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

    LENUS (Irish Health Repository)

    Limaye, Sandhya

    2009-04-15

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

  2. Flow cytometric characterization of cerebrospinal fluid cells.

    Science.gov (United States)

    de Graaf, Marieke T; de Jongste, Arjen H C; Kraan, Jaco; Boonstra, Joke G; Sillevis Smitt, Peter A E; Gratama, Jan W

    2011-09-01

    Flow cytometry facilitates the detection of a large spectrum of cellular characteristics on a per cell basis, determination of absolute cell numbers and detection of rare events with high sensitivity and specificity. White blood cell (WBC) counts in cerebrospinal fluid (CSF) are important for the diagnosis of many neurological disorders. WBC counting and differential can be performed by microscopy, hematology analyzers, or flow cytometry. Flow cytometry of CSF is increasingly being considered as the method of choice in patients suspected of leptomeningeal localization of hematological malignancies. Additionally, in several neuroinflammatory diseases such as multiple sclerosis and paraneoplastic neurological syndromes, flow cytometry is commonly performed to obtain insight into the immunopathogenesis of these diseases. Technically, the low cellularity of CSF samples, combined with the rapidly declining WBC viability, makes CSF flow cytometry challenging. Comparison of flow cytometry with microscopic and molecular techniques shows that each technique has its own advantages and is ideally combined. We expect that increasing the number of flow cytometric parameters that can be simultaneously studied within one sample, will further refine the information on CSF cell subsets in low-cellular CSF samples and enable to define cell populations more accurately. Copyright © 2011 International Clinical Cytometry Society.

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

    African Journals Online (AJOL)

    Jane

    2011-10-24

    Oct 24, 2011 ... daughter cells, leading to a characteristic flow cytometric profile where a ... cell recognition without any impact on bone marrow hemato- ... cells of various cancer cells that load CFSE concentration ... (B) Target cells (R1) were further analyzed in an FL1/FL3 dot plot, ..... hematopoietic cell transplantation.

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

    African Journals Online (AJOL)

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

  5. Technical discussions II - Flow cytometric analysis

    NARCIS (Netherlands)

    Cunningham, A; Cid, A; Buma, AGJ

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

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

    Science.gov (United States)

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

    2018-02-01

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

  7. Flow cytometric determination of micronucleus frequency.

    Science.gov (United States)

    Elhajouji, Azeddine; Lukamowicz-Rajska, Magdalena

    2013-01-01

    During the last two decades the micronucleus (MN) test has been extensively used as a genotoxicity screening tool of chemicals and in a variety of exploratory and mechanistic investigations. The MN is a biomarker for chromosomal damage or mitotic abnormalities, since it can originate from chromosome fragments or whole chromosomes that fail to be incorporated into daughter nuclei during mitosis (Fenech et al., Mutagenesis 26:125-132, 2011; Kirsch-Volders et al., Arch Toxicol 85:873-899, 2011). The simplicity of scoring, accuracy, amenability to automation by image analysis or flow cytometry, and readiness to be applied to a variety of cell types either in vitro or in vivo have made it a versatile tool that has contributed to a large extent in our understanding of key toxicological issues related to genotoxins and their effects at the cellular and organism levels. Recently, the final acceptance of the in vitro MN test guideline 487 (OECD Guideline for Testing of Chemicals, In vitro mammalian cell micronucleus test 487. In vitro mammalian cell micronucleus test (MNVIT). Organization for Economic Cooperation and Development, Paris, 2010) together with the standard in vivo MN test OECD guideline 474 (OECD Guideline for The Testing of Chemicals, Mammalian erythrocyte micronucleus test no. 474. Organization for Economic Cooperation and Development, Paris, 1997) will further position the assay as a key driver in the determination of the genotoxicity potential in exploratory research as well as in the regulatory environment. This chapter covers to some extent the protocol designs and experimental steps necessary for a successful performance of the MN test and an accurate analysis of the MN by the flow cytometry technique.

  8. Uncovering Aberrant Mutant PKA Function with Flow Cytometric FRET

    Directory of Open Access Journals (Sweden)

    Shin-Rong Lee

    2016-03-01

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

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

    Science.gov (United States)

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

    2012-01-01

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

  10. Leukocytospermia and sperm preparation - a flow cytometric study

    Directory of Open Access Journals (Sweden)

    Perticarari Sandra

    2009-01-01

    Full Text Available Abstract Background Leukocytes represent the predominant source of reactive oxygen species both in seminal plasma and in sperm suspensions and have been demonstrated to negatively influence sperm function and fertilization rate in assisted reproduction procedures. Peroxidase test is the standard method recommended by WHO to detect semen leukocytes but it may be inaccurate. The aims of this study were (i to compare the efficiency of swim-up and density-gradient centrifugation techniques in removing seminal leukocytes, (ii to examine the effect of leukocytes on sperm preparation, and (iii to compare flow cytometry and peroxidase test in determining leukocyte concentration in semen using a multiparameter flow cytometric method. Methods Semen samples from 126 male partners of couples undergoing infertility investigations were analyzed for leukocytospermia using standard optical microscopy and flow cytometry. Sixty-nine out of 126 samples were also processed using simultaneously the swim-up and density-gradient centrifugation techniques. A multiparameter flow cytometric analysis to assess simultaneously sperm concentration, sperm viability, sperm apoptosis, and leukocyte concentration was carried out on neat and prepared sperm. Results Both sperm preparation methods removed most seminal leukocytes. However, the concentration of leukocytes was significantly lower after swim-up compared to that after density-gradient centrifugation preparation. Leukocytes concentration, either initial or in prepared fractions, was not correlated with sperm parameters (optical microscopy and flow cytometry parameters after semen processing. There was no correlation between leukocyte concentration in the ejaculate and sperm recovery rate, whereas a significant correlation was found between the concentration of the residual leukocytes in prepared fractions and viable sperm recovery rate. Although the overall concordance between the flow cytometry and the optical

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

    Science.gov (United States)

    Shapiro, Howard M

    2011-01-01

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

  12. Flow cytometric and morphological analyses of Pinus pinaster somatic embryogenesis.

    Science.gov (United States)

    Marum, Liliana; Loureiro, João; Rodriguez, Eleazar; Santos, Conceição; Oliveira, M Margarida; Miguel, Célia

    2009-09-25

    An approach combining morphological profiling and flow cytometric analysis was used to assess genetic stability during the several steps of somatic embryogenesis in Pinus pinaster. Embryogenic cell lines of P. pinaster were established from immature zygotic embryos excised from seeds obtained from open-pollinated trees. During the maturation stage, phenotype of somatic embryos was characterized as being either normal or abnormal. Based upon the prevalent morphological traits, different types of abnormal embryos underwent further classification and quantification. Nuclear DNA content of maritime pine using the zygotic embryos was estimated to be 57.04 pg/2C, using propidium iodide flow cytometry. According to the same methodology, no significant differences (P< or =0.01) in DNA ploidy were detected among the most frequently observed abnormal phenotypes, embryogenic cell lines, zygotic and normal somatic embryos, and somatic embryogenesis-derived plantlets. Although the differences in DNA ploidy level do not exclude the occurrence of a low level of aneuploidy, the results obtained point to the absence of major changes in ploidy level during the somatic embryogenesis process of this economically important species. Therefore, our primary goal of true-to-typeness was assured at this level.

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

    International Nuclear Information System (INIS)

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

    1988-01-01

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

  14. Flow cytometric life cycle analysis in cellular radiation biology

    International Nuclear Information System (INIS)

    Wood, J.C.S.

    1982-01-01

    Three approaches to flow cytometric histogram analysis were developed: (1) differential histogram analysis, (2) DNA histogram analysis, and (3) multiparameter data analysis. These techniques were applied to an important unresolved problem in radiation biology. The initial responses to irradiation of a mammalian cell which occur during the first two cell cycles following the irradiation are of considerable interest to the radiation biologist. During the first two post-irradiation cell cycles, cells which ultimately will survive repair radiation-induced damage, while some cells begin to express some of the radiation-induced nuclear and chomatin damage. Caffeine- and thymidine-treated, and untreated gamma-irradiated cell populations were studied with respect to the radiation-induced G2 delay, deficient DNA synthesis, and the appearance of cells with abnormal DNA contents. It is hypothesized that the measured deficiency in DNA synthesis observed in the first post-irradiation cell cycle may be a result of daughter cells from abnormal first post-irradiation mitoses

  15. Quantifying Distribution of Flow Cytometric TCR-Vβ Usage with Economic Statistics.

    Directory of Open Access Journals (Sweden)

    Kornelis S M van der Geest

    Full Text Available Measuring changes of the T cell receptor (TCR repertoire is important to many fields of medicine. Flow cytometry is a popular technique to study the TCR repertoire, as it quickly provides insight into the TCR-Vβ usage among well-defined populations of T cells. However, the interpretation of the flow cytometric data remains difficult, and subtle TCR repertoire changes may go undetected. Here, we introduce a novel means for analyzing the flow cytometric data on TCR-Vβ usage. By applying economic statistics, we calculated the Gini-TCR skewing index from the flow cytometric TCR-Vβ analysis. The Gini-TCR skewing index, which is a direct measure of TCR-Vβ distribution among T cells, allowed us to track subtle changes of the TCR repertoire among distinct populations of T cells. Application of the Gini-TCR skewing index to the flow cytometric TCR-Vβ analysis will greatly help to gain better understanding of the TCR repertoire in health and disease.

  16. 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...

  17. Immunologic status of children with thyroid cancer living near Chernobyl (flow cytometric and electron microscopic study)

    International Nuclear Information System (INIS)

    Zak, K.P.; Gruzov, M.A.; Bolshova, B.V.; Afanasyeva, V.V.; Shlyakhovenko, V.S.; Vishnevskaya, O.A.; Tronko, N.D.

    1996-01-01

    It hag been carded out a light, election microscopic and flow cytometric study of blood leukocyte of children with malignant tumors (papillary carcinoma) of thyroid gland who were living at the moment of the accident near Chernobyl. The results obtained point out the presence of some disturbances of immune status of these children

  18. 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

  19. 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

    were defined: region 1 includes bacteria mainly associated with poor hygiene, region 2 includes psychrotrophic hygiene bacteria and region 3 includes bacteria mainly related to mastitis. The ability of the flow cytometric technique to predict the main cause of elevated bacterial counts on routine...

  20. 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

  1. 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

  2. Flow cytometric applications of tumor biology: prospects and pitfalls

    International Nuclear Information System (INIS)

    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

  3. Flow cytometric applications to tumour biology: prospects and pitfalls

    International Nuclear Information System (INIS)

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

    1980-01-01

    A brief review of cytometry instrumentation and its potential applications in tumour biology is presented. DNA distribution measurements of cells from spontaneous dog tumours 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, however, to utilize the sophisticated developments in flow cytometry instrumentation. (author)

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

    Czech Academy of Sciences Publication Activity Database

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

    2012-01-01

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

  5. Solid KHT tumor dispersal for flow cytometric cell kinetic analysis

    International Nuclear Information System (INIS)

    Pallavicini, M.G.; Folstad, L.J.; Dunbar, C.

