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Sample records for flow cytometric parasite

  1. 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

  2. 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

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

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    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

  4. 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

    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...... 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...

  5. 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, pr...

  6. Recent advances in flow cytometric cell sorting.

    Science.gov (United States)

    Osborne, Geoffrey W

    2011-01-01

    The classification and separation of one cell type or particle from others is a fundamental task in many areas of science. Numerous techniques are available to perform this task; however, electrostatic cell sorting has gained eminence over others because, when combined with the analysis capabilities of flow cytometry it provides flexible separations based on multiple parameters. Unlike competing technologies, such as gradient or magnetic separations that offer much larger total throughput, flow cytometric cell sorting permits selections based on various levels of fluorescent reporters, rather the complete presence or absence of the reporter. As such, this technology has found application in a huge range of fields. This chapter aims to describe the utility of single-cell sorting with particular emphasis given to index sorting. This is followed by two recently developed novel techniques of sorting cells or particles. The first of these is positional sorting which is useful in cell-based studies where sorting can proceed and produce meaningful results without being inherently dependant on prior knowledge of where gates should be set. Secondly, reflective plate sorting is introduced which positionally links multiwell sample and collection plates in a convenient assay format so that cells in the collection plate "reflect" those in the sample plate.

  7. Howard University Flow Cytometric Sorter For Research and Education

    Science.gov (United States)

    2015-08-04

    SECURITY CLASSIFICATION OF: Howard University’s newly acquired Fluorescence Activated Cytometric Sorter (FACS) has been integrated into the new flow...Research Administrative Services Washington, DC 20059 -0001 11-Mar-2015 ABSTRACT Number of Papers published in peer-reviewed journals : Number of Papers...published in non peer-reviewed journals : Final Report: Howard University Flow Cytometric Sorter For Research and Education Report Title Howard

  8. Two-Color Flow Cytometric Analysis of Intraerythrocytic Malaria Parasite DNA and Surface Membrane-Associated Antigen in Erythrocytes Infected with Plasmodium falciparum

    Science.gov (United States)

    1993-01-01

    Infected erythrocytes were fixea with 0.025% glutaraidehyde, followed by treatment with 1%saponiu to gain acceiss to intramembranous components and...Antigen in Erythrocytes Infected With Plasmodium falciparum 1 Kovit Pattanapanyasat,2 Rachanee Udomsangpetch, and H. Kyle Webster The Thalassemia Center...glutaral- izonts. Simultaneous measurement of dehyde followed by treatment with 1% parasite DNA and antigen in the infected saponin to gain access to

  9. Flow cytometric enumeration of marine viral populations at low abundances

    NARCIS (Netherlands)

    Mojica, K.D.A.; Evans, C.; Brussaard, C.P.D.

    2014-01-01

    Flow cytometric enumeration has advanced our ability to analyze aquatic virus samples and thereby our understanding of the ecological role viruses play in the oceans. However, low virus abundances are underestimated using the current flow cytometry (FCM) protocol. Our results revealed that low

  10. 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 automat

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

    NARCIS (Netherlands)

    Frita, R.; Rebelo, M.; Pamplona, A.; Vigario, A.M.; Mota, M.M.; Grobusch, M.P.; Haenscheid, T.

    2011-01-01

    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 accurat

  12. Technical discussions II - Flow cytometric analysis

    NARCIS (Netherlands)

    Cunningham, A; Cid, A; Buma, AGJ

    1996-01-01

    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 sp

  13. Technical discussions II - Flow cytometric analysis

    NARCIS (Netherlands)

    Cunningham, A; Cid, A; Buma, AGJ

    1996-01-01

    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 sp

  14. Flow cytometric detection of aberrant chromosomes

    Energy Technology Data Exchange (ETDEWEB)

    Gray, J.W.; Lucas, J.; Yu, L.C.; Langlois, R.

    1983-05-11

    This report describes the quantification of chromosomal aberrations by flow cytometry. Both homogeneously and heterogeneously occurring chromosome aberrations were studied. Homogeneously occurring aberrations were noted in chromosomes isolated from human colon carcinoma (LoVo) cells, stained with Hoechst 33258 and chromomycin A3 and analyzed using dual beam flow cytometry. The resulting bivariate flow karyotype showed a homogeneously occurring marker chromosome of intermediate size. Heterogeneously occurring aberrations were quantified by slit-scan flow cytometry in chromosomes isolated from control and irradiated Chinese hamster cells and stained with propidium iodide. Heterogeneously occurring dicentric chromosomes were detected by their shapes (two centrometers). The frequencies of such chromosomes estimated by slit-scan flow cytometry correlated well with the frequencies determined by visual microscopy.

  15. Flow cytometric studies of human osteosarcoma.

    Science.gov (United States)

    Mankin, H J; Gebhardt, M C; Springfield, D S; Litwak, G J; Kusazaki, K; Rosenberg, A E

    1991-09-01

    A number of recent studies have emphasized the potential value of flow cytometry as a "marker" to assess the malignity and therefore to help predict the biologic behavior of neoplasms, including bone tumors. Using propidium iodide and a home-built flow cytometer, the authors have studied the DNA distribution in 95 patients with osteosarcoma and determined the percentage of cells in diploidy, S-phase, tetraploidy, and aneuploidy. Using these values and a derived one, mean DNA concentration, it was possible to demonstrate the extent of the abnormalities observed in this group of neoplasms and show their severity as compared with the normal pattern. When the data are compared against disease-free survival and total survival, correlations were noted that, although weak, suggested that some patterns were predictive of increased risk of metastasis and death. The effect of treatment could also be assessed by evaluating the pattern before and after chemotherapy and correlating these with survival. It seems likely that with some improvement in technology, flow cytometry will be of value in the future in assessing the prognosis for osteosarcoma and predicting whether treatment has been effective.

  16. Flow cytometric detection method for DNA samples

    Energy Technology Data Exchange (ETDEWEB)

    Nasarabadi,Shanavaz (Livermore, CA); Langlois, Richard G. (Livermore, CA); Venkateswaran, Kodumudi S. (Round Rock, TX)

    2011-07-05

    Disclosed herein are two methods for rapid multiplex analysis to determine the presence and identity of target DNA sequences within a DNA sample. Both methods use reporting DNA sequences, e.g., modified conventional Taqman.RTM. probes, to combine multiplex PCR amplification with microsphere-based hybridization using flow cytometry means of detection. Real-time PCR detection can also be incorporated. The first method uses a cyanine dye, such as, Cy3.TM., as the reporter linked to the 5' end of a reporting DNA sequence. The second method positions a reporter dye, e.g., FAM.TM. on the 3' end of the reporting DNA sequence and a quencher dye, e.g., TAMRA.TM., on the 5' end.

  17. A novel flow cytometric hemozoin detection assay for real-time sensitivity testing of Plasmodium falciparum.

    Directory of Open Access Journals (Sweden)

    Maria Rebelo

    Full Text Available Resistance of Plasmodium falciparum to almost all antimalarial drugs, including the first-line treatment with artemisinins, has been described, representing an obvious threat to malaria control. In vitro antimalarial sensitivity testing is crucial to detect and monitor drug resistance. Current assays have been successfully used to detect drug effects on parasites. However, they have some limitations, such as the use of radioactive or expensive reagents or long incubation times. Here we describe a novel assay to detect antimalarial drug effects, based on flow cytometric detection of hemozoin (Hz, which is rapid and does not require any additional reagents. Hz is an optimal parasite maturation indicator since its amount increases as the parasite matures. Due to its physical property of birefringence, Hz depolarizes light, hence it can be detected using optical methods such as flow cytometry. A common flow cytometer was adapted to detect light depolarization caused by Hz. Synchronized in vitro cultures of P. falciparum were incubated for 48 hours with several antimalarial drugs. Analysis of depolarizing events, corresponding to parasitized red blood cells containing Hz, allowed the detection of parasite maturation. Moreover, chloroquine resistance and the inhibitory effect of all antimalarial drugs tested, except for pyrimethamine, could be determined as early as 18 to 24 hours of incubation. At 24 hours incubation, 50% inhibitory concentrations (IC50 were comparable to previously reported values. These results indicate that the reagent-free, real-time Hz detection assay could become a novel assay for the detection of drug effects on Plasmodium falciparum.

  18. Flow-cytometric Analysis of Bacillus anthracis Spores

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    D. V. Kamboj

    2006-11-01

    Full Text Available Flow-cytometric technique has been established as a powerful tool for detection andidentification of microbiological agents. Unambiguous and rapid detection of Bacillus anthracisspores has been reported using immunoglobulin G-fluorescein isothiocyanate conjugate againstlive spores. In addition to the high sensitivity, the present technique could differentiate betweenspores of closely related species, eg, Bacillus cereus and Bacillus subtilis using fluorescenceintensity. The technique can be used for detection of live as well as inactivated spores makingit more congenial for screening of suspected samples of bioterrorism.

  19. 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.

  20. Uncovering Aberrant Mutant PKA Function with Flow Cytometric FRET

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    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.

  1. An automated flow cytometric micronucleus assay for human lymphocytes.

    Science.gov (United States)

    Schreiber, G A; Beisker, W; Braselmann, H; Bauchinger, M; Bögl, K W; Nüsse, M

    1992-12-01

    A new flow cytometric method is presented for scoring micronuclei (MN) in human lymphocytes after in vitro gamma-irradiation. Fifty to fifty-five hours after PHA-stimulation, the frequency of micronuclei per nucleus and the fraction of cells in the second cell cycle were measured using flow cytometry. All data were automatically analysed using our DAS-software package. Eight individual linear-quadratic dose response curves derived from five donors revealed inter- and intra-individual variabilities of all curve parameters. Since also an age dependence was found for spontaneous MN-frequencies and for the linear curve parameter, a combined linear-quadratic age-dose-effect model was used to fit the data. The 90% prediction intervals show that a reliable individual dose estimation for donors aged between 23 and 54 years cannot be achieved for exposures below 1 Gy.

  2. Comparative flow cytometric analysis of immunofunctionalized nanowire and nanoparticle signatures.

    Science.gov (United States)

    Prina-Mello, Adriele; Whelan, Aine M; Atzberger, Ann; McCarthy, Joseph E; Byrne, Fiona; Davies, Gemma-Louise; Coey, J M D; Volkov, Yuri; Gun'ko, Yurii K

    2010-01-01

    Flow cytometry is one of the gold-standard techniques used in clinical medicine for quantitative immunoassaying. The continuous development of its probes, commonly fluorescent nanoparticles, is important. Lately, the introduction of quantitative multiplexed immunoassay has challenged the use of nanoparticles as probes. Functionalized fluorescent silica-based magnetic nanowires are investigated under flow cytometry as a novel probe category. The preparation and full characterization of these multimodal nanowires is reported and compared to those of silica-based magnetic nanoparticles by flow cytometry. Full characterization includes transmission electron microscopy and fluorescence microscopy imaging, flow cytometric assaying, superconducting quantum interference device (SQUID) magnetization, and Mössbauer spectroscopy measurements. This work shows that loaded silica nanowires have intrinsic geometrical advantages when compared to similar spherical particles due to their unique "flow cytometry fingerprint" when utilized as magnetic carriers for immunodetection applications. These advantages account for a 17% yield in detecting the functional binding between THP-1 and ICAM-1, by utilizing a much lower concentration than that required for the nanoparticles.

  3. Flow cytometric detection of anti-gliadin antibodies.

    Science.gov (United States)

    Presani, G; Perticarari, S; Mangiarotti, M A

    1989-05-12

    A very sensitive solid-phase fluorescent immunoassay to detect anti-alpha-gliadin IgA class antibodies is described. The solid phase consisted of polystyrene carboxylated microspheres, of 5 microns diameter, coated with alpha-gliadin. Serum-specific antibodies bound to the alpha-gliadin were measured by flow cytometry using fluorescein-conjugated anti-human IgA. 41 samples were tested and the results compared with those obtained by a standard method: an enzyme-linked immunosorbent assay (ELISA). A good correlation was found between the two techniques (r = 0.96). The sera of untreated coeliac children showed significantly higher antibody values than the sera of children on a gluten-free diet or healthy control groups. The flow cytometric method was more sensitive when the Kolgomorov/Smirnov test was used to analyse the histograms. This method provides an alternative screening test for coeliac disease and may also be used to confirm borderline results obtained in the ELISA test.

  4. Flow cytometric detection of immunoglobulin light chain in hematolymphoid immunophenotyping

    Institute of Scientific and Technical Information of China (English)

    Xu Dongsheng

    2011-01-01

    During B cell development and maturation, the antigen receptor,which is encoded by the immunoglobulin heavy-(IgH)and light chain genes,rearrange to associate one of a number of variable, diverse, and joining gene segments. A single mature Bcell expresses an IgH chain and either a kappa or lambda light chain, which is known as allelic or isotypic exclusion. In normal or reactive conditions, lymphoid cells comprise the mixtures of lymphocytes with either kappa or lambda expression. The norreal proportion of kappa to lambda (κ/λ ratio) is within the range of 0. 5 - 3. 0 in peripheral blood or bone marrow and 1.2 2.7 in lymph nodes[1].The most useful feature for diagnosing mature B cell neoplasm is light chain restriction or monotypic staining with κ or λ light chain. Currently, it is a common assumption that demonstration of light chain restriction in a B lymphocyte population is generally considered proof of monoclonality and indicates malignancy although monotypic B cell populations have been infrequently demonstrated in patients with no definitive evidence ofB cell malignancy[2-4]. The most common flow cytometric analysis for determining B cell monotype is the percent κ and λimmunoglobulin light chains.Because of the importance of light chain restriction in the diagnosis of B cell neoplasm, anti immunoglobulin antibodies (e. g. anti-κ and anti-λ) are vital tools in the detection of monotypic B cell populations. Accurate determination of surface light chain expression depends on many factors, such as proper washing procedure, lysing solution, type of antibody used, specimen type or lymphoma type[5]. This article will discuss some common problems encountered in flow cytometric(FCM)deterruination of surface immunoglobulin light chain expression inhematolymphoid immunophenotyping.%@@ During B-cell development and maturation,the antigen receptor,which is encoded by the immunoglobulin heavy-(IgH) and light-chain genes,rearrange to associate one of a number of

  5. Flow cytometric and laser scanning microscopic approaches in epigenetics research.

    Science.gov (United States)

    Szekvolgyi, Lorant; Imre, Laszlo; Minh, Doan Xuan Quang; Hegedus, Eva; Bacso, Zsolt; Szabo, Gabor

    2009-01-01

    Our understanding of epigenetics has been transformed in recent years by the advance of technological possibilities based primarily on a powerful tool, chromatin immunoprecipitation (ChIP). However, in many cases, the detection of epigenetic changes requires methods providing a high-throughput (HTP) platform. Cytometry has opened a novel approach for the quantitative measurement of molecules, including PCR products, anchored to appropriately addressed microbeads (Pataki et al. 2005. Cytometry 68, 45-52). Here we show selected examples for the utility of two different cytometry-based platforms of epigenetic analysis: ChIP-on-beads, a flow-cytometric test of local histone modifications (Szekvolgyi et al. 2006. Cytometry 69, 1086-1091), and the laser scanning cytometry-based measurement of global epigenetic modifications that might help predict clinical behavior in different pathological conditions. We anticipate that such alternative tools may shortly become indispensable in clinical practice, translating the systematic screening of epigenetic tags from basic research into routine diagnostics of HTP demand.

  6. Flow cytometric analysis of lectin binding to in vitro-cultured Perkinsus marinus surface carbohydrates

    Science.gov (United States)

    Gauthier, J.D.; Jenkins, J.A.; La Peyre, Jerome F.

    2004-01-01

    Parasite surface glycoconjugates are frequently involved in cellular recognition and colonization of the host. This study reports on the identification of Perkinsus marinus surface carbohydrates by flow cytometric analyses of fluorescein isothiocyanate-conjugated lectin binding. Lectin-binding specificity was confirmed by sugar inhibition and Kolmogorov-Smirnov statistics. Clear, measurable fluorescence peaks were discriminated, and no parasite autofluorescence was observed. Parasites (GTLA-5 and Perkinsus-1 strains) harvested during log and stationary phases of growth in a protein-free medium reacted strongly with concanavalin A and wheat germ agglutinin, which bind to glucose-mannose and N-acetyl-D-glucosamine (GlcNAc) moieties, respectively. Both P. marinus strains bound with lower intensity to Maclura pomifera agglutinin, Bauhinia purpurea agglutinin, soybean agglutinin (N-acetyl-D-galactosamine-specific lectins), peanut agglutinin (PNA) (terminal galactose specific), and Griffonia simplicifolia II (GlcNAc specific). Only background fluorescence levels were detected with Ulex europaeus agglutinin I (L-fucose specific) and Limulus polyphemus agglutinin (sialic acid specific). The lectin-binding profiles were similar for the 2 strains except for a greater relative binding intensity of PNA for Perkinsus-1 and an overall greater lectin-binding capacity of Perkinsus-1 compared with GTLA-5. Growth stage comparisons revealed increased lectin-binding intensities during stationary phase compared with log phase of growth. This is the first report of the identification of surface glycoconjugates on a Perkinsus spp. by flow cytometry and the first to demonstrate that differential surface sugar expression is growth phase and strain dependent. ?? American Society of Parasitologists 2004.

  7. Flow cytometric immunophenotypic characteristics of plasma cell leukemia

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    Barbara Kruk

    2011-04-01

    Full Text Available The aim of this prospective study was to define the flow cytometric characteristics of simultaneously investigated bone marrow and peripheral blood plasma cells antigens expression in 36 plasma cell leukemia (PCL patients. The immunophenotypic profile of plasma cells was determined with a panel of monoclonal antibodies. The antigen expression intensity was calculated as relative fluorescence intensity (RFI. Bone marrow plasma cells showed expression of particular antigens in the following proportion of cases: CD49d 100%, CD29 94%, CD54 93%, CD44 83%, CD56 60%, CD18 26%, CD11b 29%, CD11a 19%, CD117 27%, CD71 30%, CD126 100% and CD19 0%, while the expression of those antigens on peripheral blood plasma cells was present in the following percentage of patients: CD49d 100%, CD29 96%, CD54 93%, CD44 95%, CD56 56%, CD18 50%, CD11b 53%, CD11a 29%, CD117 26%, CD71 28%, CD126 100% and CD19 0%. The expression of CD54 was significantly higher than that of adhesion molecules belonging to the integrin b2 family: CD11a, CD18 and CD11b, on both bone marrow and peripheral blood cells (p < 0.01. Expression of CD18, CD11a and CD11b was differential between two cell compartments: lower on bone marrow and higher on peripheral blood cells. We found that plasma cells in the bone marrow of patients with plasma cell leukaemia showed significantly greater granularity and size than those in the peripheral blood (p = 0.0001 and p = 0.04, respectively. However, no differences in cell size or granularity were revealed between bone marrow plasma cells from patients with PCL and multiple myeloma. In conclusion, impaired expression of adhesion molecules such as CD11a/CD18 (LFA-1 or CD56 may explain hematogenic dissemination characterizing PCL. The following pattern of adhesion molecule expression according to the proportion of plasma cells expressing a given antigen in peripheral blood and bone marrow and arranged in diminishing order may be established: CD49d > CD44 > CD54

  8. Cell kinetics in a model of artificial skin. An immunohistochemical and flow cytometric analysis

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    A Casasco

    2009-12-01

    Full Text Available Bioengineered organs raised in vitro are candidate substitutes for natural organs in biological, pharmacological and clinical applications. We have studied cell kinetics in a human skin equivalent (HSE using a combined immunohistochemical and flow cytometric approach. Morphological analysis has shown that, relative to unstimulated natural skin, cell proliferation mainly occurs in the basal layer of the epidermal equivalent. Immunohistochemical and flow cytometric measurements of the growth fraction suggested a cell turnover comparable to that of natural skin. Immunohistochemical labelling indices matched well with flow cytometric data. These observations are consistent with morphological and histochemical data demonstrating normal cell differentiation and tissue architecture in HSE and suggest that such HSE may be a usefull substitute for human skin.

  9. 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

  10. 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

    A simple procedure for long-term storage of cells for flow cytometric DNA analysis was developed and tested. The cells were stored as single cells or fine-needle aspirates suspended in a citrate buffer with dimethylsulfoxide (DMSO), or as small blocks of tissue from solid tumors. The cells were...

  11. Flow cytometric sorting of paraffin-embedded tumor tissues considerably improves molecular genetic analysis

    NARCIS (Netherlands)

    Jordanova, ES; Corver, WE; Vonk, MJ; Leers, MPG; Riemersma, SA; Schuuring, E; Kluin, PM

    2003-01-01

    The characterization of genetic aberrations in paraffin-embedded tumor material is impaired by contaminating normal cells. In the present study on the genetic causes of loss of HLA expression in diffuse large B-cell lymphoma (DLBCL), we compared the efficacy of microdissection with flow cytometric s

  12. Flow cytometric determination of circulating immune complexes with the indirect granulocyte phagocytosis test

    NARCIS (Netherlands)

    Terstappen, L.W.M.M.; Grooth, de B.G.; Nolten, G.M.J.; Napel, ten C.H.H.; Berkel, van W.; Greve, J.

    1985-01-01

    A method for the determination of circulating immune complexes (CIC) was adapted for flow cytometric analysis. Human granulocytes were used to phagocytose IgG-bearing CIC of serum from systemic lupus erythematosus (SLE) patients. A method for labeling the phagocytosed CIC with FITC-conjugated anti-h

  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 orthogona...

  14. 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)...

  15. A flow-cytometric method to evaluate eosinophil-mediated uptake of probiotic Lactobacillus reuteri.

    Science.gov (United States)

    Kraemer, Laura S; Brenner, Todd A; Krumholz, Julia O; Rosenberg, Helene F

    2017-03-27

    Eosinophils are resident leukocytes of gut mucosa. Here we present a combined flow cytometric-antibiotic protection assay to identify mouse eosinophils capable of bacterial uptake, specifically, Gram-positive Lactobacillus reuteri, in studies performed ex vivo. The assay may be adapted for use in vivo.

  16. Simultaneous preparation of RNA and nuclei for Northern blot and flow cytometric analysis

    Energy Technology Data Exchange (ETDEWEB)

    Tesfaigzi, J.; Jaramillo, R. [Inhalation Toxicology Research Inst., Albuquerque, NM (United States)

    1996-11-01

    Several methods have been developed to quantify RNA synthesis during the progression of the cell cycle. The rate of RNA synthesis can be detected during different stages of the cell cycle by staining cells with agents that intercalate with nucleic acids. For example, following staining of mammalian cells with acridin orange, the green and red fluorescence that correlates with DNA and RNA content, respectively, can be analyzed by flow cytometry. Increase in RNA content during the progression of cells through the cell cycle can be measured after staining with acridin orange. RNA synthesis resulting from the stimulation of quiescent cells with various growth factors has also been demonstrated by labeling cells with bromo-uridine and using the anti-bromo-deoxyuridine antibody. These methods allow measurement of the overall RNA content in cells; however, they do not allow the measurement of the levels of specific mRNAs throughout the cell cycle. Current methods to quantify specific mRNAs generally require the preparation of a large number of cells (5--10 {times} 10{sup 6} cells) to carry out flow cytometric analyses and to isolate RNA for Northern blot analysis or solution hybridization. In this report, the authors describe a method of simultaneously preparing RNA and nuclei for Northern blot and flow cytometric analyses, respectively. The minimum number of nuclei required to obtain flow cytometric data and the effect of conserving nuclei in methanol for several days are also presented.

  17. Results of external quality control study in flow cytometric acute lymphoblastic leukemia diagnostics

    Directory of Open Access Journals (Sweden)

    A. M. Popov

    2016-01-01

    Full Text Available Comparison of interpretation of acute lymphoblastic leukemia (ALL flow cytometric diagnostics data was the aim of the study. Immunophenotyping data obtained from 10 patients with ALL were analysed separately in 26 laboratories from Russian Federation and Kazahstan. Results comparison showed four main type of discordance: B-lineage ALL diagnostics during heavy bone marrow regeneration, great variability of T-ALL interpretation, complexity of ambiguous lineage acute leukemia and, finally, very different report types, unique for each laboratory. All these problems are the serious obstacles for standardization of flow cytometric ALL diagnostics in multicenter setting. Continuation of similar QC rounds following by consecutive discussions with further development of consensus diagnostic algorithm could be the first step for standardization of ALL immunophenotyping in Russian Federation and CIS countries.

  18. Flow cytometric assessment of viability of lactic acid bacteria

    NARCIS (Netherlands)

    Bunthof, C.J.; Bloemen, K.; Breeuwer, P.; Rombouts, F.M.; Abee, T.

    2001-01-01

    The viability of lactic acid bacteria is crucial for their applications as dairy starters and as probiotics. We investigated the usefulness of flow cytometry (FCM) for viability assessment of lactic acid bacteria. The esterase substrate carboxyfluorescein diacetate (cFDA) and the dye exclusion DNA b

  19. Flow cytometric DNA ploidy analysis of ovarian granulosa cell tumors

    NARCIS (Netherlands)

    D. Chadha; C.J. Cornelisse; A. Schabert (A.)

    1990-01-01

    textabstractAbstract The nuclear DNA content of 50 ovarian tumors initially diagnosed as granulosa cell tumors was measured by flow cytometry using paraffin-embedded archival material. The follow-up period of the patients ranged from 4 months to 19 years. Thirty-eight tumors were diploid or near-dip

  20. Detachment and flow cytometric quantification of seagrass-associated bacteria.

    Science.gov (United States)

    Trevathan-Tackett, Stacey; Macreadie, Peter; Ralph, Peter; Seymour, Justin

    2014-07-01

    A new protocol was developed to detach bacteria from seagrass tissue and subsequently enumerate cells using flow cytometry (FCM). A method involving addition of the surfactant Tween 80 and vortexing resulted in maximum detachment efficiency of seagrass attached bacteria, providing a robust protocol for precisely enumerating seagrass-associated bacteria with FCM. Using this approach we detected cell concentrations between 2.0×10(5) and 8.0×10(6)cells mg(-1) DW tissue.

  1. Strategies for immunophenotyping and purifying classical Hodgkin lymphoma cells from lymph nodes by flow cytometry and flow cytometric cell sorting.

    Science.gov (United States)

    Fromm, Jonathan R; Wood, Brent L

    2012-07-01

    Flow cytometry is an established technique to immunophenotype hematopoietic neoplasms. While the diagnosis of classical Hodgkin lymphoma (CHL) has commonly been made using paraffin sections, we have recently demonstrated that the neoplastic Hodgkin and Reed-Sternberg (HRS) cells of CHL can be identified by flow cytometry. Using 6- and 9-color flow cytometric assays, CHL can be immunophenotyped with 85-90% sensitivity and nearly 100% specificity. Analysis of this data requires using established gating strategies to help in the identification of putative HRS cell populations. Interestingly, HRS cells bind to reactive T cells (HRS-T cell rosetting) and this phenomenon can be identified and utilized diagnostically by flow cytometry. In addition, the reactive T cells of CHL show characteristic immunophenotypic changes by flow cytometry and these changes can suggest a diagnosis of CHL. Finally, these principles can be employed to rapidly purify HRS cells using flow cytometric cell sorting. This manuscript provides experimental protocols for immunophenotyping CHL by flow cytometry as well as purifying the HRS cells via flow cytometric cell sorting.

  2. Flow cytometric fluorescence lifetime analysis of DNA binding fluorochromes

    Energy Technology Data Exchange (ETDEWEB)

    Crissman, Harry A.; Cui, H. H. (H. Helen); Steinkamp, J. A.

    2002-01-01

    Most flow cytometry (FCM) applications monitor fluorescence intensity to quantitate the various cellular parameters; however, the fluorescence emission also contains information relative to the fluorescence lifetime. Recent developments in FCM (Pinsky et al., 1993; Steinkamp & Crissman, 1993; Steinkamp et al., 1993), provide for the measurement of fluorescence lifetime which is also commonly referred to as fluorescence decay, or the time interval in which a fluorochrome remains in the excited state. Many unbound fluorochromes have characteristic lifetime values that are determined by their molecular structure; however, when the probe becomes bound, the lifetime value is influenced by a number of factors that affect the probe interaction with a target molecule. Monitoring the changes in the lifetime of the probe yields information relating to the molecular conformation, the functional state or activity of the molecular target. In addition, the lifetime values can be used as signatures to resolve the emissions of multiple fluorochrome labels with overlapping emission spectra that cannot be resolved by conventional FCM methodology. Such strategies can increase the number of fluorochrome combinations used in a flow cytometer with a single excitation source. Our studies demonstrate various applications of lifetime measurements for the analysis of the binding of different fluorochromes to DNA in single cells. Data presented in this session will show the utility of lifetime measurements for monitoring changes in chromatin structure associated with cell cycle progression, cellular differentiation, or DNA damage, such as induced during apoptosis. Several studies show that dyes with specificity for nucleic acids display different lifetime values when bound to DNA or to dsRNA. The Phase Sensitive Flow Cytometer is a multiparameter instrument, capable of performing lifetime measurements in conjunction with all the conventional FCM measurements. Future modifications of this

  3. Flow cytometric data analysis of circulating progenitor cell stability.

    Science.gov (United States)

    Mahar, Ernestine A; Mou, Liping; Hayek, Salim S; Quyyumi, Arshed A; Waller, Edmund K

    2017-02-01

    A recent publication by Mekonnen et al. demonstrated that among women with non-obstructive coronary artery disease, higher levels of circulating progenitor cells in the blood (CPC), were associated with impaired coronary flow reserve [1]. We performed a quality control assessment of the stability of circulating blood progenitor cells in blood samples stored at 4 °C, to determine the time period during which blood samples can be analyzed and yield consistent data for progenitor cell content. Healthy volunteers (n=6) were recruited and underwent phlebotomy, and blood was stored in EDTA tubes at 4 °C. Flow cytometry was performed to quantitate progenitor cell subsets at 0-4 h, 24 h, and 48 h post phlebotomy. All processed samples were fixed with 1% Paraformaldehyde and 1,000,000 total data events were collected. We found no significant differences in PC data for both CD34+ (P=0.68 for one-way ANOVA) and CD34+/CD133+ (P=0.74 for one-way ANOVA).

  4. Flow cytometric data analysis of circulating progenitor cell stability

    Directory of Open Access Journals (Sweden)

    Ernestine A. Mahar

    2017-02-01

    We performed a quality control assessment of the stability of circulating blood progenitor cells in blood samples stored at 4 °C, to determine the time period during which blood samples can be analyzed and yield consistent data for progenitor cell content. Healthy volunteers (n=6 were recruited and underwent phlebotomy, and blood was stored in EDTA tubes at 4 °C. Flow cytometry was performed to quantitate progenitor cell subsets at 0–4 h, 24 h, and 48 h post phlebotomy. All processed samples were fixed with 1% Paraformaldehyde and 1,000,000 total data events were collected. We found no significant differences in PC data for both CD34+ (P=0.68 for one-way ANOVA and CD34+/CD133+ (P=0.74 for one-way ANOVA.

  5. 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.

  6. 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.

  7. IVBT-documented platelet function correlates with flow cytometric data.

    Science.gov (United States)

    Hoffmann, J; Bonacker, G; Kretschmer, V; Schulzki, T; Heimanns, J

    1996-12-01

    Thrombocytopenic patients with identical platelet counts often show different bleeding tendencies owing to significant differences in the platelet function. This could be demonstrated by the in vitro bleeding test (IVBT). Using flow cytometry, we tried to find characteristics of platelet antigen expression in order to explain these differences in function. Thirty patients with bone marrow hypoplasia receiving 65 platelet transfusions (mainly from a cell separator) were observed for 3 to 29 days. Size, granulation and fluorescence of platelet-rich plasma (n = 522 samples) were evaluated using monoclonal antibodies against GP IIIb (collagen receptor), GP IIb/IIIa (fibrinogen receptor) and GP Ib (thrombin receptor). We defined separate gates for each antibody using the results from 50 normals and by laying an orthograde cross over the gate to divide the gate into four equal quadrants. The platelet populations were divided into four different groups according to the occlusion time (OT) of the IVBT and the Simplate time (ST). The thrombocytes with the most impaired function (OT > or = 485 s/ST > 30 min) had significantly less platelet fluorescence when marked with antibodies against GP IIIb and GP Ib than those with short OT and ST (OT platelet fluorescence when marked with anti-GP IIIb and anti-GP Ib than thrombocytopenic patients, who had a spontaneous platelet rise beyond 30,000 platelets/microliters a few days later. One day after platelet transfusion, significantly more platelets with high GP IIIb and Ib expression could be found. We were also able to document better transfusion efficacy of platelet concentrates with high GP IIIb and Ib expression. Finally, patients with high bleeding scores showed less GP Ib expression on the platelets than patients with low bleeding scores. In summary, the IVBT-documented platelet function clearly corresponded to an increased expression of the collagen receptor and the thrombin receptor of platelets.

  8. 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

  9. 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.

  10. 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 (.... 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...

  11. Two and three-color fluorescence flow cytometric analysis of immunoidentified viable bacteria.

    Science.gov (United States)

    Barbesti, S; Citterio, S; Labra, M; Baroni, M D; Neri, M G; Sgorbati, S

    2000-07-01

    Traditional culture methods well established in the past and still in use are not able to detect the environmental microorganisms that exist in a viable but not culturable state. A number of different fluorescence-based assays have been developed over the past decade to detect and identify viable bacteria in the environment. We have developed a simple and rapid method for measuring the number and viability of immunolabeled bacteria by means of a two/three color fluorescence flow cytometric analysis. After washing, cultured bacteria in suspension were labeled with a rabbit polyclonal antibody recognizing the wall lipopolysaccharide complex. A secondary biotinylated anti-rabbit polyclonal antibody was added allowing the cells to be labeled with the streptavidin R-phycoerythrin-Cyanine 5 (RPE-Cy5) fluorochrome. Before flow cytometric analysis, bacterial suspensions were stained with SYBR Green I and propidium iodide which stain all of the cells and the non viable ones, respectively. With the appropriate filter sets of both Bryte-HS (Bio-Rad, Hercules, CA) and FACScan (Becton Dickinson, San Jose, CA) flow cytometers, the measurement of separated green (SYBR Green I), orange-red (propidium iodide), and far red (RPE-Cy5) fluorescence was possible, allowing the enumeration of viable immunodetected bacteria. The entire protocol is completed in less than 3 h, offering numerous possibilities for rapid and precise analyses in sanitary, industrial, and environmental microbiology. Copyright 2000 Wiley-Liss, Inc.

  12. Flow cytometric methods to investigate culture heterogeneities for plant metabolic engineering.

    Science.gov (United States)

    Gaurav, Vishal; Kolewe, Martin E; Roberts, Susan C

    2010-01-01

    Plant cell cultures provide an important method for production and supply of a variety of natural products, where conditions can be easily controlled, manipulated, and optimized. Development and optimization of plant cell culture processes require both bioprocess engineering and metabolic engineering approaches. Cultures are generally highly heterogeneous, with significant variability amongst cells in terms of growth, metabolism, and productivity of key metabolites. Taxus cultures produce the important anti-cancer agent Taxol((R)) (i.e., paclitaxel) and have demonstrated significant variability amongst cell populations in culture with regard to paclitaxel accumulation, cell cycle participation, and protein synthesis. To fully understand the link between cellular metabolism and culture behavior and to enable targeted metabolic engineering approaches, cultures need to be studied at a single cell level. This chapter describes the application of plant cell flow cytometric techniques to investigate culture heterogeneity at the single cell level, in order to optimize culture performance through targeted metabolic engineering. Flow cytometric analytical methods are described to study Taxus single cells, protoplasts, and nuclei suspensions with respect to secondary metabolite accumulation, DNA content, cell size, and complexity. Reproducible methods to isolate these single particle suspensions from aggregated Taxus cultures are discussed. Methods to stain both fixed and live cells for a variety of biological markers are provided to enable characterization of cell phenotypes. Fluorescence-activated cell sorting (FACS) methods are also presented to facilitate isolation of certain plant cell culture populations for both analysis and propagation of superior cell lines for use in bioprocesses.

  13. Flow cytometric analysis of microbial contamination in food industry technological lines – initial study

    Directory of Open Access Journals (Sweden)

    Katarzyna Czaczyk

    2012-06-01

    Full Text Available Background. Flow cytometry constitutes an alternative for traditional methods of microorganisms identifi cation 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 signifi cant 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 fl ow cytometry seems to constitute an ideal tool for the assessment of microbial contamination on the surface of technological lines. Material and methods. 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 fl ow cytometer. The measured parameters were forward and side scatter of laser light signals allowing the estimation of microbial cell contents in each sample. Results. 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. Conclusions. 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 laborsaving detection of microbial contamination in food industry.

  14. Flow-Cytometric Isolation of Human Antibodies from a Nonimmune Saccharomyces cerevisiae Surface Display Library

    Energy Technology Data Exchange (ETDEWEB)

    Feldhaus, Michael (BATTELLE (PACIFIC NW LAB)); Siegel, Robert W.(BATTELLE (PACIFIC NW LAB)); Opresko, Lee (BATTELLE (PACIFIC NW LAB)); Coleman, James R.(BATTELLE (PACIFIC NW LAB)); Feldhaus, Jane M.(BATTELLE (PACIFIC NW LAB)); Yeung, Yik A.(Massachusetts Institute Of Tec); Cochran, Jennifer R.(Massachusetts Institute Of Tec); Heinzelman, Peter (Massachusetts Institute Of Tec); Colby, David (Massachusetts Institute Of Tec); Swers, Jeffrey (Massachusetts Institute Of Tec); Graff, Christilyn (Massachusetts Institute Of Tec); Wiley, H Steven (BATTELLE (PACIFIC NW LAB)); Wittrup, K D.(Massachusetts Institute Of Tec)

    2003-02-28

    A nonimmune library of 109 human antibody scFv fragments has been cloned and expressed on the surface of yeast, and nanomolar-affinity scFvs routinely obtained by magnetic bead screening and flow cytometric sorting. The yeast library can be amplified 1010-fold without measurable loss of clonal diversity, enabling effectively indefinite expansion of the library. The expression, stability, and antigen binding properties of more than 50 isolated scFv clones were assessed directly on the yeast cell surface by immunofluorescent labeling and flow cytometry, obviating separate subcloning, expression, and purification steps and thereby expediting the isolation of novel affinity reagents. The ability to use multiplex library screening demonstrates the utility of this approach for high throughput antibody isolation for proteomics applications.

  15. Procarbazine effects on spermatogenesis in golden hamster: a flow cytometric evaluation.

    Science.gov (United States)

    Weissenberg, R; Golan, R; Shochat, L; Lewin, L M

    2002-01-01

    The response of hamster testis to the administration of 450mg/kg procarbazine (PCB) over a period of 4 weeks was evaluated. Flow cytometry was used to investigate changes in cell populations in testicular single cell suspensions and to correlate these changes with those observed in histological sections. PCB caused significant decrease in testicular and epididymal weight and a drastic reduction in haploid cells and spermatogenic arrest, demonstrating variation among the test animals. The results obtained confirm previous observations concerning detrimental effects of PCB upon spermatogenesis in species such as the rat and mouse, though its effect on hamster testis is milder and does not include the germinal stem cells. The histological evaluation of the testis showed a good correlation with flow cytometric evaluation, emphasizing the usefulness of this method in providing quantitative and rapid results.

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

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Hye-Weon; Kim, In S. [School of Environmental Science and Engineering, Gwangju Institute of Science and Technology (GIST), 261 Cheomdan-gwagiro, Buk-gu, Gwangju (Korea, Republic of); Niessner, Reinhard [Chair for Analytical Chemistry, Institute of Hydrochemistry, Technische Universitaet Muenchen, Marchioninistrasse 17, 81377 Muenchen (Germany); Knopp, Dietmar, E-mail: dietmar.knopp@ch.tum.de [Chair for Analytical Chemistry, Institute of Hydrochemistry, Technische Universitaet Muenchen, Marchioninistrasse 17, 81377 Muenchen (Germany)

    2012-10-31

    Highlights: Black-Right-Pointing-Pointer First time, duplex competitive bead-based flow cytometric immunoassay was developed using ODs. Black-Right-Pointing-Pointer Antibody-coated QD detection probes and antigen-immobilized microspheres were synthesized. Black-Right-Pointing-Pointer The two model target analytes were low molecular weight compounds of microbial and chemical origin. Black-Right-Pointing-Pointer 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{sub 50} values of 5 {mu}g L{sup -1} and 1.1 {mu}g L{sup -1} and dynamic ranges of 0.52-30 {mu}g L{sup -1} and 0

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

    Science.gov (United States)

    Yazıcı, Serkan; Bülbül Başkan, Emel; Budak, Ferah; Oral, Barbaros; Adim, Şaduman Balaban; Ceylan Kalin, Zübeyde; Özkaya, Güven; Aydoğan, Kenan; Saricaoğlu, Hayriye; Tunali, Şükran

    2015-01-01

    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. PMID:26788525

  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. A Flow Cytometric and Computational Approaches to Carbapenems Affinity to the Different Types of Carbapenemases

    Science.gov (United States)

    Pina-Vaz, Cidália; Silva, Ana P.; Faria-Ramos, Isabel; Teixeira-Santos, Rita; Moura, Daniel; Vieira, Tatiana F.; Sousa, Sérgio F.; Costa-de-Oliveira, Sofia; Cantón, Rafael; Rodrigues, Acácio G.

    2016-01-01

    The synergy of carbapenem combinations regarding Enterobacteriaceae producing different types of carbapenemases was study through different approaches: flow cytometry and computational analysis. Ten well characterized Enterobacteriaceae (KPC, verona integron-encoded metallo-β-lactamases –VIM and OXA-48-like enzymes) were selected for the study. The cells were incubated with a combination of ertapenem with imipenem, meropenem, or doripenem and killing kinetic curves performed with and without reinforcements of the drugs. A cephalosporin was also used in combination with ertapenem. A flow cytometric assay with DiBAC4-(3), a membrane potential dye, was developed in order to evaluate the cellular lesion after 2 h incubation. A chemical computational study was performed to understand the affinity of the different drugs to the different types of enzymes. Flow cytometric analysis and time-kill assays showed a synergic effect against KPC and OXA-48 producing-bacteria with all combinations; only ertapenem with imipenem was synergic against VIM producing-bacteria. A bactericidal effect was observed in OXA-48-like enzymes. Ceftazidime plus ertapenem was synergic against ESBL-negative KPC producing-bacteria. Ertapenem had the highest affinity for those enzymes according to chemical computational study. The synergic effect between ertapenem and others carbapenems against different carbapenemase-producing bacteria, representing a therapeutic choice, was described for the first time. Easier and faster laboratorial methods for carbapenemase characterization are urgently needed. The design of an ertapenem derivative with similar affinity to carbapenemases but exhibiting more stable bonds was demonstrated as highly desirable. PMID:27555844

  20. A flow cytometric and computational approaches to carbapenems affinity to the different types of carbapenemases

    Directory of Open Access Journals (Sweden)

    Cidália Pina-Vaz

    2016-08-01

    Full Text Available The synergy of carbapenem combinations regarding Enterobacteriaceae producing different types of carbapenemases was study through different approaches: flow cytometry and computa-tional analysis. Ten well characterized Enterobacteriaceae (KPC, verona integron-encoded metallo-β-lactamases –VIM and OXA-48-like enzymes were selected for the study. The cells were incubated with a combination of ertapenem with imipenem, meropenem or doripenem and killing kinetic curves performed with and without reinforments of the drugs. A cephalosporin was also used in combination with ertapenem. A flow cytometric assay with DiBAC4-(3, a membrane potential dye, was developed in order to evaluate the cellular lesion after 2 h incuba-tion. A chemical computational study was performed to understand the affinity of the different drugs to the different types of enzymes. Flow cytometric analysis and time-kill assays showed a synergic effect against KPC and OXA-48 producing-bacteria with all combinations; only ertapenem with imipenem was synergic against VIM producing-bacteria. A bactericidal effect was observed in OXA-48-like enzymes. Ceftazidime plus ertapenem was synergic against ESBL-negative KPC producing-bacteria. Ertapenem had the highest affinity for those enzymes according to chemical computational study. The synergic effect between ertapenem and others carbapenems against different carbapenemase-producing bacteria, representing a therapeutic choice, was described for the first time. Easier and faster laboratorial methods for car-bapenemase characterization are urgently needed. The design of an ertapenem derivative with similar affinity to carbapenemases but exhibiting more stable bonds was demonstrated as highly desirable.

  1. 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.

  2. A flow cytometric in vivo chalone assay using retransplanted old murine JB-1 ascites tumour cells.

    Science.gov (United States)

    Barfod, N M

    1981-07-01

    A flow cytometric in vivo chalone assay is described. Transplantation of old JB-1 ascites tumour cells to new hosts induced an influx of tumour cells, with G1 DNA content, to the S phase. This induction could be reversibly and specifically blocked by injections of an ultrafiltrate of old JB-1 ascites fluid. The method described is superior to a previously published in vivo chalone assay using regenerating ascites tumours. Owing to a reduced variability in time of onset of DNA synthesis, a smaller scatter of observations is achieved and thus the number of mice per group may be reduced using the new method. In contrast to the older technique, the present one does not necessitate killing of mice during the observation period.

  3. Standardizing flow cytometric assays in long-term population-based studies

    Science.gov (United States)

    Melzer, Susanne; Bocsi, Jozsef; Tárnok, Attila

    2015-03-01

    Quantification of leukocyte subpopulations and characterization of antigen-expression pattern on the cellular surface can play an important role in diagnostics. The state of cellular immunology on the single-cell level was analyzed by polychromatic flow cytometry in a recent comparative study within the average Leipzig population (LIFE - Leipzig Research Centre for Civilization Diseases). Data of 1699 subjects were recorded over a long-time period of three years (in a total of 1126 days). To ensure compatibility of such huge data sets, quality-controls on many levels (stability of instrumentation, low intra-laboratory variance and reader independent data analysis) are essential. The LIFE study aims to analyze various cytometric pattern to reveal the relationship between the life-style, the environmental effects and the individual health. We therefore present here a multi-step quality control procedure for long-term comparative studies.

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

    Science.gov (United States)

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

    1991-06-01

    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.

  5. 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 bind

  6. 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 bind

  7. 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...

  8. Clonal heterogeneity of small-cell anaplastic carcinoma of the lung demonstrated by flow-cytometric DNA analysis

    DEFF Research Database (Denmark)

    Vindeløv, L L; Hansen, H H; Christensen, I J

    1980-01-01

    Flow-cytometric DNA analysis yields information on ploidy and proliferative characteristics of a cell population. The analysis was implemented on small-cell anaplastic carcinoma of the lung using a rapid detergent technique for the preparation of fine-needle aspirates for DNA determination...

  9. A simple and sensitive flow cytometric assay for determination of the cytotoxic activity of human natural killer cells

    NARCIS (Netherlands)

    Radosevic, Katarina; Radosevic, K.; Garritsen, Henk S.P.; Garritsen, H.S.P.; van Graft, M.; van Graft, Marja; de Grooth, B.G.; Greve, Jan

    1990-01-01

    A new, simple and sensitive flow cytometric assay for the determination of the cytotoxic activity of human natural killer cells is described. The assay is based on the use of two fluorochromes. The target cell population is stained with one fluorochrome (octadecylamine-fluorescein isothiocyanate,

  10. Identification and purification of classical Hodgkin cells from lymph nodes by flow cytometry and flow cytometric cell sorting.

    Science.gov (United States)

    Fromm, Jonathan R; Kussick, Steven J; Wood, Brent L

    2006-11-01

    We demonstrate the feasibility of using flow cytometry (FC) to identify the Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (CHL). Initial flow cytometric studies of the HRS cell line L1236 demonstrated potentially useful antigens for identifying HRS cells. L1236 cells spontaneously bound normal T cells, analogous to the T-cell rosetting of HRS cells seen in tissue sections of CHL, but these interactions could be blocked by using a cocktail of unlabeled antibodies to 4 adhesion molecules. Among 27 lymph nodes involved by CHL, FC enabled HRS cells to be identified in 89%, whereas none of 29 non-CHL neoplasms or 23 reactive lymph nodes demonstrated HRS populations. Of the CHL cases, 82% demonstrated interactions between HRS cells and T cells that could be disrupted with blocking antibodies. Flow cytometric cell sorting experiments demonstrated typical HRS cytomorphologic features among the purified cells. FC may offer an alternative to immunohistochemical analysis in confirming the diagnosis of CHL in certain cases, and, through cell sorting, it provides a means of rapidly isolating pure HRS cells.

  11. Flow cytometric scoring of micronucleated erythrocytes: an efficient platform for assessing in vivo cytogenetic damage.

    Science.gov (United States)

    Dertinger, Stephen D; Torous, Dorothea K; Hayashi, Makoto; MacGregor, James T

    2011-01-01

    The relative simplicity of the micronucleated erythrocyte endpoint has made it amenable to automated scoring approaches. Flow cytometry is one such scoring platform that has been employed successfully. This review describes the evolution and properties of flow cytometry-based scoring of micronucleated erythrocytes. The methodology has become widely applied to rodent blood specimens and the high throughput nature of the technology provides a number of advantages over manual microscopic scoring. For instance, the ability to efficiently survey many dose levels and many more cells per specimen relative to microscopy benefits studies that are designed to identify no observable effect levels or lowest observable effect levels. Furthermore, flow cytometry makes it practical to study species with low spontaneous reticulocyte (RET) counts and micronucleus (MN) frequencies, thereby facilitating integration of blood-based micronucleated reticulocyte (MN-RET) frequency measurements into experiments conducted across species of toxicological interest. This capability enhances genotoxicity assessments that have historically been made in dedicated MN tests performed in one species. Importantly, the feasibility of using MN-RET frequencies in blood from humans as an index of genetic damage in bone marrow opens a critical area of application that had not been practical previously. We conclude with recommendations for additional work that is needed to more fully realise the potential of flow cytometric in vivo MN scoring.

  12. A new spreadsheet method for the analysis of bivariate flow cytometric data

    Directory of Open Access Journals (Sweden)

    Isacke Clare M

    2004-03-01

    Full Text Available Abstract Background A useful application of flow cytometry is the investigation of cell receptor-ligand interactions. However such analyses are often compromised due to problems interpreting changes in ligand binding where the receptor expression is not constant. Commonly, problems are encountered due to cell treatments resulting in altered receptor expression levels, or when cell lines expressing a transfected receptor with variable expression are being compared. To overcome this limitation we have developed a Microsoft Excel spreadsheet that aims to automatically and effectively simplify flow cytometric data and perform statistical tests in order to provide a clearer graphical representation of results. Results To demonstrate the use and advantages of this new spreadsheet method we have investigated the binding of the transmembrane adhesion receptor CD44 to its ligand hyaluronan. In the first example, phorbol ester treatment of cells results in both increased CD44 expression and increased hyaluronan binding. By applying the spreadsheet method we effectively demonstrate that this increased ligand binding results from receptor activation. In the second example we have compared AKR1 cells transfected either with wild type CD44 (WT CD44 or a mutant with a truncated cytoplasmic domain (CD44-T. These two populations do not have equivalent receptor expression levels but by using the spreadsheet method hyaluronan binding could be compared without the need to generate single cell clones or FACS sorting the cells for matching CD44 expression. By this method it was demonstrated that hyaluronan binding requires a threshold expression of CD44 and that this threshold is higher for CD44-T. However, at high CD44-T expression, binding was equivalent to WT CD44 indicating that the cytoplasmic domain has a role in presenting the receptor at the cell surface in a form required for efficient hyaluronan binding rather than modulating receptor activity. Conclusion

  13. The influence of fixation delay on mitotic activity and flow cytometric cell cycle variables.

    Science.gov (United States)

    Bergers, E; Jannink, I; van Diest, P I; Cuesta, M A; Meyer, S; van Mourik, J C; Baak, J P

    1997-01-01

    Proliferation variables such as mitotic activity and the percentage of S-phase cells have been shown to be of prognostic value in many tumors, especially in breast cancer. However, some studies reported a decrease in mitotic activity caused by delay in fixation of the tissue. In contrast, other studies showed that the identifiability of mitotic figures decreases after fixation delay, but the total number of mitotic figures and also the percentage of S-phase cells remain unchanged. Most studies have been done on small numbers of experimental tumors, thus introducing the risk of selection bias. The aim of this study was to reinvestigate the influence of fixation delay on mitotic activity and cell cycle variables assessed by flow cytometry in an adequate number of resected human tissues to reach firmer conclusions. Resection specimens of 19 and 21 cases, respectively, for the mitotic activity estimate and the flow cytometric percentage of S-phase calculation were collected directly from the operating theater using lung, breast, and intestinal cancers and normal intestinal mucosa. The tissues were cut in pieces, and from each specimen, pieces were fixed in 4% buffered formaldehyde (for mitosis counting) as well as snap frozen (for flow cytometry) immediately after excision, as well as after a fixation delay of 1, 2, 4, 6, 8, 18, and 24 hours. Moreover, during the fixation delay, one series from each specimen was kept in the refrigerator and the second at room temperature. Thus, a total of 304 (19 X 16) and 336 (21 X 16) specimens were investigated for the mitotic activity estimate and the percentage of S-phase cells calculation, respectively. With regard to the estimation of the mitotic activity, both clear and doubtful mitotic figures were registered separately, obtaining an "uncorrected" and "corrected" (for doubtful mitotic figures) mitotic activity estimate. The percentage of S-phase cells was obtained by cell cycle analysis of flow cytometric DNA-histograms. The

  14. 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.

  15. Evaluation of Red Cell Membrane Cytoskeletal Disorders Using a Flow Cytometric Method in South Iran

    Directory of Open Access Journals (Sweden)

    Habib Alah Golafshan

    2014-03-01

    Full Text Available OBJECTIVE: The diagnosis of hereditary red blood cell (RBC membrane disorders, and in particular hereditary spherocytosis (HS and Southeast Asian ovalocytosis (SAO, is based on clinical history, RBC morphology, and other conventional tests such as osmotic fragility. However, there are some milder cases of these disorders that are difficult to diagnose. The application of eosin-5’-maleimide (EMA was evaluated for screening of RBC membrane defects along with some other anemias. We used EMA dye, which binds mostly to band 3 protein and to a lesser extent some other membrane proteins, for screening of some membrane defects such as HS. METHODS: Fresh RBCs from hematologically normal controls and patients with HS, SAO, hereditary elliptocytosis, hereditary spherocytosis with pincered cells, severe iron deficiency, thalassemia minor, and autoimmune hemolytic anemia were stained with EMA dye and analyzed for mean fluorescent intensity (MFI using a flow cytometer. RESULTS: RBCs from patients with HS and iron deficiency showed a significant reduction in MFI compared to those from normal controls (p<0.0001 and p<0.001, respectively, while macrocytic RBCs showed a significant increase in MFI (p<0.01. A significant correlation was shown between mean corpuscular volume and MFI, with the exceptions of HS and thalassemia minor. CONCLUSION: Our results showed that the flow cytometric method could be a reliable diagnostic method for screening and confirmation, with higher sensitivity and specificity (95% and 93%, respectively than conventional routine tests for HS patients prior to further specific membrane protein molecular tests.

  16. Flow cytometric quantification of T cell proliferation and division kinetics in woodchuck model of hepatitis B.

    Science.gov (United States)

    Gujar, Shashi A; Michalak, Tomasz I

    2005-01-01

    Woodchucks infected with woodchuck hepatitis virus (WHV) represent the closest natural animal model to study the immunopathogenesis of liver injury caused by essentially noncytopathic, highly human specific hepatitis B virus (HBV). The importance of antiviral T cell response in induction of hepatitis and in control of HBV replication has been demonstrated. However, the understanding of how these responses contribute to the development of different immunomorphological forms of liver disease and their outcomes remain elusive. In this study, we established and standardized a flow cytometry assay using peripheral blood mononuclear cells labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) to assess WHV-specific and mitogen-driven T lymphocyte proliferative responses in woodchucks. The assay is of significantly greater sensitivity than the adenine incorporation assay currently used when applied to measure either WHV-specific T cell responses in acute (P measuring cell division rates. The study shows that woodchuck PBMC labeled with CFSE exhibit light scatter and fluorescence profiles compatible to those of human PBMC, allowing quantitation and deconvolution of the flow cytometric data by applying the existing analytical softwares. The availability of this novel assay should facilitate a more precise and comprehensive evaluation of hepadnavirus-specific and generalized T cell responses in experimental WHV hepatitis.

  17. Flow cytometric analysis of oil palm: a preliminary analysis for cultivars and genomic DNA alteration

    Directory of Open Access Journals (Sweden)

    Warawut Chuthammathat

    2005-12-01

    Full Text Available DNA contents of oil palm (Elaeis guineensis Jacq. cultivars were analyzed by flow cytometry using different external reference plant species. Analysis using corn (Zea mays line CE-777 as a reference plant gave the highest DNA content of oil palm (4.72±0.23 pg 2C-1 whereas the DNA content was found to be lower when using soybean (Glycine max cv. Polanka (3.77±0.09 pg 2C-1 or tomato (Lycopersicon esculentum cv. Stupicke (4.25±0.09 pg 2C-1 as a reference. The nuclear DNA contents of Dura (D109, Pisifera (P168 and Tenera (T38 cultivars were 3.46±0.04, 3.24±0.03 and 3.76±0.04 pg 2C-1 nuclei, respectively, using soybean as a reference. One haploid genome of oil palm therefore ranged from 1.56 to 1.81±109 base pairs. DNA contents from one-year-old calli and cell suspension of oil palm were found to be significantly different from those of seedlings. It thus should be noted that genomic DNA alteration occurred in these cultured tissues. We therefore confirm that flow cytometric analysis could verify cultivars, DNA content and genomic DNA alteration of oil palm using soybean as an external reference standard.

  18. Comparison of multidimensional flow cytometric data by a novel data mining technique

    Science.gov (United States)

    Leary, James F.; Smith, Jacob; Szaniszlo, Peter; Reece, Lisa M.

