specifically within the CNS. Highly purified populations of microglia and nonmicroglial but CNS-associated macrophages (CD45{sup high}CD11b/c{sup +}) have been obtained directly from the adult CNS, by using flow cytometric sorting. Morphologically, freshly isolated microglia vs other CNS macrophages are quite distinct. Of the two populations recovered from the normal CNS, it is the minority CD45{sup high}CD11 b/c{sup +} transitional macrophage population, and not microglia, that is the effective APC for experimental autoimmune encephalomyelitis-inducing CD4{sup +} myelin basic protein (MBP)-reactive T cells. CD45{sup high}CD11b/c{sup +} CNS macrophages also stimulate MBP-reactive T cells without addition of MBP to culture suggesting presentation of endogenous Ag. This is the first study in which microglia vs other CNS macrophages have been analyzed for APC ability directly from the CNS, with substantial cross-contamination between the two populations eliminated. The heterogeneity of these populations in terms of APC function is clearly demonstrated. Evidence is still lacking that adult CNS microglia have the capacity to interact with and stimulate CD4{sup +} T cells to proliferate or secrete IL-2. 60 refs., 6 figs., 1 tab.

  3. Innovations in diagnosis and post-therapeutic monitoring of Chagas disease: Simultaneous flow cytometric detection of IgG1 antibodies anti-live amastigote, anti-live trypomastigote, and anti-fixed epimastigote forms of Trypanosoma cruzi.

    Science.gov (United States)

    Alessio, Glaucia Diniz; Côrtes, Denise Fonseca; Machado de Assis, Girley Francisco; Júnior, Policarpo Ademar Sales; Ferro, Eloisa Amália Vieira; Antonelli, Lis Ribeiro do Valle; Teixeira-Carvalho, Andréa; Martins-Filho, Olindo Assis; de Lana, Marta

    2014-11-01

    This study developed a remarkable methodological innovation (FC-ATE) which enables simultaneous detection of antibodies specific to the three evolutive forms of Trypanosoma cruzi: live amastigote (AMA), live trypomastigote (TRYPO), and fixed epimastigote (EPI) using a differential fluorescence staining as low (AMA), intermediate (TRYPO), and high (EPI). An outstanding performance (100%) was observed in the discrimination of the chagasic (CH) and non-chagasic (NCH) patients. In the applicability of FC-ATE in the diagnosis of Chagas disease, 100% of the CH samples presented positivity in the percentage of positive fluorescent parasites (PPFP) for all the three forms of T. cruzi. Moreover, 94% of the samples of NCH presented negative values of PPFP with AMA and TRYPO, and 88% with EPI. Samples from the NCH group with false-positive results were those belonging to the leishmaniasis patients. Considering the applicability of this technique in post-therapeutic monitoring of Chagas disease, 100% of non-treated (NT) and treated non-cured (TNC) samples were positive with the three T. cruzi evolutive forms, while a percentage of 100% from samples of the treated cured (TC) patients were negative with AMA, 93% with TRYPO and 96% with EPI. The comparison between FC-ATE and two other flow cytometric tests using the same samples of patients NT, TNC and TC showed that the three techniques presented different reactivities, although categorical correlation between the methodologies was observed. Taken together, the results obtained with the novel FC-ATE method have shown an outstanding performance in the diagnosis and post-therapeutic monitoring of Chagas disease. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Corrected Lymphocyte Percentages Reduce the Differences in Absolute CD4+ T Lymphocyte Counts between Dual-Platform and Single-Platform Flow Cytometric Approaches.

    Science.gov (United States)

    Noulsri, Egarit; Abudaya, Dinar; Lerdwana, Surada; Pattanapanyasat, Kovit

    2018-03-13

    To determine whether a corrected lymphocyte percentage could reduce bias in the absolute cluster of differentiation (CD)4+ T lymphocyte counts obtained via dual-platform (DP) vs standard single-platform (SP) flow cytometry. The correction factor (CF) for the lymphocyte percentages was calculated at 6 laboratories. The absolute CD4+ T lymphocyte counts in 300 blood specimens infected with human immunodeficiency virus (HIV) were determined using the DP and SP methods. Applying the CFs revealed that 4 sites showed a decrease in the mean bias of absolute CD4+ T lymphocyte counts determined via DP vs standard SP (-109 vs -84 cells/μL, -80 vs -58 cells/μL, -52 vs -45 cells/μL, and -32 vs 1 cells/μL). However, 2 participating laboratories revealed an increase in the difference of the mean bias (-42 vs -49 cells/μL and -20 vs -69 cells/μL). Use of the corrected lymphocyte percentage shows potential for decreasing the difference in CD4 counts between DP and the standard SP method.

  5. In vitro effects of zirconia and alumina particles on human blood monocyte-derived macrophages: X-ray microanalysis and flow cytometric studies.

    Science.gov (United States)

    Nkamgueu, E M; Adnet, J J; Bernard, J; Zierold, K; Kilian, L; Jallot, E; Benhayoune, H; Bonhomme, P

    2000-12-15

    The cytocompatibility of two particulate bioceramics, zirconia and alumina, was studied using human blood monocytes driven to differentiate into mature macrophages with granulocyte macrophage-colony-stimulating factor. Changes in individual cell elemental composition, particularly sodium and potassium content, were assessed by X-ray microanalysis of ultrathin freeze-dried sections. Phagocytosis and respiratory burst of macrophages exposed to biomaterial for 7 days were analyzed under flow cytometry using uptake of fluorescent latex beads and 2'7'-dichlorofluorescien diacetate oxidation, respectively. Zirconia and alumina particles were found to decrease the intracellular potassium/sodium ratio (an index of cell vitality) significantly (p2 times reduced by zirconia and >5 times reduced by alumina). The present study clearly demonstrates that reduction of the phagocytic capacity of macrophages associated with altered oxidative metabolism caused by biomaterial particles is characterized by changes in intracellular elemental content. Thus, investigation of cellular homeostasis by electron probe microanalysis together with analysis of functional changes may improve estimation of biomaterial cytocompatibility. Copyright 2000 John Wiley & Sons, Inc.

  6. Lymphocyte subsets show different response patterns to in vivo bound natalizumab--a flow cytometric study on patients with multiple sclerosis.

    Science.gov (United States)

    Harrer, Andrea; Pilz, Georg; Einhaeupl, Max; Oppermann, Katrin; Hitzl, Wolfgang; Wipfler, Peter; Sellner, Johann; Golaszewski, Stefan; Afazel, Shahrzad; Haschke-Becher, Elisabeth; Trinka, Eugen; Kraus, Joerg

    2012-01-01

    Natalizumab is an effective monoclonal antibody therapy for the treatment of relapsing-remitting multiple sclerosis (RRMS) and interferes with immune cell migration into the central nervous system by blocking the α(4) subunit of very-late activation antigen-4 (VLA-4). Although well tolerated and very effective, some patients still suffer from relapses in spite of natalizumab therapy or from unwanted side effects like progressive multifocal leukoencephalopathy (PML). In search of a routine-qualified biomarker on the effectiveness of natalizumab therapy we applied flow cytometry and analyzed natalizumab binding to α(4) and α(4) integrin surface levels on T-cells, B-cells, natural killer (NK) cells, and NKT cells from 26 RRMS patients under up to 72 weeks of therapy. Four-weekly infusions of natalizumab resulted in a significant and sustained increase of lymphocyte-bound natalizumab (pqualified biomarker on the basis of individual natalizumab saturation on lymphocyte subsets might be an effective tool to improve treatment safety.

  7. Flow Cytometric Determination of Cellular Sources and Frequencies of Key Cytokine-Producing Lymphocytes Directed against Recombinant LACK and Soluble Leishmania Antigen in Human Cutaneous Leishmaniasis

    Science.gov (United States)

    Bottrel, R. L. A.; Dutra, W. O.; Martins, F. A.; Gontijo, B.; Carvalho, E.; Barral-Netto, M.; Barral, A.; Almeida, R. P.; Mayrink, W.; Locksley, R.; Gollob, K. J.

    2001-01-01

    Leishmaniasis, caused by infection with the protozoan parasite Leishmania, affects millions of individuals worldwide, causing serious morbidity and mortality. This study directly determined the frequency of cells producing key immunoregulatory cytokines in response to the recombinant antigen Leishmania homolog of receptors for activated kinase C (LACK) and soluble leishmania antigen (SLA), and it determined relative contributions of these antigens to the overall cytokine profile in individuals infected for the first time with Leishmania braziliensis. All individuals presented with the cutaneous clinical form of leishmaniasis and were analyzed for proliferative responses to LACK antigen and SLA, frequency of lymphocyte subpopulations (analyzed ex vivo), and antigen-induced (LACK and SLA) cytokine production at the single-cell level (determined by flow cytometry). The following were determined. (i) The Th1-type response previously seen in patients with cutaneous leishmaniasis is due to gamma interferon (IFN-γ) production by several different sources, listed in order of contribution: CD4+ T lymphocytes, CD4−, CD8− lymphocytes, and CD8+ T lymphocytes. (ii) SLA induced a higher frequency of lymphocytes producing IFN-γ and tumor necrosis factor alpha (TNF-α) than did LACK. (iii) LACK induced an activation of monocyte populations as reflected by an increased percentage of CD14-positive cells. (iv) Neither SLA nor LACK induced detectable frequencies of cells producing interleukin-4 (IL-4) or IL-5. These data demonstrated a multifaceted immune response to SLA in human leishmaniasis involving Th1 CD4+ T lymphocytes (IFN-γ+ and IL-10−/IL-4−), Tc1 CD8+ T cells (IFN-γ+, and IL-10−/IL-4−), and a high frequency of TNF-α-producing lymphocytes. Moreover, it was determined that the recombinant antigen LACK acts as a weak inducer of Th1-type lymphocyte responses compared to SLA. PMID:11292745

  8. EPR-Spin Trapping and Flow Cytometric Studies of Free Radicals Generated Using Cold Atmospheric Argon Plasma and X-Ray Irradiation in Aqueous Solutions and Intracellular Milieu.

    Science.gov (United States)

    Uchiyama, Hidefumi; Zhao, Qing-Li; Hassan, Mariame Ali; Andocs, Gabor; Nojima, Nobuyuki; Takeda, Keigo; Ishikawa, Kenji; Hori, Masaru; Kondo, Takashi

    2015-01-01

    Electron paramagnetic resonance (EPR)-spin trapping and flow cytometry were used to identify free radicals generated using argon-cold atmospheric plasma (Ar-CAP) in aqueous solutions and intracellularly in comparison with those generated by X-irradiation. Ar-CAP was generated using a high-voltage power supply unit with low-frequency excitation. The characteristics of Ar-CAP were estimated by vacuum UV absorption and emission spectra measurements. Hydroxyl (·OH) radicals and hydrogen (H) atoms in aqueous solutions were identified with the spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M4PO), and phenyl N-t-butylnitrone (PBN). The occurrence of Ar-CAP-induced pyrolysis was evaluated using the spin trap 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS) in aqueous solutions of DNA constituents, sodium acetate, and L-alanine. Human lymphoma U937 cells were used to study intracellular oxidative stress using five fluorescent probes with different affinities to a number of reactive species. The analysis and quantification of EPR spectra revealed the formation of enormous amounts of ·OH radicals using Ar-CAP compared with that by X-irradiation. Very small amounts of H atoms were detected whereas nitric oxide was not found. The formation of ·OH radicals depended on the type of rare gas used and the yield correlated inversely with ionization energy in the order of krypton > argon = neon > helium. No pyrolysis radicals were detected in aqueous solutions exposed to Ar-CAP. Intracellularly, ·OH, H2O2, which is the recombination product of ·OH, and OCl- were the most likely formed reactive oxygen species after exposure to Ar-CAP. Intracellularly, there was no practical evidence for the formation of NO whereas very small amounts of superoxides were formed. Despite the superiority of Ar-CAP in forming ·OH radicals, the exposure to X-rays proved more lethal. The mechanism of free radical formation in aqueous solutions and an

  9. EPR-Spin Trapping and Flow Cytometric Studies of Free Radicals Generated Using Cold Atmospheric Argon Plasma and X-Ray Irradiation in Aqueous Solutions and Intracellular Milieu.

    Directory of Open Access Journals (Sweden)

    Hidefumi Uchiyama

    Full Text Available Electron paramagnetic resonance (EPR-spin trapping and flow cytometry were used to identify free radicals generated using argon-cold atmospheric plasma (Ar-CAP in aqueous solutions and intracellularly in comparison with those generated by X-irradiation. Ar-CAP was generated using a high-voltage power supply unit with low-frequency excitation. The characteristics of Ar-CAP were estimated by vacuum UV absorption and emission spectra measurements. Hydroxyl (·OH radicals and hydrogen (H atoms in aqueous solutions were identified with the spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO, 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M4PO, and phenyl N-t-butylnitrone (PBN. The occurrence of Ar-CAP-induced pyrolysis was evaluated using the spin trap 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS in aqueous solutions of DNA constituents, sodium acetate, and L-alanine. Human lymphoma U937 cells were used to study intracellular oxidative stress using five fluorescent probes with different affinities to a number of reactive species. The analysis and quantification of EPR spectra revealed the formation of enormous amounts of ·OH radicals using Ar-CAP compared with that by X-irradiation. Very small amounts of H atoms were detected whereas nitric oxide was not found. The formation of ·OH radicals depended on the type of rare gas used and the yield correlated inversely with ionization energy in the order of krypton > argon = neon > helium. No pyrolysis radicals were detected in aqueous solutions exposed to Ar-CAP. Intracellularly, ·OH, H2O2, which is the recombination product of ·OH, and OCl- were the most likely formed reactive oxygen species after exposure to Ar-CAP. Intracellularly, there was no practical evidence for the formation of NO whereas very small amounts of superoxides were formed. Despite the superiority of Ar-CAP in forming ·OH radicals, the exposure to X-rays proved more lethal. The mechanism of free radical formation in aqueous solutions and

  10. The impact of flow PRA on outcome in pediatric heart recipients in modern era: An analysis of the Pediatric Heart Transplant Study database.

    Science.gov (United States)

    Das, B B; Pruitt, E; Molina, K; Ravekes, W; Auerbach, S; Savage, A; Knox, L; Kirklin, J K; Naftel, D C; Hsu, D

    2018-02-01

    Data from patients in the Pediatric Heart Transplant Study (PHTS) registry transplanted between 2010 and 2014 were analyzed to determine the association between HLA antibody (PRA) determined by SPA using Luminex or flow cytometry with a positive retrospective cross-match and the post-transplant outcomes of acute rejection and graft survival. A total of 1459 of 1596 (91%) recipients had a PRA reported pretransplant; 26% had a PRA > 20%. Patients with a PRA > 20% were more likely to have CHD, prior cardiac surgery, ECMO support at listing, and waited longer for transplantation than patients with a PRA PRA% determined by SPA were predictive of a positive retrospective cross-match determined by flow cytometric method (P PRA > 50% determined by SPA was independently associated with worse overall graft survival after first month of transplant in both unadjusted and adjusted for all other risk factors. In this large multicenter series of pediatric heart transplant recipients, an elevated PRA determined by SPA remains a significant risk factor in the modern era. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  11. The relative utility of the autologous control and the antiglobulin test phase of the crossmatch.

    Science.gov (United States)

    Perkins, J T; Arruza, M; Fong, K; Sosler, S D; Saporito, C

    1990-01-01

    A retrospective study of pretransfusion testing records compared the utility of the antiglobulin test (AGT) phase of the crossmatch and the autologous control (autocontrol) for detecting clinically significant alloimmunization to red cells (RBCs). Of 110,780 consecutive crossmatches, 141 were positive after a negative antibody screening test; only 4 of these were due to alloantibodies of potential clinical significance, for a predictive value of a positive AGT crossmatch, after a negative antibody screen, of 2.8 percent (4/141). The frequency of potentially shortened RBC survival was 1 in 27,685 units crossmatched. During a similar period, 56,090 autocontrols were performed with the antibody screen. The autocontrol was positive on 902 samples in which the antibody screen was negative. Antibody identification performed in 684 cases generally yielded only cold or warm autoagglutinins. In 96 cases, some form of alloantibody was detected, but only 25 had potential clinical significance by our criteria. Eight of these alloantibodies had concurrently caused in vivo sensitization of RBCs and were classified as delayed hemolytic transfusion reactions. The predictive value of the autocontrol, calculated as the number of significant alloantibodies detected in autocontrol-positive, antibody-screen-negative samples, was 3.6 percent (25/684). Inspection of these cases revealed 11 in which shortened RBC survival might have resulted if the serologic abnormality had not been detected. Thus, the autocontrol had a slightly greater yield of clinically significant findings than the AGT crossmatch.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. In Kidney Transplant Recipients With a Positive Virtual Crossmatch, High PRA was Associated With Lower Incidence of Viral Infections.

    Science.gov (United States)

    Parajuli, Sandesh; Muth, Brenda L; Turk, Jennifer A; Astor, Brad C; Mohammed, Maha; Mandelbrot, Didier A; Djamali, Arjang

    2016-03-01

    There is little information on the incidence, risk factors, and outcomes associated with CMV and BK infections in sensitized patients. We examined 254 consecutive kidney transplant recipients with positive virtual crossmatch and negative flow crossmatch. A total of 111 patients (43%) developed CMV disease or BK infection or nephropathy (BKVN). Specifically, 78 patients (30.7%) developed BK infection, 19 (7.5%) had BKVN, and 33 (12.9%) presented with CMV disease. Four patients (1.5%) developed both infections. Mean time from transplant to diagnosis for BK and CMV was 4.07 ± 3.10 and 8.35 ± 5.20 months, respectively. African American (HR, 2.64; 95% CI, 1.37-5.07; P = 0.003), thymoglobulin induction (HR, 2.18; 95% CI, 1.38-3.43; P = 0.0008), DSA greater than 500 MFI at transplant (HR, 1.64; 95% CI, 1.05-2.57; P = 0.03), history of diabetes (HR, 1.62; 95% CI, 1.01-2.60; P = 0.04), CMV D+/R- (HR, 2.30; 95% CI, 1.06-5.01; P = 0.03), and acute rejection (HR, 1.49; 95% CI, 0.99-2.24; P = 0.05) were associated with increase incident of BK/CMV, whereas rituximab (HR, 0.47; 95% CI, 0.24-0.91; P = 0.02), peak PRA greater than 80% (HR, 0.48; 95% CI, 0.27-0.84; P = 0.01), and living donor transplant (HR, 0.57; 95% CI, 0.36-0.87; P = 0.01) were associated with a lower likelihood of infection. Thymoglobulin induction (HR, 2.50; 95% CI, 1.02-6.13; P = 0.04), and peak PRA greater than 80% (HR, 0.45; 95% CI, 0.23-0.86; P = 0.02) remained significant predictors of infection after multivariate adjustment. Although more than 40% of patients with a positive virtual crossmatch presented with BK infection/CMV disease, high PRA greater than 80% seemed to be protective.

  13. Preventing the Decodability Attack Based Cross-Matching in a Fuzzy Commitment Scheme

    NARCIS (Netherlands)

    Kelkboom, E.J.C.; Breebaart, Jeroen; Kevenaar, Tom A.M.; Buhan, I.R.; Veldhuis, Raymond N.J.

    Template protection techniques are used within biometric systems in order to safeguard the privacy of the system's subjects. This protection also includes unlinkability, i.e., preventing cross-matching between two or more reference templates from the same subject across different applications. In

  14. Platelet crossmatch tests using radiolabelled staphylococcal protein A or peroxidase anti-peroxidase in alloimmunised patients

    International Nuclear Information System (INIS)

    Yam, P.; Petz, L.D.; Scott, E.P.; Santos, S.

    1984-01-01

    Refractoriness to random-donor platelets as a result of alloimmunization remains a major problem in long-term platelet transfusion therapy despite the use of HLA-matched platelets. A study has been made of two methods for detection of platelet associated IgG as platelet crossmatch tests for the selection of platelet donors. These methods use radiolabelled staphylococcal protein A( 125 I-SPA) and peroxidase anti-peroxidase (PAP), respectively. One hundred and ten crossmatch tests using 125 I-SPA were performed retrospectively in 18 alloimmunized patients. The results indicated that the predictive value of a positive or a negative test was 87%; the sensitivity was 73% and the specificity was 95%. Results with the PAP test were similar. The HLA types were known for 48 donor-recipient pairs. With few exceptions, there was a correlation between the results of the platelet crossmatch tests and the effectiveness of platelet transfusion regardless of the degree of HLA match. These results indicate that platelet crossmatch tests may be valuable even when closely HLA matched donors are not available. A large-scale prospective study is warranted, particularly in highly immunized patients. (author)

  15. Does a positive pretransplant crossmatch affect long-term outcome in liver transplantation?

    LENUS (Irish Health Repository)

    Al-Sibae, Mohamad R

    2012-02-01

    Despite the historical success of liver transplantation in the face of a positive lymphocytic crossmatch, increased incidence of acute cellular rejection and graft loss have been reported in this setting. Given the potential adverse effects of antirejection treatment, especially in hepatitis C virus-positive recipients, identification of predisposing factors could allow for better surveillance, avoidance of rejection, and potentially better graft outcomes.

  16. Incomplete Antibodies May Reduce ABO Cross-Match Incompatibility: A Pilot Study

    Directory of Open Access Journals (Sweden)

    Mehmet Özen

    2018-02-01

    Full Text Available Objective: Any erythrocyte transfusion among humans having type A or B blood groups is impossible due to antibodies causing fatal transfusion complications. A cross-match test is performed to prevent immune transfusion complications before transfusion. Our hypothesis is that the fragment antibody (Fab part of the antibody (incomplete antibody may be used to prevent an immune stimulus related to the complete antibody. Therefore, we designed a pilot study to evaluate the effectiveness of these incomplete antibodies using cross-match tests. Materials and Methods: Pepsin enzyme and staphylococcal protein A columns were used to cut anti-A and anti-B monoclonal antibodies and purify their Fab (2 fragments, respectively. An Rh-positive erythrocyte suspension with purified anti-A Fab (2 solution and B Rh-positive erythrocyte suspension with purified anti-B Fab (2 solution were combined correspondingly. Cross-match tests were performed by tube and gel centrifugation methods. The agglutination levels due to the anti-A and anti-B Fab (2 antibodies and their effects on the agglutination normally observed with complete antibodies were then measured. Results: No agglutination for the purified incomplete anti-A Fab (2 with A Rh+ erythrocyte and anti-B Fab (2 with B Rh+ erythrocyte combinations was observed in the tube cross-match tests. These agglutination levels were 1+ in two wells in the gel centrifugation cross-match tests. Fab (2-treated erythrocytes were also resistant to the agglutination that normally occurs with complete antibodies. Conclusion: We determined that the Fab (2 fragments of antibodies may not only be used to obtain a mild or negative reaction when compared to complete antibodies, but they might also be used for decreasing ABO incompatibility. Incomplete antibodies might be a therapeutic option in autoimmune hemolytic anemia and they may also be used in solid organ or hematopoietic stem cell transplantation. Therefore, we have planned an

  17. Flow cytometry beads rather than the antihuman globulin method should be used to detect HLA Class I IgG antibody (PRA) in cadaveric renal regraft candidates.

    Science.gov (United States)

    Bryan, Christopher F; McDonald, Scott B; Baier, Karen A; Luger, Alan M; Aeder, Mark I; Murillo, Daniel; Muruve, Nicolas A; Nelson, Paul W; Shield, Charles F; Warady, Bradley A

    2002-01-01

    HLA Class I antibody screening can be performed by flow cytometry using a mixture of 30 distinct bead populations each coated with the Class I antigen phenotype derived from different cell lines. In this study we compared the efficacy of Class I antibody screens done by flow cytometry beads with the antihuman globulin (AHG) method for patients awaiting cadaveric renal retransplantation. Class I panel reactive antibody (PRA) screening by flow cytometric beads of 21 regraft serum samples that had all been found to be negative by AHG DTT Class I PRA, revealed that 57.1% (12 of 21) had a flow Class I PRA of > or = 10%. Furthermore, when five regraft sera with an intermediate PRA were screened (mean AHG DTT PRA = 33.2 +/- 13%) the mean flow Class I PRA almost doubled (mean flow PRA = 72.4 +/- 10.2%) (p PRA by flow beads, were divided into the three PRA categories based on their peak flow Class I PRA value (0-20%, 21-79% and > or = 80%), the incidence of a positive flow cross-match was 0%, 72% and 85% and the incidence of retransplantation was 60%, 22% and 10%, in each of these groups, respectively. These data provided our histocompatibility laboratory with the rationale to stop performing the AHG PRA and perform only the flow Class I PRA method for regraft candidates.

  18. Pre- and Post-Transfusion Alloimmunization in Dogs Characterized by 2 Antiglobulin-Enhanced Cross-match Tests.

    Science.gov (United States)

    Goy-Thollot, I; Giger, U; Boisvineau, C; Perrin, R; Guidetti, M; Chaprier, B; Barthélemy, A; Pouzot-Nevoret, C; Canard, B

    2017-09-01

    When dogs are transfused, blood compatibility testing varies widely but may include dog erythrocyte antigen (DEA) 1 typing and rarely cross-matching. Prospective study to examine naturally occurring alloantibodies against red blood cells (RBCs) and alloimmunization by transfusion using 2 antiglobulin-enhanced cross-match tests. Eighty client-owned anemic, 72 donor, and 7 control dogs. All dogs were typed for DEA 1 and some also for DEA 4 and DEA 7. Major cross-match tests with canine antiglobulin-enhanced immunochromatographic strip and gel columns were performed 26-129 days post-transfusion (median, 39 days); some dogs had an additional early evaluation 11-22 days post-transfusion (median, 16 days). Plasma from alloimmunized recipients was cross-matched against RBCs from 34 donor and control dogs. The 2 cross-match methods gave entirely concordant results. All 126 pretransfusion cross-match results for the 80 anemic recipients were compatible, but 54 dogs died or were lost to follow up. Among the 26 recipients with follow-up, 1 dog accidently received DEA 1-mismatched blood and became cross-match-incompatible post-transfusion. Eleven of the 25 DEA 1-matched recipients (44%) became incompatible against other RBC antigens. No naturally occurring anti-DEA 7 alloantibodies were detected in DEA 7- dogs. The antiglobulin-enhanced immunochromatographic strip cross-match and laboratory gel column techniques identified no naturally occurring alloantibodies against RBC antigens, but a high degree of post-transfusion alloimmunization in dogs. Cross-matching is warranted in any dog that has been previously transfused independent of initial DEA 1 typing and cross-matching results before the first transfusion event. Copyright © 2017 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

  19. Calculated PRA: initial results show benefits for sensitized patients and a reduction in positive crossmatches.

    Science.gov (United States)

    Cecka, J M; Kucheryavaya, A Y; Reinsmoen, N L; Leffell, M S

    2011-04-01

    The calculated panel reactive antibody (CPRA), which is based upon unacceptable HLA antigens listed on the waitlist form for renal transplant candidates, replaced PRA as the measure of sensitization among US renal transplant candidates on October 1, 2009. An analysis of the impact of this change 6 months after its implementation shows an 83% reduction in the number of kidney offers declined nationwide because of a positive crossmatch. The increasing acceptance and utilization of unacceptable HLA antigens to avoid offers of predictably crossmatch-positive donor kidneys has increased the efficiency of kidney allocation, resulting in a significant increase in the percentage of transplants to broadly sensitized (80+% PRA/CPRA) patients from 7.3% during the period 07/01/2001-6/30/2002 to 15.8% of transplants between 10/1/09-3/31/10. The transplant rates per 1000 active patient-years on the waitlist also increased significantly for broadly sensitized patients after October 1, 2009. These preliminary results suggest that 'virtual' positive crossmatch prediction based on contemporary tools for identifying antibodies directed against HLA antigens is effective, increases allocation efficiency and improves access to transplants for sensitized patients awaiting kidney transplantation. ©2010 The Authors Journal compilation©2010 The American Society of Transplantation and the American Society of Transplant Surgeons.

  20. In vivo cross-match by chromium-51 urinary excretion from labeled erythrocytes: A case of anti-Gerbich

    International Nuclear Information System (INIS)

    Mochizuki, T.; Tauxe, W.N.; Ramsey, G.

    1990-01-01

    We studied a patient with an alloantibody to the high-frequency red blood cell (RBC) antigen Gerbich. A nationwide search for rare Gerbich-negative blood (less than 1:45,000 donors) located only seven units, and our supply was quickly exhausted. By using an in vivo cross-matching method, we demonstrated that this anti-Gerbich did not cause RBC destruction. Regular Gerbich-positive transfusions could then proceed without hemolysis. This cross-match test was based on the determination of the urinary excretion rates of injected radioactive chromium-labeled donor erythrocytes by which it was possible to determine compatibility only 24 hr after the test was begun. The procedure provides an easy and accurate means for in vivo cross-matching of conventionally incompatible donor blood

  1. A Triple-Stain Flow Cytometric Method to Assess Plasma- and Acrosome-Membrane Integrity of Cryopreserved Bovine Sperm Immediately after Thawing in Presence of Egg-Yolk Particles

    NARCIS (Netherlands)

    Nagy, S.; Jansen, J.; Topper, E.K.; Gadella, B.M.

    2003-01-01

    Simultaneously evaluating postthaw viability and acrosome integrity of spermatozoa by flow cytometry would provide a valuable testing tool in both research and routine work. In the present study, a new triple-stain combination was developed for the simultaneous evaluation of viability and acrosome

  2. Measuring trade-offs that matter: assessing the impact of a new electronic cross-match policy on the turnaround time and the cross-match workload efficiency.

    Science.gov (United States)

    Lin, David M; Goldfinger, Dennis; Lu, Qun; Wallace, Bridget; Kosaka-Nguyen, Dawn; Wood, Alisa; Porter, Bethany; Bumerts, Pamela; Jeffery, Rebecca; Fang, Amy; Stalcup, Irene; Penaflorida, Tracy; Ziman, Alyssa

    2014-12-01

    Our traditional cross-match (XM) policy generated a significant number of XM units that were never issued. To minimize the unnecessary XM workload, we proposed a new policy where orders eligible for the electronic XM (EXM) are pended until orders to issue red blood cells (RBCs) are received. To address concerns that this new policy might unduly delay blood availability, we conducted a study to assess whether the new policy was noninferior to the traditional policy with regard to the turnaround time (TAT). We monitored the TAT and XM workload efficiency (XM-to-issue [C : I] ratio) for a total of 8 weeks split between the two policies' periods. The primary outcome was the proportion of RBC issue requests that was turned around in less than 12 minutes. Fifty percent (1133 of 2265) of issue requests were turned around in 12 minutes or less under the traditional policy compared to 43.9% (975 of 2223) under the new policy (absolute difference of 6.1%; 95% confidence interval [CI], 3.2%-9.1%; p trade-off between delays in the TAT and efficiency gains in the XM workload remained acceptable for patient care. © 2014 AABB.

  3. Comparison of a commercial blood cross-matching kit to the standard laboratory method for establishing blood transfusion compatibility in dogs.

    Science.gov (United States)

    Guzman, Leo Roa; Streeter, Elizabeth; Malandra, Allison

    2016-01-01

    To evaluate the accuracy of a commercial blood transfusion cross-match kit when compared to the standard laboratory method for establishing blood transfusion compatibility. A prospective observational in intro study performed from July 2009 to July 2013. Private referral veterinary center. Ten healthy dogs, 11 anemic dogs, and 24 previously transfused dogs. None. Forty-five dogs were enrolled in a prospective study in order to compare the standard blood transfusion cross-match technique to a commercial blood transfusion cross-matching kit. These dogs were divided into 3 different groups that included 10 healthy dogs (control group), 11 anemic dogs in need of a blood transfusion, and 24 sick dogs that were previously transfused. Thirty-five dogs diagnosed with anemia secondary to multiple disease processes were cross-matched using both techniques. All dogs cross-matched via the kit had a compatible major and minor result, whereas 16 dogs out of 45 (35%) had an incompatible cross-match result when the standard laboratory technique was performed. The average time to perform the commercial kit was 15 minutes and this was 3 times shorter than the manual cross-match laboratory technique that averaged 45-50 minutes to complete. While the gel-based cross-match kit is quicker and less technically demanding than standard laboratory cross-match procedures, microagglutination and low-grade hemolysis are difficult to identify by using the gel-based kits. This could result in transfusion reactions if the gel-based kits are used as the sole determinant of blood compatibility prior to transfusion. Based on our results, the standard manual cross-match technique remains the gold standard test to determine blood transfusion compatibility. © Veterinary Emergency and Critical Care Society 2016.

  4. In vivo crossmatching with Tc-99m-RBC's and In-111-oxine-RBC's

    International Nuclear Information System (INIS)

    Marcus, C.S.; Myhre, B.A.; Angulo, M.C.; Salk, R.D.; Essex, C.E.

    1984-01-01

    In vitro crossmatching techniques are often inadequate for patients who have received multiple prior transfusions. These patients usually have multiple antibodies to minor blood groups, not all of which are necessarily important to vivo. It becomes increasingly difficult to obtain appropriate units for transfusion, and often units are used with hopes that a minor group antibody will not be significantly active in vivo. If a transfusion reaction occurs, the unit is stopped. The authors have developed and successfully tested a method whereby 1.5 to 3c of potential donor RBC's are labeled with 25-50 μCi of Tc-99m using the BNL kits. After injection, samples are drawn at 10, 20, 60, and 120 minutes and the RBC survival is measured. If it is desirable to test 2 units simultaneously, the authors use 400 μCi Tc-99m to label an RBC aliquot of one unit and 25 μCi In-111-oxine to label the other; both labeled aliquots are injected together. The method is simple and reliable. In addition to assessing compatibility, the authors may also estimate the % viability of transfused, compatible RBC's by starting with 400 μCi of Tc-99m and multiplying % survival at 24 hours by 1.2. For 24 hr. survival measurements of IN-111-oxine-RBC's, 25 μCi is adequate and no multiplication factor is necessary. The authors have performed 13 in vivo crossmatches, 4 of which were double, in 6 patients. One documented mild transfusion reaction occurred. There were no false positive or false negative results

  5. Intestinal intraepithelial lymphocyte cytometric pattern is more accurate than subepithelial deposits of anti-tissue transglutaminase IgA for the diagnosis of celiac disease in lymphocytic enteritis.

    Directory of Open Access Journals (Sweden)

    Fernando Fernández-Bañares

    Full Text Available BACKGROUND & AIMS: An increase in CD3+TCRγδ+ and a decrease in CD3- intraepithelial lymphocytes (IEL is a characteristic flow cytometric pattern of celiac disease (CD with atrophy. The aim was to evaluate the usefulness of both CD IEL cytometric pattern and anti-TG2 IgA subepithelial deposit analysis (CD IF pattern for diagnosing lymphocytic enteritis due to CD. METHODS: Two-hundred and five patients (144 females who underwent duodenal biopsy for clinical suspicion of CD and positive celiac genetics were prospectively included. Fifty had villous atrophy, 70 lymphocytic enteritis, and 85 normal histology. Eight patients with non-celiac atrophy and 15 with lymphocytic enteritis secondary to Helicobacter pylori acted as control group. Duodenal biopsies were obtained to assess both CD IEL flow cytometric (complete or incomplete and IF patterns. RESULTS: Sensitivity of IF, and complete and incomplete cytometric patterns for CD diagnosis in patients with positive serology (Marsh 1+3 was 92%, 85 and 97% respectively, but only the complete cytometric pattern had 100% specificity. Twelve seropositive and 8 seronegative Marsh 1 patients had a CD diagnosis at inclusion or after gluten free-diet, respectively. CD cytometric pattern showed a better diagnostic performance than both IF pattern and serology for CD diagnosis in lymphocytic enteritis at baseline (95% vs 60% vs 60%, p = 0.039. CONCLUSIONS: Analysis of the IEL flow cytometric pattern is a fast, accurate method for identifying CD in the initial diagnostic biopsy of patients presenting with lymphocytic enteritis, even in seronegative patients, and seems to be better than anti-TG2 intestinal deposits.

  6. Flow cytometric analysis of mitotic cycle perturbation by chemical carcinogens in cultured epithelial cells. [Effects of benzo(a)pyrene-diol-epoxide on mitotic cycle of cultural mouse liver epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Pearlman, Andrew Leonard [Univ. of California, Berkeley, CA (United States)

    1978-08-01

    A system for kinetic analysis of mitotic cycle perturbation by various agents was developed and applied to the study of the mitotic cycle effects and dependency of the chemical carcinogen benzo(a)pyrene-diolepoxide, DE, upon a mouse lever epithelial cell line, NMuLi. The study suggests that the targets of DE action are not confined to DNA alone but may include cytoplasmic structures as well. DE was found to affect cells located in virtually every phase of the mitotic cycle, with cells that were actively synthesizing DNA showing the strongest response. However, the resulting perturbations were not confined to S-phase alone. DE slowed traversal through S-phase by about 40% regardless of the cycle phase of the cells exposed to it, and slowed traversal through G2M by about 50%. When added to G1 cells, DE delayed recruitment of apparently quiescent (G0) cells by 2 hours, and reduced the synchrony of the cohort of cells recruited into active proliferation. The kinetic analysis system consists of four elements: tissue culture methods for propagating and harvesting cell populations; an elutriation centrifugation system for bulk synchronization of cells in various phases of the mitotic cycle; a flow cytometer (FCM), coupled with appropriate staining protocols, to enable rapid analysis of the DNA distribution of any given cell population; and data reduction and analysis methods for extracting information from the DNA histograms produced by the FCM. The elements of the system are discussed. A mathematical analysis of DNA histograms obtained by FCM is presented. The analysis leads to the detailed implementation of a new modeling approach. The new modeling approach is applied to the estimation of cell cycle kinetic parameters from time series of DNA histograms, and methods for the reduction and interpretation of such series are suggested.