    1981-01-01

    A bacterial neutral protease was used to disperse KHT solid tumors into single cell suspensions suitable for routine cell kinetic analysis by flow cytometry and for clonogenic cell survival. Neutral protease disaggregation under conditions which would be suitable for routine tumor dispersal was compared with a trypsin/DNase procedure. Cell yield, clonogenic cell survival, DNA distributions of untreated and drug-perturbed tumors, rates of radioactive precursor incorporation during the cell cycle, and preferential cell cycle phase-specific cell loss were investigated. Tumors dispersed with neutral protease yielded approximately four times more cells than those dispersed with trypsin/DNase and approximately a 1.5-fold higher plating efficiency in a semisolid agar system. Quantitative analysis of DNA distributions obtained from untreated and cytosine-arabinoside-perturbed tumors produced similar results with both dispersal procedures. The rates of incorporation of tritiated thymidine during the cell cycle were also similar with neutral protease and trypsin/DNase dispersal. Preferential phase-specific cell loss was not obseved with either technique. We find that neutral protease provides good single cell suspensions of the KHT tumor for cell survival measurements and for cell kinetic analysis of drug-induced perturbations by flow cytometry. In addition, the high cell yields facilitate electronic cell sorting where large numbers of cells are often required

  6. A flow cytometric method for assessing viability of intraerythrocytic hemoparasites.

    Science.gov (United States)

    Wyatt, C R; Goff, W; Davis, W C

    1991-06-24

    We have developed a rapid, reliable method of evaluating growth and viability of intraerythrocytic protozoan hemoparasites. The assay involves the selective uptake and metabolic conversion of hydroethidine to ethidium by live parasites present in intact erythrocytes. The red fluorescence imparted by ethidium intercalated into the DNA of the parasite permits the use of flow cytometry to distinguish infected erythrocytes with viable parasites from uninfected erythrocytes and erythrocytes containing dead parasites. Comparison of the fluorochromasia technique of enumerating the number and viability of hemoparasites in cultured erythrocytes with enumeration in Giemsa-stained films and uptake of [3H]hypoxanthine demonstrated the fluorochromasia technique yields comparable results. Studies with the hemoparasite, Babesia bovis, have shown the fluorochromasia technique can also be used to monitor the effect of parasiticidal drugs on parasites in vitro. The cumulative studies with the fluorochromasia assay suggest the assay will also prove useful in investigations focused on analysis of the immune response to hemoparasites and growth in vitro.

  7. Coexpression of multidrug resistance involve proteins: a flow cytometric analysis.

    Science.gov (United States)

    Boutonnat, J; Bonnefoix, T; Mousseau, M; Seigneurin, D; Ronot, X

    1998-01-01

    Cross resistance to multiple natural cytotoxic products represents a major obstacle in myeloblastic acute leukaemia (AML). Multidrug resistance (MDR) often involves overexpression of plasma membrane drug transporter P-glycoprotein (PGP) or the resistance associated protein (MRP). Recently, a protein overexpressed in a non-PGP MDR lung cancer cell line and termed lung resistance related protein (LRP) was identified. These proteins are known to be associated with a bad prognosis in AML. We have developed a triple indirect labelling analysed by flow cytometry to detect the coexpression of these proteins. Since no cell line expressing all three antigens is known, we mixed K562 cells (resistant to Adriblastine, PGP+, MRP-, LRP-) with GLC4 cells (resistant to Adriblastine, PGP-, MRP+, LRP+) to create a model system to test the method. The antibodies used were UIC2 for PGP, MRPm6 for MRP and LRP56 for LRP. They were revealed by Fab'2 coupled with Fluoresceine-isothiocyanate, Phycoerythrin or Tricolor with isotype specificity. Cells were fixed and permeabilized after PGP labelling because MRPm6 and LRP56 recognize intracellular epitopes. PGP and LRP were easily detected. MRP is expressed at relatively low levels and was more difficult to detect because in the triple labelling the non specific staining was higher than in a single labelling. Despite the increased background in the triple labelling we were able to detect coexpression of PGP, MRP, LRP by flow cytometry. This method appears to be very useful to detect coexpression of markers in AML. Such coexpression could modify the therapeutic approach with revertants.

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

    Directory of Open Access Journals (Sweden)

    Fatemeh Atabaki pasdar

    2015-05-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-12-01

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

  10. Use of LysoTracker dyes: a flow cytometric study of autophagy.

    Science.gov (United States)

    Chikte, Shaheen; Panchal, Neelam; Warnes, Gary

    2014-02-01

    The flow cytometric use of LysoTracker dyes was employed to investigate the autophagic process and to compare this with the upregulation of autophagy marker, the microtubule-associated protein LC3B. Although the mechanism of action of LysoTracker dyes is not fully understood, they have been used in microscopy to image acidic spherical organelles, and their use in flow cytometry has not been thoroughly investigated in the study of autophagy. This investigation uses numerous autophagy-inducing agents including chloroquine (CQ), rapamycin, low serum (used to analyze patient cells as well as easier to use and significantly less costly. Copyright © 2013 International Society for Advancement of Cytometry.

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

    Science.gov (United States)

    Józwa, Wojciech; Czaczyk, Katarzyna

    2012-04-02

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

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

    Directory of Open Access Journals (Sweden)

    D Cavallo

    2010-01-01

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

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

    DEFF Research Database (Denmark)

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

    1994-01-01

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

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

    International Nuclear Information System (INIS)

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

    1996-01-01

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

  15. Quality control in the application of flow cytometric assays of genetic damage due to environmental contaminants

    International Nuclear Information System (INIS)

    McCreedy, C.D.; Jagoe, C.H.; Brisbin, I.L. Jr.; Wentworth, R.W.; Dallas, C.E.

    1995-01-01

    Clinical technologies, such as flow cytometry, are increasingly adopted by environmental toxicologists to identify resource damage associated with exposure to xenobiotics. One application of flow cytometry allows the rapid determination of the DNA content of large numbers of individual cells, and can be used to detect aneuploidy or other genetic abnormalities. The laboratory has used this methodology in studies of genetic toxicology of fish, birds, arid mammals exposed to organic pollutants, metals and radionuclides, However, without appropriate quality controls, false positive results and other artifacts can arise from sample handling and preparations, inter and intra-individual variations, instrument noise and other sources. The authors describe the routine measures this laboratory employs to maintain quality control of genomic DNA analysis, including the control of staining conditions, machine standardization, pulse-width doublet discrimination, and, in particular, the use of internal controls and the use of time as a cytometric parameter. Neglect of these controls can produce erroneous results, leading to conclusions of genetic abnormalities when none are present. Conversely, attention to these controls, routinely used in clinical settings, facilitates the interpretation of flow cytometric data and allows the application of this sensitive indicator of genotoxic effects to a variety of environmental problems

  16. Flow cytometric techniques for detection of candidate cancer stem cell subpopulations in canine tumour models.

    Science.gov (United States)

    Blacking, T M; Waterfall, M; Samuel, K; Argyle, D J

    2012-12-01

    The cancer stem cell (CSC) hypothesis proposes that tumour growth is maintained by a distinct subpopulation of 'CSC'. This study applied flow cytometric methods, reported to detect CSC in both primary and cultured cancer cells of other species, to identify candidate canine subpopulations. Cell lines representing diverse canine malignancies, and cells derived from spontaneous canine tumours, were evaluated for expression of stem cell-associated surface markers (CD34, CD44, CD117 and CD133) and functional properties [Hoecsht 33342 efflux, aldehyde dehydrogenase (ALDH) activity]. No discrete marker-defined subsets were identified within established cell lines; cells derived directly from spontaneous tumours demonstrated more heterogeneity, although this diminished upon in vitro culture. Functional assays produced variable results, suggesting context-dependency. Flow cytometric methods may be adopted to identify putative canine CSC. Whilst cell lines are valuable in assay development, primary cells may provide a more rewarding model for studying tumour heterogeneity in the context of CSC. However, it will be essential to fully characterize any candidate subpopulations to ensure that they meet CSC criteria. © 2011 Blackwell Publishing Ltd.

  17. Vortex-dislodged cells from bone marrow trephine biopsy yield satisfactory results for flow cytometric immunophenotyping.

    Science.gov (United States)

    Bommannan, K; Sachdeva, M U S; Gupta, M; Bose, P; Kumar, N; Sharma, P; Naseem, S; Ahluwalia, J; Das, R; Varma, N

    2016-10-01

    A good bone marrow (BM) sample is essential in evaluating many hematologic disorders. An unsuccessful BM aspiration (BMA) procedure precludes a successful flow cytometric immunophenotyping (FCI) in most hematologic malignancies. Apart from FCI, most ancillary diagnostic techniques in hematology are less informative. We describe the feasibility of FCI in vortex-dislodged cell preparation obtained from unfixed trephine biopsy (TB) specimens. In pancytopenic patients and dry tap cases, routine diagnostic BMA and TB samples were complemented by additional trephine biopsies. These supplementary cores were immediately transferred into sterile tubes filled with phosphate-buffered saline, vortexed, and centrifuged. The cell pellet obtained was used for flow cytometric immunophenotyping. Of 7955 BMAs performed in 42 months, 34 dry tap cases were eligible for the study. Vortexing rendered a cell pellet in 94% of the cases (32 of 34), and FCI rendered a rapid diagnosis in 100% of the cases (32 of 32) where cell pellets were available. We describe an efficient procedure which could be effectively utilized in resource-limited centers and reduce the frequency of repeat BMA procedures. © 2016 John Wiley & Sons Ltd.

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

    International Nuclear Information System (INIS)

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

    2006-01-01

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

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

    International Nuclear Information System (INIS)

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

    2012-01-01

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

  20. 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.

  1. 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 < 0.05). This flow cytometric method takes advantage of the propensity of 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. Published by Elsevier Inc.

  2. 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.

  3. Automatic analysis of flow cytometric DNA histograms from irradiated mouse male germ cells

    International Nuclear Information System (INIS)

    Lampariello, F.; Mauro, F.; Uccelli, R.; Spano, M.

    1989-01-01

    An automatic procedure for recovering the DNA content distribution of mouse irradiated testis cells from flow cytometric histograms is presented. First, a suitable mathematical model is developed, to represent the pattern of DNA content and fluorescence distribution in the sample. Then a parameter estimation procedure, based on the maximum likelihood approach, is constructed by means of an optimization technique. This procedure has been applied to a set of DNA histograms relative to different doses of 0.4-MeV neutrons and to different time intervals after irradiation. In each case, a good agreement between the measured histograms and the corresponding fits has been obtained. The results indicate that the proposed method for the quantitative analysis of germ cell DNA histograms can be usefully applied to the study of the cytotoxic and mutagenic action of agents of toxicological interest such as ionizing radiations.18 references

  4. Evaluation of Prognostic Factors Following Flow-Cytometric DNA Analysis after Cytokeratin Labelling: I. Breast Cancer

    Directory of Open Access Journals (Sweden)

    Pauline Wimberger

    2002-01-01

    Full Text Available In gynecologic oncology valid prognostic factors are necessary to estimate the course of disease and to define biologically similar subgroups for analysis of therapeutic efficacy. The presented study is a prospective study concerning prognostic significance of DNA ploidy and S‐phase fraction in breast 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 327 fresh specimens of primary breast cancer. Univariate analysis in breast cancer detected the prognostic significance of DNA‐ploidy, S‐phase fraction and CV (coefficient of variation of G0G1‐peak of tumor cells for clinical outcome, especially for nodal‐negative patients. Multivariate analysis could not confirm prognostic evidence of DNA‐ploidy and S‐phase fraction. In conclusion, in breast cancer no clinical significance for determination of DNA‐parameters was found.

  5. Flow cytometric determination of radiation-induced chromosome damage and its correlation with cell survival

    International Nuclear Information System (INIS)

    Welleweerd, J.; Wilder, M.E.; Carpenter, S.G.; Raju, M.R.

    1984-01-01

    Chinese hamster M3-1 cells were irradiated with several doses of x rays or α particles from 238 Pu. Propidium iodide-stained chromosome suspensions were prepared at different times after irradiation; cells were also assayed for survival. The DNA histograms of these chromosomes showed increased background counts with increased doses of radiation. This increase in background was cell-cycle dependent and was correlated with cell survival. The correlation between radiation-induced chromosome damage and cell survival was the same for X rays and α particles. Data are presented which indicate that flow cytometric analysis of chromosomes of irradiated cell populations can be a useful adjunct to classical cytogenic analysis of irradiation-induced chromosomal damage by virtue of its ability to express and measure chromosomal damage not seen by classical cytogenic methods

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

    International Nuclear Information System (INIS)

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

    1991-01-01

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

  7. 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 and a ...

  8. 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

  9. 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.