    2007-02-01

    Most flow/image cytometric data analysis methods look for clusters in the data corresponding to specific cell subpopulations. Comparisons between different cytometry datafiles often use human pattern recognition visualization of all the different combinations of variables ("parameters") two at a time in so-called bivariate scattergrams. Not only is this tedious, but it can miss potential clusters due to projection of higher dimensional dataspaces down onto two dimensional planes making them indiscernible as separate clusters. Novel data mining algorithms, implemented in software allow for the comparison of two or more higher dimensional datafiles without the requirement for reduction of dimensionality for human visualization. Of equal importance is the comparison of higher dimensional clusters which may move around slightly in space, yet still be "similar" according to algorithms which provide measures of similarity. This software, written in C/C++ and currently implemented in software with a Windows graphical user interface, allows for direct reading of FCS2.0 format flow cytometry datafiles of any number of parameters. In a few minutes or less, complex multiparameter data of two or more files can be compared on a personal computer or workstation. The software operates in either supervised or unsupervised mode, depending on whether the user wishes to include prior user knowledge or in a data mining discovery mode. Differences between these files can be exported as sub-datafiles which can be further analyzed using any other software that can read FCS2.0 data format.

  19. A new flow cytometric method for quantitative assessment of lymphocyte mitogenic potentials.

    Science.gov (United States)

    Yamamura, Y; Rodriguez, N; Schwartz, A; Eylar, E; Bagwell, B; Yano, N

    1995-01-01

    A new flow cytometric method was developed to quantitatively assess lymphocyte proliferation simultaneously for different subsets. The cells were stained with a fluorescent dye, PKH-26 and were stimulated with mitogens. The fluorescence intensities (FL2) of proliferating cells were measured by flow cytometry; and each subset was identified by the use of a monoclonal antibody (Mab)-fluorescein-isothiocyanate (FITC) (FL1). FL2 histograms were then analyzed by the cell proliferation model based on the ModFit software (Verity). This new method revealed information which could not be obtained by conventional mitogen assays. For example, the CD4+ and the CD4- T-subsets responded to phytohemagglutinin (PHA) quite differently from each other and it was indicated that activation of one population could significantly alter the response of the other. In addition, even within a subset, all activated cells did not proliferate uniformly. Some cells divided only once while others underwent further cellular division during the same time period. The method is, therefore, invaluable for studying the nature and the extent of interactions between different cellular subsets within a culture.

  20. Flow cytometric evaluation of the intracellular bacterium, Wolbachia pipientis, in mosquito cells

    Science.gov (United States)

    Fallon, Ann M

    2014-01-01

    Wolbachia is an obligate intracellular bacterium (Anaplasmataceae, Rickettisales) that occurs in arthropods and filarial worms, and spreads by vertical transmission in the oocyte cytoplasm. In insects, reproductive distortions associated with Wolbachia, such as cytoplasmic incompatibility in mosquitoes, have potential value for controlling pests, including species that transmit human, animal and plant diseases. Wolbachia strains that propagate as a persistent infection in insect cell lines provide an important resource for developing the genetic tools that will facilitate these applications. Here I describe conditions for flow cytometric evaluation of Wolbachia growth in persistently infected mosquito cells. Cytometry parameters were established using uninfected mosquito cells and Escherichia coli as a surrogate for Wolbachia, and quantitation was correlated with cell counts determined with a Coulter electronic cell counter and bacterial counts based on optical density. The protocol was validated by showing depletion of Wolbachia in medium containing tetracycline and rifampicin, and sensitivity of Wolbachia to treatment of host cells with paraquat, an oxidizing agent, and lumiflavin, an inhibitor of riboflavin uptake. The Wolbachia peak on the flow cytometry histogram was shown to contain Wolbachia by DNA analysis using the polymerase chain reaction, and by infection of naive recipient cells. This approach will streamline investigation of Wolbachia growth in insect cell lines and facilitate identification of culture conditions that select for Wolbachia-infected cells. PMID:25300665

  1. 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.

  2. Flow cytometric sorting coupled with exon capture sequencing identifies somatic mutations in archival lymphoma tissues.

    Science.gov (United States)

    Jiang, Nenggang; Chen, Christopher; Gong, Qiang; Shields, Kristen; Li, Yuping; Chen, YuanYuan; Song, Joo; McKeithan, Timothy W; Chan, Wing C

    2017-08-07

    The enormous number of archived formalin-fixed paraffin-embedded (FFPE) tissues available are a valuable resource of material for research. However, the use of such tissues poses many challenges, among which is the difficulty of isolating different cell populations within the tissue. In this study, we used tissue from two types of non-Hodgkin lymphoma as a model to demonstrate a method we have established and optimized to separate FFPE samples into distinct tumor and nonmalignant populations. Using FFPE reactive tonsil sections, various approaches for antigen retrieval and labeling, and the effectiveness of flow cytometric sorting were tested. We found that, among the 11 cell surface or intracellular antigen markers investigated, CD3ɛ, CD79A, LAT, PD-1, and PAX5 could be successfully labeled after antigen retrieval in Tris-EDTA buffer (pH 8.0) at 65 °C for 60 min, and 1.8-2.7 μg DNA per million cells could be extracted after sorting with DNA quality similar to that of tissue without staining or sorting. To test whether we could perform next-generation sequencing using a custom capture platform on sorted cells, we used three lymphoma cases with FFPE tissues which had been stored for 1 to 4 years. We demonstrated that the DNA from sorted cells was adequate for exon capture sequencing. By comparing the sequencing results between neoplastic and normal populations, somatic mutations could be clearly identified in the tumor population with variant frequencies as low as 11.7%.The corresponding normal fraction clearly helps in the analysis of somatic mutations and the exclusion of artifacts. This study provides an approach using flow cytometric sorting to separate different cellular populations in paraffin-embedded tissues and to unambiguously distinguish somatic mutations from germline variants or artifacts. This approach is also useful in enriching the tumor component in samples with heterogeneous components and low tumor content.Laboratory Investigation advance

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

    OpenAIRE

    Wade T. Rogers; Herbert A. Holyst

    2009-01-01

    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 i...

  4. Flow cytometric assessment of specific leucine incorporation in the open Mediterranean

    Science.gov (United States)

    Talarmin, A.; van Wambeke, F.; Catala, P.; Courties, C.; Lebaron, P.

    2011-02-01

    The surface of the Mediterranean Sea is a low-phosphate-low-chlorophyll marine area where marine heterotrophic prokaryotes significantly contribute to the biogeochemical cycles of all biogenic elements such as carbon, notably through the mineralization of dissolved organic compounds. Cell-specific leucine incorporation rates were determined in early summer in the open stratified Mediterranean Sea. The bulk leucine incorporation rate was on average 5 ± 4 pmol leu l-1 h-1 (n=30). Cell-specific 3H-leucine incorporation rates were assayed using flow cytometry coupled to cell sorting. Heterotrophic prokaryotes (Hprok) were divided into cytometric groups according to their side scatter and green fluorescence properties: high nucleic acid containing cells (HNA) with high scatter (HNA-hs) and low scatter (HNA-ls) and low nucleic acid containing cells (LNA). Cell-specific leucine incorporation rates of these cytometric groups ranged from 2 to 54, 0.9 to 11, and 1 to 12 × 10-21 mol cell-1 h-1, respectively. LNA cells represented 45 to 63% of the Hprok abundance, and significantly contributed to the bulk leucine incorporation rates, from 12 to 43%. HNA/LNA ratios of cell-specific leucine incorporation were on average 2.0 ± 0.7 (n=30). In surface layers (from 0 m down to the deep chlorophyll depth, DCM), cell-specific rates of HNA-hs were elevated (7 and 13 times greater than LNA and HNA-ls, respectively). Nevertheless, on average HNA-hs (26%) and LNA (27%) equally contributed to the bulk leucine incorporation in these layers. Prochlorococcus cells were easily sorted near the DCM and displayed cell-specific leucine incorporation rates ranging from 3 to 55 × 10-21 mol leu cell-1 h-1, i.e. as high as HNA-hs'. These sorted groups could therefore be defined as key-players in the process of leucine incorporation into proteins. The mixotrophic features of certain photosynthetic prokaryotes and the high contribution of LNA cells to leucine incorporation within the microbial

  5. Flow cytometric assessment of specific leucine incorporation in the open Mediterranean

    Directory of Open Access Journals (Sweden)

    A. Talarmin

    2011-02-01

    Full Text Available The surface of the Mediterranean Sea is a low-phosphate-low-chlorophyll marine area where marine heterotrophic prokaryotes significantly contribute to the biogeochemical cycles of all biogenic elements such as carbon, notably through the mineralization of dissolved organic compounds. Cell-specific leucine incorporation rates were determined in early summer in the open stratified Mediterranean Sea. The bulk leucine incorporation rate was on average 5 ± 4 pmol leu l−1 h−1 (n=30. Cell-specific 3H-leucine incorporation rates were assayed using flow cytometry coupled to cell sorting. Heterotrophic prokaryotes (Hprok were divided into cytometric groups according to their side scatter and green fluorescence properties: high nucleic acid containing cells (HNA with high scatter (HNA-hs and low scatter (HNA-ls and low nucleic acid containing cells (LNA. Cell-specific leucine incorporation rates of these cytometric groups ranged from 2 to 54, 0.9 to 11, and 1 to 12 × 10-21 mol cell−1 h−1, respectively. LNA cells represented 45 to 63% of the Hprok abundance, and significantly contributed to the bulk leucine incorporation rates, from 12 to 43%. HNA/LNA ratios of cell-specific leucine incorporation were on average 2.0 ± 0.7 (n=30. In surface layers (from 0 m down to the deep chlorophyll depth, DCM, cell-specific rates of HNA-hs were elevated (7 and 13 times greater than LNA and HNA-ls, respectively. Nevertheless, on average HNA-hs (26% and LNA (27% equally contributed to the bulk leucine incorporation in these layers. Prochlorococcus cells were easily sorted near the DCM and displayed cell-specific leucine incorporation rates ranging from 3 to 55 × 10-21 mol leu cell−1 h−1, i.e. as high as HNA-hs'. These sorted groups could therefore be defined as key-players in the process of leucine incorporation into proteins. The

  6. [Flow cytometric analysis of ICRF-193 influence on cell passage through mitosis].

    Science.gov (United States)

    Shatrova, A N; Aksenov, N D; Zenin, V V

    2002-01-01

    Studying the effect of topoisomerase II (topo II) inhibitors on cell passage through mitosis seems to be important for understanding the role of this enzyme during chromosome condensation and segregation. A flow cytometric assay (Zenin et al., 2001) allowed to determine the mitotic index, and to discriminate between not only cells in G2 and M phases (including metaphase and anaphase cells), but also cells in pseudo-G1 with 4c DNA content. It is shown that topo II catalytic inhibitor ICRF-193 blocks G2-M transition in a lymphoblastoid cell line GM-130. Addition of caffeine to cells abrogated a block of their entering mitosis but not the inhibitor action. Cells entered mitosis, which was proven by the presence of chromosomes in the examined specimen, and, bypassing anaphase, appeared in pseudo-G1 with 4c DNA content. We have found that in the presence of ICRF-193 cells, GM-130 and Hep-2 lines, previously blocked by nocodazole when in mitosis and then washed, pass through metaphase, enter anaphase and leave it to pass to pseudo-G1 with the 4c DNA content. Thus, by inhibiting topo II activity ICRF-193 causes abnormal mitotic transition.

  7. Establishment of multiplexed, microsphere-based flow cytometric assay for multiple human tumor markers

    Institute of Scientific and Technical Information of China (English)

    Kai SUN; Qian WANG; Xiao-hui HUANG; Mao-chuan ZHEN; Wen LI; Long-juan ZHANG

    2007-01-01

    Aim: The multiplexed, microsphere-based flow cytometric assay (MFCA) for mul- tiple human tumor markers was established for the early screening and detection of suspected cancer patients. Methods: Covalent coupling of capture antibodies directed against their respective tumor markers to fluorescent microspheres was performed by following the protocols recommended by a commercial corporation with some modifications. The coupling efficiency and cross-reactivity were iden- tified by the Luminex 100 system and associated software. The standard curve was constructed by using serial dilution of recombinant tumor marker standards and was validated by comparison with ELISA for quantifying the tumor markers in serum samples. Results: The identifications revealed that the coupling proce- dures were successful without non-specific cross-reactivity and the standard curve was highly efficient. However, it was necessary to ensure the quality con- trol of the coupling process since slight variations in the coupling procedures could profoundly affect the density of capture reagents coupled to the microspheres and consequently adversely affect the assay precision. In addition to its multi-analyte capability, the MFCA system had definite advantages, such as higher reproducibility, greater dynamic range of measurement, and considerably less preparation time and labor over the conventional "gold standard", which was the ELISA. Conclusion: The successful establishment of the MFCA system for the simultaneous detection of multiple tumor markers will provide the foundation for the further study of clinical applications.

  8. Flow cytometric quantitation of phagocytosis in heparinized complete blood with latex particles and Candida albicans

    Directory of Open Access Journals (Sweden)

    Jesús M. Egido

    1997-12-01

    Full Text Available We report a rapid method for the flow cytometric quantitation of phagocytosis in heparinized complete peripherial blood (HCPB, using commercially available phycoerythrin-conjugated latex particles of 1µm diameter. The method is faster and shows greater reproducibility than Bjerknes' (1984 standard technique using propidium iodide-stained Candida albicans, conventionally applied to the leukocytic layer of peripherial blood but here modified for HCPB. We also report a modification of Bjerknes' Intracellular Killing Test to allow its application to HCPB.Se da cuenta de un método rápido para la cuantización del flujo citométrico de la fagocitosis en sangre periférica completamente heparinizada (HCPB, mediante la utilización de partículas de látex phycoerythrin-conjugadas de 1µm de diámetro disponibles comercialmente. El método es más rápido y presenta mayor reproducibilidad que la técnica estandar de Bjerknes' (1984 utilizando propidium iodide-teñida Candida albicans, aplicada convencionalmente a la capa leucocitica de sangre periférica pero modificada por HCPB. Tambien damos cuenta de una modificación de Bjerknes' Intracellular Killing Test para permitir su aplicación a HCPB.

  9. 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.

  10. A Flow Cytometric Clonogenic Assay Reveals the Single-Cell Potency of Doxorubicin

    Science.gov (United States)

    Maass, Katie F.; Kulkarni, Chethana; Quadir, Mohiuddin A.; Hammond, Paula T.; Betts, Alison M.; Wittrup, K. Dane

    2015-01-01

    Standard cell proliferation assays use bulk media drug concentration to ascertain the potency of chemotherapeutic drugs; however, the relevant quantity is clearly the amount of drug actually taken up by the cell. To address this discrepancy, we have developed a flow cytometric clonogenic assay to correlate the amount of drug in a single cell with the cell’s ability to proliferate using a cell tracing dye and doxorubicin, a naturally fluorescent chemotherapeutic drug. By varying doxorubicin concentration in the media, length of treatment time, and treatment with verapamil, an efflux pump inhibitor, we introduced 105 – 1010 doxorubicin molecules per cell; then used a dye-dilution assay to simultaneously assess the number of cell divisions. We find that a cell’s ability to proliferate is a surprisingly conserved function of the number of intracellular doxorubicin molecules, resulting in single-cell IC50 values of 4 – 12 million intracellular doxorubicin molecules. The developed assay is a straightforward method for understanding a drug’s single-cell potency and can be used for any fluorescent or fluorescently-labeled drug, including nanoparticles or antibody-drug conjugates. PMID:26344409

  11. 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.

  12. Antiphospholipid antibody syndrome: the flow cytometric annexin A5 competition assay as a diagnostic tool.

    Science.gov (United States)

    Tomer, A; Bar-Lev, S; Fleisher, S; Shenkman, B; Friger, M; Abu-Shakra, M

    2007-10-01

    The mechanism underlying hypercoagulability in antiphospholipid antibody syndrome (APS) is uncertain. Here, we present a flow-cytometric assay (FCA) based on the hypothesis that anti-platelet-anionic-phospholipid autoantibodies (aPL) interfere with the activity of the natural anticoagulant protein annexin A5, thereby accelerating platelet procoagulant activity. This study assessed the clinical utility of the feasible FCA, which demonstrates the competition of the patient's aPL with the binding of annexin A5 to the platelet-anionic-phospholipids, in the diagnosis of APS. Sixty-two (94%) of 66 APS patients, 20 (51%) of 39 patients with systemic lupus erythematosus and two (4%) of 49 healthy individuals were positive by FCA. Compared with the anticardiolipin (aCL) assay, the relative sensitivity was 82% and the specificity 73.3%. However, 19 (25%) aCL-negative patients were positive by FCA; 12 were positive for lupus-anticoagulant (LA). Compared with LA assay, the relative sensitivity was 85% and the specificity 72.2%. However, 21 (26%) LA-negative patients were FCA-positive, 12 were positive for aCL. The FCA was particularly sensitive for APS patients with arterial (97.0%) and gestational vascular complications (100%) with overall sensitivity of 95% and specificity of 97%. Our findings suggest that the FCA is practical, sensitive and specific for the detection of clinically relevant aPL in the diagnosis of APS.

  13. Dissociation of skeletal muscle for flow cytometric characterization of immune cells in macaques.

    Science.gov (United States)

    Liang, Frank; Ploquin, Aurélie; Hernández, José DelaO; Fausther-Bovendo, Hugues; Lindgren, Gustaf; Stanley, Daphne; Martinez, Aiala Salvador; Brenchley, Jason M; Koup, Richard A; Loré, Karin; Sullivan, Nancy J

    2015-10-01

    The majority of vaccines and several treatments are administered by intramuscular injection. The aim is to engage and activate immune cells, although they are rare in normal skeletal muscle. The phenotype and function of resident as well as infiltrating immune cells in the muscle after injection are largely unknown. While methods for obtaining and characterizing murine muscle cell suspensions have been reported, protocols for nonhuman primates (NHPs) have not been well defined. NHPs comprise important in vivo models for studies of immune cell function due to their high degree of resemblance with humans. In this study, we developed and systematically compared methods to collect vaccine-injected muscle tissue to be processed into single cell suspensions for flow cytometric characterization of immune cells. We found that muscle tissue processed by mechanical disruption alone resulted in significantly lower immune cell yields compared to enzymatic digestion using Liberase. Dendritic cell subsets, monocytes, macrophages, neutrophils, B cells, T cells and NK cells were readily detected in the muscle by the classic human markers. The methods for obtaining skeletal muscle cell suspension established here offer opportunities to increase the understanding of immune responses in the muscle, and provide a basis for defining immediate post-injection vaccine responses in primates.

  14. Successful low dose insemination of flow cytometrically sorted ram spermatozoa in sheep.

    Science.gov (United States)

    de Graaf, S P; Evans, G; Maxwell, W M C; Downing, J A; O'Brien, J K

    2007-12-01

    The fertility of ram spermatozoa that had undergone flow cytometric sorting (MoFlo SX) and cryopreservation was assessed after low-dose insemination of synchronized Merino ewes. Oestrus was synchronized with progestagen-impregnated pessaries, PMSG and GnRH treatment. Ewes (n = 360) were inseminated with 1 x 10(6), 5 x 10(6) or 15 x 10(6) motile sorted frozen-thawed (S(1), S(5), or S(15) respectively) or non-sorted frozen-thawed (C(1), C(5) or C(15) respectively) spermatozoa from three rams. An additional group of ewes were inseminated with 50 x 10(6) motile non-sorted frozen-thawed spermatozoa (C(50)) to provide a commercial dose control. The percentage of ewes lambing after insemination was similar for C(50) (24/38, 63.2%), C(15) (37/54, 68.5%), S(15) (38/57, 66.7%), S(5) (37/56, 66.1%) and S(1) (32/52, 61.5%) groups (p > 0.05), but lower for C(5) (19/48, 39.6%) and C(1) (19/55, 34.5%) treatments (p sheep as a reduction in the minimum effective sperm number will allow a corresponding decrease in the associated cost per dose.

  15. Flow cytometric viability assessment of lactic acid bacteria starter cultures produced by fluidized bed drying.

    Science.gov (United States)

    Bensch, Gerald; Rüger, Marc; Wassermann, Magdalena; Weinholz, Susann; Reichl, Udo; Cordes, Christiana

    2014-06-01

    For starter culture production, fluidized bed drying is an efficient and cost-effective alternative to the most frequently used freeze drying method. However, fluidized bed drying also poses damaging or lethal stress to bacteria. Therefore, investigation of impact of process variables and conditions on viability of starter cultures produced by fluidized bed drying is of major interest. Viability of bacteria is most frequently assessed by plate counting. While reproductive growth of cells can be characterized by the number of colony-forming units, it cannot provide the number of viable-but-nonculturable cells. However, in starter cultures, these cells still contribute to the fermentation during food production. In this study, flow cytometry was applied to assess viability of Lactobacillus plantarum starter cultures by membrane integrity analysis using SYBR®Green I and propidium iodide staining. The enumeration method established allowed for rapid, precise and sensitive determination of viable cell concentration, and was used to investigate effects of fluidized bed drying and storage on viability of L. plantarum. Drying caused substantial membrane damage on cells, most likely due to dehydration and oxidative stress. Nevertheless, high bacterial survival rates were obtained, and granulates contained in the average 2.7 × 10(9) viable cells/g. Furthermore, increased temperatures reduced viability of bacteria during storage. Differences in results of flow cytometry and plate counting suggested an occurrence of viable-but-nonculturable cells during storage. Overall, flow cytometric viability assessment is highly feasible for rapid routine in-process control in production of L. plantarum starter cultures, produced by fluidized bed drying.

  16. A sensitive flow cytometric methodology for studying the binding of L. chagasi to canine peritoneal macrophages

    Directory of Open Access Journals (Sweden)

    Mosser David M

    2005-05-01

    Full Text Available Abstract Background The Leishmania promastigote-macrophage interaction occurs through the association of multiple receptors on the biological membrane surfaces. The success of the parasite infection is dramatically dependent on this early interaction in the vertebrate host, which permits or not the development of the disease. In this study we propose a novel methodology using flow cytometry to study this interaction, and compare it with a previously described "in vitro" binding assay. Methods To study parasite-macrophage interaction, peritoneal macrophages were obtained from 4 dogs and adjusted to 3 × 106 cells/mL. Leishmania (Leishmania chagasi parasites (stationary-phase were adjusted to 5 × 107 cells/mL. The interaction between CFSE-stained Leishmania chagasi and canine peritoneal macrophages was performed in polypropylene tubes to avoid macrophage adhesion. We carried out assays in the presence or absence of normal serum or in the presence of a final concentration of 5% of C5 deficient (serum from AKR/J mice mouse serum. Then, the number of infected macrophages was counted in an optical microscope, as well as by flow citometry. Macrophages obtained were stained with anti-CR3 (CD11b/CD18 antibodies and analyzed by flow citometry. Results Our results have shown that the interaction between Leishmania and macrophages can be measured by flow cytometry using the fluorescent dye CFSE to identify the Leishmania, and measuring simultaneously the expression of an important integrin involved in this interaction: the CD11b/CD18 (CR3 or Mac-1 β2 integrin. Conclusion Flow cytometry offers rapid, reliable and sensitive measurements of single cell interactions with Leishmania in unstained or phenotypically defined cell populations following staining with one or more fluorochromes.

  17. An imaging flow cytometric method for measuring cell division history and molecular symmetry during mitosis.

    Science.gov (United States)

    Filby, Andrew; Perucha, Esperanza; Summers, Huw; Rees, Paul; Chana, Prabhjoat; Heck, Susanne; Lord, Graham M; Davies, Derek

    2011-07-01

    Asymmetric cell division is an important mechanism for generating cellular diversity, however, techniques for measuring the distribution of fate-regulating molecules during mitosis have been hampered by a lack of objectivity, quantitation, and statistical robustness. Here we describe a novel imaging flow cytometric approach that is able to report a cells proliferative history and cell cycle position using dye dilution, pH3, and PI staining to then measure the spatial distribution of fluorescent signals during mitosis using CCD-derived imagery. Using Jurkat cells, resolution of the fluorescently labeled populations was comparable to traditional PMT based cytometers thus eliminating the need to sort cells with specific division histories for microscopy. Subdividing mitotic stages by morphology allowed us to determine the time spent in each cell cycle phase using mathematical modeling approaches. Furthermore high sample throughput allowed us to collect statistically relevant numbers of cells without the need to use blocking agents that artificially enrich for mitotic events. The fluorescent imagery was used to measure PKCζ protein and EEA-1+ endosome distribution during different mitotic phases in Jurkat cells. While telophase cells represented the favorable population for measuring asymmetry, asynchronously dividing cells spent approximately 43 seconds in this stage, explaining why they were present at such low frequencies. This necessitated the acquisition of large cell numbers. Interestingly we found that PKCζ was inherited asymmetrically in 2.5% of all telophasic events whereas endosome inheritance was significantly more symmetrical. Furthermore, molecular polarity at early mitotic phases was a poor indicator of asymmetry during telophase highlighting that, though rare, telophasic events represented the best candidates for asymmetry studies. In summary, this technique combines the spatial information afforded by fluorescence microscopy with the statistical

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

    Science.gov (United States)

    Jenkins, J A; Draugelis-Dale, R O; Pinkney, A E; Iwanowicz, L R; Blazer, V S

    2015-03-15

    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 fragmented DNA to be denatured under standard conditions, or 1 minute at 37 °C with 10% buffered formalin-fixed cells. The study of fixed sperm makes possible the restrospective investigation of germplasm fragmentation, spermatogenic ploidy patterns, and chromatin compaction levels from samples translocated over distance and time. The protocol provides an approach that can be modified for other species across taxa.

  19. 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 fragmented DNA to be denatured under standard conditions, or 1 minute at 37 °C with 10% buffered formalin–fixed cells. The study of fixed sperm makes possible the restrospective investigation of germplasm fragmentation, spermatogenic ploidy patterns, and chromatin compaction levels from samples translocated over distance and time. The protocol provides an approach that can be modified for other species across taxa.

  20. Navigating parasite webs and parasite flow: emerging and re-emerging parasitic zoonoses of wildlife origin.

    Science.gov (United States)

    Polley, Lydden

    2005-10-01

    Wildlife are now recognised as an important source of emerging human pathogens, including parasites. This paper discusses the linkages between wildlife, people, zoonotic parasites and the ecosystems in which they co-exist, revisits definitions for 'emerging' and 're-emerging', and lists zoonotic parasites that can be acquired from wildlife including, for some, estimates of the associated global human health burdens. The paper also introduces the concepts of 'parasite webs' and 'parasite flow', provides a context for parasites, relative to other infectious agents, as causes of emerging human disease, and discusses drivers of disease emergence and re-emergence, especially changes in biodiversity and climate. Angiostrongylus cantonensis in the Caribbean and the southern United States, Baylisascaris procyonis in California and Georgia, Plasmodium knowlesi in Sarawak, Malaysia, Human African Trypanosomiasis, Sarcoptes scabiei in carnivores, and Cryptosporidium, Giardia and Toxoplasma in marine ecosystems are presented as examples of wildlife-derived zoonotic parasites of particular recent interest. An ecological approach to disease is promoted, as is a need for an increased profile for this approach in undergraduate and graduate education in the health sciences. Synergy among scientists and disciplines is identified as critical for the study of parasites and parasitic disease in wildlife populations. Recent advances in techniques for the investigation of parasite fauna of wildlife are presented and monitoring and surveillance systems for wildlife disease are discussed. Some of the limitations inherent in predictions for the emergence and re-emergence of infection and disease associated with zoonotic parasites of wildlife are identified. The importance of public awareness and public education in the prevention and control of emerging and re-emerging zoonotic infection and disease are emphasised. Finally, some thoughts for the future are presented.

  1. Evaluation of the S phase distribution of flow cytometric DNA histograms by autoradiography and computer algorithms.

    Science.gov (United States)

    Sheck, L E; Muirhead, K A; Horan, P K

    1980-09-01

    Cell sorting and tritiated thymidine autoradiography were used to define the distribution of S phase cells in flow cytometric DNA histograms obtained from exponential mouse lymphoma cells (L5178Y). The numbers of labeled S phase cells, autoradiographically determined from cells sorted at 2-channel intervals in the G1/early S and late S/G2M regions of the histogram, were compared with the numbers of computed S phase cells in comparable 2-channel intervals as predicted by several computer algorithms used to extract cell cycle phase distributions from DNA histograms. Polynomial and multirectangle algorithms gave computed estimates of total %S in close agreement with the tritiated thymidine labeling index for the cell population, while multi-Gaussian algorithms underestimated %S. Interval autoradiographic and algorithm studies confirmed these results in that no significant differences were found between the autoradiographic S phase distribution and S phase distributions calculated by the polynomial and multirectangle models. However, S phase cells were significantly underestimated in G1/early S by a constrained multi-Gaussian model and in both G1/early S and late S/G2 by an unconstrained multi-Gaussian model. For the particular cell line (L5178Y), staining protocol (mithramycin following ethanol fixation) and instrumentation (Coulter TPS-2 cell sorter) used in this study, close agreement between computed %S and tritiated thymidine labeling index was found to be a reliable indicator of an algorithm's success in resolving S phase cells in the G1/S and S/G2 transition regions of the DNA histograms.

  2. Flow Cytometric Assessment of Bacterial Abundance in Soils, Sediments and Sludge.

    Science.gov (United States)

    Frossard, Aline; Hammes, Frederik; Gessner, Mark O

    2016-01-01

    Bacterial abundance is a fundamental measure in microbiology, but its assessment is often tedious, especially for soil, and sediment samples. To overcome this limitation, we adopted a time-efficient flow-cytometric (FCM) counting method involving cell detachment and separation from matrix particles by centrifugation in tubes receiving sample suspensions and Histodenz(®) solution. We used this approach to assess bacterial abundances in diverse soils (natural and agricultural), sediments (streams and lakes) and sludge from sand-filters in a drinking water treatment plant and compared the results to bacterial abundances determined by two established methods, epifluorescence microscopy (EM) and adenosine triphosphate (ATP) quantification. Cell abundances determined by FCM and EM correlated fairly well, although absolute cell abundances were generally lower when determined by FCM. FCM also showed significant relations with cell counts converted from ATP concentrations, although estimates derived from ATP determinations were typically higher, indicating the presence of ATP sources other than bacteria. Soil and sediment organic matter (OM) content influenced the goodness of fit between counts obtained with EM and FCM. In particular, bacterial abundance determined by FCM in samples containing less than 10% OM, such as stream sediment, was particularly well correlated with the cell counts assessed by EM. Overall, these results suggest that FCM following cell detachment and purification is a useful approach to increase sample throughput for determining bacterial abundances in soils, sediments and sludge. However, notable scatter and only partial concordance among the FCM and reference methods suggests that protocols require further improvement for assessments requiring high precision, especially when OM contents in samples are high.

  3. Novel nuclei isolation buffer for flow cytometric genome size estimation of Zingiberaceae: a comparison with common isolation buffers.

    Science.gov (United States)

    Sadhu, Abhishek; Bhadra, Sreetama; Bandyopadhyay, Maumita

    2016-11-01

    Cytological parameters such as chromosome numbers and genome sizes of plants are used routinely for studying evolutionary aspects of polyploid plants. Members of Zingiberaceae show a wide range of inter- and intrageneric variation in their reproductive habits and ploidy levels. Conventional cytological study in this group of plants is severely hampered by the presence of diverse secondary metabolites, which also affect their genome size estimation using flow cytometry. None of the several nuclei isolation buffers used in flow cytometry could be used very successfully for members of Zingiberaceae to isolate good quality nuclei from both shoot and root tissues. The competency of eight nuclei isolation buffers was compared with a newly formulated buffer, MB01, in six different genera of Zingiberaceae based on the fluorescence intensity of propidium iodide-stained nuclei using flow cytometric parameters, namely coefficient of variation of the G0/G1 peak, debris factor and nuclei yield factor. Isolated nuclei were studied using fluorescence microscopy and bio-scanning electron microscopy to analyse stain-nuclei interaction and nuclei topology, respectively. Genome contents of 21 species belonging to these six genera were determined using MB01. Flow cytometric parameters showed significant differences among the analysed buffers. MB01 exhibited the best combination of analysed parameters; photomicrographs obtained from fluorescence and electron microscopy supported the superiority of MB01 buffer over other buffers. Among the 21 species studied, nuclear DNA contents of 14 species are reported for the first time. Results of the present study substantiate the enhanced efficacy of MB01, compared to other buffers tested, in the generation of acceptable cytograms from all species of Zingiberaceae studied. Our study facilitates new ways of sample preparation for further flow cytometric analysis of genome size of other members belonging to this highly complex polyploid family.

  4. Effect of intra-cellular trafficking on flow cytometric measurement of neutrophil's oxidative status in iron deficient pregnant females.

    Science.gov (United States)

    Youssef, Soha R; Hendawy, Sherif F; Boshnak, Noha H; Sedhom, Mariana S

    2017-03-27

    Iron deficiency and iron deficiency anemia are prevalent among pregnant women particularly in developing countries. This study aimed to evaluate the iron status among Egyptian pregnant women and its impact on their neutrophil's count and antimicrobial functions. Ninety pregnant females underwent complete blood count, iron profile, flow cytometric studies for neutrophil myeloperoxidase expression & oxidative burst using dihydrorhodamine 123 (DHR) after phorbol-12-myristate-13-acetate (PMA) stimulation as well as neutrophil phagocytic and lytic indices. According to percent saturation 54/90 women (60%) were iron deficient (women were in their third trimester compared to controls. No significant difference was found between the iron deficient & sufficient groups as regards anemia despite a positive correlation between haemoglobin level and percent saturation (P=.02). Both the phagocytic and lytic indices were significantly lower among the cases compared to controls (P=.014 & .002 respectively). Cases and controls were comparable as regards flow cytometric studies of neutrophils' myeloperoxidase and oxidative burst (P>.05). No significant correlation was found between any of the iron profile parameters and the oxidative burst by flow cytometry. Functional microphage assay (phagocytic and lytic indices) may be more relevant and cost effective than flow cytometry assays of myeloperoxidase and oxidative burst in reflecting either iron status or cellular immunity in pregnancy. © 2017 Wiley Periodicals, Inc.

  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 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....

  6. Effects of Hoechst33342 staining on the viability and flow cytometric sex-sorting of frozen-thawed ram sperm.

    Science.gov (United States)

    Quan, Guo Bo; Ma, Yuan; Li, Jian; Wu, Guo Quan; Li, Dong Jiang; Ni, Yi Na; Lv, Chun Rong; Zhu, Lan; Hong, Qiong Hua

    2015-02-01

    Cytometric sorting of frozen-thawed sperm can overcome difficulties caused by the unavailability of sorting facilities on farms where semen is collected from male livestock. In order to optimize the cytometric sex-sorting procedure, effects of Hoechst33342 staining on the viability and cytometric sorting efficiency of frozen-thawed ram sperm were evaluated. The frozen-thawed sperm were stained with Hoechst33342 at various dye concentrations (80 μM, 120 μM, 160 μM, 200 μM, 240 μM, or 320 μM) for 45 min to evaluate effects of dye dose. The frozen-thawed sperm were stained with 160 μM Hoechst33342 for various durations (0 min, 15 min, 30 min, 45 min, 60 min, 75 min, or 90 min) to evaluate effects of staining duration. Sperm motility and moving velocity were analyzed using a computer-assisted sperm analysis system (CASAS). Acrosome status, membrane integrity, and distribution of phosphatidylserine (PS) in Hoechst33342-stained sperm were analyzed using flow cytometry after staining with fluorescein isothiocyanate-labeled lectin from pisum sativum (FITC-PSA), Annexin V, or propidium iodide (PI). The fertility of Hoechst33342-stained sperm was analyzed by in vitro fertilization (IVF). A high-speed cell sorter was used to evaluate effects of Hoechst33342 staining on cytometric sex-sorting of frozen-thawed sperm. The motility, moving velocity, membrane integrity, and PS distribution of Hoechst33342-stained sperm were significantly different from that of immediately thawed sperm (Pram sperm. Results of cytometric sorting indicated that frozen-thawed sperm can be efficiently sorted into two sperm populations with X and Y chromosome when the Hoechst33342 concentration was 160 μM. Moreover, when the staining duration was equal to or longer than 45 min, the frozen-thawed sperm can be successfully sorted in the presence of 160μM Hoechst33342. In conclusion, Hoechst33342 staining can detrimentally influence viability of frozen-thawed ram sperm except acrosome and in vitro

  7. Postlarval muscle growth in fish: a DNA flow cytometric and morphometric analysis.

    Science.gov (United States)

    Alfei, L; Maggi, F; Parvopassu, F; Bertoncello, G; De Vita, R

    1989-01-01

    The mechanism of postlarval fish myotomal growth was investigated in trout (Salmo gairdneri) by means of morphometric and cytofluorometric analysis. The mechanism by which new fibres are added during postlarval growth (hyperplasia) is not fully understood. In histological cross sections these new fibres have a small diameter which give the muscle a "mosaic" appearance. One hypothesis suggested that they could be derived from the proliferative activity of satellite cells. DNA cytofluorometric analysis of nuclei suspensions obtained from trout white myotomal muscle during different developmental stages (eleutherembyronic; alevin; yearling and adult) showed a consistently low S-cytometric phase during all stage in which myofibres of small diameters were present. The percentage of such small fibres, determined by morphometric analysis, suggested that satellite cells are the proliferative population. In fact, their percentages, as determined by morphometric analysis in histological section, bear a linear relationship with the S-cytometric phase percent nuclei (R = 0.927). Only in adults (67 cm in size) there was a significant decrease in the S-cytometric phase. At this stage, in histological sections, the myotomal muscle no longer had a "mosaic" appearance because of the disappearance of the small fibres. It may, therefore, be supposed that in the cm 67 adult specimens, the proliferative population is entering the G0 phase. It is known, in fact, that muscle growth proceeds only by fibre hypertrophy in trout longer than 70 cm in length (Stickland, 1983).

  8. Correlation between histological grading and ploidy status in potentially malignant disorders of the oral mucosa: A flow cytometric analysis

    Directory of Open Access Journals (Sweden)

    T Vijayavel

    2013-01-01

    Full Text Available Background: Histopathological grading of oral dysplastic lesions is the method of choice for evaluating malignant and potentially malignant disorders. Owing to inter- and intra-observer variability, determination of the DNA ploidy status of lesions may serve as an adjunct in the prediction of malignant transformation. Aim: To correlate histopathological grading and ploidy status in potentially malignant and malignant disorders of the oral mucosa. Settings and Design: A pilot study was done with 30 patients (10 patients with oral potentially malignant disorders predominantly leukoplakia, 10 patients with oral malignant lesions and 10 patients with normal mucosa. Materials and Methods: Incisional biopsy was done after isolating the biopsy site with 1% Toluidine blue staining. Two sections of the tissue were removed and sent for histopathological and Flow-cytometric analysis respectively. Histopathological diagnosis was obtained and compared with Flow-cytometric results which were graded as diploid and aneuploid. Further, the S - phase fraction, DNA index were also calculated to evaluate the severity of malignant transformation or malignancy. Statistical Analysis: The results were analyzed using Pearson Chi-Square Test. Results: There exists a significant correlation between histopathology and ploidy status in both potentially malignant and malignant group. (P = 0.002. Conclusion: The data from this study has shown that DNA Ploidy analysis can be used as a valuable tool in assessing the carcinomatous progression of potentially malignant and malignant lesions.

  9. A cluster analysis method for identification of subpopulations of cells in flow cytometric list-mode arrays

    Science.gov (United States)

    Li, Z. K.

    1985-01-01

    A specialized program was developed for flow cytometric list-mode data using an heirarchical tree method for identifying and enumerating individual subpopulations, the method of principal components for a two-dimensional display of 6-parameter data array, and a standard sorting algorithm for characterizing subpopulations. The program was tested against a published data set subjected to cluster analysis and experimental data sets from controlled flow cytometry experiments using a Coulter Electronics EPICS V Cell Sorter. A version of the program in compiled BASIC is usable on a 16-bit microcomputer with the MS-DOS operating system. It is specialized for 6 parameters and up to 20,000 cells. Its two-dimensional display of Euclidean distances reveals clusters clearly, as does its 1-dimensional display. The identified subpopulations can, in suitable experiments, be related to functional subpopulations of cells.

  10. Flow cytometric kinetic assay of calcium mobilization in whole blood platelets using Fluo-3 and CD41.

    Science.gov (United States)

    do Céu Monteiro, M; Sansonetty, F; Gonçalves, M J; O'Connor, J E

    1999-04-01

    Platelet activation plays a major role in the physiology and pathology of hemostasis. Flow cytometry is a promising approach for the structural and functional analysis of platelets. However, the choice of adequate biological parameters and most technical issues are still under discussion. A rise in cytosolic free Ca2+ is a key early event that follows platelet stimulation and precedes several activation responses, including shape change, aggregation, secretion, and expression of procoagulant activity. Our objective was to set up a fast and sensitive flow cytometric method to determine the kinetics of intracellular Ca2+ mobilization in platelets, which could be performed with the least artifactual perturbation of platelet function. Anticoagulated blood was diluted in Tyrode's buffer and incubated with Fluo-3-acetoxymethyl ester prior to staining with phycoerytrin-conjugated antiplatelet GPIIb/IIIa complex monoclonal antibody. Platelets were identified by a gate including only CD41+ events. After the determination of baseline Fluo-3 green fluorescence on a flow cytometer (EPICS XL-MCL, Coulter Electronics, Hialeah, FL), adequate agonists were added and time-dependent changes in Fluo-3 fluorescence were recorded on-line for up to 3 min. In these conditions, a very fast and transient increase of cytosolic-free Ca2+ was observed following the addition of thrombin, a strong platelet agonist. Stimulation with adenosine diphosphate (ADP), a weak agonist, also resulted in evident increase of Ca2+ levels. Our results show that this flow cytometric kinetic method provides a simple and sensitive tool to assess in vitro the time course and intensity of signal transduction responses to different platelet agonists under near physiological conditions. In this way, it may be useful to evaluate the degree of platelet reactivity and thus to monitor antiplatelet therapy.

  11. 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.

  12. Flow cytometric analyses of the viability, surface marker expression and function of lymphocytes from children following cryopreservation.

    Science.gov (United States)

    Chen, Xi; Zhang, Hui; Mou, Wenjun; Qi, Zhan; Ren, Xiaoya; Wang, Guoliang; Jiao, Hong; Kong, Xiaohui; Gui, Jingang

    2016-11-01

    Flow cytometric analysis is important for the investigation and clinical preparation of lymphocytes from children. However, the strict requirement of cell freshness and inter‑assay variability limits the application of this methodology for pediatric investigations. Therefore, it is necessary to identify a reliable cryopreservative method capable of maintaining high cell viability and proper cell function in lymphocytes from children. In the present study, eight commonly‑used cell cyropreservative methods were used, and their effects on cell viability, surface marker expression and cell function were examined. In addition, how these methods affect the distribution of T‑cell receptor Vβ subfamilies were also determined. The results of the present study provided valuable experimental evidence, based on which the optimal method for the cryopreservation of lymphocytes from children in pediatric investigations and clinical applications can be selected.

  13. Ultra-Fast and Optimized Method for the Preparation of Rodent Testicular Cells for Flow Cytometric Analysis

    Directory of Open Access Journals (Sweden)

    López-Carro Beatriz

    2009-01-01

    Full Text Available Abstract Homogeneity of cell populations is a prerequisite for the analysis of biochemical and molecular events during male gamete differentiation. Given the complex organization of the mammalian testicular tissue, various methods have been used to obtain enriched or purified cell populations, including flow cell sorting. Current protocols are usually time-consuming and may imply loss of short-lived RNAs, which is undesirable for expression profiling. We describe an optimized method to speed up the preparation of suitable testicular cell suspensions for cytometric analysis of different spermatogenic stages from rodents. The procedure takes only 15 min including testis dissection, tissue cutting, and processing through the Medimachine System (Becton Dickinson. This method could be a substitute for the more tedious and time-consuming cell preparation techniques currently in use.

  14. Ultra-Fast and Optimized Method for the Preparation of Rodent Testicular Cells for Flow Cytometric Analysis

    Directory of Open Access Journals (Sweden)

    Rodríguez-Casuriaga Rosana

    2009-03-01

    Full Text Available Abstract Homogeneity of cell populations is a prerequisite for the analysis of biochemical and molecular events during male gamete differentiation. Given the complex organization of the mammalian testicular tissue, various methods have been used to obtain enriched or purified cell populations, including flow cell sorting. Current protocols are usually time-consuming and may imply loss of short-lived RNAs, which is undesirable for expression profiling. We describe an optimized method to speed up the preparation of suitable testicular cell suspensions for cytometric analysis of different spermatogenic stages from rodents. The procedure takes only 15 min including testis dissection, tissue cutting, and processing through the Medimachine System (Becton Dickinson. This method could be a substitute for the more tedious and time-consuming cell preparation techniques currently in use.

  15. Adaptation of ubiquitin-PNA based sperm quality assay for semen evaluation by a conventional flow cytometer and a dedicated platform for flow cytometric semen analysis.

    Science.gov (United States)

    Odhiambo, J F; Sutovsky, M; DeJarnette, J M; Marshall, C; Sutovsky, P

    2011-10-01

    The purpose of semen quality evaluation is to predict the fertility potential of the sample in an objective, rapid and inexpensive manner. However, utilization of sperm quality biomarkers such as ubiquitin and lectin Arachis hypogaea agglutinin (PNA) for flow cytometric semen evaluation might eliminate the need for visual assessment by microscopy. Herein, we demonstrate a robust ubiquitin and PNA-based semen evaluation conducted on a simple, easy to operate, dedicated sperm flow cytometer, EasyCyte Plus (IMV Technologies, L'Aigle, France). Semen samples were collected periodically from two dairy bulls, which were subjected to temporary scrotal insults to induce variable semen quality. Samples were labeled with fluorescently-conjugated anti-ubiquitin antibodies (bind exclusively to the surface of defective sperm) and lectin PNA (binds to acrosomal surface in prematurely capacitated and acrosome-damaged sperm). Fluorescent properties of the samples were measured with a conventional flow cytometer (Becton Dickinson FACScan; Becton Dickinson Corp., Franklin Lakes, NJ, USA) and by the EasyCyte (IMV Technologies) instrument. Data from the two flow cytometers were positively correlated for the percentage of PNA-positive sperm with a damaged acrosome (r = 0.47; P flow cytometric semen evaluation.

  16. 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

  17. Flow cytometric measurement of calcium influx in murine T cell hybrids using Fluo-3 and an organic-anion transport inhibitor.

    Science.gov (United States)

    Baus, E; Urbain, J; Leo, O; Andris, F

    1994-07-12

    A method is described to facilitate flow cytometric analysis of calcium mobilization upon stimulation of murine T cell hybrids. In these transformed cell lines, the accuracy of cytometric measurement of free cytoplasmic calcium with Fluo-3 is compromised by the rapid loss of the intracellular dye. We have found that the addition of sulfinpyrazone, a known organic-anion transporter inhibitor in epithelial cells and in macrophages, severely impairs the leakage of the Fluo-3 probe from the cytoplasmic matrix. Under appropriate conditions, sulfinpyrazone has little effect on the cell physiology and permits the detection of calcium influx in a variety of murine T cell hybrids.

  18. Flow cytometric measurement of RNA synthesis based on bromouridine labelling and combined with measurement of DNA content or cell surface antigen

    DEFF Research Database (Denmark)

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

    1993-01-01

    that RNA synthesis increased within the first 24 hours of phytohemagglutinin (PHA) stimulation, reaching a maximum at 48 hours, when cells had entered the cell cycle. Using a new method for flow cytometric dual parameter analysis of BrUrd incorporation and a cell surface antigen, spontaneous RNA synthesis...

  19. 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...

  20. 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

    1993-01-01

    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 epifluoresce

  1. Flow Cytometric Measurement of [Ca2+]i and pHi in Conjugated Natural Killer Cells and K562 Target Cells during the Cytotoxic Process1,2

    NARCIS (Netherlands)

    van Graft, Marja; van Graft, M.; Kraan, Yvonne M.; Segers-Nolten, Gezina M.J.; Radosevic, K.; Radosevic, Katarina; de Grooth, B.G.; Greve, Jan

    1993-01-01

    We describe a flow cytometric assay that enables one to follow conjugate formation between cytotoxic cells and their target cells during the cytotoxic process. In addition, the internal calcium concentration ([Ca2+]i) and internal pH (pHi) of the conjugated cells can be monitored and directly

  2. 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.

  3. Overview of very small embryonic-like stem cells (VSELs) and methodology of their identification and isolation by flow cytometric methods.

    Science.gov (United States)

    Zuba-Surma, Ewa K; Ratajczak, Mariusz Z

    2010-01-01

    The protocols presented here describe the procedures employed to identify and isolate very small embryonic-like stem cells (VSELs) using flow cytometric technologies including fluorescence-activated cell sorting (FACS). We describe the recommended steps in detail for their successful identification and isolation from adult tissues. These protocols were initially established to isolate such cells from murine bone marrow (BM) and human cord blood (CB) and may also be employed to isolate these primitive cells from other adult organs and embryonic tissues. Here, we focus on some critical parameters/key points required for the successful identification and purification of these rare cells by employing classical flow cytometry. In the last part of this unit, we also discuss a novel flow cytometric tool, ImageStream, an imaging flow cytometer, which allows better identification and morphological analysis of sorted cells.

  4. 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.

  5. A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood.

    Science.gov (United States)

    Palmer, Clovis S; Anzinger, Joshua J; Butterfield, Tiffany R; McCune, Joseph M; Crowe, Suzanne M

    2016-08-12

    Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of monocytes induces effector functions and a concomitant shift from oxidative to glycolytic metabolism that is accompanied by increased glucose transporter expression. This increased glycolytic metabolism is also observed for trained immunity of monocytes, a form of innate immunological memory. Although in vitro protocols examining glucose transporter expression and glucose uptake by monocytes have been described, none have been examined by multi-parametric flow cytometry in whole blood. We describe a multi-parametric flow cytometric protocol for the measurement of fluorescent glucose analog 2-NBDG uptake in whole blood by total monocytes and the classical (CD14(++)CD16(-)), intermediate (CD14(++)CD16(+)) and non-classical (CD14(+)CD16(++)) monocyte subpopulations. This method can be used to examine glucose transporter expression and glucose uptake for total monocytes and monocyte subpopulations during homeostasis and inflammatory disease, and can be easily modified to examine glucose uptake for other leukocytes and leukocyte subpopulations within blood.

  6. HLA-targeted flow cytometric sorting of blood cells allows separation of pure and viable microchimeric cell populations.

    Science.gov (United States)

    Drabbels, Jos J M; van de Keur, Carin; Kemps, Berit M; Mulder, Arend; Scherjon, Sicco A; Claas, Frans H J; Eikmans, Michael

    2011-11-10

    Microchimerism is defined by the presence of low levels of nonhost cells in a person. We developed a reliable method for separating viable microchimeric cells from the host environment. For flow cytometric cell sorting, HLA antigens were targeted with human monoclonal HLA antibodies (mAbs). Optimal separation of microchimeric cells (present at a proportion as low as 0.01% in artificial mixtures) was obtained with 2 different HLA mAbs, one targeting the chimeric cells and the other the background cells. To verify purity of separated cell populations, flow-sorted fractions of 1000 cells were processed for DNA analysis by HLA-allele-specific and Y-chromosome-directed real-time quantitative PCR assays. After sorting, PCR signals of chimeric DNA markers in the positive fractions were significantly enhanced compared with those in the presort samples, and they were similar to those in 100% chimeric control samples. Next, we demonstrate applicability of HLA-targeted FACS sorting after pregnancy by separating chimeric maternal cells from child umbilical cord mononuclear cells. Targeting allelic differences with anti-HLA mAbs with FACS sorting allows maximal enrichment of viable microchimeric cells from a background cell population. The current methodology enables reliable microchimeric cell detection and separation in clinical specimens.

  7. 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.

  8. A whole blood flow cytometric determination of platelet activation by unfractionated and low molecular weight heparin in vitro.

    Science.gov (United States)

    Klein, Bernd; Faridi, Andreé; von Tempelhoff, G F; Heilmann, Lothar; Mittermayer, Christian; Rath, Werner

    2002-12-15

    The influence of unfractionated (Heparin-Natrium) and low-molecular heparin (Fragmin(R)) on platelet activation in whole blood was investigated by FACS analysis in vitro using antibodies against glycoprotein (gp) IIb/IIIa (CD 41), GMP 140 (CD 62P), gp 53 (CD 63) and fibrinogen. Samples were also labeled with anti-gp Ib (CD 42b). Neither unfractionated heparin (UFH) nor low molecular weight heparin (LMWH) led to significant (i.e., p<0.05) changes in fluorescence intensities of platelets labeled with anti-gp IIb/IIIa or anti-gp 53. Significant platelet activation due to unfractionated heparin could be observed by labeling with anti-GMP 140 (UFH: p=0.009; LMWH: p=0.16). The proportion of platelets with surface-bound fibrinogen was significantly increased (UFH: p=0.00006; LMWH: p=0.008). After incubation with heparins, activation ability of platelets by adenosine diphosphate (ADP) was significantly increased. The potentiating action of unfractionated heparin was larger. Therefore, flow cytometric results of platelet activation in patients receiving heparin should be interpreted carefully.

  9. Estimation of the Whitefly Bemisia tabaci Genome Size Based on k-mer and Flow Cytometric Analyses

    Directory of Open Access Journals (Sweden)

    Wenbo Chen

    2015-07-01

    Full Text Available Whiteflies of the Bemisia tabaci (Hemiptera: Aleyrodidae cryptic species complex are among the most important agricultural insect pests in the world. These phloem-feeding insects can colonize over 1000 species of plants worldwide and inflict severe economic losses to crops, mainly through the transmission of pathogenic viruses. Surprisingly, there is very little genomic information about whiteflies. As a starting point to genome sequencing, we report a new estimation of the genome size of the B. tabaci B biotype or Middle East-Asia Minor 1 (MEAM1 population. Using an isogenic whitefly colony with over 6500 haploid male individuals for genomic DNA, three paired-end genomic libraries with insert sizes of ~300 bp, 500 bp and 1 Kb were constructed and sequenced on an Illumina HiSeq 2500 system. A total of ~50 billion base pairs of sequences were obtained from each library. K-mer analysis using these sequences revealed that the genome size of the whitefly was ~682.3 Mb. In addition, the flow cytometric analysis estimated the haploid genome size of the whitefly to be ~690 Mb. Considering the congruency between both estimation methods, we predict the haploid genome size of B. tabaci MEAM1 to be ~680–690 Mb. Our data provide a baseline for ongoing efforts to assemble and annotate the B. tabaci genome.

  10. 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.

  11. 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...... cytometric DNA analysis. Melphalan produced a dose-related S phase accumulation in the two sensitive tumors, whereas no changes in the cell cycle distribution were found in the resistant tumor. The size of S phase accumulation correlated to the chemosensitivity in vivo. For low concentrations of melphalan...

  12. Improved flow cytometric detection of minimal residual disease in childhood acute lymphoblastic leukemia

    NARCIS (Netherlands)

    Denys, B.; van der Sluijs-Gelling, A. J.; Homburg, C.; van der Schoot, C. E.; de Haas, V.; Philippe, J.; Pieters, R.; van Dongen, J. J. M.; van der Velden, V. H. J.

    2013-01-01

    Most current treatment protocols for acute lymphoblastic leukemia (ALL) include minimal residual disease (MRD) diagnostics, generally based on PCR analysis of rearranged antigen receptor genes. Although flow cytometry (FCM) can be used for MRD detection as well, discordant FCM and PCR results are ob

  13. 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-...

  14. 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 invalu

  15. 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

  16. 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 invalu

  17. Comparison of monoclonal antibodies reactive with lymphocyte subsets in routinely fixed paraffin-embedded material: flow cytometric analyses, immunoperoxidase staining and influence of fixatives.

    Directory of Open Access Journals (Sweden)

    Yoshino,Tadashi

    1990-10-01

    Full Text Available We have attempted to clarify the characteristics of monoclonal antibodies (MAbs detecting lymphocyte subsets in fixed materials. We examined by means of flow cytometric technique influences of fixatives and reactivity with malignant lymphomas (MLs. Specific markers for T-cells were UCHL1 and OPD4, which reacted especially with helper/inducer T-cells. MT1 recognized almost all of T-cells from peripheral blood and tonsils, but reacted with a part of B-MLs. As for B-cell markers, L26 was the most reliable marker for B-MLs. L26 and MB1 antigens could not be detected on living cells flow cytometrically. LN1 reacted with a part of T-cells as well as B-cells, but fluorescent intensity of the former was apparently stronger than that of the latter. Although LN2 antigen was located mainly in the cytoplasm close to the nuclear membrane immunohistochemically, it could be detected on living cells flow cytometrically. LN2 positive cells belonged to B-cells in peripheral blood and tonsils. When fixed for relatively short time, B5 and buffered formalin were better for examining MAbs than non-buffered formalin and ethanol.

  18. Current International Flow Cytometric Practices for the Detection and Monitoring of Paroxysmal Nocturnal Haemoglobinuria (PNH) clones: A UK NEQAS Survey.

    Science.gov (United States)

    Fletcher, Matthew; Whitby, Liam; Whitby, Alison; Barnett, David

    2016-03-02

    Background Paroxysmal Nocturnal Haemoglobinuria (PNH) is a rare acquired genetic disorder, with an incidence of approximately 1.3 new cases per million population per year. Evidence from the UK National External Quality Assessment Service for Leucocyte Immunophenotyping (UK NEQAS LI) programme suggested major discrepancies on how PNH testing is undertaken. To investigate this we surveyed laboratories in the UK NEQAS LI PNH programme and report here the findings. Method A questionnaire was distributed to all centres registered in UK NEQAS LI flow cytometry programmes (n=1587). Comprising several subsections, it covered the majority of clinical flow cytometric practices. Participants completed a general section and then the subsections relevant to their laboratory repertoire. One subsection contained 34 questions regarding practices in PNH clone detection. Results A total of 105 laboratories returned results for the PNH section; the results demonstrated lack of consensus in all areas of PNH testing. Variation was seen in gating and testing strategies, sensitivity levels and final reporting of test results. Several incorrect practices were highlighted such as inappropriate antibody selection and failure to wash the red blood cells (RBCs) prior to analysis. Conclusion Despite the availability of consensus guidelines there appears to be no agreement in the detection and monitoring of PNH. We found only fourteen centres using methods compatible with the International Clinical Cytometry Society guidelines. Of specific note we found that no two laboratories used the same method. This technical variation could lead to incorrect diagnoses, highlighting the need for better adoption and understanding of consensus practices. This article is protected by copyright. All rights reserved.

  19. 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.