  7. Seasonality in molecular and cytometric diversity of marine bacterioplankton: the reshuffling of bacterial taxa by vertical mixing

    KAUST Repository

    García, Francisca C.

    2015-07-17

    The ’cytometric diversity’ of phytoplankton communities has been studied based on single-cell properties, but the applicability of this method to characterize bacterioplankton has been unexplored. Here, we analysed seasonal changes in cytometric diversity of marine bacterioplankton along a decadal time-series at three coastal stations in the Southern Bay of Biscay. Shannon-Weaver diversity estimates and Bray-Curtis similarities obtained by cytometric and molecular (16S rRNA tag sequencing) methods were significantly correlated in samples from a 3.5-year monthly time-series. Both methods showed a consistent cyclical pattern in the diversity of surface bacterial communities with maximal values in winter. The analysis of the highly resolved flow cytometry time-series across the vertical profile showed that water column mixing was a key factor explaining the seasonal changes in bacterial composition and the winter increase in bacterial diversity in coastal surface waters. Due to its low cost and short processing time as compared to genetic methods, the cytometric diversity approach represents a useful complementary tool in the macroecology of aquatic microbes.

  8. Flow Cytometric Applicability of Fluorescent Vitality Probes on Phytoplankton

    NARCIS (Netherlands)

    Peperzak, L.; Brussaard, C.P.D.

    2011-01-01

    The applicability of six fluorescent probes (four esterase probes: acetoxymethyl ester of Calcein [Calcein-AM], 5-chloromethylfluorescein diacetate [CMFDA], fluorescein diacetate [FDA], and 2',7'-dichlorofluorescein diacetate [H(2)DCFDA]; and two membrane probes: bis-(1,3-dibutylbarbituric acid)

  9. FLOW CYTOMETRIC APPLICABILITY OF FLUORESCENT VITALITY PROBES ON PHYTOPLANKTON1.

    Science.gov (United States)

    Peperzak, Louis; Brussaard, Corina P D

    2011-06-01

    The applicability of six fluorescent probes (four esterase probes: acetoxymethyl ester of Calcein [Calcein-AM], 5-chloromethylfluorescein diacetate [CMFDA], fluorescein diacetate [FDA], and 2',7'-dichlorofluorescein diacetate [H 2 DCFDA]; and two membrane probes: bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC 4 (3)] and SYTOX-Green) as vitality stains was tested on live and killed cells of 40 phytoplankton strains in exponential and stationary growth phases, belonging to 12 classes and consisting of four cold-water, 26 temperate, and four warm-water species. The combined live/dead ratios of all six probes indicated significant differences between the 12 plankton classes (P live/dead ratios of FDA and CMFDA were not significantly different from each other, and both performed better than Calcein-AM and H 2 DCFDA (P live/dead ratios) among all six probes belonged to nine genera from six classes of phytoplankton. In conclusion, FDA, CMFDA, DIBAC 4 (3), and SYTOX-Green represent a wide choice of vitality probes in the study of phytoplankton ecology, applicable in many species from different algal classes, originating from different regions and at different stages of growth. © 2011 Phycological Society of America.

  10. Comparison of Five Nuclear Isolation Buffers for Flow Cytometric ...

    African Journals Online (AJOL)

    Jocky

    2012-02-23

    Feb 23, 2012 ... factor as a measure of sample quality (%DF) and nuclear yield factor (YF) in order to compare the quantity of nuclei in suspension. The following equations were used: Estimation of DNA contents for cultivars classification. Young leaves of Deli Dura, Pisifera and hybrid Tenera trees were prepared following ...

  11. Flow Cytometric Ploidy Determination of Oral Premalignant and Malignant Lesions

    Science.gov (United States)

    1990-01-01

    identifiable lesions, some were associated with oral white lesions, either concomitant or precedent. These white lesions were referred to as leukoplakia , a...widely variable results. These authors undertook a clinical-pathologic correlation of oral leukoplakia encountered in the biopsy services of two...with oral leukoplakia for an average period of 7.2 years. All lesions were more than one centimeter in size and had been present and observed for a

  12. Flow cytometric quantification of radiation responses of murine peritoneal cells

    International Nuclear Information System (INIS)

    Tokita, N.; Raju, M.R.

    1982-01-01

    Methods have been developed to distinguish subpopulations of murine peritoneal cells, and these were applied to the measurement of early changes in peritoneal cells after irradiation. The ratio of the two major subpopulations in the peritoneal fluid, lymphocytes and macrophages, was measured rapidly by means of cell volume distribution analysis as well as by hypotonic propidium iodide (PI) staining. After irradiation, dose and time dependent changes were noted in the cell volume distributions: a rapid loss of peritoneal lymphocytes, and an increase in the mean cell volume of macrophages. The hypotonic PI staining characteristics of the peritoneal cells showed two or three distinctive G 1 peaks. The ratio of the areas of these peaks was also found to be dependent of the radiation dose and the time after irradiation. These results demonstrate that these two parameters may be used to monitor changes induced by irradiation (biological dosimetry), and to sort different peritoneal subpopulations

  13. Flow cytometric analysis of bone marrow leukocytes in neonatal dogs

    Czech Academy of Sciences Publication Activity Database

    Faldyna, M.; Šinkora, Jiří; Knotigová, P.; Řeháková, Zuzana; Morávková, Alena; Toman, M.

    2003-01-01

    Roč. 95, - (2003), s. 165-176 ISSN 0165-2427 R&D Projects: GA ČR GA524/00/0474; GA ČR GP524/02/P010 Institutional research plan: CEZ:AV0Z5020903 Keywords : cd34 * sirp * b cell Subject RIV: EC - Immunology Impact factor: 1.652, year: 2003

  14. Howard University Flow Cytometric Sorter For Research and Education

    Science.gov (United States)

    2015-08-04

    based on beta -D- galactosidase activity after transduction of Escherichia coli lacZ. Proceedings of the National Academy of Sciences of the United... beta -D- galactosidase activity after transduction of Escherichia coli lacZ. Proceedings of the National Academy of Sciences of the United States of

  15. Rapid Flow cytometric prenatal diagnosis of primary immunodeficiency (PID) disorders.

    Science.gov (United States)

    Mishra, Anju; Gupta, Maya; Dalvi, Aparna; Ghosh, Kanjaksha; Madkaikar, Manisha

    2014-04-01

    Primary Immunodeficiency diseases (PID) are a heterogeneous group of inherited disorders of immune system. Immunophenotypic evaluation of PIDs using flowcytometry provides important clues for diagnosis of these disorders, though confirmation requires identification of underlying molecular defects. Prenatal diagnosis (PND) forms an important component of management in families affected with severe PID. However, molecular diagnostic facilities for each of these diseases are not available and may not be possible to perform in all cases. In such scenario we opted for phenotypic prenatal diagnosis by cordocentesis for families with index case having immunophenotypically well characterized PID. Normal reference ranges of lymphocyte subsets, CD 18/CD11 integrins on leukocytes, MHC class II expression and oxidative burst activity of fetal neutrophils at 18 weeks of gestation were previously established on 30 cord blood samples. PND was performed in 13 families with PIDs. Maternal contamination was ruled out by VNTR analysis. Out of 13 fetuses, nine were found to be unaffected (three cases with leukocyte adhesion deficiency (LAD-I), four cases with severe combined immunodeficiency diseases (SCID), one with X-linked agammaglobulinemia (XLA), and one with chronic granulomatous disease (CGD)] and three were found to be affected (one with T-B+NK-SCID, one with MHC class II deficiency and one with LAD-I). Diagnosis was confirmed by testing the cord blood samples after delivery and further follow-up of the children. In one family diagnosis could not be offered due to maternal contamination. No procedure related complications were observed. Flowcytometry offers rapid and sensitive method for prenatal diagnosis and genetic counseling for selected phenotypically well characterized PID in cases where molecular diagnostic facilities are not available.

  16. Flow cytometric detection of viruses in the Zuari estuary, Goa

    Digital Repository Service at National Institute of Oceanography (India)

    Mitbavkar, S.; Rajaneesh, K.M.; SathishKumar, P.

    abundance 3 . Viruses are the smallest known organisms (20–200 nm) with simple biological structures consist- ing of nucleic acid, either DNA or RNA (single- or double-stranded), with a pro- tein coat (capsid). Viruses are known to infect prokaryo- tes... in the microbial loop. Because prokaryotes and autotrophic and heterotrophic protists play pivotal roles in the biogeochemical cycles and global ocean functioning, viral infections of these groups of organ- ism have important ecological conse- quences 2...

  17. Cytometric Approach for Detection of Encephalitozoon intestinalis, an Emergent Agent▿

    Science.gov (United States)

    Barbosa, Joana; Rodrigues, Acácio Gonçalves; Pina-Vaz, Cidália

    2009-01-01

    Encephalitozoon intestinalis is responsible for intestinal disease in patients with AIDS and immunocompetent patients. The infectious form is a small spore that is resistant to water treatment procedures. Its detection is very important, but detection is very cumbersome and time-consuming. Our main objective was to develop and optimize a specific flow cytometric (FC) protocol for the detection of E. intestinalis in hospital tap water and human feces. To determine the optimal specific antibody (Microspor-FA) concentration, a known concentration of E. intestinalis spores (Waterborne, Inc.) was suspended in hospital tap water and stool specimens with different concentrations of Microspor-FA, and the tap water and stool specimens were incubated under different conditions. The sensitivity limit and specificity were also evaluated. To study spore infectivity, double staining with propidium iodide (PI) and Microspor-FA was undertaken. Distinct approaches for filtration and centrifugation of the stool specimens were used. E. intestinalis spores stained with 10 μg/ml of Microspor-FA at 25°C overnight provided the best results. The detection limit was 5 × 104 spores/ml, and good specificity was demonstrated. Simultaneous staining with Microspor-FA and PI ensured that the E. intestinalis spores were dead and therefore noninfectious. With the stool specimens, better spore recovery was observed with a saturated solution of NaCl and centrifugation at 1,500 × g for 15 min. A new approach for the detection of E. intestinalis from tap water or human feces that ensures that the spores are not viable is now available and represents an important step for the prevention of this threat to public health. PMID:19439525

  18. The Belgian repository of fundamental atomic data and stellar spectra (BRASS). I. Cross-matching atomic databases of astrophysical interest

    Science.gov (United States)

    Laverick, M.; Lobel, A.; Merle, T.; Royer, P.; Martayan, C.; David, M.; Hensberge, H.; Thienpont, E.

    2018-04-01

    Context. Fundamental atomic parameters, such as oscillator strengths, play a key role in modelling and understanding the chemical composition of stars in the Universe. Despite the significant work underway to produce these parameters for many astrophysically important ions, uncertainties in these parameters remain large and can propagate throughout the entire field of astronomy. Aims: The Belgian repository of fundamental atomic data and stellar spectra (BRASS) aims to provide the largest systematic and homogeneous quality assessment of atomic data to date in terms of wavelength, atomic and stellar parameter coverage. To prepare for it, we first compiled multiple literature occurrences of many individual atomic transitions, from several atomic databases of astrophysical interest, and assessed their agreement. In a second step synthetic spectra will be compared against extremely high-quality observed spectra, for a large number of BAFGK spectral type stars, in order to critically evaluate the atomic data of a large number of important stellar lines. Methods: Several atomic repositories were searched and their data retrieved and formatted in a consistent manner. Data entries from all repositories were cross-matched against our initial BRASS atomic line list to find multiple occurrences of the same transition. Where possible we used a new non-parametric cross-match depending only on electronic configurations and total angular momentum values. We also checked for duplicate entries of the same physical transition, within each retrieved repository, using the non-parametric cross-match. Results: We report on the number of cross-matched transitions for each repository and compare their fundamental atomic parameters. We find differences in log(gf) values of up to 2 dex or more. We also find and report that 2% of our line list and Vienna atomic line database retrievals are composed of duplicate transitions. Finally we provide a number of examples of atomic spectral lines

  19. Type and screen policy in the blood bank: Is AHG cross-match still required? A study at a multispecialty corporate hospital in India

    Directory of Open Access Journals (Sweden)

    Pathak Sangeeta

    2011-01-01

    Full Text Available Background: Antibodies against only about 25-28 blood group antigens are known to cause hemolytic reactions (HTRs, and red cell antibody screening should detect such clinically significant antibodies. An extension of the antibody screening test is the ′type and screen′ done to detect clinically significant antibodies, omiting the anti-human globulin (AHG cross-match. Aim: The aim of this study was to find out if the type and screen procedure is a safe method for pre-transfusion testing when compared to the AHG cross-match currently in use in India. Materials and Methods: We evaluated data from 45373 patients for whom a total of 61668 units of packed red blood cells (PRBC were cross-matched in the AHG phase using DiaMed; ID cards. An antibody screen was carried out in all the patients using the DiaMed; ID-DiaCell I+II+III. The AHG cross-match was also carried out for all recipients, irrespective of the result of the antibody screen. The results were compared to see if there were any cases where the antibody screening was negative but the AHG cross-match showed incompatibility. Results: Not a single case was found where the antibody screen was negative and AHG cross-match showed incompatibility. In 68 cases the antibody screening was positive. Out of the 68 cases, AHG cross-match was incompatible with at least one unit of PRBC in 41 cases. Conclusion: The screening cell panel adequately detected the clinically significant antibodies in the Indian population in our study. The type and screen policy can be safe, efficient, cost-effective, and beneficial to the transfusion service in India.

  20. Peri-operative alemtuzumab (Campath-1H) and plasmapheresis for high-PRA positive lymphocyte crossmatch heart transplant: a strategy to shorten left ventricular assist device support.

    Science.gov (United States)

    Lick, Scott D; Vaidya, Smita; Kollar, Andras C; Boor, Paul J; Vertrees, Roger A

    2008-09-01

    Patients on a left ventricular assist device (LVAD) often have a high level of panel-reactive antibodies (PRA). Conventional therapy is to await a heart from a negative prospective-crossmatch donor. We transplanted three high-PRA patients with non-crossmatched hearts, using intra- and post-operative plasmapheresis and long-term T-/B-/plasma-cell therapy with alemtuzumab. Three highly sensitized patients (70%, 94% and 96% T-PRA; 63%, 24% and 73% B-PRA) were transplanted after 29, 187 and 94 days LVAD support. The first patient (Case 1) had an erroneous prospective negative crossmatch (due to an outside laboratory's use of the wrong patient's serum) with immediate allograft dysfunction. The correct serum showed a strongly positive crossmatch; plasmapheresis followed by alemtuzumab (20 mg intravenously) shortly after arrival in the ICU resulted in rapid hemodynamic improvement. Encouraged by this success, the next two patients (Cases 2 and 3) underwent LVAD explant and heart transplant with the next available ABO-identical, non-crossmatched donors, using plasmapheresis on bypass immediately before heart implant and alemtuzumab 20 mg intravenously upon ICU arrival, with uneventful courses. All three patients had positive retrospective T- and B-cell crossmatches. Maintenance immunosuppression consisted of cyclosporine and routine prednisone taper, with plasmapheresis as needed (Patient 1, x10; Patient 2, x5) based on diastolic dysfunction. Mycophenolate mofetil was started as a third agent several months post-transplant. Patients are presently New York Heart Association (NYHA) Class I at 26, 16 and 13 months post-transplant. In this small series with follow-up, immediate antibody removal with plasmapheresis, combined with alemtuzumab, a long-acting antibody to CD52 (expressed on T, B and some plasma cells), appears effective in allowing transplantation in sensitized, positive crossmatch recipients. Expanded use of this strategy could shorten LVAD support in many

  1. Multiparameter cytometric analysis of complex cellular response

    Czech Academy of Sciences Publication Activity Database

    Šimečková, Šárka; Fedr, Radek; Remšík, Jan; Kahounová, Z.; Slabáková, Eva; Souček, Karel

    2018-01-01

    Roč. 93A, č. 2 (2018), s. 239-248 ISSN 1552-4922 R&D Projects: GA MZd(CZ) NV15-28628A; GA MZd(CZ) NV15-33999A; GA MZd(CZ) NV17-28518A Institutional support: RVO:68081707 Keywords : flow-cytometry * permeabilization * apoptosis * fixation Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Cell biology Impact factor: 3.222, year: 2016

  2. Flow: Statistics, visualization and informatics for flow cytometry

    Directory of Open Access Journals (Sweden)

    Kepler Thomas B

    2008-06-01

    Full Text Available Abstract Flow is an open source software application for clinical and experimental researchers to perform exploratory data analysis, clustering and annotation of flow cytometric data. Flow is an extensible system that offers the ease of use commonly found in commercial flow cytometry software packages and the statistical power of academic packages like the R BioConductor project.

  3. Monitoring functions in managed microbial systems by cytometric bar coding.

    Science.gov (United States)

    Koch, Christin; Fetzer, Ingo; Schmidt, Thomas; Harms, Hauke; Müller, Susann

    2013-02-05

    Cytometric monitoring of microbial community dynamics can be used to estimate stability of technical microbial processes like biogas production by analysis of segregated cell abundance changes. In this study, structure variations of a biogas community were cytometrically recorded over 9 months and found to be of diagnostic value for process details. The reactor regime was intentionally disturbed with regard to substrate overload or H(2)S accumulation. A single-cell based approach called cytometric bar coding (CyBar) for fast identification of reactive subcommunities was used. Functionality of specific subcommunities was uncovered by processing CyBar data with abiotic reactor parameters using Spearman's correlation coefficient. Twenty subcommunities showed a discrete and divergent behavior. For example, a 4-fold substrate overload increased the cell number of two acidogenic index subcommunities to 176 and 193% within three days. Supplementary analyses were done using DNA fingerprinting, cloning, and sequencing. Bioreactor perturbations were shown to create cell abundance changes in subcommunities rather than variations in their phylogenetic composition. The used workflow and macros are ready-to-use tools and allow on-site monitoring and interpretation of variation in microbial community functions within a few hours.

  4. Multiparameter cytometric analysis of complex cellular response.

    Science.gov (United States)

    Šimečková, Šárka; Fedr, Radek; Remšík, Ján; Kahounová, Zuzana; Slabáková, Eva; Souček, Karel

    2018-02-01

    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. © 2017 International Society for Advancement of Cytometry.

  5. Improved method for fluorescence cytometric immunohematology testing.

    Science.gov (United States)

    Roback, John D; Barclay, Sheilagh; Hillyer, Christopher D

    2004-02-01

    A method for accurate immunohematology testing by fluorescence cytometry (FC) was previously described. Nevertheless, the use of vacuum filtration to wash RBCs and a standard-flow cytometer for data acquisition hindered efforts to incorporate this method into an automated platform. A modified procedure was developed that used low-speed centrifugation of 96-well filter plates for RBC staining. Small-footprint benchtop capillary cytometers (PCA and PCA-96, Guava Technologies, Inc.) were used for data acquisition. Authentic clinical samples from hospitalized patients were tested for ABO group and the presence of D antigen (n = 749) as well as for the presence of RBC alloantibodies (n = 428). Challenging samples with mixed-field reactions and weak antibodies were included. Results were compared to those obtained by column agglutination technology (CAT), and discrepancies were resolved by standard tube methods. Detailed investigations of FC sensitivity and reproducibility were also performed. The modified FC method with the PCA determined the correct ABO group and D type for 98.7 percent of 520 samples, compared to 98.8 percent for CAT (p > 0.05). No-type-determined (NTD) rates were 1.2 percent for both methods. In testing for unexpected alloantibodies, FC determined the correct result for 98.6 percent of 215 samples, compared to 96.3 percent for CAT (p > 0.05). When samples were automatically acquired in the 96-well plate format with the PCA-96, 98.7 percent of 229 samples had correct ABO group and D type determined by FC, compared to 97.4 percent for CAT (p > 0.05). NTD rates were 0.9 and 2.6 percent, respectively. Antibody screens were accurate for 99.1 percent of 213 samples with the PCA-96, compared to 99.5 percent for CAT (p > 0.05). Further investigations demonstrated that FC with the PCA-96 was better than CAT at detecting weak anti-A (p < 0.0001) and alloantibodies. An improved method for FC immunohematology testing has been described. This assay was comparable

  6. Vertical and longitudinal gradients in HNA-LNA cell abundances and cytometric characteristics in the Mediterranean Sea

    Directory of Open Access Journals (Sweden)

    F. Van Wambeke

    2011-07-01

    Full Text Available Heterotrophic bacterioplankton abundance and production were investigated with depth (down to bathypelagic layers and with longitude (from 4.9° E to 32.7° E along a cruise track across the Mediterranean Sea in early summer 2008. Abundances and flow cytometric characteristics (green fluorescence and side scatter signals of high nucleic acid (HNA and low nucleic acid (LNA bacterial cells were determined using flow cytometry. Contrary to what is generally observed, the relative importance of HNA cells, as a percent of total cells, (%HNA, range 30–69 % was inversely related to bacterial production (range 0.15–44 ng C l−1 h−1 although the negative relation was weak (log–log regression r2=0.19. The %HNA as well as the mean side scatter of HNA group increased significantly with depth in the meso and bathypelagic layers. Vertical stratification played an important role in influencing the distribution and characteristics of bacterial cells especially with regard to layers located above, within or below the deep chlorophyll maximum. Within a given layer, the relationships between the flow cytometric characteristics and environmental variables such as chlorophyll-a, nutrients or bacterial production changed. Overall, the relationships between HNA and LNA cells and environmental parameters differed vertically more than longitudinally.

  7. Cross-matching: A modified cross-correlation underlying threshold energy model and match-based depth perception

    Directory of Open Access Journals (Sweden)

    Takahiro eDoi

    2014-10-01

    Full Text Available Three-dimensional visual perception requires correct matching of images projected to the left and right eyes. The matching process is faced with an ambiguity: part of one eye’s image can be matched to multiple parts of the other eye’s image. This stereo correspondence problem is complicated for random-dot stereograms (RDSs, because dots with an identical appearance produce numerous potential matches. Despite such complexity, human subjects can perceive a coherent depth structure. A coherent solution to the correspondence problem does not exist for anticorrelated RDSs (aRDSs, in which luminance contrast is reversed in one eye. Neurons in the visual cortex reduce disparity selectivity for aRDSs progressively along the visual processing hierarchy. A disparity-energy model followed by threshold nonlinearity (threshold energy model can account for this reduction, providing a possible mechanism for the neural matching process. However, the essential computation underlying the threshold energy model is not clear. Here, we propose that a nonlinear modification of cross-correlation, which we term ‘cross-matching’, represents the essence of the threshold energy model. We placed half-wave rectification within the cross-correlation of the left-eye and right-eye images. The disparity tuning derived from cross-matching was attenuated for aRDSs. We simulated a psychometric curve as a function of graded anticorrelation (graded mixture of aRDS and normal RDS; this simulated curve reproduced the match-based psychometric function observed in human near/far discrimination. The dot density was 25% for both simulation and observation. We predicted that as the dot density increased, the performance for aRDSs should decrease below chance (i.e., reversed depth, and the level of anticorrelation that nullifies depth perception should also decrease. We suggest that cross-matching serves as a simple computation underlying the match-based disparity signals in

  8. Adenoid and tonsil surgeries in children: How relevant is pre-operative blood grouping and cross-matching?

    Directory of Open Access Journals (Sweden)

    Lucky Onotai

    2013-01-01

    Full Text Available Background: As a part of pre-operative evaluation, several otolaryngologists group and cross-match blood routinely for children undergoing adenoid and tonsil surgeries. This practice has generated several debates either in support or against this practice. The aim of this study is to critically evaluate the incidence of post-tonsillectomy (with or without adenoidectomy bleeding and blood transfusions in otherwise healthy children with adenoid/tonsil pathologies conducted in the University of Port Harcourt Teaching Hospital (UPTH. Patients and Methods: A descriptive retrospective study of children who underwent adenoid and tonsil surgeries in the Department of Ear, Nose and Throat (ENT surgery of UPTH from January 2003 to December 2012. Children with family history of bleeding disorders and derangement of clotting profile as well as different co-morbidity like sickle cell disease were excluded from this study. The patients′ data were retrieved from the registers of ENT out-patient clinics, theatre registers and patients case notes. Demographic data, indications for surgery, preoperative investigations, complications and management outcomes were recorded and analyzed. Results: Out of 145 children that had adenoid and tonsil surgeries; only 100 met the criteria for this study. The study subjects included 65 males and 35 females (male: female ratio 1.9:1 belonging to 0-16 years age group (mean age: 3.46 ± 2.82 years. The age group of 3-5 years had the highest (n = 40, 40% number of surgeries. Adenotonsillectomy was the commonest (n = 85, 85% surgery performed on patients who had obstructive sleep apnea (OSA. The commonest (n = 6, 6% complication was haemorrhage, and only few (n = 3, 3% patients had blood transfusion. However, mortality was recorded in some (n = 3, 3% patients. Conclusion: This study confirms that the incidence of post adenoidectomy/tonsillectomy bleeding in otherwise healthy children is low and rarely requires blood transfusion

  9. Analysis of Luminex-based algorithms to define unacceptable HLA antibodies in CDC-crossmatch negative kidney transplant recipients.

    Science.gov (United States)

    Zecher, Daniel; Bach, Christian; Preiss, Adrian; Staudner, Christoph; Utpatel, Kirsten; Evert, Matthias; Jung, Bettina; Bergler, Tobias; Böger, Carsten A; Spriewald, Bernd M; Banas, Bernhard

    2018-02-20

    HLA-specific antibodies detected by solid phase assays are increasingly used to define unacceptable HLA antigen mismatches (UAM) prior to renal transplantation. The accuracy of this approach is unclear. Day of transplant sera from 211 CDC-crossmatch-negative patients were retrospectively analyzed for donor-specific anti-HLA antibodies (DSA) using Luminex technology. HLA were defined as UAM if DSA had mean fluorescence intensity above (I) 3000 (patients retransplanted and those with DSA against HLA class I and II) or 5000 (all other patients), (II) 5000 for HLA A, B and DR and 10,000 for HLA DQ or (III) 10,000 (all HLA). We then studied the accuracy of these algorithms to identify patients with antibody-mediated rejection (AMR) and graft loss. UAM were also determined in 256 transplant candidates and virtual panel-reactive antibody (vPRA) levels calculated. At transplantation, 67/211 patients had DSA. Of these, 31 (algorithm I), 24 (II) and 17 (III) had UAM. 9 (I and II) and 8 (III) of 11 early AMR episodes and 7 (I), 6 (II) and 5 (III) of 9 graft losses occurred in UAM-positive patients during 4.9 years of follow-up. Algorithms (I) and (II) identified patients with persistently lower GFR even in the absence of overt AMR. 23-33% of waiting list patients had UAM with median vPRA of 69.2-79.1%. Algorithms (I) and (II) had comparable efficacy but were superior to (III) in identifying at-risk patients at an acceptable false positive rate. However, Luminex-defined UAM significantly restrict the donor pool of affected patients, which might prolong waiting time.

  10. Flow cytometry PRA using lymphocyte pools from random donors.

    Science.gov (United States)

    Won, Dong Il; Jung, Hee Du; Jung, Ok-Ju; Huh, Seung; Suh, Jang Soo

    2007-07-01

    Pools of lymphocytes from carefully chosen donors have been used for flow cytometry (FC) panel reactive antibody (PRA) assays. We intended to devise an FC PRA assay using mixed lymphocyte pools from a large number of randomly selected donors (RD FC PRA) to accurately predict the likelihood of a positive HLA crossmatch. Lymphocyte pools were prepared from randomly selected donors (N = 120). %PRA was calculated based on the anti-IgG FITC histogram of the T cells. The proposed RD FC PRA assay was assessed in comparison with the bead FC PRA, antiglobulin-augmented CDC (AHG-CDC) PRA assay, and the expected %PRA calculated by summing up the antigen frequencies of the known specificities. In 29 FC crossmatch positive sera, the positivity rate for the bead FC, RD FC, and AHG-CDC PRA was 100, 100, and 79%, and the mean %PRA was 77% +/- 0.205). In 19 sensitized patients with a negative FC crossmatch, the positivity rate was 21% using the RD FC PRA and 16% using the bead FC PRA, which suggested that both assays had similar abilities to detect low levels of HLA antibodies. The RD FC PRA assay allows easy panel preparation, reduces cost, and naturally reflects the probabilities of a positive crossmatch in the population to which the cadaveric donor belongs. Therefore, this new assay is expected to be useful as another approach to determine the % PRA. Copyright 2007 Clinical Cytometry Society.

  11. Cytometric analysis of shape and DNA content in mammalian sperm

    Energy Technology Data Exchange (ETDEWEB)

    Gledhill, B.L.

    1983-10-10

    Male germ cells respond dramatically to a variety of insults and are important reproductive dosimeters. Semen analyses are very useful in studies on the effects of drugs, chemicals, and environmental hazards on testicular function, male fertility and heritable germinal mutations. Sperm were analyzed by flow cytometry and slit-scan flow analysis for injury following the exposure of testes to mutagens. The utility of flow cytometry in genotoxin screening and monitoring of occupational exposure was evaluated. The technique proved valuable in separation of X- and Y-chromosome bearing sperm and the potential applicability of this technique in artificial insemination and a solution, of accurately assessing the DNA content of sperm were evaluated-with reference to determination of X- and Y-chromosome bearing sperm.

  12. Cytometric analysis of shape and DNA content in mammalian sperm

    International Nuclear Information System (INIS)

    Gledhill, B.L.

    1983-01-01

    Male germ cells respond dramatically to a variety of insults and are important reproductive dosimeters. Semen analyses are very useful in studies on the effects of drugs, chemicals, and environmental hazards on testicular function, male fertility and heritable germinal mutations. Sperm were analyzed by flow cytometry and slit-scan flow analysis for injury following the exposure of testes to mutagens. The utility of flow cytometry in genotoxin screening and monitoring of occupational exposure was evaluated. The technique proved valuable in separation of X- and Y-chromosome bearing sperm and the potential applicability of this technique in artificial insemination and a solution, of accurately assessing the DNA content of sperm were evaluated-with reference to determination of X- and Y-chromosome bearing sperm

  13. Simplified flow cytometric assay to detect minimal residual disease in childhood with acute lymphoblastic leukemia Detecção de doença residual mínima em crianças com leucemia linfoblástica aguda por citometria de fluxo

    Directory of Open Access Journals (Sweden)

    Elizabete Delbuono

    2008-08-01

    Full Text Available The detection of minimal residual disease (MRD is an important prognostic factor in childhood acute lymphoblastic leukemia (ALL providing crucial information on the response to treatment and risk of relapse. However, the high cost of these techniques restricts their use in countries with limited resources. Thus, we prospectively studied the use of flow cytometry (FC with a simplified 3-color assay and a limited antibody panel to detect MRD in the bone marrow (BM and peripheral blood (PB of children with ALL. BM and PB samples from 40 children with ALL were analyzed on days (d 14 and 28 during induction and in weeks 24-30 of maintenance therapy. Detectable MRD was defined as > 0.01% cells expressing the aberrant immunophenotype as characterized at diagnosis among total events in the sample. A total of 87% of the patients had an aberrant immunophenotype at diagnosis. On d14, 56% of the BM and 43% of the PB samples had detectable MRD. On d28, this decreased to 45% and 31%, respectively. The percentage of cells with the aberrant phenotype was similar in both BM and PB in T-ALL but about 10 times higher in the BM of patients with B-cell-precursor ALL. Moreover, MRD was detected in the BM of patients in complete morphological remission (44% on d14 and 39% on d28. MRD was not significantly associated to gender, age, initial white blood cell count or cell lineage. This FC assay is feasible, affordable and readily applicable to detect MRD in centers with limited resources.A detecção de doença residual mínima (DRM é um importante fator prognóstico na leucemia linfóide aguda (LLA infantil e fornece informações sobre a resposta ao tratamento e o risco de recaída. Entretanto, os altos custos das técnicas utilizadas limitam seu uso nos países em desenvolvimento. Desta forma, realizamos um estudo prospectivo para avaliar a citometria de fluxo (CF, utilizando três fluorescências e um painel limitado de anticorpos monoclonais, como método de detec

  14. Flow

    DEFF Research Database (Denmark)

    Knoop, Hans Henrik

    2006-01-01

    FLOW. Orden i hovedet på den fede måde Oplevelsesmæssigt er flow-tilstanden kendetegnet ved at man er fuldstændig involveret, fokuseret og koncentreret; at man oplever stor indre klarhed ved at vide hvad der skal gøres, og i hvilket omfang det lykkes; at man ved at det er muligt at løse opgaven...

  15. Supercontinuum white light lasers for flow cytometry

    OpenAIRE

    Telford, William G.; Subach, Fedor V.; Verkhusha, Vladislav V.

    2009-01-01

    Excitation of fluorescent probes for flow cytometry has traditionally been limited to a few discrete laser lines, an inherent limitation in our ability to excite the vast array of fluorescent probes available for cellular analysis. In this report, we have used a supercontinuum (SC) white light laser as an excitation source for flow cytometry. By selectively filtering the wavelength of interest, almost any laser wavelength in the visible spectrum can be separated and used for flow cytometric a...

  16. Glycerol-treated nuclear suspensions - an efficient preservation method for flow cytometric analysis of plant samples

    Czech Academy of Sciences Publication Activity Database

    Kolář, Filip; Lučanová, Magdalena; Těšitel, J.; Loureiro, J.; Suda, Jan

    2012-01-01

    Roč. 20, č. 2 (2012), s. 303-315 ISSN 0967-3849 R&D Projects: GA ČR GD206/08/H049 Institutional research plan: CEZ:AV0Z60050516 Institutional support: RVO:67985939 Keywords : cytometry * ploidy * genome size Subject RIV: EF - Botanics Impact factor: 2.847, year: 2012

  17. Long-term storage of samples for flow cytometric DNA analysis

    DEFF Research Database (Denmark)

    Vindeløv, L L; Christensen, I J; Keiding, N

    1983-01-01

    estimation by deconvolution, there was significant intraday and interday variation. Hence the most accurate results are obtained if different aliquots of a sample are measured on different days rather than on the same day. Use of the storage method thus has the potential of increasing the accuracy...

  18. Flow-cytometric measurements of somatic cell mutations in Thorotrast patients

    International Nuclear Information System (INIS)

    Umeki, Shigeko; Kyoizumi, Seishi; Kusunoki, Yoichiro; Nakamura, Nori; Sasaki, Masao; Mori, Takesaburo; Ishikawa, Yuichi; Cologne, J.B.; Akiyama, Mitoshi.

    1992-10-01

    Exposure to ionizing radiation is a well-recognized risk factor for cancer development. Because ionizing radiation can induce mutations, an accurate way of measuring somatic mutation frequencies could be a useful tool for evaluating cancer risk. In the present study, we have examined in vivo somatic mutation frequencies at the erythrocyte glycophorin A and T-cell receptor loci in 18 Thorotrast patients. These persons have been continuously irradiated with alpha particles emitted from the internal deposition of thorium dioxide and thus have increased risks of certain malignant tumors. When compared with controls, the Thorotrast patients showed a significantly higher frequency of mutants at the lymphocyte T-cell receptor loci but not at the erythrocyte glycophorin A loci. (author)

  19. DEA 1 Expression on Dog Erythrocytes Analyzed by Immunochromatographic and Flow Cytometric Techniques

    OpenAIRE

    Acierno, M.M.; Raj, K.; Giger, U.

    2014-01-01

    Background The Dog erythrocyte antigen (DEA) 1 blood group system was thought to contain types DEA 1.1 and 1.2 (and possibly 1.3 [A3]). However, DEA 1.2+ dogs are very rare and newer typing methods reveal varying degrees of DEA 1 positivity. Objectives To assess if variation in DEA 1 positivity is because of quantitative differences in surface antigen expression. To determine expression patterns in dogs over time and effects of blood storage (4?C). To evaluate DEA 1.2+ samples by DEA 1 typing...

  20. Flow cytometric analysis of lymphocytes in aplastic anemia among atomic bomb survivors

    International Nuclear Information System (INIS)

    Imamura, Nobutaka; Inada, Tominari; Asaoku, Hideki; Abe, Kazuhiro; Oguma, Nobuo; Kuramoto, Atsushi

    1986-01-01

    In 6 patients with aplastic anemia and 3 patients with pernicious anemia, lymphocyte subpopulations in the peripheral blood were measured, before and after steroid therapy, with a fluorescence-activated cell sorder using various monoclonal antibodies. The ratio of OKT4-positive lymphocytes (T4) to OKT8-positive lymphocytes (T8) in the peripheral blood was reduced in 2 patients (20 %). The T4/T8 ratio returned to normal during remission of anemia. Hematological improvement was seen after a large amount of steroid therapy in 3 patients. The number of Tac-positive cells tended to decrease and the T4/T8 ratio tended to return to normal with hematological improvement, although there was no correlation to hydrocortisone reaction. Some patients were supposed to have abnormal number of suppressor and inducer T cells. (Namekawa, K.)