  10. Quantification of circulating mature endothelial cells using a whole blood four-color flow cytometric assay.

    Science.gov (United States)

    Jacques, Nathalie; Vimond, Nadege; Conforti, Rosa; Griscelli, Franck; Lecluse, Yann; Laplanche, Agnes; Malka, David; Vielh, Philippe; Farace, Françoise

    2008-09-15

    Circulating endothelial cells (CEC) are currently proposed as a potential biomarker for measuring the impact of anti-angiogenic treatments in cancer. However, the lack of consensus on the appropriate method of CEC measurement has led to conflicting data in cancer patients. A validated assay adapted for evaluating the clinical utility of CEC in large cohorts of patients undergoing anti-angiogenic treatments is needed. We developed a four-color flow cytometric assay to measure CEC as CD31(+), CD146(+), CD45(-), 7-amino-actinomycin-D (7AAD)(-) events in whole blood. The distinctive features of the assay are: (1) staining of 1 ml whole blood, (2) use of a whole blood IgPE control to measure accurately background noise, (3) accumulation of a large number of events (almost 5 10(6)) to ensure statistical analysis, and (4) use of 10 microm fluorescent microbeads to evaluate the event size. Assay reproducibility was determined in duplicate aliquots of samples drawn from 20 metastatic cancer patients. Assay linearity was tested by spiking whole blood with low numbers of HUVEC. Five-color flow cytometric experiments with CD144 were performed to confirm the endothelial origin of the cells. CEC were measured in 20 healthy individuals and 125 patients with metastatic cancer. Reproducibility was good between duplicate aliquots (r(2)=0.948, mean difference between duplicates of 0.86 CEC/ml). Detected HUVEC correlated with spiked HUVEC (r(2)=0.916, mean recovery of 100.3%). Co-staining of CD31, CD146 and CD144 confirmed the endothelial nature of cells identified as CEC. Median CEC levels were 6.5/ml (range, 0-15) in healthy individuals and 15.0/ml (range, 0-179) in patients with metastatic carcinoma (p<0.001). The assay proposed here allows reproducible and sensitive measurement of CEC by flow cytometry and could help evaluate CEC as biomarkers of anti-angiogenic therapies in large cohorts of patients.

  11. Determination of chitin content in fungal cell wall: an alternative flow cytometric method.

    Science.gov (United States)

    Costa-de-Oliveira, Sofia; Silva, Ana P; Miranda, Isabel M; Salvador, Alexandre; Azevedo, Maria M; Munro, Carol A; Rodrigues, Acácio G; Pina-Vaz, Cidália

    2013-03-01

    The conventional methods used to evaluate chitin content in fungi, such as biochemical assessment of glucosamine release after acid hydrolysis or epifluorescence microscopy, are low throughput, laborious, time-consuming, and cannot evaluate a large number of cells. We developed a flow cytometric assay, efficient, and fast, based on Calcofluor White staining to measure chitin content in yeast cells. A staining index was defined, its value was directly related to chitin amount and taking into consideration the different levels of autofluorecence. Twenty-two Candida spp. and four Cryptococcus neoformans clinical isolates with distinct susceptibility profiles to caspofungin were evaluated. Candida albicans clinical isolate SC5314, and isogenic strains with deletions in chitin synthase 3 (chs3Δ/chs3Δ) and genes encoding predicted GlycosylPhosphatidylInositol (GPI)-anchored proteins (pga31Δ/Δ and pga62Δ/Δ), were used as controls. As expected, the wild-type strain displayed a significant higher chitin content (P relationship between chitin content and antifungal drug susceptibility phenotype was found, an association was established between the paradoxical growth effect in the presence of high caspofungin concentrations and the chitin content. This novel flow cytometry protocol revealed to be a simple and reliable assay to estimate cell wall chitin content of fungi. Copyright © 2013 International Society for Advancement of Cytometry.

  12. Flow cytometric analysis of cell-surface and intracellular antigens in leukemia diagnosis.

    Science.gov (United States)

    Knapp, W; Strobl, H; Majdic, O

    1994-12-15

    New technology allows highly sensitive flow cytometric detection and quantitative analysis of intracellular antigens in normal and malignant hemopoietic cells. With this technology, the earliest stages of myeloid and lymphoid differentiation can easily and reliably be identified using antibodies directed against (pro-)myeloperoxidase/MPO, CD22 and CD3 antigens, respectively. Particularly for the analysis of undifferentiated acute myeloblastic leukemia (AML) cells, the immunological demonstration of intracellular MPO or its enzymatically inactive proforms is highly relevant, since other myeloid marker molecules such as CD33, CD13, or CDw65 are either not restricted to the granulomonocytic lineage or appear later in differentiation. By combining MPO staining with staining for lactoferrin (LF), undifferentiated cells can be distinguished from the granulomonocytic maturation compartment in bone marrow, since LF is selectively expressed from the myelocyte stage of differentiation onward. The list of informative intracellular antigens to be used in leukemia cell analysis will certainly expand in the near future. One candidate, intracellular CD68, has already been tested by us, and results are presented. Also dealt within this article are surface marker molecules not (as yet) widely used in leukemia cell analysis but with the potential to provide important additional information. Among them are the surface structures CD15, CD15s, CDw65, CD79a (MB-1), CD79b (B29), CD87 (uPA-R), and CD117 (c-kit).

  13. 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.

  14. 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.

  15. A flow-cytometric gram-staining technique for milk-associated bacteria.

    Science.gov (United States)

    Holm, Claus; Jespersen, Lene

    2003-05-01

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

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

    NARCIS (Netherlands)

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

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

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

    Science.gov (United States)

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

    2017-10-23

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

  18. Genome size variation among and within Camellia species by using flow cytometric analysis.

    Directory of Open Access Journals (Sweden)

    Hui Huang

    Full Text Available BACKGROUND: The genus Camellia, belonging to the family Theaceae, is economically important group in flowering plants. Frequent interspecific hybridization together with polyploidization has made them become taxonomically "difficult taxa". The DNA content is often used to measure genome size variation and has largely advanced our understanding of plant evolution and genome variation. The goals of this study were to investigate patterns of interspecific and intraspecific variation of DNA contents and further explore genome size evolution in a phylogenetic context of the genus. METHODOLOGY/PRINCIPAL FINDINGS: The DNA amount in the genus was determined by using propidium iodide flow cytometry analysis for a total of 139 individual plants representing almost all sections of the two subgenera, Camellia and Thea. An improved WPB buffer was proven to be suitable for the Camellia species, which was able to counteract the negative effects of secondary metabolite and generated high-quality results with low coefficient of variation values (CV <5%. Our results showed trivial effects on different tissues of flowers, leaves and buds as well as cytosolic compounds on the estimation of DNA amount. The DNA content of C. sinensis var. assamica was estimated to be 1C = 3.01 pg by flow cytometric analysis, which is equal to a genome size of about 2940 Mb. CONCLUSION: Intraspecific and interspecific variations were observed in the genus Camellia, and as expected, the latter was larger than the former. Our study suggests a directional trend of increasing genome size in the genus Camellia probably owing to the frequent polyploidization events.

  19. 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.

  20. Flow cytometric kinetic assay of the activity of Na+/H+ antiporter in mammalian cells.

    Science.gov (United States)

    Dolz, María; O'Connor, José-Enrique; Lequerica, Juan L

    2004-10-01

    The Na(+)/H(+) exchanger (NHE) of mammalian cells is an integral membrane protein that extrudes H(+) ion in exchange for extracellular Na(+) and plays a crucial role in the regulation of intracellular pH (pHi). Thus, when pHi is lowered, NHE extrudes protons at a rate depending of pHi that can be expressed as pH units/s. To abolish the activity of other cellular pH-restoring systems, cells were incubated in bicarbonate-free Dulbecco's modified Eagle's medium buffered with HEPES. Flow cytometry was used to determine pHi with 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester or 5-(and-6)-carboxy SNARF-1 acetoxymethyl ester acetate, and the appropriate fluorescence ratios were measured. The calibration of fluorescence ratios versus pHi was established by using ionophore nigericin. The activity of NHE was calculated by a kinetic flow cytometric assay as the slope at time 0 of the best-fit curve of pHi recovery versus time after intracellular acidification with a pulse of exogenous sodium propionate. The kinetic method allowed determination of the pHi-dependent activity of NHE in cell lines and primary cell cultures. NHE activity values were demonstrated to be up to 0.016 pH units/s within the pHi range of 7.3 to 6.3. The inhibition of NHE activity by the specific inhibitor ethyl isopropyl amiloride was easily detected by this method. The assay conditions can be used to relate variations in pHi with the activity of NHE and provide a standardized method to compare between different cells, inhibitors, models of ischemia by acidification, and other relevant experimental or clinical situations.

  1. Flow cytometric detection of micronuclei by combined staining of DNA and membranes

    International Nuclear Information System (INIS)

    Wessels, J.M.; Nuesse, M.

    1995-01-01

    A new staining method is presented for flow cytometric measurement of micronuclei (MN) in cell cultures and human lymphocytes using membrane-specific fluorescent dyes in addition to DNA staining. Several combinations of fluorescent membrane and DNA dyes were studied for a better discrimination of MN from debris in a suspension of nuclei and micronuclei. For staining of membranes, the lipophilic dyes 2-hydroxyethyl-7,12,17-tris(methoxyethyl)porphycene (HEPn) and 1,6-diphenyl-1,3,5-hexatriene (DPH) were used in combination with ethidium bromide (EB), proflavine (PF), and Hoechst 33258 (HO). Due to their spectral properties, HO or EB combined with HEPn were not as suitable for the discrimination of MN from debris as was HEPn in combination with PF. With HEPn in combination with PF, however, additional noise was found at low fluorescence intensities, probably due to free fluorescent dye molecules in the solution. The optimal simultaneous staining of membranes and DNA was obtained using a combination of DPH and EB. The induction of MN in Chinese hamster and mouse NIH-3T3 cells by UV-B illumination was studied with this new staining technique. UV-B illumination (280-360 nm) induced MN in both cell lines. Chinese hamster cells were found to be more sensitive to these wavelengths. Illumination with wavelengths above 360 nm did not induce MN in either cell line. The results obtained from human lymphocytes using the combination of EB or DPH were comparable to the results obtained with the combination of EB and HO. 23 refs., 7 figs

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

    Science.gov (United States)

    Polireddy, Kishore; Khan, Mohiuddin Md Taimur; Chavan, Hemantkumar; Young, Susan; Ma, Xiaochao; Waller, Anna; Garcia, Matthew; Perez, Dominique; Chavez, Stephanie; Strouse, Jacob J; Haynes, Mark K; Bologa, Cristian G; Oprea, Tudor I; Tegos, George P; Sklar, Larry A; Krishnamurthy, Partha

    2012-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Kishore Polireddy

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

  4. Flow cytometric chemosensitivity assay using JC‑1, a sensor of mitochondrial transmembrane potential, in acute leukemia.

    Science.gov (United States)

    Yokosuka, Tomoko; Goto, Hiroaki; Fujii, Hisaki; Naruto, Takuya; Takeuchi, Masanobu; Tanoshima, Reo; Kato, Hiromi; Yanagimachi, Masakatsu; Kajiwara, Ryosuke; Yokota, Shumpei

    2013-12-01

    The purpose of the study is to establish a simple and relatively inexpensive flow cytometric chemosensitivity assay (FCCA) for leukemia to distinguish leukemic blasts from normal leukocytes in clinical samples. We first examined whether the FCCA with the mitochondrial membrane depolarization sensor, 5, 50, 6, 60-tetrachloro-1, 10, 3, 30 tetraethyl benzimidazolo carbocyanine iodide (JC-1), could detect drug-induced apoptosis as the conventional FCCA by annexin V/7-AAD detection did and whether it was applicable in the clinical samples. Second, we compared the results of the FCCA for prednisolone (PSL) with clinical PSL response in 18 acute lymphoblastic leukemia (ALL) patients to evaluate the reliability of the JC-1 FCCA. Finally, we performed the JC-1 FCCA for bortezomib (Bor) in 25 ALL or 11 acute myeloid leukemia (AML) samples as the example of the clinical application of the FCCA. In ALL cells, the results of the JC-1 FCCA for nine anticancer drugs were well correlated with those of the conventional FCCA using anti-annexin V antibody (P < 0.001). In the clinical samples from 18 children with ALL, the results of the JC-1 FCCA for PSL were significantly correlated with the clinical PSL response (P = 0.005). In ALL samples, the sensitivity for Bor was found to be significantly correlated with the sensitivity for PSL (P = 0.005). In AML samples, the Bor sensitivity was strongly correlated with the cytarabine sensitivity (P = 0.0003). This study showed the reliability of a relatively simple and the FCCA using JC-1, and the possibility for the further clinical application.