  20. High-throughput flow cytometric screening of combinatorial chemistry bead libraries for proteomics and drug discovery

    Science.gov (United States)

    Leary, James F.; Reece, Lisa M.; Yang, Xian-Bin; Gorenstein, David

    2005-04-01

    For proteomics drug discovery applications, combinatorial microbead thioaptamer libraries (one thioaptamer sequence per bead) are being created by split synthesis method, creating a "proteomics library" of protein capture beads which can be analyzed by high-throughput screening methods in this case, flow cytometry and cell sorting. Thioaptamers, oligonucleotides with thiophosphate backbone substitutions, function like antibodies in terms of recognizing specific protein sequences but have a number of advantages over antibody libraries. These proteomics beads can then be analyzed by high-speed flow cytometry and sorted to single-bead level depending on relative fluorescence brightness of fluorescently-labeled proteins, or for a specific protein from all of the molecules of cell subpopulations being analyzed. The thioaptamer sequences on a given bead showing high affinity for that protein can then be sequenced. Alternatively, the protein-capturing beads can be analyzed by MALDI-TOF mass spectrometry for analysis of the bound proteins. The beads can be thought of as equivalent to single-element positions of a proteomics chip arrays but with the advantage of being able to much more rapidly analyze hundreds of millions of possible amino acid sequences/epitopes on the basis of thioaptamer sequence affinities to select single sequences of interest. Additionally, those beads can be manipulated and isolated at the single bead level by high-throughput flow cytometry/cell sorting for subsequent sequencing of the thioaptamer sequences.

  1. 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.

  2. Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10.

    Science.gov (United States)

    Süring, Katrin; Bach, Sabine; Höflich, Conny; Straff, Wolfgang

    2016-11-19

    Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 µm in diameter in inhalable particulate matter (PM10, particle size ≤10 µm in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions. So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles >0.5 µm.

  3. Flow cytometric assessment of Streptococcus mutans viability after exposure to blue light-activated curcumin.

    Science.gov (United States)

    Manoil, Daniel; Filieri, Anna; Gameiro, Cécile; Lange, Norbert; Schrenzel, Jacques; Wataha, John C; Bouillaguet, Serge

    2014-09-01

    Streptococcus mutans biofilms are considered as primary causative agents of dental caries. Photodynamic antimicrobial chemotherapy (PACT) has been recently proposed as a strategy for inactivating dental biofilms. This study aimed to investigate the effect of blue light-activated curcumin on S. mutans viability and to explore its potential as a new anti-caries therapeutic agent. The effect of different concentrations and incubation times of photo-activated curcumin on the survival of S. mutans in planktonic and biofilm models of growth was assessed by flow cytometry. Streptococcus mutans in planktonic suspensions or biofilms formed on hydroxyapatite disks were incubated for 5 or 10min with curcumin prior to blue light activation. Bacteria were labeled with SYTO 9 and propidium iodide before viability was assessed by flow cytometry. Results were statistically analyzed using one-way ANOVA and Tukey multiple comparison intervals (α=0.05). For planktonic cultures, 0.2μM of light-activated curcumin significantly reduced S. mutans viability (p<0.05). For biofilm cultures, light-activated curcumin at concentration of 40-60μM only suppressed viability by 50% (p<0.05). Independently of the mode of growth, incubation time has no significant effect on PACT efficiency. This study indicates that blue light-activated curcumin can efficiently inactivate planktonic cultures of S. mutans whereas biofilms were more resistant to treatment. Flow cytometry allowed the detection of bacteria with damaged membranes that were unable to replicate and grow after cell sorting. Further studies seem warranted to optimize the efficacy of light-activated curcumin against S. mutans biofilms. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Flow cytometric determination of genome size in European sunbleak Leucaspius delineatus (Heckel, 1843).

    Science.gov (United States)

    Filipiak, Marta; Tylko, Grzegorz; Kilarski, Wincenty

    2012-04-01

    The aim of this study was to compare DNA content in hepatocyte and erythrocyte nuclei of the European sunbleak, Leucaspius delineatus, in relation to nuclear and cell size by means of flow cytometry and fluorescence microscopy. The DNA standards, chicken and rainbow trout erythrocytes, were prepared in parallel with both cell types, with initial separation of liver cells in pepsin solution followed by cell filtering. Standards and investigated cells were stained with a mixture of propidium iodide, citric acid, and Nonidet P40 in the presence of RNAse, and fluorescence of at least 50,000 nuclei was analyzed by flow cytometry. Average cell size was determined by flow cytometry, using fresh cell suspension in relation to latex beads of known diameter. The size of nuclei was examined on the basis of digital micrographs obtained by fluorescence microscopy after nuclei staining with DAPI. The sunbleak's erythrocyte nuclei contain 2.25 ± 0.06 pg of DNA, whereas the hepatocyte nuclei contain 2.46 ± 0.06 pg of DNA. This difference in DNA content was determined spectroscopically using isolated DNA from the two cell types. The modal diameters of the erythrocytes and hepatocytes were estimated to be 5.1 ± 0.2 and 22.3 ± 5.0 μm, respectively, and the corresponding modal dimensions of their nuclei (measured as surface area) were 15.2 and 21.4 μm(2), respectively. The nucleoplasmic index, as calculated from diameters estimated from surface area of nuclear profiles, was 2.51 for the erythrocytes compared with 0.08 for hepatocytes.

  5. Flow cytometric analysis of T lymphocyte proliferation in vivo by EdU incorporation.

    Science.gov (United States)

    Sun, Xiaojing; Zhang, Chunpan; Jin, Hua; Sun, Guangyong; Tian, Yue; Shi, Wen; Zhang, Dong

    2016-12-01

    Monitoring T lymphocyte proliferation, especially in vivo, is essential for the evaluation of adaptive immune reactions. Flow cytometry-based proliferation assays have advantages in measuring cell division of different T lymphocyte subsets at the same time by multicolor labelling. In this study, we aimed to establish the use of 5-Ethynyl-2'-deoxyuridine (EdU) incorporation in vivo to monitor T lymphocyte proliferation by flow cytometry with an adoptive transfer model. We found that fixation followed by permeabilization preserved T cell surface antigens and had no obvious effects on the fluorescence intensity of APC, PE, PE-Cy7, FITC and PerCP-Cy5.5 when the concentration of the permeabilization reagents was optimized. However, the click reaction resulted in a significant decrease in the fluorescence intensity of PE and PE-Cy7, and surface staining after the click reaction improved the fluorescence intensity. Thus, an extra step of blocking with PBS with 3% FBS between the click reaction and cell surface staining is needed. Furthermore, the percentage of EdU-positive cells increased in a dose-dependent manner, and the saturated dose of EdU was 20mg/kg. Intraperitoneal and intravenous injection had no differences in lymphocyte proliferation detection with EdU in vivo. In addition, T cell proliferation measured by EdU incorporation was comparable to BrdU but was lower than CFSE labelling. In conclusion, we optimized the protocols for EdU administration in vivo and staining in vitro, providing a feasible method for the measurement of T lymphocyte proliferation with EdU incorporation by flow cytometry in vivo.

  6. Quantitative assessment of BAX transcript and flow cytometric expression in acute myeloid leukemia: a prospective study.

    Science.gov (United States)

    Sharawat, Surender Kumar; Raina, Vinod; Kumar, Lalit; Sharma, Atul; Bakhshi, Radhika; Vishnubhatla, Sreenivas; Gupta, Ritu; Bakhshi, Sameer

    2014-10-01

    Quantitative assessment of BAX transcripts and protein in acute myeloid leukemia (AML). We quantitatively evaluated BAX gene transcripts by real-time polymerase chain reaction (TaqMan probe chemistry) and protein expression by flow cytometry. Consecutive 112 AML patients with a median age of 16 (1-59) years were recruited in the study. By flow cytometry, the percentage expression was in linear correlation with relative median fluorescent intensity (RMFI; R = 0.4425; P BAX with its RMFI (R = -0.0559; P = 0.586). The expression of the BAX at both protein and transcript level was significantly higher in AML patients as compared with normal control. RMFI of the BAX were higher in the cohort with lower white blood cell count (P = 0.029). None of the other baseline characteristics correlated with either the BAX transcript or the RMFI. BAX expression did not correlate with complete remission rate, event free, disease free, and overall survival. BAX gene expression in AML was evaluated first time with two different methods but did not correlate with the survival outcome.

  7. Flow cytometric measurement of the cellular propagation of TDP-43 aggregation.

    Science.gov (United States)

    Zeineddine, Rafaa; Whiten, Daniel R; Farrawell, Natalie E; McAlary, Luke; Hanspal, Maya A; Kumita, Janet R; Wilson, Mark R; Yerbury, Justin J

    2017-05-04

    Amyotrophic lateral sclerosis is a devastating neuromuscular degenerative disease characterized by a focal onset of motor neuron loss, followed by contiguous outward spreading of pathology including TAR DNA-binding protein of 43 kDa (TDP-43) aggregates. Previous work suggests that TDP-43 can move between cells. Here we used a novel flow cytometry technique (FloIT) to analyze TDP-43 inclusions and propagation. When cells were transfected to express either mutant G294A TDP-43 fused to GFP or wild type TDP-43fused to tomato red and then co-cultured, flow cytometry detected intact cells containing both fusion proteins and using FloIT detected an increase in the numbers of inclusions in lysates from cells expressing wild type TDP-43-tomato. Furthermore, in this same model, FloIT analyses detected inclusions containing both fusion proteins. These results imply the transfer of TDP-43 fusion proteins between cells and that this process can increase aggregation of wild-type TDP-43 by a mechanism involving co-aggregation with G294A TDP-43.

  8. Flow cytometric applicability to evaluate UV inactivation of phytoplankton in marine water samples.

    Science.gov (United States)

    Olsen, Ranveig Ottoey; Hess-Erga, Ole-Kristian; Larsen, Aud; Thuestad, Gunnar; Tobiesen, August; Hoell, Ingunn Alne

    2015-07-15

    Disinfection of microbes is of importance to prevent the spread of pathogens and non-indigenous species in the environment. Here we test the applicability of using flow cytometry (FCM) to evaluate inactivation of the phytoplankter Tetraselmis suecica after UV irradiation and labeling with the esterase substrate 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM). Non-irradiated and UV irradiated samples were analyzed with the plate count technique and FCM for 24 days. The numbers of colony forming units were used as a standard to develop a FCM protocol. Our protocol readily distinguishes live and dead cells, but challenges were encountered when determining whether UV damaged cells are dying or repairable. As damaged cells can represent a risk to aquatic organisms and/or humans, this was taken into account when developing the FCM protocol. In spite of the above mentioned challenges we argue that FCM represents an accurate and rapid method to analyze T. suecica samples.

  9. Determination of micro-litre volumes with high accuracy for flow cytometric blood cell counting

    Science.gov (United States)

    Reitz, S.; Kummrow, A.; Kammel, M.; Neukammer, J.

    2010-07-01

    We have gravimetrically calibrated the volumes dispensed by 1 mL syringes in the range between 1 µL and 100 µL using ultra-pure water. Protocols are based on series of consecutive difference measurements of masses in order to precisely compensate for evaporation, being the most important disturbing quantity. We determined expanded uncertainties of volume measurements for glass syringes of typically 0.2% (expansion factor 2) when dispensing volumes of 10 µL. For polypropylene syringes, selected with respect to the manufacturer, expanded uncertainties of 0.25% (expansion factor 2) were observed. Calibrated syringes were applied for measuring concentrations of blood cells in a flow cytometer demonstrating the capability to determine reference measurement values. Since the direct interaction of blood cells and syringe walls may lead to cell adhesion, glass syringes as well as (disposable) polypropylene syringes were calibrated.

  10. 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

    as control antibody. Without antibodies this system is suitable for analyses of natural killer cell activity. In optimization of the assay we have used effector lymphocytes from healthy donors. The most effective effector cells are CD56(+) cells. CD8(+) T cells also express CD107a in ADCC. Using the adapted......Damage of target cells by cytotoxicity, either mediated by specific lymphocytes or via antibody-dependent reactions, may play a decisive role in causing the central nervous system (CNS) lesions seen in multiple sclerosis (MS). Relevant epitopes, antibodies towards these epitopes and a reliable...... assay are all mandatory parts in detection and evaluation of the pertinence of such cytotoxicity reactions. We have adapted a flow cytometry assay detecting CD107a expression on the surface of cytotoxic effector cells to be applicable for analyses of the effect on target cells from MS patients...

  11. 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.

  12. Assessment of a five-color flow cytometric assay for verifying automated white blood cell differentials

    Institute of Scientific and Technical Information of China (English)

    HUANG Chun-mei; YU Lian-hui; PU Cheng-wei; WANG Xin; WANG Geng; SHEN Li-song; WANG Jian-zhong

    2013-01-01

    Background White blood cell (WBC) counts and differentials performed using an automated cell counter typically require manual microscopic review.However,this last step is time consuming and requires experienced personnel.We evaluated the clinical efficiency of using flow cytometry (FCM) employing a six-antibody/five-color reagent for verifying automated WBC differentials.Methods A total of 56 apparently healthy samples were assessed using a five-color flow cytometer to verify the normal reference ranges of WBC differentials.WBC differentials of 622 samples were also determined using both a cell counter and FCM.These results were then confirmed using manual microscopic methods.Results The probabilities for all of the parameters of WBC differentials exceeded the corresponding normal reference ranges by no more than 7.5%.The resulting WBC differentials were well correlated between FCM and the cell counter (r >0.88,P <0.001),except in the case of basophils.Neutrophils,lymphocytes,and eosinophils were well correlated between FCM and standard microscopic cytology assessment (r >0.80,P <0.001).The sensitivities of FCM for identification of immature granulocytes and blast cells (72.03% and 22.22%,respectively) were higher than those of the cell counter method (44.92% and 11.11%,respectively).The specificities of FCM were all above 85%,substantially better than those of the cell counter method.Conclusion These five-color FCM assays could be applied to accurately verify abnormal results of automated assessment of WBC differentials.

  13. 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.

  14. Histopathologic and Flow-Cytometric Analysis of Neoplastic and Benign “background” Tissue in Breast Carcinoma Resections

    Directory of Open Access Journals (Sweden)

    Daniel W. Visscher

    1998-01-01

    Full Text Available Two-color, multiparametric synthesis phase fraction (SPF analysis of cytokeratin-labeled epithelial cells was flow cytometrically performed on both benign (SPFb and malignant tissue samples (if available, SPFt from 132 mastectomy/lumpectomy specimens. These data were then correlated with clinicopathologic features, including (1 tumor differentiation, (2 the proportion of tumor comprised of duct carcinoma-in situ (DCIS, and (3 the histology of accompanying benign breast tissue, classified by predominant microscopic pattern as intact, normal terminal duct lobular units (NTDLU, 34% of cases, atrophic (AT, 33% of cases, proliferative fibrocystic (PFC, 26% of cases, and non-proliferative fibrocystic (NPFC, 7% of cases. SPFt was inversely correlated with extent of DCIS (DCIS =0 – 20% tumor volume – 12.7% mean SPFt, vs. DCIS >20% tumor volume – 6.4% mean SPFt, p = 0.001. SPFt also correlated with the histology of background benign breast tissue (NTDLU – 14.8% mean SPFt vs. AT – 6.9% mean SPFt vs. PFC – 12.7% mean SPFt, p = 0.05 but it did not correlate with patient age or SPFb (overall mean =0.73%. SPFb was correlated with patient age (>56 yr – 0.59% mean SPFb vs. < yr – 0.84% mean SPFb, p = 0.02, with background histology (NTDLU – 1.1% mean SPFb vs. AT – 0.43% mean SPFb vs. PFC – 0.70% mean SPFb, p < 0.02 and with the grade of the neoplasm (well/moderate – 0.58% mean vs. poorly differentiated – 0.85% mean, p = 0.04. Patients having a background of PFC were significantly older than patients with a background of NTDLU (45.2 yr vs. 60.2 yr, p = 0.01.

  15. Performance of the flow cytometric E-screen assay in screening estrogenicity of pure compounds and environmental samples

    Energy Technology Data Exchange (ETDEWEB)

    Vanparys, Caroline, E-mail: caroline.vanparys@ua.ac.be [Laboratory of Ecophysiology, Biochemistry and Toxicology, University of Antwerp, Antwerp (Belgium); Depiereux, Sophie; Nadzialek, Stephanie [Research Unit in Organismal Biology (URBO), University of Namur (FUNDP), Namur (Belgium); Robbens, Johan; Blust, Ronny [Laboratory of Ecophysiology, Biochemistry and Toxicology, University of Antwerp, Antwerp (Belgium); Kestemont, Patrick [Research Unit in Organismal Biology (URBO), University of Namur (FUNDP), Namur (Belgium); De Coen, Wim [Laboratory of Ecophysiology, Biochemistry and Toxicology, University of Antwerp, Antwerp (Belgium); European Chemicals Agency (ECHA), Helsinki (Finland)

    2010-09-15

    In vitro estrogenicity screens are believed to provide a first prioritization step in hazard characterization of endocrine disrupting chemicals. When applied to complex environmental matrices or mixture samples, they have been indicated valuable in estimating the overall estrogen-mimicking load. In this study, the performance of an adapted format of the classical E-screen or MCF-7 cell proliferation assay was profoundly evaluated to rank pure compounds as well as influents and effluents of sewage treatment plants (STPs) according to estrogenic activity. In this adapted format, flow cytometric cell cycle analysis was used to allow evaluation of the MCF-7 cell proliferative effects after only 24 h of exposure. With an average EC{sub 50} value of 2 pM and CV of 22%, this assay appears as a sensitive and reproducible system for evaluation of estrogenic activity. Moreover, estrogenic responses of 17 pure compounds corresponded well, qualitatively and quantitatively, with other in vitro and in vivo estrogenicity screens, such as the classical E-screen (R{sup 2} = 0.98), the estrogen receptor (ER) binding (R{sup 2} = 0.84) and the ER transcription activation assay (R{sup 2} = 0.87). To evaluate the applicability of this assay for complex samples, influents and effluents of 10 STPs covering different treatment processes, were compared and ranked according to estrogenic removal efficiencies. Activated sludge treatment with phosphorus and nitrogen removal appeared most effective in eliminating estrogenic activity, followed by activated sludge, lagoon and filter bed. This is well in agreement with previous findings based on chemical analysis or biological activity screens. Moreover, ER blocking experiments indicated that cell proliferative responses were mainly ER mediated, illustrating that the complexity of the end point, cell proliferation, compared to other ER screens, does not hamper the interpretation of the results. Therefore, this study, among other E-screen studies

  16. Evaluation of Prognostic Factors Following Flow-Cytometric DNA Analysis after Cytokeratin Labelling: II. Cervical and Endometrial Cancer

    Directory of Open Access Journals (Sweden)

    Pauline Wimberger

    2002-01-01

    Full Text Available In gynecologic oncology valid prognostic factors are necessary to define biologically similar subgroups for analysis of therapeutic efficacy. This study is the first published prospective study concerning prognostic significance of DNA ploidy and S‐phase fraction in cervical and endometrial cancer following enrichment of tumor cells by cytokeratin labelling. Epithelial cells were labeled by FITC‐conjugated cytokeratin antibody (CK 5, 6, 8, and CK 17 prior to flow cytometric cell cycle analysis in 91 specimens of cervical cancer and 73 samples of endometrial cancer. In cervical cancer neither DNA‐ploidy nor S‐phase fraction were relevant prognostic parameters. But CV of the G0G1‐peak showed prognostic relevance in cervical cancer cells, even in multivariate analysis. This interesting observation, however, seems to have no therapeutic consequence due to the small discrimination capacity of CV. In endometrial carcinoma, gross DNA‐aneuploidy (DNA‐index > 1.3 and a high percentage of proliferating cells (>75th percentile were univariate and multivariate highly significant prognostic factors for recurrence‐free survival. Especially DNA‐aneuploidy (DI>1.3 is one of the most important independent molecular biological prognostic factors. While diagnostic curettage we could identify risk patients even preoperatively by determination of the prognostic factors like histologic tumor type, grading, cervical involvement and DNA‐ploidy. Thereby these patients could be treated primarily in an oncologic center. In conclusion, our investigations showed that the determination of DNA‐ploidy should be done in endometrial carcinoma. In cervical cancer no clinical significance for determination of DNA‐parameters was found.

  17. Antibody affinity maturation through combining display of two-chain paired antibody and precision flow cytometric sorting.

    Science.gov (United States)

    Sun, Shuang; Yang, Xiao; Wang, Haifeng; Zhao, Yun; Lin, Yan; Ye, Chen; Fang, Xiangdong; Hang, Haiying

    2016-07-01

    Recombination of antibody light and heavy chain libraries greatly increases the size of a two-chain paired antibody library, thus easing the construction of large antibody libraries. Here, light and heavy chain variable domains paired by a coiled coil were applied to a bacterial inner membrane display system. However, the probability of the correct pairing of light and heavy chains through random recombination after each round of flow cytometric sorting and cloning was very low in the presence of mostly unmatched light and heavy chain genes, resulting in inefficient enrichment; a target antibody clone in the ratio of 1:100,000 negative control spheroplasts was unable to be enriched by six rounds of sorting and cloning by a conventional sorting strategy (sorting the top 1 %). By just sorting the top 0.000025 % of spheroplasts, we succeeded in enriching the target antibody clone mixed with negative control spheroplasts in a ratio of 1:10(8) by just one round of sorting and cloning. Furthermore, using this gating strategy, we efficiently enriched for an antibody clone with an affinity slightly better than the parent antibody clone from mixed spheroplasts which were present in the ratio of 1 better affinity clone to 10 parent clones to 10(6) negative control clones after just two rounds of sorting and cloning, suggesting that this gating strategy is highly sensitive in distinguishing between clones with a small difference in affinity and also enriching for clones with a higher affinity. Taken together, the combination of the display of a two-chain paired antibody library and the use of stringent gating has significantly increased the efficiency of the antibody maturation system.

  18. Relevance of Flow Cytometric Auto-Crossmatch to the Post-transplant Course of Kidney Transplant Recipients.

    Science.gov (United States)

    Demir, E; Yeğit, O; Erol, A; Akgül, S U; Çalışkan, B; Bayraktar, A; Çalışkan, Y; Türkmen, A; Savran, F O; Sever, M S

    2017-04-01

    The crossmatch test is essential prior to kidney transplantation (tx) to confirm compatibility between the donor and the recipient. However, its results can be misleading due to "undetectable antibodies" in the recipient's serum. To establish if undetectable autoantibodies are responsible for a positive result, an auto-crossmatch test can be performed. In this study, we aim to determine the long-term prognostic value of auto-flow cytometric auto-crossmatch (FCXM) test on kidney survival in kidney tx recipients. The primary outcome variable was reduced renal function. Secondary endpoints were incidence of biopsy-confirmed chronic antibody-mediated rejection (CAMR) and recurrent glomerulonephritis (GN). There were no differences regarding initial serum creatinine levels between the study and control groups (P = .441). Patients who had positive auto-B FCXM had a significantly reduced renal function compared with the control group (P = .016). Four patients developed biopsy-confirmed CAMR in the study group and 1 patient in the control group (P = .047). Five patients had biopsy-confirmed recurrent GN in the GN study group, and only 1 patient had recurrent GN in the GN control group (P = .026). Kidney transplant recipients with positive auto-FCXM test had significantly reduced renal function and a higher incidence of recurrent GN and CAMR compared with the control group. The findings of this study suggest a potential role of auto-antibody causing positive auto-FCXM test result, meanwhile increasing the risk of CAMR, recurrent GN, and new-onset diabetes after tx. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis.

    Science.gov (United States)

    Koopman, G; Reutelingsperger, C P; Kuijten, G A; Keehnen, R M; Pals, S T; van Oers, M H

    1994-09-01

    Apoptosis, or programmed cell death, is a general mechanism for removal of unwanted cells from the immune system. It is characterized by chromatin condensation, a reduction in cell volume, and endonuclease cleavage of DNA into oligonucleosomal length fragments. Apoptosis is also accompanied by a loss of membrane phospholipid asymmetry, resulting in the exposure of phosphatidylserine at the surface of the cell. Expression of phosphatidylserine at the cell surface plays an important role in the recognition and removal of apoptotic cells by macrophages. Here we describe a new method for the detection of apoptotic cells by flow cytometry, using the binding of fluorescein isothiocyanate-labeled annexin V to phosphatidylserine. When Burkitt lymphoma cell lines and freshly isolated germinal center B cells are cultured under apoptosis inducing conditions, all cells showing chromatin condensation strongly stain with annexin V, whereas normal cells are annexin V negative. Moreover, DNA fragmentation is only found in the annexin V-positive cells. The nonvital dye ethidium bromide was found to stain a subpopulation of the annexin V-positive apoptotic cells, increasing with time. Our results indicate that the phase in apoptosis that is characterized by chromatin condensation coincides with phosphatidylserine exposure. Importantly, it precedes membrane damage that might lead to release from the cells of enzymes that are harmful to the surrounding tissues. Annexin V may prove important in further unravelling the regulation of apoptosis.

  20. 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.

  1. Improved sensitivity in flow cytometric intracellular ionized calcium measurement using fluo-3/Fura Red fluorescence ratios.

    Science.gov (United States)

    Novak, E J; Rabinovitch, P S

    1994-10-01

    Measurement of changes in intracellular ionized calcium concentrations ([Ca2+]i) has proved to be of wide use in the study of cellular responses to activating stimuli. The fluorescent dye Indo-1 has successfully been used in flow cytometry for this purpose, and when used as a ratiometric indicator it provides optimum sensitivity and accuracy. Unfortunately, this dye requires ultraviolet (UV) excitation which is often not available. We show here that similar results can be obtained using a ratio of green to red fluorescence from the simultaneous loading of the dyes Fura Red and fluo-3. Both Fura Red and fluo-3 are excited using the commonly available blue 488 nm laser line. With appropriate concentrations of the two dyes, the magnitude of response with the fluo-3/Fura Red ratio is greater than that achieved with indo-1, while the intercellular variation in measurement is similar to that seen with indo-1. Analyses can be simultaneously combined with immunofluorescent detection of PE-labeled antibodies to enable [Ca2+]i measurement within cell subsets.

  2. Neuropathological similarities and differences between schizophrenia and bipolar disorder: a flow cytometric postmortem brain study.

    Directory of Open Access Journals (Sweden)

    Yoshitaka Hayashi

    Full Text Available Recent studies suggest that schizophrenia (SCH and bipolar disorder (BPD may share a similar etiopathology. However, their precise neuropathological natures have rarely been characterized in a comprehensive and quantitative fashion. We have recently developed a rapid, quantitative cell-counting method for frozen unfixed postmortem brains using a flow cytometer. In the present study, we not only counted stained nuclei, but also measured their sizes in the gray matter of frontopolar cortices (FPCs and inferior temporal cortices (ITCs from patients with SCH or BPD, as well as in that from normal controls. In terms of NeuN(+ neuronal nuclei size, particularly in the reduced densities of small NeuN(+ nuclei, we found abnormal distributions present in the ITC gray matter of both patient groups. These same abnormalities were also found in the FPCs of SCH patients, whereas in the FPCs of BPD patients, a reduction in oligodendrocyte lineage (olig2(+ cells was much more common. Surprisingly, in the SCH FPC, normal left-greater-than-right asymmetry in neural nuclei densities was almost completely reversed. In the BPD FPC, this asymmetry, though not obvious, differed significantly from that in the SCH FPC. These findings indicate that while similar neuropathological abnormalities are shared by patients with SCH or BPD, differences also exist, mainly in the FPC, which may at least partially explain the differences observed in many aspects in these disorders.

  3. 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.

  4. Establishing the flow cytometric assessment of myeloid cells in kidney ischemia/reperfusion injury.

    Science.gov (United States)

    Williams, Timothy M; Wise, Andrea F; Alikhan, Maliha A; Layton, Daniel S; Ricardo, Sharon D

    2014-03-01

    Polychromatic flow cytometry is a powerful tool for assessing populations of cells in the kidney through times of homeostasis, disease and tissue remodeling. In particular, macrophages have been identified as having central roles in these three settings. However, because of the plasticity of myeloid cells it has been difficult to define a specific immunophenotype for these cells in the kidney. This study developed a gating strategy for identifying and assessing monocyte and macrophage subpopulations, along with neutrophils and epithelial cells in the healthy kidney and following ischemia/reperfusion (IR) injury in mice, using antibodies against CD45, CD11b, CD11c, Ly6C, Ly6G, F4/80, CSF-1R (CD115), MHC class II, mannose receptor (MR or CD206), an alternatively activated macrophage marker, and the epithelial cell adhesion marker (EpCAM or CD326). Backgating analysis and assessment of autofluorescence was used to extend the knowledge of various cell types and the changes that occur in the kidney at various time-points post-IR injury. In addition, the impact of enzymatic digestion of kidneys on cell surface markers and cell viability was assessed. Comparisons of kidney myeloid populations were also made with those in the spleen. These results provide a useful reference for future analyses of therapies aimed at modulating inflammation and enhancing endogenous remodeling following kidney injury.

  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 functional analysis of multidrug resistance by Fluo-3: a comparison with rhodamine-123.

    Science.gov (United States)

    Koizumi, S; Konishi, M; Ichihara, T; Wada, H; Matsukawa, H; Goi, K; Mizutani, S

    1995-09-01

    Using four cell lines including drug-sensitive K562/Parent cells, P-glycoprotein (Pgp)-mediated multidrug resistant (MDR) K562/VCR, K562/ADR and revertant K562/ADR-R cells, two fluorescent agents, Fluo-3 and rhodamine-123 (Rh-123), were compared as indicators in a functional assay of MDR. Cells were incubated with 4 microM Fluo-3 or 1 microM Rh-123 for 45 min and then the intracellular accumulation of the agent was measured using a flow cytometer. Verapamil (20 microM) or cepharanthine (biscoclaurine alkaloid, 10 microM) was added just before the fluorescent agents. Efflux patterns were also studied 60 min after incubation with or without verapamil and cepharanthine. Increased intracellular accumulation and a delayed efflux pattern of Fluo-3 by verapamil and cepharanthine were demonstrated in multidrug resistant K562/VCR and K562/ADR cells, indicating that Fluo-3 is another good indicator of MDR. However, a similar, but lower, increase in uptake and a delayed efflux pattern of Fluo-3 by verapamil and cepharanthine were also demonstrated even in Pgp-non-overexpressed K562/Parent cells. In contrast, accumulation of Rh-123 was not affected by verapamil and cepharanthine. To further study the Pgp dependency of Fluo-3, another cell line, K562/NC16 expressing minimum MDR1 mRNA, was cloned. Increased uptake and a delayed efflux pattern of Fluo-3, but not Rh-123, with verapamil or cepharanthine were again demonstrated in K562/NC16 cells, indicating that intracellular accumulation of Fluo-3 may be non-specifically influenced by verapamil and cepharanthine at very low levels of Pgp-related MDR, while the influx and efflux patterns of Rh-123 may be specifically affected by Pgp overexpression.

  7. 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.

  8. 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.

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

    Science.gov (United States)

    Prest, E I; Hammes, F; Kötzsch, S; van Loosdrecht, M C M; Vrouwenvelder, J S

    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 15 min 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. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. A numerical analysis model for interpretation of flow cytometric studies of ex vivo phagocytosis.

    Directory of Open Access Journals (Sweden)

    Ted S Strom

    Full Text Available The study of ex vivo phagocytosis via flow cytometry requires that one distinguish experimentally between uptake and adsorption of fluorescently labeled targets by phagocytes. Removal of the latter quantity from the analysis is the most common means of analyzing such data. Because the probability of phagocytosis is a function of the probability of adsorption, and because partially quenched fluorescence after uptake often overlaps with that of negative controls, this approach is suboptimal at best. Here, we describe a numerical analysis model which overcomes these limitations. We posit that the random adsorption of targets to macrophages, and subsequent phagocytosis, is a function of three parameters: the ratio of targets to macrophages (m, the mean fluorescence intensity imparted to the phagocyte by the internalized target (alpha, and the probability of phagocytosis per adsorbed target (p. The potential values of these parameters define a parameter space and their values at any point in parameter space can be used to predict the fraction of adsorption(+ and [adsorption(-, phagocytosis(+] cells that might be observed experimentally. By systematically evaluating the points in parameter space for the latter two values and comparing them to experimental data, the model arrives at sets of parameter values that optimally predict such data. Using activated THP-1 cells as macrophages and platelets as targets, we validate the model by demonstrating that it can distinguish between the effects of experimental changes in m, alpha, and p. Finally, we use the model to demonstrate that platelets from a congenitally thrombocytopenic WAS patient show an increased probability of ex vivo phagocytosis. This finding correlates with other evidence that rapid in vivo platelet consumption contributes significantly to the thrombocytopenia of WAS. Our numerical analysis method represents a useful and innovative approach to multivariate analysis.

  11. 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.

  12. Flow cytometric sorting of fecal bacteria after in situ hybridization with polynucleotide probes.

    Science.gov (United States)

    Bruder, Lena M; Dörkes, Marcel; Fuchs, Bernhard M; Ludwig, Wolfgang; Liebl, Wolfgang

    2016-10-01

    The gut microbiome represents a key contributor to human physiology, metabolism, immune function, and nutrition. Elucidating the composition and genetics of the gut microbiota under various conditions is essential to understand how microbes function individually and as a community. Metagenomic analyses are increasingly used to study intestinal microbiota. However, for certain scientific questions it is sufficient to examine taxon-specific submetagenomes, covering selected bacterial genera in a targeted manner. Here we established a new variant of fluorescence in situ hybridization (FISH) combined with fluorescence-activated cell sorting (FACS), providing access to the genomes of specific taxa belonging to the complex community of the intestinal microbiota. In contrast to standard oligonucleotide probes, the RNA polynucleotide probe used here, which targets domain III of the 23S rRNA gene, extends the resolution power in environmental samples by increasing signal intensity. Furthermore, cells hybridized with the polynucleotide probe are not subjected to harsh pretreatments, and their genetic information remains intact. The protocol described here was tested on genus-specifically labeled cells in various samples, including complex fecal samples from different laboratory mouse types that harbor diverse intestinal microbiota. Specifically, as an example for the protocol described here, RNA polynucleotide probes could be used to label Enterococcus cells for subsequent sorting by flow cytometry. To detect and quantify enterococci in fecal samples prior to enrichment, taxon-specific PCR and qPCR detection systems have been developed. The accessibility of the genomes from taxon-specifically sorted cells for subsequent molecular analyses was demonstrated by amplification of functional genes. Copyright © 2016 Elsevier GmbH. All rights reserved.

  13. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Meimaridou, Anastasia, E-mail: anastasia.meimaridou@wur.nl [RIKILT-Institute of Food Safety, Wageningen UR, P.O. Box 230, 6700 AE Wageningen (Netherlands); Haasnoot, Willem; Noteboom, Linda; Mintzas, Dimitrios [RIKILT-Institute of Food Safety, Wageningen UR, P.O. Box 230, 6700 AE Wageningen (Netherlands); Pulkrabova, Jana; Hajslova, Jana [Department of Food Chemistry and Analysis, Institute of Chemical Technology Prague, Technicka 3, 166 28 Prague 6 (Czech Republic); Nielen, Michel W.F. [RIKILT-Institute of Food Safety, Wageningen UR, P.O. Box 230, 6700 AE Wageningen (Netherlands); Wageningen University, Laboratory of Organic Chemistry, Dreijenplein 8, 6703 HB Wageningen (Netherlands)

    2010-07-05

    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{sub 50} of 0.3 {mu}g L{sup -1} using a monoclonal antibody (Mab22F12) against BaP, similar to the IC{sub 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

  15. Flow-cytometric determination of genotoxic effects of exposure to petroleum in mink and sea otters

    Science.gov (United States)

    Bickham, J.W.; Mazet, J.A.; Blake, J.; Smolen, M.J.; Lou, Y.; Ballachey, B.E.

    1998-01-01

    Three experiments were conducted to investigate the genotoxic effects of crude oil on mink and sea otters, In the first experiment, the effects on mink of chronic exposure to weathered Prudhoe Bay crude oil were studied, Female mink were fed a diet that included weathered crude oil for a period of 3 weeks prior to mating, during pregnancy and until weaning. Kits were exposed through lactation and by diet after weaning until 4 months of age. Kidney and liver tissues of the kits were examined using flow cytometry (FCM) and it was found that the genome size was increased in kidney samples from the experimental group compared to the control group. This effect was probably due to some type of DNA amplification and it could have been inherited from the exposed mothers or have been a somatic response to oil exposure in the pups, No evidence of clastogenic effects, as measured by the coefficient of variation (CV) of the G(1) peak, was found in kidney or liver tissue. In the second experiment, yearling female mink were exposed either by diet or externally to crude oil or bunker C fuel oil. Evidence for clastogenic damage was found in spleen tissue for the exposure groups, but not in kidney tissue. No evidence of increased genome size was observed. In the third experiment, blood was obtained from wild-caught sea otters in Prince William Sound. The sea otters represented two populations: one from western Prince William Sound that was potentially exposed to oil from the Exxon Valdez oil spill and a reference population from eastern Prince William Sound that did not receive oil from the spill. The spill had occurred 1.5 years prior to obtaining the blood samples. Although the mean CVs did not differ between the populations, the exposed population had a significantly higher variance of CV measurements and five out of 15 animals from the exposed population had CVs higher than the 95% confidence limits of the reference population, It is concluded that FCM is a sensitive indicator

  16. 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.

  17. The effects of orange juice clarification on the physiology of Escherichia coli; growth-based and flow cytometric analysis.

    Science.gov (United States)

    Anvarian, Amir H P; Smith, Madeleine P; Overton, Tim W

    2016-02-16

    Orange juice (OJ) is a food product available in various forms which can be processed to a greater or lesser extent. Minimally-processed OJ has a high consumer perception but presents a potential microbiological risk due to acid-tolerant bacteria. Clarification of OJ (such as removal of cloud) is a common processing step in many OJ products. However, many of the antimicrobial components of OJ such as essential oils are present in the cloud fraction. Here, the effect of clarification by filtration on the viability and physiology of Escherichia coli K-12 was tested using total viable count (TVC) and flow cytometric (FCM) analysis. The latter technique was also used to monitor intracellular pH during incubation in OJ. Removal of the OJ cloud fraction was shown to have dramatic effects on bacterial viability and physiology during storage at a range of incubation temperatures. For instance, at 4 °C, a significantly lower number of healthy cells and a significantly higher number of injured cells were observed in 0.22 μm-filtered OJ at 24h post-inoculation, compared to filtered OJ samples containing particles between 0.22 μm and 11 μm in size. Similarly, there was a significant difference between the number of healthy bacteria in the 0.7 μm-filtered OJ and both 0.22 μm-filtered and 1.2 μm-filtered OJ after 24 hour incubation at 22.5 °C. This indicated that OJ cloud between 0.7 μm and 0.22 μm in size might have an adverse effect on the viability of E. coli K-12. Furthermore, FCM allowed the rapid analysis of bacterial physiology without the requirement for growth on agar plates, and revealed the extent of the viable but non-culturable (VBNC) population. For example, at 4 °C, while the FCM viable count did not substantially decrease until 48 h, decreases in TVC were observed between 0 and 48 hour incubation, due to a subset of injured bacteria entering the VBNC state, hence being unable to grow on agar plates. This study highlights the application of FCM in

  18. Flow cytometric 96-well microplate-based in vitro micronucleus assay with human TK6 cells: protocol optimization and transferability assessment.

    Science.gov (United States)

    Bryce, Steven M; Avlasevich, Svetlana L; Bemis, Jeffrey C; Tate, Matthew; Walmsley, Richard M; Saad, Frédéric; Van Dijck, Kris; De Boeck, Marlies; Van Goethem, Freddy; Lukamowicz-Rajska, Magdalena; Elhajouji, Azeddine; Dertinger, Stephen D

    2013-04-01

    An automated approach for scoring in vitro micronuclei (MN) has been described in which flow cytometric analysis is combined with compound exposure, processing, and sampling in a single 96-well plate (Bryce SM et al. [2010]: Mutat Res 703:191-199). The current report describes protocol optimization and an interlaboratory assessment of the assay's transferability and reproducibility. In a training phase, the methodology was refined and collaborating laboratories were qualified by repeatedly testing three compounds. Second, a set of 32 chemicals comprised of reference genotoxicants and presumed non-genotoxicants was tested at each of four sites. TK6 cells were exposed to 10 closely spaced compound concentrations for 1.5- to 2-cell population doublings, and were then stained and lysed for flow cytometric analysis. MN frequencies were determined by evaluating ≥ 5,000 cells per replicate well, and several indices of cytotoxicity were acquired. The prevalence of positive results varied according to the MN-fold increase used to signify a genotoxic result, as well as the endpoint used to define a cytotoxicity limit. By varying these parameters, assay sensitivity and specificity values ranged from 82 to 98%, and 86 to 97%, respectively. In a third phase, one laboratory tested a further six genotoxicants and five non-genotoxic apoptosis inducers. In these experiments assay specificity was markedly improved when top concentration selection was based on two cytotoxicity endpoints-relative survival and quantification of ethidium monoazide-positive events. Collectively, the results indicate that the miniaturized assay is transferable across laboratories. The 96-well format consumes considerably less compound than conventional in vitro MN test methods, and the high information content provided by flow cytometry helps guard against irrelevant positive results arising from overt toxicity.

  19. 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

    2014-02-01

    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.

  20. Flow cytometric method for in situ preparation of standard materials of a small defined number of microbial cells with colony-forming potentiality.

    Science.gov (United States)

    Matsuoka, Hideaki; Nakano, Koichiro; Takatani, Norimasa; Yoshida, Tomonori; Igimi, Shizunobu; Saito, Mikako

    2014-01-01

    Standard materials of a small defined number of cells with colony-forming potentiality are essential for the rational validation of food microbiological methods. An in situ flow cytometric method using viable staining with 6-carboxyfluorescein diacetate (CFDA) and tryptic soy agar (TSA) was previously proposed and its feasibility was demonstrated with five strains. In this study, this method was applied to 16 strains to support its broad applicability. The cell sorting gate was previously determined based on the CFDA stainability alone. Now the structural properties of cells designated by forward and side-scattering intensities have been introduced as the second gating criteria. Under the optimum gate condition, 100 cells have been selected and sorted on TSA. Consequently, a 95% or higher colony-forming rate has been attained for every strain. A successful application to microaerophilic Campylobacter spp. is especially of great importance because it suggests further broader applicability.

  1. 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.

  2. Detection of P-glycoprotein with a rapid flow cytometric functional assay using Fluo-3: evaluation of sensitivity, specificity and feasibility in multiparametric analysis.

    Science.gov (United States)

    Van Acker, K L; De Greef, C; Eggermont, J; Zhang, P; Vandenberghe, P; Boogaerts, M A

    1995-08-01

    The specificity and sensitivity of a flow cytometric assay simultaneously measuring expression and transport function of the multidrug resistance associated P-glycoprotein (Pgp) was evaluated. The monoclonal antibody (mAb), MRK16 was used to detect phenotypic Pgp expression while Fluo-3-AM was used as a fluorescent substrate in a Pgp functional transport assay. The specificity of the functional assay was examined in two vinblastine selected human leukemic cell lines (K562/VLB2.5 and CCRF-CEM/VLB50) with acquired Pgp overexpression. Downmodulation of Pgp function in these cell lines could be demonstrated with different substances (verapamil, vinblastine, trifluoperazine, cyclosporin A, progesterone and quinidine) and was proven to be consistently higher in the vinblastine selected cells than in their non-selected drug sensitive counterparts. Unexpectedly, modulator activity was also observed in drug sensitive K562 and CCRF-CEM cell lines despite the inability to detect Pgp in those cells by MRK16 flow cytometrically. Low level expression of the MDR1 gene encoding Pgp in sensitive K562 cells was however demonstrated with a sensitive RT-PCR procedure. The small effect of Pgp modulators in non-drug selected cells could therefore be attributed to low level basal expression of Pgp and illustrates the sensitivity of the functional assay. Also, the effect of various Pgp modulators on Pgp function was more pronounced in a subpopulation of Pgp expressing lymphocytes than in lymphocytes which did not express Pgp. Finally, a correlation was found between discrete variations in Pgp expression and Pgp function of CD4+ lymphocytes, underscoring the feasibility of the functional assay in a triple parametric procedure. The triple parametric assay holds promise to detect Pgp expression and function in clinical samples containing mixtures of malignant and non-malignant cells.

  3. Flow cytometric analysis with a fluorescently labeled formyl peptide receptor ligand as a new method to study the pharmacological profile of the histamine H2 receptor.

    Science.gov (United States)

    Werner, Kristin; Kälble, Solveig; Wolter, Sabine; Schneider, Erich H; Buschauer, Armin; Neumann, Detlef; Seifert, Roland

    2015-10-01

    The histamine H2 receptor (H2R) is a Gs protein-coupled receptor. Its activation leads to increases in the second messenger adenosine-3',5'-cyclic monophosphate (cAMP). Presently, several systems are established to characterize the pharmacological profile of the H2R, mostly requiring radioactive material, animal models, or human blood cells. This prompted us to establish a flow cytometric analysis with a fluorescently labeled formyl peptide receptor (FPR) ligand in order to investigate the H2R functionally and pharmacologically. First, we stimulated U937 promonocytes, which mature in a cAMP-dependent fashion upon H2R activation, with histamine (HA) or selective H2R agonists and measured increases in cAMP concentrations by mass spectrometry. Next, indicative for the maturation of U937 promonocytes, we assessed the FPR expression upon incubation with HA or H2R agonists. FPR expression was measured either indirectly by formyl peptide-induced changes in intracellular calcium concentrations ([Ca(2+)]i) or directly with the fluorescein-labeled FPR ligand fNleLFNleYK-Fl. HA and H2R agonists concentration-dependently induced FPR expression, and potencies and efficacies of fMLP-induced increases in [Ca(2+)]i and FPR density correlated linearly. Accordingly, flow cytometric analysis of FPR expression constitutes a simple, inexpensive, sensitive, and reliable method to characterize the H2R pharmacologically. Furthermore, we evaluated FPR expression at the mRNA level. Generally, quantitative real-time polymerase chain reaction confirmed functional data. Additionally, our study supports the concept of functional selectivity of the H2R, since we observed dissociations in the efficacies of HA and H2R agonists in cAMP accumulation and FPR expression.

  4. Detection of chromosome aneuploidy in breast lesions with fluorescence in situ hybridization: Comparison of whole nuclei to thin tissue sections and correlation with flow cytometric DNA analysis

    Energy Technology Data Exchange (ETDEWEB)

    Visscher, D.W.; Wallis, T.; Ritchie, C.A. [Wayne State Univ., Detroit, MI (United States)

    1995-09-01

    We compared flow-cytometric DNA histogram pattern to counts of 4 fluorescent-labelled centromeric probes (chromosomes 1, 7, 8, and 17) in whole nuclei (WN) and in nuclei from formalin-fixed deparaffinized thin tissue section (TS) in 25 breast lesions. In benign lesions, signal gains (i.e., trisomic nuclei) were never observed in greater than 10% of nuclei from either WN or TS preparations. Loss of signal in benign breast lesions, however, varied considerably (0-43%) between individual case and between chromosome probes. The mean incidence of signal loss in WN of benign lesions ranged from 8.9% (chromosome 7) to 14.4 % (chromosome 1) of nuclei. These signal loss frequencies exceeded those of benign lymphoid control cells. In three benign lesions, signal loss in WN (with one probe) was observed in at least 25% of nuclei. Signal losses in benign TS, on average, were 50-150% greater than in matched WN preparations (chromosome 1: 21.7%, chromosome 7: 21.5%). Malignant lesions generally, but not always, displayed fewer monosomic nuclei and more trisomic nuclei in compared to TS, compatible with a slicing (i.e., nuclear truncation) artifact. Signal counts in carcinomas correlated well with flow cytometric DNA index; however, they were also characterized by evidence of genetic instability, manifest as signal gains in a subset of nuclei (10-25%) with individual probes in diploid range cases, as well as intratumoral heterogeneity, reflected as discrepancies in probe counts between WN and TS samples. We conclude that signal losses with centromeric probes are largely, but not entirely, explained by nuclear slicing. The minimum signal loss threshold for establishment of monosomy using interphase cytogenetics is thus unclear, even in WN. Signal gains indicative of trisomy, in contrast, are reliably associated with malignancy and may reflect gross DNA aneuploidy as well as genetic instability. 10 refs., 1 fig., 3 tabs.

  5. Flow Cytometric DNA Analysis Using Cytokeratin Labeling for Identification of Tumor Cells in Carcinomas of the Breast and the Female Genital Tract

    Directory of Open Access Journals (Sweden)

    Rainer Kimmig

    2001-01-01

    Full Text Available Flow cytometric assessment of DNA‐ploidy and S‐phase fraction in malignant tumors is compromised by the heterogeneity of cell subpopulations derived from the malignant and surrounding connective tissue, e.g., tumor, stromal and inflammatory cells. To evaluate the effect on quality of DNA cell cycle analysis and determination of DNA ploidy, cytokeratin labeling of epithelial cells was used for tumor cell enrichment in breast, ovarian, cervical and endometrial cancer prior to DNA analysis. In a prospective study, tumor cell subpopulations of 620 malignant tumors were labeled by a FITC‐conjugated cytokeratin antibody (CK 5, 6, CK18 and CK 5, 6, 8 and CK 17, respectively prior to flow cytometric cell cycle analysis. Compared to total cell analysis, detection rate of DNA‐aneuploid tumors following cytokeratin labeling was increased from 62% to 76.5% in breast cancer, from 68% to 77% in ovarian cancer, from 60% to 80% in cervical cancer and from 30% to 53% in endometrial cancer. Predominantly in DNA‐diploid tumors, a significantly improved detection of S‐phase fraction of the tumor cells was shown due to the elimination of contaminating nonproliferating “normal cells”. S‐phase fraction following tumor cell enrichment was increased by 10% (mean following cytokeratin staining in ovarian and endometrial cancer, by 30% in breast cancer and even by 70% in cervical cancer compared to total cell analysis. Thus, diagnostic accuracy of DNA‐analysis was enhanced by cytokeratin labeling of tumor cells for all tumor entities investigated.

  6. 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

    of the treatment was evaluated using growth curves and flow cytometric DNA analysis. The treatment induced a dose-dependent growth delay and dose-dependent changes in the cell cycle distribution. The cell cycle changes comprised a decrease in the G1 phase, an accumulation of cells in the S phase, and an increasing...

  7. 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 flow cytometry, despite there being differences in the absolute number of cells detected. The combined approach of flow cytometric CRP measurement and cell sorting allowed an in situ analysis of microbial cell vitality and the identification of species from defined sub-populations, although the identified microbes were limited to culturable cells.

  8. 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)

  9. Development of flow cytometric protocol for nuclear DNA content estimation and determination of chromosome number in Pongamia pinnata L., a valuable biodiesel plant.

    Science.gov (United States)

    Ramesh, Aadi Moolam; Basak, Supriyo; Choudhury, Rimjhim Roy; Rangan, Latha

    2014-01-01

    The potentiality of Pongamia pinnata L. as a sustainable source of feedstock for the biodiesel industry is dependent on an extensive knowledge of the genome structure of the plant. Flow cytometry, with propidium iodide (PI) as the DNA stain, was used to estimate the nuclear DNA content of P. pinnata, with respect to Zea mays 'CE-777' as standard. The internal and pseudo-internal standardization was followed on account of the inhibitory effect of secondary compounds on PI intercalation. The antioxidants (PVP-40 and β-mercaptoethanol) were added to the nuclear isolation buffer for the reduction of inhibitory effect of P. pinnata cytosol. Nuclear DNA content estimation was done for P. pinnata leaves from different altitudes (37-117 m height from sea level) of Assam. Flow cytometry analysis indicated that the nuclear DNA content of P. pinnata is 2.66 pg with predicted 1C value of 1,300 Mb using Z. mays as standard. Coefficient of variation in flow cytometric analysis was within the limit of 5 % indicating that the results were reliable. Somatic chromosome numbers were counted from root-tip cells and was found to be 2n = 22 corresponding to the diploid level (x = 11). A decreasing trend in the nuclear DNA content was observed for the species of different altitudes.

  10. Establishment of flow cytometric in micronucleus assay in vitro%流式细胞术检测体外微核方法的建立

    Institute of Scientific and Technical Information of China (English)

    欧红梅; 周长慧; 涂宏刚; 黄鹏程; 常艳

    2015-01-01

    OBJECTIVE:Establish the flow cytometric 96-well microplate-basedin vitro micronucleus assay in CHO-K1 cells,and explore the possibility of this method for early genetic toxicity screening during drug discovery. MEHTODS:The test included treatment with and without metabolic activation. For the treatment with metabolic activation,CHO-K1 cells were treated with three different concentrations of cyclophosphamide in the S9 mixmedium for 4 h,then incubated with S9-free fresh medium for 20 h. For the treatment without metabolic activation,cells were incubated with three different concentrations of mitomycin C continuously for 24 h. In all cases,after a total of 24 h since initiation of the treatment,cells were processed for microscopic scoring or flow cytometric MN analysis. A flow cytometric method for scoring MN used EMA and SYTOX Green to label the cells in 96-well microplate,and then compared with cytokinesis-block micronucleus assay in cell culture disks based on microscopy.RESULTS:Mitomycin C and cyclophosphamide at different concerntrations caused statistically significant and dose-dependent increasess in micronucleus assay . Non-parametric Spearman's coefficients (rs) is 1.000.CONCLUSION:Similar to literature published,mitomycin C and cyclophosphamide induced positive results in flow cytometric based in vitro micronucleus assay. So the method of flow cytometric 96-well microplate-based in vitro micronucleus assay in CHO-K1 cells was established. The concordance between microscopic scoring and flow cytometricwas good,therefore this method is promising for screening and evaluating genetic toxicity of chemicals.%目的:建立96孔板流式细胞术体外微核自动化检测的方法,并探讨其用于药物早期遗传毒性筛选和遗传毒性评价的可能性。方法:试验分为+S9短时处理组(4 h)和-S9持续处理组(24 h),分别选择3个不同浓度的环磷酰胺和丝裂霉素C处理CHO-K1细胞,24 h后收获细胞。采用EMA和SYTOX Green

  11. Flow cytometric assay to assess short-term effects of personal care products on the marine microalga Tetraselmis suecica.

    Science.gov (United States)

    Seoane, Marta; Esperanza, Marta; Rioboo, Carmen; Herrero, Concepción; Cid, Ángeles

    2017-03-01

    Large quantities of personal care products (PCPs) are used daily and many of their chemical ingredients are subsequently released into marine environments. Cultures of the marine microalga Tetraselmis suecica were exposed for 24 h to three emerging compounds included in the main classes of PCPs: the UV filter benzophenone-3 (BP-3), the disinfectant triclosan (TCS) and the fragrance tonalide (AHTN). Concentrations tested, expressed as cellular quota (pg cell(-1)), ranged from 5 to 40 for BP-3, from 2 to 16 for TCS and from 1.2 to 2.4 for AHTN. A small cytometric panel was carried out to evaluate key cytotoxicity biomarkers including inherent cell properties, growth and metabolic activity and cytoplasmic membrane properties. BP-3 caused a significant increase in growth rate, metabolic activity and chlorophyll a fluorescence from 10 pg cell(-1). However, growth and esterase activity decreased in cells exposed to all TCS and AHTN concentrations, except the lowest ones. Also these two compounds provoked a significant swelling of cells, more pronounced in the case of TCS-exposed cells. Although all treated cells remained viable, changes in membrane potential were observed. BP-3 and AHTN caused a significant depolarization of cells from 10 to 1.6 pg cell(-1), respectively; however all TCS concentrations assayed caused a noticeable hyperpolarization of cells. Metabolic activity and cytoplasmic membrane potential were the most sensitive parameters. It can be concluded that the toxicological model used and the toxicological parameters evaluated are suitable to assess the toxicity of these emerging contaminants.

  12. Flow cytometric analysis of kappa and lambda light chain expression in endoscopic biopsy specimens before the diagnosis of B-cell lymphoma.

    Science.gov (United States)

    Oka, Satoko; Muroi, Kazuo; Sato, Kazuya; Fujiwara, Shin-ichiro; Oh, Iekuni; Matsuyama, Tomohiro; Ohmine, Ken; Suzuki, Takahiro; Ozaki, Katsutoshi; Mori, Masaki; Nagai, Tadashi; Fukushima, Noriyoshi; Fukushima, Noriyoshi; Tanaka, Akira; Ozawa, Keiya

    2012-01-01

    Forty-eight patients with gastrointestinal (GI) tract B-cell lymphoma (BCL) were analyzed retrospectively. The diagnosis was based on the histological examination of specimens obtained by endoscopic biopsy. Before the diagnosis was made, single-color flow cytometry was performed to analyze the expression of light chains and B-cell antigens including CD10 in the specimens. Restricted light chain (RLC) expression, a marker of B-cell clonality, was defined as κ and λ ratios of either more than 3.0 or less than 0.5. The specimens from 30 patients (62.5%) showed RLC expression. No RLC expression or RLC expression not examined was divided into two groups : those showing CD10 positivity in more than 20% of cells (4 patients, 8.3%) and those showing no positivity (14 patients, 29.2%). The cell number analyzed in the latter group was significantly smaller than that in the other two groups. Abnormal karyotypes were found in the specimens from 8 patients (16.7%). These results indicate that the flow cytometric analysis of endoscopic biopsy specimens is useful when BCL is suspected if an adequate number of cells are obtained.

  13. 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.

  14. Flow cytometric immunobead assay for fast and easy detection of PML-RARA fusion proteins for the diagnosis of acute promyelocytic leukemia.