  1. Flow cytometric analysis of RNA synthesis by detection of bromouridine incorporation

    DEFF Research Database (Denmark)

    Larsen, J K; Jensen, Peter Østrup; Larsen, J

    2001-01-01

    RNA synthesis has traditionally been investigated by a laborious and time-consuming radiographic method involving incorporation of tritiated uridine. Now a faster non-radioactive alternative has emerged, based on immunocytochemical detection. This method utilizes the brominated RNA precursor...... bromouridine, which is taken into a cell, phosphorylated, and incorporated into nascent RNA. The BrU-substituted RNA is detected by permeabilizing the cells and staining with certain anti-BrdU antibodies. This dynamic approach yields information complementing that provided by cellular RNA content analysis...

  2. Flow cytometric assay detecting cytotoxicity against human endogenous retrovirus antigens expressed on cultured multiple sclerosis cells

    DEFF Research Database (Denmark)

    Møller-Larsen, A; Brudek, T; Petersen, T

    2013-01-01

    expressing increased amounts of human endogenous retrovirus antigens. MS patients also have increased antibody levels to these antigens. The target cells are spontaneously growing peripheral blood mononuclear cells (PBMCs) of B cell lineage, expressing human endogenous retrovirus HERV epitopes...... on their surface. Polyclonal antibodies against defined peptides in the Env- and Gag-regions of the HERVs were raised in rabbits and used in antibody-dependent cell-mediated cytotoxicity (ADCC) -assays. Rituximab® (Roche), a chimeric monoclonal antibody against CD20 expressed primarily on B cells, was used...

  3. Proliferation and apoptosis in infection with infectious bursal disease virus: a flow cytometric study.

    Science.gov (United States)

    Ojeda, F; Skardova, I; Guarda, M I; Ulloa, J; Folch, H

    1997-01-01

    Programmed cell death, or apoptosis, is involved in the normal physiology of many immunocompetent organs, including lymphocytes of the bursa of Fabricius in chickens. Involvement of apoptosis has also been described in some viral diseases such as AIDS. The purpose of this work was to study the potential role of apoptosis in the pathogenesis of Gumboro disease in the bursa of Fabricius. Our results show that 1-3 days after infection of young chickens with infectious bursal disease virus, the number of apoptotic cells increases and cellularity and proliferation decrease. Because of the dynamic nature of bursal lymphocyte populations and the involvement of apoptosis in lymphocyte cell physiology, the increased level of cells undergoing apoptosis may be due to an impairment in the withdrawal of apoptotic cells. A concomitant increase in macrophages in infected bursae and a dramatic decrease in cellularity suggest that an increase in apoptosis may be an important cause of cell depletion.

  4. Image cytometric nuclear texture features in inoperable head and neck cancer: a pilot study

    International Nuclear Information System (INIS)

    Strojan-Flezar, Margareta; Lavrencak, Jaka; Zganec, Mario; Strojan, Primoz

    2011-01-01

    Image cytometry can measure numerous nuclear features which could be considered a surrogate end-point marker of molecular genetic changes in a nucleus. The aim of the study was to analyze image cytometric nuclear features in paired samples of primary tumor and neck metastasis in patients with inoperable carcinoma of the head and neck. Image cytometric analysis of cell suspensions prepared from primary tumor tissue and fine needle aspiration biopsy cell samples of neck metastases from 21 patients treated with concomitant radiochemotherapy was performed. Nuclear features were correlated with clinical characteristics and response to therapy. Manifestation of distant metastases and new primaries was associated (p<0.05) with several chromatin characteristics from primary tumor cells, whereas the origin of index cancer and disease response in the neck was related to those in the cells from metastases. Many nuclear features of primary tumors and metastases correlated with the TNM stage. A specific pattern of correlation between well-established prognostic indicators and nuclear features of samples from primary tumors and those from neck metastases was observed. Image cytometric nuclear features represent a promising candidate marker for recognition of biologically different tumor subgroups

  5. Cytometric analysis of DNA changes induced by sulfur mustard

    Energy Technology Data Exchange (ETDEWEB)

    Smith, W.J.; Sanders, K.M.; Ruddle, S.E.; Gross, C.L.

    1993-05-13

    Sulfur mustard is an alkylating agent which causes severe, potentially debilitating blisters following cutaneous exposure. Its mechanism of pathogenesis is unknown and no antidote exists to prevent its pathology. The biochemical basis of sulfur mustard's vesicating activity has been hypothesized to be a cascade of events beginning with alkylation of DNA. Using human cells in culture, we have assessed the effects of sulfur mustard on cell cycle activity using flow cytometry with propidium iodide. Two distinct patterns emerged, a Gl/S interface block at concentrations equivalent to vesicating doses (>50-micronM) and a G2 block at 10-fold lower concentrations. In addition, noticeable increases in amount of dye uptake were observed at 4 and 24 hours after sulfur mustard exposure. These increases are believed to be related to DNA repair activities and can be prevented by treatment of the cells with niacinamide, which inhibits DNA repair. Other drugs which provide alternate alkylating sites or inhibit cell cycle progression were shown to lower the cytotoxicity of sulfur mustard and to protect against its direct DNA damaging effects.

  6. Fast in-database cross-matching of high-cadence, high-density source lists with an up-to-date sky model

    Science.gov (United States)

    Scheers, B.; Bloemen, S.; Mühleisen, H.; Schellart, P.; van Elteren, A.; Kersten, M.; Groot, P. J.

    2018-04-01

    Coming high-cadence wide-field optical telescopes will image hundreds of thousands of sources per minute. Besides inspecting the near real-time data streams for transient and variability events, the accumulated data archive is a wealthy laboratory for making complementary scientific discoveries. The goal of this work is to optimise column-oriented database techniques to enable the construction of a full-source and light-curve database for large-scale surveys, that is accessible by the astronomical community. We adopted LOFAR's Transients Pipeline as the baseline and modified it to enable the processing of optical images that have much higher source densities. The pipeline adds new source lists to the archive database, while cross-matching them with the known cataloguedsources in order to build a full light-curve archive. We investigated several techniques of indexing and partitioning the largest tables, allowing for faster positional source look-ups in the cross matching algorithms. We monitored all query run times in long-term pipeline runs where we processed a subset of IPHAS data that have image source density peaks over 170,000 per field of view (500,000 deg-2). Our analysis demonstrates that horizontal table partitions of declination widths of one-degree control the query run times. Usage of an index strategy where the partitions are densely sorted according to source declination yields another improvement. Most queries run in sublinear time and a few (processing IPHAS data is 25 s.

  7. A CROSS-MATCH OF 2MASS AND SDSS. II. PECULIAR L DWARFS, UNRESOLVED BINARIES, AND THE SPACE DENSITY OF T DWARF SECONDARIES

    International Nuclear Information System (INIS)

    Geissler, Kerstin; Metchev, Stanimir; Kirkpatrick, J. Davy; Berriman, G. Bruce; Looper, Dagny

    2011-01-01

    We present the completion of a program to cross-correlate the Sloan Digital Sky Survey Data Release 1 (SDSS DR1) and Two-Micron All-Sky Survey (2MASS) Point Source Catalog in search for extremely red L and T dwarfs. The program was initiated by Metchev and collaborators, who presented the findings on all newly identified T dwarfs in SDSS DR1 and estimated the space density of isolated T0-T8 dwarfs in the solar neighborhood. In the current work, we present most of the L dwarf discoveries. Our red-sensitive (z - J ≥ 2.75 mag) cross-match proves to be efficient in detecting peculiarly red L dwarfs, adding two new ones, including one of the reddest known L dwarfs. Our search also nets a new peculiarly blue L7 dwarf and, surprisingly, two M8 dwarfs. We further broaden our analysis to detect unresolved binary L or T dwarfs through spectral template fitting to all L and T dwarfs presented here and in the earlier work by Metchev and collaborators. We identify nine probable binaries, six of which are new and eight harbor likely T dwarf secondaries. We combine this result with current knowledge of the mass ratio distribution and frequency of substellar companions to estimate an overall space density of 0.005-0.05 pc -3 for individual T0-T8 dwarfs.

  8. Comparative analysis of Luminex-based donor-specific antibody mean fluorescence intensity values with complement-dependent cytotoxicity & flow crossmatch results in live donor renal transplantation

    OpenAIRE

    Ajay Kumar Baranwal; Deepali Krishan Bhat; Sanjeev Goswami; Sanjay Kumar Agarwal; Gurvinder Kaur; Jasmeet Kaur; Narinder Mehra

    2017-01-01

    Background & objectives: Antibodies specific to donor human leucocyte antigen (HLA) play a critical role in graft rejection and graft loss. In recent years, techniques for their detection have evolved significantly providing an ever-increasing degree of sensitivity and specificity, from the conventional cell-based assays to the advanced solid-phase system based on the Luminex platform. Consensus is still evolving on the routine employment of all these methods, either stand alone or in combina...

  9. Challenges of setting up flow cytometry for diagnosis of leukemia ...

    African Journals Online (AJOL)

    The bone marrow (BM) is a complex tissue containing cells of multiple hematopoietic cell lineages in all stages of development. Flow cytometric immunophenotyping evaluates the frequencies of the various leukocyte (sub) populations in BM and blood that then helps in the diagnosis of leukemia's. The aim of this study was ...

  10. Assessment of Equine Autoimmune Thrombocytopenia (EAT by flow cytometry

    Directory of Open Access Journals (Sweden)

    Schwarzwald Colin

    2001-04-01

    Full Text Available Abstract Rationale Thrombocytopenia is a platelet associated process that occurs in human and animals as result of i decreased production; ii increased utilization; iii increased destruction coupled to the presence of antibodies, within a process know as immune-mediated thrombocytopenia (IMT; or iv platelet sequestration. Thus, the differentiation of the origin of IMT and the development of reliable diagnostic approaches and methodologies are important in the clarification of IMT pathogenesis. Therefore, there is a growing need in the field for easy to perform assays for assessing platelet morphological characteristics paired with detection of platelet-bound IgG. Objectives This study is aimed to develop and characterize a single color flow cytometric assay for detection of platelet-bound IgG in horses, in combination with flow cytometric assessment of platelet morphological characteristics. Findings The FSC and SSC evaluation of the platelets obtained from the thrombocytopenic animals shows several distinctive features in comparison to the flow cytometric profile of platelets from healthy animals. The thrombocytopenic animals displayed i increased number of platelets with high FSC and high SSC, ii a significant number of those gigantic platelets had strong fluorescent signal (IgG bound, iii very small platelets or platelet derived microparticles were found significantly enhanced in one of the thrombocytopenic horses, iv significant numbers of these microplatelet/microparticles/platelet-fragments still carry very high fluorescence. Conclusions This study describes the development and characterization of an easy to perform, inexpensive, and noninvasive single color flow cytometric assay for detection of platelet-bound IgG, in combination with flow cytometric assessment of platelet morphological characteristics in horses.

  11. Identification and detection of murine leukemia blasts by flow cytometry

    OpenAIRE

    sprotocols

    2015-01-01

    Human leukemia has been determined and classified with the help of flow cytometry for the past two decades. Past attempts to detect leukemia blasts relied on both forward and side scatter (FSC and SSC) based on cell size and granularity. However, this technique failed to show a clean separation of blasts from normal lineage cells. In 1993, Borowitz, et al developed flow cytometric analysis to distinguish human leukemia blasts from other normal lineage cells by using fluorescence-conjugated CD...

  12. Expression of Cyclins A, E and Topoisomerase II α correlates with centrosome amplification and genomic instability and influences the reliability of cytometric S-phase determination

    Directory of Open Access Journals (Sweden)

    Laytragoon-Lewin Nongnit

    2003-07-01

    Full Text Available Abstract Background The progression of normal cells through the cell cycle is meticulously regulated by checkpoints guaranteeing the exact replication of the genome during S-phase and its equal division at mitosis. A prerequisite for this achievement is synchronized DNA-replication and centrosome duplication. In this context the expression of cyclins A and E has been shown to play a principal role. Results Our results demonstrated a correlation between centrosome amplification, cell cycle fidelity and the level of mRNA and protein expression of cyclins A and E during the part of the cell cycle defined as G1-phase by means of DNA content based histogram analysis. It is shown that the normal diploid breast cell line HTB-125, the genomically relatively stable aneuploid breast cancer cell line MCF-7, and the genomically unstable aneuploid breast cancer cell line MDA-231 differ remarkably concerning both mRNA and protein expression of the two cyclins during G1-phase. In MDA-231 cells the expression of e.g. cyclin A mRNA was found to be ten times higher than in MCF-7 cells and about 500 times higher than in HTB-125 cells. Topoisomerase II α showed high mRNA expression in MDA compared to MCF-7 cells, but the difference in protein expression was small. Furthermore, we measured centrosome aberrations in 8.4% of the MDA-231 cells, and in only 1.3% of the more stable aneuploid cell line MCF-7. MDA cells showed 27% more incorporation of BrdU than reflected by S-phase determination with flow cytometric DNA content analysis, whereas these values were found to be of the same size in both HTB-125 and MCF-7 cells. Conclusions Our data indicate that the breast cancer cell lines MCF-7 and MDA-231, although both DNA-aneuploid, differ significantly regarding the degree of cell cycle disturbance and centrosome aberrations, which partly could explain the different genomic stability of the two cell lines. The results also question the reliability of cytometric DNA

  13. Flow cytometry of duodenal intraepithelial lymphocytes improves diagnosis of celiac disease in difficult cases.

    Science.gov (United States)

    Valle, Julio; Morgado, José Mario T; Ruiz-Martín, Juan; Guardiola, Antonio; Lopes-Nogueras, Miriam; García-Vela, Almudena; Martín-Sacristán, Beatriz; Sánchez-Muñoz, Laura

    2017-10-01

    Diagnosis of celiac disease is difficult when the combined results of serology and histology are inconclusive. Studies using flow cytometry of intraepithelial lymphocytes (IELs) have found that celiac patients have increased numbers of γδ IELs, along with a decrease in CD3-CD103 + IELs. The objective of this article is to assess the role of flow cytometric analysis of IELs in the diagnosis of celiac disease in difficult cases. A total of 312 patients with suspicion of celiac disease were included in the study. Duodenal biopsy samples were used for histological assessment and for flow cytometric analysis of IELs. In 46 out of 312 cases (14.7%) the combination of serology and histology did not allow the confirmation or exclusion of celiac disease. HLA typing had been performed in 42 of these difficult cases. Taking into account HLA typing and the response to a gluten-free diet, celiac disease was excluded in 30 of these cases and confirmed in the remaining 12. Flow cytometric analysis of IELs allowed a correct diagnosis in 39 out of 42 difficult cases (92.8%) and had a sensitivity of 91.7% (95% CI: 61.5% to 99.8%) and a specificity of 93.3% (95% CI: 77.9% to 99.2%) for the diagnosis of celiac disease in this setting. Flow cytometric analysis of IELs is useful for the diagnosis of celiac disease in difficult cases.

  14. Ratiometric fluorescence polarization as a cytometric functional parameter: theory and practice

    Energy Technology Data Exchange (ETDEWEB)

    Yishai, Yitzhak; Fixler, Dror; Cohen-Kashi, Meir; Zurgil, Naomi; Deutsch, Mordechai [The Biophysical Interdisciplinary Jerome Schottenstein Center for the Research and the Technology of the Cellome, Department of Physics, Bar-Ilan University, Ramat-Gan 52900 (Israel)

    2003-08-07

    The use of ratiometric fluorescence polarization (RFP) as a functional parameter in monitoring cellular activation is suggested, based on the physical phenomenon of fluorescence polarization dependency on emission wavelengths in multiple (at least binary) solutions. The theoretical basis of this dependency is thoroughly discussed and examined via simulation. For simulation, aimed to imitate a fluorophore-stained cell, real values of the fluorescence spectrum and polarization of different single fluorophore solutions were used. The simulation as well as the experimentally obtained values of RFP indicated the high sensitivity of this measure. Finally, the RFP parameter was utilized as a cytometric measure in three exemplary cellular bioassays. In the first, the apoptotic effect of oxLDL in a human Jurkat FDA-stained T cell line was monitored by RFP. In the second, the interaction between cell surface membrane receptors of human T lymphocyte cells was monitored by RFP measurements as a complementary means to the fluorescence resonance energy transfer (FRET) technique. In the third bioassay, cellular thiol level of FDA- and CMFDA-labelled Jurkat T cells was monitored via RFP.

  15. Functional and cytometric examination of different human lung epithelial cell types as drug transport barriers.

    Science.gov (United States)

    Min, Kyoung Ah; Rosania, Gus R; Kim, Chong-Kook; Shin, Meong Cheol

    2016-03-01

    To develop inhaled medications, various cell culture models have been used to examine the transcellular transport or cellular uptake properties of small molecules. For the reproducible high throughput screening of the inhaled drug candidates, a further verification of cell architectures as drug transport barriers can contribute to establishing appropriate in vitro cell models. In the present study, side-by-side experiments were performed to compare the structure and transport function of three lung epithelial cells (Calu-3, normal human bronchial primary cells (NHBE), and NL-20). The cells were cultured on the nucleopore membranes in the air-liquid interface (ALI) culture conditions, with cell culture medium in the basolateral side only, starting from day 1. In transport assays, paracellular transport across all three types of cells appeared to be markedly different with the NHBE or Calu-3 cells, showing low paracellular permeability and high TEER values, while the NL-20 cells showed high paracellular permeability and low TEER. Quantitative image analysis of the confocal microscope sections further confirmed that the Calu-3 cells formed intact cell monolayers in contrast to the NHBE and NL-20 cells with multilayers. Among three lung epithelial cell types, the Calu-3 cell cultures under the ALI condition showed optimal cytometric features for mimicking the biophysical characteristics of in vivo airway epithelium. Therefore, the Calu-3 cell monolayers could be used as functional cell barriers for the lung-targeted drug transport studies.

  16. The utility of flow sorting to identify chromosomes carrying a single copy transgene in wheat

    Czech Academy of Sciences Publication Activity Database

    Cápal, Petr; Endo, Takashi R.; Vrána, Jan; Kubaláková, Marie; Karafiátová, Miroslava; Komínková, Eva; Mora-Ramirez, I.; Weschke, W.; Doležel, Jaroslav

    2016-01-01

    Roč. 12, APR 25 (2016), s. 24 ISSN 1746-4811 R&D Projects: GA MŠk(CZ) LO1204; GA ČR GBP501/12/G090 Institutional support: RVO:61389030 Keywords : Transgene localization * Flow cytometric sorting * Single chromosome amplification Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.510, year: 2016

  17. Genome-size variation in switchgrass (Panicum virgatum): flow cytometry and cytology reveal rampant aneuploidy

    Science.gov (United States)

    Switchgrass (Panicum virgatum L.), a native perennial dominant of the prairies of North America, has been targeted as a model herbaceous species for biofeedstock development. A flow-cytometric survey of a core set of 11 primarily upland polyploid switchgrass accessions indicated that there was con...

  18. Flow cytometric assessment of activation of peripheral blood platelets in dogs with normal platelet count and asymptomatic thrombocytopenia.

    Science.gov (United States)

    Żmigrodzka, M; Guzera, M; Winnicka, A

    2016-01-01

    Platelets play a crucial role in hemostasis. Their activation has not yet been evaluated in healthy dogs with a normal and low platelet count. The aim of this study was to determine the influence of activators on platelet activation in dogs with a normal platelet count and asymptomatic thrombocytopenia. 72 clinically healthy dogs were enrolled. Patients were allocated into three groups. Group 1 consisted of 30 dogs with a normal platelet count, group 2 included 22 dogs with a platelet count between 100 and 200×109/l and group 3 consisted of 20 dogs with a platelet count lower than 100×109/l. Platelet rich-plasma (PRP) was obtained from peripheral blood samples using tripotassium ethylenediaminetetraacetic acid (K3-EDTA) as anticoagulant. Next, platelets were stimulated using phorbol-12-myristate-13-acetate or thrombin, stabilized using procaine or left unstimulated. The expression of CD51 and CD41/CD61 was evaluated. Co-expression of CD41/CD61 and Annexin V served as a marker of platelet activation. The expression of CD41/CD61 and CD51 did not differ between the 3 groups. Thrombin-stimulated platelets had a significantly higher activity in dogs with a normal platelet count than in dogs with asymptomatic thrombocytopenia. Procaine inhibited platelet activity in all groups. In conclusion, activation of platelets of healthy dogs in vitro varied depending on the platelet count and platelet activator.

  19. Development of a flow cytometric immunoassay for recombinant bovine somatotropin-induced antibodies in serum of dairy cows

    NARCIS (Netherlands)

    Smits, N.G.E.; Bremer, M.G.E.G.; Ludwig, S.K.J.; Nielen, M.W.F.

    2012-01-01

    Administration of recombinant bovine somatotropin (rbST) to enhance milk production in dairy cows is banned within the European Union. Therefore, methods for pinpointing rbST abuse are required. Due to the problematic detection of rbST itself in serum, methods are also focused on detecting changes

  20. Transmission Electron Microscopic Morphological Study and Flow Cytometric Viability Assessment of Acinetobacter baumannii Susceptible to Musca domestica cecropin

    OpenAIRE

    Gui, Shuiqing; Li, Rongjiang; Feng, Yongwen; Wang, Sanming

    2014-01-01

    Multidrug-resistant (MDR) Acinetobacter baumannii infections are difficult to treat owing to the extremely limited armamentarium. Expectations about antimicrobial peptides' use as new powerful antibacterial agents have been raised on the basis of their unique mechanism of action. Musca domestica cecropin (Mdc), a novel antimicrobial peptide from the larvae of Housefly (Musca domestica), has potently active against Gram-positive and Gram-negative bacteria standard strain. Here we evaluated the...

  1. Studies of the ABO and FORS Histo-Blood Group Systems: Focus on Flow Cytometric and Genetic Analysis

    OpenAIRE

    Hult, Annika

    2013-01-01

    ABO is the clinically most important blood group system and its antigens are carbohydrate moieties present on the surface of the red blood cell (RBC) but also on other tissues throughout the body. The ABO gene encodes an enzyme, a glycosyltransferase (GT),that adds a terminal monosaccharide to the precursor structure, H antigen, to define the A or B antigens. Blood group O is due to a non-functional GT that leaves the precursor unchanged. Weak expression of ABO antigens can be acquired or be ...

  2. Flow cytometric chromosome sorting from diploid progenitors of bread wheat, T. urartu, Ae. speltoides and Ae. tauschii

    Czech Academy of Sciences Publication Activity Database

    Molnár, I.; Kubaláková, Marie; Šimková, Hana; Farkas, A.; Cseh, A.; Megyeri, M.; Vrána, Jan; Molnár-Láng, M.; Doležel, Jaroslav

    2014-01-01

    Roč. 127, č. 5 (2014), s. 1091-1104 ISSN 0040-5752 R&D Projects: GA ČR GBP501/12/G090; GA MŠk(CZ) LO1204 Grant - others:GA MŠk(CZ) ED0007/01/01 Program:ED Institutional support: RVO:61389030 Keywords : SYNTHETIC HEXAPLOID WHEAT * AEGILOPS-TRITICUM GROUP * GENETIC-LINKAGE MAP Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.790, year: 2014

  3. Stereologic, histopathologic, flow cytometric, and clinical parameters in the prognostic evaluation of 74 patients with intraoral squamous cell carcinomas

    DEFF Research Database (Denmark)

    Bundgaard, T; Sørensen, Flemming Brandt; Gaihede, M

    1992-01-01

    , tumor size, and the TNM classification. RESULTS: The investigation showed a significant difference between the volume-weighted mean nuclear volume (nuclear vv) of oral leukoplakia (n = 29) and oral squamous cell carcinomas (P = 0.001). The value of the parameters as prognostic indicators of survival...

  4. Single and multigland disease in primary hyperparathyroidism: Clinical follow-up, histopathology, and flow cytometric DNA analysis

    NARCIS (Netherlands)

    H.J. Bonjer (Jaap); H.A. Bruining (Hajo); J.C. Birkenhäger (Jan); Y. Nishiyama (Yoshihiko); M.A. Jones (Michael); C. Bruce Bagwell (C.)

    1992-01-01

    textabstractTwo-hundred seventy-four patients with primary hyperparathyroidism had selective removal of enlarged parathyroid glands. Biopsies were taken from all parathyroid glands. Normal-size glands were not resected irrespective of their histological appearance. After a mean follow-up of 13.5

  5. A rapid flow cytometric method for determining the cellular composition of bronchoalveolar lavage fluid cells in mouse models of asthma

    NARCIS (Netherlands)

    van Rijt, Leonie S.; Kuipers, Harmjan; Vos, Nanda; Hijdra, Daniëlle; Hoogsteden, Henk C.; Lambrecht, Bart N.

    2004-01-01

    Mouse models of allergic asthma are increasingly used to study the immunopathology of this complex disorder. The degree and type of airway inflammation is often studied by determination of differential cell counts on cytospins of bronchoalveolar lavage fluid (BALF) cells stained with May-Grünwald

  6. Immuno-flow cytometric detection of the ichthyotoxic dinoflagellates Gyrodinium aureolum and Gymnodinium nagasakiense : Independence of physiological state

    NARCIS (Netherlands)

    Vrieling, EG; vandePoll, WH; Vriezekolk, G; Gieskes, WWC

    The ichthyotoxic dinoflagellates Gyrodinium aureolum and Gymnodinium nagasakiense were cultured under different environmental conditions to test possible variability in immunochemical labelling intensity of cell-surface antigens using species-specific monoclonal antibodies. Variation of antigen

  7. A modified method of flow cytometric seed screen simplifies the quantification of progeny classes with different ploidy levels

    Czech Academy of Sciences Publication Activity Database

    Krahulcová, Anna; Suda, Jan

    2006-01-01

    Roč. 50, č. 3 (2006), s. 457-460 ISSN 0006-3134 R&D Projects: GA AV ČR IAA6005203 Institutional research plan: CEZ:AV0Z60050516 Keywords : facultative apomixis * reproduction routes * polyhaploids Subject RIV: EF - Botanics Impact factor: 1.198, year: 2006

  8. Single-laboratory validation of a multiplex flow cytometric immunoassay for the simultaneous detection of coccidiostats in eggs and feed

    NARCIS (Netherlands)

    Bienenmann-Ploum, M.E.; Vincent, U.; Campbell, K.; Huet, A.C.; Haasnoot, W.; Delahaut, P.; Stolker, A.A.M.; Elliott, C.T.; Nielen, M.W.F.

    2013-01-01

    Coccidiostats are authorized in the European Union (EU) to be used as poultry feed additives. Maximum (residue) levels (M(R)Ls) have been set within the EU for consumer and animal protection against unintended carry-over, and monitoring is compulsory. This paper describes the single-laboratory

  9. Naturalized plants have smaller genomes than their non-invading relatives: a flow cytometric analysis of the Czech alien flora

    Czech Academy of Sciences Publication Activity Database

    Kubešová, M.; Moravcová, Lenka; Suda, Jan; Jarošík, V.; Pyšek, Petr

    2010-01-01

    Roč. 82, č. 1 (2010), s. 81-96 ISSN 0032-7786 R&D Projects: GA ČR GA206/09/0563; GA ČR GD206/08/H049; GA MŠk LC06073 Institutional research plan: CEZ:AV0Z60050516 Keywords : cytometry * ploidy * genome size Subject RIV: EF - Botanics Impact factor: 2.792, year: 2010

  10. Restorative proctocolectomy: histological assessment and cytometric DNA analysis of ileal pouch biopsies.

    Science.gov (United States)

    Pronio, A; Montesani, C; Vecchione, A; Giovagnoli, M G; Giarnieri, E; Nardi, F; Nigri, G; Ribotta, G

    1997-01-01

    The pathological changes and the risk of developing cancer in the ileal pouch mucosa of patients who received restorative proctocolectomy with ileal pouch-anal anastomosis (IPAA) were studied. The presence or absence of remaining rectal mucosa below the IPAA in both patients with stapled and handsewn IPAA was also examined. Endoscopy of the ileal pouch was performed on 38 patients at 4, 12, 18 and 36 months after restorative proctocolectomy with ileal pouch. Mucosal biopsy specimens were taken from the ileal reservoir in order to assess the histological incidence of inflammation. In 23 patients, biopsies were taken to perform cytometric DNA analysis. Clinical symptoms of pouchitis (over six evacuations in 24 hours, night-time evacuations, leakage of feces, bloody diarrhea, abdominal pain and fever) were recorded and correlated with the histological findings. Biopsies were also sampled below the ileo-anal anastomosis (IPAA) in order to identify residual rectal mucosa. Results of histological assessment showed various degrees of chronic inflammation increasing over time (from 42 to 60%) while the presence of both acute and chronic inflammation of the reservoir was less frequent (from 18 to 30%). Villous atrophy was present in 39-68% of patients and the grade of villous atrophy was correlated to the grade of inflammation. Clinical pouchitis was present in 3 to 8% of cases at the different controls and it was always associated with the highest grade of histological inflammation and severe villous atrophy. No significant alteration of the DNA cellular content was observed. Very low incidence of aneuploidy (0.7-1% Ex.R.) has been reported in three cases. However, we found dysplasia in only one patient who underwent surgical treatment for familial polyposis coli. IPAA evaluation showed no residual rectal mucosa in 40% of cases with stapled IPAA; in the remaining 60%, we found a small amount of rectal mucosa (maximum 1 cm). We did not find rectal mucosa after handsewn IPAA

  11. Supercontinuum white light lasers for flow cytometry

    Science.gov (United States)

    Telford, William G.; Subach, Fedor V.; Verkhusha, Vladislav V.

    2009-01-01

    Excitation of fluorescent probes for flow cytometry has traditionally been limited to a few discrete laser lines, an inherent limitation in our ability to excite the vast array of fluorescent probes available for cellular analysis. In this report, we have used a supercontinuum (SC) white light laser as an excitation source for flow cytometry. By selectively filtering the wavelength of interest, almost any laser wavelength in the visible spectrum can be separated and used for flow cytometric analysis. The white light lasers used in this study were integrated into a commercial flow cytometry platform, and a series of high-transmission bandpass filters used to select wavelength ranges from the blue (~480 nm) to the long red (>700 nm). Cells labeled with a variety of fluorescent probes or expressing fluorescent proteins were then analyzed, in comparison with traditional lasers emitting at wavelengths similar to the filtered SC source. Based on a standard sensitivity metric, the white light laser bandwidths produced similar excitation levels to traditional lasers for a wide variety of fluorescent probes and expressible proteins. Sensitivity assessment using fluorescent bead arrays confirmed that the SC laser and traditional sources resulted in similar levels of detection sensitivity. Supercontinuum white light laser sources therefore have the potential to remove a significant barrier in flow cytometric analysis, namely the limitation of excitation wavelengths. Almost any visible wavelength range can be made available for excitation, allowing access to virtually any fluorescent probe, and permitting “fine-tuning” of excitation wavelength to particular probes. PMID:19072836

  12. Image cytometric evaluation of nuclear texture features and DNA content of the reticular form of oral lichen planus.

    Science.gov (United States)

    Rode, Matjaz; Flezar, Margareta Strojan; Kogoj-Rode, Mirela; Us-Krasovec, Marija

    2006-10-01

    To analyze image cytometric chromatin changes reflected in nuclear texture features and DNA ploidy of oral lichen planus in relation to the normal buccal mucosa and buccal mucosa expressing malignancy-associated changes in cancer patients. Twenty-eight patients with the reticular form of oral lichen planus, with a follow-up period of 25 years, 50 healthy controls and 50 lung cancer patients were included in the study. Scrapings of buccal mucosa were suspended in transport medium. Monolayer filter preparations were Feulgen-thionin stained. Image cytometric analysis was performed by Cyto-Savant. All oral lichen planus specimens in our study were diploid. In univariate analysis, differences between the normal buccal mucosa and oral lichen planus were found in several nuclear texture features, which gave an 80% correct classification rate in multivariate analysis. In the second part of the study, the classifier that recognizes malignancy-associated changes on the buccal mucosa of patients with lung cancer correctly recognized > 80% of oral lichen planus samples as normal buccal mucosa. Our results indicate that chromatin changes in oral lichen planus exist compared to normal cells; however, the chromatin structure of the reticular form of oral lichen planus does not express malignancy-associated changes and is more similar to normal squamous cells.

  13. Integration of genetic and physical maps of the chickpea (Cicer arietinum L.) genome using flow-sorted chromosomes

    Czech Academy of Sciences Publication Activity Database

    Zatloukalová, Pavlína; Hřibová, Eva; Kubaláková, Marie; Suchánková, Pavla; Šimková, Hana; Adoración, C.; Kahl, G.; Millán, T.; Doležel, Jaroslav

    2011-01-01

    Roč. 19, č. 6 (2011), s. 729-739 ISSN 0967-3849 R&D Projects: GA MŠk(CZ) LC06004 Institutional research plan: CEZ:AV0Z50380511 Keywords : BAC-FISH * Chromosome isolation * Flow cytometric sorting Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.087, year: 2011

  14. Detection of Xanthomonas axonopodis pv. phaseoli in bean seeds by flow cytometry, immunostaining and direct viable counting

    NARCIS (Netherlands)

    Tebaldi, N.D.; Peters, J.; Chitarra, L.G.; Souza, R.M.; Zouwen, van der P.S.; Bergervoet, J.H.W.; Wolf, van der J.M.

    2010-01-01

    Flow cytometric analysis of immuno-stained cells (immuno-FCM) was compared to immunofluorescence microscopy (IF) and dilution plating on a semi-selective medium, for quantitative detection of Xanthomonas axonopodis pv. phaseoli (Xap) in bean seed extracts. Cell concentrations of Xap between 103-107

  15. Flow cytogenetic studies in chromosomes and whole cells for the detection of clastogenic effects

    International Nuclear Information System (INIS)

    Otto, F.J.; Oldiges, H.

    1980-01-01

    Flow cytometric measurements of the chromosomal DNA content have been used to develop a screening method for the detection of chemically- or physically-induced cytogenetic damage. The reproducibility of this flow cytogenetic assay was shown in a series of subcultures of a Chinese hamster cell clone. The accuracy and sensitivity was tested in cultures treated with chemical mutagens and x-rays. The clastogenic effectiveness was quantified and the dose-effect relationship was established by the increase of the coefficient of variation of the peak of the largest chromosome type in the flow histograms. Since structural chromosome aberrations cause an unequal division of the DNA at mitosis, it is expected that clastogenic effects can be detected also in whole cells of growing populations as an increased dispersion of the cellular DNA content. In order to test this feature, high resolution flow cytometric measurements were performed in x-irradiated hamster cells in vitro and mouse bone marrow cells in vivo

  16. A high-throughput method for detection of DNA in chloroplasts using flow cytometry

    Directory of Open Access Journals (Sweden)

    Oldenburg Delene J

    2007-03-01

    Full Text Available Abstract Background The amount of DNA in the chloroplasts of some plant species has been shown recently to decline dramatically during leaf development. A high-throughput method of DNA detection in chloroplasts is now needed in order to facilitate the further investigation of this process using large numbers of tissue samples. Results The DNA-binding fluorophores 4',6-diamidino-2-phenylindole (DAPI, SYBR Green I (SG, SYTO 42, and SYTO 45 were assessed for their utility in flow cytometric analysis of DNA in Arabidopsis chloroplasts. Fluorescence microscopy and real-time quantitative PCR (qPCR were used to validate flow cytometry data. We found neither DAPI nor SYTO 45 suitable for flow cytometric analysis of chloroplast DNA (cpDNA content, but did find changes in cpDNA content during development by flow cytometry using SG and SYTO 42. The latter dye provided more sensitive detection, and the results were similar to those from the fluorescence microscopic analysis. Differences in SYTO 42 fluorescence were found to correlate with differences in cpDNA content as determined by qPCR using three primer sets widely spaced across the chloroplast genome, suggesting that the whole genome undergoes copy number reduction during development, rather than selective reduction/degradation of subgenomic regions. Conclusion Flow cytometric analysis of chloroplasts stained with SYTO 42 is a high-throughput method suitable for determining changes in cpDNA content during development and for sorting chloroplasts on the basis of DNA content.

  17. Monodisperse Water-in-Oil-in-Water (W/O/W Double Emulsion Droplets as Uniform Compartments for High-Throughput Analysis via Flow Cytometry

    Directory of Open Access Journals (Sweden)

    Jing Yan

    2013-12-01

    Full Text Available Here we report the application of monodisperse double emulsion droplets, produced in a single step within partially hydrophilic/partially hydrophobic microfluidic devices, as defined containers for quantitative flow cytometric analysis. Samples with varying fluorophore concentrations were generated, and a clear correlation between dye concentration and fluorescence signals was observed.