  5. Ligand receptor dynamics at streptavidin-coated particle surfaces: A flow cytometric and spectrofluorimetric study

    Energy Technology Data Exchange (ETDEWEB)

    Buranda, T. [Univ. of New Mexico School of Medicine, Albuquerque, NM (United States)]|[Univ. of New Mexico, Albuquerque, NM (United States); Jones, G.M. [Univ. of New Mexico School of Medicine, Albuquerque, NM (United States); Nolan, J.P.; Keij, J. [Los Alamos National Labs., NM (United States); Lopez, G.P. [Univ. of New Mexico, Albuquerque, NM (United States); Sklar, L.A. [Univ. of New Mexico School of Medicine, Albuquerque, NM (United States)]|[Los Alamos National Lab., NM (United States)

    1999-04-29

    The authors have studied the binding of 5-((N-(5-(N-(6-(biotinoyl)amino)hexanoyl)amino)pentyl)thioureidyl)fluorescein (fluorescein biotin) to 6.2 {micro}m diameter, streptavidin-coated polystyrene beads using a combination of fluorimetric and flow cytometric methods. They have determined the average number of binding sites per bead, the extent of fluorescein quenching upon binding to the bead, and the association and dissociation kinetics. The authors estimate the site number to be {approx}1 million per bead. The binding of the fluorescein biotin ligand occurs in steps where the insertion of the biotin moiety into one receptor pocket is followed immediately by the capture of the fluorescein moiety by a neighboring binding pocket; fluorescence quenching is a consequence of this secondary binding. At high surface coverage, the dominant mechanism of quenching appears to be via the formation of nonfluorescent nearest-neighbor aggregates. At early times, the binding process is characterized by biphasic association and dissociation kinetics which are remarkably dependent on the initial concentration of the ligand. The rate constant for binding to the first receptor pocket of a streptavidin molecule is {approx}(1.3 {+-} 0.3) {times} 10{sup 7} 1{sup {minus}1} S{sup {minus}1}. The rate of binding of a second biotin may be reduced due to steric interference. The early time dissociative behavior is in sharp contrast to the typical stability associated with this system. The early time dissociative behavior is in sharp contrast to the typical stability associated with this system. The dissociation rate constant is as high as 0.05 s{sup {minus}1} shortly after binding, but decreases by 3 orders of magnitude after 3 h of binding. Potential sources for the time dependence of the dissociation rate constant are discussed.

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

    Science.gov (United States)

    Carson, Christine F; Inglis, Timothy JJ

    2018-01-01

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

  7. 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 G 0 /G 1 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

  8. Analytical validation of a flow cytometric protocol for quantification of platelet microparticles in dogs.

    Science.gov (United States)

    Cremer, Signe E; Krogh, Anne K H; Hedström, Matilda E K; Christiansen, Liselotte B; Tarnow, Inge; Kristensen, Annemarie T

    2018-06-01

    Platelet microparticles (PMPs) are subcellular procoagulant vesicles released upon platelet activation. In people with clinical diseases, alterations in PMP concentrations have been extensively investigated, but few canine studies exist. This study aims to validate a canine flow cytometric protocol for PMP quantification and to assess the influence of calcium on PMP concentrations. Microparticles (MP) were quantified in citrated whole blood (WB) and platelet-poor plasma (PPP) using flow cytometry. Anti-CD61 antibody and Annexin V (AnV) were used to detect platelets and phosphatidylserine, respectively. In 13 healthy dogs, CD61 + /AnV - concentrations were analyzed with/without a calcium buffer. CD61 + /AnV - , CD61 + /AnV + , and CD61 - /AnV + MP quantification were validated in 10 healthy dogs. The coefficient of variation (CV) for duplicate (intra-assay) and parallel (inter-assay) analyses and detection limits (DLs) were calculated. CD61 + /AnV - concentrations were higher in calcium buffer; 841,800 MP/μL (526,000-1,666,200) vs without; 474,200 MP/μL (278,800-997,500), P < .05. In WB, PMP were above DLs and demonstrated acceptable (<20%) intra-assay and inter-assay CVs in 9/10 dogs: 1.7% (0.5-8.9) and 9.0% (0.9-11.9), respectively, for CD61 + /AnV - and 2.4% (0.2-8.7) and 7.8% (0.0-12.8), respectively, for CD61 + /AnV + . Acceptable CVs were not seen for the CD61 - /AnV + MP. In PPP, quantifications were challenged by high inter-assay CV, overlapping DLs and hemolysis and lipemia interfered with quantification in 5/10 dogs. Calcium induced higher in vitro PMP concentrations, likely due to platelet activation. PMP concentrations were reliably quantified in WB, indicating the potential for clinical applications. PPP analyses were unreliable due to high inter-CV and DL overlap, and not obtainable due to hemolysis and lipemia interference. © 2018 American Society for Veterinary Clinical Pathology.

  9. Novel flow cytometric analysis of the progress and route of internalization of a monoclonal anti-carcinoembryonic antigen (CEA) antibody.

    Science.gov (United States)

    Ford, C H; Tsaltas, G C; Osborne, P A; Addetia, K

    1996-03-01

    A flow cytometric method of studying the internalization of a monoclonal antibody (Mab) directed against carcinoembryonic antigen (CEA) has been compared with Western blotting, using three human colonic cancer cell lines which express varying amounts of the target antigen. Cell samples incubated for increasing time intervals with fluoresceinated or unlabelled Mab were analyzed using flow cytometry or polyacrylamide gel electrophoresis and Western blotting. SDS/PAGE analysis of cytosolic and membrane components of solubilized cells from the cell lines provided evidence of non-degraded internalized anti-CEA Mab throughout seven half hour intervals, starting at 5 min. Internalized anti-CEA was detected in the case of high CEA expressing cell lines (LS174T, SKCO1). Very similar results were obtained with an anti-fluorescein flow cytometric assay. Given that these two methods consistently provided comparable results, use of flow cytometry for the detection of internalized antibody is suggested as a rapid alternative to most currently used methods for assessing antibody internalization. The question of the endocytic route followed by CEA-anti-CEA complexes was addressed by using hypertonic medium to block clathrin mediated endocytosis.

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

    International Nuclear Information System (INIS)

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

    1990-01-01

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

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

    International Nuclear Information System (INIS)

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

    1993-01-01

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

  12. Flow cytometric and radioisotopic determinations of platelet survival time in normal cats and feline leukemia virus-infected cats

    Energy Technology Data Exchange (ETDEWEB)

    Jacobs, R.M.; Boyce, J.T.; Kociba, G.J.

    1986-01-01

    This study demonstrates the potential usefulness of a flow cytometric technique to measure platelet survival time in cats utilizing autologous platelets labeled in vitro with fluorescein isothiocyanate (FITC). When compared with a 51Cr method, no significant differences in estimated survival times were found. Both the 51Cr and FITC-labeling procedures induced similar changes in platelet shape and collagen-induced aggregation. Platelets labeled with FITC had significantly greater volumes compared with those of glutaraldehyde-fixed platelets. These changes were primarily related to the platelet centrifugation and washing procedures rather than the labels themselves. This novel technique potentially has wide applicability to cell circulation time studies as flow cytometry equipment becomes more readily available. Problems with the technique are discussed. In a preliminary study of the platelet survival time in feline leukemia virus (FeLV)-infected cats, two of three cats had significantly reduced survival times using both flow cytometric and radioisotopic methods. These data suggest increased platelet turnover in FeLV-infected cats.

  13. Flow cytometric and radioisotopic determinations of platelet survival time in normal cats and feline leukemia virus-infected cats

    International Nuclear Information System (INIS)

    Jacobs, R.M.; Boyce, J.T.; Kociba, G.J.

    1986-01-01

    This study demonstrates the potential usefulness of a flow cytometric technique to measure platelet survival time in cats utilizing autologous platelets labeled in vitro with fluorescein isothiocyanate (FITC). When compared with a 51Cr method, no significant differences in estimated survival times were found. Both the 51Cr and FITC-labeling procedures induced similar changes in platelet shape and collagen-induced aggregation. Platelets labeled with FITC had significantly greater volumes compared with those of glutaraldehyde-fixed platelets. These changes were primarily related to the platelet centrifugation and washing procedures rather than the labels themselves. This novel technique potentially has wide applicability to cell circulation time studies as flow cytometry equipment becomes more readily available. Problems with the technique are discussed. In a preliminary study of the platelet survival time in feline leukemia virus (FeLV)-infected cats, two of three cats had significantly reduced survival times using both flow cytometric and radioisotopic methods. These data suggest increased platelet turnover in FeLV-infected cats

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

    Science.gov (United States)

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

    2011-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Yoichi Kakuta, MD, PhD

    2018-01-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    International Nuclear Information System (INIS)

    Marcusson-Staahl, Maritha; Cederbrant, Karin

    2003-01-01

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

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

    Science.gov (United States)

    Fröhling, Antje; Schlüter, Oliver

    2015-01-01

    Since heat sensitivity of fruits and vegetables limits the application of thermal inactivation processes, new emerging inactivation technologies have to be established to fulfill 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 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 parameters. PMID

  19. 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

  20. 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.

  1. Flow cytometric evaluation of peripheral blood and bone marrow and fine-needle aspirate samples from multiple sites in dogs with multicentric lymphoma.

    Science.gov (United States)

    Joetzke, Alexa E; Eberle, Nina; Nolte, Ingo; Mischke, Reinhard; Simon, Daniela

    2012-06-01

    To determine whether the extent of disease in dogs with lymphoma can be assessed via flow cytometry and to evaluate the suitability of fine-needle aspirates from the liver and spleen of dogs for flow cytometric examination. 44 dogs with multicentric B-cell (n = 35) or T-cell lymphoma (9) and 5 healthy control dogs. Procedures-Peripheral blood and bone marrow samples and fine-needle aspirates of lymph node, liver, and spleen were examined via flow cytometry. Logarithmically transformed T-cell-to-B-cell percentage ratio (log[T:B]) values were calculated. Thresholds defined by use of log(T:B) values of samples from control dogs were used to determine extranodal lymphoma involvement in lymphoma-affected dogs; results were compared with cytologic findings. 12 of 245 (5%) samples (9 liver, 1 spleen, and 2 bone marrow) had insufficient cellularity for flow cytometric evaluation. Mean log(T:B) values of samples from dogs with B-cell lymphoma were significantly lower than those of samples from the same site in dogs with T-cell lymphoma and in control dogs. In dogs with T-cell lymphoma, the log(T:B) of lymph node, bone marrow, and spleen samples was significantly higher than in control dogs. Of 165 samples assessed for extranodal lymphoma involvement, 116 (70%) tested positive via flow cytometric analysis; results agreed with cytologic findings in 133 of 161 (83%) samples evaluated via both methods. Results suggested that flow cytometry may aid in detection of extranodal lymphoma involvement in dogs, but further research is needed. Most fine-needle aspirates of liver and spleen were suitable for flow cytometric evaluation.

  2. Microscopic and flow cytometric study of micronuclei in iododeoxyuridine labelled cells irradiated with soft X-rays

    International Nuclear Information System (INIS)

    Ludwikow, G.; Staalnacke, C.G.; Johanson, K.J.; Sundell-Bergman, S.; Richter, S.; Swedish Univ. of Agricultural Sciences, Uppsala; Uppsala Univ.