    Science.gov (United States)

    Dekking, E H A; van der Velden, V H J; Varro, R; Wai, H; Böttcher, S; Kneba, M; Sonneveld, E; Koning, A; Boeckx, N; Van Poecke, N; Lucio, P; Mendonça, A; Sedek, L; Szczepański, T; Kalina, T; Kanderová, V; Hoogeveen, P; Flores-Montero, J; Chillón, M C; Orfao, A; Almeida, J; Evans, P; Cullen, M; Noordijk, A L; Vermeulen, P M; de Man, M T; Dixon, E P; Comans-Bitter, W M; van Dongen, J J M

    2012-09-01

    The PML-RARA fusion protein is found in approximately 97% of patients with acute promyelocytic leukemia (APL). APL can be associated with life-threatening bleeding complications when undiagnosed and not treated expeditiously. The PML-RARA fusion protein arrests maturation of myeloid cells at the promyelocytic stage, leading to the accumulation of neoplastic promyelocytes. Complete remission can be obtained by treatment with all-trans-retinoic acid (ATRA) in combination with chemotherapy. Diagnosis of APL is based on the detection of t(15;17) by karyotyping, fluorescence in situ hybridization or PCR. These techniques are laborious and demand specialized laboratories. We developed a fast (performed within 4-5 h) and sensitive (detection of at least 10% malignant cells in normal background) flow cytometric immunobead assay for the detection of PML-RARA fusion proteins in cell lysates using a bead-bound anti-RARA capture antibody and a phycoerythrin-conjugated anti-PML detection antibody. Testing of 163 newly diagnosed patients (including 46 APL cases) with the PML-RARA immunobead assay showed full concordance with the PML-RARA PCR results. As the applied antibodies recognize outer domains of the fusion protein, the assay appeared to work independently of the PML gene break point region. Importantly, the assay can be used in parallel with routine immunophenotyping for fast and easy diagnosis of APL.

  15. A flow cytometric comparison of Indo-1 to fluo-3 and Fura Red excited with low power lasers for detecting Ca(2+) flux.

    Science.gov (United States)

    Bailey, Sheree; Macardle, Peter J

    2006-04-20

    Indo-1 and high-power water-cooled lasers have been the standard for flow cytometric based Ca(2+) flux measurements. With advances in technology and the availability of low-power air-cooled lasers, there is interest in alternative protocols. Here, we have compared Indo-1 with the combination of fluo-3 and Fura Red calcium indicator dyes using low-power air-cooled lasers as the excitation source. The reagents were examined in parallel to detect Ca(2+) flux in peripheral blood T lymphocytes and in a T lymphoblastoid cell line. Ca(2+) flux was detected with a FACSVantage SE equipped with an Omnichrome Series 74 Helium-Cadmium, or a Spectra Physics 177-G1202 Argon ion air-cooled laser. Following determination of optimal loading conditions, Ca(2+) flux was examined in response to membrane receptor stimulation or intracellular Ca(2+) mobilization. Dose dependent Ca(2+) flux to anti-CD3 and thapsigargin was detected with either Indo-1 or with fluo-3 and Fura Red. The profile of the Ca(2+) flux detected by Indo-1 or with fluo-3 and Fura Red appeared similar, with the combination of fluo-3 and Fura Red more sensitive under the particular test conditions. The results clearly demonstrated that Indo-1 could be usefully excited with a low-power air-cooled laser. The alternative use of fluo-3 and Fura Red does not require the availability of a UV capable laser and produced equivalent data.

  16. Identification of New Rat Bone Marrow-Derived Population of Very Small Stem Cell with Oct-4A and Nanog Expression by Flow Cytometric Platforms

    Directory of Open Access Journals (Sweden)

    Anna Labedz-Maslowska

    2016-01-01

    Full Text Available Very small embryonic-like stem cells (VSELs represent a unique rare population of adult stem cells (SCs sharing several structural, genetic, biochemical, and functional properties with embryonic SCs and have been identified in several adult murine and human tissues. However, rat bone marrow- (BM- derived SCs closely resembling murine or human VSELs have not been described. Thus, we employed multi-instrumental flow cytometric approach including classical and imaging cytometry and we established that newly identified population of nonhematopoietic cells expressing CD106 (VCAM-I antigen contains SCs with very small size, expressing markers of pluripotency (Oct-4A and Nanog on both mRNA and protein levels that indicate VSEL population. Based on our experience in both murine and human VSEL isolation procedures by fluorescence-activated cell sorting (FACS, we also optimized sorting protocol for separation of CD45−/Lin−/CD106+ rat BM-derived VSELs from wild type and eGFP-expressing rats, which are often used as donor animals for cell transplantations in regenerative studies in vivo. Thus, this is a first study identifying multiantigenic phenotype and providing sorting protocols for isolation VSELs from rat BM tissue for further examining of their functional properties in vitro as well as regenerative capacity in distinct in vivo rat models of tissue injury.

  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. The level of heparin-induced antibodies in correlation with the result of the flow cytometric functional assay in the patients with suspected HIT.

    Science.gov (United States)

    Maličev, Elvira; Maček Kvanka, Marjeta; Klemenc, Polona; Rožman, Primož

    2017-09-13

    Heparin can induce the formation of antibodies against a heparin complex with a platelet factor 4 (PF4), leading to platelet activation and the development of heparin-induced thrombocytopaenia (HIT). Because screening ELISA does not discriminate between platelet activating and non-activating anti-heparin/PF4 antibodies, each positive result is confirmed by an additional functional assay. We analysed 1004 sera of patients with suspected HIT. Optical density (OD) values of ELISA-positive results were correlated with the risk for a positive result with our functional flow cytometric assay. Only 10.7% were ELISA positive and 59.8% of those were positive with the functional assay. The positive functional assay was found in 23.4% of patients with OD2.0. Although our results showed that higher ELISA OD values increasethe possibility of the presence of platelet-activating anti-heparin/PF4 antibodies - , there is no need for improving ELISA cut-off value for positive result. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  19. Flow cytometric assessment of circulating platelet and erythrocytes microparticles in young thalassemia major patients: relation to pulmonary hypertension and aortic wall stiffness.

    Science.gov (United States)

    Tantawy, Azza A G; Adly, Amira A M; Ismail, Eman A R; Habeeb, Nevin M

    2013-06-01

    Heart disease is the leading cause of mortality and morbidity in β-thalassemia major (β-TM). Aggregability of abnormal red cells and membrane-derived microparticles (MPs) stemming from activated platelets and erythrocytes are responsible for thrombotic risk. We measured platelet and erythrocyte MPs (PMPs and ErMPs) in 60 young β-TM patients compared with 40 age- and sex-matched healthy controls and assessed their relation to clinicopathological characteristics and aortic elastic properties. Patients were studied stressing on transfusion history, splenectomy, thrombotic events, chelation therapy, hematological and coagulation profiles, flow cytometric measurement of PMPs (CD41b(+) ) and ErMPs (glycophorin A(+) ) as well as echocardiographic assessment of aortic elastic properties. Aortic stiffness index and pulmonary artery pressure were significantly higher, whereas aortic strain and distensibility were lower in TM patients than controls (P 2500 μg/L (P < 0.001). Compliant patients on chelation therapy had lower MPs levels than non-compliant patients (P < 0.001). PMPs and ErMPs were positively correlated to markers of hemolysis, serum ferritin, D-dimer, vWF Ag, and aortic stiffness, whereas negatively correlated to hemoglobin level and aortic distensibility (P < 0.05). We suggest that increased MPs may be implicated in vascular dysfunction, pulmonary hypertension risk, and aortic wall stiffness observed in thalassemia patients. Their quantification could provide utility for early detection of cardiovascular abnormalities and monitoring the biological efficacy of chelation therapy.

  20. A Simple Flow-Cytometric Method Measuring B Cell Surface Immunoglobulin Avidity Enables Characterization of Affinity Maturation to Influenza A Virus

    Science.gov (United States)

    Frank, Gregory M.; Ince, William L.; Gibbs, James S.; Khurana, Surender; Wheatley, Adam K.; Max, Edward E.; McDermott, Adrian B.; Golding, Hana; Stevens, James; Bennink, Jack R.

    2015-01-01

    ABSTRACT Antibody (Ab) affinity maturation enables an individual to maintain immunity to an increasing number of pathogens within the limits of a total Ig production threshold. A better understanding of this process is critical for designing vaccines that generate optimal Ab responses to pathogens. Our study describes a simple flow-cytometric method that enumerates virus-specific germinal center (GC) B cells as well as their AC50, a measure of Ab avidity, defined as the antigen concentration required to detect 50% of specific B cells. Using a model of mouse Ab responses to the influenza A virus hemagglutinin (IAV HA), we obtained data indicating that AC50 decreases with time postinfection in an affinity maturation-dependent process. As proof of principle of the utility of the method, our data clearly show that relative to intranasal IAV infection, intramuscular immunization against inactivated IAV in adjuvant results in a diminished GC HA B cell response, with increased AC50 correlating with an increased serum Ab off-rate. Enabling simultaneous interrogation of both GC HA B cell quantity and quality, this technique should facilitate study of affinity maturation and rational vaccine design. PMID:26242629

  1. Rapid detection of BCR-ABL fusion genes using a novel combined LUX primer, in-cell RT-PCR and flow cytometric method.

    Science.gov (United States)

    Shi, Yan; Li, Li-Zhen; Sun, Jian-Zhi; Zhang, Ti; Peng, Jun; Xu, Cong-Gao

    2008-01-01

    Currently, quantitative and semiquantitative assays for minimal residual disease detection include fluorescence in situ hybridisation, multiparameter flow cytometric immunophenotyping and real-time quantitative polymerase chain reaction (RQ-PCR). We have developed a new approach to detect hybrid breakpoint cluster region and Abelson proto-oncogene (BCR-ABL) transcripts inside suspension cells using in situ RT-PCR and light upon extension (LUX) primer, followed by rapid quantitative analysis with flow cytometry. After cellular permeabilization and fixation of single cell suspension, the neoplastic mRNA was reverse transcribed and amplified by PCR with LUX primer. The results demonstrated that a strong positive yellow-green signal was observed in 99-100% cells of K562 cell line, only the red nucleus was detected in NB4 cell line and normal controls. The technique has been utilised to study 12 patients with chronic myeloid leukemia, and the results were compared with those of BCR-ABL fusion mRNA by RT-PCR and BCR-ABL fusion gene of the interphase cells by fluorescence in situ hybridization (FISH). In the five diagnosed patients, 90-98% cells were strongly positive. Four patients, including three patients treated with interferon-alpha and hydroxyurea and one patient treated with imatinib mesylate, had 26-82.5% positive cells. Three patients treated with imatinib mesylate were negative. The in situ RT-PCR results demonstrated complete concordance with the results of I-FISH and RT-PCR. A fluorescence signal was detectable at 1/10(4) cells and became negative below this threshold with flow cytometry. The results of the present study suggest that (1) LUX primers can be used to efficiently detect BCR-ABL fusion mRNA by in-cell RT-PCR; (2) the novel technique is a specific and sensitive way of detecting fusion gene with potential clinical usefulness.

  2. Flow cytometric detection of neutrophil-associated immunoglobulin in patients with or without neutropenia and establishment of the reference interval.

    Science.gov (United States)

    Hwang, Keumrock; Park, Chan-Jeoung; Huh, Hee Jin; Han, Sang Hee; Jang, Seongsoo; Chi, Hyun-Sook

    2011-01-01

    We measured neutrophil-associated immunoglobulin (NAIg) levels using flow cytometry to establish the reference interval for NAIg and to estimate NAIg in patients with or without neutropenia. Peripheral blood from 152 individuals was analyzed for NAIg detection by flow cytometry. Using fluorescescent-conjugated anti-CD10 monoclonal antibody and anti-human immunoglobulins, proportions of NAIgG, NAIgA, and NAIgM bound to neutrophils were measured. Reference intervals for NAIg were set as NAIgG reference intervals defined herein, patients with neutropenia or adverse transfusion reactions may be evaluated in a clinically relevant manner.

  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) a

  4. Development of a multiplex flow cytometric microsphere immunoassay for mycotoxins and evaluation of its application in feed

    NARCIS (Netherlands)

    Peters, J.; Ploum, M.E.; Rijk, de T.C.; Haasnoot, W.

    2011-01-01

    A multi-mycotoxin immunoassay—using the MultiAnalyte Profiling (xMAP) technology—is developed and evaluated. This technology combines a unique color-coded microsphere suspension array, with a dedicated flow cytometer. We aimed for the combined detection of aflatoxins, ochratoxin A, deoxynivalenol,

  5. 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, ...

  6. Quantitation of minimal disease levels in chronic lymphocytic leukemia using a sensitive flow cytometric assay improves the prediction of outcome and can be used to optimize therapy.

    Science.gov (United States)

    Rawstron, A C; Kennedy, B; Evans, P A; Davies, F E; Richards, S J; Haynes, A P; Russell, N H; Hale, G; Morgan, G J; Jack, A S; Hillmen, P

    2001-07-01

    Previous studies have suggested that the level of residual disease at the end of therapy predicts outcome in chronic lymphocytic leukemia (CLL). However, available methods for detecting CLL cells are either insensitive or not routinely applicable. A flow cytometric assay was developed that can differentiate CLL cells from normal B cells on the basis of their CD19/CD5/CD20/CD79b expression. The assay is rapid and can detect one CLL cell in 10(4) to 10(5) leukocytes in all patients. We have compared this assay to conventional assessment in 104 patients treated with CAMPATH-1H and/or autologous transplant. During CAMPATH-1H therapy, circulating CLL cells were rapidly depleted in responding patients, but remained detectable in nonresponders. Patients with more than 0.01 x 10(9)/L circulating CLL cells always had significant (> 5%) marrow disease, and blood monitoring could be used to time marrow assessments. In 25 out of 104 patients achieving complete remission by National Cancer Institute (NCI) criteria, the detection of residual bone marrow disease at more than 0.05% of leukocytes in 6 out of 25 patients predicted significantly poorer event-free (P =.0001) and overall survival (P =.007). CLL cells are detectable at a median of 15.8 months (range, 5.5-41.8) posttreatment in 9 out of 18 evaluable patients with less than 0.05% CLL cells at end of treatment. All patients with detectable disease have progressively increasing disease levels on follow-up. The use of sensitive techniques, such as the flow assay described here, allow accurate quantitation of disease levels and provide an accurate method for guiding therapy and predicting outcome. These results suggest that the eradication of detectable disease may lead to improved survival and should be tested in future studies.

  7. Development of a five-plex flow cytometric immunoassay for the simultaneous detection of six coccidiostats in feed and eggs

    OpenAIRE

    Bienenmann-Ploum, Monique E.; Huet, Anne-Catherine; Campbell, Katrina; Fodey, Terence L.; Vincent, Ursula; Haasnoot, Willem; Delahaut, Philippe; Elliott, Christopher T; Nielen, Michel W. F.

    2012-01-01

    Coccidiostats are the only veterinary drugs still permitted to be used as feed additives to treat poultry for coccidiosis. To protect consumers, maximum levels for their presence in food and feed have been set by the European Union (EU). To monitor these coccidiostats, a rapid and inexpensive screening method would be a useful tool. The development of such a screening method, using a flow cytometry-based immunoassay, is described. The assay uses five sets of colour-coded paramagnetic microsph...

  8. Flow cytometric evaluation of antibiotic effects on viability and mitochondrial function of refrigerated spermatozoa of Nile tilapia

    Science.gov (United States)

    Segovia, M.; Jenkins, J.A.; Paniagua-Chavez, C.; Tiersch, T.R.

    2000-01-01

    Improved techniques for storage and evaluation of fish sperm would enhance breeding programs around the world. The goal of this study was to test the effect of antibiotics on refrigerated sperm from Nile tilapia (Oreochromis niloticus) by use of flow cytometry with 2 dual-staining protocols for objective assessment of sperm quality. Concentrations of 1 x 109 sperm/mL were suspended in Ringer's buffer at 318 mOsmol/kg (pH 8.0). The fluorescent stains Sybr 14 (10 ??M), propidium iodide (2.4 mM), and rhodamine 123 (0.13 ??M) were used to assess cell viability and mitochondrial function. Three concentrations of ampicillin, gentamicin, and an antibiotic/antimycotic solution were added to fresh spermatozoa. Motility estimates and flow cytometry measurements were made daily during 7 d of refrigerated storage (4 ??C). The highest concentrations of gentamicin and antibiotic/antimycotic and all 3 concentrations of ampicillin significantly reduced sperm viability. The highest of each of the 3 antibiotic concentrations significantly reduced mitochondrial function. This study demonstrates that objective sperm quality assessments can be made using flow cytometry and that addition of antibiotics at appropriate concentrations can lengthen refrigerated storage time for tilapia spermatozoa. With minor modifications, these protocols can be adapted for use with sperm from other species and with other tissue types.

  9. Direct detection of red blood cell fragments: a new flow cytometric method to evaluate hemolysis in blood pumps.

    Science.gov (United States)

    Linneweber, J; Chow, T W; Takano, T; Maeda, T; Nonaka, K; Schulte-Eistrup, S; Kawahito, S; Elert, O; Moake, J L; Nosé, Y

    2001-01-01

    Pump induced hemolysis is presently evaluated by measuring plasma free hemoglobin (fHb). However, this method has disadvantages because quantification of fHb depends on hematocrit (HCT) and hemoglobin (Hb) levels. The aim of this work was to devise a hemoglobin independent method, capable of quantifying cell trauma directly by measuring the number of red blood cell (RBC) fragments. Whole blood flow cytometry was used to quantify circulating RBC fragments derived from a roller pump (Sarns, Inc. Model 2 M 6,002) and a centrifugal pump (Gyro C1E3, Kyocera Corp.). The pumps were tested in a mock circuit for 2 hr (5 L/min flow against 100 mm Hg pressure head). Red blood cell fragments were quantified by a phycoerythrin (PE) labeled glycophorin A antibody specific for erythrocytes. Red blood cell fragments were smaller than the intact RBC population and overlapped in size with the platelet population (based on forward- and side-light scattering measurements). For the roller pump, the values for RBC fragments increased from 1,090 +/- 260/microl at 0 min to 14,880 +/- 5,900/microl after 120 min. In contrast, using the centrifugal pump, there was little increase in RBC fragments (from 730 +/- 270/microl at 0 min to 1,400 +/- 840/microl after 120 min). Flow cytometry can be used for the rapid, sensitive, hemoglobin independent evaluation of pump induced RBC trauma.

  10. Parasites

    Centers for Disease Control (CDC) Podcasts

    2010-05-06

    In this podcast, a listener wants to know what to do if he thinks he has a parasite or parasitic disease.  Created: 5/6/2010 by National Center for Emerging and Zoonotic Infectious Diseases (NCEZID).   Date Released: 5/6/2010.

  11. 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...

  12. Development of a five-plex flow cytometric immunoassay for the simultaneous detection of six coccidiostats in feed and eggs.

    Science.gov (United States)

    Bienenmann-Ploum, Monique E; Huet, Anne-Catherine; Campbell, Katrina; Fodey, Terence L; Vincent, Ursula; Haasnoot, Willem; Delahaut, Philippe; Elliott, Christopher T; Nielen, Michel W F

    2012-09-01

    Coccidiostats are the only veterinary drugs still permitted to be used as feed additives to treat poultry for coccidiosis. To protect consumers, maximum levels for their presence in food and feed have been set by the European Union (EU). To monitor these coccidiostats, a rapid and inexpensive screening method would be a useful tool. The development of such a screening method, using a flow cytometry-based immunoassay, is described. The assay uses five sets of colour-coded paramagnetic microspheres for the detection of six selected priority coccidiostats. Different coccidiostats, with and without carrier proteins, were covalently coupled onto different bead sets and tested in combination with polyclonal antisera and with a fluorescent-labelled secondary antibody. The five optimal combinations were selected for this multiplex and a simple-to-use sample extraction method was applied for screening blank and spiked eggs and feed samples. A very good correlation (r ranging from 0.995 to 0.999) was obtained with the responses obtained in two different flow cytometers (Luminex 100 and FLEXMAP 3D). The sensitivities obtained were in accordance with the levels set by the EU as the measured limits of detection for narasin/salinomycin, lasalocid, diclazuril, nicarbazin (4,4'-dinitrocarbanilide) and monensin in eggs were 0.01, 0.1, 0.5, 53 and 0.1 μg/kg and in feed 0.1, 0.2, 0.3, 9 and 1.5 μg/kg, respectively.

  13. Level 2 validation of a flow cytometric method for detection of Escherichia coli O157:H7 in raw spinach.

    Science.gov (United States)

    Williams, Anna J; Cooper, Willie M; Summage-West, Christine V; Sims, Lillie M; Woodruff, Robert; Christman, Jessica; Moskal, Ted J; Ramsaroop, Shawn; Sutherland, John B; Alusta, Pierre; Wilkes, Jon G; Buzatu, Dan A

    2015-12-23

    The Bacteriological Analytical Manual (BAM) method currently used by the United States Food and Drug Administration (FDA) to detect Escherichia coli O157:H7 in spinach was systematically compared to a new flow cytometry based method. This Food and Drug Administration (FDA) level 2 external laboratory validation study was designed to determine the latter method's sensitivity and speed for analysis of this pathogen in raw spinach. Detection of target cell inoculations with a low cell count is critical, since enterohemorrhagic strains of E. coli require an infective dose of as few as 10 cells (Schmid-Hempel and Frank, 2007). Although, according to the FDA, the infectious dose is unknown (Food and Drug Administration, 1993). Therefore, the inoculation level into the spinach, a total of 2.0±2.6 viable E. coli O157 cells, was specified to yield between 25% and 75% detection by the new method, out of 20 samples (10 positives and 10 negatives). This criterion was met in that the new method detected 60% of the nominally positive samples; the corresponding sensitivity of the reference method was 50%. For both methods the most likely explanation for false negatives was that no viable cells were actually introduced into the sample. In this validation study, the flow cytometry method was equal to the BAM in sensitivity and far superior in speed. Published by Elsevier B.V.

  14. Flow cytometric comparison of platelets from a whole blood and finger-prick sample: impact of 24 hours storage.

    Science.gov (United States)

    Swanepoel, Albe C; Stander, Andre; Pretorius, Etheresia

    2013-03-01

    In this study, we investigate the validity and laboratory utility of flow cytometry when analyzing platelet activation by studying CD41, CD42b, CD62P and CD63. We compare flow cytometry results from citrated whole-blood and finger-prick samples directly after collection and also after storing both a finger-prick and whole-blood sample for 24 hours. Citrated whole-blood and finger-prick samples were taken from three healthy individuals on two occasions, and a total of 60,000 cells were analyzed for each of the four phycoerythrin-labeled monoclonal antibodies. Half of each sample was analyzed immediately after sampling while the other half was kept in the fridge at 6 °C for 24 hours before analysis. No significant difference was found between the sampling methods or the period of time before analysis. Results therefore suggest that an appropriately prepared finger-prick sample can be used for platelet function analysis, and samples can be stored for 24 hours in the fridge at 6 °C before analysis.

  15. Flow Cytometric Determination of the Expression of gp 51 Protein of Bovine Leukaemia Virus in Experimentally Infected Sheep

    Directory of Open Access Journals (Sweden)

    Szczotka Maria

    2014-10-01

    Full Text Available The study was performed on lambs experimentally infected with bovine leukaemia virus (BLV. The presence of BLV antibodies in sera of infected animals was detected by agar gel immunodiffusion test and ELISA. Proviral DNA was detected by PCR and nested PCR. Dual-colour flow cytometry analysis was performed with the use of specific monoclonal antibodies against lymphocyte CD markers and gp51 viral envelope protein, followed by incubation with fluorescent-labelled secondary antibodies conjugated with FITC or PE. Gp51 viral envelope protein was detected in tumours caused by BLV infection. The BLV infection resulted in depletion of CD4+ lymphocytes, increase in CD8+ lymphocytes, and decrease in CD4+ to CD8+ ratio in infected sheep. Proliferation of IgM+ CD19+ cells was also detected. These cells had an immature character without tendency to differentiate, and their vitality was prolonged. Flow cytometry enabled detection of gp51 expression in sheep blood lymphocytes at the early stages of the infection, before detection of serum antibodies using ELISA.

  16. 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.

  17. 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.

  18. 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....... Furthermore, peripheral lymphocytes from L133 exhibited a significantly higher expression of CD44 and CD45 throughout the experiment. Interestingly, also vaccine-induced differences were observed in L133 as immune chickens had a significantly higher CD45 expression on their lymphocytes than the naïve controls....... Immune chickens from both lines had a significantly higher frequency of circulating γδ T cells than the naïve controls both after vaccination and challenge. Finally, the proliferative capacity of peripheral CD4+ and CD8+ cells specific for NDV was addressed 3 weeks after vaccination and 1 week after...

  19. 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.

  20. Flow cytometric analysis of the graft-versus-Leukemia-effect after hematopoietic stem cell transplantation in mice.

    Science.gov (United States)

    Schmidt, Felix; Hilger, Nadja; Oelkrug, Christoper; Svanidze, Ellen; Ruschpler, Peter; Eichler, Wolfram; Boldt, Andreas; Emmrich, Frank; Fricke, Stephan

    2015-04-01

    Acute Graft-versus-Host-Disease (aGvHD) is one of the major complications following allogeneic hematopoietic stem cell transplantation (HSCT). Although rather helpful, the use of conventional immunosuppressive drugs leads to general immunosuppression and is toxic. The effects of CD4(+) T-cells, in respect to the development of aGvHD, can be altered by administration of antihuman CD4 monoclonal antibodies, here MAX.16H5 IgG1 . This approach must be tested for possible interference with the Graft-versus-Leukemia-Effect (GvL). Thus, in vitro experiments were conducted, exposing P815 leukemic cells to bone marrow and splenocytes from cd4(-/-) -C57Bl/6 mice transgenic for human CD4 and HLA-DR3 (triple transgenic mice, [TTG]) as well as previously irradiated splenocytes from Balb/c(wt) mice. Using flow cytometry, the vitality of the various malignant and graft cells was analyzed over the course of 4 days. The survival rate of P815 cells did not change significantly when exposed to MAX.16H5 IgG1 , neither did the viability of the graft cells. This provides evidence that MAX.16H5 IgG1 does not impair the GvL effect in vitro. Additionally, P815-Balb/c(wt) leukemic mice were transplanted with P815(GFP) cells, bone marrow, and splenocytes from TTG mice with and without MAX.16H5 IgG1 . Without transplantation, P815(GFP) leukemic cells could be detected by flow cytometry in the liver, the bone marrow, and the spleen of recipients. The antibodies prevented aGvHD while leaving the GvL effect intact. These findings indicate no negative effect of MAX.16H5 IgG1 on the GvL effect in vitro and in vivo after HSCT in a murine model.

  1. Flow cytometric determination of stem/progenitor content in epithelial tissues: an example from nonsmall lung cancer and normal lung.

    Science.gov (United States)

    Donnenberg, Vera S; Landreneau, Rodney J; Pfeifer, Melanie E; Donnenberg, Albert D

    2013-01-01

    Single cell analysis and cell sorting has enabled the study of development, growth, differentiation, repair and maintenance of "liquid" tissues and their cancers. The application of these methods to solid tissues is equally promising, but several unique technical challenges must be addressed. This report illustrates the application of multidimensional flow cytometry to the identification of candidate stem/progenitor populations in non-small cell lung cancer and paired normal lung tissue. Seventeen paired tumor/normal lung samples were collected at the time of surgical excision and processed immediately. Tissues were mechanically and enzymatically dissociated into single cell suspension and stained with a panel of antibodies used for negative gating (CD45, CD14, CD33, glycophorin A), identification of epithelial cells (intracellular cytokeratin), and detection of stem/progenitor markers (CD44, CD90, CD117, CD133). DAPI was added to measure DNA content. Formalin fixed paraffin embedded tissue samples were stained with key markers (cytokeratin, CD117, DAPI) for immunofluorescent tissue localization of populations detected by flow cytometry. Disaggregated tumor and lung preparations contained a high proportion of events that would interfere with analysis, were they not eliminated by logical gating. We demonstrate how inclusion of doublets, events with hypodiploid DNA, and cytokeratin+ events also staining for hematopoietic markers reduces the ability to quantify epithelial cells and their precursors. Using the lung cancer/normal lung data set, we present an approach to multidimensional data analysis that consists of artifact removal, identification of classes of cells to be studied further (classifiers) and the measurement of outcome variables on these cell classes. The results of bivariate analysis show a striking similarity between the expression of stem/progenitor markers on lung tumor and adjacent tumor-free lung.

  2. Immune toxicity of TiO₂ under hypoxia in the green-lipped mussel Perna viridis based on flow cytometric analysis of hemocyte parameters.

    Science.gov (United States)

    Wang, Youji; Hu, Menghong; Li, Qiongzhen; Li, Jiale; Lin, Daohui; Lu, Weiqun

    2014-02-01

    The combined effects of DO and TiO2 (mixed rutile/anatase phase, 7/3) on immune responses in Perna viridis were examined. Mussels were exposed to six combinations of oxygen levels (hypoxia: 1.5 mg O2l(-1), normoxia: 6.0 mg O2 l(-1)) and TiO2 concentrations (0, 2.5 mg l(-1) and 10 mg l(-1)) for 216 h. Mussels were sampled after 24h, 48h, 120 h and 216 h, and immune parameters of hemocytes, including mortality, phagocytosis, non-specific esterase, ROS production, lysosomal content and total hemocyte count were investigated using flow cytometric assay. Hemocyte mortality was higher under hypoxia than normoxia, and increased with TiO2 concentrations, but no interaction was found between DO and TiO2. Phagocytosis was reduced under hypoxia and decreased with TiO2 exposure, and the interactive effect between time and TiO2 was observed. The percentage of hemocytes showing non-specific esterase activity was lower under hypoxia, and decreased as TiO2 concentration increased with the significant interactive effect of DO and TiO2. ROS production and lysosomal content were lower under hypoxia and reduced as concentration of TiO2 increased, and interactive effect of DO and TiO2 on ROS was evident. THC was significantly affected by the interactive effect between TiO2 and DO, with higher values under normoxia in the presence of TiO2. The present study demonstrated that immune functions of P. viridis were influenced by both nano-TiO2 and hypoxia with some synergistic effects between the two stressors. This implies that DO has to be considered in the evaluation of the toxicity of nano-materials to bivalves. © 2013.

  3. Equal overall rejection rate in pre-transplant flow-cytometric cross-match negative and positive adult recipients in liver transplantation.

    Science.gov (United States)

    Matinlauri, Irma H; Höckerstedt, Krister A; Isoniemi, Helena M

    2005-10-01

    T cell IgG flow-cytometric cross-matches (FCXM) using 48 stored pre-transplant patient serum samples and 40 stored serum samples collected 3 wk after liver transplantation and frozen spleen cells of cadaveric donors in 48 consecutive liver transplantations were performed retrospectively. T cell IgG FCXM using pre-transplant serum samples was compared with 46 complement-dependent lymphocytotoxic cross-matches (CDCXM) performed at the time of transplantation. Clinical relevance of these tests was evaluated in relation to acute rejection, 1-, 3- and 5-yr graft and patient survival. The incidence of positive FCXM was 33% (16 of 48) and 13% (six of 46) by CDCXM. The median time of acute rejection was 29 d after transplantation in FCXM positive group (range 13-101 d) and 22 d in FCXM negative group (range 7-157 d, NS). Rejection rate was similar in 16 pre-transplant FCXM positive patients (eight of 16, 50%) compared with six pre-transplant CDCXM positive patients (three of six, 50%; NS). Recipients having graft rejection tended to be more often pre-transplant FCXM positive (eight of 21, 38%) than CDCXM positive (three of 21, 14%), but the difference was not significant (p > 0.1). No difference was found in the positive predictive value in relation to acute rejection between positive FCXM and CDCXM (69% vs. 50%; NS). Furthermore there was no correlation between post-transplant positive FCXM and acute rejection. No difference was found between pre-transplant T cell IgG FCXM positive and negative recipients in relation to graft or patient survival. Our findings are supportive for little risk associated with preformed donor-specific antibodies in liver transplantation.

  4. 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.

  5. Protease substrate profiling using bacterial display of self-blocking affinity proteins and flow-cytometric sorting.

    Science.gov (United States)

    Sandersjöö, Lisa; Jonsson, Andreas; Löfblom, John

    2017-01-01

    Proteases are involved in fundamental biological processes and are important tools in both biotechnological and biomedical research. An important property of proteases is to discriminate among potential substrates. Here, a new method for substrate profiling of proteases is presented. The substrates are displayed between two anti-idiotypic affinity domains on the Gram-positive bacterium Staphylococcus carnosus. The first domain functions as a reporter tag and has affinity for a labeled reporter protein, whereas the second domain blocks the reporter tag from interacting with the reporter protein. Site-specific proteolysis of the substrate results in release of the blocking domain, enabling the reporter tag to bind the labeled reporter protein. Proteolysis is therefore reflected in reporter binding, which is quantified by flow cytometry. First, the method with tobacco etch virus protease (TEVp) is evaluated and then the substrate preference of matrix metalloprotease-1 (MMP-1) is determined using two libraries of around three million substrates each. Identified substrate peptides contained the previously reported motif (PXXXHy ) and on-cell determination of apparent kcat /KM revealed that the enriched substrate peptides are hydrolyzed six to eight-fold more efficiently than a previously reported substrate peptide. The method thus works as intended and the authors believe it has potential as an efficient tool for substrate profiling.

  6. Picoplankton Community Composition by CARD-FISH and Flow Cytometric Techniques: A Preliminary Study in Central Adriatic Sea Water

    Directory of Open Access Journals (Sweden)

    Anita Manti

    2012-01-01

    Full Text Available Data concerning picoplanktonic community composition and abundance in the Central Adriatic Sea are presented in an effort to improve the knowledge of bacterioplankton and autotrophic picoplankton and their seasonal changes. Flow cytometry analyses revealed the presence of two distinct bacteria populations: HNA and LNA cells. HNA cells showed an explicit correlation with viable and actively respiring cells. The study of viability and activity may increase our knowledge of the part that contributes really to the remineralization and bacterial biomass production. Authotrophic picoplankton abundance, especially picocyanobacteria, was strongly influenced by seasonality, indicating that light availability and water temperature are very important regulating factors. In terms of total carbon biomass, the main contribution came from heterotrophic bacteria with a lower contribution from autotrophic picoplankton. CARD-FISH evidenced, within the Eubacteria domain, the dominance of members of the phyla Alphaproteobacteria, with a strong contribution from SAR11clade, followed by Cytophaga-Flavobacterium and Gammaproteobacteria. The bacterial groups detected contributed differently depending when the sample was taken, suggesting possible seasonal patterns. This study documents for the first time picoplankton community composition in the Central Adriatic Sea using two different approaches, FCM and CARD-FISH, and could provide preliminary data for future studies.

  7. 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.

  8. Bivariate flow cytometric analysis of DNA content versus immunopositivity for ribonucleotide reductase M1 subunit in the cell cycle.

    Science.gov (United States)

    Mangiarotti, R; Bottone, M G; Danova, M; Pellicciari, C

    1998-06-01

    Ribonucleotide reductase (RR) is a cytoplasmatic enzyme catalyzing the reduction of all four ribonucleotides to their corresponding deoxyribonucleotides. Its activity strongly correlates to the rate of DNA synthesis. By using a specific monoclonal antibody against the large M1 subunit of RR, we assessed the expression of M1-RR versus DNA content by dual-parameter flow cytometry. The aim of this paper was to compare the variations in the immunopositivity for M1-RR during the cell cycle to the positivity for other cell cycle markers identifying either proliferating cells (Ki-67 and PCNA) or quiescent cells (statin). To do this, normal human embryonic fibroblasts in different growth conditions as well as several other mammalian cell lines (rat C6 glioma cells; mouse 3T3 fibroblasts and B16 melanoma cells; human epithelial EUE cells and mammary carcinoma MCF-7 cells) were used. The expression of M1-RR antigen was found to correlate positively with the expression of Ki-67 and PCNA, and negatively with the expression of statin. During early G1 phase, M1-RR becomes detectable by specific antibodies relatively later compared to PCNA and Ki-67; therefore, the lack of immunopositivity for M1-RR cannot be taken as an absolute indication of cell quiescence in G0.

  9. Detection of DNA damage in individual cells by flow cytometric analysis using anti-DNA monoclonal antibody

    Energy Technology Data Exchange (ETDEWEB)

    Frankfurt, O.S. (Roswell Park Memorial Institute, Buffalo, NY (USA))

    1987-06-01

    A new method for the measurement of DNA damage in individual cells treated with alkylating agents is described. The method is based on the binding of anti-DNA monoclonal antibody to DNA in situ. Binding of antibody was evaluated by flow cytometry with indirect immunofluorescence. No binding of antibody to DNA in non-treated HeLa S3 cells was detected. Treatment of cells with HN2 or L-phenylalanine mustard induced binding of antibody to DNA in situ. Binding of antibody was observed after treating cells with doses of drugs which reduced the surviving fraction below 20%. Intensity of binding increased in proportion to the drug dose. In HN2-treated cells a cell subset with the lowest antibody binding was observed among cells in G1 phase. Binding of antibody to DNA in HN2-treated cells was eliminated by single-strand (ss) specific S1 nuclease. In competition assay, antibody was inhibited by thermally denatured DNA, but not by native double-stranded (ds) DNA, RNA, nucleosides and deoxyribohomopolymers. Immunoreactivity of cells with the monoclonal antibody F7-26 may be a useful probe for the assessment of cell damage induced by alkylating agents, especially in heterogeneous cell populations.

  10. FLOW CYTOMETRIC DETECTION OF SUBHAPLOID NUCLEI IN HUMAN SPERM AS A MEASURE OF DNA FRAGMENTATION AND APOPTOSIS.

    Science.gov (United States)

    Gröbner, S; Franz, M; Hoberg, U; Wetzka, B; Schweizer, T

    2015-01-01

    The use of assisted reproductive technologies (ARTs) is increasing worldwide. In order to predict the rate of pregnancy after ART the DNA fragmentation index (DFI) of ejaculated spermatocytes may be a better marker than conventional semen quality parameters. Spermatocytes with fragmented DNA are associated with apoptotic stages and are characterized by a low DNA content. The subhaploid nuclei of DNA-damaged spermatocytes can be easily detected by flow cytometry. We here analyzed the percentage of subhaploid nuclei of semen samples from 163 patients aged 26 to 74 years who consulted one of the ten centres for reproductive medicine which routinely send sperm samples to our laboratory in order to determine special sperm parameters. The percentage of subhaploid nuclei indicating the DFI of spermatocytes did not correlate with age and sperm volume, but inversely correlated with sperm concentration and the percentage of motile spermatocytes. This is in concordance with previous studies which demonstrated that DNA damage of spermatozoa correlates with conventional semen quality parameters. Since DNA-damaged spermatocytes are associated with an impaired outcome of assisted conception technologies, this method could help to monitor sperm quality of subfertile men after measures to increase sperm quality and to improve selection criteria of cryopreserved sperm samples in assisted reproduction medicine.

  11. 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.

  12. Flow cytometric assessment of reactive oxygen species generations that are directly related to cellular ZnO nanoparticle uptake.

    Science.gov (United States)

    Yoo, Hyun Ju; Yoon, Tae Hyun

    2014-07-01

    In this study, a simple flow cytometry protocol to evaluate nanoparticle associated biological response was proposed. Particularly, we have evaluated the effect of surface charge on the cellular nanoparticle associations and nanoparticle-induced apoptosis. Significant enhancement in side scattering intensity was observed for the HeLa cells treated with positively charged (PLL)ZnO nanoparticles, suggesting that the (PLL)ZnO nanoparticles may induce cell death via adsorption and endocytosis of the nanoparticles. On the other hand, the negatively charged (PAA)ZnO nanoparticle seems to cause cell death process indirectly via the released Zn ions, with less contribution from cellular association of nanoparticles. Time- and dose-dependent studies on cellular association of ZnO nanoparticles, and ZnO associated reactive oxygen species generation were also performed for the HeLa cells exposed to the (PLL)ZnO nanoparticle. For those cells associated with (PLL)ZnO nanoparticle, a significant enhancement in reactive oxygen species generation was observed even at a lower concentration (10 ppm), which was not observable for the results with the whole cell population. By using this approach, we are able to distinguish biological responses (e.g., reactive oxygen species (ROS) generation) directly related to the cellular associations of NPs from those indirectly related to the cellular associations of NPs, such as the cytotoxicity caused by the NP released metal ions.

  13. Ultrastructural and flow cytometric analyses of lipid accumulation in microalgae: Annual report, Solar Energy Research Institute, Aquatic Species Program

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-01-01

    Lipid accumulation in three species of microalgae was investigated with flow cytometry (FCM) and transmission electron microscopy (TEM). Previous studies using batch cultures of 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 lipid 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., 33 figs.

  14. Flow cytometric analysis of the H2O2-induced increase in intracellular Ca2+ concentration of rat thymocytes.

    Science.gov (United States)

    Okazaki, E; Chikahisa, L; Kanemaru, K; Oyama, Y

    1996-08-01

    The effect of hydrogen peroxide (H2O2) on the intracellular Ca2+ concentration ([Ca2+]i) of rat thymocytes was examined by a flow cytometer and two fluorescent dyes, fluo-3-AM and ethidium bromide, a dye impermeant to intact membranes, to characterize the H2O2-induced increase in [Ca2+]i. H2O2 at concentrations greater than 30 microM dose-dependently increased the [Ca2+]i of thymocytes which were not stained with ethidium. Removal of external Ca2+ greatly reduced the degree of H2O2-induced increase in [Ca2+]i. However, H2O2 still increased the [Ca2+]i under the external Ca(2+)-free condition. Diethylmaleate, which is known to produce a chemical depletion of cellular nonprotein thiol, significantly increased the [Ca2+]i. Dithiothreitol, which is used to protect cellular nonprotein thiol, slightly decreased the [Ca2+]i, but greatly reduced the H2O2-induced increase in [Ca2+]i. Therefore, it is considered that H2O2 may increase the [Ca2+]i through a mechanism related to the effects of H2O2 on the cellular nonprotein thiol.

  15. Quality control of flow cytometric immunophenotyping%流式细胞术表型分析的质量控制

    Institute of Scientific and Technical Information of China (English)

    吴丽娟; 许东升

    2011-01-01

    FCM表型分析已经从外周血淋巴细胞的双参数定量测定发展到当今对血液细胞病理学包括骨髓、淋巴结分析的5个或更多参数的定性测定,其中白血病和淋巴瘤的免疫表型分析在血液淋巴系统恶性疾病诊断、分类及病情监测上都是对形态学检验的一种重要补充.FCM 5个或更多参数分析的复杂性以及数据的解释依赖于仪器、试剂、程序的标准化及校准.此外,在美国的流式细胞学实验室还必须保留有关水平测试、样品制备、方法学的准确性、特异性、灵敏度和精密度的相关资料.CLSI和UCCC建议每个流式细胞学实验室需要对定性和定量检测程序进行验证.本文对美国临床流式细胞学实验室FCM检验的相关验证、室内与室间质量控制措施进行介绍.%Flow cytometric immunophenotyping has evolved from two-parameter quantitative measurement of peripheral blood lymphocytes to five-or more parameter qualitative evaluation of bone marrow and lymph node in hematopathology.Leukemia/lymphoma immunophenotyping represents an important addition to histomorphology in the diagnosis,classification and monitoring of hematolymphoid neoplasms.The complexity of five-or more parameter analyses and the interpretation of the data rely on standardization and validation of the instrument,the reagent and the procedure.In addition,clinical flow cytometry laboratories in U.S.A are required to document proficiency testing,sample preparation as well as accuracy,specificity,sensitivity and precision of methodology.CLSI and UCCC recommend that each laboratory should validate its own qualitative and quantitative procedures.This paper introduces the procedures for validation and quality control in a clinical flow cytometry laboratory in the United States.

  16. Flow cytometric analysis of the inhibition of human basophil activation by histamine high dilutions – a replication study

    Directory of Open Access Journals (Sweden)

    Chantal Wälchli

    2012-09-01

    perform in order to reduce the possibility of artifacts but was omitted in the former study. Conclusions: Laboratory independent replication of homeopathic basic research experiments is still a challenge. Assuming that the results formerly obtained with this model were not due to systematic errors, the quest identifying the crucial factors for successful reproducibility is open for future research. Keywords: Human basophils; histamine; high dilutions; flow cytometry Reference: [1] Sainte-Laudy J, Belon P. Improvement of flow cytometric analysis of basophil activation inhibition by high histamine dilutions. A novel basophil specific marker: CD 203c. Homeopathy. 2006;95:3-8.

  17. Evaluation of zinc oxide nanoparticles toxicity on marine algae chlorella vulgaris through flow cytometric, cytotoxicity and oxidative stress analysis.

    Science.gov (United States)

    Suman, T Y; Radhika Rajasree, S R; Kirubagaran, R

    2015-03-01

    The increasing industrial use of nanomaterials during the last decades poses a potential threat to the environment and in particular to organisms living in the aquatic environment. In the present study, the toxicity of zinc oxide nanoparticles (ZnO NPs) was investigated in Marine algae Chlorella vulgaris (C. vulgaris). High zinc dissociation from ZnONPs, releasing ionic zinc in seawater, is a potential route for zinc assimilation and ZnONPs toxicity. To examine the mechanism of toxicity, C. vulgaris were treated with 50mg/L, 100mg/L, 200mg/L and 300 mg/L ZnO NPs for 24h and 72h. The detailed cytotoxicity assay showed a substantial reduction in the viability dependent on dose and exposure. Further, flow cytometry revealed the significant reduction in C. vulgaris viable cells to higher ZnO NPs. Significant reductions in LDH level were noted for ZnO NPs at 300 mg/L concentration. The activity of antioxidant enzyme superoxide dismutase (SOD) significantly increased in the C. vulgaris exposed to 200mg/L and 300 mg/L ZnO NPs. The content of non-enzymatic antioxidant glutathione (GSH) significantly decreased in the groups with a ZnO NPs concentration of higher than 100mg/L. The level of lipid peroxidation (LPO) was found to increase as the ZnO NPs dose increased. The FT-IR analyses suggested surface chemical interaction between nanoparticles and algal cells. The substantial morphological changes and cell wall damage were confirmed through microscopic analyses (FESEM and CM).

  18. Applications of immuno-magnetic bead and immunofluorescent flow cytometric techniques for the quantitative detection of HAB microalgae

    Institute of Scientific and Technical Information of China (English)

    HUANG Jian; WEN Ruobing; BAO Zhenmin; SUI Zhenghong; SUN Ningbo; KANG Kyoungho

    2012-01-01

    Over the last severaldecades,harmful algal blooms (HABs) havebecome a serious environmental problem in many parts of the world.A rapid and accurate detection process for HAB algae has yet to be developed.Heterosigma akashiwo is one of the most important HABs species in China.The objective of this study was to develop an immunologic technique that can rapidly and sensitively count H.akashiwo cells.Five HABs species (Alexandrium catenella,Thalassiosira sp.,Cryptomonas sp.,Alexandrium tamarense and Symbiodinium sp.,) were used in this study to evaluate the analysis process we developed.A polyclonal antibody with high titers against H.akashiwo was obtained by injecting H.akashiwo cells into rabbits.Immuno-magnetic beads (IMB) were produced via conjugated polyclonal antibodies with magnetic beads and applied to isolate and count H.akashiwo cells from the culture.Results show that 66.7%-91.6% of the cells were captured from unialgal culture by IMBs,and only 5.3%-12.5% of the four other HAB microalgae species were captured,indicating that the constructed IMBs combined specifically with the H.akashiwo cells.At the same time,flow cytometry (FCM) sorting was exploited to screen H.akashiwo cells after labeling with FITC conjugated polyclonal antibodies.Using the FCM technique,91.7% of the targeted cells were sorted out from mixed microalgae samples in just a few minutes.These results indicate that both antibody-involved IMB and antibody-based FCM techniques are highly effective at detecting and quantifying HAB species.These techniques,especially immuno-magnetic separation,have low associated cost,and are fast and simple processes compared with other techniques currently in use.

  19. Flow-cytometric analysis of T-lymphocyte subsets after different treatment methods in patients with pericoronitis.

    Science.gov (United States)

    Orbak, Recep; Dayi, Ertunç

    2003-02-01

    The aim of this study was to determine whether there was any change in T-lymphocyte subsets in patients with periocoronitis after the application of different treatment methods. Twenty-six patients with acute pericoronitis were included in the study. In every phase of the treatment (pretreatment, postcurettage, and postextraction), the biopsy samples were taken from the gingival tissues at sites of pericoronitis. Then, CD4(+) and CD8(+) lymphocyte and CD4(+)/CD8(+) ratio values were determined using flow cytometry in the biopsy samples. At the same time, gingival index (Löe-Silness) and plaque index (Silness-Löe) scores were recorded to assess the periodontal status in patients. To determine the correlation between the clinical measurements and the laboratory results obtained before the treatment, after curettage, and after extraction, we conducted an analysis using a paired t-test. The normal values in peripheral blood of CD4(+) and CD8(+) lymphocytes are 25% to 29% and 19% to 48%, respectively. However, the CD4(+) and CD8(+) lymphocyte values in the patients with acute pericoronitis were found to be 22.12% +/- 6.15% and 7.69% +/- 4.12%, respectively. These values are lower than the normal values. The CD4(+) lymphocyte value increased to 31.06% +/- 7.09% postcurettage and to 32.24% +/- 3.11% postextraction. The CD8(+) lymphocyte value increased to 16.21% +/- 5.27% postcurettage and to 18.25% +/- 3.13% postextraction. The CD4/CD8 ratio increased postcurettage and postextraction. This increase was statistically significant (P pericoronitis pathobiology.

  20. Modeling of inter-sample variation in flow cytometric data with the joint clustering and matching procedure.

    Science.gov (United States)

    Lee, Sharon X; McLachlan, Geoffrey J; Pyne, Saumyadipta

    2016-01-01

    We present an algorithm for modeling flow cytometry data in the presence of large inter-sample variation. Large-scale cytometry datasets often exhibit some within-class variation due to technical effects such as instrumental differences and variations in data acquisition, as well as subtle biological heterogeneity within the class of samples. Failure to account for such variations in the model may lead to inaccurate matching of populations across a batch of samples and poor performance in classification of unlabeled samples. In this paper, we describe the Joint Clustering and Matching (JCM) procedure for simultaneous segmentation and alignment of cell populations across multiple samples. Under the JCM framework, a multivariate mixture distribution is used to model the distribution of the expressions of a fixed set of markers for each cell in a sample such that the components in the mixture model may correspond to the various populations of cells, which have similar expressions of markers (that is, clusters), in the composition of the sample. For each class of samples, an overall class template is formed by the adoption of random-effects terms to model the inter-sample variation within a class. The construction of a parametric template for each class allows for direct quantification of the differences between the template and each sample, and also between each pair of samples, both within or between classes. The classification of a new unclassified sample is then undertaken by assigning the unclassified sample to the class that minimizes the distance between its fitted mixture density and each class density as provided by the class templates. For illustration, we use a symmetric form of the Kullback-Leibler divergence as a distance measure between two densities, but other distance measures can also be applied. We show and demonstrate on four real datasets how the JCM procedure can be used to carry out the tasks of automated clustering and alignment of cell

  1. Improved flow cytometric method to enumerate residual cells: minimal linear detection limits for platelets, erythrocytes, and leukocytes.

    Science.gov (United States)

    Pichler, J; Printz, D; Scharner, D; Trbojevic, D; Siekmann, J; Fritsch, G

    2002-08-15

    Increasing demand for quality control of blood products requires more sensitive methods to enumerate residual cells. Presently, the reported threshold (in cells per microliter) is 400 for red blood cells, 30-500 for platelets, and 1 for leukocytes. To examine precision and linearity in enumerating residual platelets and red blood cells, EDTA-anticoagulated blood from healthy donors was serially diluted with serum, stained in TruCount tubes using a no-lyse/no-wash procedure and a monoclonal antibody cocktail against the CD42a (FL1) and glycophorin-A (FL2) epitopes, and analyzed by flow cytometry. Leukocyte counts were determined in separate tubes. Cell preparation and analysis were performed once for 20 blood samples each and 20 times using the same specimen. Acquisition from the same tube was performed separately for platelets (threshold on FL1) and red blood cells (threshold on FL2). Multiparameter analysis was used for data evaluation. Linear results were obtained for platelets per microliter between 3,410 and 5 and for red blood cells per microliter between 54,000 and 3. For the lower cell concentrations, the coefficient of variation was 16.7% for platelets and 10.9% for red blood cells. The presented method allows the distinction between physiologically intact and ghost red blood cells. The method represents a reliable, sensitive, and accurate approach to quantify platelets and red blood cells in diluted blood. It can be applied to enumerate residual cells in plasma products and meets the increasing demand for quality control in blood components.

  2. Applications of immuno-magnetic bead and immunofluorescent flow cytometric techniques for the quantitative detection of HAB microalgae

    Science.gov (United States)

    Huang, Jian; Wen, Ruobing; Bao, Zhenmin; Sui, Zhenghong; Sun, Ningbo; Kang, Kyoungho

    2012-05-01

    Over the last several decades, harmful algal blooms (HABs) have become a serious environmental problem in many parts of the world. A rapid and accurate detection process for HAB algae has yet to be developed. Heterosigma akashiwo is one of the most important HABs species in China. The objective of this study was to develop an immunologic technique that can rapidly and sensitively count H. akashiwo cells. Five HABs species ( Alexandrium catenella, Thalassiosira sp., Cryptomonas sp., Alexandrium tamarense and Symbiodinium sp.), were used in this study to evaluate the analysis process we developed. A polyclonal antibody with high titers against H. akashiwo was obtained by injecting H. akashiwo cells into rabbits. Immuno-magnetic beads (IMB) were produced via conjugated polyclonal antibodies with magnetic beads and applied to isolate and count H. akashiwo cells from the culture. Results show that 66.7%-91.6% of the cells were captured from unialgal culture by IMBs, and only 5.3%-12.5% of the four other HAB microalgae species were captured, indicating that the constructed IMBs combined specifically with the H. akashiwo cells. At the same time, flow cytometry (FCM) sorting was exploited to screen H. akashiwo cells after labeling with FITC conjugated polyclonal antibodies. Using the FCM technique, 91.7% of the targeted cells were sorted out from mixed microalgae samples in just a few minutes. These results indicate that both antibody-involved IMB and antibody-based FCM techniques are highly effective at detecting and quantifying HAB species. These techniques, especially immuno-magnetic separation, have low associated cost, and are fast and simple processes compared with other techniques currently in use.

  3. Flow cytometric analysis on tri-n-butyltin-induced increase in annexin V binding to membranes of rat thymocytes.

    Science.gov (United States)

    Nakata, M; Oyama, Y; Okada, Y; Yamazaki, Y; Chikahisa, L; Satoh, M

    1999-10-01

    Effects of tri-n-butyltin chloride (TBT) on rat thymocytes were examined by using a flow cytometer and three fluorescent dyes (annexin V-FITC, ethidium bromide and fluo-3-AM) to further characterize its cytotoxic action. TBT at concentrations of 100 nM or greater, time- and dose-dependently increased the population of annexin V-positive live cells in the cell suspension. Most of cells became to be annexin V-positive within 60 min after the start of application of 300 nM TBT. Some of annexin V-positive live cells were further stained with ethidium, indicating that some of the cells were killed, in continued presence of TBT at 300 nM or greater. When the cells were exposed to 300 nM TBT only for 15 min, the population of annexin V-positive live cells increased after removal of TBT from incubation medium. TBT-induced increase in the population of annexin V-positive live cells was partly attenuated under Ca(2+)-free condition, although that was not the case for the dead cells. TBT at 30 nM or greater increased [Ca(2+)]i in a dose-dependent manner. Triethyltin and trimethyltin even at 1 μM did not increase the [Ca(2+)]i and the population of annexin V-positive live cells. The population of annexin V-positive live cells increased as the [Ca(2+)]i was increased by ionomycin, a calcium ionophore. Results suggest an involvement of Ca(2+) in some of TBT-induced cytotoxicity.

  4. Radio-protective effect of vitamin E on spermatogenesis in mice exposed to γ-irradiation: a flow cytometric study

    Institute of Scientific and Technical Information of China (English)

    C.Songthaveesin; J.Saikhun; Y.Kitiyanant; K.Pavasuthipaisit

    2004-01-01

    Aim: To investigate the effect of vitamin E on the radioprotection of spermatogenesis and chromatin condensation of spermatozoa during passage through the epididymis in mice exposed to irradiation. Methods: Adult outbred male ICR mice were orally administered natural vitamin E (VE,D-a-tocopheryl acetate) at 400 IU/kg for 7 days before exposure to 1 Gy of y-irradiation. The animals were sacrificed at day 1,7,14,21, 28, 35 and 70 post-irradiation (IR) and the percentage of testicular germ cells and epididymal sperm chromatin condensation was analyzed using flow cytometry. Results: Serum D-a-tocopheryl acetate levels were 47.4±3.2μg/dL in the treatedgroup, yet it could not be detected in the control group. The testicular weight of irradiated mice pretreated with VE+IR was significantly (P<0.05) higher than that of those without VE treatment (IR) at day 14 and 21 post-irradiation. The percentage of primary spermatocytes (4C) in the VE+IR group was comparable to the controls but significantly (P<0.05) higher than those in the IR group from day 7 to 35 post-irradiation. The percentage of round spermatids (1C) in the VE+IR group was also significantly (P<0.05) higher than those in the IR group at day 28 postirradiation. The primary spermatocytes:spermatogonia ratio in the IR group was significantly (P<0.05) declined at day 7 to 35 post-irradiation when compared to the VE+IR and control groups. The round spermatid:spermatogonia ratio in the VE+IR group was significantly (P<0.05) higher than that of the IR group at day 14 and 28 post-irradiation.The chromatin condensation of epididymal spermatozoa measured by propidium iodide uptake was not affected by 1 Gy of γ-irradiation. Conclusion: The administration of VE prior to irradiation protects spermatogenic cells fromradiation. (Asian J Androl 2004 Dec; 6: 331-336)

  5. Effects of trematode parasitism on the shell morphology of snails from flow and nonflow environments.

    Science.gov (United States)

    Gustafson, Kyle D; Bolek, Matthew G

    2016-03-01

    The primary function of the gastropod shell is protection. However, shells that function well in one environment may be maladaptive in another. Upon infection, the snail shell protects internal parasites and it is to the parasite's advantage to optimize, or not interfere with, shell functionality. However, parasites, particularly trematodes, are often pathogenic and it is not clear if parasitism will induce environment-dependent or -independent changes to gastropod shells. We conducted a field study and a complementary laboratory experiment to examine the effects of trematode parasitism on shell characteristics (shape, size, and crush resistance) of Physa acuta snails in flow and nonflow environments using geometric morphometrics and crush assays. Field results indicate wetland (nonflow) snails had large, crush resistant shells with narrow apertures and tall spires. In contrast, stream (flow) snails had small, weak shells with wide apertures and short spires. Parasitism had no apparent effect on the crush resistance of wetland snails but significantly reduced the crush resistance of stream snails. Parasitism had no significant effect on overall shell shape in stream or wetland snails. Similar to the results of our field study, nonflow tank snails had significantly more crush resistant shells than flow tank snails. Additionally, the shapes of flow and nonflow tank snails significantly differed where nonflow tank snails resembled wetland snails and flow tank snails resembled stream snails. For laboratory snails, parasitism reduced crush resistance regardless of flow/nonflow treatment. Our results demonstrate that habitat and/or flow treatment was the primary factor affecting P. acuta shell morphology and that trematode parasitism played a secondary role.