  18. Morphologic, cytometric and functional characterization of the common octopus (Octopus vulgaris) hemocytes.

    Science.gov (United States)

    Castellanos-Martínez, S; Prado-Alvarez, M; Lobo-da-Cunha, A; Azevedo, C; Gestal, C

    2014-05-01

    The hemocytes of Octopus vulgaris were morphologically and functionally characterized. Light and electron microscopy (TEM and SEM), and flow cytometry analyses revealed the existence of two hemocyte populations. Large granulocytes showed U-shaped nucleus, a mean of 11.6 μm±1.2 in diameter with basophilic granules, polysaccharide and lysosomic deposits in the cytoplasm. Small granulocytes measured a mean of 8.1 μm±0.7 in diameter, and have a round nucleus occupying almost the entire cell and few or not granules in the cytoplasm. Flow cytometry analysis showed that large granulocytes are the principal cells that develop phagocytosis of latex beads (rising up to 56%) and ROS after zymosan stimulation. Zymosan induced the highest production of both ROS and NO. This study is the first tread towards understanding the O. vulgaris immune system by applying new tools to provide a most comprehensive morpho-functional study of their hemocytes. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Applications of Imaging Flow Cytometry for Microalgae.

    Science.gov (United States)

    Hildebrand, Mark; Davis, Aubrey; Abbriano, Raffaela; Pugsley, Haley R; Traller, Jesse C; Smith, Sarah R; Shrestha, Roshan P; Cook, Orna; Sánchez-Alvarez, Eva L; Manandhar-Shrestha, Kalpana; Alderete, Benjamin

    2016-01-01

    The ability to image large numbers of cells at high resolution enhances flow cytometric analysis of cells and cell populations. In particular, the ability to image intracellular features adds a unique aspect to analyses, and can enable correlation between molecular phenomena resulting in alterations in cellular phenotype. Unicellular microalgae are amenable to high-throughput analysis to capture the diversity of cell types in natural samples, or diverse cellular responses in clonal populations, especially using imaging cytometry. Using examples from our laboratory, we review applications of imaging cytometry, specifically using an Amnis(®) ImageStream(®)X instrument, to characterize photosynthetic microalgae. Some of these examples highlight advantages of imaging flow cytometry for certain research objectives, but we also include examples that would not necessarily require imaging and could be performed on a conventional cytometer to demonstrate other concepts in cytometric evaluation of microalgae. We demonstrate the value of these approaches for (1) analysis of populations, (2) documentation of cellular features, and (3) analysis of gene expression.

  20. Flow Cytometry Is a Powerful Tool for Assessment of the Viability of Fungal Conidia in Metalworking Fluids.

    Science.gov (United States)

    Vanhauteghem, D; Demeyere, K; Callaert, N; Boelaert, A; Haesaert, G; Audenaert, K; Meyer, E

    2017-08-15

    Fungal contamination of metalworking fluids (MWF) is a dual problem in automated processing plants because resulting fungal biofilms obstruct cutting, drilling, and polishing machines. Moreover, some fungal species of MWF comprise pathogens such as Fusarium solani Therefore, the development of an accurate analytical tool to evaluate conidial viability in MWF is important. We developed a flow cytometric method to measure fungal viability in MWF using F. solani as the model organism. To validate this method, viable and dead conidia were mixed in several proportions and flow was cytometrically analyzed. Subsequently, we assessed the fungicidal activity of two commercial MWF using flow cytometry (FCM) and compared it with microscopic analyses and plating experiments. We evaluated the fungal growth in both MWF after 7 days using quantitative PCR (qPCR) to assess the predictive value of FCM. Our results showed that FCM distinguishes live from dead conidia as early as 5 h after exposure to MWF, whereas the microscopic germination approach detected conidial viability much later and less accurately. At 24 h, microscopic analyses of germinating conidia and live/dead analyses by FCM correlated well, although the former consistently underestimated the proportion of viable conidia. In addition, the reproducibility and sensitivity of the flow cytometric method were high and allowed assessment of the fungicidal properties of two commercial MWF. Importantly, the obtained flow cytometric results on viability of F. solani conidia at both early time points (5 h and 24 h) correlated well with fungal biomass measurements assessed via a qPCR methodology 7 days after the start of the experiment. IMPORTANCE This result shows the predictive power of flow cytometry (FCM) in assessing the fungicidal capacity of MWF formulations. It also implies that FCM can be implemented as a rapid detection tool to estimate the viable fungal load in an industrial processing matrix (MWF). Copyright © 2017

  1. Supercontinuum white light lasers for flow cytometry.

    Science.gov (United States)

    Telford, William G; Subach, Fedor V; Verkhusha, Vladislav V

    2009-05-01

    Excitation of fluorescent probes for flow cytometry has traditionally been limited to a few discrete laser lines, an inherent limitation in our ability to excite the vast array of fluorescent probes available for cellular analysis. In this report, we have used a supercontinuum (SC) white light laser as an excitation source for flow cytometry. By selectively filtering the wavelength of interest, almost any laser wavelength in the visible spectrum can be separated and used for flow cytometric analysis. The white light lasers used in this study were integrated into a commercial flow cytometry platform, and a series of high-transmission bandpass filters used to select wavelength ranges from the blue (approximately 480 nm) to the long red (>700 nm). Cells labeled with a variety of fluorescent probes or expressing fluorescent proteins were then analyzed, in comparison with traditional lasers emitting at wavelengths similar to the filtered SC source. Based on a standard sensitivity metric, the white light laser bandwidths produced similar excitation levels to traditional lasers for a wide variety of fluorescent probes and expressible proteins. Sensitivity assessment using fluorescent bead arrays confirmed that the SC laser and traditional sources resulted in similar levels of detection sensitivity. Supercontinuum white light laser sources therefore have the potential to remove a significant barrier in flow cytometric analysis, namely the limitation of excitation wavelengths. Almost any visible wavelength range can be made available for excitation, allowing access to virtually any fluorescent probe, and permitting "fine-tuning" of excitation wavelength to particular probes. (c) 2008 International Society for Advancement of Cytometry.

  2. Platelet function investigation by flow cytometry

    DEFF Research Database (Denmark)

    Pedersen, Oliver Heidmann; Nissen, Peter H; Hvas, Anne-Mette

    2017-01-01

    Flow cytometry is an increasingly used method for platelet function analysis because it has some important advantages compared with other platelet function tests. Flow cytometric platelet function analyses only require a small sample volume (3.5 mL); however, to expand the field of applications, e...... assays. To examine the influence of sample volume, blood was collected from 20 healthy individuals in 1.0 mL, 1.8 mL, and 3.5 mL tubes. To examine the influence of the needle size on pre-activation, blood was drawn from another 13 healthy individuals with both a 19- and 21-gauge needle. For the reference...

  3. PATHOMORPHOLOGICAL AND CYTOMETRIC PARAMETERS OF BLOOD RED CELLS OF AGE-2 PRUSSIAN CARP (CARASSIUS AURATUS GIBELIO (BLOCH, 1782 IN THE CONDITIONS OF INTOXICATION WITH COPPER IONS

    Directory of Open Access Journals (Sweden)

    T. Sharamok

    2017-09-01

    Full Text Available Purpose. To detect the effect of elevated copper ion concentrations (10 aquaculture Maximum Permissible Limits on morphological and cytometric parameters of erythrocytes of age-2 Prussian carp (Carassius gibelio Bloch, 1782 in experimental and natural conditions. Methodology. During the work, summarized results of studies performed in 2015-2016 were used. Morphological and cytometric parameters of Prussian carp erythrocytes were determined in the conditions of natural habitats (Zaporizhzhia reservoir and an experiment. Copper ion concentration both in the experiment and natural conditions was similar and was 0.01 mg/L (10 aquaculture Maximum Permissible Limits. Experimental studies were performed during 21 days. In the control aquarium, fish were kept in the settled tap water; while in the experimental aquaria, intoxication of fish with copper ions was modelled by introducing CuSO4 in water. Blood smears were examined under 40x and 100 x magnifications with the use of microphotography (digital camera Sciencelab T500 5.17 M. Findings. The performed hematological studies showed that under the conditions of experimental chronic intoxication with copper ions (0.01 mg/L, age-2 Prussian carp had an increase in the share of immature forms of erythrocytes, increase in the number of erythrocytes with pathological signs (cell wall destruction, atypical forms, increase in the nucleus-cytoplasm ration, but the difference in cytometric parameters of erythrocytes between experimental and control fish was not significant. When comparing the morphometric parameters of erythrocytes of fish kept in experimental and natural conditions with similar copper ion concentrations (0.01 mg/L, a significant increase in the nucleus areas of mature erythrocytes was detected and, correspondingly, an increase in the nucleus-cytoplasm ratio of erythrocytes (by almost 30% in fish in experimental conditions compared to fish, which lived in the Zaporizhzhia reservoir. An increase

  4. Flow immunocytochemistry of marker expression in cells from body cavity fluids.

    Science.gov (United States)

    Krishan, Awtar; Ganjei-Azar, Parvin; Hamelik, Ronald; Sharma, Deepti; Reis, Isildinha; Nadji, Mehrdad

    2010-02-01

    Diagnostic cytology based on the examination of cells from body cavity fluids misses approximately 50% of patients with a proven malignancy. In an earlier study, we used immunohistochemical detection of epithelial membrane antigen expression with flow cytometric detection of DNA aneuploidy to reduce the number of false negatives. In the present study, we have combined DNA flow cytometry with flow cytometric detection of marker expression to analyze cells from body cavity fluids. Seventy-nine specimens of ascites and pleural fluids were analyzed by diagnostic cytology, DNA flow cytometry, and for the expression of the following markers: Ber-EP4, progesterone (PR), MUC4, and thyroid transcription factor-1 (TTF-1). DNA index of equal to or greater than 1.2 was seen in 33/79 (41.7%) of the samples. Statistical analysis of 79 samples in which data from cytology, DNA aneuploidy, and expression of at least one of the markers was available showed that by combining data from positive marker expression with that of aneuploidy, the sensitivity was increased from 58.5 to 100%. In contrast, out of the 38 samples designated as non-malignant by diagnostic cytology, nine had aneuploid DNA content and 16 of the diploid samples had a positive marker expression. Specificity was reduced from 74.7 to 31.6% due to the presence of aneuploidy and marker expression in these samples. ALDH1(pos)/CD44(pos)/CD24(neg) expression has been reported to be associated with human breast tumor stem cells. Some of our samples had cells with this phenotype. Flow cytometry offers the advantage of rapid multiparametric analysis of DNA aneuploidy and marker expression in cells from body cavity fluids based on the analysis of a large number of cells without observer bias. By further developing the use of specific markers and aneuploidy, it may be possible to refine flow cytometric analysis for rapid detection of malignant cells in body cavity fluids.

  5. Mixed and inhomogeneous expression profile of Th1/Th2 related cytokines detected by cytometric bead array in the saliva of patients with oral lichen planus.

    Science.gov (United States)

    Wei, Wei; Sun, Qianqian; Deng, Yiwen; Wang, Yufeng; Du, Guanhuan; Song, Chencheng; Li, Chenxi; Zhu, Mengxue; Chen, Guangjie; Tang, Guoyao

    2018-03-06

    The aim of this study was to measure T helper (Th) 1/Th2-related cytokine expression in saliva from patients with oral lichen planus (OLP), compared with healthy controls (HC group) and controls with recurrent aphthous ulcers (RAU group). Saliva was collected from 41 patients with OLP, 14 HCs, and 14 controls with RAU for Th1/Th2-related cytokines analysis with cytometric bead array. Disease activity in OLP was recorded by reticulation/keratosis, erythema, and ulceration scores. Interleukin (IL)-6, IL-10, interferon-γ (IFN-γ), and IFN-γ/IL-4 in saliva were significantly higher in the OLP group than in the HC group. A positive and significant correlation among IL-6, IL-10, and reticulation/keratosis, erythema, and ulceration scores in the OLP group was revealed. Significantly increased IL-4, IL-5, IL-6, IL-10, tumor necrosis factor-α and IFN-γ/IL-4 were found in the RAU group. Salivary cytokine profiles analyzed by cytometric bead array may provide a convenient research approach to OLP. Data indicated complicated Th1/Th2-related cytokine profile changes, rather than simple dominance model, in OLP. IL-10 and especially IL-6 may provide a surrogate endpoint for monitoring OLP. Copyright © 2018 Elsevier Inc. All rights reserved.

  6. Detection of bacteriophage-infected cells of Lactococcus lactis using flow cytometry

    DEFF Research Database (Denmark)

    Michelsen, Ole; Cuesta-Dominguez, Álvaro; Albrektsen, Bjarne

    2007-01-01

    Bacteriophage infection in dairy fermentation constitutes a serious problem worldwide. We have studied bacteriophage infection in Lactococcus lactis by using the flow cytometer. The first effect of the infection of the bacterium is a change from cells in chains toward single cells. We interpret...... describe a new method for detection of phage infection in Lactococcus lactis dairy cultures. The method is based on flow cytometric detection of cells with low-density cell walls. The method allows fast and early detection of phage-infected bacteria, independently of which phage has infected the culture...

  7. Fluorescence-intensity multiplexing: simultaneous seven-marker, two-color immunophenotyping using flow cytometry.

    Science.gov (United States)

    Bradford, Jolene A; Buller, Gayle; Suter, Michael; Ignatius, Michael; Beechem, Joseph M

    2004-10-01

    Conventional immuno-based multiparameter flow cytometric analysis has been limited by the requirement of a dedicated detection channel for each antibody-fluorophore set. To address the need to resolve multiple biological targets simultaneously, flow cytometers with as many as 10-15 detection channels have been developed. In this study, a new Zenon immunolabeling technology is developed that allows for multiple antigen detection per detection channel using a single fluorophore, through a unique method of fluorescence-intensity multiplexing. By varying the Zenon labeling reagent-to-antibody molar ratio, the fluorescence intensity of the antibody-labeled cellular targets can be used as a unique identifier. Although demonstrated in the present study with lymphocyte immunophenotyping, this approach is broadly applicable for any immuno-based multiplexed flow cytomety assay. Lymphocyte immunophenotyping of 38 clinical blood specimens using CD3, CD4, CD8, CD16, CD56, CD19, and CD20 antibodies was performed using conventional flow cytometric analysis and fluorescence-intensity multiplexing analysis. Conventional analysis measures a single antibody-fluorophore per photomultiplier tube (PMT). Fluorescence-intensity multiplex analysis simultaneously measures seven markers with two PMTs, using Zenon labeling reagent-antibody complexes in a single tube: CD19, CD4, CD8, and CD16 antibodies labeled with Zenon Alexa Fluor 488 Mouse IgG(1) labeling reagent and CD56, CD3, and CD20 antibodies labeled with Zenon R-Phycoerythrin (R-PE) Mouse IgG(1) or IgG(2b) labeling reagents. The lymphocyte immunophenotyping results from fluorescence-intensity multiplexing using Zenon labeling reagents in a single tube were comparable to results from conventional flow cytometric analysis. Simultaneous evaluation of multiple antigens using a single fluorophore can be performed using antibodies labeled with varying ratios of a Zenon labeling reagent. Labeling two sets of antibodies with different Zenon

  8. Generation of highly productive polyclonal and monoclonal tobacco suspension lines from a heterogeneous transgenic BY-2 culture through flow cytometric sorting

    OpenAIRE

    Kirchhoff, Janina

    2012-01-01

    Over the last 20 years, plant cells gained increasing interest as host systems for the production of human and animal pharmaceuticals. Transgenic tobacco suspension cultures are an especially promising production host because of their beneficial growth, low maintenance requirement and the possibility of contained cultivation in bioreactors under controlled conditions. The excellent product quality of plant-produced proteins was demonstrated for a multitude of proteins, but the heterogeneous a...

  9. Detection of HIV-RNA-positive monocytes in peripheral blood of HIV-positive patients by simultaneous flow cytometric analysis of intracellular HIV RNA and cellular immunophenotype.

    Science.gov (United States)

    Patterson, B K; Mosiman, V L; Cantarero, L; Furtado, M; Bhattacharya, M; Goolsby, C

    1998-04-01

    Determinations of plasma HIV viral RNA copy numbers help to define the kinetics of HIV-1 infection in vivo and to monitor antiretroviral therapy. However, questions remain regarding the identity of various infected cell types contributing to this free virus pool and to the in vivo lifecycle of HIV during disease progression. Characterization of a novel fluorescence in situ hybridization (FISH) assay employing a pool of labeled oligonucleotide probes directed against HIV RNA was done followed by coupling of the FISH assay with simultaneous surface immunophenotyping to address these questions. In vitro characterizations of this assay using tumor necrosis factor-alpha stimulated and unstimulated ACH-2 cells demonstrated the ability to detect < 5% HIV RNA positive cells with a sensitivity of < 30 RNA copies per cell. Peripheral blood mononuclear cells from 39 HIV-seropositive patients on no, single, combination, or triple drug therapy and 8 HIV-seronegative patients were examined. The majority of HIV-positive patients (24/39) harbored monocytes positive for HIV RNA and a significantly higher fraction of patients with high plasma viral load carried positive monocytes (13/16) than did patients in the low plasma viral load group (11/23). These results demonstrate the effectiveness of a novel FISH assay for identifying and monitoring HIV-infected cell populations in the peripheral blood of HIV-positive patients. In addition, monocytes are a major source of cellular HIV virus in the peripheral blood of HIV patients, even with progression of disease.

  10. The value of flow cytometric analysis of platelet glycoprotein expression of CD34+ cells measured under conditions that prevent P-selectin-mediated binding of platelets

    NARCIS (Netherlands)

    Dercksen, M. W.; Weimar, I. S.; Richel, D. J.; Breton-Gorius, J.; Vainchenker, W.; Slaper-Cortenbach, C. M.; Pinedo, H. M.; von dem Borne, A. E.; Gerritsen, W. R.; van der Schoot, C. E.

    1995-01-01

    In the present study, we show by adhesion assays and ultrastructural studies that platelets can bind to CD34+ cells from human blood and bone marrow and that this interaction interferes with the accurate detection of endogenously expressed platelet glycoproteins (GPs). The interaction between these

  11. Further evaluation of the prognostic value of morphometric and flow cytometric parameters in breast-cancer patients with long follow-up.

    Science.gov (United States)

    Uyterlinde, A M; Baak, J P; Schipper, N W; Peterse, H; Matze, E; Meijer, C J

    1990-01-15

    The added prognostic value of cellular DNA content compared with single and combined morphometric factors and classical parameters such as tumor size, nodal status, histologic grade and estrogen receptor (ER) content was investigated in 225 consecutive breast-cancer patients with long follow-up. Of all features investigated, the MPI (multivariate prognostic index) had the strongest prognostic value [Mantel-Cox (MC) = 48.2, p less than 0.00005]. The results further showed that neither age nor ER content had significant prognostic value, but the DNA index (DI) as a single parameter had (though weak) prognostic significance (MC = 5.9, p = 0.015); a similar result was obtained with the percentage of S-phase cells (MC = 6.1, p = 0.013). The DI had (restricted) additional prognostic value to the morphometric features (MPI plus DI Mantel-Cox 53.0, p less than 0.0001). The percentage of S-phase cells had no additional prognostic value over the MPI. On the other hand, the additional value of the DI over tumor size and nodal status was much more impressive (MC = 41.0 and 40.7), although it did not reach the prognostic significance of the MPI. Prediction of disease outcome with a linear combination of quantitative microscopical parameters of the primary tumor alone [MAI (mitotic activity index), DI and mean nuclear area] was very accurate, even without considering lymph-node status (MC 30.8, p less than 0.0005). Grade had no additional value to the MPI at all (p = 0.76). This could be especially important for lymph-node-negative patients in whom the prognostic value of the MPI and the MAI are confirmed.

  12. Effect of lecithin nanoliposome or soybean lecithin supplemented by pomegranate extract on post-thaw flow cytometric, microscopic and oxidative parameters in ram semen.

    Science.gov (United States)

    Mehdipour, Mahdieh; Daghigh Kia, Hossein; Nazari, Maryam; Najafi, Abouzar

    2017-10-01

    This investigation was carried out to study the effect of soybean lecithin 1.5% (wt/vol) (0, 2.5, 5 and 7.5 mg l -1 pomegranate extract (PE)) or PE-loaded lecithin nanoliposome (0, 2.5, 5 and 7.5 mg l -1 ) to Tris-based extender. Sperm motility (CASA), viability, membrane integrity (HOS test), abnormalities, mitochondrial activity, apoptosis status, lipid peroxidation, total antioxidant capacity (TAC)) and antioxidant activities (GPX, SOD) were investigated following freeze-thawing. No significant differences were detected in motility parameters, viability, membrane integrity, and mitochondria activity after thawing sperm between soybean lecithin and lecithin nanoliposomes. It was shown that PE5 significantly improved sperm total and progressive motility, membrane integrity, viability, mitochondria activity, TAC and reduced lipid peroxidation (malondialdehyde concentration). Moreover, the percentage of apoptotic sperm in PE5 extenders was significantly the lowest among other treatments. Sperm abnormalities, SOD and GPX were not affected by the antioxidant supplements. For apoptotic status, no differences were observed between soybean lecithin and lecithin nanoliposome. We showed that lecithin nanoliposome extender can be a beneficial alternative extender to protect ram sperm during cryopreservation without any adverse effects. It was also observed that regarding pomegranate concentration, PE5 can improve the quality of ram semen after thawing. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Flow cytometric detection of growth factor receptors in autografts and analysis of growth factor concentrations in autologous stem cell transplantation: possible significance for platelet recovery

    DEFF Research Database (Denmark)

    Schiødt, I; Jensen, Charlotte Harken; Kjaersgaard, E

    2000-01-01

    In order to improve prediction of hematopoietic recovery, we conducted a pilot study, analyzing the significance of growth factor receptor expression in autografts as well as endogenous growth factor levels in blood before, during and after stem cell transplantation. Three early acting (stem cell...

  14. Genotype-dependent variability in flow cytometric evaluation of reduced nicotinamide adenine dinucleotide phosphate oxidase function in patients with chronic granulomatous disease.

    Science.gov (United States)

    Vowells, S J; Fleisher, T A; Sekhsaria, S; Alling, D W; Maguire, T E; Malech, H L

    1996-01-01

    We studied phagocyte reduced nicotinamide adenine dinucleotide phosphate function to evaluate production of reactive oxygen species in both X-linked and autosomal forms of chronic granulomatous disease. We found a consistent and significant difference between the activated granulocyte response of the X-linked (gp91-phagocyte oxidase) form of chronic granulomatous disease (n = 18) and that of the most common autosomal recessive (p47-phagocyte oxidase) form of the disease (n = 17). The data indicate that mutations in the p47-phagocyte oxidase component of the reduced nicotinamide adenine dinucleotide phosphate oxidase component do not completely prevent oxidation despite severe defects in superoxide generation.

  15. Flow Rounding

    OpenAIRE

    Kang, Donggu; Payor, James

    2015-01-01

    We consider flow rounding: finding an integral flow from a fractional flow. Costed flow rounding asks that we find an integral flow with no worse cost. Randomized flow rounding requires we randomly find an integral flow such that the expected flow along each edge matches the fractional flow. Both problems are reduced to cycle canceling, for which we develop an $O(m \\log(n^2/m))$ algorithm.

  16. Scalable clustering algorithms for continuous environmental flow cytometry.

    Science.gov (United States)

    Hyrkas, Jeremy; Clayton, Sophie; Ribalet, Francois; Halperin, Daniel; Armbrust, E Virginia; Howe, Bill

    2016-02-01

    Recent technological innovations in flow cytometry now allow oceanographers to collect high-frequency flow cytometry data from particles in aquatic environments on a scale far surpassing conventional flow cytometers. The SeaFlow cytometer continuously profiles microbial phytoplankton populations across thousands of kilometers of the surface ocean. The data streams produced by instruments such as SeaFlow challenge the traditional sample-by-sample approach in cytometric analysis and highlight the need for scalable clustering algorithms to extract population information from these large-scale, high-frequency flow cytometers. We explore how available algorithms commonly used for medical applications perform at classification of such a large-scale, environmental flow cytometry data. We apply large-scale Gaussian mixture models to massive datasets using Hadoop. This approach outperforms current state-of-the-art cytometry classification algorithms in accuracy and can be coupled with manual or automatic partitioning of data into homogeneous sections for further classification gains. We propose the Gaussian mixture model with partitioning approach for classification of large-scale, high-frequency flow cytometry data. Source code available for download at https://github.com/jhyrkas/seaflow_cluster, implemented in Java for use with Hadoop. hyrkas@cs.washington.edu Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  17. Standardization of 8-color flow cytometry across different flow cytometer instruments: A feasibility study in clinical laboratories in Switzerland.

    Science.gov (United States)

    Glier, Hana; Heijnen, Ingmar; Hauwel, Mathieu; Dirks, Jan; Quarroz, Stéphane; Lehmann, Thomas; Rovo, Alicia; Arn, Kornelius; Matthes, Thomas; Hogan, Cassandra; Keller, Peter; Dudkiewicz, Ewa; Stüssi, Georg; Fernandez, Paula

    2017-07-29

    The EuroFlow Consortium developed a fully standardized flow cytometric approach from instrument settings, through antibody panel, reagents and sample preparation protocols, to data acquisition and analysis. The Swiss Cytometry Society (SCS) promoted a study to evaluate the feasibility of using such standardized measurements of 8-color data across two different flow cytometry platforms - Becton Dickinson (BD) FACSCanto II and Beckman Coulter (BC) Navios, aiming at increasing reproducibility and inter-laboratory comparability of immunophenotypic data in clinical laboratories in Switzerland. The study was performed in two phases, i.e. a learning phase (round 1) and an analytical phase (rounds 2 and 3) consisting of a total of three rounds. Overall, 10 laboratories using BD FACSCanto II (n=6) or BC Navios (n=4) flow cytometers participated. Each laboratory measured peripheral blood samples from healthy donors stained with a uniform antibody panel of reagents - EuroFlow Lymphoid Screening Tube (LST) - applying the EuroFlow standardized protocols for instrument setup and sample preparation (www.EuroFlow.org). All data files were analyzed centrally and median fluorescence intensity (MedFI) values for individual markers on defined lymphocyte subsets were recorded; variability from reference MedFI values was assessed using performance scores. Data troubleshooting and discussion of the results with the participants followed after each round at SCS meetings. The results of the learning phase demonstrated that standardized instrument setup and data acquisition are feasible in routine clinical laboratories without previous experience with EuroFlow. During the analytical phase, highly comparable data were obtained at the different laboratories using either BD FACSCanto II or BC Navios. The coefficient of variation of MedFI for 7 of 11 markers performed repeatedly below 30%. In the last study round, 89% of participants scored over 90% MedFI values within the acceptance criteria

  18. DNA flow cytometry of human spermatozoa: consistent stoichiometric staining of sperm DNA using a novel decondensation protocol.

    Science.gov (United States)

    Kovács, Tamás; Békési, Gyöngyi; Fábián, Akos; Rákosy, Zsuzsa; Horváth, Gábor; Mátyus, László; Balázs, Margit; Jenei, Attila

    2008-10-01

    Rapid flow cytometric measurement of the frequency of aneuploid human sperms is in increasing demand but development of an exploitable method is hindered by difficulties of stoichiometric staining of sperm DNA. An aggressive decondensation protocol is needed after which cell integrity still remains intact. We used flow cytometry to examine the effect of lithium diiodosalicylate (LIS, chaotropic agent) on fluorescence intensity of propidium iodide-treated human spermatozoa from 10 normozoospermic men. When flow cytometric identification of diploid spermatozoa was achieved, validation was performed after sorting by three-color FISH. In contrast with the extremely variable histograms of nondecondensed sperms, consistent identification of haploid and diploid spermatozoa was possible if samples were decondensed with LIS prior to flow cytometry. A 76-fold enrichment of diploid sperms was observed in the sorted fractions by FISH. A significant correlation was found between the proportion of sorted cells and of diploid sperms by FISH. Application of LIS during the preparation of sperm for flow cytometry appears to ensure the stoichiometric staining of sperm DNA, making quantification of aneuploid sperm percentage possible. To our knowledge this is the first report in terms of separating spermatozoa with confirmedly abnormal chromosomal content. High correlation between the proportion of cells identified as having double DNA content by flow cytometry and diploid sperm by FISH allows rapid calculation of diploidy rate. Copyright 2008 International Society for Advancement of Cytometry.

  19. Accurately estimating breast cancer survival in Spain: cross-matching local cancer registries with the National Death Index Estimación precisa de la supervivencia de cáncer de mama en España: emparejamiento de los registros locales de cáncer con el Índice Nacional de Defunciones

    Directory of Open Access Journals (Sweden)

    M. Carmen Martos

    2009-07-01

    Full Text Available OBJECTIVES: To assess the impact of using data from the National Death Index (NDI of Spain to estimate breast cancer survival rates among residents of Girona and Zaragoza diagnosed in 1995-1999. METHODS: This was an observational, longitudinal epidemiologic study, using two population- based cancer registries. Data collected were of female residents of Girona or Zaragoza who had been diagnosed with breast cancer in 1995-1999. Observed and relative 5-year survival rates were estimated, first using the information available from the Girona and Zaragoza cancer registries, and then with the inclusion of NDI data. The 5-year relative survival rate and corresponding 95% Confidence Intervals were estimated using the Hakulinen method. The Kaplan-Maier method and Log Rank test were used to compare survival curves. RESULTS: No statistically significant difference in survival curves was observed in Girona for the data obtained before and after cross-matching with the NDI. However, there was a significant difference in Zaragoza. A comparison of the relative survival rates of each of the two registries before NDI cross-matching showed differences of 3.9% (5-year and 16.1% (10-year between the two, whereas after the cross-match, the difference was only 0.5% (5-year and 1.2% (10-year. CONCLUSIONS: In Spain it is imperative that there be systematic use of NDI data to supplement cancer registries, so that comparisons of relative survival rates between registries can be improved.OBJETIVO: Evaluar el efecto de utilizar los datos del Índice Nacional de Defunciones (IND de España para estimar las tasas de supervivencia de cáncer de mama en las mujeres residentes en Girona y Zaragoza que recibieron el diagnóstico de cáncer de mama en 1995-1999. MÉTODOS: Se realizó un estudio epidemiológico observacional y longitudinal basado en el empleo de los registros de cáncer de mujeres residentes en Girona y Zaragoza que habían recibido el diagnóstico de cáncer de

  20. Reticulocyte maturity index by flow cytometry: its applicability in radioinduced bone marrow aplasia

    International Nuclear Information System (INIS)

    Dubner, D.; Gisone, P.; Perez, M.R.

    1995-01-01

    Flow cytometric reticulocyte quantification was assayed in ten patients undergoing bone marrow transplantation (BMT) with previous conditioning chemotherapy and total body irradiation (TBI). A reticulocyte maturity index (RMI) was determined taking into account the RNA content. With de aim of testing the utility of RMI as an early predictor of functional recovery in marrow aplasia, other haematological indicators as neutrophils count were comparatively evaluated. Mean time elapsed between BMT and engraftment evidence by RMI was 17,6 days. In six patients the RMI was the earliest indicator of functional recovery. The applicability of this assay in the following of radioinduced bone marrow aplasia is discussed. (author). 4 refs., 4 figs., 2 tabs

  1. Use of flow cytometry for the possible identification of radio-induced changes in DNA of animal cells

    International Nuclear Information System (INIS)

    Spano, M.; Leonardi, M.; Cordelli, E.

    1991-01-01

    Since DNA is the main cellular target of ionizing irradiation, methods fit for analyzing DNA alterations should be able to discern irradiated versus control cells. Flow cytometry allows the rapid measurement of DNA content of single chromosomes or cell nuclei at very high resolution on a statistically significant sample. Alterations of chromatine structure can also be analyzed by flow cytometry. Briefly, evaluation of in situ DNA resistance to denaturation can be evaluated by flow cytometric analysis of different staining pattern of single versus double strange regions of DNA. In the present work both approaches were used with the aim to recognize cells derived from an irradiated sample of breast chicken. Although flow cytometry has been demonstrated to be a useful tool to detect DNA alterations and has been widely used to detect damages on DNA induced by several physical and chemical agents, it was unable to detect clastogenic effects induced by electrons on DNA of chicken breast cells. Heavily irradiated nuclei, even if challenged by denaturating treatments that partially collapse chromatine organization, do not present differences from non irradiated samples after flow cytometric DNA content measurement. (16 refs)

  2. Cytometric comparisons between circulating tumor cells from prostate cancer patients and the prostate-tumor-derived LNCaP cell line

    Science.gov (United States)

    Lazar, Daniel C.; Cho, Edward H.; Luttgen, Madelyn S.; Metzner, Thomas J.; Loressa Uson, Maria; Torrey, Melissa; Gross, Mitchell E.; Kuhn, Peter

    2012-02-01

    Many important experiments in cancer research are initiated with cell line data analysis due to the ease of accessibility and utilization. Recently, the ability to capture and characterize circulating tumor cells (CTCs) has become more prevalent in the research setting. This ability to detect, isolate and analyze CTCs allows us to directly compare specific protein expression levels found in patient CTCs to cell lines. In this study, we use immunocytochemistry to compare the protein expression levels of total cytokeratin (CK) and androgen receptor (AR) in CTCs and cell lines from patients with prostate cancer to determine what translational insights might be gained through the use of cell line data. A non-enrichment CTC detection assay enables us to compare cytometric features and relative expression levels of CK and AR by indirect immunofluorescence from prostate cancer patients against the prostate cancer cell line LNCaP. We measured physical characteristics of these two groups and observed significant differences in cell size, fluorescence intensity and nuclear to cytoplasmic ratio. We hope that these experiments will initiate a foundation to allow cell line data to be compared against characteristics of primary cells from patients.

  3. A 'fragile cell' sub-population revealed during cytometric assessment of Saccharomyces cerevisiae viability in lipid-limited alcoholic fermentation.

    Science.gov (United States)

    Delobel, P; Pradal, M; Blondin, B; Tesniere, C

    2012-11-01

    To show that in anaerobic fermentation with limiting lipid nutrients, cell preparation impacts the viability assessment of yeast cells, and to identify the factors involved. Saccharomyces cerevisiae viability was determined using propidium iodide staining and the flow cytometry. Analyses identified intact cells, dead cells and, under certain conditions, the presence of a third subpopulation of apparently damaged cells. This intermediate population could account for up to 40% of the entire cell population. We describe, analyse and discuss the effects of different solutions for cell resuspension on the respective proportion of these three populations, in particular that of the intermediate population. We show that this intermediate cell population forms in the absence of Ca(2+)/Mg(2+). Cell preparation significantly impacts population viability assessment by FCM. The intermediate population, revealed under certain conditions, could be renamed as 'fragile cells'. For these cells, Ca(2+) and Mg(2+) reduce cell membrane permeability to PI. This is the first study that analyses and discusses the factors influencing the formation of an intermediate population when studying viability in yeast alcoholic fermentation. With a wider application in biological research, this study provides important support to the relatively new questioning of propidium iodide staining as a universal cell death indicator. © 2012 The Authors. Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.

  4. Flow cytometry-based DNA hybridization and polymorphism analysis

    Energy Technology Data Exchange (ETDEWEB)

    Cai, H.; Kommander, K.; White, P.S.; Nolan, J.P.

    1998-07-01

    Functional analysis of the humane genome, including the quantification of differential gene expression and the identification of polymorphic sites and disease genes, is an important element of the Human Genome Project. Current methods of analysis are mainly gel-based assays that are not well-suited to rapid genome-scale analyses. To analyze DNA sequence on a large scale, robust and high throughput assays are needed. The authors are developing a suite of microsphere-based approaches employing fluorescence detection to screen and analyze genomic sequence. The approaches include competitive DNA hybridization to measure DNA or RNA targets in unknown samples, and oligo ligation or extension assays to analyze single-nucleotide polymorphisms. Apart from the advances of sensitivity, simplicity, and low sample consumption, these flow cytometric approaches have the potential for high throughput multiplexed analysis using multicolored microspheres and automated sample handling.

  5. Flow visualization

    CERN Document Server

    Merzkirch, Wolfgang

    1974-01-01

    Flow Visualization describes the most widely used methods for visualizing flows. Flow visualization evaluates certain properties of a flow field directly accessible to visual perception. Organized into five chapters, this book first presents the methods that create a visible flow pattern that could be investigated by visual inspection, such as simple dye and density-sensitive visualization methods. It then deals with the application of electron beams and streaming birefringence. Optical methods for compressible flows, hydraulic analogy, and high-speed photography are discussed in other cha

  6. Coexpression of CD69 and HLADR activation markers on synovial fluid T lymphocytes of patients affected by rheumatoid arthritis: a three-colour cytometric analysis.