    1990-01-01

    Iododeoxyuridine labelled (IUdR(+)) and unlabelled (IUdR(-)) CHO cells irradiated with 2 Gy of soft X-rays showed only minor differences in the kinetics of micronuclei formation during the first 20 hours postirradiation period. Between 20 to 40 hours, the IUdR(-) cells showed approximately a constant number of micronuclei while the number of micronuclei in IUdR(+) cells was still increasing. The frequency of micronuclei was higher in IUdR(+) cells compared to IUdR(-) cells at 24 hours after irradiation with various doses up to 4.0 Gy. Dose modifying factors were found to be 1.3 (microscopic evaluation) and 1.8 (flow cytometric evaluation). Flow cytometry with use of two parameters, fluorescence from propidium iodide and light scattering, seems to be a good tool to estimate the frequency of micronuclei in CHO cells in the dose range up to about 4 Gy. At higher doses perturbation of the cell cycle and the appearance of dying cells will influence the results. (orig.)

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

    Science.gov (United States)

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

    1997-05-01

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

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

    Science.gov (United States)

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

    2011-07-01

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

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

    International Nuclear Information System (INIS)

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

    2011-01-01

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

  6. 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.

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Science.gov (United States)

    Suzuki, Hiroshi; Toyooka, Tatsushi; Ibuki, Yuko

    2007-04-15

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

  9. Flow cytometric analysis of immunoglobulin heavy chain expression in B-cell lymphoma and reactive lymphoid hyperplasia

    Science.gov (United States)

    Grier, David D; Al-Quran, Samer Z; Cardona, Diana M; Li, Ying; Braylan, Raul C

    2012-01-01

    The diagnosis of B-cell lymphoma (BCL) is often dependent on the detection of clonal immunoglobulin (Ig) light chain expression. In some BCLs, the determination of clonality based on Ig light chain restriction may be difficult. The aim of our study was to assess the utility of flow cytometric analysis of surface Ig heavy chain (HC) expression in lymphoid tissues in distinguishing lymphoid hyperplasias from BCLs, and also differentiating various BCL subtypes. HC expression on B-cells varied among different types of hyperplasias. In follicular hyperplasia, IgM and IgD expression was high in mantle cells while germinal center cells showed poor HC expression. In other hyperplasias, B cell compartments were blurred but generally showed high IgD and IgM expression. Compared to hyperplasias, BCLs varied in IgM expression. Small lymphocytic lymphomas had lower IgM expression than mantle cell lymphomas. Of importance, IgD expression was significantly lower in BCLs than in hyperplasias, a finding that can be useful in differentiating lymphoma from reactive processes. PMID:22400070

  10. Long-term preservation of Tetraselmis indica (Chlorodendrophyceae, Chlorophyta) for flow cytometric analysis: Influence of fixative and storage temperature.

    Science.gov (United States)

    Naik, Sangeeta Mahableshwar; Anil, Arga Chandrashekar

    2017-08-01

    Immediate enumeration of phytoplankton is seldom possible. Therefore, fixation and subsequent storage are required for delayed analysis. This study investigated the influence of glutaraldehyde (GA) concentrations (0.25%, 0.5%, and 1%) and storage temperatures (-80°C LN2 , -80°C, -20°C, and 5°C) on Tetraselmis indica for flow cytometric analysis. Cell recovery, granularity, and membrane permeability were independent of GA concentration whereas cell size and chlorophyll autofluorescence were concentration dependent. After an initial cell loss (16-19%), no cell loss was observed when samples were stored at 5°C. Cell recovery was not influenced by storage temperature until 4months but later samples preserved at -80°C LN2 , -80°C, and -20°C resulted in ~41% cell loss. Although maximum cell recovery with minimal effect on cell integrity was obtained at 5°C, autofluorescence was retained better at -80°C LN2 and -80°C. This suggests that in addition to fixative, the choice of storage temperature is equally important. Thus for long-term preservation, especially to retain autofluorescence, the use of lower concentration (0.25%) of GA when stored at a lower temperature (-80°C LN2 and -80°C) while a higher concentration (1%) of GA when stored at a higher temperature (5°C) is recommended. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. 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.

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

    International Nuclear Information System (INIS)

    Jensen, R.H.; Bigbee, W.L.; Langlois, R.G.; Grant, S.G.; Pleshanov, P.G.; Chirkov, A.A.; Pilinskaya, M.A.

    1990-01-01

    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 accident at Chernobyl, Ukraine, USSR, a number of people were exposed to whole body doses of ionizing 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. All data indicate that this assay might well be used as a biodosimeter to estimate radiation dose and also as an element to be used for estimating the risk of each individual to develop cancer due to radiation exposure. 17 refs., 5 figs

  13. Evaluation of flow cytometric HIT assays in relation to an IgG-Specific immunoassay and clinical outcome.

    Science.gov (United States)

    Kerényi, Adrienne; Beke Debreceni, Ildikó; Oláh, Zsolt; Ilonczai, Péter; Bereczky, Zsuzsanna; Nagy, Béla; Muszbek, László; Kappelmayer, János

    2017-09-01

    Heparin-induced thrombocytopenia (HIT) is a severe side effect of heparin treatment caused by platelet activating IgG antibodies generated against the platelet factor 4 (PF4)-heparin complex. Thrombocytopenia and thrombosis are the leading clinical symptoms of HIT. The clinical pretest probability of HIT was evaluated by the 4T score system. Laboratory testing of HIT was performed by immunological detection of antibodies against PF4-heparin complex (EIA) and two functional assays. Heparin-dependent activation of donor platelets by patient plasma was detected by flow cytometry. Increased binding of Annexin-V to platelets and elevated number of platelet-derived microparticles (PMP) were the indicators of platelet activation. EIA for IgG isotype HIT antibodies was performed in 405 suspected HIT patients. Based on negative EIA results, HIT was excluded in 365 (90%) of cases. In 40 patients with positive EIA test result functional tests were performed. Platelet activating antibodies were detected in 17 cases by Annexin V binding. PMP count analysis provided nearly identical results. The probability of a positive flow cytometric assay result was higher in patients with elevated antibody titer. 71% of patients with positive EIA and functional assay had thrombosis. EIA is an important first line laboratory test in the diagnosis of HIT; however, HIT must be confirmed by a functional test. Annexin V binding and PMP assays using flow cytometry are functional HIT tests convenient in a clinical diagnostic laboratory. The positive results of functional assays may predict the onset of thrombosis. © 2016 International Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.

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

    Science.gov (United States)

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

    2017-06-01

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

  15. Flow cytometric osmotic fragility test and eosin-5'-maleimide dye-binding tests are better than conventional osmotic fragility tests for the diagnosis of hereditary spherocytosis.

    Science.gov (United States)

    Arora, R D; Dass, J; Maydeo, S; Arya, V; Radhakrishnan, N; Sachdeva, A; Kotwal, J; Bhargava, M

    2018-03-24

    Hereditary spherocytosis (HS) is the most common inherited hemolytic anemia with heterogeneous clinico-laboratory manifestations. We evaluated the flow-cytometric tests: eosin-5'-maleimide (EMA) and flow-cytometric osmotic fragility test (FOFT) and the conventional osmotic fragility tests (OFT) for the diagnosis of hereditary spherocytosis (HS). One hundred two suspected HS patients underwent EMA, FOFT, incubated OFT (IOFT), and room temperature OFT (RT-OFT). In addition, 10 cases of immune hemolytic anemia (IHA) were included, and performance of the above 4 tests was evaluated. For EMA and FOFT, 5 normal controls were assessed together with the patients and cutoffs were calculated using receiver-operator-characteristics curve (ROC) analysis. The best cutoff for %EMA decrease was 12.5%, and for FOFT, %residual red cells (%RRC) was 25.6%. The sensitivity and specificity of RT-OFT was 62.06% and 86.3%, respectively, while that of IOFT was 79.31% and 87.67%, respectively. Both flow cytometric tests performed better. Sensitivity and specificity of EMA was 86.2% and 93.9% respectively, and that of FOFT was 96.6% and 98.63%, respectively. The combination of the FOFT with IOFT or EMA dye-binding test yields a sensitivity of 100%, but with EMA, it had a higher specificity. Hb/MCHC was a predictor of the severity of the disease while %EMA decrease and %RRC did not correlate with severity of the disease. Flow-cytometric osmotic fragility test is the best possible single test followed by EMA for diagnosis of HS. A combination of FOFT and EMA can correctly diagnose 100% patients. These tests are likely to replace conventional OFTs in future. © 2018 John Wiley & Sons Ltd.

  16. Automated flow cytometric analysis across large numbers of samples and cell types.

    Science.gov (United States)

    Chen, Xiaoyi; Hasan, Milena; Libri, Valentina; Urrutia, Alejandra; Beitz, Benoît; Rouilly, Vincent; Duffy, Darragh; Patin, Étienne; Chalmond, Bernard; Rogge, Lars; Quintana-Murci, Lluis; Albert, Matthew L; Schwikowski, Benno

    2015-04-01

    Multi-parametric flow cytometry is a key technology for characterization of immune cell phenotypes. However, robust high-dimensional post-analytic strategies for automated data analysis in large numbers of donors are still lacking. Here, we report a computational pipeline, called FlowGM, which minimizes operator input, is insensitive to compensation settings, and can be adapted to different analytic panels. A Gaussian Mixture Model (GMM)-based approach was utilized for initial clustering, with the number of clusters determined using Bayesian Information Criterion. Meta-clustering in a reference donor permitted automated identification of 24 cell types across four panels. Cluster labels were integrated into FCS files, thus permitting comparisons to manual gating. Cell numbers and coefficient of variation (CV) were similar between FlowGM and conventional gating for lymphocyte populations, but notably FlowGM provided improved discrimination of "hard-to-gate" monocyte and dendritic cell (DC) subsets. FlowGM thus provides rapid high-dimensional analysis of cell phenotypes and is amenable to cohort studies. Copyright © 2015. Published by Elsevier Inc.

  17. 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

  18. Synthesis of new, UV-photoactive dansyl derivatives for flow cytometric studies on bile acid uptake.

    Science.gov (United States)

    Rohacova, Jana; Marin, M Luisa; Martínez-Romero, Alicia; O'Connor, José-Enrique; Gomez-Lechon, M Jose; Donato, M Teresa; Castell, Jose V; Miranda, Miguel A

    2009-12-07

    Four new fluorescent derivatives of cholic acid have been synthesized; they incorporate a dansyl moiety at 3alpha-, 3beta-, 7alpha- or 7beta- positions. These cholic acid analogs are UV photoactive and also exhibit green fluorescence. In addition, they have been demonstrated to be suitable for studying the kinetics of bile acid transport by flow cytometry.

  19. 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...

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

    OpenAIRE

    Katarzyna Czaczyk; Wojciech Juzwa

    2012-01-01

    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 process...

  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 Detection of PrPSc in Neurons and Glial Cells from Prion-Infected Mouse Brains.

    Science.gov (United States)

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

    2018-01-01

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

  3. Flow cytometric monitoring of influenza A virus infection in MDCK cells during vaccine production

    Directory of Open Access Journals (Sweden)

    Reichl Udo

    2008-04-01

    Full Text Available Abstract Background In cell culture-based influenza vaccine production the monitoring of virus titres and cell physiology during infection is of great importance for process characterisation and optimisation. While conventional virus quantification methods give only virus titres in the culture broth, data obtained by fluorescence labelling of intracellular virus proteins provide additional information on infection dynamics. Flow cytometry represents a valuable tool to investigate the influences of cultivation conditions and process variations on virus replication and virus yields. Results In this study, fluorescein-labelled monoclonal antibodies against influenza A virus matrix protein 1 and nucleoprotein were used for monitoring the infection status of adherent Madin-Darby canine kidney cells from bioreactor samples. Monoclonal antibody binding was shown for influenza A virus strains of different subtypes (H1N1, H1N2, H3N8 and host specificity (human, equine, swine. At high multiplicity of infection in a bioreactor, the onset of viral protein accumulation in adherent cells on microcarriers was detected at about 2 to 4 h post infection by flow cytometry. In contrast, a significant increase in titre by hemagglutination assay was detected at the earliest 4 to 6 h post infection. Conclusion It is shown that flow cytometry is a sensitive and robust method for the monitoring of viral infection in fixed cells from bioreactor samples. Therefore, it is a valuable addition to other detection methods of influenza virus infection such as immunotitration and RNA hybridisation. Thousands of individual cells are measured per sample. Thus, the presented method is believed to be quite independent of the concentration of infected cells (multiplicity of infection and total cell concentration in bioreactors. This allows to perform detailed studies on factors relevant for optimization of virus yields in cell cultures. The method could also be used for process

  4. Flow cytometric measurement of DNA level and steroid hormone receptor assay in breast cancer

    International Nuclear Information System (INIS)

    Zubrikhina, G.N.; Kuz'mina, Eh.V.; Bassalyk, L.S.; Murav'eva, N.I.