  6. 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.

  7. Rapid Multiplexed Flow Cytometric Assay for Botulinum Neurotoxin Detection Using an Automated Fluidic Microbead-Trapping Flow Cell for Enhanced Sensitivity

    Energy Technology Data Exchange (ETDEWEB)

    Ozanich, Richard M.; Bruckner-Lea, Cindy J.; Warner, Marvin G.; Miller, Keith D.; Antolick, Kathryn C.; Marks, James D.; Lou, Jianlong; Grate, Jay W.

    2009-07-15

    A bead-based sandwich immunoassay for botulinum neurotoxin serotype A (BoNT/A) has been developed and demonstrated using a recombinant 50 kDa fragment (BoNT/A-HC-fragment) of the BoNT/A heavy chain (BoNT/A-HC) as a structurally valid simulant. Three different anti-BoNT/A antibodies were attached to three different fluorescent dye encoded flow cytometry beads for multiplexing. The assay was conducted in two formats: a manual microcentrifuge tube format and an automated fluidic system format. Flow cytometry detection was used for both formats. The fluidic system used a novel microbead-trapping flow cell to capture antibody-coupled beads with subsequent sequential perfusion of sample, wash, dye-labeled reporter antibody, and final wash solutions. After the reaction period, the beads were collected for analysis by flow cytometry. Sandwich assays performed on the fluidic system gave median fluorescence intensity signals on the flow cytometer that were 2-4 times higher than assays performed manually in the same amount of time. Limits of detection were estimated at 1 pM (~50 pg/mL for BoNT/A-HC-fragment) for the 15 minute fluidic assay.

  8. A rapid and robust tri-color flow cytometry assay for monitoring malaria parasite development.

    Science.gov (United States)

    Malleret, Benoît; Claser, Carla; Ong, Alice Soh Meoy; Suwanarusk, Rossarin; Sriprawat, Kanlaya; Howland, Shanshan Wu; Russell, Bruce; Nosten, Francois; Rénia, Laurent

    2011-01-01

    Microscopic examination of Giemsa-stained thin blood smears remains the gold standard method used to quantify and stage malaria parasites. However, this technique is tedious, and requires trained microscopists. We have developed a fast and simple flow cytometry method to quantify and stage, various malaria parasites in red blood cells in whole blood or in vitro cultured Plasmodium falciparum. The parasites were stained with dihydroethidium and Hoechst 33342 or SYBR Green I and leukocytes were identified with an antibody against CD45. Depending on the DNA stains used, samples were analyzed using different models of flow cytometers. This protocol, which does not require any washing steps, allows infected red blood cells to be distinguished from leukocytes, as well as allowing non-infected reticulocytes and normocytes to be identified. It also allows assessing the proportion of parasites at different developmental stages. Lastly, we demonstrate how this technique can be applied to antimalarial drug testing.

  9. 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)

  10. Parasitic Currents in Diffuse-Interface Two-Phase Flow Simulations

    Science.gov (United States)

    Milani, Pedro; Mirjalili, Seyedshahabaddin; Mani, Ali

    2016-11-01

    Two phase flow phenomena are important in a wide range of applications, such as bubble generation in ocean waves and droplet dynamics in fuel injectors. Several methods can be used to simulate such phenomena. The focus of this study is the diffuse-interface method, in which the interface is described via a mixing energy and spans a few computational cells, while surface tension is modeled as a force density term on the right-hand side of the momentum equation. The advantages of this method include the ability to easily simulate complex geometries since it does not require special treatment around the interface, and to conserve mass exactly. However, this method suffers from parasitic currents, an unphysical velocity field generated close to the interface due to numerical imprecisions in the surface tension term. This can be a serious problem in low speed flows, where the parasitic currents are significant compared to the velocity scale of the problem. In this study, we consider a wide range of diffuse-interface schemes for two-phase flows, including different options for discrete representation of the surface tension force. By presenting an assessment of each method's performance in scenarios involving parasitic currents, we develop accuracy estimates and guidelines for selection among these models. Supported by the ONR.

  11. 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

  12. 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 w

  13. 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.

  14. Flow Cytometric Panel-Reactive Antibody Results and the Ability to Find Transfusion-Compatible Platelets after Antibody-Desensitization for Allogeneic Bone Marrow Transplant.

    Science.gov (United States)

    Rosenbaum, Eric R; Pandey, Soumya; Harville, Terry O; Drobena, Gina A; Cottler-Fox, Michele

    2016-12-01

    Panel reactive antibody (PRA) reduction protocols are used to decrease anti-HLA antibodies with concomitant PRA monitoring as a measure of successful treatment prior to organ and haploidentical blood and marrow transplant (BMT). We hypothesized that the more sensitive flow cytometry (FC) based assays for PRA [FlowPRA(®) and Luminex(®) based Single Antigen Bead (SAB)] would also correlate with the ability to find compatible platelets for allosensitized recipients. A female patient with myelodysplastic syndrome and a high HLA class I PRA [>90% PRA and cPRA by complement-dependent cytotoxicity (CDC) assay and Flow PRA] required allogeneic BMT. Baseline HLA Class I and class II antigen typing was performed and a matched sibling donor was identified. Although baseline anti-HLA class I and class II antibodies measured by FC and CDC revealed no donor specific antibodies (DSA), the decision was made to attempt antibody desensitization to facilitate platelet transfusion during BMT. FC and CDC assays were performed to determine anti-HLA class I antibodies and cPRA/%PRA prior to starting desensitization and at the end of desensitization. Over the course of desensitization and BMT, a total of 194 apheresis platelet units underwent cross-match (XM) using Capture-P(®). We compared temporally-related PRA results with platelet XM results. High PRA by FC or CDC assays correlates with a high % of XM-positive (incompatible) platelet units. When the CDC PRA fell to 2% after desensitization, platelet XM incompatibility fell from 100% to 63% positive (incompatible). When the FC PRA fell to 5% the positive platelet XM fell to 5%. Antibody desensitization facilitated platelet transfusion. PRA determination by FC appeared better correlated than determination by CDC with the ability to find XM-compatible platelets. © 2016 by the Association of Clinical Scientists, Inc.

  15. 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.

  16. Fourier transform infra-red spectroscopy and flow cytometric assessment of the antibacterial mechanism of action of aqueous extract of garlic (Allium sativum) against selected probiotic Bifidobacterium strains.

    Science.gov (United States)

    Booyens, Jemma; Thantsha, Mapitsi Silvester

    2014-08-06

    It is generally reported that garlic (Allium sativum) harms pathogenic but not beneficial bacteria. Although numerous studies supporting the alleged garlic effects on pathogens are available, there are limited studies to prove this claim for beneficial bacteria. We have recently shown that garlic exhibits antibacterial activity against probiotic bifidobacteria. The aim of the current study was to elucidate the mechanism of action of garlic clove extract (GCE) on Bifidobacterium bifidum LMG 11041, B. longum LMG 13197 and B. lactis Bb12 using Fourier transform infrared (FT-IR) spectroscopy and flow cytometry. Cultures (1 × 108 CFU ml-1) were individually incubated for 6 h at 37°C in garlic clove extract containing allicin at a corresponding predetermined minimum bactericidal concentration for each strain. For FTIR, an aliquot of each culture was deposited on CaF2 slide and vacuum dried. The slides were immediately viewed using a Bruker Vertex 70 V FT-IR spectrometer equipped with a Hyperion microscope and data analyzed using OPUS software (version 6, Bruker). Spectra were smoothed with a Savitsky-Goly function algorithim, base-line corrected and normalized. Samples for flow cytometry were stained using the Live/Dead BacLight bacterial viability kit L7012. Data compensation and analysis was performed using a BD FACSAria and FlowJo (version 7.6.1). Fourier transform infrared spectroscopy showed changes in spectral features of lipids and fatty acids in cell membranes, proteins, polysaccharides and nucleic acids. Spectral data as per principle component analysis (PCA) revealed segregation of control and GCE-treated cells for all the tested bifidobacteria. Flow cytometry not only showed increase in numbers of membrane damaged and possibly lysed cells after GCE treatment, but also displayed diffuse light scatter patterns for GCE treated cells, which is evidence for changes to the size, granularity and molecular content of the cells. Garlic has multiple target sites in

  17. Flow Cytometric Immunobead Assay for Detection of BCR-ABL1 Fusion Proteins in Chronic Myleoid Leukemia: Comparison with FISH and PCR Techniques.

    Directory of Open Access Journals (Sweden)

    Anna Grazia Recchia

    Full Text Available Chronic Myeloid Leukemia (CML is characterized by a balanced translocation juxtaposing the Abelson (ABL and breakpoint cluster region (BCR genes. The resulting BCR-ABL1 oncogene leads to increased proliferation and survival of leukemic cells. Successful treatment of CML has been accompanied by steady improvements in our capacity to accurately and sensitively monitor therapy response. Currently, measurement of BCR-ABL1 mRNA transcript levels by real-time quantitative PCR (RQ-PCR defines critical response endpoints. An antibody-based technique for BCR-ABL1 protein recognition could be an attractive alternative to RQ-PCR. To date, there have been no studies evaluating whether flow-cytometry based assays could be of clinical utility in evaluating residual disease in CML patients. Here we describe a flow-cytometry assay that detects the presence of BCR-ABL1 fusion proteins in CML lysates to determine the applicability, reliability, and specificity of this method for both diagnosis and monitoring of CML patients for initial response to therapy. We show that: i CML can be properly diagnosed at onset, (ii follow-up assessments show detectable fusion protein (i.e. relative mean fluorescent intensity, rMFI%>1 when BCR-ABL1IS transcripts are between 1-10%, and (iii rMFI% levels predict CCyR as defined by FISH analysis. Overall, the FCBA assay is a rapid technique, fully translatable to the routine management of CML patients.

  18. Flow Cytometric Immunobead Assay for Detection of BCR-ABL1 Fusion Proteins in Chronic Myleoid Leukemia: Comparison with FISH and PCR Techniques.

    Science.gov (United States)

    Recchia, Anna Grazia; Caruso, Nadia; Bossio, Sabrina; Pellicanò, Mariavaleria; De Stefano, Laura; Franzese, Stefania; Palummo, Angela; Abbadessa, Vincenzo; Lucia, Eugenio; Gentile, Massimo; Vigna, Ernesto; Caracciolo, Clementina; Agostino, Antolino; Galimberti, Sara; Levato, Luciano; Stagno, Fabio; Molica, Stefano; Martino, Bruno; Vigneri, Paolo; Di Raimondo, Francesco; Morabito, Fortunato

    2015-01-01

    Chronic Myeloid Leukemia (CML) is characterized by a balanced translocation juxtaposing the Abelson (ABL) and breakpoint cluster region (BCR) genes. The resulting BCR-ABL1 oncogene leads to increased proliferation and survival of leukemic cells. Successful treatment of CML has been accompanied by steady improvements in our capacity to accurately and sensitively monitor therapy response. Currently, measurement of BCR-ABL1 mRNA transcript levels by real-time quantitative PCR (RQ-PCR) defines critical response endpoints. An antibody-based technique for BCR-ABL1 protein recognition could be an attractive alternative to RQ-PCR. To date, there have been no studies evaluating whether flow-cytometry based assays could be of clinical utility in evaluating residual disease in CML patients. Here we describe a flow-cytometry assay that detects the presence of BCR-ABL1 fusion proteins in CML lysates to determine the applicability, reliability, and specificity of this method for both diagnosis and monitoring of CML patients for initial response to therapy. We show that: i) CML can be properly diagnosed at onset, (ii) follow-up assessments show detectable fusion protein (i.e. relative mean fluorescent intensity, rMFI%>1) when BCR-ABL1IS transcripts are between 1-10%, and (iii) rMFI% levels predict CCyR as defined by FISH analysis. Overall, the FCBA assay is a rapid technique, fully translatable to the routine management of CML patients.

  19. Epstein-Barr Virus, Human Papillomavirus, and Flow Cytometric Cell Cycle Kinetics in Nasopharyngeal Carcinoma and Inverted Papilloma among Egyptian Patients

    Directory of Open Access Journals (Sweden)

    S. K. Kassim

    1998-01-01

    Full Text Available It is widely accepted that the Epstein-Barr virus is etiologically associated with the development of nasopharyngeal carcinoma. The human papillomavirus is also associated with inverted papilloma. We used the polymerase chain reaction technique to detect both viruses in both types of tumors. Flow cytometry was also used to study the DNA pattern and proliferative behavior of the tumors in relation to the viruses. EBV was detected in 13/20 (65% of NPC specimens, and in none of IP (n = 10 or control specimens (n = 10. This indicates the contribution of EBV as an etiologic factor in NPC. Five cases of NPC (25% were positive for HPV 16, two of them were EBV positive. Four HPV 16 positive cases were found among cases with inverted papilloma, but none among the control cases. Flow cytometry revealed that all NPC, IP, and control samples were diploid except one aneuploid NPC sample. Proliferative capacity (PC of primary tumors was predictive of tumor recurrence in NPC. Using 13.6% as a cut-off point for PC, we were able to discriminate between high risk and low risk groups with 100% sensitivity and 86% specificity. PC can be used as a baseline prognostic parameter in NPC, making it possible to modify courses of treatment in an attempt to inhibit tumor recurrence.

  20. Flow cytometric analysis of hemetopoietic progenitor cells in peripheral blood stem cell harvest from patients with CD34 positive acute leukemia.

    Science.gov (United States)

    Miyazaki, T; Matsuda, I; Oguri, M; Amaya, H; Kiyosaki, M; Hamada, A; Tamaki, S; Tashiro, E; Kudo, Y; Taniguchi, O; Nakamura, T; Tomoyasu, S

    2001-01-01

    We analyzed CD34 positive cells in peripheral blood stem cell harvest (PBSCH) using flow cytometry. PBSCH from CD34 positive acute myelogeous leukemia (AML-M2) patient contained 1.87% CD34 positive cells, of which 1.21% was represented by MRD.PBSCH from CD34 positive acute lymphoblast leukemia (ALL) patient contained 3.14% CD34 positive cells, of which 0.11% was accounted for by minimal residual disease (MRD). If PBSCH from CD34 positive acute leukemia patient is analyzed for CD34 monoclonal antibody alone, the presence of CD34 positive MRD may escape attention so that CD34 positive hematopoietic progenitor cells may be overestimated. To avoid this risk, it is necessary to analyze PBSCH using both CD34 monoclonal antibody and characteristic markers of leukemia cells that were found pre-treatment.

  1. Single-colour flow cytometric assay to determine NK cell-mediated cytotoxicity and viability against non-adherent human tumor cells.

    Science.gov (United States)

    Thakur, Ajit; Zaman, Abeyat; Hummel, Jeff; Jones, Kim; Hortelano, Gonzalo

    2012-03-01

    A flow cytometry-based cytotoxicity (FCC) assay was developed using a single fluorophore, calcein-acetoxymethyl diacetylester (calcein-AM), to measure NK cell-mediated cytotoxicity. Non-adherent human K562 and U937 target cells were individually labelled with calcein-AM and co-incubated with effector NK cells to measure calcein loss, and therefore calculate target cell cytotoxicity. This FCC assay also provided a measure of sample viability. Notably, cell viability measured by traditional calcein/7-amino-actinomycin D (7-AAD) double labelling and Trypan Blue methods were comparable to the viability calculated using calcein-loss FCC. This FCC assay may also be used with various effector and target cell types and as a multi-parameter tool to measure viability and immunophenotype cells for tissue engineering purposes.

  2. Flow cytometric detection and quantification of CD56 (neural cell adhesion molecule, NCAM) expression in diffuse large B cell lymphomas and review of the literature.

    Science.gov (United States)

    Stacchini, Alessandra; Barreca, Antonella; Demurtas, Anna; Aliberti, Sabrina; di Celle, Paola Francia; Novero, Domenico

    2012-02-01

    To report unusual CD56 (neural cell adhesion molecule, NCAM) expression on diffuse large B cell lymphoma (DLBCL). CD56 expression was first detected and quantified on tissues obtained from five cases of DLBCL by flow cytometry (FC), then confirmed by immunohistochemistry. The CD56 expression pattern was heterogeneous among the cases [the molecular equivalent of soluble fluorochrome (MESF) level ranged from 2214 to 133 466]. All were CD10 and Bcl-6 positive, suggesting their germinal centre origin; one was also CD5 positive. An extranodal presentation occurred in three of five cases. CD56 expression in B cell lymphoma is a rare occurrence. FC is able to identify aberrant immunophenotypes that can be useful in the identification and monitoring of B cell lymphoma subtypes. The presence of CD56 reported by the literature on certain DLBCL with extranodal presentation might be related to mechanisms involved in growth and expansion. © 2012 Blackwell Publishing Limited.

  3. Evaluation of a multi-endpoint assay in rats, combining the bone-marrow micronucleus test, the Comet assay and the flow-cytometric peripheral blood micronucleus test.

    Science.gov (United States)

    Bowen, Damian E; Whitwell, James H; Lillford, Lucinda; Henderson, Debbie; Kidd, Darren; Mc Garry, Sarah; Pearce, Gareth; Beevers, Carol; Kirkland, David J

    2011-05-18

    With the publication of revised draft ICH guidelines (Draft ICH S2), there is scope and potential to establish a combined multi-end point in vivo assay to alleviate the need for multiple in vivo assays, thereby reducing time, cost and use of animals. Presented here are the results of an evaluation trial in which the bone-marrow and peripheral blood (via MicroFlow(®) flow cytometry) micronucleus tests (looking at potential chromosome breakage and whole chromosome loss) in developing erythrocytes or young reticulocytes were combined with the Comet assay (measuring DNA strand-breakage), in stomach, liver and blood lymphocytes. This allowed a variety of potential target tissues (site of contact, site of metabolism and peripheral distribution) to be assessed for DNA damage. This combination approach was performed with minimal changes to the standard and regulatory recommended sampling times for the stand-alone assays. A series of eight in vivo genotoxins (2-acetylaminofluorene, benzo[a]pyrene, carbendazim, cyclophosphamide, dimethylnitrosamine, ethyl methanesulfonate, ethyl nitrosourea and mitomycin C), which are known to act via different modes of action (direct- and indirect-acting clastogens, alkylating agents, gene mutagens, cross-linking and aneugenic compounds) were tested. Male rats were dosed at 0, 24 and 45 h, and bone marrow and peripheral blood (micronucleus endpoint), liver, whole blood and stomach (Comet endpoint) were sampled at three hours after the last dose. Comet and micronucleus responses were as expected based on available data for conventional (acute) stand-alone assays. All compounds were detected as genotoxic in at least one of the endpoints. The importance of evaluating both endpoints was highlighted by the uniquely positive responses for certain chemicals (benzo[a]pyrene and 2-acetylaminofluorene) with the Comet endpoint and certain other chemicals (carbendazim and mitomycin C) with the micronucleus endpoint. The data generated from these

  4. Accurate detection of the tumor clone in peripheral T-cell lymphoma biopsies by flow cytometric analysis of TCR-Vβ repertoire.

    Science.gov (United States)

    Salameire, Dimitri; Solly, Françoise; Fabre, Blandine; Lefebvre, Christine; Chauvet, Martine; Gressin, Rémy; Corront, Bernadette; Ciapa, Agnès; Pernollet, Martine; Plumas, Joël; Macintyre, Elizabeth; Callanan, Mary B; Leroux, Dominique; Jacob, Marie-Christine

    2012-09-01

    Multiparametric flow cytometry has proven to be a powerful method for detection and immunophenotypic characterization of clonal subsets, particularly in lymphoproliferative disorders of the B-cell lineage. Although in theory promising, this approach has not been comparably fulfilled in mature T-cell malignancies. Specifically, the T-cell receptor-Vβ repertoire analysis in blood can provide strong evidence of clonality, particularly when a single expanded Vß family is detected. The purpose of this study was to determine the relevance of this approach when applied to biopsies, at the site of tumor involvement. To this end, 30 peripheral T-cell lymphoma and 94 control biopsies were prospectively studied. Vβ expansions were commonly detected within CD4+ or CD8+ T cells (97% of peripheral T-cell lymphoma and 54% of non-peripheral T-cell lymphoma cases); thus, not differentiating malignant from reactive processes. Interestingly, we demonstrated that using a standardized evaluation, the detection of a high Vβ expansion was closely associated with diagnosis of peripheral T-cell lymphoma, with remarkable specificity (98%) and sensitivity (90%). This approach also identified eight cases of peripheral T-cell lymphoma that were not detectable by other forms of immunophenotyping. Moreover, focusing Vβ expression analysis to T-cell subsets with aberrant immunophenotypes, we demonstrated that the T-cell clone might be heterogeneous with regard to surface CD7 or CD10 expression (4/11 cases), providing indication on 'phenotypic plasticity'. Finally, among the wide variety of Vβ families, the occurrence of a Vβ17 expansion in five cases was striking. To our knowledge, this is the first report demonstrating the power of T-cell receptor-Vβ repertoire analysis by flow cytometry in biopsies as a basis for peripheral T-cell lymphoma diagnosis and precise T-cell clone identification and characterization.

  5. 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.

  6. In vitro holding and PLD-repair. Pt. 2. A flow cytometric and electron microscopic analysis of some mammalian cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Stevenson, A.F.G. (Institute for Basic Research in Developmental Disabilities, Staten Island, NY (USA)); Palackal, T. (State Univ. of New York, Brooklyn, NY (USA). Div. of Radiobiology); Lange, C.S. (Kiel Univ. (Germany, F.R.). Abt. Frauenheilkunde)

    1989-12-01

    The repair of potentially lethal damage (PLDR) in mammalian cells is expected to be better in quiescent cultures since PLD is supposedly fixed during cycle progression. Plateau phase cultures, therefore, serve as models because of assumed mitotic quiescence. Four established cell lines (V79, CHO, L5178Y and HELA) and one euploid cell strain IMR-90 have been analysed by flow cytometry and electron microscopy to address questions on quiescence in the plateau phase and the effect of holding (induction of quiescence by nutrient privation). In contrast to commonly held views, our results indicate that the quiescent fraction in cultures from transformed cells is exceedingly low (1% or less). Plateau phase cultures of transformed cells are constantly turning over. Euploid cells like the IMR-90 show true quiescence in the plateau phase. Holding causes typical cytopathological changes. These changes have been ultrastructurely characterised. Resistant sub-populations of cells can be selected out under holding-conditions. Such selected cells show completely different radiobiological characteristics, which raise questions on the interpretation of data on PLDR. (orig.).

  7. Final results of a multicenter trial addressing role of CSF flow cytometric analysis in NHL patients at high risk for CNS dissemination.

    Science.gov (United States)

    Benevolo, Giulia; Stacchini, Alessandra; Spina, Michele; Ferreri, Andrés J M; Arras, Marcella; Bellio, Laura; Botto, Barbara; Bulian, Pietro; Cantonetti, Maria; Depaoli, Lorella; Di Renzo, Nicola; Di Rocco, Alice; Evangelista, Andrea; Franceschetti, Silvia; Godio, Laura; Mannelli, Francesco; Pavone, Vincenzo; Pioltelli, Pietro; Vitolo, Umberto; Pogliani, Enrico M

    2012-10-18

    This prospective study compared diagnostic and prognostic value of conventional cytologic (CC) examination and flow cytometry (FCM) of baseline samples of cerebrospinal fluid (CSF) in 174 patients with newly diagnosed aggressive non-Hodgkin lymphoma (NHL). FCM detected a neoplastic population in the CSF of 18 of 174 patients (10%), CC only in 7 (4%; P < .001); 11 patients (14%) were discordant (FCM(+)/CC(-)). At a median follow-up of 46 months, there were 64 systemic progressions and 10 CNS relapses, including 2 patients with both systemic and CNS relapses. Two-year progression-free and overall survival were significantly higher in patients with FCM(-) CSF (62% and 72%) compared with those FCM(+) CSF (39% and 50%, respectively), with a 2-year CNS relapse cumulative incidence of 3% (95% confidence interval [CI], 0-7) versus 17% (95% CI, 0-34; P = .004), respectively. The risk of CNS progression was significantly higher in FMC(+)/CC(-) versus FCM(-)/CC(-) patients (hazard ratio = 8.16, 95% CI, 1.45-46). In conclusion, FCM positivity in the CSF of patients with high-risk NHL is associated with a significantly higher CNS relapse risk and poorer outcome. The combination of IV drugs with a higher CNS bioavailability and intrathecal chemotherapy is advisable to prevent CNS relapses in FCM(+) patients.

  8. Clinical performance of human papillomavirus E6, E7 mRNA flow cytometric assay compared to human papillomavirus DNA typing.

    Science.gov (United States)

    Kottaridi, Christine; Tsiodras, Sotirios; Spathis, Aris; Chranioti, Aikaterini; Pappas, Asimakis; Kassanos, Dimitrios; Panayiotides, Ioannis; Karakitsos, Petros

    2011-12-01

    To use flow cytometry to screen cervical samples for the overexpression of human papillomavirus (HPV) E6 and E7 mRNA and compare the performance of this assay with an HPV DNA array for the detection of high-grade cervical lesions. Cervical samples were analyzed for HPV DNA by clinical arrays, and the overexpression of E6 and E7 viral oncogenes was monitored using an HPV mRNA detection kit that quantifies the intracellular HPV E6 and E7 mRNA on a cell-by-cell basis. HPV positivity increased with severity of histologic lesions. On the basis of histology-confirmed CIN 2+ cases the specificity of HPV assay was 73.9% (95% CI 66.07, 80.88), whereas it was 39.3% (95% CI 31.85, 47.1) for the DNA assay. The HPV assay provides an early predictor of persistent HPV infection and may improve cervical cancer screening by increasing the specificity of detecting high-grade lesions.

  9. The effect of anti-rosetting agents against malaria parasites under physiological flow conditions.

    Directory of Open Access Journals (Sweden)

    Yvonne Adams

    Full Text Available Rosetting remains the dominant malaria parasite adhesion phenotype associated with severe disease and pathogenicity in Africa. The formation of rosettes, whereby a Plasmodium falciparum infected erythrocyte (IE adheres to two or more non-IEs, is thought to facilitate the occlusion of microvascular blood vessels by adhering to host endothelial cells and other bound IEs. Current methods of determining the rosette-disrupting capabilities of antibodies/drugs have focused on static assays. As IEs in vivo are exposed to shear stresses within the microvasculature, the effect of flow conditions on rosetting requires further examination. This study establishes a new rosetting flow assay using a closed perfusion system together with inverted fluorescence microscopy and image analysis, and confirms previous reports that rosettes exist under shear stresses equivalent to those present in the microvasculature (0.5-1.0 dyn/cm(2. Furthermore, we tested the effectiveness of rosette-disrupting PfEMP1 antibodies, heparin and fucoidan over a range of concentrations on two P. falciparum strains, and found no statistically significant differences between the results of static and flow assays. The new flow assay is a valuable addition to the tools available to study rosetting. However, the static assay has good predictive value and remains useful as the standard screening test for rosette-disrupting interventions.

  10. Genotoxicity of doxorubicin in F344 rats by combining the comet assay, flow-cytometric peripheral blood micronucleus test, and pathway-focused gene expression profiling.

    Science.gov (United States)

    Manjanatha, Mugimane G; Bishop, Michelle E; Pearce, Mason G; Kulkarni, Rohan; Lyn-Cook, Lascelles E; Ding, Wei

    2014-01-01

    Doxorubicin (DOX) is an antineoplastic drug effective against many human malignancies. DOX's clinical efficacy is greatly limited because of severe cardiotoxicity. To evaluate if DOX is genotoxic in the heart, ~7-week-old, male F344 rats were administered intravenously 1, 2, and 3 mg/kg bw DOX at 0, 24, 48, and 69 hr and the Comet assays in heart, liver, kidney, and testis and micronucleus (MN) assay in the peripheral blood (PB) erythrocytes using flow cytometry were conducted. Rats were euthanized at 72 hr and PB was removed for the MN assay and single cells were isolated from multiple tissues for the Comet assays. None of the doses of DOX induced a significant DNA damage in any of the tissues examined by the alkaline Comet assay. Contrastingly, the glycosylase enzymes-modified Comet assay showed a significant dose dependent increase in the oxidative DNA damage in the cardiac tissue (P ≤ 0.05). In the liver, only the top dose induced significant increase in the oxidative DNA damage (P ≤ 0.05). The histopathology showed no severe cardiotoxicity but non-neoplastic lesions were present in both untreated and treated samples. A severe toxicity likely occurred in the bone marrow because no viable reticulocytes could be screened for the MN assay. Gene expression profiling of the heart tissues showed a significant alteration in the expression of 11 DNA damage and repair genes. These results suggest that DOX is genotoxic in the heart and the DNA damage may be induced primarily via the production of reactive oxygen species.

  11. 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

  12. 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

  13. Identification of Streptococcus pneumoniae serotype 11E, serovariant 11Av and mixed populations by high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR spectroscopy and flow cytometric serotyping assay (FCSA.

    Directory of Open Access Journals (Sweden)

    Romina Camilli

    Full Text Available BACKGROUND: Recent studies have identified Streptococcus pneumoniae serotype 11E and serovariant 11Av among isolates previously typed as 11A by classical serotyping methods. Serotype 11E and serovariant 11Av differ from serotype 11A by having totally or partially inactive wcjE, a gene in cps locus coding for an O-acetyl transferase. Serotype 11E is rare among carriage isolates but common among invasive isolates suggesting that it survives better during invasion. Aim of this work was to investigate the epidemiology of serotype 11A in a pneumococcal collection using a new serotyping approach based on High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance (HR-MAS NMR spectroscopy to distinguish serotypes 11A and 11E. METHODS: A collection of 48 (34 invasive and 14 carriage S. pneumoniae isolates from Italy, previously identified as serotype 11A by the Quellung reaction, were investigated by wcjE sequencing, HR-MAS NMR spectroscopy and the reference flow cytometric serotyping assay (FCSA based on monoclonal antibodies. RESULTS: HR-MAS NMR spectra from serotypes 11A and 11E showed different NMR peaks indicating that HR-MAS NMR could be used to distinguish these serotypes, although HR-MAS NMR could not distinguish serotype 11Av from serotype 11E unambiguously. Thirty-eight isolates were confirmed to be serotype 11A, 8 isolates with a mutated wcjE were serotype 11E, 1 isolate belonged to serovariant 11Av, and 1 isolate was a mixed population 11A/11Av. All 11E isolates were identified among invasive isolates. CONCLUSIONS: We proved that HR-MAS NMR can be of potential use for pneumococcal serotyping. The detection of serotype 11E among invasive isolates in our collection, supports previous epidemiological studies suggesting that mutations in wcjE can represent a mechanism promoting pneumococcal survival during invasion. The discovery of a spectrum of immunochemical diversity within established serotypes should stimulate efforts to develop new

  14. Simultaneous determination of phagocytosis of Plasmodium falciparum-parasitized and non-parasitized red blood cells by flow cytometry

    Directory of Open Access Journals (Sweden)

    Gallo Valentina

    2012-12-01

    Full Text Available Abstract Background Severe falciparum malaria anaemia (SMA is a frequent cause of mortality in children and pregnant women. The most important determinant of SMA appears to be the loss of non-parasitized red blood cells (np-RBCs in excess of loss of parasitized (p- RBCs at schizogony. Based on data from acute SMA where excretion of haemoglobin in urine and increased plasma haemoglobin represented respectively less than 1% and 0.5% of total Hb loss, phagocytosis appears to be the predominant mechanism of removal of np- and p-RBC. Estimates indicate that np-RBCs are cleared in approximately 10-fold excess compared to p-RBCs. An even larger removal of np-RBCs has been described in vivax malaria anaemia. Estimates were based on two single studies both performed on neurosyphilitic patients who underwent malaria therapy. As the share of np-RBC removal is likely to vary between wide limits, it is important to assess the contribution of both np- and p-RBC populations to overall RBC loss, and disclose the mechanism of such variability. As available methods do not discriminate between the removal of np- vs p-RBCs, the purpose of this study was to set up a system allowing the simultaneous determination of phagocytosis of p- and np-RBC in the same sample. Methods and Results Phagocytosis of p- and np-RBCs was quantified in the same sample using double-labelled target cells and the human phagocytic cell-line THP-1, pre-activated by TNF and IFNγ to enhance their phagocytic activity. Target RBCs were double-labelled with fluorescent carboxyfluorescein-succinimidyl ester (CF-SE and the DNA label ethidium bromide (EB. EB, a DNA label, allowed to discriminate p-RBCs that contain parasitic DNA from the np-RBCs devoid of DNA. FACS analysis of THP-1 cells fed with double-labelled RBCs showed that p- and np-RBCs were phagocytosed in different proportions in relation to parasitaemia. Conclusions The assay allowed the analysis of phagocytosis rapidly and with low

  15. Flow cytometric analysis of peripheral blood leukemic cells in relapse of acute leukemia%急性白血病复发外周血白血病细胞的流式细胞术检验分析

    Institute of Scientific and Technical Information of China (English)

    杨莉; 何浩明

    2015-01-01

    Objective To analyse status of peripheral blood leukemic cells detected by flow cytometry in patients with acute leu‐kemia(AL) ,and to provide references for evaluating clinical efficacy and prognosis of AL .Methods The peripheral blood specimens of 87 cases of patients with AL ,including 53 cases of patients with acute myelocytic leukemia and 34 cases of patients with acute lymphoblastic leukemia ,were detected by using flow cytometry ,morphological changes in bone marrow cells were detected ,as well . Results The sensitivity ,specificity and positive predictive value in determination of acute myelocytic leukemia was 95 .6% ,34 .5%and 81 .3% respectively ,and those in acute lymphoblastic leukemia was 87 .3% ,45 .6% and 68 .9% respectively ,statistically signif‐icant differences were found in sensitivity ,specificity and positive predictive value (P<0 .05) .A total of 19 cases with negative mini‐mal residual disease had recurrence(26 .31% ) after 24 months ,and 68 cases with positive minimal residual disease had recurrence (86 .76% ) after 24 months ,and the recurrence rate between the two groups was statistically significant (P<0 .05) .Among all pa‐tients with positive minimal residual disease ,the recurrence rate in patients with high expression level of minimal residual disease (88 .23% ) was higher than that in patients with low expression level of minimal residual disease (47 .09% ) ,and the difference was statistically significant (P<0 .05) .Conclusion Flow cytometric analysis of peripheral blood leukemic cells may has significance for diagnosing relapse of AL and guiding clinical medication .%目的:分析急性白血病(AL)患者外周血白血病细胞流式细胞术检测情况,为分析AL的临床疗效和预后提供参考。方法采用流式细胞术检测87例A L患者(急性髓细胞白血病53例、急性淋巴细胞白血病34例)外周血标本,同时检测骨髓细胞形态学变化。结果急性髓细胞白

  16. Cytometric Catheter for Neurosurgical Applications

    Energy Technology Data Exchange (ETDEWEB)

    Evans III, Boyd Mccutchen [ORNL; Allison, Stephen W [ORNL; Fillmore, Helen [ORNL; Broaddus, William C [ORNL; Dyer, Rachel L [ORNL; Gillies, George [ORNL

    2010-01-01

    Implantation of neural progenitor cells into the central nervous system has attracted strong interest for treatment of a variety of pathologies. For example, the replacement of dopamine-producing (DA) neural cells in the brain appears promising for the treatment of patients affected by Parkinson's disease. Previous studies of cell-replacement strategies have shown that less than 90% of implanted cells survive longer than 24 - 48 hours following the implantation procedure. However, it is unknown if these cells were viable upon delivery, or if they were affected by other factors such as brain pathology or an immune response. An instrumented cell-delivery catheter has been developed to assist in answering these questions by facilitating quantification and monitoring of the viability of the cells delivered. The catheter uses a fiber optic probe to perform flourescence-based cytometric measurments on cells exiting the port at the catheter tip. The current implementation of this design is on a 3.2 mm diameter catheter with 245 micrometer diameter optical fibers. Results of fluorescence testing data are presented and show that the device can characterize the quantity of cell densities ranging from 60,000 cells/ml to 600,000 cells/ml with a coefficient of determination of 0.93.

  17. Children and Parasitic Diseases

    Science.gov (United States)

    ... CDC.gov . Parasites About Parasites Animals Blood Food Insects Water Education and Training CDC Bottle Bioassay References ... flowing water. It can cause itching and impaired vision in children, and lead to blindness in adulthood. ...

  18. Cytometric patterns reveal growth states of Shewanella putrefaciens.

    Science.gov (United States)

    Melzer, Susanne; Winter, Gudrun; Jäger, Kathrin; Hübschmann, Thomas; Hause, Gerd; Syrowatka, Frank; Harms, Hauke; Tárnok, Attila; Müller, Susann

    2015-05-01

    Bacterial growth is often difficult to estimate beyond classical cultivation approaches. Low cell numbers, particles or coloured and dense media may disturb reliable growth assessment. Further difficulties appear when cells are attached to surfaces and detachment is incomplete. Therefore, flow cytometry was tested and used for analysis of bacterial growth on the single-cell level. Shewanella putrefaciens was cultivated as a model organism in planktonic or biofilm culture. Materials of smooth and rough surfaces were used for biofilm cultivation. Both aerobic and anaerobic as well as feast and famine conditions were applied. Visualization of growth was also done using Environmental Scanning and Phase Contrast Microscopy. Bioinformatic tools were applied for data interpretation. Cytometric proliferation patterns based on distributions of DNA contents per cell corresponded distinctly to the various lifestyles, electron acceptors and substrates tested. Therefore, cell cycling profiles of S. putrefaciens were found to mirror growth conditions. The cytometric patterns were consistently detectable with exception of some biofilm types whose resolution remained challenging. Corresponding heat maps proved to be useful for clear visualization of growth behaviour under all tested conditions. Therefore, flow cytometry in combination with bioinformatic tools proved to be powerful means to determine various growth states of S. putrefaciens, even in constrained environments. The approach is universal and will also be applicable for other bacterial species. © 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  19. Oral squamous cell carcinoma. Cytometric parameters of prognostic interest.

    Science.gov (United States)

    Saiz-Bustillo, Ramón; Corchero-Martín, Guadalupe; García-Montesinos-Perea, Belén; Gonzalez-Terán, Tomás; Sánchez-Santolino, Sergio

    2005-01-01

    The present study was made in order to find possible prognostic factors in oral squamous cell carcinoma, given that it is a frequent disease (3-4% of all malignant tumors) and is the cause of a high morbidity and mortality which justifies any attempt to contribute something towards the understanding of this pathology. 81 oral squamous cell carcinomas, treated with the same procedure, and retrieved from the archive of the Hospital Universitario Marqués de Valdecilla (Santander) were studied. Flow cytometry was carried out on 67 of the samples. No statistically significant differences were found between the cellular proliferative index and the mitotic index, ploidy and the S-phase factor. Likewise, none of the cytometric variables studied presented any association with the appearance of local relapse, distant metastases or survival. These variables cannot be used as a prognostic factors in squamous cell carcinomas of the oral cavity.

  20. 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 i

  1. Multicentric study underlining the interest of adding CD5, CD7 and CD56 expression assessment to the flow cytometric Ogata score in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms.

    Science.gov (United States)

    Bardet, Valérie; Wagner-Ballon, Orianne; Guy, Julien; Morvan, Céline; Debord, Camille; Trimoreau, Franck; Benayoun, Emmanuel; Chapuis, Nicolas; Freynet, Nicolas; Rossi, Cédric; Mathis, Stéphanie; Gourin, Marie-Pierre; Toma, Andréa; Béné, Marie C; Feuillard, Jean; Guérin, Estelle

    2015-04-01

    Although numerous recent publications have demonstrated interest in multiparameter flow cytometry in the investigation of myelodysplastic disorders, it is perceived by many laboratory hematologists as difficult and expensive, requiring a high level of expertise. We report a multicentric open real-life study aimed at evaluating the added value of the technically simple flow cytometry score described by the Ogata group for the diagnosis of myelodysplastic syndromes. A total of 652 patients were recruited prospectively in four different centers: 346 myelodysplastic syndromes, 53 myelodysplastic/myeloproliferative neoplasms, and 253 controls. The Ogata score was assessed using CD45 and CD34 staining, with the addition of CD10 and CD19. Moreover, labeling of CD5, CD7 and CD56 for the evaluation of myeloid progenitors and monocytes was tested on a subset of 294 patients. On the whole series, the specificity of Ogata score reached 89%. Respective sensitivities were 54% for low-risk myelodysplastic syndromes, 68% and 84% for type 1 and type 2 refractory anemia with excess of blasts, and 72% for myelodysplastic/myeloproliferative neoplasms. CD5 expression was poorly informative. When adding CD56 or CD7 labeling to the Ogata score, sensitivity rose to 66% for low-risk myelodysplastic syndromes, to 89% for myelodysplastic/myeloproliferative neoplasms and to 97% for refractory anemia with excess of blasts. This large multicenter study confirms the feasibility of Ogata scoring in routine flow cytometry diagnosis but highlights its poor sensitivity in low-risk myelodysplastic syndromes. The addition of CD7 and CD56 in flow cytometry panels improves the sensitivity but more sophisticated panels would be more informative. Copyright© Ferrata Storti Foundation.

  2. Flow cytometric detection of micronuclei and cell cycle alterations in fish-derived cells after exposure to three model genotoxic agents: mitomycin C, vincristine sulfate and benzo(a)pyrene.

    Science.gov (United States)

    Sánchez, P; Llorente, M T; Castaño, A

    2000-02-16

    The measurement of cytogenetic alterations in vitro is considered an initial step in the risk assessment procedures for genotoxic agents. The concern about genotoxic pollutants in natural fish population makes the use of fish-derived cells an useful tool for these purposes. The technological improvements in well-established cytogenetic endpoints, such as micronuclei (MN) estimations by means of flow cytometry, have been proposed in the later years using mammalian cells. In this work, we test the capability of flow cytometry to evaluate MN induction and cell cycle alterations in an established fish cell line (RTG-2) using three agent-inductor models at different concentrations and exposure periods. For mitomycin C, an inverse relationship between length of exposure period and concentrations was observed. A dose-response relationship was observed after exposing RTG-2 cells to vincristine sulfate and benzo(a)pyrene. As this study shows, RTG-2 cells respond to clastogenic and aneugenic effects of the tested chemicals through the induction of MN at similar doses to mammalian cells and without the addition of exogenous metabolic activity. The possibility to check cell cycle alterations, in the same sample, gives the opportunity to evaluate early signals of cytotoxicity. The use of flow cytometry improves the assay by means of its speed and objectivity, which makes the assay very useful for genotoxicity assessment of aquatic chemicals.

  3. Parasitic Diseases

    Science.gov (United States)

    ... water, a bug bite, or sexual contact. Some parasitic diseases are easily treated and some are not. Parasites ... can be seen with the naked eye. Some parasitic diseases occur in the United States. Contaminated water supplies ...

  4. Immune complex stimulation of human neutrophils involves a novel Ca2+/H+ exchanger that participates in the regulation of cytoplasmic pH: flow cytometric analysis of Ca2+/pH responses by subpopulations.

    Science.gov (United States)

    Bernardo, John; Hartlaub, Hilary; Yu, Xin; Long, Heidi; Simons, Elizabeth R

    2002-12-01

    The activation of human phagocytic leukocytes by immune complexes (IC) or opsonized microbes via their Fc and complement receptors has been well-described. The mechanisms involved in this process are complex and depend on the receptors involved. The biochemical events that lead to the destruction of invading organisms in turn display varying degrees of interdependence, but the controlling elements that lead to the ultimate killing of ingested organisms within phagosomes by lysosomal enzymes and reactive oxygen intermediates are still not completely understood. We have addressed these mechanisms by following and correlating the kinetics of responses by individual cells, using multiparameter flow cytometry. Using nonopsonized IC as stimuli, we document here the presence of a novel Ca(2)(+)/H(+) voltage-independent channel in human neutrophils, which helps to control their cytoplasmic pH.

  5. Flow-cytometric method for observing the effects of pulsed electromagnetic fields on the growth of rat bone marrow mesenchymal stem cells%流式细胞仪观察脉冲电磁场干预骨髓间充质干细胞的生长

    Institute of Scientific and Technical Information of China (English)

    黄钊; 苏伟; 崔向荣; 覃万安

    2011-01-01

    BACKGROUND: Compared with the morphology, DNA electrophoresis and other methods, flow-cytometric method has more advantages on the detection of cell phenotype, proliferation rate and cell cycle of rat bone marrow mesenchymal stem cells (BMSCs). OBJECTIVE: To observe and discuss the effect of specific pulsed electromagnetic fields stimulation on the proliferation and cell cycle of rat BMSCs with the flow-cytometric method (FCM). METHODS: Rat BMSCs were exposed to pulsed electromagnetic fields (frequency for 1 kHz, magnetic flux density for 0.05 mT, power density for 5 mW/cm2). BMSCs without exposure to pulsed electromagnetic fields were used as controls. Expression of CD29, CD31, CD44 CD45, CD105 were detect in the control group. The proliferation rate and cell cycle of passage 3 cells were detected at 3, 6, 9, 12 days. RESULTS AND CONCLUSION: CD29, CD44, CD105 in the 3rd passage non-stimulated BMSCs was positively expressed and the CD31, CD45 was negatively expressed (P < 0.05). The survival rate of passage 3 BMSCs and percentage of S phase following intervention of pulsed electromagnetic fields were greater than those in the control group (P < 0.05). These results indicated that the specific pulsed electromagnetic fields can promote the growth and proliferation of BMSCs to some extent.%背景:应用流式细胞仪检测细胞表型、存活或凋亡细胞计数及细胞周期,较形态学观察、DNA电泳等检测方法更具优势.目的:采用流式细胞仪分选检测特定脉冲电磁场干预对大鼠骨髓间充质干细胞生长周期及其增殖效率的影响.方法:使用振荡频率1 kHz,磁感应强度0.05 mT、功率密度5 mW/cm2的脉冲电磁场照射大鼠骨髓间充质干细胞,以未经磁场干预的细胞为对照.采用流式细胞仪检测未经磁场干预细胞表面抗原CD29、CD31、CD44、CD45和CD105表达率;于传第3代后第3,6,9,12天测定不同干预细胞增殖率及生长周期.结果与结论:未经磁场干预的第3代

  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, A.L.

    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 G/sub 2/M by about 50%. When added to G/sub 1/ cells, DE delayed recruitment of apparently quiescent (G/sub 0/) 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. 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.

  8. 应用流式微球检测黄曲霉毒素B1方法的建立%Determination of aflatoxin B1 based on a flow cytometric microsphere immunoassay

    Institute of Scientific and Technical Information of China (English)

    李泳宁; 吴海燕; 郑允权; 郭养浩

    2012-01-01

    采用活性酯法,将AFB1-BSA人工抗原交联于含有羧基表面的荧光微球,通过与游离AFB1竞争抗AFB1单克隆抗体后,再与异硫氰酸荧光素标记二抗的反应,建立基于微球的间接竞争免疫检测方法.检测结果表明,流式细胞仪检测AFB1的检测限为0.03 ng·mL-1,检测范围为0.05 ~ 1.0 ng· mL-1.与黄曲霉毒素B2、黄曲霉毒素G1、黄曲霉毒素G2、黄曲霉毒素M1和黄曲霉毒素M2的交叉反应率均低于1.0%.在玉米样品中的加标回收率为87% ~ 103%,变异系数为6.1%~8.4%.%Carboxyl - modified microspheres internally dyed with fluroscent dye were conjugated with the artificial antigen AFB, - BSA. Aflatoxin B, ( AFB,) was used as a positive control to compete with the AFB, - BSA antigen on the surface of the microspheres for the anti - AFB, McAb. A fluorescein isothiocyanate labeled IgG reporter antibody was added to react specifically with the anti - AFB, McAb on the microspheres. The detection limit of AFB, reached 0.03 ng · mL-1, with a good linearity ranging 0.05 ~ 1.0 ng mL-1. The cross - reactivity rates were less than 1.0% with other toxins such as aflatoxins B2, aflatoxins G,, aflatoxins G2, aflatoxins M, and aflatoxins M2. The recovery of AFB, from artificially contaminated corn samples was from 89% to 92% , with CVs from 6.8% to 9.0%. A novel method for the determination of aflatoxin B, by an indirect competitive immunoassay with a flow cytometer has been developed.

  9. Analysis of the Fine-Scale Population Structure of “Candidatus Accumulibacter phosphatis” in Enhanced Biological Phosphorus Removal Sludge, Using Fluorescence In Situ Hybridization and Flow Cytometric Sorting▿

    Science.gov (United States)

    Kim, Jeong Myeong; Lee, Hyo Jung; Kim, Sun Young; Song, Jae Jun; Park, Woojun; Jeon, Che Ok

    2010-01-01

    To investigate the fine-scale diversity of the polyphosphate-accumulating organisms (PAO) “Candidatus Accumulibacter phosphatis” (henceforth referred to as “Ca. Accumulibacter”), two laboratory-scale sequencing batch reactors (SBRs) for enhanced biological phosphorus removal (EBPR) were operated with sodium acetate as the sole carbon source. During SBR operations, activated sludge always contained morphologically different “Ca. Accumulibacter” strains showing typical EBPR performances, as confirmed by the combined technique of fluorescence in situ hybridization (FISH) and microautoradiography (MAR). Fragments of “Ca. Accumulibacter” 16S rRNA genes were retrieved from the sludge. Phylogenetic analyses together with sequences from the GenBank database showed that “Ca. Accumulibacter” 16S rRNA genes of the EBPR sludge were clearly differentiated into four “Ca. Accumulibacter” clades, Acc-SG1, Acc-SG2, Acc-SG3, and Acc-SG4. The specific FISH probes Acc444, Acc184, Acc72, and Acc119 targeting these clades and some helpers and competitors were designed by using the ARB program. Microbial characterization by FISH analysis using specific FISH probes also clearly indicated the presence of different “Ca. Accumulibacter” cell morphotypes. Especially, members of Acc-SG3, targeted by probe Acc72, were coccobacillus-shaped cells with a size of approximately 2 to 3 μm, while members of Acc-SG1, Acc-SG2, and Acc-SG4, targeted by Acc444, Acc184, and Acc119, respectively, were coccus-shaped cells approximately 1 μm in size. Subsequently, cells targeted by each FISH probe were sorted by use of a flow cytometer, and their polyphosphate kinase 1 (ppk1) gene homologs were amplified by using a ppk1-specific PCR primer set for “Ca. Accumulibacter.” The phylogenetic tree based on sequences of the ppk1 gene homologs was basically congruent with that of the 16S rRNA genes, but members of Acc-SG3 with a distinct morphology comprised two different ppk1 genes

  10. Flow Cytometric Evidence for Hydroxyl Radical-induced Apoptosis in Tobacco Protoplasts%羟自由基诱导的烟草原生质体的凋亡:流式细胞法的新证据

    Institute of Scientific and Technical Information of China (English)

    雷晓勇; 廖旭东; 张贵友; 戴尧仁

    2003-01-01

    用1.0 mmol/L FeSO4/0.5 mmol/L H202处理烟草(Nicotiana tabacum L.cultivar BY 2)原生质体,发现羟自由基能够诱导烟草原生质体的凋亡.具体表现为细胞核皱缩、DNA Ladder、TUNEL阳性反应等典型的凋亡特征.在动物细胞凋亡过程中,线粒体起着非常重要的作用,其中膜电位(△ψm)的变化以及由其引起的位于线粒体膜上的通透性孔(PTP)的开放与Cyt c的释放有关.另外,在动物凋亡细胞中,磷脂酰丝氨酸(phosphatidyl serine,PS)会从细胞膜内侧向外翻转.为了判断植物细胞凋亡过程中膜电位的变化情况以及PS的外翻程度,我们采用了流式细胞法.结果表明,随着处理时间的延长,烟草原生质体线粒体的膜电位逐渐降低;膜内PS大量外翻.说明由羟自由基和烟草原生质体组成的凋亡体系是一种可靠的凋亡组合,可以用来对植物细胞凋亡机理做进一步研究.%Protoplasts prepared from tobacco (Nicotiana tabacum L., cultivar BY-2) suspension cellshave similar morphological characteristics to those in animal cells. The hallmarks of apoptosis such ascondensation and peripheral distribution of nuclei, TUNEL positive reaction, and DNA ladders were ob-served when tobacco protoplasts were treated with the hydroxyl radical generating system (1.0 mmol/LFeSO4/0.5 mmol/L H2O2). In animals, the loss of transmembrane potential (△ψm ) and the exposure ofphospholipid phosphatidylserine (PS) are believed to be the main apoptosis events. To test whether thesesignificant processes take place in plants, flow cytometry was used to detect annexin V binding andchanges in △ψm. Results showed that the PS turned out from inner membrane and △ψm graduallydecreased during the apoptosis. All these apoptotic characteristics proved that hydroxyl radicals cancause typical programmed cell death (PCD) in tobacco protoplasts and this design can be served as aneffective experiment system to explore the mechanism of plant apoptosis.

  11. 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.

  12. 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.

    and microalgae, the most abundant organisms in the ocean and also micro- zooplankton 2 . They have been implicated in phytoplankton mortality and the de- cline of phytoplankton blooms 1 . Marine phytoplankton is responsible for up to half of the total primary...

  13. Flow cytometric immunophenotyping test for staging/monitoring neuroblastoma patients

    National Research Council Canada - National Science Library

    Warzynski, Michael J; Graham, David M; Axtell, Richard A; Higgins, James V; Hammers, Yuki A

    2002-01-01

    .... Following the “rare event” philosophy of selecting one negative and two positive antigens, we initially tried a “cocktail” of CD45 − CD56 very bright+ neuron‐specific enolase (NSE) cytoplasmic...

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

    Science.gov (United States)

    1990-01-01

    However, subjectivity remains an inherent part of the diagnostic process.9 According to Dabelsteen,10 subjectivity is most apparent in the determination...of Silverman 16 and Pindborg43 and Mincer et al.21 that light microscopio features of premalignancy are often not present in original biopsy specimens...was caused either in whole or in part by the presence of doublets was eliminated by syringina,. filtering and visually examining the population. In

  15. 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) tri

  16. 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

  17. Parasites: Water

    Science.gov (United States)

    ... Tropical Diseases Laboratory Diagnostic Assistance [DPDx] Parasites Home Water Language: English Español (Spanish) Recommend on Facebook Tweet Share Compartir Parasites can live in natural water sources. When outdoors, treat your water before drinking ...

  18. 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.

  19. Parasitic Colitis

    Science.gov (United States)

    Hechenbleikner, Elizabeth M.; McQuade, Jennifer A.

    2015-01-01

    Over one billion people worldwide harbor intestinal parasites. Parasitic intestinal infections have a predilection for developing countries due to overcrowding and poor sanitation but are also found in developed nations, such as the United States, particularly in immigrants or in the setting of sporadic outbreaks. Although the majority of people are asymptomatically colonized with parasites, the clinical presentation can range from mild abdominal discomfort or diarrhea to serious complications, such as perforation or bleeding. Protozoa and helminths (worms) are the two major classes of intestinal parasites. Protozoal intestinal infections include cryptosporidiosis, cystoisosporiasis, cyclosporiasis, balantidiasis, giardiasis, amebiasis, and Chagas disease, while helminth infections include ascariasis, trichuriasis, strongyloidiasis, enterobiasis, and schistosomiasis. Intestinal parasites are predominantly small intestine pathogens but the large intestine is also frequently involved. This article highlights important aspects of parasitic infections of the colon including epidemiology, transmission, symptoms, and diagnostic methods as well as appropriate medical and surgical treatment. PMID:26034403

  20. Parasitic Colitis

    OpenAIRE

    Hechenbleikner, Elizabeth M.; McQuade, Jennifer A.

    2015-01-01

    Over one billion people worldwide harbor intestinal parasites. Parasitic intestinal infections have a predilection for developing countries due to overcrowding and poor sanitation but are also found in developed nations, such as the United States, particularly in immigrants or in the setting of sporadic outbreaks. Although the majority of people are asymptomatically colonized with parasites, the clinical presentation can range from mild abdominal discomfort or diarrhea to serious complication...

  1. Cat parasites

    OpenAIRE

    Vošická, Kristýna

    2016-01-01

    The content of this bachelor thesis describes a different variety of cat parasites. This study discovers that the most infected group of the outdoor cats due to the fact that these animals are not provided with the same care as the household pets. Those cats are usually not vaccinated, not rid of worms, no one takes care of their fur and so they tend to become a host for the parasites. There are several kinds of parasites which attack cats. Among those belong the skin parasites like a cat fle...

  2. Fish parasites

    DEFF Research Database (Denmark)

    This book contains 22 chapters on some of the most important parasitic diseases in wild and farmed fish. International experts give updated reviews and provide solutions to the problems......This book contains 22 chapters on some of the most important parasitic diseases in wild and farmed fish. International experts give updated reviews and provide solutions to the problems...

  3. 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.

  4. Dispersal and gene flow in the rare, parasitic Large Blue butterfly Maculinea arion

    DEFF Research Database (Denmark)

    Ugelvig, Line Vej; Andersen, Anne; Boomsma, Jacobus Jan

    2012-01-01

    decline throughout Europe and extinction in Britain followed by reintroduction of a seed population from the Swedish island of Öland. We find that populations are highly structured genetically, but that gene flow occurs over distances 15 times longer than the maximum distance recorded from mark...