    Science.gov (United States)

    AFELTRA, ANTONELLA; GALEAZZI, MAURO; DOMENICO SEBASTIANI, GIAN; MARIA FERRI, GIOVANNI; CACCAVO, DOMENICO; ASSUNTA ADDESSI, MARIA; MARCOLONGO, ROBERTO; BONOMO, LORENZO

    1997-01-01

    The aim of the present study was to evaluate the coexpression of very early (CD69), early (CD25) and late (HLADR) antigens and to analyse the mean fluorescence intensity (MFI) of such activation markers on synovial fluid (SF) and peripheral blood (PB) lymphocytes of patients affected by rheumatoid arthritis (RA) and other types of chronic synovitis (OCS). A three colour cytometric analysis was performed using a peridinin chlorophyll protein conjugated anti-CD3 antibody in combination with fluorescein isothiocyanate or phycoerythrin labelled anti-CD69, anti-HLADR, anti-CD25 monoclonal antibodies (mAbs). A T cell gating method was utilized, so that three sets of bivariant dot plot quadrant displays were obtained (CD69/HLADR, CD69/CD25, CD25/HLADR). A large percentage of SF T lymphocytes in RA showed the coexpression of very early and late activation antigens (CD3 + CD69 + HLADR +), whereas CD3 + CD69 + CD25 + bearing cells and CD3 + CD25 + HLADR + lymphocytes were only a small percentage. Similar results were obtained in patients with OCS, although to a lesser extent. No statistically significant differences in MFI of CD69 and HLADR positive SF T cells between RA and OCS were observed. The CD69 + CD25-HLADR + T cell subset is the most commonly represented in the synovial environment, among those we have evaluated; this phenotype may be characteristic of chronic inflammatory arthritis. PMID:9462230

  7. Multicolor Flow Cytometry for the Diagnosis of Primary Immunodeficiency Diseases.

    Science.gov (United States)

    Takashima, Takehiro; Okamura, Miko; Yeh, Tzu-Wen; Okano, Tsubasa; Yamashita, Motoi; Tanaka, Keisuke; Hoshino, Akihiro; Mitsuiki, Noriko; Takagi, Masatoshi; Ishii, Eiichi; Imai, Kohsuke; Kanegane, Hirokazu; Morio, Tomohiro

    2017-07-01

    Primary immunodeficiency diseases (PIDDs) are rare inherited diseases that impair the human immune system. We established a multicolor flow cytometric assay to comprehensively evaluate the immune status and immunological characteristics of patients with PIDDs. Fifty-nine normal controls and 75 patients with PIDDs, including X-linked severe combined immunodeficiency (X-SCID), X-linked agammaglobulinemia (XLA), X-linked hyper IgM syndrome (X-HIGM), ataxia telangiectasia (AT), Wiskott-Aldrich syndrome (WAS), hyper IgE syndrome (HIES), and chronic mucocutaneous candidiasis disease (CMCD), were enrolled in this study. Immunophenotyes were evaluated by multicolor flow cytometry using seven different panels that allowed the detection of major leukocyte populations in peripheral blood. Multicolor flow cytometry revealed distinct leukocyte populations and immunological features of patients with X-SCID, XLA, X-HIGM, AT, WAS, HIES, and CMCD. Immunophenotyping by multicolor flow cytometry is useful to evaluate immune status and contributes to the diagnosis and management of patients with PIDDs.

  8. Flow regimes

    International Nuclear Information System (INIS)

    Liles, D.R.

    1982-01-01

    Internal boundaries in multiphase flow greatly complicate fluid-dynamic and heat-transfer descriptions. Different flow regimes or topological configurations can have radically dissimilar interfacial and wall mass, momentum, and energy exchanges. To model the flow dynamics properly requires estimates of these rates. In this paper the common flow regimes for gas-liquid systems are defined and the techniques used to estimate the extent of a particular regime are described. Also, the current computer-code procedures are delineated and introduce a potentially better method is introduced

  9. Lipid nanoparticles assessment by flow cytometry.

    Science.gov (United States)

    Bryła, Anna; Juzwa, Wojciech; Weiss, Marek; Lewandowicz, Grażyna

    2017-03-30

    Liposomes are promising carriers for drugs and bioactive compounds. Size and structure are their crucial parameters. Thus, it is essential to assess individual vesicles as prepared. Currently available techniques fail to measure liposome's size and structure simultaneously, with a high throughput. To solve this problem, we have developed a novel, flow cytometric method quantifying liposomes. Firstly, the following fluorescent staining combinations were tested: DiD/TO, Rh123/DiD, Syto9/DiD. Further, chosen fluorochromes were used to compare three populations of vesicles: raw (R), obtained by thin film hydration and extruded ones (populations E10 and E21). Dynamic light scattering (DLS) was used for determination of average diameter and size distribution of nanocarriers. Structural differences between the raw and the extruded liposomes, as well as additional information concerning vesicles size were acquired employing atomic force microscopy (AFM). DLS analysis indicated that, three distinct populations of vesicles were obtained. Liposomes were characterized by mean diameter of 323nm, 220nm and 170nm for population R, E10 and E21 respectively. All the populations were stable and revealed zeta potential of -29mV. AFM confirmed that raw and extruded liposomes were differed in structure. DiD/TO was the optimal fluorochrome combination that enabled to resolve distinctly the sub-populations of liposomes. Results obtained by flow cytometry were in a good agreement with those from DLS and AFM. It was proved that, flow cytometry, when proper fluorescent dyes are used, is an adequate method for liposomes assessment. The proposed method enables fast and reliable analysis of liposomes in their native environment. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Flow cytometry detection of planktonic cells with polycyclic aromatic hydrocarbons sorbed to cell surfaces

    KAUST Repository

    Cerezo, Maria I.

    2017-02-17

    Polycyclic aromatic hydrocarbons are very important components of oil pollution. These pollutants tend to sorb to cell surfaces, exerting toxic effects on organisms. Our study developed a flow cytometric method for the detection of PAHs sorbed to phytoplankton by exploiting their spectral characteristics. We discriminated between cells with PAHs from cells free of PAHs. Clear discrimination was observed with flow cytometer provided with 375 or 405nm lasers in addition to the standard 488nm laser necessary to identify phytoplankton. Using this method, we measured the relationship between the percentages of phytoplankton organisms with PAHs, with the decrease in the growth rate. Moreover, the development of this method could be extended to facilitate the study of PAHs impact on cell cultures from a large variety of organisms.

  11. Improved and Reproducible Flow Cytometry Methodology for Nuclei Isolation from Single Root Meristem

    Directory of Open Access Journals (Sweden)

    Thaís Cristina Ribeiro Silva

    2010-01-01

    Full Text Available Root meristems have increasingly been target of cell cycle studies by flow cytometric DNA content quantification. Moreover, roots can be an alternative source of nuclear suspension when leaves become unfeasible and for chromosome analysis and sorting. In the present paper, a protocol for intact nuclei isolation from a single root meristem was developed. This proceeding was based on excision of the meristematic region using a prototypical slide, followed by short enzymatic digestion and mechanical isolation of nuclei during homogenization with a hand mixer. Such parameters were optimized for reaching better results. Satisfactory nuclei amounts were extracted and analyzed by flow cytometry, producing histograms with reduced background noise and CVs between 3.2 and 4.1%. This improved and reproducible technique was shown to be rapid, inexpensive, and simple for nuclear extraction from a single root tip, and can be adapted for other plants and purposes.

  12. Prognostic relevance of DNA flow cytometry in breast cancer revisited: The 25-year experience of the Portuguese Institute of Oncology of Lisbon

    Science.gov (United States)

    Pinto, António E.; Pereira, Teresa; Silva, Giovani L.; André, Saudade

    2017-01-01

    The potential prognostic significance of DNA flow cytometric measurements (DNA ploidy and S-phase fraction) in breast cancer remains in dispute. Inconclusive data, primarily due to the lack of consistent standardization and quality control programs, have limited its translation into clinical practice. The aim of the present review, based on the 25-year experience of the Portuguese Institute of Oncology of Lisbon, is to assess the clinical relevance and application of DNA flow cytometry for the prognosis of breast cancer. Overall, data from Portuguese Institute of Oncology of Lisbon indicate that DNA flow cytometry provides significant prognostic information that is biologically relevant and clinically useful for the management of patients with breast cancer. Furthermore, this data has demonstrated the independent value of DNA aneuploidy as a prognostic indicator of poor clinical outcome in various subgroups of patients with early or locally advanced breast cancer at short- and long-term follow-up. Notably, aneuploidy identifies subsets of patients with grade (G)1 or G2 tumours who exhibit a poor clinical outcome. These patients may benefit from adjuvant chemotherapy, particularly those with luminal A and luminal B/human epidermal growth factor-2-negative endocrine-responsive breast cancer. In conclusion, data from Portuguese Institute of Oncology of Lisbon reinforces the clinical importance and utility of DNA flow cytometric analysis, particularly DNA ploidy, in the prognostic assessment and therapeutic planning for patients with breast cancer. PMID:28454358

  13. Ploidy level variability in South American fescues (festuca L., poaceae): use of flow cytometry in up to 5 1/2-year-old caryopses and herbarium specimens.

    Science.gov (United States)

    Smarda, P; Stancík, D

    2006-01-01

    Ploidy levels and chromosome numbers for 24 species of Festuca L. from 29 sites in Bolivia, Colombia, Ecuador and Venezuela are given. The ploidy level of 22 species is reported for the first time. A higher proportion of tetraploids in northern South America and the high frequency of polyploids in the whole continent are documented. In combination with chromosome counts, ploidy level was determined using flow cytometry in 4- to 5 1/2 -year-old herbarium specimens and mature caryopses. Flow cytometric determination from seeds was more reliable than determination from herbarium specimens. In herbarium specimens, the youngest, fresh green leaves, still hidden in sheaths, seem to be most suitable for cytometric determination. In old, brownish leaves, or poorly preserved herbarium specimens, the degradation of DNA signal in flow histograms was documented. DNA content measured in seeds was always higher than that measured in herbarium specimens, which may be caused by the presence of different cytosolic compounds. Differences of about 15% in relative DNA content of F. sodiroana and F. vaginalis was found in simultaneous measurements in seeds.

  14. Measurement of separase proteolytic activity in single living cells by a fluorogenic flow cytometry assay.

    Directory of Open Access Journals (Sweden)

    Wiltrud Haaß

    Full Text Available ESPL1/Separase, an endopeptidase, is required for centrosome duplication and separation of sister-chromatides in anaphase of mitosis. Overexpression and deregulated proteolytic activity of Separase as frequently observed in human cancers is associated with the occurrence of supernumerary centrosomes, chromosomal missegregation and aneuploidy. Recently, we have hypothesized that increased Separase proteolytic activity in a small subpopulation of tumor cells may serve as driver of tumor heterogeneity and clonal evolution in chronic myeloid leukemia (CML. Currently, there is no quantitative assay to measure Separase activity levels in single cells. Therefore, we have designed a flow cytometry-based assay that utilizes a Cy5- and rhodamine 110 (Rh110-biconjugated Rad21 cleavage site peptide ([Cy5-D-R-E-I-M-R]2-Rh110 as smart probe and intracellular substrate for detection of Separase enzyme activity in living cells. As measured by Cy5 fluorescence the cellular uptake of the fluorogenic peptide was fast and reached saturation after 210 min of incubation in human histiocytic lymphoma U937 cells. Separase activity was recorded as the intensity of Rh110 fluorescence released after intracellular peptide cleavage providing a linear signal gain within a 90-180 min time slot. Compared to conventional cell extract-based methods the flow cytometric assay delivers equivalent results but is more reliable, bypasses the problem of vague loading controls and unspecific proteolysis associated with whole cell extracts. Especially suited for the investigaton of blood- and bone marrow-derived hematopoietic cells the flow cytometric Separase assay allows generation of Separase activity profiles that tell about the number of Separase positive cells within a sample i.e. cells that currently progress through mitosis and about the range of intercellular variation in Separase activity levels within a cell population. The assay was used to quantify Separase proteolytic

  15. Flow assurance

    Energy Technology Data Exchange (ETDEWEB)

    Mullins, O.C.; Dong, C. [Schlumberger-Doll Research Center, Cambridge, MA (United States); Elshahawi, H. [Shell Exploration and Production Company, The Hague (Netherlands)

    2008-07-01

    This study emphasized the need for considering flow assurance for producing oil and gas, particularly in high cost areas such as deepwater. Phase behaviour studies, sticking propensities, and interfacial interactions have been investigated in many laboratory studies using asphaltenes, wax, hydrates, organic and inorganic scale, and even diamondoids. However, the spatial variation of reservoir fluids has received little attention, despite the fact that it is one of the most important factors affecting flow assurance. This issue was difficult to address in a systematic way in the past because of cost constraints. Today, reservoir fluid variation and flow assurance can be considered at the outset of a project given the technological advances in downhole fluid analysis. This study described the origins of reservoir fluid compositional variations and the controversies surrounding them. It also described the indispensable chemical analytical technology. The impact of these reservoir fluid compositional variations on flow assurance considerations was also discussed. A methodology that accounts for these variations at the outset in flow assurance evaluation was also presented.

  16. Application of bead array technology to simultaneous detection of human leucocyte antigen and human platelet antigen antibodies.

    Science.gov (United States)

    Fujiwara, K; Shimano, K; Tanaka, H; Sekine, M; Kashiwase, K; Uchikawa, M; Satake, M; Nakajima, K

    2009-04-01

    Detection of antibodies against human leucocyte antigens (HLA) and human platelet antigens (HPA) is crucial for patients refractory to platelet transfusion therapy. However, a reliable and high-throughput method for HLA cross-matching and detecting HPA antibodies has not yet been described. Immunocomplex capture fluorescence analysis (ICFA) was developed for high-throughput, simultaneous detection of HLA and HPA antibodies. Microarray beads were separately coupled with monoclonal antibodies specific for CD36, CD41, CD42b, CD49b, CD61 and HLA class I antigens. Platelets reacting with patient serum were lysed and the lysates were incubated with the bead mixture to specifically capture antigen-antibody complexes via the epitopes on platelet glycoproteins or HLA antigens. The beads capturing immunocomplexes were then subjected to flow cytometric analysis. Immunocomplex capture fluorescence analysis was validated using 50 serum samples containing HLA antibodies and 20 serum samples containing HPA antibodies. The method enabled the detection of all the HLA antibodies with a sensitivity comparable to that of the purified HLA antigen-coated pooled-bead assay (FlowPRA, One Lambda, Canoga Park, CA, USA). The method also enabled the detection of all the HPA antibodies with a sensitivity higher than that of the mixed passive haemagglutination. In this study, we developed a rapid, simple and reliable method for the simultaneous analysis of HLA and HPA antibodies. ICFA can also be used as an alternative to the lymphocyte cytotoxicity test for HLA cross-matching.

  17. Functional characterization of neotropical snakes peripheral blood leukocytes subsets: Linking flow cytometry cell features, microscopy images and serum corticosterone levels.

    Science.gov (United States)

    de Carvalho, Marcelo Pires Nogueira; Queiroz-Hazarbassanov, Nicolle Gilda Teixeira; de Oliveira Massoco, Cristina; Sant'Anna, Sávio Stefanini; Lourenço, Mariana Mathias; Levin, Gabriel; Sogayar, Mari Cleide; Grego, Kathleen Fernandes; Catão-Dias, José Luiz

    2017-09-01

    Reptiles are the unique ectothermic amniotes, providing the key link between ectothermic anamniotes fish and amphibians, and endothermic birds and mammals; becoming an important group to study with the aim of providing significant knowledge into the evolutionary history of vertebrate immunity. Classification systems for reptiles' leukocytes have been described by their appearance rather than function, being still inconsistent. With the advent of modern techniques and the establishment of analytical protocols for snakes' blood by flow cytometry, we bring a qualitative and quantitative assessment of innate activities presented by snakes' peripheral blood leukocytes, thereby linking flow cytometric features with fluorescent and light microscopy images. Moreover, since corticosterone is an important immunomodulator in reptiles, hormone levels of all blood samples were measured. We provide novel and additional information which should contribute to better understanding of the development of the immune system of reptiles and vertebrates. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Granular flow

    DEFF Research Database (Denmark)

    Mitarai, Namiko; Nakanishi, Hiizu

    2012-01-01

    Granular material is a collection of macroscopic particles that are visible with naked eyes. The non-equilibrium nature of the granular materials makes their rheology quite different from that of molecular systems. In this minireview, we present the unique features of granular materials focusing ...... on the shear flow of dry granular materials and granule-liquid mixture....

  19. Lubrication Flows.

    Science.gov (United States)

    Papanastasiou, Tasos C.

    1989-01-01

    Discusses fluid mechanics for undergraduates including the differential Navier-Stokes equations, dimensional analysis and simplified dimensionless numbers, control volume principles, the Reynolds lubrication equation for confined and free surface flows, capillary pressure, and simplified perturbation techniques. Provides a vertical dip coating…

  20. Sex-sorting sperm using flow cytometry/cell sorting.

    Science.gov (United States)

    Garner, Duane L; Evans, K Michael; Seidel, George E

    2013-01-01

    The sex of mammalian offspring can be predetermined by flow sorting relatively pure living populations of X- and Y-chromosome-bearing sperm. This method is based on precise staining of the DNA of sperm with the nucleic acid-specific fluorophore, Hoechst 33342, to differentiate between the subpopulations of X- and Y-sperm. The fluorescently stained sperm are then sex-sorted using a specialized high speed sorter, MoFlo(®) SX XDP, and collected into biologically supportive media prior to reconcentration and cryopreservation in numbers adequate for use with artificial insemination for some species or for in vitro fertilization. Sperm sorting can provide subpopulations of X- or Y-bearing bovine sperm at rates in the 8,000 sperm/s range while maintaining; a purity of 90% such that it has been applied to cattle on a commercial basis. The sex of offspring has been predetermined in a wide variety of mammalian species including cattle, swine, horses, sheep, goats, dogs, cats, deer, elk, dolphins, water buffalo as well as in humans using flow cytometric sorting of X- and Y-sperm.

  1. A Curvature Flow Unifying Symplectic Curvature Flow And Pluriclosed Flow

    OpenAIRE

    Dai, Song

    2013-01-01

    Streets and Tian introduced pluriclosed flow and symplectic curvature flow in recent years. Here we construct a curvature flow to unify these two flows. We show the short time existence of our flow and exhibit an obstruction to long time existence.

  2. Monitoring of Legionella pneumophila viability after chlorine dioxide treatment using flow cytometry.

    Science.gov (United States)

    Mustapha, Pascale; Epalle, Thibaut; Allegra, Séverine; Girardot, Françoise; Garraud, Olivier; Riffard, Serge

    2015-04-01

    The viability of three Legionella pneumophila strains was monitored after chlorine dioxide (ClO2) treatment using a flow cytometric assay. Suspensions of L. pneumophila cells were submitted to increasing concentrations of ClO2. Culturable cells were still detected when using 4 mg/L, but could no longer be detected after exposure to 6 mg/L of ClO2, although viable but not culturable (VBNC) cells were found after exposure to 4-5 mg/L of ClO2. When testing whether these VBNC were infective, two of the strains were resuscitated after co-culture with Acanthamoeba polyphaga, but neither of them could infect macrophage-like cells. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  3. Using flow cytometry for counting natural planktonic bacteria and understanding the structure of planktonic bacterial communities

    Directory of Open Access Journals (Sweden)

    Josep M. Gasol

    2000-06-01

    Full Text Available Flow cytometry is rapidly becoming a routine methodology in aquatic microbial ecology. The combination of simple to use bench-top flow cytometers and highly fluorescent nucleic acid stains allows fast and easy determination of microbe abundance in the plankton of lakes and oceans. The different dyes and protocols used to stain and count planktonic bacteria as well as the equipment in use are reviewed, with special attention to some of the problems encountered in daily routine practice such as fixation, staining and absolute counting. One of the main advantages of flow cytometry over epifluorescence microscopy is the ability to obtain cell-specific measurements in large numbers of cells with limited effort. We discuss how this characteristic has been used for differentiating photosynthetic from non-photosynthetic prokaryotes, for measuring bacterial cell size and nucleic acid content, and for estimating the relative activity and physiological state of each cell. We also describe how some of the flow cytometrically obtained data can be used to characterize the role of microbes on carbon cycling in the aquatic environment and we prospect the likely avenues of progress in the study of planktonic prokaryotes through the use of flow cytometry.

  4. Flow Game

    DEFF Research Database (Denmark)

    Olsen, Jesper Lind

    2003-01-01

    Flow Game er et dialogspil, der kan bruges som ledelsesværktøj, ledertræning, samtaletræning, coachingtræning og ideudvikling m.m. Gennem dilemmakort provokeres en dialog og teori-U inspireret afklaring- og udviklingsproces, hvor der enten arbejdes på en gruppes eller et individs vision/innovatio......Flow Game er et dialogspil, der kan bruges som ledelsesværktøj, ledertræning, samtaletræning, coachingtræning og ideudvikling m.m. Gennem dilemmakort provokeres en dialog og teori-U inspireret afklaring- og udviklingsproces, hvor der enten arbejdes på en gruppes eller et individs vision...

  5. Media Flow

    DEFF Research Database (Denmark)

    Kabel, Lars

    2016-01-01

    News and other kinds of journalistic stories, 16-17 hours a day, all year round, on all platforms, also the moderated social media. The key research thesis behind this article is that the continuous and speedy stream of news stories and media content now is becoming the centre of the production...... processes and the value creation in converged multimedia newsrooms. The article identify new methods and discuss editorial challenges in handling media flow....

  6. Network Flows

    Science.gov (United States)

    1988-12-01

    n Vlog n ), which is a linear time algorithm for all but the sparsest classes of shortest path problems. 3.4. Label Correcting Algorithms Label...11962] (Programming , Games and Transportation Networks), Iri (1969] (Network Flows, Transportation and Scheduling), Hu [1969] (Integer Programming and...though the improvements are not as dramatic as they have been for E>inic’s and the FIFO preflow push algorithms. For example, the 0(nm + n^ Vlog U

  7. Filamentary Flows

    Science.gov (United States)

    Kirk, Helen

    2014-07-01

    Observations over the last few years, especially those from the Herschel Space Telescope have shown that filaments are intimately connected with dense cores. Both theory and observations are starting to suggest that filaments may provide an important reservoir of material both in the accretion of relatively isolated dense cores found along filaments, as well cluster-forming cores, which tend to be found at the junction of multiple filaments. I will review some of the recent evidence of gas flows in filaments, highlighting results from the young Serpens South cluster-forming region. I will also touch on recent analysis of numerical simulations which suggests a similar behavior.

  8. Detection of acute lymphoblastic leukemia involvement in pleural fluid in an adult patient with ataxia telangiectasia by flow cytometry method.

    Science.gov (United States)

    Keklik, Muzaffer; Koker, M Yavuz; Sivgin, Serdar; Camlica, Demet; Pala, Cigdem; Cetin, Mustafa; Kaynar, Leylagul; Unal, Ali; Eser, Bulent

    2014-09-01

    Ataxia-telangiectasia (AT) is a rare multisystem, neurodegenerative genetic disorder. Patients should be closely monitored due to risk of malignancy development. Due to its wide clinical heterogeneity, it often leads physicians to an inaccurate or missed diagnosis, and insight into this rare disease is important. Pediatric patients may develop lymphomas and acute lymphoblastic leukemia (ALL). However, in adults, there are limited numbers of reports regarding association of AT and ALL. Rarely, ALL cases may present with pleural fluid involvement. In our study, we presented an adult case with AT, in which ALL involvement was detected in pleural fluid by flow cytometry (FC). A 20-years old male presented to emergency department with fever, shortness of breath and cough, as he had been followed with a diagnosis of AT. The following findings were detected in laboratory tests: Hb, 11.5 g/L; WBC, 36 × 10(9)/L; Plt: 140 × 10(9)/L. Blastic cells were observed in peripheral blood smear. On chest radiography, pleural fluid appearance was observed. On thorax CT, pleural fluid was detected in both hemithorax. Cytoplasmic CD3(+) and superficial CD3 (+), CD45 (+), CD5 (+), CD7 (+) and CD38 (+) was found in the flow cytometric evaluation of peripheral blood. Superficial CD3 (+), CD2 (+), CD5 (+) and CD7 (+) were found in flow cytometric evaluation of pleural fluid. These findings were considered as consistent with pleural involvement of T-ALL. FC is a potentially useful diagnostic tool for clinical practice and it is a convenience method which has an important role in detection of ALL in patients with pleural fluid.

  9. Plant cytokinin analogues with inhibitory activity on cyclin-dependent kinases exert their antiproliferative effect through induction of apoptosis initiated by the mitochondrial pathway: Determination by a multiparametric flow cytometric analysis

    Czech Academy of Sciences Publication Activity Database

    Vermeulen, K.; Strnad, Miroslav; Havlíček, Libor; Onckelen, H.; Lenjou, M.; Nijs, G.; Bockstaele, D. R.; Berneman, Z. N.

    2002-01-01

    Roč. 30, č. 10 (2002), s. 1107-1114 ISSN 0301-472X R&D Projects: GA MŠk OC 844.10 Institutional research plan: CEZ:AV0Z5038910 Keywords : PLASMA MEMBRANE * PURINE ANALOGS * CELLS Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.366, year: 2002

  10. Dose-dependent effect of 17 beta-estradiol determined by growth curves and flow cytometric DNA analysis of a human breast carcinoma (T61) grown in nude mice

    DEFF Research Database (Denmark)

    Brünner, N; Spang-Thomsen, M; Vindeløv, L

    1985-01-01

    An estrogen and progesterone receptor-positive human breast carcinoma (T61) grown in nude mice was exposed to 1.0, 0.1, 0.01, and 0.001 mg 17 beta-estradiol. These doses resulted in serum peak concentrations (day 1) of estradiol ranging from 3.5 X 10(-8) to 6.9 X 10(-10) M. The effect of the trea......An estrogen and progesterone receptor-positive human breast carcinoma (T61) grown in nude mice was exposed to 1.0, 0.1, 0.01, and 0.001 mg 17 beta-estradiol. These doses resulted in serum peak concentrations (day 1) of estradiol ranging from 3.5 X 10(-8) to 6.9 X 10(-10) M. The effect...

  11. Dose-dependent effect of 17 beta-estradiol determined by growth curves and flow cytometric DNA analysis of a human breast carcinoma (T61) grown in nude mice

    DEFF Research Database (Denmark)

    Brünner, N; Spang-Thomsen, M; Vindeløv, L

    1985-01-01

    An estrogen and progesterone receptor-positive human breast carcinoma (T61) grown in nude mice was exposed to 1.0, 0.1, 0.01, and 0.001 mg 17 beta-estradiol. These doses resulted in serum peak concentrations (day 1) of estradiol ranging from 3.5 X 10(-8) to 6.9 X 10(-10) M. The effect...... fraction of polyploid cells. The results suggest that estradiol induces a dose-dependent cell killing effect in the T61 human breast carcinoma. The correlation between the treatment-induced growth delay and the effect on the cell cycle distribution indicates that the changes in the cell cycle...... are a reflection of the estradiol-induced cell destruction. Since no tumor growth stimulation could be observed even at very low serum estradiol concentrations, the T61 human breast carcinoma may represent a new aspect in the study of human breast cancer....

  12. Automated Blood Sample Preparation Unit (ABSPU) for Portable Microfluidic Flow Cytometry.

    Science.gov (United States)

    Chaturvedi, Akhil; Gorthi, Sai Siva

    2017-02-01

    Portable microfluidic diagnostic devices, including flow cytometers, are being developed for point-of-care settings, especially in conjunction with inexpensive imaging devices such as mobile phone cameras. However, two pervasive drawbacks of these have been the lack of automated sample preparation processes and cells settling out of sample suspensions, leading to inaccurate results. We report an automated blood sample preparation unit (ABSPU) to prevent blood samples from settling in a reservoir during loading of samples in flow cytometers. This apparatus automates the preanalytical steps of dilution and staining of blood cells prior to microfluidic loading. It employs an assembly with a miniature vibration motor to drive turbulence in a sample reservoir. To validate performance of this system, we present experimental evidence demonstrating prevention of blood cell settling, cell integrity, and staining of cells prior to flow cytometric analysis. This setup is further integrated with a microfluidic imaging flow cytometer to investigate cell count variability. With no need for prior sample preparation, a drop of whole blood can be directly introduced to the setup without premixing with buffers manually. Our results show that integration of this assembly with microfluidic analysis provides a competent automation tool for low-cost point-of-care blood-based diagnostics.

  13. Distinct neutrophil subpopulations phenotype by flow cytometry in myelodysplastic syndromes.

    Science.gov (United States)

    Vikentiou, Myrofora; Psarra, Katerina; Kapsimali, Violetta; Liapis, Konstantinos; Michael, Michalis; Tsionos, Konstantinos; Lianidou, Evi; Papasteriades, Chryssa

    2009-03-01

    The cardinal feature of myelodysplastic syndromes (MDS) is dysplasia involving one or more myeloid cell lineages. In the present study, we used 4-color flow cytometric analysis to investigate dysgranulopoiesis in bone marrow specimens from 65 patients with MDS. The antigen expression patterns of total neutrophil granulocytes (TNG) and of the two distinct neutrophil granulocytic subpopulations (NGSs), NGS-1 (dimmer CD45 expression) and NGS-2 (stronger CD45 expression) identified on the side scatter (SS) vs. CD45-intensity plot, were studied. The neutrophil granulocytes from patients with MDS showed characteristic antigen expression aberrancies which were more pronounced in NGS-2 subpopulation. Studying separately the NGS-2 subpopulation with the CD16/MPO/LF combination, the low CD16(+)/MPO(+) and low CD16(+)/LF(+) percentages seemed to discriminate between lower-risk and higher-risk patients with MDS in most occasions. Furthermore, a detailed assessment of the NGS-1 and NGS-2 immunophenotypic patterns revealed early dysplastic changes, not otherwise observed by standard TNG analysis, especially in cases of lower-risk MDS.

  14. Flow cytometry of DNA in mouse sperm and testis nuclei

    Energy Technology Data Exchange (ETDEWEB)

    Meistrich, M.L. (Univ. of Texas, Houston); Lake, S.; Steinmetz, L.L.; Gledhill, B.L.

    1978-01-01

    Mutations that occur in spermatogenic cells may be expressed as changes in DNA content, but developmentally-dependent alteration of its staining properties complicates the quantitation of DNA in individual germ cells. These alterations have been studied with flow cytometric techniques. Nuclei from mouse testis cells and sperm were stained by the acriflavine--Feulgen method. The fluorescence intensity frequency distribution of nuclei of testis cells was characterized by 2 major and 5 minor peaks. Nuclei sorted from the various peaks with a fluorescence-activated cell sorter were identified microscopically. These data were confirmed by generation of peaks with nuclei prepared from cell suspensions enriched in specific cell types. One of the major peaks corresponded to round spermatid nuclei. The other major peak, located at 0.6 of the fluorescence intensity of the round nuclei, corresponded to elongated spermatid nuclei. Purified nuclei of epididymal and vas deferens spermatozoa displayed asymmetric fluorescence distributions. A minor peak at 0.8 the intensity of the round spermatid nuclei was tentatively assigned to elongating spermatids. 2 of the minor peaks, located at 1.7 and 2.0 times the fluorescence intensity of the round nuclei, corresponded to clumps of 2 haploid and diploid nuclei.

  15. Multiplex flow cytometry barcoding and antibody arrays identify surface antigen profiles of primary and metastatic colon cancer cell lines.

    Directory of Open Access Journals (Sweden)

    Kumar Sukhdeo

    Full Text Available Colon cancer is a deadly disease affecting millions of people worldwide. Current treatment challenges include management of disease burden as well as improvements in detection and targeting of tumor cells. To identify disease state-specific surface antigen signatures, we combined fluorescent cell barcoding with high-throughput flow cytometric profiling of primary and metastatic colon cancer lines (SW480, SW620, and HCT116. Our multiplexed technique offers improvements over conventional methods by permitting the simultaneous and rapid screening of cancer cells with reduced effort and cost. The method uses a protein-level analysis with commercially available antibodies on live cells with intact epitopes to detect potential tumor-specific targets that can be further investigated for their clinical utility. Multiplexed antibody arrays can easily be applied to other tumor types or pathologies for discovery-based approaches to target identification.

  16. Quality assessment program for EuroFlow protocols: summary results of four-year (2010-2013) quality assurance rounds.

    Science.gov (United States)

    Kalina, Tomas; Flores-Montero, Juan; Lecrevisse, Quentin; Pedreira, Carlos E; van der Velden, Vincent H J; Novakova, Michaela; Mejstrikova, Ester; Hrusak, Ondrej; Böttcher, Sebastian; Karsch, Dennis; Sędek, Łukasz; Trinquand, Amelie; Boeckx, Nancy; Caetano, Joana; Asnafi, Vahid; Lucio, Paulo; Lima, Margarida; Helena Santos, Ana; Bonaccorso, Paola; van der Sluijs-Gelling, Alita J; Langerak, Anton W; Martin-Ayuso, Marta; Szczepański, Tomasz; van Dongen, Jacques J M; Orfao, Alberto

    2015-02-01

    Flow cytometric immunophenotyping has become essential for accurate diagnosis, classification, and disease monitoring in hemato-oncology. The EuroFlow Consortium has established a fully standardized "all-in-one" pipeline consisting of standardized instrument settings, reagent panels, and sample preparation protocols and software for data analysis and disease classification. For its reproducible implementation, parallel development of a quality assurance (QA) program was required. Here, we report on the results of four consecutive annual rounds of the novel external QA EuroFlow program. The novel QA scheme aimed at monitoring the whole flow cytometric analysis process (cytometer setting, sample preparation, acquisition and analysis) by reading the median fluorescence intensities (MedFI) of defined lymphocytes' subsets. Each QA participant applied the predefined reagents' panel on blood cells of local healthy donors. A uniform gating strategy was applied to define lymphocyte subsets and to read MedFI values per marker. The MedFI values were compared with reference data and deviations from reference values were quantified using performance score metrics. In four annual QA rounds, we analyzed 123 blood samples from local healthy donors on 14 different instruments in 11 laboratories from nine European countries. The immunophenotype of defined cellular subsets appeared sufficiently standardized to permit unified (software) data analysis. The coefficient of variation of MedFI for 7 of 11 markers performed repeatedly below 30%, average MedFI in each QA round ranged from 86 to 125% from overall median. Calculation of performance scores was instrumental to pinpoint standardization failures and their causes. Overall, the new EuroFlow QA system for the first time allowed to quantify the technical variation that is introduced in the measurement of fluorescence intensities in a multicentric setting over an extended period of time. EuroFlow QA is a proficiency test specific for

  17. Flow cytometry as a novel tool for structural and functional characterization of isolated yeast vacuoles.

    Science.gov (United States)

    Rodrigues, Jorge; Silva, Rui D; Noronha, Henrique; Pedras, Andreia; Gerós, Hernâni; Côrte-Real, Manuela

    2013-05-01

    The yeast vacuole is functionally analogous to the mammalian lysosome. Both play important roles in fundamental cellular processes such as protein degradation, detoxification, osmoregulation, autophagy and apoptosis which, when deregulated in humans, can lead to several diseases. Some of these vacuolar roles are difficult to study in a cellular context, and therefore the use of a cell-free system is an important approach to gain further insight into the different molecular mechanisms required for vacuolar function. In the present study, the potentialities of flow cytometry for the structural and functional characterization of isolated yeast vacuoles were explored. The isolation protocol resulted in a yeast vacuolar fraction with a degree of purity suitable for cytometric analysis. Moreover, isolated vacuoles were structurally and functionally intact and able to generate and maintain electrochemical gradients of ions across the vacuolar membrane, as assessed by flow cytometry. Proton and calcium gradients were dissipated by NH4Cl and calcimycin, respectively. These results established flow cytometry as a powerful technique for the characterization of isolated vacuoles. The protocols developed in this study can also be used to enhance our understanding of several molecular mechanisms underlying the development of lysosome-related diseases, as well as provide tools to screen for new drugs that can modulate these processes, which have promising clinical relevance.

  18. Quantitative analysis of gold and carbon nanoparticles in mammalian cells by flow cytometry light scattering

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Gang [Nanjing University, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences (China); Liu, Naicheng; Wang, Zhenheng [Nanjing University, Department of Orthopedics, Jinling Hospital, School of Medicine (China); Shi, Tongguo; Gan, Jingjing; Wang, Zhenzhen; Zhang, Junfeng, E-mail: jfzhang@nju.edu.cn [Nanjing University, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences (China)

    2017-02-15

    Nanoparticle-based applications for diagnostics and therapeutics have been extensively studied. These applications require a profound understanding of the fate of nanoparticles (NPs) in cellular environments. However, until now, few analytical methods are available and most of them rely on fluorescent properties or special elements of NPs; therefore, for NPs without observable optical properties or special elements, the existing methods are hardly applicable. In this study, we introduce a flow cytometry light scattering (FCLS)-based approach that quantifies in situ NPs accurately in mammalian cells. Continuous cells of heterogeneous human epithelial colorectal adenocarcinoma (Caco-2 cells), mouse peritoneal macrophages (MPM), and human adenocarcinomic alveolar basal epithelia (A549 cells) were cultured with NPs with certain concentrations and size. The intensity of the flow cytometric side scattered light, which indicates the quantity of NPs in the cells, was analyzed. The result shows an accurate size- and dose-dependent uptake of Au NPs (5, 30, 250 nm) in Caco-2 cells. The size- and dose- dependence of Au NPs (5, 30, 250 nm) and carbon NPs (50, 500 nm) in cells was validated by transmission electron microscope (TEM). This paper demonstrates the great potential of flow cytometry light scattering in the quantitative study of the size and dose effect on in situ metallic or non-metallic NPs in mammalian cells.