    1989-01-01

    DNA level measured by flow cytometry and estrogen and progesteron receptors assayed in tissue samples obtained from 85 malignant and 16 benign lesions of the breast. All the benign tumors revealed 2c DNA content and most of them were receptor-negative, while 74.1% of breast carcinomas displayed aneuploidy. Three patients (3.5%) had two lines of aneuploid cells. Many aneuploid tumors were receptor-negative. Preoperative radiation treatmet (14-20 Gy) did not significantly influence the level of steroid hormone receptors in tumors. Estrogen receptor level was higher in menopausal patients than in premenopausal ones

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

    Science.gov (United States)

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

    2016-09-01

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

  6. Flow cytometric analysis of regulatory T cells during hyposensitization of acquired allergic contact dermatitis.

    Science.gov (United States)

    Fraser, Kathleen; Abbas, Mariam; Hull, Peter R

    2014-01-01

    We previously demonstrated that repeated intradermal steroid injections administered at weekly intervals into positive patch-test sites induce hyposensitization and desensitization. To examine changes in CD4CD25CD127lo/ regulatory T cells during the attenuation of the patch-test response. Ten patients with known allergic contact dermatitis were patch tested weekly for 10 weeks. The patch-test site was injected intradermally with 2 mg triamcinolone. At weeks 1 and 7, a biopsy was performed on the patch-test site in 6 patients, and flow cytometry was performed assessing CD4CD25CD127lo/ regulatory T cells. Secondary outcomes were clinical score, reaction size, erythema, and temperature. Statistical analysis included regression, correlation, and repeated-measures analysis of variance. The percentage of CD4CD25CD127lo/ regulatory T cells, measured by flow cytometry, increased from week 1 to week 7 by an average of 19.2%. The average grade of patch-test reaction decreased from +++ (vesicular reaction) to ++ (palpable erythema). The mean drop in temperature following treatment was 0.28°C per week. The mean area decreased 8.6 mm/wk over 10 weeks. Intradermal steroid injections of weekly patch-test reactions resulted in hyposensitization of the allergic contact dermatitis reaction. CD4CD25CD127lo/ regulatory T cells showed a tendency to increase; however, further studies are needed to determine if this is significant.

  7. Flow-cytometric analysis of mouse embryonic stem cell lipofection using small and large DNA constructs.

    Science.gov (United States)

    McLenachan, Samuel; Sarsero, Joseph P; Ioannou, Panos A

    2007-06-01

    Using the lipofection reagent LipofectAMINE 2000 we have examined the delivery of plasmid DNA (5-200 kb) to mouse embryonic stem (mES) cells by flow cytometry. To follow the physical uptake of lipoplexes we labeled DNA molecules with the fluorescent dye TOTO-1. In parallel, expression of an EGFP reporter cassette in constructs of different sizes was used as a measure of nuclear delivery. The cellular uptake of DNA lipoplexes is dependent on the uptake competence of mES cells, but it is largely independent of DNA size. In contrast, nuclear delivery was reduced with increasing plasmid size. In addition, linear DNA is transfected with lower efficiency than circular DNA. Inefficient cytoplasmic trafficking appears to be the main limitation in the nonviral delivery of large DNA constructs to the nucleus of mES cells. Overcoming this limitation should greatly facilitate functional studies with large genomic fragments in embryonic stem cells.

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

    International Nuclear Information System (INIS)

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

    1984-01-01

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

  9. Whole blood flow cytometric analysis of Ureaplasma-stimulated monocytes from pregnant women.

    Science.gov (United States)

    Friedland, Yael D; Lee-Pullen, Tracey F; Nathan, Elizabeth; Watts, Rory; Keelan, Jeffrey A; Payne, Matthew S; Ireland, Demelza J

    2015-06-01

    We hypothesised that circulating monocytes of women with vaginal colonisation with Ureaplasma spp., genital microorganisms known to cause inflammation-driven preterm birth, would elicit a tolerised cytokine response to subsequent in vitro Ureaplasma parvum serovar 3 (UpSV3) stimulation. Using multi-parameter flow cytometry, we found no differences with regard to maternal colonisation status in the frequency of TNF-α-, IL-6-, IL-8- and IL-1β-expressing monocytes in response to subsequent UpSV3 stimulation (P > 0.10 for all cytokines). We conclude that vaginal Ureaplasma spp. colonisation does not specifically tolerise monocytes of pregnant women towards decreased responses to subsequent stimulation. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2014-01-01

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

  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. 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

  13. 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.

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

    International Nuclear Information System (INIS)

    Vanparys, Caroline; Depiereux, Sophie; Nadzialek, Stephanie; Robbens, Johan; Blust, Ronny; Kestemont, Patrick; De Coen, Wim

    2010-01-01

    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 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 2 = 0.98), the estrogen receptor (ER) binding (R 2 = 0.84) and the ER transcription activation assay (R 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, supports the use of

  15. 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.

  16. Flow cytometric analysis of lymphocyte subset in patients with neutropenia among atomic bomb survivors

    Energy Technology Data Exchange (ETDEWEB)

    Imamura, Nobutaka; Kimura, Akiro [Hiroshima Univ. (Japan). Research Inst. for Radiation Biology and Medicine

    1998-12-01

    In 51 patients (atomic bomb survivors 50, unexposed persons 1) who have had neutropenia for two years or more under indistinct cause, cell surface antigen was analyzed by flow cytometry. Twenty-nine cases of survivors were diagnosed as NK cell leukemia or NK cell cytosis because analysis data showed CD3(-), CD56(+) and CD57(+/-). Six cases were diagnosed as NK like T cell hypercytosis because analysis data showed CD3(+), CD56(+/-) and CD57(+). As for 15 cases, CD56(+) cell number was in range of 15.96{+-}5.35 of a normal person, and no relation with NK cell was recognized. But, CD4/CD8 ratio was higher than 2.1, and gain of T helper cell was recognized. One unexposed persons was diagnosed as chronic NK cell leukemia because analysis data showed CD3(-), CD56(+) and CD57(+). Anti-neutrophil antibody wasn't recognized. Cytotoxic activity for K562 and Raji cell line showed high value compared with that of a normal person. Epstein Barr virus wasn't detected. (K.H.)

  17. Stability of eosin-5'-maleimide dye used in flow cytometric analysis for red cell membrane disorders.

    Science.gov (United States)

    Mehra, Simmi; Tyagi, Neetu; Dorwal, Pranav; Pande, Amit; Jain, Dharmendra; Sachdev, Ritesh; Raina, Vimarsh

    2015-06-01

    The eosin-5'-maleimide (EMA) binding test using flow cytometry is a common method to measure reduced mean channel fluorescence (MCF) of EMA-labeled red blood cells (RBCs) from patients with red cell membrane disorders. The basic principle of the EMA-RBC binding test involves the covalent binding of EMA to lysine-430 on the first extracellular loop of band 3 protein. In the present study, the MCF of EMA was analyzed for samples derived from 12 healthy volunteers (controls) to determine the stability (i.e., the percentage decrease in fluorescence) of EMA over a period of 1 year. Comparison of periodical MCF readings over time, that is, at 2-month intervals, showed that there were no significant changes in mean channel fluorescence for up to 6 months; however, there was a significant decrease in MCF at 8 months. For optimal dye utilization, EMA remained stable only for up to 6 months. Therefore, we recommend reconstitution of the dye every 6 months when implementing this test and storage at -80℃ in dark conditions.

  18. Flow cytometric analysis of cell killing by the jumper ant venom peptide pilosulin 1.

    Science.gov (United States)

    King, M A; Wu, Q X; Donovan, G R; Baldo, B A

    1998-08-01

    Pilosulin 1 is a synthetic 56-amino acid residue polypeptide that corresponds to the largest allergenic polypeptide found in the venom of the jumper ant Myrmecia pilosula. Initial experiments showed that pilosulin 1 lysed erythrocytes and killed proliferating B cells. Herein, we describe how flow cytometry was used to investigate the cytotoxicity of the peptide for human white blood cells. Cells were labeled with fluorochrome-conjugated antibodies, incubated with the peptide and 7-aminoactinomycin D (7-AAD), and then analyzed. The effects of varying the peptide concentration, serum concentration, incubation time, and incubation temperature were measured, and the cytotoxicity of pilosulin 1 was compared with that of the bee venom peptide melittin. The antibodies and the 7-AAD enabled the identification of cell subpopulations and dead cells, respectively. It was possible, using the appropriate mix of antibodies and four-color analysis, to monitor the killing of three or more cell subpopulations simultaneously. We found that 1) pilosulin 1 killed cells within minutes, with kinetics similar to those of melittin; 2) pilosulin 1 was a slightly more potent cytotoxic agent than melittin; 3) both pilosulin 1 and melittin were more potent against mononuclear leukocytes than against granulocytes; and 4) serum inhibited killing by either peptide.

  19. Flow cytometric assessment of DNA damage in the fish Catla catla (Ham.) exposed to gamma radiation

    International Nuclear Information System (INIS)

    Anbumani, S.; Mohankumar, Mary N.; Selvanayagam, M.

    2012-01-01

    Environmental mutagens such as ionizing radiation and chemicals induce DNA damage in a wide variety of organisms. The International Commission on Radiological Protection (lCRP) has recently emphasized the need to protect non-human biota from the potential effects of ionizing radiation. Radiation exposures to non-humans can occur as a result of low-level radioactive discharges into the environment. Molecular genetic effects at low-level radiation exposures are largely unexplored and systematic studies using sensitive biomarkers are required to assess DNA damage in representative non-human species. The objective of the study was to detect DNA damage in the fish Catla catla exposed to gamma radiation using flow cytometry at different time intervals. Increases in the coefficient of variation (CV) of the G 0 /G 1 peak, indicating abnormal DNA distributions were observed in fish exposed to gamma radiation than in controls. Significant increase in the CV was observed from day 12-90 and thereafter decreased. This increase in CV might be due to DNA damage in the cell populations at G 0 /G 1 phase or deletions and duplications caused by improper repair of chromosomes in the cell-cycle machinery. Ionizing radiation induced cell-cycle perturbations and apoptosis were also observed after gamma radiation exposure. (author)

  20. Identification of ataxia telangiectasia heterozygotes by flow cytometric analysis of X-ray damage

    International Nuclear Information System (INIS)

    Rudolph, N.S.

    1989-01-01

    Flow cytometry was used to identify heterozygotes for the autosomal recessive DNA-repair deficiency disease ataxia telangiectasia (AT). Confluent G 0 /G 1 fibroblasts from 4 homozygotes (at/at), 5 obligate heterozygates (at/+) and 7 presumed normal (+/+) were X-irradiated with 200 Rad and subcultured immediately in medium containing 5-bromodeoxyuridine (BrdU). Cells were harvested 72 h later and stained with fluoresceinated anti-BrdU antibody to identify cells that had entered S phase. They were counterstained with propidium iodide to measure total DNA content. On the basis of relative release from G 0 /G 1 , the at/+ strains as a group were distinguished from both the presumed +/+ strains and at/at strains, although the individual values for some strains did show overlap between genotypes. When 10 cell strains were coded and analyzed in 'blind' experiments, all 4 heterozygotes were correctly assigned. By a similar assay in which exponentially growing cultures were pulsed briefly with BrdU 8 h after irradiation with 400 Rad and then harvested immediately, presumed +/+ cells as a group could be distinguished from at/at cells but not from at/- cells. This combination of assays assists in the identification of all 3 AT genotypes. This should be of both basic and diagnostic use, particularly in families known to segregate AT. (author). 37 refs.; 3 figs.; 5 tabs

  1. 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...