  5. Flow cytometric monitoring of minimal residual diseases in patients with acute leukemia after allogeneic hemapoietic stem cell transplantation%急性白血病异基因造血干细胞移植后流式细胞术监测微量残留病的意义

    Institute of Scientific and Technical Information of China (English)

    高雁群; 孙媛; 张耀臣; 纪树荃; 陆道培; 吴彤; 王卉; 童春容; 张维婕; 王静波; 卢岳; 赵艳丽; 周葭蕤

    2012-01-01

    目的 研究急性白血病异基因造血干细胞移植(allo-HSCT)后采用流式细胞术(FCM)监测微量残留病(MRD)的意义.方法 自2007年1月至2008年1月采用FCM对102例初诊时未检测出白血病基因和染色体改变的急性白血病allo-HSCT后患者进行骨髓MRD检测(移植后1、2、3、6、12个月,部分高危患者增加检测频率),观察MRD结果与临床转归的关系,对有意义的MRD增高患者予以临床干预并采用FCM监测疗效.MRD> 0.01%为阳性.结果 ①移植后MRD持续阴性者71例,均为血液学完全缓解(CR),仅3例髓外复发,其无病生存( DFS)及总生存(0S)率分别为66.2%及90.1%.②移植后MRD阳性者27例,经过干预治疗(化疗加供者淋巴细胞输注、多种细胞因子诱导的杀伤细胞和NK细胞治疗),11例患者转阴,其DFS及OS率分别为63.6%及72.7%.另外16例血液学复发,其DFS及OS率分别为11.1%及25.0%.从MRD增高至血液学复发的中位时间为48(7~69)d.③移植后直接血液学复发者共4例,均死亡.结论 移植后采用FCM检测MRD:①MRD持续阴性组患者其DFS及OS率均明显高于MRD阳性组.②移植后出现MRD阳性的患者,通过干预性治疗,MRD再次转阴后,其DFS及OS率仍然高于持续阳性组.③移植后直接血液学复发的患者,其DFS及OS率极低,预后极差.采用FCM监测急性白血病allo-HSCT后MRD是一种敏感、特异、快速、简便的方法,可及时提示复发倾向,便于早期干预治疗,降低血液学复发风险,提高allo-HSCT后患者的DFS率.%Objective To study the significance of flow cytometric monitoring minimal residual diseases (MRD) in patients with acute leukemia (AL) after allogeneic hemapoietic stem cell transplantation (HSCT).Methods From January 2007 and January 2008 MRD were detected by flow cytometry (FCM) in 402 bone marrow (BM) in 102 AL patients without leukemic gene and chromosomal changes at first diagnosis after HSCT( 1,2,3,6,12 months after HSCT

  6. Diagnosis of Parasitic Diseases

    Science.gov (United States)

    ... Laboratory Diagnostic Assistance [DPDx] Parasites Home Diagnosis of Parasitic Diseases Recommend on Facebook Tweet Share Compartir On this ... the United States cannot diagnose parasites? How are parasitic diseases diagnosed? Many kinds of lab tests are available ...

  7. Monitoring Plasmodium falciparum growth and development by UV flow cytometry using an optimized Hoechst-thiazole orange staining strategy.

    Science.gov (United States)

    Grimberg, Brian T; Erickson, John J; Sramkoski, R Michael; Jacobberger, James W; Zimmerman, Peter A

    2008-06-01

    The complex life cycle of Plasmodium falciparum (Pf) makes it difficult to limit infections and reduce the risk of severe malaria. Improved understanding of Pf blood-stage growth and development would provide new opportunities to evaluate and interfere with successful completion of the parasite's life cycle. Cultured blood stage Pf was incubated with Hoechst 33342 (HO) and thiazole orange (TO) to stain DNA and total nucleic acids, respectively. Correlated HO and TO fluorescence emissions were then measured by flow cytometry. Complex bivariate data patterns were analyzed by manual cluster gating to quantify parasite life cycle stages. The permutations of viable staining with both reagents were tested for optimal detection of parasitized RBC (pRBC). Pf cultures were exposed to HO and TO simultaneously to achieve optimal staining of pRBC and consistent quantification of early and late stages of the replicative cycle (rings through schizonts). Staining of Pf nucleic acids allows for analysis of parasite development in the absence of fixatives, lysis, or radioactivity to enable examination of erythrocytes from parasite invasion through schizont rupture using sensitive and rapid assay procedures. Investigation of the mechanisms by which anti-malarial drugs and antibodies act against different Pf lifecycle stages will be aided by this cytometric strategy. (c) 2008 International Society for Advancement of Cytometry.

  8. Isolation of Plasmodium falciparum by flow-cytometry: implications for single-trophozoite genotyping and parasite DNA purification for whole-genome high-throughput sequencing of archival samples

    Directory of Open Access Journals (Sweden)

    Boissière Anne

    2012-05-01

    Full Text Available Abstract Background Flow cytometry and cell sorting are powerful tools enabling the selection of particular cell types within heterogeneous cell mixtures. These techniques, combined with whole genome amplification that non-specifically amplify small amounts of starting DNA, offer exciting new opportunities for the study of malaria genetics. Among them, two are tested in this paper: (1 single cell genotyping and (2 parasite DNA purification for subsequent whole genome sequencing using shotgun technologies. Methods The method described allows isolation of Plasmodium falciparum trophozoites, genotyping and whole genome sequencing from the blood of infected patients. For trophozoite isolation, parasite and host nuclei are stained using propidium iodide (PI followed by flow cytometry and cell sorting to separate trophozoites from host cells. Before genotyping or sequencing, whole genome amplification is used to increase the amount of DNA within sorted samples. The method has been specifically designed to deal with frozen blood samples. Results and conclusion The results demonstrate that single trophozoite genotyping is possible and that cell sorting can be successfully applied to reduce the contaminating host DNA for subsequent whole genome sequencing of parasites extracted from infected blood samples.

  9. Protozoan Parasites.

    Science.gov (United States)

    Custodio, Haidee

    2016-02-01

    • Stool antigen detection for Cryptosporidium sp, Giardia lamblia and Entamoeba histolytica are now commercially available, have better sensitivity and specificity than the traditional stool microscopy, and are less dependent on personnel skill. Tests employing newer techniques with faster turnaround time are also available for diagnosing trichomoniasis.• Nitazoxanide, the only U.S. Food and Drug Administration-approved medication for therapy of cryptosporidiosis, is effective among immunocompetent patients. However, on the basis of strong evidence from multiple clinical trials, nitazoxanide is considered ineffective among immunocompromised patients. (14) • Giardiasis can be asymptomatic or have a chronic course leading to malabsorption and failure to thrive. It can be treated with metronidazole, tinidazole, or nitazoxanide. On the basis of growing observational studies, postinfectious and extraintestinal manifestations of giardiasis occur, but the mechanisms are unclear. Given the high prevalence of giardiasis, public health implications need to be defined. (16) • Eradicating E histolytica from the gastrointestinal tract requires only intraluminal agent therapy. Therapy for invasive illnesses requires use of imidazole followed by intraluminal agents to eliminate persistent intraluminal parasites. • Malaria is considered the most lethal parasitic infection, with Plasmodium falciparum as the predominant cause of mortality. P vivax and P ovale can be dormant in the liver, and primaquine is necessary to resolve infection by P vivax and P ovale. • Among immunocompetent patients, infection with Toxoplasma gondii may be asymptomatic, involve localized lymphadenopathy, or cause ocular infection. In immunocompromised patients, reactivation or severe infection is not uncommon. On the basis of limited observational studies (there are no well-controlled randomized trials), therapy is recommended for acute infection during pregnancy to prevent transmission to the

  10. Parasitism in bivalves from an Arctic ecosystem

    Science.gov (United States)

    Petersen, G. Høpner

    1984-03-01

    In arctic ecosystems parasitism seems less conspicuous than in more diverse tropical ecosystems. However, several host-parasite relations may be a burden in Arctic ecosystems and modify energy-flow patterns. Four different localities of the shallow shelf off Godhavn, West Greenland, were studied with regard to biomass, production and life cycles of selected bivalve and polychaete species. The following important parasite-bivalve associations were found: the athecate hydroid Monobrachium parasitum on Macoma calcarea; the digenetic trematode Gymnophallus somateriae in Hiatella byssifera; an unidentified species of turbellarian in Hiatella byssifera; and the nemertean Malacobdella grossa in Mya truncata. In the bivalves Mytilus edulis, Serripes groenlandicus and Cardium ciliatum only few parasites were detected. Parasitism seems to follow the general trend observed in Arctic ecosystems which exhibit low species diversities and high numbers of individuals.

  11. Parasitic Diseases: Glossary

    Science.gov (United States)

    ... multicellular organism that is generally visible to the naked eye in its adult stages. Helminths can be ... with parasites such as Cyclospora and Cryptosporidium . (Also see " coccidian " and " sporulation .") Back To Top P Parasite: ...

  12. Women and Parasitic Diseases

    Science.gov (United States)

    ... Resources How to Find A Physician Diagnosis of Parasitic Diseases Statistics More Information Get Email Updates To receive ... often need special consideration when being treated for parasitic diseases in order to avoid harm to the fetus, ...

  13. Immunity to parasitic infection

    National Research Council Canada - National Science Library

    Lamb, Tracey J

    2012-01-01

    ... be manipulated to develop therapeutic interventions against parasitic infection. For easy reference, the most commonly studied parasites are examined in individual chapters written by investigators at the forefront of their field...

  14. Contribuição da citometria de fluxo para o diagnóstico e prognóstico das síndromes mielodisplásicas The application of flow cytometric analysis of bone marrow cells for the diagnosis and prognosis of myelodysplastic syndromes

    Directory of Open Access Journals (Sweden)

    Irene Lorand-Metze

    2006-09-01

    Full Text Available O diagnóstico das síndromes mielodisplásicas (SMD é baseado nos achados de citopenias no sangue periférico, na morfologia (atipias das células hemopoiéticas na medula óssea e no cariótipo. Em uma proporção considerável de casos, porém, o grau de atipias encontrado é discreto e sujeito a interpretações subjetivas. Além disso, alterações citogenéticas são encontradas apenas em 30%-80% dos casos. A citometria de fluxo multiparamétrica é uma técnica rápida, reproduzível e relativamente barata, capaz de objetivar alterações funcionais do clone SMD na maioria dos casos, o que permite o diagnóstico diferencial com patologias não-clonais que cursam com citopenias periféricas. Várias alterações têm sido descritas na expressão de antígenos ligados a linhagem e maturação celular nas três séries hemopoiéticas. Protocolos de três ou quatro cores analisando-se as séries eritroblástica, mielomonocítica e blastos têm sido propostos e conseguem resolver o diagnóstico diferencial em praticamente todos os casos. A citometria de fluxo também é útil para o acompanhamento dos pacientes, já que a progressão do clone neoplásico é acompanhada por um aumento do número de alterações fenotípicas e de células CD34+ além da diminuição de marcadores pró-apoptóticos.The diagnosis of MDS is based on the presence of peripheral cytopenias together with cell atypias in bone marrow precursors and cytogenetic abnormalities. However, in several cases, the cell atypias are discrete, and/or the karyotype is normal, precluding a clear-cut diagnosis. Multiparametric flow cytometry is a fast, reproducible and relatively inexpensive technique, which is able to disclose changes in the expression of lineage and maturation related antigens. Several of such abnormalities have been described in MDS. Three or four-color protocols have been used to analyze erythroblasts, granulocytes, monocytes and blasts, permitting, in most of the

  15. 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.

  16. Paradigms for parasite conservation.

    Science.gov (United States)

    Dougherty, Eric R; Carlson, Colin J; Bueno, Veronica M; Burgio, Kevin R; Cizauskas, Carrie A; Clements, Christopher F; Seidel, Dana P; Harris, Nyeema C

    2016-08-01

    Parasitic species, which depend directly on host species for their survival, represent a major regulatory force in ecosystems and a significant component of Earth's biodiversity. Yet the negative impacts of parasites observed at the host level have motivated a conservation paradigm of eradication, moving us farther from attainment of taxonomically unbiased conservation goals. Despite a growing body of literature highlighting the importance of parasite-inclusive conservation, most parasite species remain understudied, underfunded, and underappreciated. We argue the protection of parasitic biodiversity requires a paradigm shift in the perception and valuation of their role as consumer species, similar to that of apex predators in the mid-20th century. Beyond recognizing parasites as vital trophic regulators, existing tools available to conservation practitioners should explicitly account for the unique threats facing dependent species. We built upon concepts from epidemiology and economics (e.g., host-density threshold and cost-benefit analysis) to devise novel metrics of margin of error and minimum investment for parasite conservation. We define margin of error as the risk of accidental host extinction from misestimating equilibrium population sizes and predicted oscillations, while minimum investment represents the cost associated with conserving the additional hosts required to maintain viable parasite populations. This framework will aid in the identification of readily conserved parasites that present minimal health risks. To establish parasite conservation, we propose an extension of population viability analysis for host-parasite assemblages to assess extinction risk. In the direst cases, ex situ breeding programs for parasites should be evaluated to maximize success without undermining host protection. Though parasitic species pose a considerable conservation challenge, adaptations to conservation tools will help protect parasite biodiversity in the face of

  17. Flow cytometric test using eosin-5′-maleimide (EMA) labelling of red blood for diagnosis of hereditary spherocytosis%流式细胞术检测伊红-5′-马来酰亚胺标记红细胞在80例遗传性球形红细胞增多症中的诊断价值

    Institute of Scientific and Technical Information of China (English)

    王继英; 郑彬; 赵玉平; 陈雪晶; 刘燕; 薄丽津; 郑以州; 张凤奎; 汝昆

    2015-01-01

    目的 探讨伊红-5′-马来酰亚胺(EMA)为标记流式细胞术检测遗传性球形红细胞的灵敏性和特异性,并对试剂和样本稳定性进行验证.方法 对80例遗传性球形红细胞增多症(HS)和44例非HS患者外周血样本全部采用EMA标记流式细胞术检测、红细胞渗透脆性试验和酸化甘油溶解试验三种方法进行检测,比较三种方法的灵敏性和特异性,探讨EMA标记流式细胞术检测的可行性.并观察EMA及样本在不同储存条件下的稳定性.结果 通过检测124例样本得出,EMA标记流式细胞术检测的灵敏性和特异性分别为0.925和0.954,红细胞渗透脆性试验分别为0.950和0.455,酸化甘油溶解试验为1.000和0.318.红细胞渗透脆性试验和酸化甘油溶解试验的灵敏性稍高于EMA流式检测方法,但特异性差,不能明确区分球形红细胞增多是否为HS.EMA对温度很敏感,-80℃储存180 d较4℃存储1d稳定.HS样本的稳定性较好,4℃放置6d、室温条件放置3d不会影响结果的判定.结论 EMA标记流式细胞术检测HS具有良好的灵敏性和特异性,-80℃储存EMA较为稳定,须在样本4℃放置6d内、室温条件放置3d内完成检测.%Objective To investigate the sensitivity and specificity of eosin-5′-maleimide (EMA) assay for the diagnosis of hereditary spherocytosis (HS),and to verify the stability of reagent and samples.Methods EMA flow cytometry test,NaC1-osmotic fragility test and acidified glycerol lysis test were performed using peripheral blood samples from 80 patients with HS and 44 patients with other blood diseases,the sensitivity and specificity of the three methods were compared,and the feasibility of EMA binding test was estimated.The stability of EMA reagent and HS samples stored at different temperatures were tested.Results Among the 124 tested samples,the sensitivity and specificity of EMA binding test was 0.925 and 0.954,that of NaCl-osmotic fragility test was 0.950 and 0.455,and

  18. 多参数流式细胞术在多发性骨髓瘤及其微小残留疾病的免疫表型分析%Immunophenotypic analysis of multiparametric flow cytometric in multiple myeloma and minimal residual disease

    Institute of Scientific and Technical Information of China (English)

    许艳丽; 王顺清; 毛平; 杜庆华

    2015-01-01

    Objective To investigate the detectable significance of multiparameter flow cytometry (MFC) for the first visiting and minimal residual disease (MRD) in the patients with multiple myeloma .Methods MFC was used to identify the plasma cells by the expression of CD138 or CD38 antigen in 74 patients with multiple myeloma .By combining surface antigens like CD45 ,CD56 , CD19 ,CD20 ,CD117 and the cytoplasm Kappa and Lambda light chain ,the aberrant myeloma cells were differentiated from normal plasma cells .Results In the 44 first visiting cases ,the positive expression of CD138 can be detected in all cases ,while the expres‐sion of CD19 was negative and 42 cases (95% ) were negative or weak positive expression for CD45 .The detection rates of CD38 , CD56 ,CD20 and CD117 were 98% ,93% ,45% and 41% ,respectively .The cytoplasm Kappa and Lambda light chains were showed the limited expression .Of the patients with MM ,14 cases were used for evaluating the change of immunophenotype at first visiting and during the treatment process ,among them ,11 cases(79% ) appeared the changes in at least one of aberrant phenotypes .4 cases (29% ) had the significant enhancement of antigen marker fluorescence intensities after chemotherapy and 7 cases (50% ) had sig‐nificant decrease of antigen marker fluorescence intensities after chemotherapy .CD45 ,CD19 and cytoplasm immunoglobulin light chains were the most stable marker ,no obvious antigen marker changes were found during the treatment ,while there was a signifi‐cant antigen density change in 2 cases of CD38 (14% ) ,7 cases of CD56 (50% ) ,4 cases of CD20 (29% ) and 2 cases of CD117 (14% ) .Of the 30 cases for evaluating MRD immunophenotype ,the abnormal myeloma cells were detected in 25 cases .In 5 cases ,no expression of limited Kappa and Lambda light chains was found and the ratio of Kappa and Lambda was 0 .5 - 2 ,which were identi‐fied as negative for MRD .Conclusion The multiparameter flow cytometry has important

  19. 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

  20. Flow karyotyping and sorting of human chromosomes

    Energy Technology Data Exchange (ETDEWEB)

    Gray, J.W.; Lucas, J.; Peters, D.; Pinkel, D.; Trask, B.; van den Engh, G.; Van Dilla, M.A.

    1986-07-16

    Flow cytometry and sorting are becoming increasingly useful as tools for chromosome classfication and for the detection of numerical and structural chromosome aberrations. Chromosomes of a single type can be purified with these tools to facilitate gene mapping or production of chromosome specific recombinant DNA libraries. For analysis of chromosomes with flow cytometry, the chromosomes are extracted from mitotic cells, stained with one or more fluorescent dyes and classified one-by-one according to their dye content(s). Thus, the flow approach is fundamentally different than conventional karyotyping where chromosomes are classified within the context of a metaphase spread. Flow sorting allows purification of chromosomes that can be distinguished flow cytometrically. The authors describe the basic principles of flow cytometric chromosome classification i.e. flow karyotyping, and chromosome sorting and describe several applications. 30 refs., 8 figs.

  1. Parasites: evolution's neurobiologists.

    Science.gov (United States)

    Adamo, Shelley Anne

    2013-01-01

    For millions of years, parasites have altered the behaviour of their hosts. Parasites can affect host behaviour by: (1) interfering with the host's normal immune-neural communication, (2) secreting substances that directly alter neuronal activity via non-genomic mechanisms and (3) inducing genomic- and/or proteomic-based changes in the brain of the host. Changes in host behaviour are often restricted to particular behaviours, with many other behaviours remaining unaffected. Neuroscientists can produce this degree of selectivity by targeting specific brain areas. Parasites, however, do not selectively attack discrete brain areas. Parasites typically induce a variety of effects in several parts of the brain. Parasitic manipulation of host behaviour evolved within the context of the manipulation of other host physiological systems (especially the immune system) that was required for a parasite's survival. This starting point, coupled with the fortuitous nature of evolutionary innovation and evolutionary pressures to minimize the costs of parasitic manipulation, likely contributed to the complex and indirect nature of the mechanisms involved in host behavioural control. Because parasites and neuroscientists use different tactics to control behaviour, studying the methods used by parasites can provide novel insights into how nervous systems generate and regulate behaviour. Studying how parasites influence host behaviour will also help us integrate genomic, proteomic and neurophysiological perspectives on behaviour.

  2. Implementation of erythroid lineage analysis by flow cytometry in diagnostic models for myelodysplastic syndromes

    Science.gov (United States)

    Cremers, Eline M.P.; Westers, Theresia M.; Alhan, Canan; Cali, Claudia; Visser-Wisselaar, Heleen A.; Chitu, Dana A.; van der Velden, Vincent H.J.; te Marvelde, Jeroen G.; Klein, Saskia K.; Muus, Petra; Vellenga, Edo; de Greef, Georgina E.; Legdeur, Marie-Cecile C.J.C.; Wijermans, Pierre W.; Stevens-Kroef, Marian J.P.L.; da Silva-Coelho, Pedro; Jansen, Joop H.; Ossenkoppele, Gert J.; van de Loosdrecht, Arjan A.

    2017-01-01

    Flow cytometric analysis is a recommended tool in the diagnosis of myelodysplastic syndromes. Current flow cytometric approaches evaluate the (im)mature myelo-/monocytic lineage with a median sensitivity and specificity of ~71% and ~93%, respectively. We hypothesized that the addition of erythroid lineage analysis could increase the sensitivity of flow cytometry. Hereto, we validated the analysis of erythroid lineage parameters recommended by the International/European LeukemiaNet Working Group for Flow Cytometry in Myelodysplastic Syndromes, and incorporated this evaluation in currently applied flow cytometric models. One hundred and sixty-seven bone marrow aspirates were analyzed; 106 patients with myelodysplastic syndromes, and 61 cytopenic controls. There was a strong correlation between presence of erythroid aberrancies assessed by flow cytometry and the diagnosis of myelodysplastic syndromes when validating the previously described erythroid evaluation. Furthermore, addition of erythroid aberrancies to two different flow cytometric models led to an increased sensitivity in detecting myelodysplastic syndromes: from 74% to 86% for the addition to the diagnostic score designed by Ogata and colleagues, and from 69% to 80% for the addition to the integrated flow cytometric score for myelodysplastic syndromes, designed by our group. In both models the specificity was unaffected. The high sensitivity and specificity of flow cytometry in the detection of myelodysplastic syndromes illustrates the important value of flow cytometry in a standardized diagnostic approach. The trial is registered at www.trialregister.nl as NTR1825; EudraCT n.: 2008-002195-10 PMID:27658438

  3. Parasite Zoonoses and Wildlife: Emerging Issues

    Directory of Open Access Journals (Sweden)

    Andrew Smith

    2009-02-01

    Full Text Available The role of wildlife as important sources, reservoirs and amplifiers of emerging human and domestic livestock pathogens, in addition to well recognized zoonoses of public health significance, has gained considerable attention in recent years. However, there has been little attention given to the transmission and impacts of pathogens of human origin, particularly protozoan, helminth and arthropod parasites, on wildlife. Substantial advances in molecular technologies are greatly improving our ability to follow parasite flow among host species and populations and revealing valuable insights about the interactions between cycles of transmission. Here we present several case studies of parasite emergence, or risk of emergence, in wildlife, as a result of contact with humans or anthropogenic activities. For some of these parasites, there is growing evidence of the serious consequences of infection on wildlife survival, whereas for others, there is a paucity of information about their impact.

  4. Cystatins of parasitic organisms.

    Science.gov (United States)

    Klotz, Christian; Ziegler, Thomas; Daniłowicz-Luebert, Emilia; Hartmann, Susanne

    2011-01-01

    The cystatin superfamily comprises several groups of protease inhibitors. In this chapter we will focus on I25 family members, which consist predominantly of the type 2 cystatins. Recently, a wealth of information on these molecules and their activities has been described. Parasite cystatins are shown to have dual functions via interaction with both parasite and host proteases. Thereby, parasite cystatins are not only essentially involved in the regulation of physiological processes during parasite development, but also represent important pathogenicity factors. Interestingly, some studies indicate that parasite cystatins evolved exceptional immuno-modulatory properties. these capacities could be exploited to interfere with unwanted immune responses in unrelated human inflammatory diseases. We highlight the different biological roles of parasite cystatins and the anticipated future developments.

  5. Foodborne parasites from wildlife

    DEFF Research Database (Denmark)

    Kapel, Christian Moliin Outzen; Fredensborg, Brian Lund

    2015-01-01

    The majority of wild foods consumed by humans are sourced from intensively managed or semi-farmed populations. Management practices inevitably affect wildlife density and habitat characteristics, which are key elements in the transmission of parasites. We consider the risk of transmission...... of foodborne parasites to humans from wildlife maintained under natural or semi-natural conditions. A deeper understanding will be useful in counteracting foodborne parasites arising from the growing industry of novel and exotic foods....

  6. Parasites, Plants, and People.

    Science.gov (United States)

    Johnson, Marion; Moore, Tony

    2016-06-01

    Anthelminthic resistance is acknowledged worldwide and is a major problem in Aotearoa New Zealand, thus alternative parasite management strategies are imperative. One Health is an initiative linking animal, human, and environmental health. Parasites, plants, and people illustrate the possibilities of providing diverse diets for stock thereby lowering parasite burdens, improving the cultural wellbeing of a local community, and protecting the environment. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Neglected Parasitic Infections: Toxocariasis

    Centers for Disease Control (CDC) Podcasts

    2012-01-05

    This podcast is an overview of the Clinician Outreach and Communication Activity (COCA) Call: Neglected Parasitic Infections in the United States. Neglected Parasitic Infections are a group of diseases that afflict vulnerable populations and are often not well studied or diagnosed. A subject matter expert from CDC's Division of Parasitic Diseases and Malaria describes the epidemiology, diagnosis, and treatment of toxocariasis.  Created: 1/5/2012 by Center for Global Health, Division of Parasitic Diseases and Malaria (DPDM); Emergency Risk Communication Branch (ERCB)/Joint Information Center (JIC), Office of Public Health Preparedness and Response (OPHPR).   Date Released: 1/9/2012.

  8. Parasites in food webs: the ultimate missing links.

    Science.gov (United States)

    Lafferty, Kevin D; Allesina, Stefano; Arim, Matias; Briggs, Cherie J; De Leo, Giulio; Dobson, Andrew P; Dunne, Jennifer A; Johnson, Pieter T J; Kuris, Armand M; Marcogliese, David J; Martinez, Neo D; Memmott, Jane; Marquet, Pablo A; McLaughlin, John P; Mordecai, Erin A; Pascual, Mercedes; Poulin, Robert; Thieltges, David W

    2008-06-01

    Parasitism is the most common consumer strategy among organisms, yet only recently has there been a call for the inclusion of infectious disease agents in food webs. The value of this effort hinges on whether parasites affect food-web properties. Increasing evidence suggests that parasites have the potential to uniquely alter food-web topology in terms of chain length, connectance and robustness. In addition, parasites might affect food-web stability, interaction strength and energy flow. Food-web structure also affects infectious disease dynamics because parasites depend on the ecological networks in which they live. Empirically, incorporating parasites into food webs is straightforward. We may start with existing food webs and add parasites as nodes, or we may try to build food webs around systems for which we already have a good understanding of infectious processes. In the future, perhaps researchers will add parasites while they construct food webs. Less clear is how food-web theory can accommodate parasites. This is a deep and central problem in theoretical biology and applied mathematics. For instance, is representing parasites with complex life cycles as a single node equivalent to representing other species with ontogenetic niche shifts as a single node? Can parasitism fit into fundamental frameworks such as the niche model? Can we integrate infectious disease models into the emerging field of dynamic food-web modelling? Future progress will benefit from interdisciplinary collaborations between ecologists and infectious disease biologists.

  9. Parasites in food webs: the ultimate missing links

    Science.gov (United States)

    Lafferty, Kevin D.; Allesina, Stefano; Arim, Matias; Briggs, Cherie J.; De Leo, Giulio A.; Dobson, Andrew P.; Dunne, Jennifer A.; Johnson, Pieter T.J.; Kuris, Armand M.; Marcogliese, David J.; Martinez, Neo D.; Memmott, Jane; Marquet, Pablo A.; McLaughlin, John P.; Mordecai, Eerin A.; Pascual, Mercedes; Poulin, Robert; Thieltges, David W.

    2008-01-01

    Parasitism is the most common consumer strategy among organisms, yet only recently has there been a call for the inclusion of infectious disease agents in food webs. The value of this effort hinges on whether parasites affect food-web properties. Increasing evidence suggests that parasites have the potential to uniquely alter food-web topology in terms of chain length, connectance and robustness. In addition, parasites might affect food-web stability, interaction strength and energy flow. Food-web structure also affects infectious disease dynamics because parasites depend on the ecological networks in which they live. Empirically, incorporating parasites into food webs is straightforward. We may start with existing food webs and add parasites as nodes, or we may try to build food webs around systems for which we already have a good understanding of infectious processes. In the future, perhaps researchers will add parasites while they construct food webs. Less clear is how food-web theory can accommodate parasites. This is a deep and central problem in theoretical biology and applied mathematics. For instance, is representing parasites with complex life cycles as a single node equivalent to representing other species with ontogenetic niche shifts as a single node? Can parasitism fit into fundamental frameworks such as the niche model? Can we integrate infectious disease models into the emerging field of dynamic food-web modelling? Future progress will benefit from interdisciplinary collaborations between ecologists and infectious disease biologists.

  10. Flow cytometric detection of some activation and proliferation markers in human hematopoietic cell lines.

    Science.gov (United States)

    Glasová, M; Koníková, E; Kusenda, J; Babusíková, O

    1996-01-01

    Simultaneous surface marker/DNA, cytoplasmic/DNA or nuclear/DNA staining was used to study proliferation of hematopoietic cell lines (MOLT4, BJAB, P3HR1). Different fixation/permeabilization methods (paraformaldehyde with metanol or Tween 20 or saponin, buffered formaldehyde-acetone) were used providing optimal results of the double stainings. There was a significant increase of S phase and proliferation index (PI) of CD71+ and Ki67+ MOLT4 cells in comparison with their negative counterparts. This indicates their close connection with proliferation. Unlike that, the correlation between the expression of CD38 and S phase or PI was not significant either in MOLT4 or in P3HRI cells. For cytoplasmic markers CD3 (in MOLT4 cells) and CD22 (in BJAB cells) statistically significant (cCD3) and not significant (cCD22) correlation was demonstrated between their expression and S phase or PI. Molecular equivalents of soluble fluorescein values for CD71 were always higher than for CD38. The density of these cell surface markers in addition to the percentage of their expression is of considerable significance for their evaluation as activation or proliferation markers.

  11. Flow Cytometric Analysis of DNA Content in Parotid Tumor and Its Contiguous Acini

    Institute of Scientific and Technical Information of China (English)

    ZHU Shengrong; SHAO Lenan; CHEN Weimin; WU Huihua; WANG Xiuli; CHEN Xinming

    2000-01-01

    To investigate the relationship between proliferative capacity of salivary gland cells in contiguous acini of parotid tumors and recurrent neoplasma, DNA contents of 30 fresh specimens of parotid were studied by using cytometry in tumors, normal and shallow or deep lobe acini of the masses. The results showed that the DI was 1.369, S % 16.95, PI 26.18 in malignant tumors;DI was 1.171, S % 12.41, PI 15.54 in recurrent pleomorphic adenoma; DI was 1.141, S % 12.74, PI 13.07 in pleomorphic adenoma, DI was 0.999, S % 5.10, PI 8.00 in normal acini. Analysis of variance showed there was a significant difference (P<0.01). The average DNA contents of shallow on deep lobe of contiguous tumors was 1.08 in DI, 10. 65 in S %, 13.49 in PI in malignant tumor, 1.06 in DI, 8.96 in S % and 9.85 in PI in pleomorphic adenoma, which were all higher than in normal acini (P>0.05). It was concluded that the levels of DI and S % of parotid tumor and its contiguous acini are related to degree of malignancy or recurrent condition of the tumors, suggesting contiguous acini of parotid tumors had the strong capacity of proliferation,which might play an important role in recurrent or malignant change of the parotid tumors.

  12. Joint modeling and registration of cell populations in cohorts of high-dimensional flow cytometric data.

    Science.gov (United States)

    Pyne, Saumyadipta; Lee, Sharon X; Wang, Kui; Irish, Jonathan; Tamayo, Pablo; Nazaire, Marc-Danie; Duong, Tarn; Ng, Shu-Kay; Hafler, David; Levy, Ronald; Nolan, Garry P; Mesirov, Jill; McLachlan, Geoffrey J

    2014-01-01

    In biomedical applications, an experimenter encounters different potential sources of variation in data such as individual samples, multiple experimental conditions, and multivariate responses of a panel of markers such as from a signaling network. In multiparametric cytometry, which is often used for analyzing patient samples, such issues are critical. While computational methods can identify cell populations in individual samples, without the ability to automatically match them across samples, it is difficult to compare and characterize the populations in typical experiments, such as those responding to various stimulations or distinctive of particular patients or time-points, especially when there are many samples. Joint Clustering and Matching (JCM) is a multi-level framework for simultaneous modeling and registration of populations across a cohort. JCM models every population with a robust multivariate probability distribution. Simultaneously, JCM fits a random-effects model to construct an overall batch template--used for registering populations across samples, and classifying new samples. By tackling systems-level variation, JCM supports practical biomedical applications involving large cohorts. Software for fitting the JCM models have been implemented in an R package EMMIX-JCM, available from http://www.maths.uq.edu.au/~gjm/mix_soft/EMMIX-JCM/.

  13. Generation, culture and flow-cytometric characterization of primary mouse macrophages.

    Science.gov (United States)

    Schleicher, Ulrike; Bogdan, Christian

    2009-01-01

    Macrophages are not only host cells for many pathogens, but also fulfill several key functions in the innate and adaptive immune response, including the release of pro- and anti-inflammatory cytokines, the generation of organic and inorganic autacoids, the phagocytosis and killing of intracellular microorganisms or tumor cells, and the degradation and presentation of antigens. Several of these functions are shared by other immune cells, including dendritic cells, granulocytes, NK cells, and/or T lymphocytes. Thus, the analysis of macrophage functions in vitro using primary mouse cell populations requires standardized methods for the generation and culture of macrophages that guarantee high cell purity as well as the absence of stimulatory microbial contaminants. This chapter presents methodology to achieve these aims.

  14. Expanding the potential of standard flow cytometry by extracting fluorescence lifetimes from cytometric pulse shifts

    National Research Council Canada - National Science Library

    Cao, Ruofan; Naivar, Mark A; Wilder, Mark; Houston, Jessica P

    2014-01-01

    Fluorescence lifetime measurements provide information about the fluorescence relaxation, or intensity decay, of organic fluorophores, fluorescent proteins, and other inorganic molecules that fluoresce...

  15. Ciliate ingestion and digestion: flow cytometric measurements and regrowth of a digestion-resistant campylobacter jejuni

    Science.gov (United States)

    We developed a method to measure ingestion and digestion rates of bacterivorous protists feeding on pathogenic bacteria. We tested this method using the enteric bacteria Campylobacter jejuni and a freshwater colpodid ciliate. Campylobacter and a non-pathogenic bacteria isolated from the environment ...

  16. Measurement of Transient Permeability of Sp2/0 Myeloma Cells: Flow Cytometric Study

    Directory of Open Access Journals (Sweden)

    Novickij Vitalij

    2016-12-01

    Full Text Available Electroporation is an electric field induced phenomenon occurring when the permeability of the cell membrane is increased due to the excess of critical transmembrane potential. Fluorescent dye assays are frequently used for evaluation of the permeabilization rate, however, the protocols vary, which negatively affects the repeatability of the results. In this work we have designed experiments to investigate the protocols and threshold concentrations of the Propidium Iodide (PI and YO-PRO-1 (YP fluorescent dyes for evaluation of mammalian cell permeabilization induced by electroporation. The Sp2/0 mouse myeloma cells were used and the bursts of 100 μs × 8 electrical pulses of 0.8-2 kV/cm were applied. It has been shown that the dye concentration has an influence on the detectable permeabilization, and the concentrations below 30 μM for PI and 1 μM for YP should be avoided for measurement of electropermeabilization efficacy due to unreliable fluorescence signals. Further, based on the experimental data, the permeabilization curve for the Sp2/0 myeloma cells in the 0.8-2 kV/cm range has been presented.

  17. Flow Cytometric Testing of Green Fluorescent Protein-Tagged Lactobacillus rhamnosus GG for Response to Defensins

    OpenAIRE

    De Keersmaecker, Sigrid C. J.; Braeken, Kristien; Verhoeven, Tine L. A.; Perea Vélez, Mónica; Lebeer, Sarah; Vanderleyden, Jos; Hols, Pascal

    2006-01-01

    Lactobacillus rhamnosus GG is of general interest as a probiotic. Although L. rhamnosus GG is often used in clinical trials, there are few genetic tools to further determine its mode of action or to develop it as a vehicle for heterologous gene expression in therapy. Therefore, we developed a reproducible, efficient electroporation procedure for L. rhamnosus GG. The best transformation efficiency obtained was 104 transformants per μg of DNA. We validated this protocol by tagging L. rhamnosus ...

  18. Preanalytical requirements for flow cytometric evaluation of platelet activation: choice of anticoagulant.

    Science.gov (United States)

    Mody, M; Lazarus, A H; Semple, J W; Freedman, J

    1999-06-01

    Accurate assessment of in vivo or in vitro platelet activation requires optimal preanalytical conditions to prevent artefactual in vitro activation of the platelets. The choice of anticoagulant is one of the critical preanalytical conditions as anticoagulants exert different effects on the activation of platelets ex vivo. We tested the effectiveness of Diatube-H (also known as CTAD; sodium citrate, theophylline, adenosine and dipyridamole) and citrate vacutainer tubes in preventing artefactual activation of platelets and preserving functional reserve. Platelet surface expression of the CD62P (reflecting alpha granule release), CD63 (reflecting lysosomal release) and modulation of normal platelet membrane glycoproteins CD41a and CD42b, were measured in whole blood and in isolated platelets immediately after collection and at 6, 24 and 48 h after venipuncture. Samples taken into Diatube-H showed less spontaneous platelet activation than did those taken into citrate. To measure in vitro platelet functional reserve, thrombin was added as agonist to blood stored for varying periods up to 48 h. Although Diatube-H suppressed in vitro platelet activation for up to 4 h, in samples kept for 6-24 h before thrombin addition, the inhibitory effect was lost and platelets responded fully to agonist activation. Hence, Diatube-H preserved platelets and allowed for measurement of in vivo platelet activation as well as thrombin-induced in vitro platelet activation after 6-24 h, in both whole blood and isolated platelets.

  19. 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...

  20. 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

    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...... molecules, but not the class II, was correlated with lymphoid infiltration and the Jass classification. Expression of these surface molecules was not correlated with the Dukes' stage. The tumour cells were generally equipped to present antigens to the effector arm of the immune system since HLA class I...... is expressed, but the tumour cells were not optimal in stimulating an immune response, since HLA class II and CD58 were only marginally expressed and CD80 and CD54 were absent....

  1. Efficient quantification and characterization of bacterial outer membrane derived nano-particles with flow cytometric analysis.

    Science.gov (United States)

    Wieser, Andreas; Storz, Enno; Liegl, Gabriele; Peter, Annabell; Pritsch, Michael; Shock, Jonathan; Wai, Sun Nyunt; Schubert, Sören

    2014-11-01

    There currently exists no efficient and easy method for size profiling and counting of membranous nano-scale particles, such as bacterial outer membrane vesicles (OMVs). We present here a cost-effective and fast method capable of profiling and counting small sample volumes of nano-scale membranous vesicles with standard laboratory equipment without the need for any washing steps. OMV populations of different bacterial species are compared and even subpopulations of OMVs can be identified after a simple labelling procedure. Counting is possible over three orders of magnitude without any changes to the protocol. Protein contaminations do not alter the described measurements. Copyright © 2014 Elsevier GmbH. All rights reserved.

  2. Downregulation of pro-inflammatory cytokines by lupeol measured using cytometric bead array immunoassay.

    Science.gov (United States)

    Ahmad, Sheikh Fayaz; Pandey, Anjali; Kour, Kiranjeet; Bani, Sarang

    2010-01-01

    The objective of the study was to investigate the activity of Lupeol (LUP) on proinflammatory and anti-inflammatory cytokines in the pleural exudate from male swiss albino mice. We applied Cytometric bead array technology for simultaneously measurement of these cytokines in pleurisy induced mice treated with lupeol in graded oral doses. Cytometric bead array uses the sensitivity of amplified fluorescence detection by flowcytometer to measure soluble analytes in a particle based immune assay. This assay can accurately quantitate 5 cytokines in a 50 microlitre sample volume. Oral administration of LUP at doses of 25, 50, 100 and 200 mg/kg p.o. produced dose related inhibition of IL-2, IFN-gamma and TNF-alpha in the pleural exudate with the most significant effect at 100 mg/kg oral dose. LUP had a non significant inhibitory effect on the levels of IL-4 and IL-5.

  3. Parasites and marine invasions

    Science.gov (United States)

    Torchin, M.E.; Lafferty, K.D.; Kuris, A.M.

    2002-01-01

    Introduced marine species are a major environmental and economic problem. The rate of these biological invasions has substantially increased in recent years due to the globalization of the world's economies. The damage caused by invasive species is often a result of the higher densities and larger sizes they attain compared to where they are native. A prominent hypothesis explaining the success of introduced species is that they are relatively free of the effects of natural enemies. Most notably, they may encounter fewer parasites in their introduced range compared to their native range. Parasites are ubiquitous and pervasive in marine systems, yet their role in marine invasions is relatively unexplored. Although data on parasites of marine organisms exist, the extent to which parasites can mediate marine invasions, or the extent to which invasive parasites and pathogens are responsible for infecting or potentially decimating native marine species have not been examined. In this review, we present a theoretical framework to model invasion success and examine the evidence for a relationship between parasite presence and the success of introduced marine species. For this, we compare the prevalence and species richness of parasites in several introduced populations of marine species with populations where they are native. We also discuss the potential impacts of introduced marine parasites on native ecosystems.

  4. PARASITES OF FISH

    Science.gov (United States)

    The intent of this chapter is to describe the parasites of importance to fishes maintained and used in laboratory settings. In contrast to the frist edition, the focus will be only on those parasites that pose a serious threat to or are common in fishes held in these confined en...

  5. Parasites from the Past

    DEFF Research Database (Denmark)

    Søe, Martin Jensen; Fredensborg, Brian Lund; Nejsum, Peter

    will investigate how the diversity of food-borne parasitic infections has changed with cultural and dietary habits, hunting practice and intensity of animal husbandry. This is done by isolating and typing ancient DNA remains from parasite eggs found in archeological samples from across Denmark....

  6. Parasite infections revisited

    NARCIS (Netherlands)

    Wiegertjes, G.F.; Forlenza, M.; Joerink, M.; Scharsack, J.P.

    2005-01-01

    Studying parasites helps reveal basic mechanisms in immunology. For long this has been recognized for studies on the immune system of mice and man. But it is not true for immunological studies on fish. To support this argument we discuss selected examples of parasite infections not only in warm-bloo

  7. Automation in high-content flow cytometry screening.

    Science.gov (United States)

    Naumann, U; Wand, M P

    2009-09-01

    High-content flow cytometric screening (FC-HCS) is a 21st Century technology that combines robotic fluid handling, flow cytometric instrumentation, and bioinformatics software, so that relatively large numbers of flow cytometric samples can be processed and analysed in a short period of time. We revisit a recent application of FC-HCS to the problem of cellular signature definition for acute graft-versus-host-disease. Our focus is on automation of the data processing steps using recent advances in statistical methodology. We demonstrate that effective results, on par with those obtained via manual processing, can be achieved using our automatic techniques. Such automation of FC-HCS has the potential to drastically improve diagnosis and biomarker identification.

  8. AIDS - associated parasitic diarrhoea

    Directory of Open Access Journals (Sweden)

    Arora D

    2009-01-01

    Full Text Available Since the advent of human immunodeficiency virus infection, with its profound and progressive effect on the cellular immune system, a group of human opportunistic pathogens has come into prominence. Opportunistic parasitic infection can cause severe morbidity and mortality. Because many of these infections are treatable, an early and accurate diagnosis is important. This can be accomplished by a variety of methods such as direct demonstration of parasites and by serological tests to detect antigen and/or specific antibodies. However, antibody response may be poor in these patients and therefore immunodiagnostic tests have to be interpreted with caution. Cryptosporidium parvum , Isospora belli , Cyclospora cayetanensis , Microsporidia, Entamoeba histolytica and Strongyloides stercoralis are the commonly detected parasites. Detection of these parasites will help in proper management of these patients because drugs are available for most of these parasitic infections.

  9. 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.

  10. [The specific features of diagnosis of mixed-phenotype acute leukemia: A combination of B-cell antigen expressions according to the results of flow cytometry and morphological markers of myeloid differentiation in blast cells: A clinical case].

    Science.gov (United States)

    Gritsaev, S V; Kostroma, I I; Ryadnova, G M; Tiranova, S A; Chubukina, Zh V; Balashova, V A; Zenina, M N; Martynkevich, I S; Potikhonova, N A; Abdulkadyrov, K M

    2015-01-01

    This rare type of acute leukemia, blast cells of which express myeloid and/or lymphoid markers, is mainly diagnosed using flow cytometric findings. The paper describes a clinical case of mixed-phenotype acute leukemia, in which B-cell lymphoid antigen expressions were revealed by a flow cytometric technique, while bone marrow morphological specimens showed the signs of myeloid differentiation specific to blast cells. It is concluded that there is a need for a comprehensive examination of patients with new-onset acute leukemia and for an aggregate analysis of flow cytometric results with morphological and cytochemical findings.

  11. Pitting of malaria parasites and spherocyte formation

    Directory of Open Access Journals (Sweden)

    Gichuki Charity W

    2006-07-01

    Full Text Available Abstract Background A high prevalence of spherocytes was detected in blood smears of children enrolled in a case control study conducted in the malaria holoendemic Lake Victoria basin. It was speculated that the spherocytes reflect intraerythrocytic removal of malarial parasites with a concurrent removal of RBC membrane through a process analogous to pitting of intraerythrocytic inclusion bodies. Pitting and re-circulation of RBCs devoid of malaria parasites could be a host mechanism for parasite clearance while minimizing the anaemia that would occur were the entire parasitized RBC removed. The prior demonstration of RBCs containing ring-infected erythrocyte surface antigen (pf 155 or RESA but no intracellular parasites, support the idea of pitting. Methods An in vitro model was developed to examine the phenomenon of pitting and spherocyte formation in Plasmodium falciparum infected RBCs (iRBC co-incubated with human macrophages. In vivo application of this model was evaluated using blood specimens from patients attending Kisumu Ditrict Hospital. RBCs were probed with anti-RESA monoclonal antibody and a DNA stain (propidium iodide. Flow cytometry and fluorescent microscopy was used to compare RBCs containing both the antigen and the parasites to those that were only RESA positive. Results Co-incubation of iRBC and tumor necrosis factor-alpha activated macrophages led to pitting (14% ± 1.31% macrophages with engulfed trophozoites as opposed to erythrophagocytosis (5.33% ± 0.95% (P Conclusion It is proposed that in malaria holoendemic areas where prevalence of asexual stage parasites approaches 100% in children, RBCs with pitted parasites are re-circulated and pitting may produce spherocytes.

  12. [Parasitism and ecological parasitology].

    Science.gov (United States)

    Balashov, Iu S

    2011-01-01

    Parasitism as one of the life modes is a general biological phenomenon and is a characteristic of all viruses, many taxa of bacteria, fungi, protists, metaphytes, and metazoans. Zooparasitology is focused on studies of parasitic animals, particularly, on their taxonomy, anatomy, life cycles, host-parasite relations, biocoenotic connections, and evolution. Ecological parasitology is a component of ecology, as the scientific study of the relation of living organisms with each other and their surroundings. In the present paper, critical analysis of the problems, main postulates, and terminology of the modern ecological parasitology is given.

  13. Potential Use of Quantum Dots in Flow Cytometry

    Directory of Open Access Journals (Sweden)

    Raquel Ibáñez-Peral

    2008-12-01

    Full Text Available QDs may offer significant advantages in environmental and bead-based applications where the target cells need to be discriminated above background fluorescence. We have examined the possible applications of QDs for flow cytometric measurements (FCM by studying their excitation - emission spectra and their binding to paramagnetic beads. We labelled beads with either QDs or a commonly-used fluorochrome (FITC and studied their fluorescence intensity by FCM. Flow cytometric comparisons indicated that the minimum fluorophore concentration required for detection of QDs above autofluorescent background was 100-fold less than for FITC.

  14. Metabolomics and protozoan parasites.

    Science.gov (United States)

    Paget, Timothy; Haroune, Nicolas; Bagchi, Sushmita; Jarroll, Edward

    2013-06-01

    In this review, we examine the state-of-the-art technologies (gas and liquid chromatography, mass spectroscopy and nuclear magnetic resonance, etc.) in the well-established area of metabolomics especially as they relate to protozoan parasites.

  15. Transfection of malaria parasites.

    Science.gov (United States)

    Waters, A P; Thomas, A W; van Dijk, M R; Janse, C J

    1997-10-01

    The stable genetic transformation of three phylogenetically diverse species of Plasmodium, the parasitic etiological agent of malaria, is now possible. The parasite is haploid throughout the vast majority of its life cycle. Therefore with the single selectable marker activity and protocols currently available, it is possible not only to express introduced transgenes but also to study the effects of site-specific homologous recombination such as gene knockout. Transgene expression will allow the detailed study of many aspects of the cellular biology of malaria parasites, for example, the mechanisms underlying drug resistance and protein trafficking. We describe here the methods for propagation of the two animal models (Plasmodium berghei and Plasmodium knowlesi) and for transfection of these two species and the human parasite, Plasmodium falciparum. Examples of transgene expression are given.

  16. Pathoecology of Chiribaya parasitism

    Directory of Open Access Journals (Sweden)

    Martinson Elizabeth

    2003-01-01

    Full Text Available The excavations of Chiribaya culture sites in the Osmore drainage of southern Peru focused on the recovery of information about prehistoric disease, including parasitism. The archaeologists excavated human, dog, guinea pig, and llama mummies. These mummies were analyzed for internal and external parasites. The results of the analysis and reconstruction of prehistoric life from the excavations allows us to interpret the pathoecology of the Chiribaya culture.

  17. Parasites and human evolution.

    Science.gov (United States)

    Perry, George H

    2014-01-01

    Our understanding of human evolutionary and population history can be advanced by ecological and evolutionary studies of our parasites. Many parasites flourish only in the presence of very specific human behaviors and in specific habitats, are wholly dependent on us, and have evolved with us for thousands or millions of years. Therefore, by asking when and how we first acquired those parasites, under which environmental and cultural conditions we are the most susceptible, and how the parasites have evolved and adapted to us and we in response to them, we can gain considerable insight into our own evolutionary history. As examples, the tapeworm life cycle is dependent on our consumption of meat, the divergence of body and head lice may have been subsequent to the development of clothing, and malaria hyperendemicity may be associated with agriculture. Thus, the evolutionary and population histories of these parasites are likely intertwined with critical aspects of human biology and culture. Here I review the mechanics of these and multiple other parasite proxies for human evolutionary history and discuss how they currently complement our fossil, archeological, molecular, linguistic, historical, and ethnographic records. I also highlight potential future applications of this promising model for the field of evolutionary anthropology.

  18. Parasites in marine food webs

    Science.gov (United States)

    Lafferty, Kevin D.

    2013-01-01

    Most species interactions probably involve parasites. This review considers the extent to which marine ecologists should consider parasites to fully understand marine communities. Parasites are influential parts of food webs in estuaries, temperate reefs, and coral reefs, but their ecological importance is seldom recognized. Though difficult to observe, parasites can have substantial biomass, and they can be just as common as free-living consumers after controlling for body mass and trophic level. Parasites have direct impacts on the energetics of their hosts and some affect host behaviors, with ecosystem-level consequences. Although they cause disease, parasites are sensitive components of ecosystems. In particular, they suffer secondary extinctions due to biodiversity loss. Some parasites can also return to a system after habitat restoration. For these reasons, parasites can make good indicators of ecosystem integrity. Fishing can indirectly increase or decrease parasite populations and the effects of climate change on parasites are likely to be equally as complex.

  19. 流式细胞术在感染鼠疟原虫活红细胞检测中的应用%Application of Flow Cytometry in the Detection of Mouse Malaria Parasites in Living Cells

    Institute of Scientific and Technical Information of China (English)

    刘毅; 奚卫

    2012-01-01

    Peripheral blood samples were obtained from Hasmodium berghei-infected mice, and stained with Vybrant DyeCycle Green and anti-mouse CD71 PE. With normal mouse as negative control, the blood samples were tested by flow cytometry at 0, 30, and 60min after staining. There was no positive signal in the erythrocytes from negative control and the unstained erythrocytes from the infected mouse. However, the positive signal was detected in the stained erythrocytes from the infected mouse. The results showed an adequate consistency by intra-class correlation coefficieni (ICC) test (ICC=0.999, P<0.01). Flow cytometry with the use of Vybrant DyeCycle Green and anti-mouse CD71 PE ir staining living cells can be used for the detection of malaria parasite-infected blood. In a given period, there is nc negative impact on the detection of unfixed cells.%采集感染伯氏疟原虫的ICR小鼠外周血,未经固定的红细胞用Vybrant DyeCycle Green染料和抗小鼠CD71 PE荧光抗体进行双染色(实验组).以健康小鼠外周血作阴性对照.分别在染色后0、30和60 min利用流式细胞仪进行检测,比较各次结果的一致性.结果显示,阴性对照(健康小鼠血样)与空白对照(感染疟原虫鼠血未作染色)未检出感染红细胞的疟原虫,实验组可检出感染红细胞的疟原虫.组内相关系数(ICC)检验结果显示,3个时间点检测结果的一致性较好(ICC=0.999,P<0.01).表明未经固定的红细胞用Vybrant DyeCycle Green染料和抗小鼠CD71 PE荧光抗体染色经流式细胞术检测可区分感染疟原虫红细胞.采集的红细胞在一定时间内未作固定对检测结果无明显影响.

  20. Cytometric analysis of surface molecules of leucocytes and phagocytic activity of granulocytes and monocytes/macrophages in cows with pyometra.

    Science.gov (United States)

    Brodzki, P; Kostro, K; Brodzki, A; Niemczuk, K; Lisiecka, U

    2014-10-01

    Pyometra is a serious problem in dairy cow herds, causing large economic losses due to infertility. The development of pyometra depends mainly on the immunological status of the cow. The aim of the study was a comparative evaluation of selected indicators involving non-specific and specific immunity in cows with pyometra and in cows without inflammation of the uterus. The study was performed in 20 cows, which were divided into two groups: pyometra group and healthy group, each comprising 10 cows, based on the results of cytological and ultrasonographic tests. A flow cytometric analysis was performed for the surface molecules CD4, CD8, CD14, CD21, CD25 and CD4(+) CD25(+) on leucocytes, and the phagocytic activity was determined from granulocytes and monocytes/macrophages in the peripheral blood and uterine washings, respectively. It was demonstrated that the percentage of phagocytic granulocytes and monocytes/macrophages in both the peripheral blood and uterine washings was significantly lower in cows with pyometra compared with the healthy group (p < 0.001). Significantly (p ≤ 0.001) lower percentage of CD4(+) , CD14(+) , CD25(+) and CD4(+) CD25(+) phenotype leucocytes was also observed in the peripheral blood of cows from the pyometra group, along with a significantly higher (p < 0.001) percentage of CD8(+) and CD21(+) lymphocytes as compared to the healthy group. The results of work indicate that disfunction of cell immunity coexisting with pyometra may be caused by a bacterial infection and the presence of blocking agents (IL-10), released by the increasing number of CD8(+) lymphocytes what leads to the advanced inflammation of uterus.

  1. Clinical utility of flow cytometry in the study of erythropoiesis and nonclonal red cell disorders.

    Science.gov (United States)

    Chesney, Alden; Good, David; Reis, Marciano

    2011-01-01

    Erythropoiesis involves proliferation and differentiation of small population of hematopoietic stem cells resident in the bone marrow into mature red blood cells. The determination of the cellular composition of the blood is a valuable tool in the diagnosis of diseases and monitoring of therapy. Flow cytometric analysis is increasingly being used to characterize the heterogeneous cell populations present in the blood and the hematopoietic cell differentiation and maturation pathways of the bone marrow. Here we discuss the role of flow cytometry in the study of erythropoiesis and nonclonal red blood cell disorders. First, we discuss flow cytometric analysis of reticulocytes. Next, we review salient quantitative methods that can be used for detection of fetal-maternal hemorrhage (FMH). We also discuss flow cytometric analysis of high hemoglobin F (HbF) in Sickle Cell Disease (SCD), hereditary spherocytosis (HS), red cell survival and red cell volume. We conclude by discussing cell cycle of erythroid cells.

  2. Internal parasites of reptiles.

    Science.gov (United States)

    Raś-Noryńska, Małgorzata; Sokół, Rajmund

    2015-01-01

    Nowadays a growing number of exotic reptiles are kept as pets. The aim of this study was to determine the species of parasites found in reptile patients of veterinary practices in Poland. Fecal samples obtained from 76 lizards, 15 turtles and 10 snakes were examined by flotation method and direct smear stained with Lugol's iodine. In 63 samples (62.4%) the presence of parasite eggs and oocysts was revealed. Oocysts of Isospora spp. (from 33% to 100% of the samples, depending on the reptilian species) and Oxyurids eggs (10% to 75%) were predominant. In addition, isolated Eimeria spp. oocysts and Giardia intestinalis cysts were found, as well as Strongylus spp. and Hymenolepis spp. eggs. Pet reptiles are often infected with parasites, some of which are potentially dangerous to humans. A routine parasitological examination should be done in such animals.

  3. Foodborne protozoan parasites.

    Science.gov (United States)

    Dawson, David

    2005-08-25

    This report addresses Cryptosporidium, Giardia, Cyclospora, and more briefly, Toxoplasma as the main parasitic protozoa of concern to food production worldwide. Other parasitic protozoa may be spread in food or water but are not considered as great a risk to food manufacture. The protozoan parasites Cryptosporidium, Giardia, and Cyclospora have proven potential to cause waterborne and foodborne disease. Toxoplasma gondii has been considered a risk in specific cases, but humans are not its primary host. Cryptosporidium and Giardia are widespread in the environment, particularly the aquatic environment, and major outbreaks of cryptosporidiosis and giardiasis have occurred as a result of contaminated drinking water. Large outbreaks of waterborne cyclosporiasis have not been identified. Cryptosporidium, Giardia, and Cyclospora have potential significance in the preparation and consumption of fresh produce and in catering practice, in which ready-to-eat foods may be served that have not received heat treatment. None of the three organisms Cryptosporidium, Giardia, and Cyclospora has been shown to be a problem for heat processed food or tap water that has undergone appropriate treatment at a water treatment works. All three are sensitive to standard pasteurisation techniques. Although humans are not a primary host for T. gondii, the potential exists for both waterborne and foodborne toxoplasmosis. Parasitic protozoa do not multiply in foods, but they may survive in or on moist foods for months in cool, damp environments. Their ecology makes control of these parasites difficult. For general control of parasitic protozoa in the food chain, the following steps are necessary: - Follow good hygienic practice in food service and catering industries.- Minimise dissemination of cysts and oocysts in the farming environment and via human waste management.- Include these microorganisms in Hazard Analysis Critical Control Point (HACCP) plans of water suppliers, industries or sectors

  4. Parasite zoonoses and wildlife: One Health, spillover and human activity.

    Science.gov (United States)

    Thompson, R C Andrew

    2013-11-01

    This review examines parasite zoonoses and wildlife in the context of the One Health triad that encompasses humans, domestic animals, wildlife and the changing ecosystems in which they live. Human (anthropogenic) activities influence the flow of all parasite infections within the One Health triad and the nature and impact of resulting spillover events are examined. Examples of spillover from wildlife to humans and/or domestic animals, and vice versa, are discussed, as well as emerging issues, particularly the need for parasite surveillance of wildlife populations. Emphasis is given to Trypanosoma cruzi and related species in Australian wildlife, Trichinella, Echinococcus, Giardia, Baylisascaris, Toxoplasma and Leishmania.