  19. A flow cytometry method for testing the susceptibility of Cryptococcus spp. to amphotericin B.

    Science.gov (United States)

    Benaducci, Tatiane; Matsumoto, Marcelo Teruyuki; Sardi, Janaina Cássia Orlandi; Fusco-Almeida, Ana Marisa; Mendes-Giannini, Maria José Soares

    2015-01-01

    Human fungal infections have increased at an alarming rate in recent years, particularly in immunocompromised individuals. Cryptococcosis is the second most prevalent systemic fungal infection worldwide, and the most prevalent systemic infection in immunocompromised individuals, representing more than 70% of cases. The incidence of cryptococcosis is high in people with HIV/acquired immunodeficiency syndrome (AIDS), with recent estimates indicating that there are one million cases of cryptococcal meningitis globally per year in AIDS patients. The aim of this research was to develop a rapid flow cytometric antifungal susceptibility test and to compare the results with the standard methods. A reference strain and clinical isolates of Cryptococcus neoformans and Cryptococcus gattii were tested for susceptibility to amphotericin B by flow cytometry using propidium iodide as indicator of viability. Flow cytometry (FC) results were compared with the minimum inhibitory concentration (MIC) values determined by microdilution. The antifungal activity of amphotericin B ranged from MICs of 0.06 to 2μg/ml for the 11 isolates studied. The same results were found by FC. The FC method allows same-day results, assisting in the selection of appropriate antifungal therapies. These results demonstrate an excellent correlation between FC and the classic methods of testing for susceptibility to antifungal agents. This rapid diagnosis method makes it possible to quickly administer effective therapeutic interventions, often saving lives. Copyright © 2013 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.

  20. Flow of Aqueous Humor

    Science.gov (United States)

    ... Home Flow of Aqueous Humor Flow of Aqueous Humor Most, but not all, forms of glaucoma are ... remains normal when some of the fluid (aqueous humor) produced by the eye's ciliary body flows out ...

  1. Cytometric evaluation of intracellular IFN-γ and IL-4 levels in thyroid follicular cells from patients with autoimmune thyroid diseases

    Directory of Open Access Journals (Sweden)

    Bossowski Artur

    2011-09-01

    Full Text Available Abstract Background In recent few years is underlined that altered balance of pro- and anti-inflammatory cytokines play an important role in the pathogenesis of AITD. The aim of this study was to estimate intracellular INF-γ and IL-4 levels in thyroid-infiltrating lymphocytes and thyrocytes isolated from thyroid tissues in 54 adolescent patients aged 8-21 years, with Graves' disease (GD; n = 18, Hashimoto's thyroiditis (HT; n = 18 and non-toxic multinodular goiter (NTMG; n = 18. Methods Fresh thyroid tissues were taken on culture medium RPMI -1640, it was mechanically prepared. In next step were added cell activators -12- myristate 13- the acetate (PMA and Ionomycin as well as the inhibitor of transportation of proteins - Breferdin A. They were cultured 24 hours in 50 ml flasks at 37°C in a 5-95% CO2-air water-saturated atmosphere. After that, thyrocytes were identified by mouse mAb directed against human TPO epitope 64 conjugated with rabbit anti-mouse antibodies IgG (Fab'2 labeled by FITC. After incubation at room temperature to each of samples added reagent A fixative the cellular membrane. In next step into the cell suspensions were added reagent B to permeabilization of cellular membrane and specific anti-IL-4-PE or anti-IFN-γ-PE mAbs. Identification of intracellular cytokines in T lymphocytes was performed in the same procedure with application of anti-CD4-PerCP and anti-CD8-PerCP mAbs specific for T lymphocytes. The cells were analyzed in a flow cytometry (Coulter EPICS XL. Results In examined group of patients with GD we observed statistically significant higher mean percentage of cells with phenotype CD4+IL-4 (p Conclusions We conclude that human thyrocytes in autoimmune thyroid disorders could be a source of cytokine production and that their activation influences local interaction with T lymphocytes inflowing to the thyroid gland.

  2. Laboratory Tests of Multiplex Detection of PCR Amplicons Using the Luminex 100 Flow Analyzer

    Energy Technology Data Exchange (ETDEWEB)

    Venkateswaran, K.S.; Nasarabadi, S.; Langlois, R.G.

    2000-05-05

    Lawrence Livermore National Laboratory (LLNL) demonstrated the power of flow cytometry in detecting the biological agents simulants at JFT III. LLNL pioneered in the development of advanced nucleic acid analyzer (ANM) for portable real time identification. Recent advances in flow cytometry provide a means for multiplexed nucleic acid detection and immunoassay of pathogenic microorganisms. We are presently developing multiplexed immunoassays for the simultaneous detection of different simulants. Our goal is to build an integrated instrument for both nucleic acid analysis and immuno detection. In this study we evaluated the Luminex LX 100 for concurrent identification of more than one PCR amplified product. ANAA has real-time Taqman fluorescent detection capability for rapid identification of field samples. However, its multiplexing ability is limited by the combination of available fluorescent labels. Hence integration of ANAA with flow cytometry can give the rapidity of ANAA amplification and the multiplex capability of flow cytometry. Multiplexed flow cytometric analysis is made possible using a set of fluorescent latex microsphere that are individually identified by their red and infrared fluorescence. A green fluorochrome is used as the assay signal. Methods were developed for the identification of specific nucleic acid sequences from Bacillus globigii (Bg), Bacillus thuringensis (Bt) and Erwinia herbicola (Eh). Detection sensitivity using different reporter fluorochromes was tested with the LX 100, and also different assay formats were evaluated for their suitability for rapid testing. A blind laboratory trial was carried out December 22-27, 1999 to evaluate bead assays for multiplex identification of Bg and Bt PCR products. This report summarizes the assay development, fluorochrome comparisons, and the results of the blind trial conducted at LLNL for the laboratory evaluation of the LX 100 flow analyzer.

  3. Relaminarization of fluid flows

    International Nuclear Information System (INIS)

    Narasimha, R.; Sreenivasan, K.R.

    1979-01-01

    The mechanisms of the relaminarization of turbulent flows are investigated with a view to establishing any general principles that might govern them. Three basic archetypes of reverting flows are considered: the dissipative type, the absorptive type, and the Richardson type exemplified by a turbulent boundary layer subjected to severe acceleration. A number of other different reverting flows are then considered in the light of the analysis of these archetypes, including radial Poiseuille flow, convex boundary layers, flows reverting by rotation, injection, and suction, as well as heated horizontal and vertical gas flows. Magnetohydrodynamic duct flows are also examined. Applications of flow reversion for turbulence control are discussed

  4. Intelligent Flow Control Valve

    Science.gov (United States)

    Kelley, Anthony R (Inventor)

    2015-01-01

    The present invention is an intelligent flow control valve which may be inserted into the flow coming out of a pipe and activated to provide a method to stop, measure, and meter flow coming from the open or possibly broken pipe. The intelligent flow control valve may be used to stop the flow while repairs are made. Once repairs have been made, the valve may be removed or used as a control valve to meter the amount of flow from inside the pipe. With the addition of instrumentation, the valve may also be used as a variable area flow meter and flow controller programmed based upon flowing conditions. With robotic additions, the valve may be configured to crawl into a desired pipe location, anchor itself, and activate flow control or metering remotely.

  5. Tricyclic antidepressant-induced lipidosis in human peripheral monocytes in vitro, as well as in a monocyte-derived cell line, as monitored by spectrofluorimetry and flow cytometry after staining with Nile red.

    Science.gov (United States)

    Xia, Z; Appelkvist, E L; DePierre, J W; Nässberger, L

    1997-05-15

    Human mono- and lymphocytes from peripheral blood and the monoblastoid cell line U-937 were used in this in vitro study of drug-induced lipidosis. Mono- and lymphocytes were exposed for 4 days to three different tricyclic antidepressants (TCAs), imipramine (25 microM), clomipramine (10 microM) and citalopram (80 microM). The lipophilic fluorophore Nile red, which stains intracellular lipid structures selectively, was used as a lipid probe. Fluorescence microscopy, spectrofluorimetry and flow cytometry were used to detect cellular lipidosis, as verified by electron microscopy. Our results demonstrate that imipramine, clomipramine and citalopram induce lipidosis in monocytes and U-937 cells, but not in lymphocytes. An accurate quantitation of induced intracellular lipidosis can be achieved by spectrofluorimetric and flow cytometric analysis.

  6. A Flow Cytometry Protocol to Estimate DNA Content in the Yellowtail Tetra Astyanax altiparanae

    Directory of Open Access Journals (Sweden)

    Pedro L. P. Xavier

    2017-09-01

    Full Text Available The production of triploid yellowtail tetra Astyanax altiparanae is a key factor to obtain permanently sterile individuals by chromosome set manipulation. Flow cytometric analysis is the main tool for confirmation of the resultant triploids individuals, but very few protocols are specific for A. altiparanae species. The current study has developed a protocol to estimate DNA content in this species. Furthermore, a protocol for long-term storage of dorsal fins used for flow cytometry analysis was established. The combination of five solutions with three detergents (Nonidet P-40 Substitute, Tween 20, and Triton X-100 at 0.1, 0.2, and 0.4% concentration was evaluated. Using the best solution from this first experiment, the addition of trypsin (0.125, 0.25, and 0.5% and sucrose (74 mM and the effects of increased concentrations of the detergents at 0.6 and 1.2% concentration were also evaluated. After adjustment of the protocol for flow cytometry, preservation of somatic tissue or isolated nuclei was also evaluated by freezing (at −20°C and fixation in saturated NaCl solution, acetic methanol (1:3, ethanol, and formalin at 10% for 30 or 60 days of storage at 25°C. Flow cytometry analysis in yellowtail tetra species was optimized using the following conditions: lysis solution: 9.53 mM MgCl2.7H20; 47.67 mM KCl; 15 mM Tris; 74 mM sucrose, 0.6% Triton X-100, pH 8.0; staining solution: Dulbecco's PBS with DAPI 1 μg mL−1; preservation procedure: somatic cells (dorsal fin samples frozen at −20°C. Using this protocol, samples may be stored up to 60 days with good accuracy for flow cytometry analysis.

  7. Diurnal Variations of Circulating Extracellular Vesicles Measured by Nano Flow Cytometry.

    Directory of Open Access Journals (Sweden)

    Kirsty M Danielson

    Full Text Available The identification of extracellular vesicles (EVs as intercellular conveyors of biological information has recently emerged as a novel paradigm in signaling, leading to the exploitation of EVs and their contents as biomarkers of various diseases. However, whether there are diurnal variations in the size, number, and tissue of origin of blood EVs is currently not known, and could have significant implications when using EVs as biomarkers for disease progression. Currently available technologies for the measurement of EV size and number are either time consuming, require specialized equipment, or lack sufficient accuracy across a range of EV sizes. Flow cytometry represents an attractive alternative to these methods; however, traditional flow cytometers are only capable of measuring particles down to 500 nm, which is significantly larger than the average and median sizes of plasma EVs. Utilizing a Beckman Coulter MoFlo XDP flow cytometer with NanoView module, we employed nanoscale flow cytometry (termed nanoFCM to examine the relative number and scatter distribution of plasma EVs at three different time points during the day in 6 healthy adults. Analysis of liposomes and plasma EVs proved that nanoFCM is capable of detecting biologically-relevant vesicles down to 100 nm in size. With this high resolution configuration, we observed variations in the relative size (FSC/SSC distributions and concentration (proportions of EVs in healthy adult plasma across the course of a day, suggesting that there are diurnal variations in the number and size distribution of circulating EV populations. The use of nanoFCM provides a valuable tool for the study of EVs in both health and disease; however, additional refinement of nanoscale flow cytometric methods is needed for use of these instruments for quantitative particle counting and sizing. Furthermore, larger scale studies are necessary to more clearly define the diurnal variations in circulating EVs, and thus

  8. Diurnal Variations of Circulating Extracellular Vesicles Measured by Nano Flow Cytometry.

    Science.gov (United States)

    Danielson, Kirsty M; Estanislau, Jessica; Tigges, John; Toxavidis, Vasilis; Camacho, Virginia; Felton, Edward J; Khoory, Joseph; Kreimer, Simion; Ivanov, Alexander R; Mantel, Pierre-Yves; Jones, Jennifer; Akuthota, Praveen; Das, Saumya; Ghiran, Ionita

    2016-01-01

    The identification of extracellular vesicles (EVs) as intercellular conveyors of biological information has recently emerged as a novel paradigm in signaling, leading to the exploitation of EVs and their contents as biomarkers of various diseases. However, whether there are diurnal variations in the size, number, and tissue of origin of blood EVs is currently not known, and could have significant implications when using EVs as biomarkers for disease progression. Currently available technologies for the measurement of EV size and number are either time consuming, require specialized equipment, or lack sufficient accuracy across a range of EV sizes. Flow cytometry represents an attractive alternative to these methods; however, traditional flow cytometers are only capable of measuring particles down to 500 nm, which is significantly larger than the average and median sizes of plasma EVs. Utilizing a Beckman Coulter MoFlo XDP flow cytometer with NanoView module, we employed nanoscale flow cytometry (termed nanoFCM) to examine the relative number and scatter distribution of plasma EVs at three different time points during the day in 6 healthy adults. Analysis of liposomes and plasma EVs proved that nanoFCM is capable of detecting biologically-relevant vesicles down to 100 nm in size. With this high resolution configuration, we observed variations in the relative size (FSC/SSC distributions) and concentration (proportions) of EVs in healthy adult plasma across the course of a day, suggesting that there are diurnal variations in the number and size distribution of circulating EV populations. The use of nanoFCM provides a valuable tool for the study of EVs in both health and disease; however, additional refinement of nanoscale flow cytometric methods is needed for use of these instruments for quantitative particle counting and sizing. Furthermore, larger scale studies are necessary to more clearly define the diurnal variations in circulating EVs, and thus further inform

  9. Panel development for multicolor flow-cytometry testing of proliferation and immunophenotype in hMSCs.

    Science.gov (United States)

    Bradford, Jolene A; Clarke, Scott T

    2011-01-01

    Adult human mesenchymal stem cells (hMSC) are rare fibroblast-like cells capable of differentiation into a variety of cell tissues which include bone, cartilage, muscle, ligament, tendon, and adipose. Normal adult bone marrow and adipose tissue are the most common sources of these cells. The International Society for Cellular Therapy (ISCT) has proposed a set of standards to define hMSC for laboratory investigations and preclinical studies: adherence to plastic in standard culture conditions; in vitro differentiation into osteoblasts, adipocytes, and chondroblasts; and specific surface antigen expression. Direct measurement of proliferation combined with simultaneous detection of the ISCT-consensus immunophenotypic profile provides data that is used to determine the differentiation status and health of the cells. Flow cytometry provides a powerful technology that is routinely used to simultaneously and rapidly measure multiple parameters in a single sample. This chapter describes a flow cytometric panel for the simultaneous detection of immunophenotypic profile, proliferative capacity, and DNA content measurement in hMSC. Because a relatively small number of cells are needed with this approach, measurements can be made with minimal impact on expansion potential. The ability to assess antigen expression and proliferative status enables the investigator to make informed decisions on expansion and harvesting.

  10. Measurement of lipid accumulation in Chlorella vulgaris via flow cytometry and liquid-state ¹H NMR spectroscopy for development of an NMR-traceable flow cytometry protocol.

    Directory of Open Access Journals (Sweden)

    Michael S Bono

    Full Text Available In this study, we cultured Chlorella vulgaris cells with a range of lipid contents, induced via nitrogen starvation, and characterized them via flow cytometry, with BODIPY 505/515 as a fluorescent lipid label, and liquid-state 1H NMR spectroscopy. In doing so, we demonstrate the utility of calibrating flow cytometric measurements of algal lipid content using triacylglyceride (TAG, also known as triacylglycerol or triglyceride content per cell as measured via quantitative 1H NMR. Ensemble-averaged fluorescence of BODIPY-labeled cells was highly correlated with average TAG content per cell measured by bulk NMR, with a linear regression yielding a linear fit with r2 = 0.9974. This correlation compares favorably to previous calibrations of flow cytometry protocols to lipid content measured via extraction, and calibration by NMR avoids the time and complexity that is generally required for lipid quantitation via extraction. Flow cytometry calibrated to a direct measurement of TAG content can be used to investigate the distribution of lipid contents for cells within a culture. Our flow cytometry measurements showed that Chlorella vulgaris cells subjected to nitrogen limitation exhibited higher mean lipid content but a wider distribution of lipid content that overlapped the relatively narrow distribution of lipid content for replete cells, suggesting that nitrogen limitation induces lipid accumulation in only a subset of cells. Calibration of flow cytometry protocols using direct in situ measurement of TAG content via NMR will facilitate rapid development of more precise flow cytometry protocols, enabling investigation of algal lipid accumulation for development of more productive algal biofuel feedstocks and cultivation protocols.

  11. Flow Element Models

    DEFF Research Database (Denmark)

    Heiselberg, Per; Nielsen, Peter V.

    Air distribution in ventilated rooms is a flow process that can be divided into different elements such as supply air jets, exhaust flows, thermal plumes, boundary layer flows, infiltration and gravity currents. These flow elements are isolated volumes where the air movement is controlled...... by a restricted number of parameters, and the air movement is fairly independent of the general flow in the enclosure. In many practical situations, the most convenient· method is to design the air distribution system using flow element theory....

  12. Dynamic power flow controllers

    Energy Technology Data Exchange (ETDEWEB)

    Divan, Deepakraj M.; Prasai, Anish

    2017-03-07

    Dynamic power flow controllers are provided. A dynamic power flow controller may comprise a transformer and a power converter. The power converter is subject to low voltage stresses and not floated at line voltage. In addition, the power converter is rated at a fraction of the total power controlled. A dynamic power flow controller controls both the real and the reactive power flow between two AC sources having the same frequency. A dynamic power flow controller inserts a voltage with controllable magnitude and phase between two AC sources; thereby effecting control of active and reactive power flows between two AC sources.

  13. Introduction to compressible fluid flow

    CERN Document Server

    Oosthuizen, Patrick H

    2013-01-01

    IntroductionThe Equations of Steady One-Dimensional Compressible FlowSome Fundamental Aspects of Compressible FlowOne-Dimensional Isentropic FlowNormal Shock WavesOblique Shock WavesExpansion Waves - Prandtl-Meyer FlowVariable Area FlowsAdiabatic Flow with FrictionFlow with Heat TransferLinearized Analysis of Two-Dimensional Compressible FlowsHypersonic and High-Temperature FlowsHigh-Temperature Gas EffectsLow-Density FlowsBibliographyAppendices

  14. FC-TRIPLEX Chagas/Leish IgG1: a multiplexed flow cytometry method for differential serological diagnosis of chagas disease and leishmaniasis.

    Directory of Open Access Journals (Sweden)

    Andréa Teixeira-Carvalho

    Full Text Available Differential serological diagnosis of Chagas disease and leishmaniasis is difficult owing to cross-reactivity resulting from the fact that the parasites that cause these pathologies share antigenic epitopes. Even with optimized serological assays that use parasite-specific recombinant antigens, inconclusive test results continue to be a problem. Therefore, new serological tests with high sensitivity and specificity are needed. In the present work, we developed and evaluated the performance of a new flow cytometric serological method, referred to as FC-TRIPLEX Chagas/Leish IgG1, for the all-in-one classification of inconclusive tests. The method uses antigens for the detection of visceral leishmaniasis, localized cutaneous leishmaniasis, and Chagas disease and is based on an inverted detuned algorithm for analysis of anti-Trypanosomatidae IgG1 reactivity. First, parasites were label with fluorescein isothiocyanate or Alexa Fluor 647 at various concentrations. Then serum samples were serially diluted, the dilutions were incubated with suspensions of mixed labeled parasites, and flow cytometric measurements were performed to determine percentages of positive fluorescent parasites. Using the new method, we obtained correct results for 76 of 80 analyzed serum samples (95% overall performance, underscoring the outstanding performance of the method. Moreover, we found that the fluorescently labeled parasite suspensions were stable during storage at room temperature, 4 °C, and -20 °C for 1 year. In addition, two different lots of parasite suspensions showed equivalent antigen recognition; that is, the two lots showed equivalent categorical segregation of anti-Trypanosomatidae IgG1 reactivity at selected serum dilutions. In conclusion, we have developed a sensitive and selective method for differential diagnosis of Chagas disease, visceral leishmaniasis, and localized cutaneous leishmaniasis.

  15. Use of quantitative flow cytometry to measure ex vivo immunostimulant activity of echinacea: the case for polysaccharides.

    Science.gov (United States)

    Pillai, Segaran; Pillai, Christine; Mitscher, Lester A; Cooper, Raymond

    2007-01-01

    When directly exposed to various echinacea fractions, human leukocytes ex vivo are strongly stimulated to proliferate and to produce immunostimulation and inflammatory cytokines. A comparison of fractions containing lipoidal small molecules and high-molecular-weight water-soluble polysaccharides indicates that the latter are substantially more potent as immunostimulants. Echinacea purpurea (L.) Moench, E. angustifolia DC, and E. pallida (Nutt.), Nutt. extracts, and each plant part contain significantly potent constituents. Flow cytometric techniques were utilized. This study was undertaken to determine whether flow cytometry could measure immunostimulant activity present in echinacea and, if so, which species produced more activity, which plant part was the most active, and whether the organic soluble or the aqueous extractables were more active. Ex vivo human clinical material was employed. Echinacea extracts were analyzed using flow cytometric techniques. The immunostimulation assays were measured in triplicate. Samples dissolved in dimethyl sulfoxide (DMSO) were added to 200 microL of heparinized blood mixed with 50 muL of phosphate buffer, vortexed, and incubated to allow adequate time for immune-cell stimulation. Fifty (50) microL of the stimulated blood samples were added to each of a reagent cocktail consisting of 20 microL of CD4FITC/CD69PE/CD3PerCP expressed on the helper/inducer T-lymphocyte subset; CD8FITC/CD69/PE/ CD3PerCP expressed on the human suppresser/cytotoxic T-lymphocytes and on a subset of natural killer lymphocytes; CD19FITC/CD69PE/CD45PerCP expressed on B-lymphocytes; or CD56FITC/CD69PE/CD45PerCP expressed on NK lymphocytes. Four hundred and fifty (450) microL of 1 X FACS lysing solution was added and incubated in the dark (rt, 30 minutes) and then subjected to flow cytometric analysis. All reported readings are the average of several determinations. Positive controls consisted of phorbol myristyl acetate (PMA) (50 ng/mL), phytohemagglutinin

  16. Flow cytometry-assisted rapid isolation of recombinant Plasmodium berghei parasites exemplified by functional analysis of aquaglyceroporin

    Science.gov (United States)

    Kenthirapalan, Sanketha; Waters, Andrew P.; Matuschewski, Kai; Kooij, Taco W.A.

    2012-01-01

    The most critical bottleneck in the generation of recombinant Plasmodium berghei parasites is the mandatory in vivo cloning step following successful genetic manipulation. This study describes a new technique for rapid selection of recombinant P. berghei parasites. The method is based on flow cytometry to isolate isogenic parasite lines and represents a major advance for the field, in that it will speed the generation of recombinant parasites as well as cut down on animal use significantly. High expression of GFP during blood infection, a prerequisite for robust separation of transgenic lines by flow cytometry, was achieved. Isogenic recombinant parasite populations were isolated even in the presence of a 100-fold excess of wild-type (WT) parasites. Aquaglyceroporin (AQP) loss-of-function mutants and parasites expressing a tagged AQP were generated to validate this approach. aqp− parasites grow normally within the WT phenotypic range during blood infection of NMRI mice. Similarly, colonization of the insect vector and establishment of an infection after mosquito transmission were unaffected, indicating that AQP is dispensable for life cycle progression in vivo under physiological conditions, refuting its use as a suitable drug target. Tagged AQP localized to perinuclear structures and not the parasite plasma membrane. We suggest that flow-cytometric isolation of isogenic parasites overcomes the major roadblock towards a genome-scale repository of mutant and transgenic malaria parasite lines. PMID:23137753

  17. Organocatalysis in continuous flow

    NARCIS (Netherlands)

    Berg, van den S.A.

    2016-01-01

    Continuous flow chemistry is an enabling technique in organic chemistry. Advantages include extremely fast mixing and heat transfer capabilities as well as rapid screening of reaction conditions. Combining continuous flow chemistry with solid-supported organocatalysis presents challenges that have

  18. Review of zonal flows

    International Nuclear Information System (INIS)

    Diamond, P.H.; Itoh, S.-I.; Itoh, K.; Hahm, T.S.

    2004-10-01

    A comprehensive review of zonal flow phenomena in plasmas is presented. While the emphasis is on zonal flows in laboratory plasmas, zonal flows in nature are discussed as well. The review presents the status of theory, numerical simulation and experiments relevant to zonal flows. The emphasis is on developing an integrated understanding of the dynamics of drift wave - zonal flow turbulence by combining detailed studies of the generation of zonal flows by drift waves, the back-interaction of zonal flows on the drift waves, and the various feedback loops by which the system regulates and organizes itself. The implications of zonal flow phenomena for confinement in, and the phenomena of fusion devices are discussed. Special attention is given to the comparison of experiment with theory and to identifying direction for progress in future research. (author)

  19. Practical flow cytometry

    National Research Council Canada - National Science Library

    Shapiro, Howard M

    2003-01-01

    ... ... Conflict: Resolution ... 1.3 Problem Number One: Finding The Cell(s) ... Flow Cytometry: Quick on the Trigger ... The Main Event ... The Pulse Quickens, the Plot Thickens ... 1.4 Flow Cytometry: ...

  20. Peak flow meter (image)

    Science.gov (United States)

    A peak flow meter is commonly used by a person with asthma to measure the amount of air that can be ... become narrow or blocked due to asthma, peak flow values will drop because the person cannot blow ...

  1. Load flow optimization and optimal power flow

    CERN Document Server

    Das, J C

    2017-01-01

    This book discusses the major aspects of load flow, optimization, optimal load flow, and culminates in modern heuristic optimization techniques and evolutionary programming. In the deregulated environment, the economic provision of electrical power to consumers requires knowledge of maintaining a certain power quality and load flow. Many case studies and practical examples are included to emphasize real-world applications. The problems at the end of each chapter can be solved by hand calculations without having to use computer software. The appendices are devoted to calculations of line and cable constants, and solutions to the problems are included throughout the book.

  2. Hyperspectral imaging flow cytometer

    Science.gov (United States)

    Sinclair, Michael B.; Jones, Howland D. T.

    2017-10-25

    A hyperspectral imaging flow cytometer can acquire high-resolution hyperspectral images of particles, such as biological cells, flowing through a microfluidic system. The hyperspectral imaging flow cytometer can provide detailed spatial maps of multiple emitting species, cell morphology information, and state of health. An optimized system can image about 20 cells per second. The hyperspectral imaging flow cytometer enables many thousands of cells to be characterized in a single session.

  3. Granular media : flow & agitations

    NARCIS (Netherlands)

    Dijksman, Joshua Albert

    2009-01-01

    This thesis is about weakly driven granular flows and suspensions. Chapter 1 is an overview of the current knowledge of slow granular flows in so-called split-bottom geometries, which in essence consist of a disk rotating at the bottom of a container. In chapter 2 we study dry granular flows in this

  4. OpenFlow cookbook

    CERN Document Server

    Smiler S, Kingston

    2015-01-01

    This book is intended for network protocol developers, SDN controller application developers, and academics who would like to understand and develop their own OpenFlow switch or OpenFlow controller in any programming language. With basic understanding of OpenFlow and its components, you will be able to follow the recipes in this book.

  5. Thermal flow micro sensors

    NARCIS (Netherlands)

    Elwenspoek, Michael Curt

    1999-01-01

    A review is given on sensors fabricated by silicon micromachining technology using the thermal domain for the measurement of fluid flow. Attention is paid especially to performance and geometry of the sensors. Three basic types of thermal flow sensors are discussed: anemometers, calorimetric flow

  6. Separation of flow

    CERN Document Server

    Chang, Paul K

    2014-01-01

    Interdisciplinary and Advanced Topics in Science and Engineering, Volume 3: Separation of Flow presents the problem of the separation of fluid flow. This book provides information covering the fields of basic physical processes, analyses, and experiments concerning flow separation.Organized into 12 chapters, this volume begins with an overview of the flow separation on the body surface as discusses in various classical examples. This text then examines the analytical and experimental results of the laminar boundary layer of steady, two-dimensional flows in the subsonic speed range. Other chapt

  7. Signal flow analysis

    CERN Document Server

    Abrahams, J R; Hiller, N

    1965-01-01

    Signal Flow Analysis provides information pertinent to the fundamental aspects of signal flow analysis. This book discusses the basic theory of signal flow graphs and shows their relation to the usual algebraic equations.Organized into seven chapters, this book begins with an overview of properties of a flow graph. This text then demonstrates how flow graphs can be applied to a wide range of electrical circuits that do not involve amplification. Other chapters deal with the parameters as well as circuit applications of transistors. This book discusses as well the variety of circuits using ther

  8. Lateral flow strip assay

    Science.gov (United States)

    Miles, Robin R [Danville, CA; Benett, William J [Livermore, CA; Coleman, Matthew A [Oakland, CA; Pearson, Francesca S [Livermore, CA; Nasarabadi, Shanavaz L [Livermore, CA

    2011-03-08

    A lateral flow strip assay apparatus comprising a housing; a lateral flow strip in the housing, the lateral flow strip having a receiving portion; a sample collection unit; and a reagent reservoir. Saliva and/or buccal cells are collected from an individual using the sample collection unit. The sample collection unit is immersed in the reagent reservoir. The tip of the lateral flow strip is immersed in the reservoir and the reagent/sample mixture wicks up into the lateral flow strip to perform the assay.

  9. Annular Flow Distribution test

    Energy Technology Data Exchange (ETDEWEB)

    Kielpinski, A.L. (ed.) (Westinghouse Savannah River Co., Aiken, SC (United States)); Childerson, M.T.; Knoll, K.E.; Manolescu, M.I.; Reed, M.J. (Babcock and Wilcox Co., Alliance, OH (United States). Research Center)

    1990-12-01

    This report documents the Babcock and Wilcox (B W) Annular Flow Distribution testing for the Savannah River Laboratory (SRL). The objective of the Annular Flow Distribution Test Program is to characterize the flow distribution between annular coolant channels for the Mark-22 fuel assembly with the bottom fitting insert (BFI) in place. Flow rate measurements for each annular channel were obtained by establishing hydraulic similarity'' between an instrumented fuel assembly with the BFI removed and a reference'' fuel assembly with the BFI installed. Empirical correlations of annular flow rates were generated for a range of boundary conditions.

  10. Annular Flow Distribution test

    International Nuclear Information System (INIS)

    Kielpinski, A.L.; Childerson, M.T.; Knoll, K.E.; Manolescu, M.I.; Reed, M.J.

    1990-12-01

    This report documents the Babcock and Wilcox (B ampersand W) Annular Flow Distribution testing for the Savannah River Laboratory (SRL). The objective of the Annular Flow Distribution Test Program is to characterize the flow distribution between annular coolant channels for the Mark-22 fuel assembly with the bottom fitting insert (BFI) in place. Flow rate measurements for each annular channel were obtained by establishing ''hydraulic similarity'' between an instrumented fuel assembly with the BFI removed and a ''reference'' fuel assembly with the BFI installed. Empirical correlations of annular flow rates were generated for a range of boundary conditions

  11. Physics of zonal flows

    International Nuclear Information System (INIS)

    Itoh, K.; Fujisawa, A.; Itoh, S.-I.; Yagi, M.; Nagashima, Y.; Diamond, P.H.; Tynan, G.R.; Hahm, T.S.

    2006-01-01

    Zonal flows, which means azimuthally symmetric band-like shear flows, are ubiquitous phenomena in nature and the laboratory. It is now widely recognized that zonal flows are a key constituent in virtually all cases and regimes of drift wave turbulence, indeed, so much so that this classic problem is now frequently referred to as ''drift wave-zonal flow turbulence.'' In this review, new viewpoints and unifying concepts are presented, which facilitate understanding of zonal flow physics, via theory, computation and their confrontation with the results of laboratory experiment. Special emphasis is placed on identifying avenues for further progress. (author)

  12. 46 CFR 154.546 - Excess flow valve: Closing flow.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 5 2010-10-01 2010-10-01 false Excess flow valve: Closing flow. 154.546 Section 154.546... and Process Piping Systems § 154.546 Excess flow valve: Closing flow. (a) The rated closing flow of vapor or liquid cargo for an excess flow valve must be specially approved by the Commandant (CG-522). (b...

  13. Group flow, complex flow, unit vector flow, and the (2+ϵ)-flow conjecture

    DEFF Research Database (Denmark)

    Thomassen, Carsten

    2014-01-01

    If F is a (possibly infinite) subset of an abelian group Γ, then we define f(F,Γ) as the smallest natural number such that every f(F,Γ)-edge-connected (finite) graph G has a flow where all flow values are elements in F. We prove that f(F,Γ) exists if and only if some odd sum of elements in F equals...... some even sum. We discuss various instances of this problem. We prove that every 6-edge-connected graph has a flow whose flow values are the three roots of unity in the complex plane. If the edge-connectivity 6 can be reduced, then it can be reduced to 4, and the 3-flow conjecture follows. We prove...... that every 14-edge-connected graph has a flow whose flow values are the five roots of unity in the complex plane. Any such flow is balanced modulo 5. So, if the edge-connectivity 14 can be reduced to 9, then the 5-flow conjecture follows, as observed by F. Jaeger. We use vector flow to prove that, for each...

  14. Genome-size Variation in Switchgrass (Panicum virgatum: Flow Cytometry and Cytology Reveal Rampant Aneuploidy

    Directory of Open Access Journals (Sweden)

    Denise E. Costich

    2010-11-01

    Full Text Available Switchgrass ( L., a native perennial dominant of the prairies of North America, has been targeted as a model herbaceous species for biofeedstock development. A flow-cytometric survey of a core set of 11 primarily upland polyploid switchgrass accessions indicated that there was considerable variation in genome size within each accession, particularly at the octoploid (2 = 8 = 72 chromosome ploidy level. Highly variable chromosome counts in mitotic cell preparations indicated that aneuploidy was more common in octoploids (86.3% than tetraploids (23.2%. Furthermore, the incidence of hyper- versus hypoaneuploidy is equivalent in tetraploids. This is clearly not the case in octoploids, where close to 90% of the aneuploid counts are lower than the euploid number. Cytogenetic investigation using fluorescent in situ hybridization (FISH revealed an unexpected degree of variation in chromosome structure underlying the apparent genomic instability of this species. These results indicate that rapid advances in the breeding of polyploid biofuel feedstocks, based on the molecular-genetic dissection of biomass characteristics and yield, will be predicated on the continual improvement of our understanding of the cytogenetics of these species.

  15. Flow-Based Single Cell Deposition for High-Throughput Screening of Protein Libraries.

    Directory of Open Access Journals (Sweden)

    Cassandra Stowe

    Full Text Available The identification and engineering of proteins having refined or novel characteristics is an important area of research in many scientific fields. Protein modelling has enabled the rational design of unique proteins, but high-throughput screening of large libraries is still required to identify proteins with potentially valuable properties. Here we report on the development and evaluation of a novel fluorescent activated cell sorting based screening platform. Single bacterial cells, expressing a protein library to be screened, are electronically sorted and deposited onto plates containing solid nutrient growth media in a dense matrix format of between 44 and 195 colonies/cm2. We show that this matrix format is readily applicable to machine interrogation (<30 seconds per plate and subsequent bioinformatic analysis (~60 seconds per plate thus enabling the high-throughput screening of the protein library. We evaluate this platform and show that bacteria containing a bioluminescent protein can be spectrally analysed using an optical imager, and a rare clone (0.5% population can successfully be identified, picked and further characterised. To further enhance this screening platform, we have developed a prototype electronic sort stream multiplexer, that when integrated into a commercial flow cytometric sorter, increases the rate of colony deposition by 89.2% to 24 colonies per second. We believe that the screening platform described here is potentially the foundation of a new generation of high-throughput screening technologies for proteins.

  16. A neural flow estimator

    DEFF Research Database (Denmark)

    Jørgensen, Ivan Harald Holger; Bogason, Gudmundur; Bruun, Erik

    1995-01-01

    This paper proposes a new way to estimate the flow in a micromechanical flow channel. A neural network is used to estimate the delay of random temperature fluctuations induced in a fluid. The design and implementation of a hardware efficient neural flow estimator is described. The system...... is implemented using switched-current technique and is capable of estimating flow in the μl/s range. The neural estimator is built around a multiplierless neural network, containing 96 synaptic weights which are updated using the LMS1-algorithm. An experimental chip has been designed that operates at 5 V...