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

    KAUST Repository

    Prest, Emmanuelle I E C; Hammes, Frederik A.; Kö tzsch, Stefan; van Loosdrecht, Mark C.M.; Vrouwenvelder, Johannes S.

    2013-01-01

    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.

  3. 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.

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

    Science.gov (United States)

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

    2014-03-01

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

  5. 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.

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

    KAUST Repository

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

    2017-01-01

    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.

  7. Flow-cytometric determination of high-density-lipoprotein binding sites on human leukocytes

    International Nuclear Information System (INIS)

    Schmitz, G.; Wulf, G.; Bruening, T.A.; Assmann, G.

    1987-01-01

    In this method, leukocytes were isolated from 6 mL of EDTA-blood by density-gradient centrifugation and subsequently incubated with rhodamine isothiocyanate (RITC)-conjugated high-density lipoproteins (HDL). The receptor-bound conjugate particles were determined by fluorescent flow cytometry and compared with 125 I-labeled HDL binding data for the same cells. Human granulocytes express the highest number of HDL binding sites (9.4 x 10(4)/cell), followed by monocytes (7.3 x 10(4)/cell) and lymphocytes (4.0 x 10(4)/cell). Compared with conventional analysis of binding of 125 I-labeled HDL in tissue-culture dishes, the present determination revealed significantly lower values for nonspecific binding. In competition studies, the conjugate competes for the same binding sites as 125 I-labeled HDL. With the use of tetranitromethane-treated HDL3, which fails to compete for the HDL receptor sites while nonspecific binding is not affected, we could clearly distinguish between 37 degrees C surface binding and specific 37 degrees C uptake of RITC-HDL3, confirming that the HDL receptor leads bound HDL particles into an intracellular pathway rather than acting as a docking type of receptor. Patients with familial dysbetalipoproteinemia showed a significantly higher number of HDL binding sites in the granulocyte population but normal in lymphocytes and monocytes, indicating increased uptake of cholesterol-containing lipoproteins. In patients with familial hypercholesterolemia, HDL binding was increased in all three cell types, indicating increased cholesterol uptake and increased cholesterol synthesis. The present method allows rapid determination of HDL binding sites in leukocytes from patients with various forms of hyper- and dyslipoproteinemias

  8. 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.

  9. Gastric lymphomas in Turkey. Analysis of prognostic factors with special emphasis on flow cytometric DNA content.

    Science.gov (United States)

    Aydin, Z D; Barista, I; Canpinar, H; Sungur, A; Tekuzman, G

    2000-07-01

    In contrast to DNA ploidy, to the authors' knowledge the prognostic significance of S-phase fraction (SPF) in gastric lymphomas has not been determined. In the current study, the prognostic significance of various parameters including SPF and DNA aneuploidy were analyzed and some distinct epidemiologic and biologic features of gastric lymphomas in Turkey were found. A series of 78 gastric lymphoma patients followed at Hacettepe University is reported. DNA flow cytometry was performed for 34 patients. The influence of various parameters on survival was investigated with the log rank test. The Cox proportional hazards model was fitted to identify independent prognostic factors. The median age of the patients was 50 years. There was no correlation between patient age and tumor grade. DNA content analysis revealed 4 of the 34 cases to be aneuploid with DNA index values < 1.0. The mean SPF was 33.5%. In the univariate analysis, surgical resection of the tumor, modified Ann Arbor stage, performance status, response to first-line chemotherapy, lactate dehydrogenase (LDH) level, and SPF were important prognostic factors for disease free survival (DFS). The same parameters, excluding LDH level, were important for determining overall survival (OS). In the multivariate analysis, surgical resection of the tumor, disease stage, performance status, and age were found to be important prognostic factors for OS. To the authors' knowledge the current study is the first to demonstrate the prognostic significance of SPF in gastric lymphomas. The distinguishing features of Turkish gastric lymphoma patients are 1) DNA indices of aneuploid cases that all are < 1.0, which is a unique feature; 2) a lower percentage of aneuploid cases; 3) a higher SPF; 4) a younger age distribution; and 5) lack of an age-grade correlation. The authors conclude that gastric lymphomas in Turkey have distinct biologic and epidemiologic characteristics. Copyright 2000 American Cancer Society.

  10. A Novel Tool for High-Throughput Screening of Granulocyte-Specific Antibodies Using the Automated Flow Cytometric Granulocyte Immunofluorescence Test (Flow-GIFT

    Directory of Open Access Journals (Sweden)

    Xuan Duc Nguyen

    2011-01-01

    Full Text Available Transfusion-related acute lung injury (TRALI is a severe complication related with blood transfusion. TRALI has usually been associated with antibodies against leukocytes. The flow cytometric granulocyte immunofluorescence test (Flow-GIFT has been introduced for routine use when investigating patients and healthy blood donors. Here we describe a novel tool in the automation of the Flow-GIFT that enables a rapid screening of blood donations. We analyzed 440 sera from healthy female blood donors for the presence of granulocyte antibodies. As positive controls, 12 sera with known antibodies against anti-HNA-1a, -b, -2a; and -3a were additionally investigated. Whole-blood samples from HNA-typed donors were collected and the test cells isolated using cell sedimentation in a Ficoll density gradient. Subsequently, leukocytes were incubated with the respective serum and binding of antibodies was detected using FITC-conjugated antihuman antibody. 7-AAD was used to exclude dead cells. Pipetting steps were automated using the Biomek NXp Multichannel Automation Workstation. All samples were prepared in the 96-deep well plates and analyzed by flow cytometry. The standard granulocyte immunofluorescence test (GIFT and granulocyte agglutination test (GAT were also performed as reference methods. Sixteen sera were positive in the automated Flow-GIFT, while five of these sera were negative in the standard GIFT (anti—HNA 3a, n = 3; anti—HNA-1b, n = 1 and GAT (anti—HNA-2a, n = 1. The automated Flow-GIFT was able to detect all granulocyte antibodies, which could be only detected in GIFT in combination with GAT. In serial dilution tests, the automated Flow-GIFT detected the antibodies at higher dilutions than the reference methods GIFT and GAT. The Flow-GIFT proved to be feasible for automation. This novel high-throughput system allows an effective antigranulocyte antibody detection in a large donor population in order to prevent TRALI due to transfusion of

  11. A novel tool for high-throughput screening of granulocyte-specific antibodies using the automated flow cytometric granulocyte immunofluorescence test (Flow-GIFT).

    Science.gov (United States)

    Nguyen, Xuan Duc; Dengler, Thomas; Schulz-Linkholt, Monika; Klüter, Harald

    2011-02-03

    Transfusion-related acute lung injury (TRALI) is a severe complication related with blood transfusion. TRALI has usually been associated with antibodies against leukocytes. The flow cytometric granulocyte immunofluorescence test (Flow-GIFT) has been introduced for routine use when investigating patients and healthy blood donors. Here we describe a novel tool in the automation of the Flow-GIFT that enables a rapid screening of blood donations. We analyzed 440 sera from healthy female blood donors for the presence of granulocyte antibodies. As positive controls, 12 sera with known antibodies against anti-HNA-1a, -b, -2a; and -3a were additionally investigated. Whole-blood samples from HNA-typed donors were collected and the test cells isolated using cell sedimentation in a Ficoll density gradient. Subsequently, leukocytes were incubated with the respective serum and binding of antibodies was detected using FITC-conjugated antihuman antibody. 7-AAD was used to exclude dead cells. Pipetting steps were automated using the Biomek NXp Multichannel Automation Workstation. All samples were prepared in the 96-deep well plates and analyzed by flow cytometry. The standard granulocyte immunofluorescence test (GIFT) and granulocyte agglutination test (GAT) were also performed as reference methods. Sixteen sera were positive in the automated Flow-GIFT, while five of these sera were negative in the standard GIFT (anti-HNA 3a, n = 3; anti-HNA-1b, n = 1) and GAT (anti-HNA-2a, n = 1). The automated Flow-GIFT was able to detect all granulocyte antibodies, which could be only detected in GIFT in combination with GAT. In serial dilution tests, the automated Flow-GIFT detected the antibodies at higher dilutions than the reference methods GIFT and GAT. The Flow-GIFT proved to be feasible for automation. This novel high-throughput system allows an effective antigranulocyte antibody detection in a large donor population in order to prevent TRALI due to transfusion of blood products.

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

    International Nuclear Information System (INIS)

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

    2010-01-01

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

  13. 2006 Bethesda International Consensus recommendations on the immunophenotypic analysis of hematolymphoid neoplasia by flow cytometry: optimal reagents and reporting for the flow cytometric diagnosis of hematopoietic neoplasia.

    Science.gov (United States)

    Wood, Brent L; Arroz, Maria; Barnett, David; DiGiuseppe, Joseph; Greig, Bruce; Kussick, Steven J; Oldaker, Teri; Shenkin, Mark; Stone, Elizabeth; Wallace, Paul

    2007-01-01

    Immunophenotyping by flow cytometry has become standard practice in the evaluation and monitoring of patients with hematopoietic neoplasia. However, despite its widespread use, considerable variability continues to exist in the reagents used for evaluation and the format in which results are reported. As part of the 2006 Bethesda Consensus conference, a committee was formed to attempt to define a consensus set of reagents suitable for general use in the diagnosis and monitoring of hematopoietic neoplasms. The committee included laboratory professionals from private, public, and university hospitals as well as large reference laboratories that routinely operate clinical flow cytometry laboratories with an emphasis on lymphoma and leukemia immunophenotyping. A survey of participants successfully identified the cell lineage(s) to be evaluated for each of a variety of specific medical indications and defined a set of consensus reagents suitable for the initial evaluation of each cell lineage. Elements to be included in the reporting of clinical flow cytometric results for leukemia and lymphoma evaluation were also refined and are comprehensively listed. The 2006 Bethesda Consensus conference represents the first successful attempt to define a set of consensus reagents suitable for the initial evaluation of hematopoietic neoplasia. Copyright 2007 Clinical Cytometry Society.

  14. 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.

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

    Science.gov (United States)

    Rose, Jonathan A; Wanner, Nicholas; Cheong, Hoi I; Queisser, Kimberly; Barrett, Patrick; Park, Margaret; Hite, Corrine; Naga Prasad, Sathyamangla V; Erzurum, Serpil; Asosingh, Kewal

    2016-01-01

    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.

  16. Automated analysis of flow cytometric data for measuring neutrophil CD64 expression using a multi-instrument compatible probability state model.