  5. On biomass of parasitic plants

    Energy Technology Data Exchange (ETDEWEB)

    He, J H [Modern Textile Institute, Donghua University, 1882 Yan' an Xilu Road, Shanghai 200051 (China); Mo, L-F [School of Information Engineering, Zhejiang Forestry College, Lin' an 311300, Zhejiang (China)], E-mail: jhhe@dhu.edu.cn

    2008-02-15

    An extremely simple and elementary but rigorous derivation of maximal biomass of parasitic plants is given using an assumption that metabolic rate of the parasite should not be larger than that of its host organ.

  6. On biomass of parasitic plants

    Science.gov (United States)

    He, J. H.; Mo, L.-F.

    2008-02-01

    An extremely simple and elementary but rigorous derivation of maximal biomass of parasitic plants is given using an assumption that metabolic rate of the parasite should not be larger than that of its host organ

  7. Enteric parasites and AIDS

    Directory of Open Access Journals (Sweden)

    Sérgio Cimerman

    1999-11-01

    Full Text Available OBJECTIVE: To report on the importance of intestinal parasites in patients with AIDS, showing relevant data in the medical literature, with special emphasis on epidemiology, diagnosis and treatment of enteroparasitosis, especially cryptosporidiasis, isosporiasis, microsporidiasis and strongyloidiasis. DESIGN: Narrative review.

  8. Parasites and the skin

    African Journals Online (AJOL)

    2009-06-11

    Jun 11, 2009 ... remind you of those rare and wonderful infestations that you might never see. ... from a burrow, mounted on a glass slide. The findings are ... Parasitic infections may be confined to the skin or may have skin involvement as part ...

  9. Parasites and Foodborne Illness

    Science.gov (United States)

    ... called "Crypto", is a one-celled, microscopic shelled parasite and a significant cause of waterborne and foodborne illness worldwide. It is found in the intestines of many herd animals including cows, sheep, goats, deer, and elk. The illness could be intestinal, ...

  10. Ungulate malaria parasites

    Science.gov (United States)

    Templeton, Thomas J.; Asada, Masahito; Jiratanh, Montakan; Ishikawa, Sohta A.; Tiawsirisup, Sonthaya; Sivakumar, Thillaiampalam; Namangala, Boniface; Takeda, Mika; Mohkaew, Kingdao; Ngamjituea, Supawan; Inoue, Noboru; Sugimoto, Chihiro; Inagaki, Yuji; Suzuki, Yasuhiko; Yokoyama, Naoaki; Kaewthamasorn, Morakot; Kaneko, Osamu

    2016-01-01

    Haemosporida parasites of even-toed ungulates are diverse and globally distributed, but since their discovery in 1913 their characterization has relied exclusively on microscopy-based descriptions. In order to bring molecular approaches to bear on the identity and evolutionary relationships of ungulate malaria parasites, we conducted Plasmodium cytb-specific nested PCR surveys using blood from water buffalo in Vietnam and Thailand, and goats in Zambia. We found that Plasmodium is readily detectable from water buffalo in these countries, indicating that buffalo Plasmodium is distributed in a wider region than India, which is the only area in which buffalo Plasmodium has been reported. Two types (I and II) of Plasmodium sequences were identified from water buffalo and a third type (III) was isolated from goat. Morphology of the parasite was confirmed in Giemsa-reagent stained blood smears for the Type I sample. Complete mitochondrial DNA sequences were isolated and used to infer a phylogeny in which ungulate malaria parasites form a monophyletic clade within the Haemosporida, and branch prior to the clade containing bird, lizard and other mammalian Plasmodium. Thus it is likely that host switching of Plasmodium from birds to mammals occurred multiple times, with a switch to ungulates independently from other mammalian Plasmodium. PMID:26996979

  11. Parasitic Diseases With Cutaneous Manifestations.

    Science.gov (United States)

    Ash, Mark M; Phillips, Charles M

    2016-01-01

    Parasitic diseases result in a significant global health burden. While often thought to be isolated to returning travelers, parasitic diseases can also be acquired locally in the United States. Therefore, clinicians must be aware of the cutaneous manifestations of parasitic diseases to allow for prompt recognition, effective management, and subsequent mitigation of complications. This commentary also reviews pharmacologic treatment options for several common diseases.

  12. microRNAs in parasites and parasite infection.

    Science.gov (United States)

    Zheng, Yadong; Cai, Xuepeng; Bradley, Janette E

    2013-03-01

    miRNAs, a subclass of small regulatory RNAs, are present from ancient unicellular protozoans to parasitic helminths and parasitic arthropods. The miRNA-silencing mechanism appears, however, to be absent in a number of protozoan parasites. Protozoan miRNAs and components of their silencing machinery possess features different from other eukaryotes, providing some clues on the evolution of the RNA-induced silencing machinery. miRNA functions possibly associate with neoblast biology, development, physiology, infection and immunity of parasites. Parasite infection can alter host miRNA expression that can favor both parasite clearance and infection. miRNA pathways are, thus, a potential target for the therapeutic control of parasitic diseases.

  13. Role of parasites in cancer.

    Science.gov (United States)

    Mandong, B M; Ngbea, J A; Raymond, Vhriterhire

    2013-01-01

    In areas of parasitic endemicity, the occurrence of cancer that is not frequent may be linked with parasitic infection. Epidemiological correlates between some parasitic infections and cancer is strong, suggesting a strong aetiological association. The common parasites associated with human cancers are schistosomiasis, malaria, liver flukes (Clonorchis sinenses, Opistorchis viverrini). To review the pathology, literature and methods of diagnosis. Literature review from peer reviewed Journals cited in PubMed and local journals. Parasites may serve as promoters of cancer in endemic areas of infection.

  14. Metazoan Parasites of Antarctic Fishes.

    Science.gov (United States)

    Oğuz, Mehmet Cemal; Tepe, Yahya; Belk, Mark C; Heckmann, Richard A; Aslan, Burçak; Gürgen, Meryem; Bray, Rodney A; Akgül, Ülker

    2015-06-01

    To date, there have been nearly 100 papers published on metazoan parasites of Antarctic fishes, but there has not yet been any compilation of a species list of fish parasites for this large geographic area. Herein, we provide a list of all documented occurrences of monogenean, cestode, digenean, acanthocephalan, nematode, and hirudinean parasites of Antarctic fishes. The list includes nearly 250 parasite species found in 142 species of host fishes. It is likely that there are more species of fish parasites, which are yet to be documented from Antarctic waters.

  15. 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.

  16. INTESTINAL PARASITES IN IRAN

    Directory of Open Access Journals (Sweden)

    K. Mohammad

    1995-12-01

    Full Text Available The purpose of this study was to investigate the status and epidemiology of Intestinal Parasites in Iran. The information was driven from an extensive Health Survey which was done by the Ministry of Health and Medical Education, deputy of Research Affairs in 1990-92. Sampling fraction was 1 per 1000 of individuals aged between 2 and 69, the sampling method was cluster sampling and each cluster consisted of 7 families. Formal-ether was the method of finding parasites which included: Oxior, Ascariasis, Giardiasis, Entamoeba-histolytica, Tinea, Strongyloidiasis, Ancylostoma, and Trichocephaliasis. The highest prevalence rate belonged to Giardiasis with 14.4% and the lowest one belonged to Tinea and Ancylostoma with 0.2%. The prevalence rate in rural area was significantly lower than urban area (p<0.0001.

  17. Nutrition and parasite interaction.

    Science.gov (United States)

    Coop, R L; Holmes, P H

    1996-01-01

    This overview focuses on the interaction between nutritional status and gastrointestinal nematode infection in ruminants and considers: (i) the influence of the parasite on host metabolism; and (ii) the effect of host nutrition on the establishment and survival of parasite populations, the development of the host-immune response and the pathophysiology of infection. Gastrointestinal nematodes reduce voluntary feed intake and efficiency of feed utilisation, a key feature being an increased endogenous loss of protein into the gastrointestinal tract. Overall there is movement of protein from productive processes into repair of the gastrointestinal tract, synthesis of plasma proteins and mucoprotein production. Although reduction in feed intake is a major factor contributing to the reduced performance of parasitised ruminants, the underlying mechanisms of the anorexia are poorly understood. Supplementation of the diet with additional protein does not appear to affect initial establishment of nematode infections but the pathophysiological consequences are generally more severe on lower planes of protein nutrition. The main effect of protein supplementation is to increase the rate of acquisition of immunity and increase resistance to reinfection and this has been associated with an enhanced cellular immune response in the gastrointestinal mucosa. The unresponsiveness of the young lamb can be improved by dietary protein supplementation. Recent trials have shown that growing sheep offered a free choice between a low and a high protein ration are able to modify their diet selection in order to alleviate the increase in protein requirements which result from gastrointestinal nematode infection. Studies on the influence of nutrition on the expression of genotype have shown that the benefits of a superior genotype are not lost on a low protein diet whereas a high protein diet can partially emeliorate the disadvantages of an inferior genotype. In addition to dietary protein

  18. [Emerging parasitic diseases].

    Science.gov (United States)

    Weibel Galluzzo, C; Wagner, N; Michel, Y; Jackson, Y; Chappuis, F

    2014-05-07

    Travels, migration and circulation of goods facilitate the emergence of new infectious diseases often unrecognized outside endemic areas. Most of emerging infections are of viral origin. Muscular Sarcocystis infection, an acute illness acquired during short trips to Malaysia, and Chagas disease, a chronic illness with long incubation period found among Latin American migrants, are two very different examples of emerging parasitic diseases. The former requires a preventive approach for travelers going to Malaysia and must be brought forth when they return with fever, myalgia and eosinophilia, while the latter requires a proactive attitude to screen Latin American migrant populations that may face difficulties in accessing care.

  19. Unexpected hosts: imaging parasitic diseases.

    Science.gov (United States)

    Rodríguez Carnero, Pablo; Hernández Mateo, Paula; Martín-Garre, Susana; García Pérez, Ángela; Del Campo, Lourdes

    2017-02-01

    Radiologists seldom encounter parasitic diseases in their daily practice in most of Europe, although the incidence of these diseases is increasing due to migration and tourism from/to endemic areas. Moreover, some parasitic diseases are still endemic in certain European regions, and immunocompromised individuals also pose a higher risk of developing these conditions. This article reviews and summarises the imaging findings of some of the most important and frequent human parasitic diseases, including information about the parasite's life cycle, pathophysiology, clinical findings, diagnosis, and treatment. We include malaria, amoebiasis, toxoplasmosis, trypanosomiasis, leishmaniasis, echinococcosis, cysticercosis, clonorchiasis, schistosomiasis, fascioliasis, ascariasis, anisakiasis, dracunculiasis, and strongyloidiasis. The aim of this review is to help radiologists when dealing with these diseases or in cases where they are suspected. Teaching Points • Incidence of parasitic diseases is increasing due to migratory movements and travelling. • Some parasitic diseases are still endemic in certain regions in Europe. • Parasitic diseases can have complex life cycles often involving different hosts. • Prompt diagnosis and treatment is essential for patient management in parasitic diseases. • Radiologists should be able to recognise and suspect the most relevant parasitic diseases.

  20. 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.

  1. Parasitism and calfhood diseases.

    Science.gov (United States)

    Herlich, H; Douvres, F W

    1977-02-01

    That animals can and do acquire an effective immunity against helminth parasites has been demonstrated extensively experimentally, and the fact that domestic animals such as cattle, sheep, and horses become adults while maintaining good health in spite of constant exposure to reinfection long has suggested that immunity must be important to such survival. Although our attempts to date to vaccinate calves against helminth parasites have either failed or been unsatisfactory because of the pathosis induced by the experimental vaccines, the results are not surprising or discouraging. In contrast to the long history of immunization research on bacterial and viral diseases, only within a relatively short time have serious efforts been directed at exploiting hostal immunity for prevention and control of helminthic diseases. Unlike the comparatively simple structures of viruses and bacteria, helminths are complex multicellular animals with vast arrays of antigens and complicated physiological and immunological interactions with their hosts. Much more fundamental information on helminth-bovine interactions, on helminth antigens, and on cattle antibody systems must be developed before progress on control of cattle helminths by vaccination can be meaningful.

  2. 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

  3. Emerging food-borne parasites.

    Science.gov (United States)

    Dorny, P; Praet, N; Deckers, N; Gabriel, S

    2009-08-07

    Parasitic food-borne diseases are generally underrecognised, however they are becoming more common. Globalization of the food supply, increased international travel, increase of the population of highly susceptible persons, change in culinary habits, but also improved diagnostic tools and communication are some factors associated with the increased diagnosis of food-borne parasitic diseases worldwide. This paper reviews the most important emerging food-borne parasites, with emphasis on transmission routes. In a first part, waterborne parasites transmitted by contaminated food such as Cyclospora cayetanensis, Cryptosporidium and Giardia are discussed. Also human fasciolosis, of which the importance has only been recognised in the last decades, with total numbers of reported cases increasing from less than 3000 to 17 million, is looked at. Furthermore, fasciolopsiosis, an intestinal trematode of humans and pigs belongs to the waterborne parasites as well. A few parasites that may be transmitted through faecal contamination of foods and that have received renewed attention, such as Toxoplasma gondii, or that are (re-)emerging, such as Trypanosoma cruzi and Echinococcus spp., are briefly reviewed. In a second part, meat-borne parasite infections are reviewed. Humans get infected by eating raw or undercooked meat infected with cyst stages of these parasites. Meat inspection is the principal method applied in the control of Taenia spp. and Trichinella spp. However, it is often not very sensitive, frequently not practised, and not done for T. gondii and Sarcocystis spp. Meat of reptiles, amphibians and fish can be infected with a variety of parasites, including trematodes (Opisthorchis spp., Clonorchis sinensis, minute intestinal flukes), cestodes (Diphyllobothrium spp., Spirometra), nematodes (Gnathostoma, spp., anisakine parasites), and pentastomids that can cause zoonotic infections in humans when consumed raw or not properly cooked. Another important zoonotic food

  4. Repetitive elements in parasitic protozoa

    Directory of Open Access Journals (Sweden)

    Clayton Christine

    2010-05-01

    Full Text Available Abstract A recent paper published in BMC Genomics suggests that retrotransposition may be active in the human gut parasite Entamoeba histolytica. This adds to our knowledge of the various types of repetitive elements in parasitic protists and the potential influence of such elements on pathogenicity. See research article http://www.biomedcentral.com/1471-2164/11/321

  5. Two types of parasitic assimilation

    Directory of Open Access Journals (Sweden)

    Peter Jurgec

    2013-02-01

    Full Text Available This paper shows that consonant harmony and parasitic vowel harmony are more similar than previously assumed. I provide a unified and restrictive analysis of parasitic assimilation using feature spreading constraints. In particular, I attribute the differences between the attested and unattested patterns to two types of markedness constraints—alignment and agreement.

  6. Parasitic zoonotic diseases in Turkey

    Directory of Open Access Journals (Sweden)

    Nazmiye Altintas

    2008-12-01

    Full Text Available Zoonoses and zoonotic diseases are becoming more common and they are now receiving increased attention across the world. Zoonotic parasites are found in a wide variety of protozoa, cestodes, nematodes, trematodes and arthropods worldwide and many zoonotic parasites have assumed an important role. The importance of some parasitic zoonoses has increased in recent years due to the fact that they can be agents of opportunistic infections. Although a number of zoonotic parasites are often found and do cause serious illnesses in Turkey, some are more common and these diseases are more important as they cause serious public health problems, such as leishmaniasis, toxoplasmosis, cryptosporidiosis, echinococcosis, trichinellosis and toxocariasis. Information on these zoonotic diseases is provided here as these are the most important zoonotic parasitic diseases in Turkey.

  7. Regulatory T Cells and Parasites

    Directory of Open Access Journals (Sweden)

    TP. Velavan

    2011-01-01

    Full Text Available Human host encounters a wide array of parasites; however, the crucial aspect is the failure of the host immune system to clear these parasites despite antigen recognition. In the recent past, a new immunological concept has emerged, which provides a framework to better understand several aspects of host susceptibility to parasitic infection. It is widely believed that parasites are able to modulate the magnitude of effector responses by inducing regulatory T cell (Tregs population and several studies have investigated whether this cell population plays a role in balancing protective immunity and pathogenesis during parasite infection. This review discusses the several mechanism of Treg-mediated immunosuppression in the human host and focuses on the functional role of Tregs and regulatory gene polymorphisms in infectious diseases.

  8. [Parasitic factor and cancer].

    Science.gov (United States)

    Khamidullin, R I; Aglullin, I R; Rakhmanin, Iu A; Pogorel'tsev, V I; Khamidullin, A R; Galkina, I V; Khamidullin, I R; Sultanaeva, E G

    2011-01-01

    There is opinion in the literature as to that liver trematode infections, such as opisthorchiasis, clonorchiasis, fascioliasis, and metorchiasis, can induce cancer of the liver pancreas, intestine - this all is clinically observed. The authors were the first in world practice to show the development of a hepatic blastomatous process in animals (albino rats, cats) with opisthorchiasis in 13%; cancer developed in 28 and 56% with the use of a hepatotropic carcinogen and combined (opisthorchiasis + a carcinogen) exposure, respectively. Throughout his life, a human being can easily catch these trematodes that have carcinogenic activity and these diseases concurrent with household and food carcinogens can give rise to tumors in the liver pancreas and intestine. Timely diagnosis and specific anthelmintic therapy are necessary to prevent parasitic cancer.

  9. Integrated parasite management

    DEFF Research Database (Denmark)

    Clausen, Jesper Hedegaard; Madsen, Henry; Van, Phan Thi

    2015-01-01

    Fishborne zoonotic trematodes (FZT) are an emerging problem and there is now a consensus that, in addition to wild-caught fish, fish produced in aquaculture present a major food safety risk, especially in Southeast Asia where aquaculture is important economically. Current control programs target ...... that target critical control points in the aquaculture production cycle identified from a thorough understanding of FZT and host biology and epidemiology. We present recommendations for an integrated parasite management (IPM) program for aquaculture farms.......Fishborne zoonotic trematodes (FZT) are an emerging problem and there is now a consensus that, in addition to wild-caught fish, fish produced in aquaculture present a major food safety risk, especially in Southeast Asia where aquaculture is important economically. Current control programs target...

  10. How have fisheries affected parasite communities?

    Science.gov (United States)

    Wood, Chelsea L.; Lafferty, Kevin D.

    2015-01-01

    To understand how fisheries affect parasites, we conducted a meta-analysis of studies that contrasted parasite assemblages in fished and unfished areas. Parasite diversity was lower in hosts from fished areas. Larger hosts had a greater abundance of parasites, suggesting that fishing might reduce the abundance of parasites by selectively removing the largest, most heavily parasitized individuals. After controlling for size, the effect of fishing on parasite abundance varied according to whether the host was fished and the parasite's life cycle. Parasites of unfished hosts were more likely to increase in abundance in response to fishing than were parasites of fished hosts, possibly due to compensatory increases in the abundance of unfished hosts. While complex life cycle parasites tended to decline in abundance in response to fishing, directly transmitted parasites tended to increase. Among complex life cycle parasites, those with fished hosts tended to decline in abundance in response to fishing, while those with unfished hosts tended to increase. However, among directly transmitted parasites, responses did not differ between parasites with and without fished hosts. This work suggests that parasite assemblages are likely to change substantially in composition in increasingly fished ecosystems, and that parasite life history and fishing status of the host are important in predicting the response of individual parasite species or groups to fishing.

  11. Host responses to interspecific brood parasitism: a by-product of adaptations to conspecific parasitism?

    Science.gov (United States)

    2014-01-01

    Background Why have birds evolved the ability to reject eggs? Typically, foreign egg discrimination is interpreted as evidence that interspecific brood parasitism (IP) has selected for the host’s ability to recognize and eliminate foreign eggs. Fewer studies explore the alternative hypothesis that rejection of interspecific eggs is a by-product of host defenses, evolved against conspecific parasitism (CP). We performed a large scale study with replication across taxa (two congeneric Turdus thrushes), space (populations), time (breeding seasons), and treatments (three types of experimental eggs), using a consistent design of egg rejection experiments (n = 1057 nests; including controls), in areas with potential IP either present (Europe; native populations) or absent (New Zealand; introduced populations). These comparisons benefited from the known length of allopatry (one and a half centuries), with no gene flow between native and introduced populations, which is rarely available in host-parasite systems. Results Hosts rejected CP at unusually high rates for passerines (up to 60%). CP rejection rates were higher in populations with higher conspecific breeding densities and no risks of IP, supporting the CP hypothesis. IP rejection rates did not covary geographically with IP risk, contradicting the IP hypothesis. High egg rejection rates were maintained in the relatively long-term isolation from IP despite non-trivial rejection costs and errors. Conclusions These egg rejection patterns, combined with recent findings that these thrushes are currently unsuitable hosts of the obligate parasitic common cuckoo (Cuculus canorus), are in agreement with the hypothesis that the rejection of IP is a by-product of fine-tuned egg discrimination evolved due to CP. Our study highlights the importance of considering both IP and CP simultaneously as potential drivers in the evolution of egg discrimination, and illustrates how populations introduced to novel ecological contexts

  12. Parasites in algae mass culture

    Directory of Open Access Journals (Sweden)

    Todd William Lane

    2014-06-01

    Full Text Available Parasites are now known to be ubiquitous across biological systems and can play an important role in modulating algal populations. However, there is a lack of extensive information on their role in artificial ecosystems such as algal production ponds and photobioreactors. Parasites have been implicated in the demise of algal blooms. Because individual mass culture systems often tend to be unialgal and a select few algal species are in wide scale application, there is an increased potential for parasites to have a devastating effect on commercial scale monoculture. As commercial algal production continues to expand with a widening variety of applications, including biofuel, food and pharmaceuticals, the parasites associated with algae will become of greater interest and potential economic impact. A number of important algal parasites have been identified in algal mass culture systems in the last few years and this number is sure to grow as the number of commercial algae ventures increases. Here, we review the research that has identified and characterized parasites infecting mass cultivated algae, the techniques being proposed and or developed to control them, and the potential impact of parasites on the future of the algal biomass industry.

  13. Miniaturized flow cytometer with 3D hydrodynamic particle focusing and integrated optical elements applying silicon photodiodes

    NARCIS (Netherlands)

    Rosenauer, M.; Buchegger, W.; Finoulst, I.; Verhaert, P.D.E.M.; Vellekoop, M.

    2010-01-01

    In this study, the design, realization and measurement results of a novel optofluidic system capable of performing absorbance-based flow cytometric analysis is presented. This miniaturized laboratory platform, fabricated using SU-8 on a silicon substrate, comprises integrated polymer-based waveguide

  14. 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...

  15. Parasites on parasites: coupled fluctuations in stacked contact processes

    CERN Document Server

    Court, Steven J; Allen, Rosalind J

    2012-01-01

    We present a model for host-parasite dynamics which incorporates both vertical and horizontal transmission as well as spatial structure. Our model consists of stacked contact processes (CP), where the dynamics of the host is a simple CP on a lattice while the dynamics of the parasite is a secondary CP which sits on top of the host-occupied sites. In the simplest case, where infection does not incur any cost, we uncover a novel effect: a nonmonotonic dependence of parasite prevalence on host turnover. Inspired by natural examples of hyperparasitism, we extend our model to multiple levels of parasites and identify a transition between the maintenance of a finite and infinite number of levels, which we conjecture is connected to a roughening transition in models of surface-growth.

  16. Development of a five-plex flow cytometric immunoassay for the simultaneous detection of six coccidiostats in feed and eggs

    NARCIS (Netherlands)

    Bienenmann-Ploum, M.E.; Huet, A.C.; Campbell, K.; Fodey, T.L.; Vincent, U.; Delahaut, P.; Elliot, C.T.; Nielen, M.W.F.

    2012-01-01

    Coccidiostats are the only veterinary drugs still permitted to be used as feed additives to treat poultry for coccidiosis. To protect consumers, maximum levels for their presence in food and feed have been set by the European Union (EU). To monitor these coccidiostats, a rapid and inexpensive

  17. Two new monoclonal antibodies for biochemical and flow cytometric analyses of human interferon regulatory factor-3 activation, turnover, and depletion.

    Science.gov (United States)

    Rustagi, Arjun; Doehle, Brian P; McElrath, M Juliana; Gale, Michael

    2013-02-01

    Interferon regulatory factor-3 (IRF-3) is a master transcription factor that drives the host intracellular innate immune response to virus infection. The importance of IRF-3 in innate immune responses is highlighted by the fact that pathogenic viruses have developed strategies for antagonism of IRF-3. Several tools exist for evaluation of viral regulation of IRF-3 activation and function, but high-quality monoclonal antibodies that mark the differential activation states of human IRF-3 are lacking. To study IRF-3 activation, turnover, and depletion in a high-throughput manner in the context of virus infection, we have developed two new monoclonal antibodies to human IRF-3. These antibodies detect IRF-3 in virus-infected cells in a wide variety of assays and provide a new tool to study virus-host interactions and innate immune signaling.

  18. Flow cytometric immunophenotyping of feline bone marrow cells and haematopoietic progenitor cells using anti-human antibodies.

    Science.gov (United States)

    Araghi, Atefeh; Nassiri, Seyed Mahdi; Atyabi, Nahid; Rahbarghazi, Reza; Mohammadi, Elham

    2014-04-01

    There is a paucity of species-specific antibodies available for feline haematopoietic conditions. The purpose of this study was to broaden the panel of antibodies available for use in the immunophenotypic characterisation of feline haematopoietic cells by testing clones of anti-human monoclonal antibodies (mAbs) on normal, neoplastic and cultured feline haematopoietic progenitors to determine cross-reactivity to feline counterparts. In this study, 24 clones of anti-human mAbs were tested on normal or neoplastic feline bone marrow and peripheral blood cells. Six of these mAbs, including anti-cluster of differentiation (CD)61, anti-CD18, anti-CD14, anti-CD235a, anti-CD41 and anti-CD29, cross-reacted with normal feline bone marrow cells, whereas anti-CD33 and anti-CD117 cross-reacted with the blast cells in the bone marrow of two cats with myelodysplastic syndrome, and anti-CD71, anti-235a, anti-41 and anti-42 cross-reacted with immature erythroid cells in a cat with erythroleukaemia. In a feline immunodeficiency virus-positive cat, bone marrow cells were labelled with anti-CD33, anti-14 and anti-45. Anti-CD18, anti-CD14, anti-CD41 and anti-CD61 also reacted with the peripheral blood cells of the healthy cats. The feline haematopoietic progenitors formed colonies in the methylcellulose-based semisolid medium with significant enrichment of colony-forming unit-granulocyte, monocyte and burst-forming unit-erythroid. A panel of six anti-feline mAbs (anti-CD21-like, anti-T lymphocytes, anti-CD172a, anti-granulocyte, anti-CD45-like and anti-CD18) and eight anti-human antibodies (anti-CD71, anti-CD33, anti-CD235a, anti-CD41, anti-CD61, anti-CD117, anti-CD38 and anti-CD34) were used for the immunophenotypic characterisation of the feline bone marrow progenitors. CD45, CD33, CD235a and CD18 were expressed by the feline haematopoietic progenitor cells, with the highest expression level for CD45.

  19. Selection of fluorescent probes for flow cytometric viability assessment of Listeria monocytogenes exposed to membrane-active and oxidizing disinfectants

    NARCIS (Netherlands)

    Luppens, S.B.I.; Barbaras, B.; Breeuwer, P.; Rombouts, F.M.; Abee, T.

    2003-01-01

    The aim of this study was to select fluorescence methods for use as alternatives to plate counting to assess the viability of Listeria monocytogenes cells exposed to benzalkonium chloride (BAC) and hydrogen peroxide, two disinfectants with different mechanisms of action. A further aim of this study

  20. Successful laparoscopic insemination with a very low number of flow cytometrically sorted boar sperm in field conditions.

    Science.gov (United States)

    del Olmo, David; Parrilla, Inmaculada; Sanchez-Osorio, Jonatan; Gomis, Jesus; Angel, Miguel A; Tarantini, Tatiana; Gil, Maria A; Cuello, Cristina; Vazquez, Jose L; Roca, Jordi; Vaquez, Juan M; Martinez, Emilio A

    2014-01-15

    The aim of this study was to develop a useful procedure for laparoscopic insemination (LI) with sex-sorted boar spermatozoa that yields adequate fertility results in farm conditions. In experiment 1, we evaluated the effects of single (oviducts) and double (oviducts and tips of the uterine horns) LI with X-sorted sperm on the reproductive performance of sows. Sows (N = 109) were inseminated once as follows: (1) single LI with 0.5 × 10(6) unsorted sperm per oviduct; (2) single LI with 0.5 × 10(6) sex-sorted sperm per oviduct; or (3) double LI with 0.5 × 10(6) sex-sorted sperm per oviduct and 0.5 × 10(6) sex-sorted sperm per uterine horn. The farrowing rates were lower (P sperm (43.2% and 61.9% for the single and double insemination groups, respectively) than in sows from the unsorted group (91.3%). Within the sex-sorted groups, the farrowing rate tended (P = 0.09) to be greater in sows inseminated using double LI. There were no differences in the litter size among groups. In experiment 2, we evaluated the effect of the number of sex-sorted sperm on the reproductive performance of sows when using double LI. Sows (N = 109) were inseminated with sex-sorted sperm once using double LI with: (1) 0.5 × 10(6) sperm per oviduct and 1 × 10(6) sperm per uterine horn; or (2) 1 × 10(6) sperm per oviduct and 2 × 10(6) sperm per uterine horn. Similarly high pregnancy (90%) and farrowing (80%) rates were achieved in both groups. The sows inseminated with the highest number of sperm tended (P = 0.09) to have more piglets (10.8 ± 0.7 vs. 9.2 ± 0.6). A high female proportion (number of female births divided by the total of all births ≥0.92) was obtained in both experiments using X-sorted sperm. Our results indicate that the double LI procedure, using between 3 and 6 × 10(6) sex-sorted sperm per sow produces adequate fertility at the farm level, making sperm-sexing technology potentially applicable in elite breeding units.

  1. Handling of boar spermatozoa during and after flow cytometric sex-sorting process to improve their in vitro fertilizing ability.

    Science.gov (United States)

    del Olmo, D; Parrilla, I; Gil, M A; Maside, C; Tarantini, T; Angel, M A; Roca, J; Martinez, E A; Vazquez, J M

    2013-09-01

    The objective of this study was to develop an adequate sperm handling protocol in order to obtain a sex-sorted sperm population with an optimal fertilizing ability. For this purpose, different aspects of the sorting procedure were examined. The effects of the high dilution rates (experiment 1), type of collection medium used (experiment 2), and sheath fluid composition (experiment 3) on sorted boar sperm quality and function were evaluated. Sperm quality was assessed by motility and viability tests, whereas sperm function was evaluated by an in vitro fertilization assay which determined the penetration and polyspermy rates as well as the mean number of sperm penetrating each oocyte. In experiment 1, the results obtained indicated that the high dilution rates did not cause a decrease either in the sperm quality parameters evaluated or the in vitro fertilization ability of spermatozoa. In experiment 2, although sperm quality was not affected, fertilizing ability was compromised after sorting, regardless of the collection medium that was used. In the experiment 3, all groups displayed adequate sperm quality values, but higher in vitro fertility parameters were obtained for spermatozoa sorted in presence of EDTA in the sheath fluid and egg yolk (EY) in the collection media when compared with those sorted in absence of these protective agents. No differences in penetration rates between unsorted highly diluted (control) and sorted sperm in the presence of EDTA and EY were observed. In conclusion, fertilizing ability was compromised in sex-sorted sperm. The addition of EDTA to sheath fluid and EY to collection medium improved boar sperm fertilizing ability, and both agents should be included as essential media components in future studies.

  2. 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

    -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.......Producing equal amounts of male and female offspring has long been considered an evolutionarily stable strategy. Nevertheless, exceptions to this general rule (i.e. male and female biases) are documented in many taxa, making sex allocation an important domain in current evolutionary biology...... research. Pinpointing the underlying mechanism of sex ratio bias is challenging owing to the multitude of potential sex ratio-biasing factors. In the dwarf spider, Oedothorax gibbosus, infection with the bacterial endosymbiont Wolbachia results in a female bias. However, pedigree analysis reveals...

  3. 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.

  4. Central neurocytoma : morphological, flow cytometric, polymerase chain reaction, fluorescence in situ hybridization, and karyotypic analyses - Case report

    NARCIS (Netherlands)

    Jay, RM; Edwards, KA; Hoving, E; Rutka, J; Becker, L; Zielenska, M; Teshima, Teruki

    1999-01-01

    The results of cytogenetic and molecular genetic analysis of a central neurocytoma are presented. Central neurocytomas are intriguing neoplasms that exhibit primarily neuronal, but also glial characteristics, which indicate an origin from a pluripotential neuroglial precursor. The authors describe a

  5. 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 valid

  6. Development of a five-plex flow cytometric immunoassay for the simultaneous detection of six coccidiostats in feed and eggs

    NARCIS (Netherlands)

    Bienenmann-Ploum, M.E.; Huet, A.C.; Campbell, K.; Fodey, T.L.; Vincent, U.; Delahaut, P.; Elliot, C.T.; Nielen, M.W.F.

    2012-01-01

    Coccidiostats are the only veterinary drugs still permitted to be used as feed additives to treat poultry for coccidiosis. To protect consumers, maximum levels for their presence in food and feed have been set by the European Union (EU). To monitor these coccidiostats, a rapid and inexpensive screen

  7. Flow cytometric assessment of antigen-specific proliferation in peripheral chicken T cells by CFSE dilution

    DEFF Research Database (Denmark)

    Dalgaard, Tina; Norup, Liselotte Rothmann; Rubbenstroth, Dennis

    2010-01-01

    fetal calf serum with serum-free medium. It was rendered probable that antigen-specific cellular immunity can be assessed by this method as NDV-vaccinated chickens showed a significantly higher proliferative capacity than age-matched naïve controls. Furthermore it was shown that the recall stimulation...

  8. 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

    1997-01-01

    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 abun

  9. Synanthropic birds and parasites.

    Science.gov (United States)

    Dipineto, Ludovico; Borrelli, Luca; Pepe, Paola; Fioretti, Alessandro; Caputo, Vincenzo; Cringoli, Giuseppe; Rinaldi, Laura

    2013-12-01

    This paper describes the parasitologic findings for 60 synanthropic bird carcasses recovered in the Campania region of southern Italy. Birds consisted of 20 yellow-legged gulls (Larus michahellis), 15 rock pigeons (Columba livia), 15 common kestrels (Falco tinnunculus), and 10 carrion crows (Corvus corone). Each carcass was examined to detect the presence of ectoparasites and then necropsied to detect helminths. Ectoparasites occurred in 100% of the birds examined. In particular, chewing lice were recovered with a prevalence of 100%, whereas Pseudolynchia canariensis (Hippoboscidae) were found only in pigeons with a prevalence of 80%. Regarding endoparasites, a total of seven helminth species were identified: three nematodes (Ascaridia columbae, Capillaria columbae, Physaloptera alata), one cestoda (Raillietina tetragona), one trematoda (Cardiocephalus longicollis), and two acanthocephalans (Centrorhynchus globocaudatus and Centrorhynchus buteonis). The findings of the present study add data to the parasitologic scenario of synanthropic birds. This is important because parasitic infection can lead to serious health problems when combined with other factors and may affect flying performance and predatory effectiveness.

  10. Parasites in pet reptiles

    Science.gov (United States)

    2011-01-01

    Exotic reptiles originating from the wild can be carriers of many different pathogens and some of them can infect humans. Reptiles imported into Slovenia from 2000 to 2005, specimens of native species taken from the wild and captive bred species were investigated. A total of 949 reptiles (55 snakes, 331 lizards and 563 turtles), belonging to 68 different species, were examined for the presence of endoparasites and ectoparasites. Twelve different groups (Nematoda (5), Trematoda (1), Acanthocephala (1), Pentastomida (1) and Protozoa (4)) of endoparasites were determined in 26 (47.3%) of 55 examined snakes. In snakes two different species of ectoparasites were also found. Among the tested lizards eighteen different groups (Nematoda (8), Cestoda (1), Trematoda (1), Acanthocephala (1), Pentastomida (1) and Protozoa (6)) of endoparasites in 252 (76.1%) of 331 examined animals were found. One Trombiculid ectoparasite was determined. In 563 of examined turtles eight different groups (Nematoda (4), Cestoda (1), Trematoda (1) and Protozoa (2)) of endoparasites were determined in 498 (88.5%) animals. In examined turtles three different species of ectoparasites were seen. The established prevalence of various parasites in reptiles used as pet animals indicates the need for examination on specific pathogens prior to introduction to owners. PMID:21624124

  11. MENGENAL PARASIT FILARIA

    Directory of Open Access Journals (Sweden)

    Tri Ramadhani

    2012-11-01

    Full Text Available Filariasis atau kaki gajah adalah penyakit menular yang disebabkan karena infeksi cacing filaria yang hidup disaluran dan kelenjar getah bening (limfe serta menyebabkan gejala akut, kronis. Filariasis mulai dikenal di Indonesia tahun 1889 sejak Haga dan Van Eecke menemukan kasus pembesaran scrotum di Jakarta. Penyakit tersebut dapat menular kepada orang lain dengan perantara gigitan nyamuk. Seluruh wilayah Indonesia berpotensi untuk terjangkitnya penyakit tersebut, hal ini mengingat cacing sebagai penyebabnya dan nyamuk penularnya tersebar luas. Keadaan ini didukung oleh kerusakan lingkungan, seperti banjir, penebangan hutan dan lainnya yang memperluas tempat berkembangbiaknya nyamuk. Meskipun filariasis tidak mematikan secara langsung, dengan adanya demam dan bisul-bisul (abses yang hilang timbul, dan gejala menahun berupa pembesaran/elefantiasis yang merupakan cacat menetap akan sangat mengganggu. Secara ekonomis keadaan tersebut sangat merugikan, karena mengurangi produktivitas masyarakat, serta diperlukan biaya pengobatan dan perawatan yang tidak mudah dan tidak murah.Di Indonesia filariasis limfatik di sebabkan oleh tiga spesies cacing filaria yaitu Brugia malayi,B.timori dan Wuchereria bancrofti, yang terbagi lagi menjadi 6 tipe secara epidemiologi.Tiap parasit mempunyai siklus hidup yang kompleks dan infeksi pada manusia tidak akan berhasil kecuali jika terjadi pemaparan larva infektif untuk waktu yang lama. Setelah terjadi pemaparan, dibutuhkan waktu bertahun-tahun sebelum timbulnya perubahan patologis yang nyata pada manusia. Periodisitas dalam sirkulasi setiap mikrofilaria akan berbeda, tergantung dari spesiesnya.

  12. Tropical parasitic diseases and women.

    Science.gov (United States)

    Okwa, O O

    2007-12-01

    Tropical parasitic diseases constitute the greatest threat to the health and socio-economic status of women as a gender and social group. There are some gender specific ways in which parasitic diseases affect women in contrast to men due to differences in exposure, occupational risk, sociocultural behavior, gender roles and practices. These parasitic diseases confer some social stigma, which affects the health seeking behavior of women. Women are therefore important in the control of these parasitic diseases and they are key agents of change, if they are included in community control programs. Women need more attention in endemic areas as a group that had been neglected. This deprived and excluded group have got vital role to play, as discussed in this review.

  13. Pervasiveness of parasites in pollinators.

    Science.gov (United States)

    Evison, Sophie E F; Roberts, Katherine E; Laurenson, Lynn; Pietravalle, Stéphane; Hui, Jeffrey; Biesmeijer, Jacobus C; Smith, Judith E; Budge, Giles; Hughes, William O H

    2012-01-01

    Many pollinator populations are declining, with large economic and ecological implications. Parasites are known to be an important factor in the some of the population declines of honey bees and bumblebees, but little is known about the parasites afflicting most other pollinators, or the extent of interspecific transmission or vectoring of parasites. Here we carry out a preliminary screening of pollinators (honey bees, five species of bumblebee, three species of wasp, four species of hoverfly and three genera of other bees) in the UK for parasites. We used molecular methods to screen for six honey bee viruses, Ascosphaera fungi, Microsporidia, and Wolbachia intracellular bacteria. We aimed simply to detect the presence of the parasites, encompassing vectoring as well as actual infections. Many pollinators of all types were positive for Ascosphaera fungi, while Microsporidia were rarer, being most frequently found in bumblebees. We also detected that most pollinators were positive for Wolbachia, most probably indicating infection with this intracellular symbiont, and raising the possibility that it may be an important factor in influencing host sex ratios or fitness in a diversity of pollinators. Importantly, we found that about a third of bumblebees (Bombus pascuorum and Bombus terrestris) and a third of wasps (Vespula vulgaris), as well as all honey bees, were positive for deformed wing virus, but that this virus was not present in other pollinators. Deformed wing virus therefore does not appear to be a general parasite of pollinators, but does interact significantly with at least three species of bumblebee and wasp. Further work is needed to establish the identity of some of the parasites, their spatiotemporal variation, and whether they are infecting the various pollinator species or being vectored. However, these results provide a first insight into the diversity, and potential exchange, of parasites in pollinator communities.

  14. Molecular diagnostics and parasitic disease.

    Science.gov (United States)

    Vasoo, Shawn; Pritt, Bobbi S

    2013-09-01

    Molecular parasitology represents an emerging field in microbiology diagnostics. Although most assays use nonstandardized, laboratory-developed methods, a few commercial systems have recently become available and are slowly being introduced into larger laboratories. In addition, a few methodologies show promise for use in field settings in which parasitic infections are endemic. This article reviews the available techniques and their applications to major parasitic diseases such as malaria, leishmaniasis, and trichomoniasis.

  15. Parasites and altruism: converging roads.

    Science.gov (United States)

    Zuk, Marlene; Borrello, Mark E

    2013-01-01

    W.D. Hamilton was most known for his work on two topics: social evolution and parasites. Although at first glance these seem to be disparate interests, they share many attributes and have logical connections within evolutionary biology. Nevertheless, Hamilton's contributions in these areas met with very different receptions, with his place in the field of social evolution assured, but his work on the role of parasites perceived as more specialized. We take an historical approach to examine the reasons for this difference.

  16. Pervasiveness of parasites in pollinators.

    Directory of Open Access Journals (Sweden)

    Sophie E F Evison

    Full Text Available Many pollinator populations are declining, with large economic and ecological implications. Parasites are known to be an important factor in the some of the population declines of honey bees and bumblebees, but little is known about the parasites afflicting most other pollinators, or the extent of interspecific transmission or vectoring of parasites. Here we carry out a preliminary screening of pollinators (honey bees, five species of bumblebee, three species of wasp, four species of hoverfly and three genera of other bees in the UK for parasites. We used molecular methods to screen for six honey bee viruses, Ascosphaera fungi, Microsporidia, and Wolbachia intracellular bacteria. We aimed simply to detect the presence of the parasites, encompassing vectoring as well as actual infections. Many pollinators of all types were positive for Ascosphaera fungi, while Microsporidia were rarer, being most frequently found in bumblebees. We also detected that most pollinators were positive for Wolbachia, most probably indicating infection with this intracellular symbiont, and raising the possibility that it may be an important factor in influencing host sex ratios or fitness in a diversity of pollinators. Importantly, we found that about a third of bumblebees (Bombus pascuorum and Bombus terrestris and a third of wasps (Vespula vulgaris, as well as all honey bees, were positive for deformed wing virus, but that this virus was not present in other pollinators. Deformed wing virus therefore does not appear to be a general parasite of pollinators, but does interact significantly with at least three species of bumblebee and wasp. Further work is needed to establish the identity of some of the parasites, their spatiotemporal variation, and whether they are infecting the various pollinator species or being vectored. However, these results provide a first insight into the diversity, and potential exchange, of parasites in pollinator communities.

  17. Parasitic Effects on Memristor Dynamics

    Science.gov (United States)

    Itoh, Makoto; Chua, Leon O.

    2016-06-01

    In this paper, we show that parasitic elements have a significant effect on the dynamics of memristor circuits. We first show that certain 2-terminal elements such as memristors, memcapacitors, and meminductors can be used as nonvolatile memories, if the principle of conservation of state variables hold by open-circuiting, or short-circuiting, their terminals. We also show that a passive memristor with a strictly-increasing constitutive relation will eventually lose its stored flux when we switch off the power if there is a parasitic capacitance across the memristor. Similarly, a memcapacitor (resp., meminductor) with a positive memcapacitance (resp., meminductance) will eventually lose their stored physical states when we switch off the power, if it is connected to a parasitic resistance. We then show that the discontinuous jump that circuit engineers assumed to occur at impasse points of memristor circuits contradicts the principles of conservation of charge and flux at the time of the discontinuous jump. A parasitic element can be used to break an impasse point, resulting in the emergence of a continuous oscillation in the circuit. We also define a distance, a diameter, and a dimension, for each circuit element in order to measure the complexity order of the parasitic elements. They can be used to find higher-order parasitic elements which can break impasse points. Furthermore, we derived a memristor-based Chua’s circuit from a three-element circuit containing a memristor by connecting two parasitic memcapacitances to break the impasse points. We finally show that a higher-order parasitic element can be used for breaking the impasse points on two-dimensional and three-dimensional constrained spaces.

  18. Adaptations in the energy metabolism of parasites

    NARCIS (Netherlands)

    van Grinsven, K.W.A.

    2009-01-01

    For this thesis fundamental research was performed on the metabolic adaptations found in parasites. Studying the adaptations in parasite metabolisms leads to a better understanding of parasite bioenergetics and can also result in the identification of new anti-parasitic drug targets. We focussed on

  19. Adaptations in the energy metabolism of parasites

    NARCIS (Netherlands)

    van Grinsven, K.W.A.

    2009-01-01

    For this thesis fundamental research was performed on the metabolic adaptations found in parasites. Studying the adaptations in parasite metabolisms leads to a better understanding of parasite bioenergetics and can also result in the identification of new anti-parasitic drug targets. We focussed on

  20. Tachinidae Parasitic on the Lymantriidae in Japan

    OpenAIRE

    Paul W., Schaefer; Hiroshi, SHIMA; Asian Parasite Laboratory, Agricultural Research, Science and Education Administration, U.S. Department of Agriculture:(Present address)Beneficial Insects Research Laboratory; Biological Laboratory, College of General Education, Kyushu University

    1981-01-01

    Recorded tachinid parasites and 23 new host records are listed for 15 lymantriid species in Japan. A parasite-host list is included and a new species of Carcelia parasitic on Leucoma candida in Hokkaido is described and illustrated. A key to adult tachinid parasites of the Lymantriidae in Japan is provided.

  1. [Intestinal parasitic diseases as a global health problem].

    Science.gov (United States)

    Chacín-Bonilla, Leonor

    2013-03-01

    In today's world, parasitic disease agents are not restricted by geography or economy, and have become a significant global threat. The increasing globalization of the fresh produce market and greater international trade and travels, have contributed to the spread of these organisms in the industrialized world. Parasitic protozoa cause waterborne and foodborne outbreaks of diarrhea. The unprecedented flow of people introduces cultural and behavior patterns around the world; the increasing tendency to eat raw or undercooked meat and seafood, favors the dissemination of several parasitic pathogens. Climate changes are predicted to cause a global increase in soil-transmitted helminthiases. The multidisciplinary study of these agents, and the interaction among scientists, global health organizations and governments are imperative to reduce the burden of these diseases and improve the life of a large segment of the world population.

  2. Parasitic Pneumonia and Lung Involvement

    Directory of Open Access Journals (Sweden)

    Attapon Cheepsattayakorn

    2014-01-01

    Full Text Available Parasitic infestations demonstrated a decline in the past decade as a result of better hygiene practices and improved socioeconomic conditions. Nevertheless, global immigration, increased numbers of the immunocompromised people, international traveling, global warming, and rapid urbanization of the cities have increased the susceptibility of the world population to parasitic diseases. A number of new human parasites, such as Plasmodium knowlesi, in addition to many potential parasites, have urged the interest of scientific community. A broad spectrum of protozoal parasites frequently affects the respiratory system, particularly the lungs. The diagnosis of parasitic diseases of airway is challenging due to their wide varieties of clinical and roentgenographic presentations. So detailed interrogations of travel history to endemic areas are critical for clinicians or pulmonologists to manage this entity. The migrating adult worms can cause mechanical airway obstruction, while the larvae can cause airway inflammation. This paper provides a comprehensive review of both protozoal and helminthic infestations that affect the airway system, particularly the lungs, including clinical and roentgenographic presentations, diagnostic tests, and therapeutic approaches.

  3. Paleoparasitology: the origin of human parasites

    OpenAIRE

    Adauto Araujo; Karl Reinhard; Luiz Fernando Ferreira; Elisa Pucu; Pedro Paulo Chieffi

    2013-01-01

    Parasitism is composed by three subsystems: the parasite, the host, and the environment. There are no organisms that cannot be parasitized. The relationship between a parasite and its host species most of the time do not result in damage or disease to the host. However, in a parasitic disease the presence of a given parasite is always necessary, at least in a given moment of the infection. Some parasite species that infect humans were inherited from pre-hominids, and were shared with other ph...

  4. Paleoparasitology: the origin of human parasites.

    Science.gov (United States)

    Araújo, Adauto; Reinhard, Karl; Ferreira, Luiz Fernando; Pucu, Elisa; Chieffi, Pedro Paulo

    2013-09-01

    Parasitism is composed by three subsystems: the parasite, the host, and the environment. There are no organisms that cannot be parasitized. The relationship between a parasite and its host species most of the time do not result in damage or disease to the host. However, in a parasitic disease the presence of a given parasite is always necessary, at least in a given moment of the infection. Some parasite species that infect humans were inherited from pre-hominids, and were shared with other phylogenetically close host species, but other parasite species were acquired from the environment as humans evolved. Human migration spread inherited parasites throughout the globe. To recover and trace the origin and evolution of infectious diseases, paleoparasitology was created. Paleoparasitology is the study of parasites in ancient material, which provided new information on the evolution, paleoepidemiology, ecology and phylogenetics of infectious diseases.

  5. Paleoparasitology: the origin of human parasites

    Directory of Open Access Journals (Sweden)

    Adauto Araujo

    2013-09-01

    Full Text Available Parasitism is composed by three subsystems: the parasite, the host, and the environment. There are no organisms that cannot be parasitized. The relationship between a parasite and its host species most of the time do not result in damage or disease to the host. However, in a parasitic disease the presence of a given parasite is always necessary, at least in a given moment of the infection. Some parasite species that infect humans were inherited from pre-hominids, and were shared with other phylogenetically close host species, but other parasite species were acquired from the environment as humans evolved. Human migration spread inherited parasites throughout the globe. To recover and trace the origin and evolution of infectious diseases, paleoparasitology was created. Paleoparasitology is the study of parasites in ancient material, which provided new information on the evolution, paleoepidemiology, ecology and phylogenetics of infectious diseases.

  6. Parasites as prey in aquatic food webs: implications for predator infection and parasite transmission

    NARCIS (Netherlands)

    Thieltges, D.W.; Amundsen, P.-A.; Hechinger, R.F.; Johnson, P.T.J.; Lafferty, K.D.; Mouritsen, K.N.; Preston, D.L.; Reise, K.; Zander, C.D.; Poulin, R.

    2013-01-01

    While the recent inclusion of parasites into food-web studies has highlighted the role of parasites as consumers, there is accumulating evidence that parasites can also serve as prey for predators. Here we investigated empirical patterns of predation on parasites and their relationships with parasit

  7. Parasites as prey in aquatic food webs: implications for predator infection and parasite transmission

    NARCIS (Netherlands)

    Thieltges, D.W.; Amundsen, P.-A.; Hechinger, R.F.; Johnson, P.T.J.; Lafferty, K.D.; Mouritsen, K.N.; Preston, D.L.; Reise, K.; Zander, C.D.; Poulin, R.

    2013-01-01

    While the recent inclusion of parasites into food-web studies has highlighted the role of parasites as consumers, there is accumulating evidence that parasites can also serve as prey for predators. Here we investigated empirical patterns of predation on parasites and their relationships with parasit

  8. Update on pathology of ocular parasitic disease

    OpenAIRE

    2016-01-01

    Parasites are a group of eukaryotic organisms that may be free-living or form a symbiotic or parasitic relationship with the hosts. Consisting of over 800,000 recognized species, parasites may be unicellular (Protozoa) or multicellular (helminths and arthropods). The association of parasites with human population started long before the emergence of civilization. Parasitic zoonotic diseases are prevalent worldwide including India. Appropriate epidemiological data are lacking on existing zoono...

  9. Population structure of a vector-borne plant parasite.

    Science.gov (United States)

    Yule, Kelsey M; Koop, Jennifer A H; Alexandre, Nicolas M; Johnston, Lauren R; Whiteman, Noah K

    2016-07-01

    Parasites are among the most diverse groups of life on Earth, yet complex natural histories often preclude studies of their speciation processes. The biology of parasitic plants facilitates in situ collection of data on both genetic structure and the mechanisms responsible for that structure. Here, we studied the role of mating, dispersal and establishment in host race formation of a parasitic plant. We investigated the population genetics of a vector-borne desert mistletoe (Phoradendron californicum) across two legume host tree species (Senegalia greggii and Prosopis velutina) in the Sonoran desert using microsatellites. Consistent with host race formation, we found strong host-associated genetic structure in sympatry, little genetic variation due to geographic site and weak isolation by distance. We hypothesize that genetic differentiation results from differences in the timing of mistletoe flowering by host species, as we found initial flowering date of individual mistletoes correlated with genetic ancestry. Hybrids with intermediate ancestry were detected genetically. Individuals likely resulting from recent, successful establishment events following dispersal between the host species were detected at frequencies similar to hybrids between host races. Therefore, barriers to gene flow between the host races may have been stronger at mating than at dispersal. We also found higher inbreeding and within-host individual relatedness values for mistletoes on the more rare and isolated host species (S. greggii). Our study spanned spatial scales to address how interactions with both vectors and hosts influence parasitic plant structure with implications for parasite virulence evolution and speciation.

  10. 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

  11. Parasites, pets, and people.

    Science.gov (United States)

    Marx, M B

    1991-03-01

    It is important for the family physician to understand that patients' relationships with their pets play an important role in helping maintain mental and physical health yet provide the potential for causing illness in the patient. Toxocara canis (dog roundworm) and Toxocara cati (cat roundworm) are the ascarids most commonly responsible for VLM and ocular larva migrans in humans. These roundworms live in their adult stage in the small intestine of the dog and cat where their eggs are passed in the feces. The eggs containing the infective larva are very sticky, thus an infant crawling around on the floor can easily pick these up on fingers that almost invariably end up in the mouth. Infections are usually mild and asymptomatic but with a persistent eosinophilia. Ocular larva migrans is the form usually occurring in older children and adults. Some public health veterinarians recommend that a puppy or kitten should not be obtained as a companion for a child who is not old enough to read, thus bypassing the crawling and toddler stages. Hookworm eggs, shed in the feces of infected dogs or cats, develop into the infective second stage within a week. Humans are usually infected when bare areas of skin such as bare feet or the torso come in contact with soil contaminated with the larvae. The second-stage larvae are able to penetrate the intact skin of humans and the foot pads of dogs and cats. In the United States, the common dog hookworm, A. caninum, is a widespread parasite. Human intestinal ancylostomiasis caused by this species is rare, with only six cases recorded in the literature. Infection in humans or animals by the common tapeworm of dogs and cats (Dipylidium caninum) requires ingestion of the intermediate host, the dog or cat flea containing the larva (cysticercoids) of the agent. Many cases in humans are asymptomatic. Dipylidiasis affects mainly infants and young children who may swallow a flea that hops up while the infant is crawling on the floor or fondling

  12. Protein palmitoylation in protozoan parasites.

    Science.gov (United States)

    Corvi, Maria Martha; Berthiaume, Luc Gerard; De Napoli, Maximiliano Gabriel

    2011-06-01

    Palmitoylation plays an important role in the regulation of the localization and function of the modified protein. Although many aspects of protein palmitoylation have been identified in mammalian and yeast cells, little information is available of this modification in protozoan parasites. Protein palmitoylation has been described for a few set of proteins in E.tenella, P. falciparum, T. gondii, G. lamblia and T. cruzi. Interestingly, in all these parasites palmitoylated proteins appears to be involved in vital processes such as invasion and motility. In addition, most of these parasites contain in their genomes genes that encode for putative palmitoyl-acyl transferases, the enzymes catalyzing the palmitoylation reaction. Although protein palmitoylation could be playing key roles in invasion and motility in a variety of parasites, little is known about this important reversible modification of proteins that typically plays a role in membrane tethering. As such, this review will focus on the main features of protein palmitoylation as well as provide an overview of the state of knowledge of this modification in protozoan parasites.

  13. Host-parasite interactions: Marine bivalve molluscs and protozoan parasites, Perkinsus species.

    Science.gov (United States)

    Soudant, Philippe; E Chu, Fu-Lin; Volety, Aswani

    2013-10-01

    This review assesses and examines the work conducted to date concerning host and parasite interactions between marine bivalve molluscs and protozoan parasites, belonging to Perkinsus species. The review focuses on two well-studied host-parasite interaction models: the two clam species, Ruditapes philippinarum and R. decussatus, and the parasite Perkinsus olseni, and the eastern oyster, Crassostrea virginica, and the parasite Perkinsus marinus. Cellular and humoral defense responses of the host in combating parasitic infection, the mechanisms (e.g., antioxidant enzymes, extracellular products) employed by the parasite in evading host defenses as well as the role of environmental factors in modulating the host-parasite interactions are described.