  17. Hemoglobinopathy screening by osmotic fragility test based on flow cytometer or naked eye.

    Science.gov (United States)

    Mohapatra, Rinkle; Warang, Prashant; Ghosh, Kanjaksha; Colah, Roshan

    2016-05-01

    Diagnosis of hemoglobin (Hb) disorders is based mostly on abnormal red blood cell (RBC) indices, elevated levels of HbA2, HbF, or any other Hb on the Variant high performance liquid chromatography (HPLC) system, and confirmation by molecular methods. However, large scale population screening is of prime importance and requires a simple, accurate, and cost effective technique. We have tried to compare the sensitivity of the widely used Naked Eye Single Tube Red Cell Osmotic Fragility Test (NESTROFT) and the osmotic fragility described as % residual RBCs through flow cytometry for population screening. The count of residual red cells was measured sequentially in real-time using flow cytometry. NESTROFT was performed using a 0.36% buffered saline. HbA2 and HbF levels along with other abnormal Hbs were determined on the Variant HPLC System. Molecular studies were done to confirm the diagnosis. The normal group showed a significantly lower percentage of residual RBCs (48.08 ± 11.87) as compared to cases (β thalassemia trait-82.97 ± 12.20, α thalassemia trait-72.58 ± 8.34, and HbS trait-85.00 ± 4.05). The sensitivity and specificity of NESTROFT was high for both β thalassemia traits (98.33 and 96.72%, respectively) and α thalassemia traits (100 and 96.72%, respectively) but very low sensitivity for HbS traits (54.84%). Flow cytometric osmotic fragility was a more sensitive method to discriminate normal from the group of hemoglobinopathy carriers as compared to NESTROFT which missed majority of HbS carriers. However, in view of feasibility and cost effectiveness, NESTROFT could still be used for population screening of thalassemia. © 2014 International Clinical Cytometry Society. © 2014 International Clinical Cytometry Society.

  18. FLOCK cluster analysis of mast cell event clustering by high-sensitivity flow cytometry predicts systemic mastocytosis.

    Science.gov (United States)

    Dorfman, David M; LaPlante, Charlotte D; Pozdnyakova, Olga; Li, Betty

    2015-11-01

    In our high-sensitivity flow cytometric approach for systemic mastocytosis (SM), we identified mast cell event clustering as a new diagnostic criterion for the disease. To objectively characterize mast cell gated event distributions, we performed cluster analysis using FLOCK, a computational approach to identify cell subsets in multidimensional flow cytometry data in an unbiased, automated fashion. FLOCK identified discrete mast cell populations in most cases of SM (56/75 [75%]) but only a minority of non-SM cases (17/124 [14%]). FLOCK-identified mast cell populations accounted for 2.46% of total cells on average in SM cases and 0.09% of total cells on average in non-SM cases (P < .0001) and were predictive of SM, with a sensitivity of 75%, a specificity of 86%, a positive predictive value of 76%, and a negative predictive value of 85%. FLOCK analysis provides useful diagnostic information for evaluating patients with suspected SM, and may be useful for the analysis of other hematopoietic neoplasms. Copyright© by the American Society for Clinical Pathology.

  19. Detection of Sp110 by Flow Cytometry and Application to Screening Patients for Veno-occlusive Disease with Immunodeficiency.

    Science.gov (United States)

    Marquardsen, Florian A; Baldin, Fabian; Wunderer, Florian; Al-Herz, Waleed; Mikhael, Raymond; Lefranc, Gérard; Baz, Zeina; Rezaee, Fariba; Hanna, Rabi; Kfir-Erenfeld, Shlomit; Stepensky, Polina; Meyer, Benedikt; Jauch, Annaise; Bigler, Marc B; Burgener, Anne-Valérie; Higgins, Rebecca; Navarini, Alexander A; Church, Joeseph A; Chou, Janet; Geha, Raif; Notarangelo, Luigi D; Hess, Christoph; Berger, Christoph T; Bloch, Donald B; Recher, Mike

    2017-10-01

    Mutations in Sp110 are the underlying cause of veno-occlusive disease with immunodeficiency (VODI), a combined immunodeficiency that is difficult to treat and often fatal. Because early treatment is critically important for patients with VODI, broadly usable diagnostic tools are needed to detect Sp110 protein deficiency. Several factors make establishing the diagnosis of VODI challenging: (1) Current screening strategies to identify severe combined immunodeficiency are based on measuring T cell receptor excision circles (TREC). This approach will fail to identify VODI patients because the disease is not associated with severe T cell lymphopenia at birth; (2) the SP110 gene contains 17 exons, making it a challenge for Sanger sequencing. The recently developed next-generation sequencing (NGS) platforms that can rapidly determine the sequence of all 17 exons are available in only a few laboratories; (3) there is no standard functional assay to test for the effects of novel mutations in Sp110; and (4) it has been difficult to use flow cytometry to identify patients who lack Sp110 because of the low level of Sp110 protein in peripheral blood lymphocytes. We report here a novel flow cytometric assay that is easily performed in diagnostic laboratories and might thus become a standard assay for the evaluation of patients who may have VODI. In addition, the assay will facilitate investigations directed at understanding the function of Sp110.

  20. Shifts in the fluorescence lifetime of EGFP during bacterial phagocytosis measured by phase-sensitive flow cytometry

    Science.gov (United States)

    Li, Wenyan; Houston, Kevin D.; Houston, Jessica P.

    2017-01-01

    Phase-sensitive flow cytometry (PSFC) is a technique in which fluorescence excited state decay times are measured as fluorescently labeled cells rapidly transit a finely focused, frequency-modulated laser beam. With PSFC the fluorescence lifetime is taken as a cytometric parameter to differentiate intracellular events that are challenging to distinguish with standard flow cytometry. For example PSFC can report changes in protein conformation, expression, interactions, and movement, as well as differences in intracellular microenvironments. This contribution focuses on the latter case by taking PSFC measurements of macrophage cells when inoculated with enhanced green fluorescent protein (EGFP)-expressing E. coli. During progressive internalization of EGFP-E. coli, fluorescence lifetimes were acquired and compared to control groups. It was hypothesized that fluorescence lifetimes would correlate well with phagocytosis because phagosomes become acidified and the average fluorescence lifetime of EGFP is known to be affected by pH. We confirmed that average EGFP lifetimes consistently decreased (3 to 2 ns) with inoculation time. The broad significance of this work is the demonstration of how high-throughput fluorescence lifetime measurements correlate well to changes that are not easily tracked by intensity-only cytometry, which is affected by heterogeneous protein expression, cell-to-cell differences in phagosome formation, and number of bacterium engulfed.

  1. Glucocorticoids and irradiation-induced apoptosis in normal murine bone marrow B-lineage lymphocytes as determined by flow cytometry

    Energy Technology Data Exchange (ETDEWEB)

    Garvy, B.A.; Telford, W.G.; King, L.E.; Fraker, P.J. (Michigan State Univ., East Lansing, MI (United States))

    1993-06-01

    A substantial proportion of murine bone marrow B220[sup +] and IgM[sup +] cells were induced to undergo apoptosis when exposed to glucocorticoids or ionizing radiation in vitro. Two-colour flow cytometric analysis of the cell cycle indicated that a distinct subpopulation of cells formed to the left of G[sub o]/G[sub 1] in the hypodiploid or A[sub o] region previously shown to contain apoptotic cells with fragmented DNA. Indeed, 45-65% of all B220[sup +] or IgM[sup +] cells of the marrow were found in this apoptotic region 12 hr after treatment with dexamethasone (Dex) or exposure to 500 rads or irradiation. Zinc sulphate, a frequently cited inhibitor of apoptosis, prevented accumulation of cells exposed to glucocorticoids or ionizing radiation in the A[sub o] region as did the glucocorticoid receptor antagonist RU 38486. Although Dex was more potent, corticosterone and cortisol also induced significant degrees of apoptosis in B220[sup +] and IgM[sup +] marrow cells at physiological concentrations. These results demonstrate that freshly isolated B-lineage cells of the murine bone marrow readily undergo apoptosis upon exposure to glucocorticoids and ionizing radiation and suggest that apoptosis may play a role in the regulation of lymphopoiesis. The data also show the value of flow cytometry to the study of apoptosis in subsets of cells within a heterogenous population such as the bone marrow which heretofore was exceedingly difficult to evaluate. (Author).

  2. Genome size estimations on Ulmus minor Mill., Ulmus glabra Huds., and Celtis australis L. using flow cytometry.

    Science.gov (United States)

    Loureiro, J; Rodriguez, E; Gomes, A; Santos, C

    2007-07-01

    The Ulmaceae family is composed of nearly 2000 species widely distributed in the northern hemisphere. Despite their wide distribution area, there are only four native species in the Iberian Peninsula. In this work the genome size of three of those species (ULMUS MINOR, U. GLABRA, and CELTIS AUSTRALIS) was estimated using flow cytometry. The nuclear DNA content of C. AUSTRALIS was estimated as 2.46 +/- 0.061 pg/2C, of U. MINOR as 4.25 +/- 0.158 pg/2C, and of U. GLABRA as 4.37 +/- 0.103 pg/2C of DNA. No statistically significant differences were detected among individuals of the same species. These species revealed to be problematic for flow cytometric analyses, due to the release of mucilaginous compounds into the nuclear suspension. Despite that, the modified protocol here presented ensured high quality analyses (low coefficient of variation and background debris and nuclear fluorescence stability), opening good perspectives on its application to estimate the genome size of species with similar problems.

  3. Flow visualization using bubbles

    International Nuclear Information System (INIS)

    Henry, J.P.

    1974-01-01

    Soap bubbles were used for visualizing flows. The tests effected allowed some characteristics of flows around models in blow tunnels to be precised at mean velocities V 0 5 . The velocity of a bubble is measured by chronophotography, the bulk envelope of the trajectories is also registered [fr

  4. Airport Network Flow Simulator

    Science.gov (United States)

    1978-10-01

    The Airport Network Flow Simulator is a FORTRAN IV simulation of the flow of air traffic in the nation's 600 commercial airports. It calculates for any group of selected airports: (a) the landing and take-off (Type A) delays; and (b) the gate departu...

  5. Flow cytometry protocols

    National Research Council Canada - National Science Library

    Jaroszeski, Mark J; Heller, Richard

    1998-01-01

    ... are individually analyzed, and it is typical for flow cytometers to quantitatively process thousands of individual particles in a matter of seconds. This a powerful analytic feat particularly if one relates it to the time required to examine several thousand individual cells using a microscope. This leaves little doubt regarding why the field of flow cytometry has...

  6. Flow Around Steep Topography

    Science.gov (United States)

    2015-09-30

    Flow around steep topography T. M. Shaun Johnston Scripps Institution of Oceanography University of California, San Diego 9500 Gilman Drive, M...tall, steep, submarine topography and islands. During the Flow Encountering Abrupt Topography (FLEAT) DRI, investigators will determine: • Whether...estimates from making accurate statistical/deterministic predictions at ᝺ km resolution around submarine topography and islands? How can we

  7. Biomimetic Flow Sensors

    NARCIS (Netherlands)

    Casas, J.; Liu, Chang; Krijnen, Gijsbertus J.M.

    2012-01-01

    Biomimetic flow sensors are biologically inspired devices that measure the speed and direction of fluids. This survey starts by describing the role and functioning of airflow-sensing hairs in arthropods and in fishes, carries on with the biomimetic MEMS implementations, both for air and water flow

  8. Flow og anerkendelse

    DEFF Research Database (Denmark)

    Kristensen, René

    2006-01-01

    Artiklen handler om pædagogisk perspektivering af flow i forhold til anerkendelse og kreativitet. Procedural hukommelsesdannelse inddrages i forbindelse med flowforståelsen......Artiklen handler om pædagogisk perspektivering af flow i forhold til anerkendelse og kreativitet. Procedural hukommelsesdannelse inddrages i forbindelse med flowforståelsen...

  9. Flow i hverdagen

    DEFF Research Database (Denmark)

    Andersen, Frans Ørsted; Hanssen, Nina

    Bogen afspejler et eksplorativt dansk-norsk forskningsprojekt med fokus på hverdagslivets psykiske sider, herunder størrelser som stress og flow.......Bogen afspejler et eksplorativt dansk-norsk forskningsprojekt med fokus på hverdagslivets psykiske sider, herunder størrelser som stress og flow....

  10. Delays of Interconnected Flows

    Science.gov (United States)

    Sorger, U.; Suchanecki, Z.

    2011-07-01

    A rigorous approach to flows of particles in networks is presented. Under the assumption of independence of the transversal flows the asymptotic distributions of inter-delay times between particles are shown to be log-normal. In the case of dependent transversal traffic the ARCH and GARCH time series models, as well as martingale approach, have been applied.

  11. AUTO-EXPANSIVE FLOW

    Science.gov (United States)

    Physics suggests that the interplay of momentum, continuity, and geometry in outward radial flow must produce density and concomitant pressure reductions. In other words, this flow is intrinsically auto-expansive. It has been proposed that this process is the key to understanding...

  12. Optimised Renormalisation Group Flows

    CERN Document Server

    Litim, Daniel F

    2001-01-01

    Exact renormalisation group (ERG) flows interpolate between a microscopic or classical theory and the corresponding macroscopic or quantum effective theory. For most problems of physical interest, the efficiency of the ERG is constrained due to unavoidable approximations. Approximate solutions of ERG flows depend spuriously on the regularisation scheme which is determined by a regulator function. This is similar to the spurious dependence on the ultraviolet regularisation known from perturbative QCD. Providing a good control over approximated ERG flows is at the root for reliable physical predictions. We explain why the convergence of approximate solutions towards the physical theory is optimised by appropriate choices of the regulator. We study specific optimised regulators for bosonic and fermionic fields and compare the optimised ERG flows with generic ones. This is done up to second order in the derivative expansion at both vanishing and non-vanishing temperature. An optimised flow for a ``proper-time ren...

  13. Mere om flow

    DEFF Research Database (Denmark)

    Wohlgemuth, Ole

    2016-01-01

    "Begrebet flow, som det er formuleret af den ungarskefødte, amerikanske psykolog Mihaly Csikszentmihalyi", "beskriver en koncentreret tilstand af opslugthed i forbindelse med en oplevelse eller aktivitet. Flow betragtes som den tilstand, hvor læring foregår mest meningsfuldt, intensivt og...... grundlæggende; flow er endog blevet beskrevet som "de positive følelser kroppen belønner os med, når vi lærer noget vigtigt". Flow har sit udspring i positiv psykologi, som er et begreb indenfor den psykologiske forskning, der fokuserer på spørgsmålet om, hvad skal der til for at mennesker og samfund bedre kan...... trives og blomstre frem for et fokus på sygdomsfremkaldende og negative sammenhænge." ... "flow er en væsentlig tilstand at være fokuseret på og arbejde mod i forbindelse med formidling af naturen."...

  14. Vega flow assurance system

    Energy Technology Data Exchange (ETDEWEB)

    Larsen, Marit; Munaweera, Sampath

    2010-07-01

    Vega is a gas condensate field located at the west coast of Norway and developed as a tie-in to the Gjoea platform. Operator is Statoil, production startup is estimated to the end of 2010. Flow assurance challenges are high reservoir pressure and temperature, hydrate and wax control, liquid accumulation and monitoring the well/template production rates. The Vega Flow Assurance System (FAS) is a software that supports monitoring and operation of the field. The FAS is based FlowManagerTM designed for real time systems. This is a flexible tool with its own steady state multiphase- and flow assurance models. Due to the long flowlines lines and the dynamic behavior, the multiphase flow simulator OLGA is also integrated in the system. Vega FAS will be used as: - An online monitoring tool - An offline what-if simulation and validation tool - An advisory control system for well production allocation. (Author)

  15. Environmental stratified flows

    CERN Document Server

    Armenio, Vincenzo

    2006-01-01

    The volume covers the theory of stratified flows, from basic concepts to recent developments in the area of environmental fluid mechanics. The state of art of numerical techniques suited for stratified flows is given. Results of very recent researches in the areas of environmental stratified flows are discussed with details. The topics are treated in four separate chapters. The volume gives a unified view of stratified turbulent flows, from small-scale mixing, to large-scale environmental phenomena, also including detailed discussion on interaction between turbulence and internal gravity waves. The book will serve as an important review tool for all the scientists involved in the investigation of small scale as well as geophysical stratified flows.

  16. Flow regime analysis of non-Newtonian duct flows

    Science.gov (United States)

    Speetjens, Michel; Rudman, Murray; Metcalfe, Guy

    2006-01-01

    Reoriented duct flows of generalized Newtonian fluids are an idealization of non-Newtonian fluid flow in industrial in-line mixers. Based on scaling analysis and computation we find that non-Newtonian duct flows have several limit behaviors, in the sense that such flows can become (nearly) independent of one or more of the rheological and dynamical control parameters, simplifying the general flow and mixing problem. These limit flows give several levels of modeling complexity to the full problem of non-Newtonian duct flow. We describe the sets of simplified flow models and their corresponding regions of validity. This flow-model decomposition captures the essential rheological and dynamical characteristics of the reoriented duct flows and enables a more efficient and systematic study and design of flow and mixing of non-Newtonian fluids in ducts. Key aspects of the flow-model decomposition are demonstrated via a specific, but representative, duct flow.

  17. Flow in bedrock canyons.

    Science.gov (United States)

    Venditti, Jeremy G; Rennie, Colin D; Bomhof, James; Bradley, Ryan W; Little, Malcolm; Church, Michael

    2014-09-25

    Bedrock erosion in rivers sets the pace of landscape evolution, influences the evolution of orogens and determines the size, shape and relief of mountains. A variety of models link fluid flow and sediment transport processes to bedrock incision in canyons. The model components that represent sediment transport processes are increasingly well developed. In contrast, the model components being used to represent fluid flow are largely untested because there are no observations of the flow structure in bedrock canyons. Here we present a 524-kilometre, continuous centreline, acoustic Doppler current profiler survey of the Fraser Canyon in western Canada, which includes 42 individual bedrock canyons. Our observations of three-dimensional flow structure reveal that, as water enters the canyons, a high-velocity core follows the bed surface, causing a velocity inversion (high velocities near the bed and low velocities at the surface). The plunging water then upwells along the canyon walls, resulting in counter-rotating, along-stream coherent flow structures that diverge near the bed. The resulting flow structure promotes deep scour in the bedrock channel floor and undercutting of the canyon walls. This provides a mechanism for channel widening and ensures that the base of the walls is swept clear of the debris that is often deposited there, keeping the walls nearly vertical. These observations reveal that the flow structure in bedrock canyons is more complex than assumed in the models presently used. Fluid flow models that capture the essence of the three-dimensional flow field, using simple phenomenological rules that are computationally tractable, are required to capture the dynamic coupling between flow, bedrock erosion and solid-Earth dynamics.

  18. Lava flows are fractals

    Science.gov (United States)

    Bruno, B. C.; Taylor, G. J.; Rowland, S. K.; Lucey, P. G.; Self, S.

    1992-01-01

    Results are presented of a preliminary investigation of the fractal nature of the plan-view shapes of lava flows in Hawaii (based on field measurements and aerial photographs), as well as in Idaho and the Galapagos Islands (using aerial photographs only). The shapes of the lava flow margins are found to be fractals: lava flow shape is scale-invariant. This observation suggests that nonlinear forces are operating in them because nonlinear systems frequently produce fractals. A'a and pahoehoe flows can be distinguished by their fractal dimensions (D). The majority of the a'a flows measured have D between 1.05 and 1.09, whereas the pahoehoe flows generally have higher D (1.14-1.23). The analysis is extended to other planetary bodies by measuring flows from orbital images of Venus, Mars, and the moon. All are fractal and have D consistent with the range of terrestrial a'a and have D consistent with the range of terrestrial a'a and pahoehoe values.

  19. The use of isotypic control antibodies in the analysis of CD3+ and CD3+, CD4+ lymphocyte subsets by flow cytometry. Are they really necessary?

    Science.gov (United States)

    Sreenan, J J; Tbakhi, A; Edinger, M G; Tubbs, R R

    1997-02-01

    Isotypic control reagents are defined as irrelevant antibodies of the same immunoglobulin class as the relevant reagent antibody in a flow cytometry panel. The use of the isotypic control antibody has been advocated as a necessary quality control measure in analysis of flow cytometry. The purpose of this study was to determine the necessity of an isotypic control antibody in the analysis of CD3+ and CD3+, CD4+ lymphocyte subsets. We performed a prospective study of 46 consecutive patient samples received for lymphocyte subset analysis to determine the need for the isotypic control. For each sample, a sham buffer (autocontrol) and isotypic control reagent were stained for three-color immunofluorescence, processed, and identically analyzed with Attractors software. The Attractors software allowed independent, multiparametric, simultaneous gating; was able to identically and reproducibly process each list mode file; and yielded population data in spreadsheet form. Statistical analysis (Fisher's z test) revealed no difference between the CD3+ autocontrol and CD3+ isotypic control (correlation = 1, P autocontrol and the CD3+, CD4+ isotypic control (correlation = 1, P < .0001). The elimination of the isotypic control reagent resulted in a total cost savings of $3.36 per test. Additionally, the subtraction of isotypic background can artifactually depress population enumeration. The use of an isotypic control antibody is not necessary to analyze flow cytometric data that result in discrete cell populations, such as CD3+ and CD3+, CD4+ lymphocyte subsets. The elimination of this unnecessary quality control measure results in substantial cost savings.

  20. The use of a spaceflight-compatible device to perform WBC surface marker staining and whole-blood mitogenic activation for cytokine detection by flow cytometry

    Science.gov (United States)

    Crucian, B. E.; Sams, C. F.

    1999-01-01

    Significant changes have recently been described regarding circulating peripheral immune cells immediately following spaceflight. Existing methods for immunophenotype staining of peripheral blood in terrestrial labs do not meet the constraints for flight on the Space Shuttle. We have recently described the development and use of the Whole Blood Staining Device (WBSD), a simple device for staining flow cytometry specimens during spaceflight. When preparing samples with the WBSD, all liquids are safely contained as the cells are moved through staining, lysis and fixation steps. Here we briefly review the use of the WBSD, and then describe another versatile adaptation, a modification to perform intracellular staining of cytokines for detection by flow cytometry. Alterations in cytokine production have been reported both in ground-based simulated microgravity culture and in astronaut samples returning from spaceflight. Data regarding microgravity effects on cytokine production for specific subpopulations of cells is lacking. Flow cytometric cytokine analysis offers the unique ability to perform simultaneous surface marker analysis and positively identity cytokine producing subsets of cells. The utilization of the WBSD provides the ability to perform rapid and routine mitogenic activation during spaceflight coupled with the ability to perform simultaneous surface marker analysis. The only external requirements for this procedure are an in-flight 37-degree incubator and the capacity for 4-degree storage.

  1. Flow in rod bundles

    International Nuclear Information System (INIS)

    Hazi, G.; Mayer, G.

    2005-01-01

    For power upgrading VVER-440 reactors we need to know exactly how the temperature measured by the thermocouples is related to the average outlet temperature of the fuel assemblies. Accordingly, detailed knowledge on mixing process in the rod bundles and in the fuel assembly head have great importance. Here we study the hydrodynamics of rod bundles based on the results of direct numerical and large eddy simulation of flows in subchannels. It is shown that secondary flow and flow pulsation phenomena can be observed using both methodologies. Some consequences of these observations are briefly discussed. (author)

  2. Initiation of slug flow

    International Nuclear Information System (INIS)

    Hanratty, T.J.; Woods, B.D.

    1995-01-01

    The initiation of slug flow in a horizontal pipe can be predicted either by considering the stability of a slug or by considering the stability of a stratified flow. Measurements of the shedding rate of slugs are used to define necessary conditions for the existence of a slug. Recent results show that slugs develop from an unstable stratified flow through the evolution of small wavelength waves into large wavelength waves that have the possibility of growing to form a slug. The mechanism appears to be quite different for fluids with viscosities close to water than for fluids with large viscosities (20 centipoise)

  3. Magnetic vortex filament flows

    International Nuclear Information System (INIS)

    Barros, Manuel; Cabrerizo, Jose L.; Fernandez, Manuel; Romero, Alfonso

    2007-01-01

    We exhibit a variational approach to study the magnetic flow associated with a Killing magnetic field in dimension 3. In this context, the solutions of the Lorentz force equation are viewed as Kirchhoff elastic rods and conversely. This provides an amazing connection between two apparently unrelated physical models and, in particular, it ties the classical elastic theory with the Hall effect. Then, these magnetic flows can be regarded as vortex filament flows within the localized induction approximation. The Hasimoto transformation can be used to see the magnetic trajectories as solutions of the cubic nonlinear Schroedinger equation showing the solitonic nature of those

  4. Cash flow statement

    Directory of Open Access Journals (Sweden)

    Pavlović Miloš

    2013-01-01

    Full Text Available A cash flow is "bloodstream" of business and without constant cash flow a company would not be able to function. The cash flow statement is statutory financial report that provides information to the interested parties on cash inflows and outflows from operating, investing and financing activities during the accounting period. This paper will discuss the origin and significance of the cash slow statement; in addition, we will define the main categories of this statement and present the methodology of its composition in accordance with IAS 7.

  5. Refrigeration. Two-Phase Flow. Flow Regimes and Pressure Drop

    DEFF Research Database (Denmark)

    Knudsen, Hans-Jørgen Høgaard

    2002-01-01

    The note gives the basic definitions used in two-phase flow. Flow regimes and flow regimes map are introduced. The different contributions to the pressure drop are stated together with an imperical correlation from the litterature.......The note gives the basic definitions used in two-phase flow. Flow regimes and flow regimes map are introduced. The different contributions to the pressure drop are stated together with an imperical correlation from the litterature....

  6. Using FRET to quantify changes in integrin structures in human leukocytes induced by chemoattractants with multi-frequency flow cytometry

    Science.gov (United States)

    Sambrano, Jesus; Smagley, Yelena; Chigaev, Alexandre; Sklar, Larry A.; Houston, Jessica P.

    2017-02-01

    Flow cytometry for single cell counting uses optical measurements to report multiple cell features such as cell morphology, cell phenotype, and microenvironmental changes. Time-resolved flow cytometry is a unique method that involves the detection of the average fluorescence lifetime as a cytometric parameter. Measuring the average fluorescence lifetime is helpful when discriminating between more than one emission signal from a single cell because of spectrally overlapping emission. In this contribution, we present preliminary measurements toward a study that advances simple time-resolved flow cytometry and introduces a technique to measure fluorescence lifetime values from single cells labeled with a Forster Resonance Energy Transfer (FRET) pair. Specifically, donor fluorophore fluorescein isothiocyanate (FITC) fluorescence lifetime is measured to identify its proximity to the acceptor fluorophore. We hypothesize that our time-resolved flow cytometry approach can resolve changes in FRET in order to study integrin structures on the surface of leukocyte cells. Our results show that FITC has an average lifetime of 4.2 +/-0.1 nsec, and an average fluorescence lifetime of 2.4 nsec +/-0.2 nsec when engaged in FRET. After the release of FRET (e.g. dequenched) the average fluorescence lifetime of FITC was measured to be 3.1 +/- 0.5 nsec. Phasor graphs reveal large distributions of fluorescence lifetimes on a per cell basis, suggesting the existence of multiple fluorescence lifetimes. These data suggest more than one integrin conformation occurs throughout the cell population. The impact of this work is the addition of quantitative information for FRET efficiency values and determination of FRET calculations using high-throughput data.

  7. Assessment of sperm function parameters and DNA fragmentation in ejaculated alpaca sperm (Lama pacos) by flow cytometry.

    Science.gov (United States)

    Cheuquemán, C; Merino, O; Giojalas, L; Von Baer, A; Sánchez, R; Risopatrón, J

    2013-06-01

    Flow cytometry has been shown to be an accurate and highly reproducible tool for the analysis of sperm function. The main objective of this study was to assess sperm function parameters in ejaculated alpaca sperm by flow cytometry. Semen samples were collected from six alpaca males and processed for flow cytometric analysis of sperm viability and plasma membrane integrity using SYBR-14⁄PI staining; acrosomal membrane integrity using FITC-conjugated Pisum Sativum Agglutinin⁄PI labelling; mitochondrial membrane potential (Δψm) by staining with JC-1 and DNA Fragmentation Index (DFI) by TUNEL. The results indicate that the mean value for sperm viability was 57 ± 8 %. Spermatozoa with intact acrosome membrane was 87.9 ± 5%, and viable sperm with intact acrosomal membrane was 46.8 ± 9%, high mitochondrial membrane potential (Δψm) was detected in 66.32 ± 9.51% of spermatozoa and mean DFI value was 0.91 ± 0.9%. The DFI was inversely correlated with high Δψm (p = 0.04; r = -0.41) and with plasma membrane integrity (p = 0.01; r = -0.47). To our knowledge, this is the first report of the assessment on the same sample of several parameters of sperm function in ejaculated alpaca sperm by flow cytometry. © 2012 Blackwell Verlag GmbH.

  8. Complex Flow Workshop Report

    Energy Technology Data Exchange (ETDEWEB)

    none,

    2012-05-01

    This report documents findings from a workshop on the impacts of complex wind flows in and out of wind turbine environments, the research needs, and the challenges of meteorological and engineering modeling at regional, wind plant, and wind turbine scales.

  9. Reversed extension flow

    DEFF Research Database (Denmark)

    Nielsen, Jens Kromann; Rasmussen, Henrik K.

    2008-01-01

    Afilament stretching rheometer (FSR) was used for measuring the start-up of uni-axial elongational flow followed by reversed bi-axial flow, both with a constant elongational rate. A narrow molecular mass distribution linear polystyrene with a molecular weight of 145 kg / mole wis subjected...... to the start-up of elongation for three Hencky strain units and subsequently the reversed flow. The integral molecular stress function formulation within the 'interchain pressure' concept agrees with the experiments. In the experiments the Hencky strain at which the str~ss becomes zero (the recovery strain......) in the reversed flow has been identified. The recovery strain is found to increase with elongational rate, and has a maximum value of approximately 1.45. The Doi Edwards model using any stretch evolution equation is not able to predict the correct level of the recovery strain....

  10. Web life: Ice Flows

    Science.gov (United States)

    2016-11-01

    Computer and video gamers of a certain vintage will have fond memories of Lemmings, a game in which players must shepherd pixelated, suicidal rodents around a series of obstacles to reach safety. At first glance, Ice Flows is strikingly similar.

  11. Turbulent flow computation

    National Research Council Canada - National Science Library

    Drikakis, D; Geurts, Bernard

    2002-01-01

    ... discretization 3 A test-case: turbulent channel flow 4 Conclusions 75 75 82 93 98 4 Analysis and control of errors in the numerical simulation of turbulence Sandip Ghosal 1 Introduction 2 Source...

  12. Flow Cytometry Section

    Data.gov (United States)

    Federal Laboratory Consortium — The primary goal of the Flow Cytometry Section is to provide the services of state-of-the-art multi-parameter cellular analysis and cell sorting for researchers and...

  13. Flow cytometry bioinformatics.

    Directory of Open Access Journals (Sweden)

    Kieran O'Neill

    Full Text Available Flow cytometry bioinformatics is the application of bioinformatics to flow cytometry data, which involves storing, retrieving, organizing, and analyzing flow cytometry data using extensive computational resources and tools. Flow cytometry bioinformatics requires extensive use of and contributes to the development of techniques from computational statistics and machine learning. Flow cytometry and related methods allow the quantification of multiple independent biomarkers on large numbers of single cells. The rapid growth in the multidimensionality and throughput of flow cytometry data, particularly in the 2000s, has led to the creation of a variety of computational analysis methods, data standards, and public databases for the sharing of results. Computational methods exist to assist in the preprocessing of flow cytometry data, identifying cell populations within it, matching those cell populations across samples, and performing diagnosis and discovery using the results of previous steps. For preprocessing, this includes compensating for spectral overlap, transforming data onto scales conducive to visualization and analysis, assessing data for quality, and normalizing data across samples and experiments. For population identification, tools are available to aid traditional manual identification of populations in two-dimensional scatter plots (gating, to use dimensionality reduction to aid gating, and to find populations automatically in higher dimensional space in a variety of ways. It is also possible to characterize data in more comprehensive ways, such as the density-guided binary space partitioning technique known as probability binning, or by combinatorial gating. Finally, diagnosis using flow cytometry data can be aided by supervised learning techniques, and discovery of new cell types of biological importance by high-throughput statistical methods, as part of pipelines incorporating all of the aforementioned methods. Open standards, data

  14. RCA and information flow

    International Nuclear Information System (INIS)

    2005-01-01

    RCA is an inter-governmental agreement among IAEA Member States in Asia and the Pacific. The Member States are Australia, Bangladesh, China, India, Indonesia, Japan, Korea, Malaysia, Mongolia, Myanmar, New Zealand, Pakistan, The Philippines, Singapore, Sri Lanka, Thailand and Viet Nam. The RCA Thematic Sectors are Agriculture, Human Health, Industry, Environment, Energy, Research Reactors, Radiation Safety, Electronic Networking and Outreach. This PowerPoint presentation gives an overview about the RCA projects, the information flow mechanism and the information flow programme

  15. Flow assurance study

    OpenAIRE

    Böser, W.; Belfroid, S.P.C.

    2013-01-01

    Generally large scale carbon capture projects require pipeline systems for the transporting of the CO2 from its point of capture to the storage site. The article will give information on the proposed operational management system. This has to work for all process situations, ranging from steady flow at varying injection conditions and flow rates, to start-up and shutdown procedures and also for emergency shutdown at the platform. In all these operational situations the phase behaviour of CO2 ...

  16. Electrochemical flow capacitors

    Science.gov (United States)

    Gogotsi, Yury; Presser, Volker; Kumbur, Emin Caglan

    2015-10-27

    The present invention generally relates to devices for energy storage technologies, and more particularly to electrochemical flow capacitor systems and applications. In some aspects, these flow capacitors have at least one electrode comprising a non-stationary solid or semi-solid composition comprising supercapacitive particles and an electrolytic solvent in electrical communication with at least one current collector, and energy is stored and/or released by charging and/or discharging the electrode(s).

  17. Flow energy conversion system

    International Nuclear Information System (INIS)

    Sargsyan, R.A.

    2011-01-01

    A cost-effective hydropower system called here Flow Energy Converter was developed, patented, manufactured and tested for water pumping, electricity generation and other purposes especially useful for the rural communities. The system consists of water-driven turbine with plane-surface blades, power transmission means and pump and/or generator. Working sample of the Flow Energy Converter was designed and manufactured at the Institute of Radio Physics and Electronics

  18. Stability of parallel flows

    CERN Document Server

    Betchov, R

    2012-01-01

    Stability of Parallel Flows provides information pertinent to hydrodynamical stability. This book explores the stability problems that occur in various fields, including electronics, mechanics, oceanography, administration, economics, as well as naval and aeronautical engineering. Organized into two parts encompassing 10 chapters, this book starts with an overview of the general equations of a two-dimensional incompressible flow. This text then explores the stability of a laminar boundary layer and presents the equation of the inviscid approximation. Other chapters present the general equation

  19. Epigenetic targeting in acute myeloid leukemia: use of flow cytometry in monitoring therapeutic effects.

    Science.gov (United States)

    Ryningen, Anita; Bruserud, Øystein

    2007-12-01

    Flow cytometric techniques have emerged as a powerful tool in hematology allowing fast, sensitive and reproducible multi-parametric analyses at the single cell level of heterogeneous samples. Small subsets of cells can be studied with high degree of accuracy, and a broad and constantly increasing specter of antibodies is available. Flow cytometry has therefore become the method of choice for evaluation of therapeutic effects at single cell level. These methodological approaches can easily be used to study hematological malignancies, and the future use of this strategy in other malignancies will depend on the development of laboratory techniques to prepare suspensions of viable cells also from tumor biopsies. The selection of biological parameters for evaluation of treatment effects should probably be based on (i) molecular markers involved in cancer-associated genetic abnormalities; (ii) other molecular markers showing altered expression in the malignant cells and thought to be involved in leukemogenesis or having a prognostic impact; (ii) functional assays known to reflect biological characteristics that are important in carcinogenesis (e.g. cell cycle distribution, functional evaluation of apoptosis regulation). These molecules will in addition often represent the therapeutic targets when new anticancer drugs are developed. In this review we use treatment of acute myeloid leukemia with histone deacetylase inhibitors as an example. Based on the criteria mentioned above we suggest that the monitoring of therapeutic effects on the cancer cells in these patients should include differentiation status, histone acetylation, cell cycle distribution, pro- and anti-apoptotic signaling balance and intracellular levels of various transcription factors.

  20. A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry.

    Science.gov (United States)

    Gong, J; Traganos, F; Darzynkiewicz, Z

    1994-05-01

    In cells undergoing apoptosis (programmed cell death), a fraction of nuclear DNA is fragmented to the size equivalent of DNA in mono- or oligonucleosomes. When such DNA is analyzed by agarose gel electrophoresis it generates the characteristic "ladder" pattern of discontinuous DNA fragments. Such a pattern of DNA degradation generally serves as a marker of the apoptotic mode of cell death. We developed a simple, rapid, and selective procedure for extraction of the degraded, low-molecular-weight DNA from apoptotic cells. The cells are prefixed in 70% ethanol, DNA is extracted with 0.2 M phosphate-citrate buffer at pH 7.8, and the extract is sequentially treated with RNase A and proteinase K and then subjected to electrophoresis. The ladder pattern was detected from DNA extracted from 1-2 x 10(6) HL-60 cells, of which as few as 8% were apoptotic, by flow cytometric criteria, as well as from blood and bone marrow samples from leukemic patients undergoing chemotherapy. The method is rapid and uses nontoxic reagents (no phenol, chloroform, etc.). This approach permits the analysis of DNA extracted from the very same cell population that is subjected to measurements by flow cytometry to estimate DNA ploidy, the cell cycle distribution of nonapoptotic cells, the percentage of apoptotic cells, or other parameters. Furthermore, the cells may be stored in 70% ethanol for at least several weeks before analysis without any significant DNA degradation. Treatment with ethanol also inactivates several pathogens, thereby increasing the safety of sample handling. The method is applicable to clinical samples, which can be fixed in ethanol and then stored and/or safety transported prior to analysis.

  1. Flow with Negative Differential Viscosity

    OpenAIRE

    川口, 明彦; Akihiko, KAWAGUCHI; 京大人環; Graduate School of Human and Enviromental Studies, Kyoto University

    2000-01-01

    Only a monotonous flow appears to the movement of the incompressible flow body in a porous medium under a simple condition according to Darcy's law. However, the character of the flow changes greatly if we think about the model by which the temperature dependency in the coefficient of viscosity is considered. Becoming of the inclination of pressure deifference-flow velocity specific characteristics nagative if we think about the one-dimensnional flow under a suitable condition, that is, "Flow...

  2. Report of the results of the International Clinical Cytometry Society and American Society for Clinical Pathology workload survey of clinical flow cytometry laboratories.

    Science.gov (United States)

    Wolniak, Kristy; Goolsby, Charles; Choi, Sarah; Ali, Asma; Serdy, Nina; Stetler-Stevenson, Maryalice

    2017-11-01

    Thorough review of current workload, staffing, and testing practices in clinical laboratories allows for optimization of laboratory efficiency and quality. This information is largely missing with regard to clinical flow cytometry laboratories. The purpose of this survey is to provide comprehensive, current, and accurate data on testing practices and laboratory staffing in clinical laboratories performing flow cytometric studies. Survey data was collected from flow cytometry laboratories through the ASCP website. Data was collected on the workload during a 1-year time period of full-time and part-time technical and professional (M.D./D.O./Ph.D. or equivalent) flow cytometry employees. Workload was examined as number of specimens and tubes per full time equivalent (FTE) technical and professional staff. Test complexity, test result interpretation, and reporting practices were also evaluated. There were 205 respondent laboratories affiliated predominantly with academic and health system institutions. Overall, 1,132 FTE employees were reported with 29% professional FTE employees and 71% technical. Fifty-one percent of the testing performed was considered high complexity and 49% was low complexity. The average number of tubes per FTE technologist was 1,194 per year and the average number of specimens per FTE professional was 1,659 per year. The flow cytometry reports were predominantly written by pathologists (57%) and were typically written as a separate report (58%). This survey evaluates the overall status of the current practice of clinical flow cytometry and provides a comprehensive dataset as a framework to help laboratory departments, directors, and managers make appropriate, cost-effective staffing decisions. © 2016 International Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.

  3. Flow rate calibration. III. The use of stabilized biostandards to calibrate the flow rate and calculate absolute CD4+ T-cell counts.

    Science.gov (United States)

    Walker, Clare L; Whitby, Liam; Granger, Viv; Storie, Ian; Reilly, John T; Barnett, David

    2006-05-01

    We have previously reported a flow rate calibration method for the determination of absolute CD4(+) T-lymphocyte counts that removes the need for the addition of latex beads to each sample. However, a limitation with this approach is that a calibration factor (CF) needs to be applied to adjust for differences in viscosity between latex bead suspensions and biological specimens. We have also demonstrated the value of using stabilized whole blood samples in external quality assessment (EQA) studies; such samples have a stable absolute lymphocyte count for over 1 year, at 4 degrees C. It was successfully demonstrated that this material can be used as a flow rate biocalibration (FRB) material for use as a flow cytometric control to provide a sample with a known CD4(+) T-lymphocyte count. Such material has advantages over latex bead technology as it can act as a full process control as well as having the same matrix and viscosity characteristics as the test material, thus removing the need for a CF. In this study, we have analyzed 268 consecutive normal, abnormal, and HIV(+) samples using FRB, incorporating the PanLeucoGating approach and compared this to the MultiSet method, defined as the predicate. Percentage similarity statistics revealed the following: 0-3,000 CD4(+) cells/mul mean percentage difference (MPD; bias) 1.2%, 95% CI of 5.6-8%; 0-200 CD4(+) cells/microl MPD of 1.25%, 95% CI of 11.63-14.13%; 201-500 CD4(+) cells/microl MPD of 1%, 95% CI of 4.6-6.6%. This study demonstrates that stabilized whole blood can be used for FRB. It has the advantage of being a full process control, in addition to costing less than latex beads with highly comparable results. As bench top flow cytometers are extremely stable, this is a low cost and robust alternative to bead based methods for generating absolute CD4 counts. Copyright 2006 International Society for Analytical Cytology.

  4. Planetary heat flow measurements.

    Science.gov (United States)

    Hagermann, Axel

    2005-12-15

    The year 2005 marks the 35th anniversary of the Apollo 13 mission, probably the most successful failure in the history of manned spaceflight. Naturally, Apollo 13's scientific payload is far less known than the spectacular accident and subsequent rescue of its crew. Among other instruments, it carried the first instrument designed to measure the flux of heat on a planetary body other than Earth. The year 2005 also should have marked the launch of the Japanese LUNAR-A mission, and ESA's Rosetta mission is slowly approaching comet Churyumov-Gerasimenko. Both missions carry penetrators to study the heat flow from their target bodies. What is so interesting about planetary heat flow? What can we learn from it and how do we measure it?Not only the Sun, but all planets in the Solar System are essentially heat engines. Various heat sources or heat reservoirs drive intrinsic and surface processes, causing 'dead balls of rock, ice or gas' to evolve dynamically over time, driving convection that powers tectonic processes and spawns magnetic fields. The heat flow constrains models of the thermal evolution of a planet and also its composition because it provides an upper limit for the bulk abundance of radioactive elements. On Earth, the global variation of heat flow also reflects the tectonic activity: heat flow increases towards the young ocean ridges, whereas it is rather low on the old continental shields. It is not surprising that surface heat flow measurements, or even estimates, where performed, contributed greatly to our understanding of what happens inside the planets. In this article, I will review the results and the methods used in past heat flow measurements and speculate on the targets and design of future experiments.

  5. Oscillatory flow chemical reactors

    Directory of Open Access Journals (Sweden)

    Slavnić Danijela S.

    2014-01-01

    Full Text Available Global market competition, increase in energy and other production costs, demands for high quality products and reduction of waste are forcing pharmaceutical, fine chemicals and biochemical industries, to search for radical solutions. One of the most effective ways to improve the overall production (cost reduction and better control of reactions is a transition from batch to continuous processes. However, the reactions of interests for the mentioned industry sectors are often slow, thus continuous tubular reactors would be impractically long for flow regimes which provide sufficient heat and mass transfer and narrow residence time distribution. The oscillatory flow reactors (OFR are newer type of tube reactors which can offer solution by providing continuous operation with approximately plug flow pattern, low shear stress rates and enhanced mass and heat transfer. These benefits are the result of very good mixing in OFR achieved by vortex generation. OFR consists of cylindrical tube containing equally spaced orifice baffles. Fluid oscillations are superimposed on a net (laminar flow. Eddies are generated when oscillating fluid collides with baffles and passes through orifices. Generation and propagation of vortices create uniform mixing in each reactor cavity (between baffles, providing an overall flow pattern which is close to plug flow. Oscillations can be created by direct action of a piston or a diaphragm on fluid (or alternatively on baffles. This article provides an overview of oscillatory flow reactor technology, its operating principles and basic design and scale - up characteristics. Further, the article reviews the key research findings in heat and mass transfer, shear stress, residence time distribution in OFR, presenting their advantages over the conventional reactors. Finally, relevant process intensification examples from pharmaceutical, polymer and biofuels industries are presented.

  6. Load flow analysis using decoupled fuzzy load flow under critical ...

    African Journals Online (AJOL)

    user

    The conventional load flow methods like Newton-Raphson load flow (NRLF), Fast Decoupled load flow (FDLF) provide poor performance under critical conditions such as high R/X ratio, heavily loading condition etc. Exploiting the decoupling properties of power system, reliable fuzzy load flow is developed to overcome the ...

  7. Tomographic multiphase flow measurement

    International Nuclear Information System (INIS)

    Sætre, C.; Johansen, G.A.; Tjugum, S.A.

    2012-01-01

    Measurement of multiphase flow of gas, oil and water is not at all trivial and in spite of considerable achievements over the past two decades, important challenges remain (). These are related to reducing measurement uncertainties arising from variations in the flow regime, improving long term stability and developing new means for calibration, adjustment and verification of the multiphase flow meters. This work focuses on the first two issues using multi gamma beam (MGB) measurements for identification of the type of flow regime. Further gamma ray tomographic measurements are used for reference of the gas/liquid distribution. For the MGB method one Am-241 source with principal emission at 59.5 keV is used because this relatively low energy enables efficient collimation and thereby shaping of the beams, as well as compact detectors. One detector is placed diametrically opposite the source whereas the second is positioned to the side so that this beam is close to the pipe wall. The principle is then straight forward to compare the measured intensities of these detectors and through that identify the flow pattern, i.e. the instantaneous cross-sectional gas-liquid distribution. The measurement setup also includes Compton scattering measurements, which can provide information about the changes in the water salinity for flow segments with high water liquid ratio and low gas fractions. By measuring the transmitted intensity in short time slots (<100ms), rapid regime variations are revealed. From this we can select the time sections suitable for salinity measurements. Since the salinity variations change at the time scale of hours, a running average can be performed to increase the accuracy of the measurements. Recent results of this work will be presented here. - Highlights: ► Multiphase flow gas-fraction and flow regime measurements by multi gamma ray beams. ► High-speed gamma ray tomograph as reference for the flow pattern and gas fraction. ► Dual modality

  8. Radiotracer techniques for measuring fluid flow and calibrating flow meters

    International Nuclear Information System (INIS)

    Cooper, E.L.

    1987-08-01

    Radiotracer techniques can be used to measure accurately both gas and liquid flow rates under operating conditions in a wide range of flow systems. They are ideally suited for calibrating flow meters as well as for measuring unmetered flows in industrial plants. Applications of these techniques range from measuring the flows of fuels and process fluids for energy and mass balance studies to measuring the flows of liquid and airborne effluents for pollution control. This report describes the various radiotracer techniques which can be used to measure fluid flows. The range of application and inherent accuracy of each technique is discussed

  9. Upscaling of Forchheimer flows

    KAUST Repository

    Aulisa, Eugenio

    2014-08-01

    In this work we propose upscaling method for nonlinear Forchheimer flow in heterogeneous porous media. The generalized Forchheimer law is considered for incompressible and slightly-compressible single-phase flows. We use recently developed analytical results (Aulisa et al., 2009) [1] and formulate the resulting system in terms of a degenerate nonlinear flow equation for the pressure with the nonlinearity depending on the pressure gradient. The coarse scale parameters for the steady state problem are determined so that the volumetric average of velocity of the flow in the domain on fine scale and on coarse scale are close. A flow-based coarsening approach is used, where the equivalent permeability tensor is first evaluated following streamline methods for linear cases, and modified in order to take into account the nonlinear effects. Compared to previous works (Garibotti and Peszynska, 2009) [2], (Durlofsky and Karimi-Fard) [3], this approach can be combined with rigorous mathematical upscaling theory for monotone operators, (Efendiev et al., 2004) [4], using our recent theoretical results (Aulisa et al., 2009) [1]. The developed upscaling algorithm for nonlinear steady state problems is effectively used for variety of heterogeneities in the domain of computation. Direct numerical computations for average velocity and productivity index justify the usage of the coarse scale parameters obtained for the special steady state case in the fully transient problem. For nonlinear case analytical upscaling formulas in stratified domain are obtained. Numerical results were compared to these analytical formulas and proved to be highly accurate. © 2014.

  10. Micromodel foam flow study

    Energy Technology Data Exchange (ETDEWEB)

    Chambers, K.T.; Radke, C.J.

    1990-10-01

    Foams are often utilized as part of enhanced oil recovery techniques. This report presents the results of a micromodel foam flow study. Micromodels are valuable tools in uncovering capillary phenomena responsible for lamellae generation and coalescence during foam flow in porous media. Among the mechanisms observed are snap-off, weeping-flow breakup, and lamella division and leave behind. Coalescence mechanisms include dynamic capillary-pressure-induced lamella drainage and gas diffusion. These phenomena are sensitive to the mode of injection, the local capillary environment, and the geometry of the pore structure. An important consideration in presenting a tractable model of foam flow behavior is the ability to identify the pore-level mechanisms having the greatest impact on foam texture. The predominant mechanisms will vary depending upon the application for foam as an enhanced oil recovery (EOR) fluid. Both simultaneous gas and surfactant injection and surfactant alternating with gas injection (SAG) have been used to create foam for mobility control in EOR projects. The model developed is based on simultaneous gas and surfactant injection during steady-state conditions into a Berea sandstone core. The lamellae generation and coalescence mechanisms included in this model are snap-off, lamella division, and dynamic capillary-pressure-induced lamella drainage. This simplified steady-state model serves as a foundation for developing more complete rate expressions and for extending the population balance to handle transient foam flow behavior. 70 refs., 30 figs.

  11. Use of flow cytometry for the adhesion analysis of Streptococcus pyogenes mutant strains to epithelial cells: investigation of the possible role of surface pullulanase and cysteine protease, and the transcriptional regulator Rgg

    Directory of Open Access Journals (Sweden)

    Finne Jukka

    2006-02-01

    Full Text Available Abstract Background Flow cytometry based adherence assay is a potentially powerful but little used method in the study of bacterial binding to host structures. We have previously characterized a glycoprotein-binding activity in Streptococcus pyogenes called 'strepadhesin' binding to thyroglobulin, submaxillar mucin, fetuin and asialofetuin. We have identified surface-associated pullulanase (PulA and cysteine protease (SpeB as carriers of strepadhesin activity. In the present paper, we investigated the use of flow cytometry as a method to study the binding of Rgg, SpeB and PulA knock-out strains to cultured human epithelial cells. Results Streptococcal mutants were readily labelled with CFDA-SE and their binding to epithelial cells could be effectively studied by flow cytometry. A strain deficient in Rgg expression showed increased binding to the analyzed epithelial cell lines of various origin. Inactivation of SpeB had no effect on the adhesion, while PulA knock-out strains displayed decreased binding to the cell lines. Conclusion These results suggest that the flow cytometric assay is a valuable tool in the analysis of S. pyogenes adherence to host cells. It appears to be an efficient and sensitive tool for the characterization of interactions between the bacteria and the host at the molecular level. The results also suggest a role for Rgg regulated surface molecules, like PulA, in the adhesion of S. pyogenes to host cells.

  12. Turbine flow meter response in two-phase flows

    International Nuclear Information System (INIS)

    Shim, W.J.; Dougherty, T.J.; Cheh, H.Y.

    1996-01-01

    The purpose of this paper is to suggest a simple method of calibrating turbine flow meters to measure the flow rates of each phase in a two-phase flow. The response of two 50.8 mm (2 inch) turbine flow meters to air-water, two-phase mixtures flowing vertically in a 57 mm I.D. (2.25 inch) polycarbonate tube has been investigated for both upflow and downflow. The flow meters were connected in series with an intervening valve to provide an adjustable pressure difference between them. Void fractions were measured by two gamma densitometers, one upstream of the flow meters and the other downstream. The output signal of the turbine flow meters was found to depend only on the actual volumetric flow rate of the gas, F G , and liquid, F L , at the location of the flow meter

  13. Allogeneic Mesenchymal Stem Cell Treatment Induces Specific Alloantibodies in Horses

    Directory of Open Access Journals (Sweden)

    Sean D. Owens

    2016-01-01

    Full Text Available Background. It is unknown whether horses that receive allogeneic mesenchymal stem cells (MSCs injections develop specific humoral immune response. Our goal was to develop and validate a flow cytometric MSC crossmatch procedure and to determine if horses that received allogeneic MSCs in a clinical setting developed measurable antibodies following MSC administration. Methods. Serum was collected from a total of 19 horses enrolled in 3 different research projects. Horses in the 3 studies all received unmatched allogeneic MSCs. Bone marrow (BM or adipose tissue derived MSCs (ad-MSCs were administered via intravenous, intra-arterial, intratendon, or intraocular routes. Anti-MSCs and anti-bovine serum albumin antibodies were detected via flow cytometry and ELISA, respectively. Results. Overall, anti-MSC antibodies were detected in 37% of the horses. The majority of horses (89% were positive for anti-bovine serum albumin (BSA antibodies prior to and after MSC injection. Finally, there was no correlation between the amount of anti-BSA antibody and the development of anti-MSC antibodies. Conclusion. Anti allo-MSC antibody development was common; however, the significance of these antibodies is unknown. There was no correlation between either the presence or absence of antibodies and the percent antibody binding to MSCs and any adverse reaction to a MSC injection.

  14. Designing reliability information flows

    International Nuclear Information System (INIS)

    Petkova, Valia T.; Lu Yuan; Ion, Roxana A.; Sander, Peter C.

    2005-01-01

    It is well-known [Reliab. Eng. Syst. Saf. 75 (2002) 295] that in modern development processes it is essential to have an information flow structure that facilitates fast feedback from product users (customers) to departments at the front end, in particular development and production. As information is only relevant if it is used when taking decisions, this paper presents a guideline for building field feedback information flows that facilitate the decision taking during the product creation and realisation process. The guideline takes into consideration that the type of decisions depends on the span-of-control, therefore following Parsons [Structure and Process in Modern Societies (1990)] the span-of-control is subdivided into the following three levels: strategic, tactic, and executive. The guideline is illustrated with a case in which it is used for analysing the quality of existing field feedback flows

  15. Particles in flows

    CERN Document Server

    Galdi, Giovanni; Nečasová, Šárka

    2017-01-01

    This book aims to face particles in flows from many different, but essentially interconnected sides and points of view. Thus the selection of authors and topics represented in the chapters, ranges from deep mathematical analysis of the associated models, through the techniques of their numerical solution, towards real applications and physical implications. The scope and structure of the book as well as the selection of authors was motivated by the very successful summer course and workshop "Particles in Flows'' that was held in Prague in the August of 2014. This meeting revealed the need for a book dealing with this specific and challenging multidisciplinary subject, i.e. particles in industrial, environmental and biomedical flows and the combination of fluid mechanics, solid body mechanics with various aspects of specific applications.

  16. Choked flow through cracks

    International Nuclear Information System (INIS)

    Feburie, V.; Giot, M.; Granger, S.; Seynhaeve, J.M.

    1992-06-01

    The leaks through steam-generator cracks are the subject of a research carried out in cooperation between EDF and UCL. A software called ECREVISSE to predict the mass flow rate has been developed and has been successfully validated. The purpose of the paper is to present the mathematical model used in ECREVISSE as well as some comparison between the results and the presently available data. The model takes into account the persistence of some metastable liquid in the crack and the special flow pattern which appears in such particular geometry. Although the model involves the use of several correlations (friction, heat transfer), no adjustment of parameters against the data has been needed, neither in the single-phase part of the flow, or in the two-phase part. (authors). 8 figs., 1 tab., 20 refs

  17. Robust Optical Flow Estimation

    Directory of Open Access Journals (Sweden)

    Javier Sánchez Pérez

    2013-10-01

    Full Text Available n this work, we describe an implementation of the variational method proposed by Brox etal. in 2004, which yields accurate optical flows with low running times. It has several benefitswith respect to the method of Horn and Schunck: it is more robust to the presence of outliers,produces piecewise-smooth flow fields and can cope with constant brightness changes. Thismethod relies on the brightness and gradient constancy assumptions, using the information ofthe image intensities and the image gradients to find correspondences. It also generalizes theuse of continuous L1 functionals, which help mitigate the effect of outliers and create a TotalVariation (TV regularization. Additionally, it introduces a simple temporal regularizationscheme that enforces a continuous temporal coherence of the flow fields.

  18. Fluid Flow at Branching Junctions

    OpenAIRE

    Sochi, Taha

    2013-01-01

    The flow of fluids at branching junctions plays important kinematic and dynamic roles in most biological and industrial flow systems. The present paper highlights some key issues related to the flow of fluids at these junctions with special emphasis on the biological flow networks particularly blood transportation vasculature.

  19. The squeezing flow

    Directory of Open Access Journals (Sweden)

    Nilankush Acharya

    2016-06-01

    Full Text Available The present article investigates the squeezing flow of two types of nanofluids such as Cu-water and Cu-kerosene between two parallel plates in the presence of magnetic field. The governing non-linear partial differential equations are transformed into ordinary differential equations by applying suitable similarity transformation and then solved numerically using RK-4 method with shooting technique and analytically using differential transformation method (DTM. The influence of arising relevant parameters on flow characteristics has been discussed through graphs and tables. A comparative study has been taken into account between existing results and present work and it is found to be in excellent harmony.

  20. Three-Dimensional Flows

    CERN Document Server

    Araujo, Vitor; Viana, Marcelo

    2010-01-01

    In this book, the authors present the elements of a general theory for flows on three-dimensional compact boundaryless manifolds, encompassing flows with equilibria accumulated by regular orbits. The book aims to provide a global perspective of this theory and make it easier for the reader to digest the growing literature on this subject. This is not the first book on the subject of dynamical systems, but there are distinct aspects which together make this book unique. Firstly, this book treats mostly continuous time dynamical systems, instead of its discrete counterpart, exhaustively treated

  1. Flow Analysis Software Toolkit

    Science.gov (United States)

    Watson, Velvin; Castagnera, Karen; Plessel, Todd; Merritt, Fergus; Kelaita, Paul; West, John; Sandstrom, Tim; Clucas, Jean; Globus, AL; Bancroft, Gordon; hide

    1993-01-01

    Flow Analysis Software Toolkit (FAST) computer program provides software environment facilitating visualization of data. Collection of separate programs (modules) running simultaneously and helps user to examine results of numerical and experimental simulations. Intended for graphical depiction of computed flows, also assists in analysis of other types of data. Combines capabilities of such programs as PLOT3D, RIP, SURF, and GAS into one software environment with modules sharing data. All modules have consistent, highly interactive graphical user interface. Modular construction makes it flexible and extensible. Environment custom-configured, and new modules developed and added as needed. Written in ANSI compliant FORTRAN 77 and C language.

  2. Mechanics of fluid flow

    CERN Document Server

    Basniev, Kaplan S; Chilingar, George V 0

    2012-01-01

    The mechanics of fluid flow is a fundamental engineering discipline explaining both natural phenomena and human-induced processes, and a thorough understanding of it is central to the operations of the oil and gas industry.  This book, written by some of the world's best-known and respected petroleum engineers, covers the concepts, theories, and applications of the mechanics of fluid flow for the veteran engineer working in the field and the student, alike.  It is a must-have for any engineer working in the oil and gas industry.

  3. Using Crossmatch tests for serological compatibility assessment intra- and interspecific at dogs and cats

    Directory of Open Access Journals (Sweden)

    Sergiu Adrian Muntean

    2016-11-01

    Conclusions: The intraspecific evaluations revealed a high level of blood compatibility in the case of dogs unsensitivized through previous blood transfusions, yet without excluding the possibility of some atypical sensitivization for clinical interest. Having all the interspecific tests exclusively highly positive, we can not sustain a probable xenotransfusion.

  4. Flow Cytometry Detection of Bacterial Cell Entrapment within the Chitosan Hydrogel and Antibacterial Property of Extracted Chitosan

    Directory of Open Access Journals (Sweden)

    Nafise Sadat Majidi

    2016-09-01

    Full Text Available Background:   Chitosan is unbranched polysaccharide composed of D-glucosamine and N-acetyl-D-glucosamine. Chitosan, derived from shrimp shell, has broad antimicrobial properties against Gram-negative, Gram-positive bacteria and fungi. Methods:  Chitosan was extracted from shrimp shell and studied for cell entrapment and anti-bacterial properties. The hydrogel chitosan was used as the beads for cell entrapment and chitosan beads were designed to deliver cells and nutrients. These data confirmed with flow cytometric analyses.                 Results:   Experimental results exhibited that internal diffusion through the chitosan matrix was the main mechanism for whole gelation by TPP (Tri-polyphosphate. The minimum inhibitory concentration (MIC for chitosan against Staphylococcus aureus and Escherichia coli was 16 and 32 μg/ml respectively. Conclusion:  Despite the antimicrobial properties of chitosan, trapped bacteria in the gel network were alive and were chelated indicating that their access to the outside was limited.

  5. Transient two-phase flow

    International Nuclear Information System (INIS)

    Hsu, Y.Y.

    1974-01-01

    The following papers related to two-phase flow are summarized: current assumptions made in two-phase flow modeling; two-phase unsteady blowdown from pipes, flow pattern in Laval nozzle and two-phase flow dynamics; dependence of radial heat and momentum diffusion; transient behavior of the liquid film around the expanding gas slug in a vertical tube; flooding phenomena in BWR fuel bundles; and transient effects in bubble two-phase flow. (U.S.)

  6. Using Crossflow for Flow Measurements and Flow Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Gurevich, A.; Chudnovsky, L.; Lopeza, A. [Advanced Measurement and Analysis Group Inc., Ontario (Canada); Park, M. H. [Sungjin Nuclear Engineering Co., Ltd., Gyeongju (Korea, Republic of)

    2016-10-15

    Ultrasonic Cross Correlation Flow Measurements are based on a flow measurement method that is based on measuring the transport time of turbulent structures. The cross correlation flow meter CROSSFLOW is designed and manufactured by Advanced Measurement and Analysis Group Inc. (AMAG), and is used around the world for various flow measurements. Particularly, CROSSFLOW has been used for boiler feedwater flow measurements, including Measurement Uncertainty Recovery (MUR) reactor power uprate in 14 nuclear reactors in the United States and in Europe. More than 100 CROSSFLOW transducers are currently installed in CANDU reactors around the world, including Wolsung NPP in Korea, for flow verification in ShutDown System (SDS) channels. Other CROSSFLOW applications include reactor coolant gross flow measurements, reactor channel flow measurements in all channels in CANDU reactors, boiler blowdown flow measurement, and service water flow measurement. Cross correlation flow measurement is a robust ultrasonic flow measurement tool used in nuclear power plants around the world for various applications. Mathematical modeling of the CROSSFLOW agrees well with laboratory test results and can be used as a tool in determining the effect of flow conditions on CROSSFLOW output and on designing and optimizing laboratory testing, in order to ensure traceability of field flow measurements to laboratory testing within desirable uncertainty.

  7. Is flow verification necessary

    International Nuclear Information System (INIS)

    Beetle, T.M.

    1986-01-01

    Safeguards test statistics are used in an attempt to detect diversion of special nuclear material. Under assumptions concerning possible manipulation (falsification) of safeguards accounting data, the effects on the statistics due to diversion and data manipulation are described algebraically. A comprehensive set of statistics that is capable of detecting any diversion of material is defined in terms of the algebraic properties of the effects. When the assumptions exclude collusion between persons in two material balance areas, then three sets of accounting statistics are shown to be comprehensive. Two of the sets contain widely known accountancy statistics. One of them does not require physical flow verification - comparisons of operator and inspector data for receipts and shipments. The third set contains a single statistic which does not require physical flow verification. In addition to not requiring technically difficult and expensive flow verification, this single statistic has several advantages over other comprehensive sets of statistics. This algebraic approach as an alternative to flow verification for safeguards accountancy is discussed in this paper

  8. Flow on noisy terrains

    DEFF Research Database (Denmark)

    Tsirogiannis, Konstantinos; Haverkort, Herman

    2011-01-01

    Computing watersheds on triangulated terrain models in a robust manner is a difficult task as it is sensitive to noise that appears in the elevation values of the input. This is amplified by the existence of many very small watersheds (corresponding to spurious minima) that obscure the overall hy...... to use a robust flow model together with exact arithmetic....

  9. Erosion in extruder flow

    Science.gov (United States)

    Kaufman, Miron; Fodor, Petru S.

    A detailed analysis of the fluid flow in Tadmor's unwound channel model of the single screw extruder is performed by combining numerical and analytical methods. Using the analytical solution for the longitudinal velocity field (in the limit of zero Reynolds number) allows us to devote all the computational resources solely for a detailed numerical solution of the transversal velocity field. This high resolution 3D model of the fluid flow in a single-screw extruder allows us to identify the position and extent of Moffatt eddies that impede mixing. We further consider the erosion of particles (e.g. carbon-black agglomerates) advected by the polymeric flow. We assume a particle to be made of primary fragments bound together. In the erosion process a primary fragment breaks out of a given particle. Particles are advected by the laminar flow and they disperse because of the shear stresses imparted by the fluid. The time evolution of the numbers of particles of different sizes is described by the Bateman coupled differential equations used to model radioactivity. Using the particle size distribution we compute an entropic fragmentation index which varies from 0 for a monodisperse system to 1 for an extreme poly-disperse system.

  10. ECAL Energy Flow Calibration

    CERN Multimedia

    CERN. Geneva

    2015-01-01

    My talk will be covering my work as a whole over the course of the semester. The focus will be on using energy flow calibration in ECAL to check the precision of the corrections made by the light monitoring system used to account for transparency loss within ECAL crystals due to radiation damage over time.

  11. Flow Control and Diagnostics

    Indian Academy of Sciences (India)

    A multi-component wind tunnel balance, working with fibre-optic sensors, was already demonstrated by the authors in ... often for mapping surface pressure fields on wind tunnel models. The paint is a fluorescent ... When a gas turbine engine operates close to its peak performance, flow within the com- pressor can become ...

  12. Flow Injection Analysis

    DEFF Research Database (Denmark)

    Hansen, Elo Harald

    1998-01-01

    Learning objectives:* To provide an introduction to automated assays* To describe the basic principles of FIA * To demonstrate the capabilities of FIA in relation to batch assays and conventional continuous flow systems* To show that FIA allows one to augment existing analytical techniques* To show...

  13. Flow assurance study

    NARCIS (Netherlands)

    Böser, W.; Belfroid, S.P.C.

    2013-01-01

    Generally large scale carbon capture projects require pipeline systems for the transporting of the CO2 from its point of capture to the storage site. The article will give information on the proposed operational management system. This has to work for all process situations, ranging from steady flow

  14. Quaternions and ideal flows

    Energy Technology Data Exchange (ETDEWEB)

    Eshraghi, H [Iran University of Science and Technology (IUST), School of Physics, Institute for Studies in Theoretical Physics and Mathematics, Tehran (Iran, Islamic Republic of); Gibbon, J D [Department of Mathematics, Imperial College London, London SW7 2AZ (United Kingdom)

    2008-08-29

    After a review of some of the recent works by Holm and Gibbon on quaternions and their application to Lagrangian flows, particularly the incompressible Euler equations and the equations of ideal MHD, this paper investigates the compressible and relativistic Euler equations using these methods.

  15. Water Flow Experiments

    Indian Academy of Sciences (India)

    This is a simple exercise in elementary fluid dynamics for the undergraduate and the secondary school level. Here, we explore the flow of water through an orifice at the bottom of a cylindri- cal bottle/tank, first through a tube attached to the bottom of the bottle/tank and then without the tube. The experiment is easy to perform.

  16. Lateral flow assays

    NARCIS (Netherlands)

    Posthuma-Trumpie, G.A.; Amerongen, van A.

    2012-01-01

    A simple version of immunochemical-based methods is the Lateral Flow Assay (LFA). It is a dry chemistry technique (reagents are included); the fluid from the sample runs through a porous membrane (often nitrocellulose) by capillary force. Typically the membrane is cut as a strip of 0.5*5 cm. In most

  17. Probabilistic Load Flow

    DEFF Research Database (Denmark)

    Chen, Peiyuan; Chen, Zhe; Bak-Jensen, Birgitte

    2008-01-01

    This paper reviews the development of the probabilistic load flow (PLF) techniques. Applications of the PLF techniques in different areas of power system steady-state analysis are also discussed. The purpose of the review is to identify different available PLF techniques and their corresponding...

  18. Polystyrene beads coated with antibodies directed to HLA class I intracytoplasmic domain: the use in quantitative measurement of peptide-HLA class I binding by flow cytometry.

    Science.gov (United States)

    Chersi, A; Rosano, L; Tanigaki, N

    2000-12-01

    Protein-reactive, conformation-independent anti-peptide antibodies were raised in rabbits against a C-terminal sequence SDSAQGSDVSLA, common to most HLA-A and -B locus products. Antibodies were coupled to 4.5-microm polystyrene beads through the Fc portion by the use of protein A. The antibody-coupled beads showed a high capacity to bind HLA-A and -B proteins as well as their alpha chains by the intracytoplasmic domain, keeping the extracellular domains solvent exposed. The density of HLA class I proteins bound on the beads was approximately the same as that on cultured B cells. The antibody beads made it possible to quantitate peptide-HLA class I binding, i.e., in vitro HLA class I assembly by flow cytometry. The assembly rate determined by the provisionally called flow cytometric HLA class I assay was 15%-19% for the reassembly of dissociated HLA class I proteins with the released selfpeptides. With single synthetic peptides, the highest rate so far obtained was 6.5%. The assay specificity and reproducibility were satisfactory.

  19. A complementary role of multiparameter flow cytometry and high-throughput sequencing for minimal residual disease detection in chronic lymphocytic leukemia: an European Research Initiative on CLL study.

    LENUS (Irish Health Repository)

    Rawstron, A C

    2016-04-01

    In chronic lymphocytic leukemia (CLL) the level of minimal residual disease (MRD) after therapy is an independent predictor of outcome. Given the increasing number of new agents being explored for CLL therapy, using MRD as a surrogate could greatly reduce the time necessary to assess their efficacy. In this European Research Initiative on CLL (ERIC) project we have identified and validated a flow-cytometric approach to reliably quantitate CLL cells to the level of 0.0010% (10(-5)). The assay comprises a core panel of six markers (i.e. CD19, CD20, CD5, CD43, CD79b and CD81) with a component specification independent of instrument and reagents, which can be locally re-validated using normal peripheral blood. This method is directly comparable to previous ERIC-designed assays and also provides a backbone for investigation of new markers. A parallel analysis of high-throughput sequencing using the ClonoSEQ assay showed good concordance with flow cytometry results at the 0.010% (10(-4)) level, the MRD threshold defined in the 2008 International Workshop on CLL guidelines, but it also provides good linearity to a detection limit of 1 in a million (10(-6)). The combination of both technologies would permit a highly sensitive approach to MRD detection while providing a reproducible and broadly accessible method to quantify residual disease and optimize treatment in CLL.

  20. Enceladus' Enigmatic Heat Flow

    Science.gov (United States)

    Howett, C.; Spencer, J. R.; Spencer, D.; Verbiscer, A.; Hurford, T.; Segura, M.

    2013-12-01

    Accurate knowledge of Enceladus' heat flow is important because it provides a vital constraint on Enceladus' tidal dissipation mechanisms, orbital evolution, and the physical processes that generate the plumes. In 2011 we published an estimate of the current heat flow from Enceladus' active south polar terrain: 15.8 +/- 3.1 GW (Howett et al., 2011). This value was calculated by first estimating by modeling, and then removing, the passive component from 17 to 1000 micron observations made of the entire south polar terrain by Cassini's Composite Infrared Spectrometer (CIRS). The heat flow was then directly calculated from the residual, assumed endogenic, component. The derived heat flow of 15.8 GW was surprisingly high, about 10 times greater than that predicted by steady-state tidal heating (Meyer and Wisdom, 2007). CIRS has also returned high spatial resolution observations of Enceladus' active south polar terrain. Two separate observations are used: 9 to 16 micron observations taken over nearly the complete south polar terrain and a single 17 to 1000 micron scan over Damascus, Baghdad and Cairo. The shorter wavelength observations are only sensitive to high temperature emission (>70 K), and so longer wavelength observations are required (despite their limited spatial coverage) to estimate the low temperature emission from the stripes. Analysis of these higher resolution observations tells a different story of Enceladus' endogenic heat flow: the preliminary estimate of the heat flow from the active tiger stripes using these observations is 4.2 GW. An additional 0.5 GW must be added to this number to account for the latent heat release by the plumes (Ingersoll and Pankine 2009), giving a total preliminary estimate of 4.9 GW. The discrepancy in these two numbers is significant and we are currently investigating the cause. One possible reason is that there is significantly higher endogenic emission from the regions between the tiger stripes than we currently estimate