  14. COMPARATIVE ANALYSES CONCERNING PARASITIC DIVERSITY OF COMMON CARP AND KOI CARP

    Directory of Open Access Journals (Sweden)

    DANIELA RADU

    2013-12-01

    Full Text Available The experiments carried on ponds of CCDP Nucet, researches being axled on comparative and comprehensive analysis of pathologic incidences on common and Japanese carp. This researches present theoretical and empirical convenience, because allow to elaborate of prophylaxis and disease control on. The ichthyo-pathologic exam was carried on for 271 exemplars of common carp, subspecies Cyprinus carpio carpio and 31 exemplars of ornamental carp (koi carp. There were identified 30 species of allogenic parasites, 9 species of parasites characteristic for carp as well as some common species of parasites with broad dissemination on cultured fish. All analyzed exemplars of showed parasitic infestation, but with a different extent of extensively and intensively. The most important epidemiologic factors from the analyzed ponds that increase the parasitic invasions were represented by the presence of infested fish, rearing in policulture of species and ages, as well as by the high density, water quality, thermic and flow fluctuations.

  15. Taming Parasites by Tailoring Them

    Directory of Open Access Journals (Sweden)

    Bingjian Ren

    2017-07-01

    Full Text Available The next-generation gene editing based on CRISPR (clustered regularly interspaced short palindromic repeats has been successfully implemented in a wide range of organisms including some protozoan parasites. However, application of such a versatile game-changing technology in molecular parasitology remains fairly underexplored. Here, we briefly introduce state-of-the-art in human and mouse research and usher new directions to drive the parasitology research in the years to come. In precise, we outline contemporary ways to embolden existing apicomplexan and kinetoplastid parasite models by commissioning front-line gene-tailoring methods, and illustrate how we can break the enduring gridlock of gene manipulation in non-model parasitic protists to tackle intriguing questions that remain long unresolved otherwise. We show how a judicious solicitation of the CRISPR technology can eventually balance out the two facets of pathogen-host interplay.

  16. Parasitic diseases of the pleura.

    Science.gov (United States)

    Lal, Chitra; Huggins, John Terrill; Sahn, Steven A

    2013-05-01

    Parasitic infections are prevalent in certain parts of the world and may cause pleural involvement, which often goes unrecognized. Common parasites involving the pleura include Entamoeba histolytica, Echinococcus granulosus and Paragonimus westermani. Amebiasis can cause empyema with "anchovy sauce" pus, reactive pleural effusions and bronchopleural fistula with hydropneumothorax. Echinococcosis may result in pleural thickening, pneumothorax, secondary pleural hydatidosis and pleural effusions. Paragonimiasis may cause chylous and cholesterol pleural effusions, pleural thickening and pneumothorax. Less commonly, pulmonary eosinophilia, or Loeffler's syndrome, caused by Ascaris lumbricoides, Ancylostoma duodenale and Necator americanus and tropical pulmonary eosinophilia caused by Wuchereria bancrofti and Brugia malayi may involve the pleura. This article provides a comprehensive review of parasitic infections involving the pleura. A high index of suspicion in the appropriate clinical setting is required to facilitate prompt diagnosis and treatment of these diseases.

  17. Parasites and immunotherapy: with or against?

    Science.gov (United States)

    Yousofi Darani, Hossein; Yousefi, Morteza; Safari, Marzieh; Jafari, Rasool

    2016-06-01

    Immunotherapy is a sort of therapy in which antibody or antigen administrates to the patient in order to treat or reduce the severity of complications of disease. This kind of treatment practiced in a wide variety of diseases including infectious diseases, autoimmune disorders, cancers and allergy. Successful and unsuccessful immunotherapeutic strategies have been practiced in variety of parasitic infections. On the other hand parasites or parasite antigens have also been considered for immunotherapy against other diseases such as cancer, asthma and multiple sclerosis. In this paper immunotherapy against common parasitic infections, and also immunotherapy of cancer, asthma and multiple sclerosis with parasites or parasite antigens have been reviewed.

  18. Canine and feline parasitic zoonoses in China.

    Science.gov (United States)

    Chen, Jia; Xu, Min-Jun; Zhou, Dong-Hui; Song, Hui-Qun; Wang, Chun-Ren; Zhu, Xing-Quan

    2012-07-28

    Canine and feline parasitic zoonoses have not been given high priority in China, although the role of companion animals as reservoirs for zoonotic parasitic diseases has been recognized worldwide. With an increasing number of dogs and cats under unregulated conditions in China, the canine and feline parasitic zoonoses are showing a trend towards being gradually uncontrolled. Currently, canine and feline parasitic zoonoses threaten human health, and cause death and serious diseases in China. This article comprehensively reviews the current status of major canine and feline parasitic zoonoses in mainland China, discusses the risks dogs and cats pose with regard to zoonotic transmission of canine and feline parasites, and proposes control strategies and measures.

  19. The Geological Record of Parasitic Nematode Evolution.

    Science.gov (United States)

    Poinar, George O

    2015-01-01

    This chapter discusses the evolutionary history of nematode parasites of invertebrates, vertebrates and plants based on fossil remains in amber, stone and coprolites dating from the Palaeozoic to the Holocene. The earliest parasitic nematode is a primitive plant parasite from the Devonian. Fossil invertebrate-parasitic nematodes first appeared in the Early Cretaceous, while the earliest fossil vertebrate-parasitic nematodes are from Upper Triassic coprolites. Specific examples of fossil nematode parasites over time are presented, along with views on the origin and evolution of nematodes and their hosts. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Parasites, emerging disease and wildlife conservation.

    Science.gov (United States)

    Thompson, R C A; Lymbery, A J; Smith, A

    2010-08-15

    In this review some emerging issues of parasite infections in wildlife, particularly in Australia, are considered. We discuss the importance of understanding parasite biodiversity in wildlife in terms of conservation, the role of wildlife as reservoirs of parasite infection, and the role of parasites within the broader context of the ecosystem. Using a number of parasite species, the value of undertaking longitudinal surveillance in natural systems using non-invasive sampling and molecular tools to characterise infectious agents is illustrated in terms of wildlife health, parasite biodiversity and ecology.

  1. Oncogenic Brain Metazoan Parasite Infection

    Directory of Open Access Journals (Sweden)

    Angela N. Spurgeon

    2013-01-01

    Full Text Available Multiple observations suggest that certain parasitic infections can be oncogenic. Among these, neurocysticercosis is associated with increased risk for gliomas and hematologic malignancies. We report the case of a 71-year-old woman with colocalization of a metazoan parasite, possibly cysticercosis, and a WHO grade IV neuroepithelial tumor with exclusively neuronal differentiation by immunohistochemical stains (immunopositive for synaptophysin, neurofilament protein, and Neu-N and not for GFAP, vimentin, or S100. The colocalization and temporal relationship of these two entities suggest a causal relationship.

  2. Genetic relationship between parasitized and non-parasitized Haloxyion ammodendron in the Alxa Desert

    Institute of Scientific and Technical Information of China (English)

    Xu-Mei WANG; Dong-Ye YANG; yong-Zhen TIAN; peng-Fei TU; Qi-Shi SUN; Xiao-Bo LI

    2009-01-01

    Haloxylon ammodendron (C. A. Mey.) Bunge is a host for the holoparasitic plant Cistanche deserticola Y. C. Ma, the original source of medicinal material known as Herba Cistanchis. The inter-simple sequence repeat marker was used to assess the genetic variations and relationships among six accessions of H. ammodendron with a total of 120 individuals collected from three localities in the Alxa Desert, Inner Mongolia, China. At each locality, individuals both parasitized (PP) by C. deserticola and non-parasitized (NP) were sampled. The results showed that Nei's gene diversity and Shannon's index of PP accessions were higher, but were not significantly different, from those of NP accessions. An unweighted pair-group method arithmetic average dendrogram showed two clusters, one that included all PP accessions, and the other the NP accessions. Genetic differentiation therefore existed between PP and NP accessions, which might be attributed to low gene flow between the NP and PP groups (Nm< 1). However, the relationship between genetic distance and geographic distance within each group, although not statistically significant in this study, might be associated with high gene flow in both the NP and PP groups.

  3. Geographic genetic differentiation of a malaria parasite, Plasmodium mexicanum, and its lizard host, Sceloporus occidentalis.

    Science.gov (United States)

    Fricke, Jennifer M; Vardo-Zalik, Anne M; Schall, Jos J

    2010-04-01

    Gene flow, and resulting degree of genetic differentiation among populations, will shape geographic genetic patterns and possibly local adaptation of parasites and their hosts. Some studies of Plasmodium falciparum in humans show substantial differentiation of the parasite in locations separated by only a few kilometers, a paradoxical finding for a parasite in a large, mobile host. We examined genetic differentiation of the malaria parasite Plasmodium mexicanum, and its lizard host, Sceloporus occidentalis, at 8 sites in northern California, with the use of variable microsatellite markers for both species. These lizards are small and highly territorial, so we expected local genetic differentiation of both parasite and lizard. Populations of P. mexicanum were found to be differentiated by analysis of 5 markers (F(st) values >0.05-0.10) over distances as short as 230-400 m, and greatly differentiated (F(st) values >0.25) for sites separated by approximately 10 km. In contrast, the lizard host had no, or very low, levels of differentiation for 3 markers, even for sites >40 km distant. Thus, gene flow for the lizard was great, but despite the mobility of the vertebrate host, the parasite was locally genetically distinct. This discrepancy could result if infected lizards move little, but their noninfected relatives were more mobile. Previous studies on the virulence of P. mexicanum for fence lizards support this hypothesis. However, changing prevalence of the parasite, without changes in density of the lizard, could also result in this pattern.

  4. Sacral Rachipagus Parasite: A Case Report.

    Science.gov (United States)

    Rattan, Kamal Nain; Singh, Jasbir; Dalal, Poonam; Sonika, Pallavi; Rattan, Ananta

    2016-01-01

    We are reporting a case of sacral rachipagus parasite which was vaginally delivered as a large irregular mass attached to the sacral region by a vascular pedicle. This case was managed successfully by surgical excision of parasite.

  5. O&P (Ova and Parasite) Test

    Science.gov (United States)

    ... Was this page helpful? Also known as: O&P Formal name: Parasitic Examination, stool Related tests: Gastrointestinal ... is it used? The ova and parasite (O&P) exam is used to detect the presence of ...

  6. Parasites and food: ripe for exploitation.

    Science.gov (United States)

    Thompson, R C Andrew

    2014-01-01

    Parasites are often exploited for emotive or political purposes. This is especially so for a number of foodborne parasitic zoonoses, where this exploitation may not necessarily best serve the public good.

  7. Can Parasites Really Reveal Environmental Impact?

    Science.gov (United States)

    This review assesses the usefulness of parasites as bioindicators of environmental impact. Relevant studies published in the past decade were compiled; factorial meta-analysis demonstrated significant effects and interactions between parasite levels and the presence and concentra...

  8. Sacral Rachipagus Parasite: A Case Report

    Directory of Open Access Journals (Sweden)

    Kamal Nain Rattan

    2016-03-01

    Full Text Available We are reporting a case of sacral rachipagus parasite which was vaginally delivered as a large irregular mass attached to the sacral region by a vascular pedicle. This case was managed successfully by surgical excision of parasite.

  9. Parasitic Nematode Interactions with Mammals and Plants

    NARCIS (Netherlands)

    Jasmer, D.P.; Goverse, A.; Smant, G.

    2003-01-01

    Parasitic nematodes that infect humans, animals, and plants cause serious diseases that are deleterious to human health and agricultural productivity. Chemical and biological control methods have reduced the impact of these parasites. However, surviving environmental stages lead to persistent

  10. Parasitic Diseases - Multiple Languages: MedlinePlus

    Science.gov (United States)

    ... Are Here: Home → Multiple Languages → All Health Topics → Parasitic Diseases URL of this page: https://medlineplus.gov/languages/ ... V W XYZ List of All Topics All Parasitic Diseases - Multiple Languages To use the sharing features on ...

  11. Copepoda parasites in economically important fish, Mugilidae ...

    African Journals Online (AJOL)

    FUNMILAYO

    parasites are ecto-parasites which negatively affect the appearance and reduced production of species of economically ... pathogenic problems in cultured mullet fish in marine and brackish ..... pollution resulting from the human settlement.

  12. Antimicrobial peptide action on parasites.

    Science.gov (United States)

    Torrent, Marc; Pulido, David; Rivas, Luis; Andreu, David

    2012-08-01

    Diseases caused by protozoan parasites can pose a severe thread to human health and are behind some serious neglected tropical diseases like malaria and leishmaniasis. Though several different drugs have been developed in order to eradicate these diseases, a successful candidate has not yet been discovered. Among the most active compounds tested, antimicrobial peptides (AMPs) are particularly appealing because of their wide spectrum of action. AMPs have been described to perturb protozoan homeostasis by disrupting the cellular membranes but also by interfering with key processes in the parasite metabolism. In this review we describe the diverse mechanisms of action of AMPs on protozoan targets and how they can be exploited to treat diseases. Moreover, we describe with detail the antimicrobial action of AMPs on two major parasitical infections: leishmaniasis and malaria. All the features reviewed here show that AMPs are promising drugs to target protozoan parasites and that further understanding of the mechanism of action of these compounds will lead to improved drugs that could be worth to test in a clinical phase.

  13. Zoology: Invertebrates that Parasitize Invertebrates.

    Science.gov (United States)

    Giribet, Gonzalo

    2016-07-11

    The genome of an orthonectid, a group of highly modified parasitic invertebrates, is drastically reduced and compact, yet it shows the bilaterian gene toolkit. Phylogenetic analyses place the enigmatic orthonectids within Spiralia, although their exact placement remains uncertain. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Parasitism and mutualism in Wolbachia

    DEFF Research Database (Denmark)

    Bordenstein, Seth R; Paraskevopoulos, Charalampos; Dunning Hotopp, Julie C;

    2009-01-01

    Ecological and evolutionary theories predict that parasitism and mutualism are not fixed endpoints of the symbiotic spectrum. Rather, parasitism and mutualism may be host or environment dependent, induced by the same genetic machinery, and shifted due to selection. These models presume the existe......Ecological and evolutionary theories predict that parasitism and mutualism are not fixed endpoints of the symbiotic spectrum. Rather, parasitism and mutualism may be host or environment dependent, induced by the same genetic machinery, and shifted due to selection. These models presume...... the existence of genetic or environmental variation that can spur incipient changes in symbiotic lifestyle. However, for obligate intracellular bacteria whose genomes are highly reduced, studies specify that discrete symbiotic associations can be evolutionarily stable for hundreds of millions of years...... in symbiotic lifestyle with a comprehensive, phylogenomic analysis. Contrary to previous claims, we show unequivocally that the transition in lifestyle cannot be reconstructed with current methods due to long-branch attraction (LBA) artifacts of the distant Anaplasma and Ehrlichia outgroups. Despite the use...

  15. Decoys in Predation and Parasitism

    NARCIS (Netherlands)

    Wilkinson, Michael H.F.

    2003-01-01

    Predator-prey or host-parasite dynamics can be altered by the presence of other species through several mechanisms. One such mechanism is the ‘‘decoy effect,’’ which itself can take a variety of forms. In its simplest form, the third species, which is inedible to the predator, nonetheless interferes

  16. Decoys in Predation and Parasitism

    NARCIS (Netherlands)

    Wilkinson, Michael H.F.

    2003-01-01

    Predator-prey or host-parasite dynamics can be altered by the presence of other species through several mechanisms. One such mechanism is the ‘‘decoy effect,’’ which itself can take a variety of forms. In its simplest form, the third species, which is inedible to the predator, nonetheless interferes

  17. The kinomes of apicomplexan parasites

    OpenAIRE

    Miranda-Saavedra, D.; Gabaldón, T.; Barton, G; Langsley, G; Doerig, C.

    2012-01-01

    Protein phosphorylation plays a fundamental role in the biology of apicomplexan parasites. Many apicomplexan protein kinases are substantially different from their mammalian orthologues, and thus constitute a landscape of potential drug targets. Here, we integrate genomic, biochemical, genetic and evolutionary information to provide an integrated and up-to-date analysis of twelve apicomplexan kinomes. All kinome sequences are available through the Kinomer database.

  18. Emerging parasitic diseases of sheep.

    Science.gov (United States)

    Taylor, M A

    2012-09-30

    There have been changes in the emergence and inability to control of a number of sheep parasitic infections over the last decade. This review focuses on the more globally important sheep parasites, whose reported changes in epidemiology, occurrence or failure to control are becoming increasingly evident. One of the main perceived driving forces is climate change, which can have profound effects on parasite epidemiology, especially for those parasitic diseases where weather has a direct effect on the development of free-living stages. The emergence of anthelmintic-resistant strains of parasitic nematodes and the increasing reliance placed on anthelmintics for their control, can exert profound changes on the epidemiology of those nematodes causing parasitic gastroenteritis. As a consequence, the effectiveness of existing control strategies presents a major threat to sheep production in many areas around the world. The incidence of the liver fluke, Fasciola hepatica, is inextricably linked to high rainfall and is particularly prevalent in high rainfall years. Over the last few decades, there have also been increasing reports of other fluke associated diseases, such as dicroceliosis and paramphistomosis, in a number of western European countries, possibly introduced through animal movements, and able to establish with changing climates. External parasite infections, such as myiasis, can cause significant economic loss and presents as a major welfare problem. The range of elevated temperatures predicted by current climate change scenarios, result in an elongated blowfly season with earlier spring emergence and a higher cumulative incidence of fly strike. Additionally, legislative decisions leading to enforced changes in pesticide usage and choices have resulted in increased reports and spread of ectoparasitic infections, particularly mite, lice and tick infestations in sheep. Factors, such as dip disposal and associated environmental concerns, and, perhaps more

  19. One Health: parasites and beyond.

    Science.gov (United States)

    Blake, Damer P; Betson, Martha

    2017-01-01

    The field of parasitism is broad, encompassing relationships between organisms where one benefits at the expense of another. Traditionally the discipline focuses on eukaryotes, with the study of bacteria and viruses complementary but distinct. Nonetheless, parasites vary in size and complexity from single celled protozoa, to enormous plants like those in the genus Rafflesia. Lifecycles range from obligate intracellular to extensive exoparasitism. Examples of parasites include high-profile medical and zoonotic pathogens such as Plasmodium, veterinary pathogens of wild and captive animals and many of the agents which cause neglected tropical diseases, stretching to parasites which infect plants and other parasites (e.g. Kikuchi et al. 2011; Hotez et al. 2014; Blake et al. 2015; Hemingway, 2015; Meekums et al. 2015; Sandlund et al. 2015). The breadth of parasitology has been matched by the variety of ways in which parasites are studied, drawing upon biological, chemical, molecular, epidemiological and other expertise. Despite such breadth bridging between disciplines has commonly been problematic, regardless of extensive encouragement from government agencies, peer audiences and funding bodies promoting multidisciplinary research. Now, progress in understanding and collaboration can benefit from establishment of the One Health concept (Zinsstag et al. 2012; Stark et al. 2015). One Health draws upon biological, environmental, medical, veterinary and social science disciplines in order to improve human, animal and environmental health, although it remains tantalizingly difficult to engage many relevant parties. For infectious diseases traditional divides have been exacerbated as the importance of wildlife reservoirs, climate change, food production systems and socio-economic diversity have been recognized but often not addressed in a multidisciplinary manner. In response the 2015 Autumn Symposium organized by the British Society for Parasitology (BSP; https

  20. Canine and feline parasitic zoonoses in China

    OpenAIRE

    2012-01-01

    Abstract Canine and feline parasitic zoonoses have not been given high priority in China, although the role of companion animals as reservoirs for zoonotic parasitic diseases has been recognized worldwide. With an increasing number of dogs and cats under unregulated conditions in China, the canine and feline parasitic zoonoses are showing a trend towards being gradually uncontrolled. Currently, canine and feline parasitic zoonoses threaten human health, and cause death and serious diseases in...

  1. Review of Parasitic Zoonoses in Egypt

    OpenAIRE

    2014-01-01

    This review presents a comprehensive picture of the zoonotic parasitic diseases in Egypt, with particular reference to their relative prevalence among humans, animal reservoirs of infection, and sources of human infection. A review of the available literature indicates that many parasitic zoonoses are endemic in Egypt. Intestinal infections of parasitic zoonoses are widespread and are the leading cause of diarrhea, particularly among children and residents of rural areas. Some parasitic zoono...

  2. Parasite control in transhumant situations.

    Science.gov (United States)

    Eckert, J; Hertzberg, H

    1994-08-01

    Transhumance is defined as 'seasonal moving of livestock to regions of different climate'. It is an integral part of livestock production in many parts of the world and takes several forms including moving of livestock from lowland to mountainous pastures or from dry to humid areas. The impact of transhumance on parasite populations of livestock and on parasite control is described, mainly using examples from Europe. The epidemiology of trichostrongylidosis of cattle, mainly caused by Ostertagia ostertagi and Cooperia oncophora, is characterised by prolonged survival of overwintered infective larvae until the end of June. Cattle moved to such contaminated pastures in a transhumant grazing system are exposed to these larvae and may be protected, during the second half of the grazing season until autumn, by a late application (June/July) of an intraruminal drug-release device. Community pastures used in a transhumant system with mixed grazing of young cattle originating from various farms may enhance transmission of dictyocaulosis. Therefore, specific prophylactic measures are required. Hill sheep nematode populations may differ from those in lowland sheep in that Haemonchus contortus generally plays a minor role in hill sheep in which Ostertagia circumcincta and Nematodirus spp. predominate. Infections with Fasciola hepatica and Dicrocoelium dendriticum can be acquired on mountainous pastures by cattle, sheep and other livestock grazing in a transhumant system as intermediate hosts of these parasites may find suitable habitats in these regions. There is evidence that in the prealpine and alpine area both parasites are mainly transmitted in two-season cycles. Further examples for the impact of transhumance on parasite-host inter-relationships include cysticercosis in cattle, echinococcosis, psoroptic manage in sheep, tick-borne fever of cattle, and hypodermosis in cattle. These are described and discussed.

  3. A rapid and robust selection procedure for generating drug-selectable marker-free recombinant malaria parasites.

    Science.gov (United States)

    Manzoni, Giulia; Briquet, Sylvie; Risco-Castillo, Veronica; Gaultier, Charlotte; Topçu, Selma; Ivănescu, Maria Larisa; Franetich, Jean-François; Hoareau-Coudert, Bénédicte; Mazier, Dominique; Silvie, Olivier

    2014-04-23

    Experimental genetics have been widely used to explore the biology of the malaria parasites. The rodent parasites Plasmodium berghei and less frequently P. yoelii are commonly utilised, as their complete life cycle can be reproduced in the laboratory and because they are genetically tractable via homologous recombination. However, due to the limited number of drug-selectable markers, multiple modifications of the parasite genome are difficult to achieve and require large numbers of mice. Here we describe a novel strategy that combines positive-negative drug selection and flow cytometry-assisted sorting of fluorescent parasites for the rapid generation of drug-selectable marker-free P. berghei and P. yoelii mutant parasites expressing a GFP or a GFP-luciferase cassette, using minimal numbers of mice. We further illustrate how this new strategy facilitates phenotypic analysis of genetically modified parasites by fluorescence and bioluminescence imaging of P. berghei mutants arrested during liver stage development.

  4. Parasitic infections of the gastrointestinal tract.

    Science.gov (United States)

    Noyer, C M; Brandt, L J

    1999-08-01

    Parasitic infections of the gastrointestinal tract are a major cause of morbidity and mortality worldwide. Increased international travel means that gastroenterologists are now more likely to care for patients with parasitic diseases. This article reviews various aspects of the more common intestinal parasites and their infections, including epidemiology, life cycle, pathogenesis, clinical manifestations, diagnosis, and treatment.

  5. Immigration, parasitic infection, and United States religiosity.

    Science.gov (United States)

    Wall, Jaimie N; Shackelford, Todd K

    2012-04-01

    Fincher & Thornhill (F&T) present a powerful case for the relationship between parasite-stress and religiosity. We argue, however, that the United States may be more religious than can be accounted for by parasite-stress. This greater religiosity might be attributable to greater sensitivity to immigration, which may hyperactivate evolved mechanisms that motivate avoidance of potential carriers of novel parasites.

  6. Postcolonial Ecologies of Parasite and Host: Making Parasitism Cosmopolitan.

    Science.gov (United States)

    Anderson, Warwick

    2016-04-01

    The interest of F. Macfarlane Burnet in host-parasite interactions grew through the 1920s and 1930s, culminating in his book, Biological Aspects of Infectious Disease (1940), often regarded as the founding text of disease ecology. Our knowledge of the influences on Burnet's ecological thinking is still incomplete. Burnet later attributed much of his conceptual development to his reading of British theoretical biology, especially the work of Julian Huxley and Charles Elton, and regretted he did not study Theobald Smith's Parasitism and Disease (1934) until after he had formulated his ideas. Scholars also have adduced Burnet's fascination with natural history and the clinical and public health demands on his research effort, among other influences. I want to consider here additional contributions to Burnet's ecological thinking, focusing on his intellectual milieu, placing his research in a settler society with exceptional expertise in environmental studies and pest management. In part, an ''ecological turn'' in Australian science in the 1930s, derived to a degree from British colonial scientific investments, shaped Burnet's conceptual development. This raises the question of whether we might characterize, in postcolonial fashion, disease ecology, and other studies of parasitism, as successful settler colonial or dominion science.

  7. 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.

  8. Relationship between sperm viability as determined by flow cytometry and nonreturn rate of dairy bulls.

    Science.gov (United States)

    Christensen, Preben; Boelling, Dorothee; Pedersen, Kurt Myrup; Korsgaard, Inge Riis; Jensen, Just

    2005-01-01

    A newly developed flow cytometric method for determination of sperm concentration and viability was tested in an insemination trial with cryopreserved bull sperm to establish the relationship between sperm viability and nonreturn rates. Semen for experimental inseminations was produced from 157 young sires (114 Holstein and 43 Jersey), each contributing 4 experimental semen collections. Straws containing approximately 15 x 10(6) motile sperm before freezing were used in 118,680 experimental inseminations performed by 254 artificial insemination technicians in 6352 Danish herds. Statistical analysis based on 44,946 experimental first inseminations showed that the major part (95.4%) of variation in the 56-day nonreturn rate (NRR56) was residual. Only 0.38% of the total variation in NRR56 was due to bulls and differences between ejaculate within bull. However, bulls were preselected, and a relatively high insemination dose was used. Correlations between sperm viability as assessed by flow cytometry and NRR56 was slightly lower than observed for microscopic assessment of sperm motility. However, flow cytometry makes it possible to achieve an objective and precise determination of sperm viability. It was therefore possible to calculate the effect on NRR56 provided selection of semen is based on the flow cytometric method. Three freezing extenders were used in this experiment, but a significant difference in NRR56 was not observed. Flow cytometric results for 1 extender (Biociphos Plus) indicated poorer sperm survival during postthaw incubation compared with Triladyl extender with whole and with clarified egg yolk.

  9. The adaptive significance of inquiline parasite workers

    DEFF Research Database (Denmark)

    Sumner, Seirian; Nash, David R; Boomsma, Jacobus J

    2003-01-01

    Social parasites exploit the socially managed resources of their host's society. Inquiline social parasites are dependent on their host throughout their life cycle, and so many of the traits inherited from their free-living ancestor are removed by natural selection. One trait that is commonly lost...... is the worker caste, the functions of which are adequately fulfilled by host workers. The few inquiline parasites that have retained a worker caste are thought to be at a transitional stage in the evolution of social parasitism, and their worker castes are considered vestigial and non-adaptive. However...... a vital role in ensuring the parasite's fitness. We show that the presence of these parasite workers has a positive effect on the production of parasite sexuals and a negative effect on the production of host sexuals. This suggests that inquiline workers play a vital role in suppressing host queen...

  10. 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.

  11. Moonlighting enzymes in parasitic protozoa.

    Science.gov (United States)

    Collingridge, Peter W; Brown, Robert W B; Ginger, Michael L

    2010-08-01

    Enzymes moonlight in a non-enzymatic capacity in a diverse variety of cellular processes. The discovery of these non-enzymatic functions is generally unexpected, and moonlighting enzymes are known in both prokaryotes and eukaryotes. Importantly, this unexpected multi-functionality indicates that caution might be needed on some occasions in interpreting phenotypes that result from the deletion or gene-silencing of some enzymes, including some of the best known enzymes from classic intermediary metabolism. Here, we provide an overview of enzyme moonlighting in parasitic protists. Unequivocal and putative examples of moonlighting are discussed, together with the possibility that the unusual biological characteristics of some parasites either limit opportunities for moonlighting to arise or perhaps contribute to the evolution of novel proteins with clear metabolic ancestry.

  12. Parasitic interference in nulling interferometry

    CERN Document Server

    Matter, Alexis; Danchi, William C; Lopez, Bruno; Absil, Olivier

    2013-01-01

    Nulling interferometry aims to detect faint objects close to bright stars. Its principle is to produce a destructive interference along the line-of-sight so that the stellar flux is rejected, while the flux of the off-axis source can be transmitted. In practice, various instrumental perturbations can degrade the nulling performance. Any imperfection in phase, amplitude, or polarization produces a spurious flux that leaks to the interferometer output and corrupts the transmitted off-axis flux. One of these instrumental pertubations is the crosstalk phenomenon, which occurs because of multiple parasitic reflections inside transmitting optics, and/or diffraction effects related to beam propagation along finite size optics. It can include a crosstalk of a beam with itself, and a mutual crosstalk between different beams. This can create a parasitic interference pattern, which degrades the intrinsic transmission map - or intensity response - of the interferometer. In this context, we describe how this instrumental ...

  13. Gene targeting in malaria parasites.

    Science.gov (United States)

    Ménard, R; Janse, C

    1997-10-01

    Gene targeting, which permits alteration of a chosen gene in a predetermined way by homologous recombination, is an emerging technology in malaria research. Soon after the development of techniques for stable transformation of red blood cell stages of Plasmodium falciparum and Plasmodium berghei, genes of interest were disrupted in the two species. The main limitations of gene targeting in malaria parasites result from the intracellular growth and slow replication of these parasites. On the other hand, the technology is facilitated by the very high rate of homologous recombination following transformation with targeting constructs (approximately 100%). Here, we describe (i) the vector design and the type of mutation that may be generated in a target locus, (ii) the selection and screening strategies that can be used to identify clones with the desired modification, and (iii) the protocol that was used for disrupting the circumsporozoite protein (CS) and thrombospondin-related anonymous protein (TRAP) genes of P. berghei.

  14. Transfusion-transmitted parasitic infections

    Directory of Open Access Journals (Sweden)

    Singh Gagandeep

    2010-01-01

    Full Text Available The transmission of parasitic organisms through transfusion is relatively rare. Of the major transfusion-transmitted diseases, malaria is a major cause of TTIP in tropical countries whereas babesiosis and Chagas′ disease pose the greatest threat to donors in the USA In both cases, this is due to the increased number of potentially infected donors. There are no reliable serologic tests available to screen donors for any of these organisms and the focus for prevention remains on adherence to donor screening guidelines that address travel history and previous infection with the etiologic agent. One goal is the development of tests that are able to screen for and identify donors potentially infectious for parasitic infections without causing the deferral of a large number of non-infectious donors or significantly increasing costs. Ideally, methods to inactivate the infectious organism will provide an element of added safety to the blood supply.

  15. 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.

  16. PARASITE MYCOPOPULATION OF SOYBEAN GRAIN

    OpenAIRE

    Jasenka Ćosić; Karolina Vrandečić; Draženka Jurković; Ivan Ereš; Jelena Poštić

    2008-01-01

    Disease appearance on soybean can influence quality and quantity of yield. Different spieces of saprophyte and parasite fungi can be isolated from stems, pods and grain of soybean. The aim of the research was to evaluate the incidence of important disease on natural soybean grain over the period of 4 years (2004-2007) of experiment held on the location Sopot-Vinkovci and included 9 cultivars of soybean. The following plant pathogenic fungi were identified: Peronospora, Sclerotinia, Cercospora...

  17. Workshop on Cytokines and Parasites

    Science.gov (United States)

    1988-03-01

    Research Institute, Seattle, WA) demonstra- ted that GM-CSF, greater than 500 U/ml, induced the intracellular destruction of Trypanosoma cruzi by...increases the uptake of Trypanosoma cruzi by ,crophaqes, and induces a modest killing of the intracellular parasite. Dr. Gerald Byrne (University of...diseases by describing an exceedingly complex interaction of TNF and Trypanosoma musculi. TNF administered to mice early during disease actually enhanced

  18. Accumulation of persistent organic pollutants in parasites.

    Science.gov (United States)

    Yen Le, T T; Rijsdijk, Laurie; Sures, Bern; Hendriks, A Jan

    2014-08-01

    Organisms are simultaneously exposed to various stressors, including parasites and pollutants, that may interact with each other. Research on the accumulation of organic compounds in host-parasite systems is scant compared to studies on parasite-metal interactions and mainly focuses on intestinal endoparasites. We reviewed factors that determine the accumulation of persistent organic pollutants (POPs) in host-parasite systems. The wet/dry weight-based concentration of POPs in these parasites is usually lower than that in host tissues because of lower lipid contents in the parasites. However, the fractionation of the pollutants into parasites and their hosts may vary, depending on developmental stages in the life cycle of the parasites. Developmental stages determine the trophic relationship and the taxon of the parasite in the host-parasite systems because of different feeding strategies between the stages. Lipid-corrected concentrations of organic chemicals in the host are usually higher than those in the endoparasites studied. This phenomenon is attributed to a number of physiological and behavioural processes, such as feeding selectivity and strategy and excretion. Moreover, no significant relationship was found between the accumulation factor (i.e. the ratio between the lipid-corrected concentrations in parasites and in their hosts) for polychlorinated biphenyls and either hydrophobicity or molecular size. At the intermediate hydrophobicity, larger and more lipophilic compounds are accumulated at higher levels in both parasites and the host than smaller and less lipophilic compounds. The bioaccumulation of POPs in parasites is affected by some other abiotic, e.g. temperature, and biotic factors, e.g. the number of host species infected by parasites. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. Gene Expression in Trypanosomatid Parasites

    Directory of Open Access Journals (Sweden)

    Santiago Martínez-Calvillo

    2010-01-01

    Full Text Available The parasites Leishmania spp., Trypanosoma brucei, and Trypanosoma cruzi are the trypanosomatid protozoa that cause the deadly human diseases leishmaniasis, African sleeping sickness, and Chagas disease, respectively. These organisms possess unique mechanisms for gene expression such as constitutive polycistronic transcription of protein-coding genes and trans-splicing. Little is known about either the DNA sequences or the proteins that are involved in the initiation and termination of transcription in trypanosomatids. In silico analyses of the genome databases of these parasites led to the identification of a small number of proteins involved in gene expression. However, functional studies have revealed that trypanosomatids have more general transcription factors than originally estimated. Many posttranslational histone modifications, histone variants, and chromatin modifying enzymes have been identified in trypanosomatids, and recent genome-wide studies showed that epigenetic regulation might play a very important role in gene expression in this group of parasites. Here, we review and comment on the most recent findings related to transcription initiation and termination in trypanosomatid protozoa.

  20. Fauna Europaea: Helminths (Animal Parasitic

    Directory of Open Access Journals (Sweden)

    David Gibson

    2014-09-01

    Full Text Available Fauna Europaea provides a public web-service with an index of scientific names (including important synonyms of all living European land and freshwater animals, their geographical distribution at country level (up to the Urals, excluding the Caucasus region, and some additional information. The Fauna Europaea project covers about 230,000 taxonomic names, including 130,000 accepted species and 14,000 accepted subspecies, which is much more than the originally projected number of 100,000 species. This represents a huge effort by more than 400 contributing specialists throughout Europe and is a unique (standard reference suitable for many users in science, government, industry, nature conservation and education. Helminths parasitic in animals represent a large assemblage of worms, representing three phyla, with more than 200 families and almost 4,000 species of parasites from all major vertebrate and many invertebrate groups. A general introduction is given for each of the major groups of parasitic worms, i.e. the Acanthocephala, Monogenea, Trematoda (Aspidogastrea and Digenea, Cestoda and Nematoda. Basic information for each group includes its size, host-range, distribution, morphological features, life-cycle, classification, identification and recent key-works. Tabulations include a complete list of families dealt with, the number of species in each and the name of the specialist responsible for data acquisition, a list of additional specialists who helped with particular groups, and a list of higher taxa dealt with down to the family level. A compilation of useful references is appended.

  1. Eosinophilic fasciitis after parasite infection

    Directory of Open Access Journals (Sweden)

    Marta Oliveira

    2016-03-01

    Full Text Available Eosinophilic fasciitis is a systemic inflammatory disease characterized by symmetrical swelling and skin induration of the distal portions of the arms and/or legs, evolving into a scleroderma-like appearance, accompanied by peripheral blood eosinophilia. It is a rare disease with a poorly understood etiology. Corticosteroid treatment remains the standard therapy, either taken alone or in association with an immunosuppressive drug. This paper presents a case of a male patient with palpebral edema and marked eosinophilia, diagnosed with intestinal parasitic infection in October 2006. He was treated with an antiparasitic drug, but both the swelling and the analytical changes remained. This was followed by a skin and muscle biopsy, which turned out to be compatible with eosinophilic fasciitis. There was progressive worsening of the clinical state, with stiffness of the abdominal wall and elevated inflammatory parameters, and the patient was referred to the Immunology Department, medicated with corticosteroids and methotrexate. Over the years there were therapeutic adjustments and other causes were excluded. Currently the patient continues to be monitored, and there is no evidence of active disease. The case described in this article is interesting because of the diagnosis of eosinophilic fasciitis probably associated/coexisting with a parasite infection. This case report differs from others in that there is an uncommon cause associated with the onset of the disease, instead of the common causes such as trauma, medication, non-parasitic infections or cancer.

  2. Eosinophilic fasciitis after parasite infection.

    Science.gov (United States)

    Oliveira, Marta; Patinha, Fabia; Marinho, Antonio

    2016-01-01

    Eosinophilic fasciitis is a systemic inflammatory disease characterized by symmetrical swelling and skin induration of the distal portions of the arms and/or legs, evolving into a scleroderma-like appearance, accompanied by peripheral blood eosinophilia. It is a rare disease with a poorly understood etiology. Corticosteroid treatment remains the standard therapy, either taken alone or in association with an immunosuppressive drug. This paper presents a case of a male patient with palpebral edema and marked eosinophilia, diagnosed with intestinal parasitic infection in October 2006. He was treated with an antiparasitic drug, but both the swelling and the analytical changes remained. This was followed by a skin and muscle biopsy, which turned out to be compatible with eosinophilic fasciitis. There was progressive worsening of the clinical state, with stiffness of the abdominal wall and elevated inflammatory parameters, and the patient was referred to the Immunology Department, medicated with corticosteroids and methotrexate. Over the years there were therapeutic adjustments and other causes were excluded. Currently the patient continues to be monitored, and there is no evidence of active disease. The case described in this article is interesting because of the diagnosis of eosinophilic fasciitis probably associated/coexisting with a parasite infection. This case report differs from others in that there is an uncommon cause associated with the onset of the disease, instead of the common causes such as trauma, medication, non-parasitic infections or cancer.

  3. Parasites as prey in aquatic food webs: implications for predator infection and parasite transmission

    Science.gov (United States)

    Thieltges, David W.; Amundsen, Per-Arne; Hechinger, Ryan F.; Johnson, Pieter T.J.; Lafferty, Levin D.; Mouritsen, Kim N.; Preston, Daniel L.; Reise, Karsten; Zander, C. Dieter; Poulin, Robert

    2013-01-01

    While the recent inclusion of parasites into food-web studies has highlighted the role of parasites as consumers, there is accumulating evidence that parasites can also serve as prey for predators. Here we investigated empirical patterns of predation on parasites and their relationships with parasite transmission in eight topological food webs representing marine and freshwater ecosystems. Within each food web, we examined links in the typical predator–prey sub web as well as the predator–parasite sub web, i.e. the quadrant of the food web indicating which predators eat parasites. Most predator– parasite links represented ‘concomitant predation’ (consumption and death of a parasite along with the prey/host; 58–72%), followed by ‘trophic transmission’ (predator feeds on infected prey and becomes infected; 8–32%) and predation on free-living parasite life-cycle stages (4–30%). Parasite life-cycle stages had, on average, between 4.2 and 14.2 predators. Among the food webs, as predator richness increased, the number of links exploited by trophically transmitted parasites increased at about the same rate as did the number of links where these stages serve as prey. On the whole, our analyses suggest that predation on parasites has important consequences for both predators and parasites, and food web structure. Because our analysis is solely based on topological webs, determining the strength of these interactions is a promising avenue for future research.

  4. Single antigen flow beads for identification of human leukocyte antigen antibody specificities in hypersensitized patients with chronic renal failure

    OpenAIRE

    Soyöz, Mustafa; Kılıçaslan-Ayna, Tülay; Özkızılcık-Koçyiğit, Aslı; Güleç, Derya; Pirim, İbrahim

    2016-01-01

    Aims of this study Aims of this study were to identify class I and class II antibodies in highly sensitized patients by flow cytometry single antigen bead (FC-SAB) assay and to evaluate according to donor HLA type in order to increase their kidney transplantation chance. Material and methods We analyzed 60 hypersensitive patients of 351 individuals, who applied to our laboratory for PRA test in November 2013-December 2014. Flow cytometric PRA screening and single antigen bead commercial kits ...

  5. RNA trafficking in parasitic plant systems

    Directory of Open Access Journals (Sweden)

    Megan L LeBlanc

    2012-08-01

    Full Text Available RNA trafficking in plants contributes to local and long-distance coordination of plant development and response to the environment. However, investigations of mobile RNA identity and function are hindered by the inherent difficulty of tracing a given molecule of RNA from its cell of origin to its destination. Several methods have been used to address this problem, but all are limited to some extent by constraints associated with accurately sampling phloem sap or detecting trafficked RNA. Certain parasitic plant species form symplastic connections to their hosts and thereby provide an additional system for studying RNA trafficking. The haustorial connections of Cuscuta and Phelipanche species are similar to graft junctions in that they are able to transmit mRNAs, viral RNAs, siRNAs and proteins from the host plants to the parasite. In contrast to other graft systems, these parasites form connections with host species that span a wide phylogenetic range, such that a high degree of nucleotide sequence divergence may exist between host and parasites and allow confident identification of most host RNAs in the parasite system. The ability to identify host RNAs in parasites, and vice versa, will facilitate genomics approaches to understanding RNA trafficking. This review discusses the nature of host parasite connections and the potential significance of host RNAs for the parasite. Additional research on host-parasite interactions is needed to interpret results of RNA trafficking studies, but parasitic plants may provide a fascinating new perspective on RNA trafficking.

  6. Sex Steroids Effects on the Molting Process of the Helminth Human Parasite Trichinella spiralis

    Directory of Open Access Journals (Sweden)

    Romel Hernández-Bello

    2011-01-01

    Full Text Available We evaluated the in vitro effects of estradiol, progesterone, and testosterone on the molting process, which is the initial and crucial step in the development of the muscular larvae (ML or L1 to adult worm. Testosterone had no significative effect on the molting rate of the parasite, however, progesterone decreased the molting rate about a 50% in a concentration- and time-independent pattern, while estradiol had a slight effect (10%. The gene expression of caveolin-1, a specific gene used as a marker of parasite development, showed that progesterone and estradiol downregulated its expression, while protein expression was unaffected. By using flow citometry, a possible protein that is recognized by a commercial antiprogesterone receptor antibody was detected. These findings may have strong implications in the host-parasite coevolution, in the sex-associated susceptibility to this infection and could point out to possibilities to use antihormones to inhibit parasite development.

  7. A novel approach to parasite population genetics: experimental infection reveals geographic differentiation, recombination and host-mediated population structure in Pasteuria ramosa, a bacterial parasite of Daphnia.

    Science.gov (United States)

    Andras, J P; Ebert, D

    2013-02-01

    The population structure of parasites is central to the ecology and evolution of host-parasite systems. Here, we investigate the population genetics of Pasteuria ramosa, a bacterial parasite of Daphnia. We used natural P. ramosa spore banks from the sediments of two geographically well-separated ponds to experimentally infect a panel of Daphnia magna host clones whose resistance phenotypes were previously known. In this way, we were able to assess the population structure of P. ramosa based on geography, host resistance phenotype and host genotype. Overall, genetic diversity of P. ramosa was high, and nearly all infected D. magna hosted more than one parasite haplotype. On the basis of the observation of recombinant haplotypes and relatively low levels of linkage disequilibrium, we conclude that P. ramosa engages in substantial recombination. Isolates were strongly differentiated by pond, indicating that gene flow is spatially restricted. Pasteuria ramosa isolates within one pond were segregated completely based on the resistance phenotype of the host-a result that, to our knowledge, has not been previously reported for a nonhuman parasite. To assess the comparability of experimental infections with natural P. ramosa isolates, we examined the population structure of naturally infected D. magna native to one of the two source ponds. We found that experimental and natural infections of the same host resistance phenotype from the same source pond were indistinguishable, indicating that experimental infections provide a means to representatively sample the diversity of P. ramosa while reducing the sampling bias often associated with studies of parasite epidemics. These results expand our knowledge of this model parasite, provide important context for the large existing body of research on this system and will guide the design of future studies of this host-parasite system.

  8. Canine and feline parasitic zoonoses in China

    Directory of Open Access Journals (Sweden)

    Chen Jia

    2012-07-01

    Full Text Available Abstract Canine and feline parasitic zoonoses have not been given high priority in China, although the role of companion animals as reservoirs for zoonotic parasitic diseases has been recognized worldwide. With an increasing number of dogs and cats under unregulated conditions in China, the canine and feline parasitic zoonoses are showing a trend towards being gradually uncontrolled. Currently, canine and feline parasitic zoonoses threaten human health, and cause death and serious diseases in China. This article comprehensively reviews the current status of major canine and feline parasitic zoonoses in mainland China, discusses the risks dogs and cats pose with regard to zoonotic transmission of canine and feline parasites, and proposes control strategies and measures.

  9. Control and prevention of emerging parasitic zoonoses.

    Science.gov (United States)

    Chomel, Bruno B

    2008-09-01

    Emerging zoonoses have been defined as zoonoses that are newly recognised or newly evolved, or that have occurred previously but show an increase in incidence or expansion in geographical, host or vector range. Among parasitic zoonoses, protozoa are particularly likely to emerge. Control and prevention of emerging parasitic zoonoses are complex tasks that require an integrative and multidisciplinary approach. Reduction of parasite burden is certainly a major objective but cannot be set alone. Therefore, environmental and ecological modifications need to be implemented to reduce not only the parasitic load, but also the risk of parasite transmission. Finally, education and behavioral changes are critical for the success of both control and prevention of these diseases. However, without appropriate financial resources specifically allocated at the local and national levels as well as through international cooperation, control and prevention of these emerging parasitic diseases will not be possible.

  10. The evolution of parasitism in plants.

    Science.gov (United States)

    Westwood, James H; Yoder, John I; Timko, Michael P; dePamphilis, Claude W

    2010-04-01

    The multiple independent origins of plant parasitism suggest that numerous ancestral plant lineages possessed the developmental flexibility to meet the requirements of a parasitic life style, including such adaptations as the ability to recognize host plants, form an invasive haustorium, and regulate the transfer of nutrients and other molecules between two different plants. In this review, we focus on the Orobanchaceae, which are unique among the parasitic plants in that extant member species include the full range of host dependence from facultative to obligate parasites. The recent emergence of genomic resources for these plants should provide new insights into parasitic plant evolution and enable the development of novel genetic strategies for controlling parasitic weeds.

  11. PARASITES INFECTIONS OF GOLDFISH (Carassius auratus L.

    Directory of Open Access Journals (Sweden)

    Emil Gjurčević

    2006-01-01

    Full Text Available Removing fish from their natural environment, and placing them in aquariums, where large number is concentrated on small space, causes not only stress but increases the possibility of disease. In these unnatural conditions but often adequate for parasite reproduction, parasites can cause diseases leading to death. In our work we investigated parasites presence in goldfish (Carassius auratus L. kept in aquarium, from three different pet shops. The study showed presence of: Trypanoplasma sp., Trichodina sp., Ichthyophthirius multifiliis, Myxoboulus sp., Dactylogyrus sp. and Gyrodactylus sp. Considering the number of parasites found in examined fish, it can be possible that parasites can cause mortality in goldfish. Therefore, special caution has to be on quarantine and healthcare while importing especially exotic aquarium fish that may be infected with exotic parasites. In case of disease, proper treatment in due time has to be conducted.

  12. Parasitic diseases in the abdomen: imaging findings.

    Science.gov (United States)

    Lim, Jae Hoon

    2008-01-01

    Parasitic diseases of the liver and biliary tract include echinococcosis, schistosomiasis, toxocariasis, clonorchiasis, and opisthorchiasis, affecting millions people in some endemic areas. Amebiasis and ascariasis are believed to be the most common bowel lumen indwelling parasitic diseases, affecting billions people worldwide, but sometimes these parasites migrate inadvertently to the liver and biliary tract, resulting in liver abscess or obstructive jaundice. Imaging findings of these parasitic diseases are fairly characteristic and easy to recognize if radiologists are aware of the findings, especially in endemic areas. Because of increased immigration and frequent travelling, some patients with "exotic" parasitic diseases may be encountered in non-endemic areas, and the diagnosis may be delayed or difficult, and it is often made only after operation. This feature section was designed to provide the detailed imaging features of common parasitic diseases affecting the abdominal organs and peritoneal cavity, based on pathology-image correlation.

  13. Increased electron donor and electron acceptor characters enhance the adhesion between oil droplets and cells of Yarrowia lipolytica as evaluated by a new cytometric assay.

    Science.gov (United States)

    Aguedo, Mario; Waché, Yves; Mazoyer, Virginie; Sequeira-Le Grand, Anabelle; Belin, Jean-Marc

    2003-05-07

    The adhesion of methyl ricinoleate droplets to cells of the yeast Yarrowia lipolytica was investigated. A new cytometric method, relying on the double staining of fatty globules with Nile Red and of cells with Calcofluor, enabled us to quantify methyl ricinoleate droplet adhesion to cells precultured on a hydrophilic or on a hydrophobic carbon source. In this last case, droplet adsorption was enhanced and a MATS (microbial adhesion to solvents) test revealed that this increase was due to Lewis acid-base interactions and not to an increase in the hydrophobic properties of the cell surface. These preliminary results demonstrate that the developed cytometric method is promising for various applications concerning the study of interactions between microorganisms and an emulsified hydrophobic substrates.

  14. Interplay between fungicides and parasites: Tebuconazole, but not copper, suppresses infection in a Daphnia-Metschnikowia experimental model.

    Science.gov (United States)

    Cuco, Ana P; Abrantes, Nelson; Gonçalves, Fernando; Wolinska, Justyna; Castro, Bruno B

    2017-01-01

    Natural populations are commonly exposed to complex stress scenarios, including anthropogenic contamination and their biological enemies (e.g., parasites). The study of the pollutant-parasite interplay is especially important, given the need for adequate regulations to promote improved ecosystem protection. In this study, a host-parasite model system (Daphnia spp. and the microparasitic yeast Metschnikowia bicuspidata) was used to explore the reciprocal effects of contamination by common agrochemical fungicides (copper sulphate and tebuconazole) and parasite challenge. We conducted 21-day life history experiments with two host clones exposed to copper (0.00, 25.0, 28.8 and 33.1 μg L-1) or tebuconazole (0.00, 154, 192 and 240 μg L-1), in the absence or presence of the parasite. For each contaminant, the experimental design consisted of 2 Daphnia clones × 4 contaminant concentrations × 2 parasite treatments × 20 replicates = 320 experimental units. Copper and tebuconazole decreased Daphnia survival or reproduction, respectively, whilst the parasite strongly reduced host survival. Most importantly, while copper and parasite effects were mostly independent, tebuconazole suppressed infection. In a follow-up experiment, we tested the effect of a lower range of tebuconazole concentrations (0.00, 6.25, 12.5, 25.0, 50.0 and 100 μg L-1) crossed with increasing parasite challenge (2 Daphnia clones × 6 contaminant concentrations × 2 parasite levels × 20 replicates = 480 experimental units). Suppression of infection was confirmed at environmentally relevant concentrations (> 6.25 μg L-1), irrespective of the numbers of parasite challenge. The ecological consequences of such a suppression of infection include interferences in host population dynamics and diversity, as well as community structure and energy flow across the food web, which could upscale to ecosystem level given the important role of parasites.

  15. Interplay between fungicides and parasites: Tebuconazole, but not copper, suppresses infection in a Daphnia-Metschnikowia experimental model

    Science.gov (United States)

    Cuco, Ana P.; Abrantes, Nelson; Gonçalves, Fernando; Wolinska, Justyna

    2017-01-01

    Natural populations are commonly exposed to complex stress scenarios, including anthropogenic contamination and their biological enemies (e.g., parasites). The study of the pollutant-parasite interplay is especially important, given the need for adequate regulations to promote improved ecosystem protection. In this study, a host-parasite model system (Daphnia spp. and the microparasitic yeast Metschnikowia bicuspidata) was used to explore the reciprocal effects of contamination by common agrochemical fungicides (copper sulphate and tebuconazole) and parasite challenge. We conducted 21-day life history experiments with two host clones exposed to copper (0.00, 25.0, 28.8 and 33.1 μg L-1) or tebuconazole (0.00, 154, 192 and 240 μg L-1), in the absence or presence of the parasite. For each contaminant, the experimental design consisted of 2 Daphnia clones × 4 contaminant concentrations × 2 parasite treatments × 20 replicates = 320 experimental units. Copper and tebuconazole decreased Daphnia survival or reproduction, respectively, whilst the parasite strongly reduced host survival. Most importantly, while copper and parasite effects were mostly independent, tebuconazole suppressed infection. In a follow-up experiment, we tested the effect of a lower range of tebuconazole concentrations (0.00, 6.25, 12.5, 25.0, 50.0 and 100 μg L-1) crossed with increasing parasite challenge (2 Daphnia clones × 6 contaminant concentrations × 2 parasite levels × 20 replicates = 480 experimental units). Suppression of infection was confirmed at environmentally relevant concentrations (> 6.25 μg L-1), irrespective of the numbers of parasite challenge. The ecological consequences of such a suppression of infection include interferences in host population dynamics and diversity, as well as community structure and energy flow across the food web, which could upscale to ecosystem level given the important role of parasites. PMID:28231278

  16. 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.

  17. In vitro leukocyte response of three-spined sticklebacks (Gasterosteus aculeatus) to helminth parasite antigens.

    Science.gov (United States)

    Franke, Frederik; Rahn, Anna K; Dittmar, Janine; Erin, Noémie; Rieger, Jennifer K; Haase, David; Samonte-Padilla, Irene E; Lange, Joseph; Jakobsen, Per J; Hermida, Miguel; Fernández, Carlos; Kurtz, Joachim; Bakker, Theo C M; Reusch, Thorsten B H; Kalbe, Martin; Scharsack, Jörn P

    2014-01-01

    Helminth parasites of teleost fish have evolved strategies to evade and manipulate the immune responses of their hosts. Responsiveness of fish host immunity to helminth antigens may therefore vary depending on the degree of host-parasite counter-adaptation. Generalist parasites, infective for a number of host species, might be unable to adapt optimally to the immune system of a certain host species, while specialist parasites might display high levels of adaptation to a particular host species. The degree of adaptations may further differ between sympatric and allopatric host-parasite combinations. Here, we test these hypotheses by in vitro exposure of head kidney leukocytes from three-spined sticklebacks (Gasterosteus aculeatus) to antigens from parasites with a broad fish host range (Diplostomum pseudospathaceum, Triaenophorus nodulosus), a specific fish parasite of cyprinids (Ligula intestinalis) and parasites highly specific only to a single fish species as second intermediate host (Schistocephalus pungitii, which does not infect G. aculeatus, and Schistocephalus solidus, infecting G. aculeatus). In vitro responses of stickleback leukocytes to S. solidus antigens from six European populations, with S. solidus prevalence from parasites and among specialists, from parasites that do not infect G. aculeatus to a G. aculeatus-infecting species. Generalist parasites seem to maintain their ability to infect different host species at the costs of relatively higher immunogenicity compared to specialist parasites. In a comparison of sympatric and allopatric combinations of stickleback leukocytes and antigens from S. solidus, magnitudes of in vitro responses were dependent on the prevalence of the parasite in the population of origin, rather than on sympatry. Antigens from Norwegian (prevalence 30-50%) and Spanish (40-66%) S. solidus induced generally higher in vitro responses compared to S. solidus from two German (<1%) populations. Likewise, leukocytes from

  18. Association of parasitic infections and cancers.

    Science.gov (United States)

    Khurana, S; Dubey, M L; Malla, N

    2005-04-01

    Recent advances in the fields of molecular biology, epidemiology and infectious diseases have led to significant revelations to clarify the relationship between cancer and infective agents. This article reviews the relationship between parasitic infections and carcinogenesis and the possible mechanisms involved. Few parasites, e.g., Schistosoma haematobium and Opisthorchis viverrini have been found to be strongly associated with bladder cancer and cholangiocarcinoma respectively. The evidence for the association of several other parasites and cancers has also been postulated.

  19. LOG PERIODIC DIPOLE ARRAY WITH PARASITIC ELEMENTS

    Science.gov (United States)

    The design and measured characteristics of dipole and monopole versions of a log periodic array with parasitic elements are discussed. In a dipole...array with parasitic elements, these elements are used in place of every alternate dipole, thereby eliminating the need of a twisted feed arrangement...for the elements to obtain log periodic performance of the anntenna. This design with parasitic elements lends itself to a monopole version of the

  20. [Anisakiasis - a little-known parasitic zoonosis].

    Science.gov (United States)

    Bardoň, Jan; Harna, Jiří; Pijáček, Martin

    2013-06-01

    Parasites of the family Anisakidae cause enteric parasitic zoonoses developing after consumption of inadequately cooked marine fish. Cases of such diseases are reported mainly from Japan or other countries where raw or uncooked fish are traditionally consumed. The presented short communication briefly reports detection of larvae of Pseudoterranova spp., parasites of the family Anisakidae, in a fresh chilled angler-fish (Lophius piscatorius) bought at a retail store in the Czech Republic.