    Science.gov (United States)

    Wong, Linda; Hill, Beth L; Hunsberger, Benjamin C; Bagwell, C Bruce; Curtis, Adam D; Davis, Bruce H

    2015-01-01

    Leuko64™ (Trillium Diagnostics) is a flow cytometric assay that measures neutrophil CD64 expression and serves as an in vitro indicator of infection/sepsis or the presence of a systemic acute inflammatory response. Leuko64 assay currently utilizes QuantiCALC, a semiautomated software that employs cluster algorithms to define cell populations. The software reduces subjective gating decisions, resulting in interanalyst variability of state modeling (PSM). Four hundred and fifty-seven human blood samples were processed using the Leuko64 assay. Samples were analyzed on four different flow cytometer models: BD FACSCanto II, BD FACScan, BC Gallios/Navios, and BC FC500. A probability state model was designed to identify calibration beads and three leukocyte subpopulations based on differences in intensity levels of several parameters. PSM automatically calculates CD64 index values for each cell population using equations programmed into the model. GemStone software uses PSM that requires no operator intervention, thus totally automating data analysis and internal quality control flagging. Expert analysis with the predicate method (QuantiCALC) was performed. Interanalyst precision was evaluated for both methods of data analysis. PSM with GemStone correlates well with the expert manual analysis, r(2) = 0.99675 for the neutrophil CD64 index values with no intermethod bias detected. The average interanalyst imprecision for the QuantiCALC method was 1.06% (range 0.00-7.94%), which was reduced to 0.00% with the GemStone PSM. The operator-to-operator agreement in GemStone was a perfect correlation, r(2) = 1.000. Automated quantification of CD64 index values produced results that strongly correlate with expert analysis using a standard gate-based data analysis method. PSM successfully evaluated flow cytometric data generated by multiple instruments across multiple lots of the Leuko64 kit in all 457 cases. The probability-based method provides greater objectivity, higher

  17. Clinical evaluation of the endothelial tie-2 crossmatch in ABO compatible and ABO incompatible renal transplants.

    Science.gov (United States)

    Kafetzi, Maria L; Boletis, John N; Melexopoulou, Christine A; Tsakris, Athanassios; Iniotaki, Aliki G; Doxiadis, Ilias I N

    2013-11-01

    The necessity of detection of other than the classical major histocompatibility complex (MHC) and MHC class I-related chain A (MICA) directed antibodies prior to organ transplantation has already been repeatedly reported. A commercial flow cytometric endothelial crossmatch (CM) using isolated peripheral blood tie-2 positive cells provides a tool to detect non-MHC antibodies in addition to antibodies directed to MHC class I and II. The vast majority of circulating tie-2 positive cells expresses HLA-DR but not the A, B blood group antigens. Tie-2 cells are circulating surrogate endothelial cells. In this retrospective study we evaluated the endothelial CM in 51 renal transplantations, 30 with ABO compatible grafts and 21 with ABO incompatible grafts. Fifteen of the ABO compatible recipients (group A) developed unexplained rejection episodes (RE) while the remaining 15 had no RE (group B). Five cases of group A and none of group B had a positive tie-2 CM before transplantation (p=0.042). A positive tie-2 CM was also correlated with graft failure in ABO compatible transplants (p=0.02). No significant correlation was found between a positive pre-transplant tie-2 CM and RE in the ABO incompatible group. This study strongly suggest that a positive tie-2 CM may predict post-transplantation complications in ABO compatible grafts while negative reactions are not predictive. The test is not significantly correlated with RE in ABO incompatible grafts possibly due to applied desensitization. Copyright © 2013 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

  18. Simultaneous use of multiplex ligation-dependent probe amplification assay and flow cytometric DNA ploidy analysis in patients with acute leukemia.

    Science.gov (United States)

    Reyes-Núñez, Virginia; Galo-Hooker, Evelyn; Pérez-Romano, Beatriz; Duque, Ricardo E; Ruiz-Arguelles, Alejandro; Garcés-Eisele, Javier

    2018-01-01

    The aim of this work was to simultaneously use multiplex ligation-dependent probe amplification (MLPA) assay and flow cytometric DNA ploidy analysis (FPA) to detect aneuploidy in patients with newly diagnosed acute leukemia. MLPA assay and propidium iodide FPA were used to test samples from 53 consecutive patients with newly diagnosed acute leukemia referred to our laboratory for immunophenotyping. Results were compared by nonparametric statistics. The combined use of both methods significantly increased the rate of detection of aneuploidy as compared to that obtained by each method alone. The limitations of one method are somehow countervailed by the other and vice versa. MPLA and FPA yield different yet complementary information concerning aneuploidy in acute leukemia. The simultaneous use of both methods might be recommended in the clinical setting. © 2017 International Clinical Cytometry Society. © 2017 International Clinical Cytometry Society.

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

    Science.gov (United States)

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

    2017-04-01

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

  20. 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.

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

    International Nuclear Information System (INIS)

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

    2013-01-01

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

  2. A Fast, Easy, and Customizable Eight-Color Flow Cytometric Method for Analysis of the Cellular Content of Bronchoalveolar Lavage Fluid in the Mouse.

    Science.gov (United States)

    Daubeuf, François; Becker, Julien; Aguilar-Pimentel, Juan Antonio; Ebel, Claudine; Hrabě de Angelis, Martin; Hérault, Yann; Frossard, Nelly

    2017-06-19

    The cell composition of bronchoalveolar lavage fluid (BAL) is an important indicator of airway inflammation. It is commonly determined by cytocentrifuging leukocytes on slides, then staining, identifying, and counting them as eosinophils, neutrophils, macrophages, or lymphocytes according to morphological criteria under light microscopy, where it is not always easy to distinguish macrophages from lymphocytes. We describe here a one-step, easy-to-use, and easy-to-customize 8-color flow cytometric method for performing differential cell count and comparing it to morphological counts on stained cytospins. This method identifies BAL cells by a simultaneous one-step immunolabeling procedure using antibodies to identify T cells, B cells, neutrophils, eosinophils, and macrophages. Morphological analysis of flow-sorted cell subsets is used to validate this protocol. An important advantage of this basic flow cytometry protocol is the ability to customize it by the addition of antibodies to study receptor expression at leukocyte cell surfaces and identify subclasses of inflammatory cells as needed. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  3. 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)

  4. 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.

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

    International Nuclear Information System (INIS)

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

    2013-01-01

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

  6. Cytometric analysis of irradiation damaged chromosomes

    International Nuclear Information System (INIS)

    Wilder, M.E.; Raju, M.R.

    1982-01-01

    Irradiation of cells in interphase results in dose-dependent damage to DNA which is discernable by flow-cytometric analysis of chromosomes. The quantity (and possibly the quality) of chromosomal changes is different in survival-matched doses of x and α irradiation. It may, therefore, be possible to use these methods for analysis of dose and type of exposure in unknown cases

  7. 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, ...

  8. 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)

  9. Chromosome specific DNA hybridization in suspension for flow cytometric detection of chimerism in bone marrow transplantation and leukemia

    NARCIS (Netherlands)

    G.J.A. Arkesteijn (Ger); C.A.J. Erpelinck (Claudia); A.C.M. Martens (Anton); A. Hagenbeek (Anton)

    1995-01-01

    textabstractFlow cytometry was used to measure the fluorescence intensity of nuclei that were subjected to fluorescent in situ hybridization in suspension with chromosome specific DNA probes. Paraformaldehyde-fixed nuclei were protein digested with trypsin and hybridized simultaneously with a

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

    Czech Academy of Sciences Publication Activity Database

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

    2006-01-01

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

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

    Czech Academy of Sciences Publication Activity Database

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

    2004-01-01

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

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

    Science.gov (United States)

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

    2015-07-01

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

  13. Antibody-modified iron oxide nanoparticles for efficient magnetic isolation and flow cytometric determination of L. pneumophila

    International Nuclear Information System (INIS)

    Bloemen, Maarten; Verbiest, Thierry; Denis, Carla; Meester, Luc De; Peeters, Miet; Gils, Ann; Geukens, Nick

    2015-01-01

    We report on the design of superparamagnetic nanoparticles capable of selectively isolating targeted bacteria (Legionella pneumophila, serogroup 1) from aqueous solutions. The surface of magnetite nanoparticles (NP) was functionalized with a heterobifunctional poly(ethylene glycol) ligand containing reactive groups for covalent coupling of polyclonal antibodies against L. pneumophila. These bioconjugates were used to label and magnetically isolate L. pneumophila. Flow cytometry revealed high separation and efficiency in this regard. The strain specificity and efficiency of the magnetic NP was tested with recombinant strains of E. coli (expressing the red fluorescent protein) and L. pneumophila (expressing the green fluorescent protein). The detection limit of the method (by flow cytometry) is 10 4 cells∙mL -1 . The results indicate that the new multifunctional NPs are capable of selectively attracting pathogens from a complex mixture and with high efficiency. This, conceivably, paves the way to pre-concentration protocols for numerous other pathogens. (author)

  14. 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.

  15. Predictive value of the flow cytometric PCNA - assay (proliferating cell nuclear antigen) in head and neck tumors after accelerated-hyperfractionated radiochemotherapy

    Energy Technology Data Exchange (ETDEWEB)

    Wenz, F; Lohr, F; Rudat, V; Dietz, A; Flentje, M; Wannenmacher, M

    1995-07-01

    Purpose/Objective: Proliferation of surviving tumor cells during fractionated radiotherapy may limit tumor control, especially in rapidly proliferating tumors. It has been widely accepted, that this may play a major role in head and neck tumors. Several methods for the assessment of tumor proliferation have been developed, however, most of them are either laborious, invasive or potentially toxic. Today, the gold standard is the flow cytometric BrdUrd assay. We present a flow cytometric method for detection of PCNA, which is an intranuclear proliferation associated protein, in solid human head and neck tumors and how these data correlate with outcome. Materials and Methods: Pretherapeutic biopsies of 20 inoperable patients with squamous cell carcinoma of the head and neck (T3-4N2M0) were examined. The tissue was disaggregated with pepsin/HCl, antibody staining was performed using the clone PC10. Biparametric flow cytometry was performed after a FITC conjugated secondary antibody and propidiumjodine staining was applied. The PCNA-index (i.e. percentage PCNA-positive cells), the DNA-index and the S-phase fraction (SPF, euploid tumors only) were determined. The therapy consisted of combined accelerated-hyperfractionated radiochemotherapy (66 Gy in 5 wks, concomittant boost of 1.6 Gy/d in wks 4+5, Carboplatin in wks 1+5). The median follow-up time was 14 mths (5 - 28), the clinical partners (V.R., A.D.) were 'blinded' towards the PCNA-values. Results: 13 patients suffered from disease progession and 11 died. The actuarial median survival and disease free survival (DFS) were 14.4 and 10.7 mths, respectively. The PCNA-values ranged from 3.2 to 70% (median 9%), there were 7 aneuploid and 13 euploid tumors. SFP in the euploid tumors ranged from 4 to 14.5% (median 10.5%). Neither SFP nor ploidy had a significant influence on the outcome. The patients were divided according to their PCNA-value in higher (n=10) and lower (n=10) than the median. The survival and DFS were 13

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

    Science.gov (United States)

    Galbraith, David W

    2009-08-01

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

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

    DEFF Research Database (Denmark)

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

    2010-01-01

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

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

    DEFF Research Database (Denmark)

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

    1992-01-01

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

  19. Flow cytometric examination of apoptotic effect on brain tissue in postnatal period created by intrauterine oxcarbazepine and gabapentin exposure.

    Science.gov (United States)

    Erisgin, Z; Tekelioglu, Y

    For epileptics, pregnancy contains the balance between no seizure period and antiepileptic use having the least teratogenicity risk. The purpose is to analyse with flow cytometry the apoptotic effects on postnatal brain tissue caused by prenatal use of second generation antiepileptics oxcarbazepine (OXC) and gabapentin (GBP) having different effect mechanisms. 30 (n = 5 each group) Wistar albino male rats (45-days-old) are used. First 3 groups are exposed to OXC (100 mg/kg/day), GBP (50 mg/kg/day), and saline, respectively on the 1st-5th prenatal days (preimplantation-implantation period) while the second 3 groups are exposed to the same substances on the 6th-15th prenatal days (organogenesis), respectively. After sacrifice, brain tissue samples were made into suspension with mechanic and enzymatic digestion and examined with flow cytometry. While apoptosis rate appeared high in rats exposed to OXC on the 1st-5th (p effect in three treatment groups, while difference was not significant for PSS and GBP groups (p = 0.847 and p = 0.934), apoptosis rate was significantly high for OXC on the 6th-15th days compared to the 1st-5th days (p < 0.001). It is observed that the use of OXC causes neurotoxicity during preimplantation, implantation and, especially, organogenesis period (neurogenesis) whereas GBP does not (Fig. 3, Ref. 32).

  20. 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.

  1. Flow cytometric characterization of phenotype, DNA indices and p53 gene expression in 55 cases of acute leukemia.

    Science.gov (United States)

    Powari, Manish; Varma, Neelam; Varma, Subhash; Marwaha, Ram Kumar; Sandhu, Harpreet; Ganguly, Nirmal Kumar

    2002-06-01

    To characterize the phenotype of acute leukemia cases using flow cytometry, to detect mixed lineage cases and to use DNA index determination, including S-phase fraction (SPF) and p53 detection, to find if there was any correlation of SPF and p53 expression with outcome. Fifty-five cases of acute leukemia were enrolled in this study. A complete hemogram and routine bone marrow examination, including cytochemistry, was done. Mycloperoxidase-negative cases were evaluated on a flow cytometer using monoclonal antibodies. DNA indices were determined by flow cytometry in all cases, and p53 was detected immunohistochemically using the alkaline phosphatase/antialkaline phosphatase technique. Acute myeloblastic leukemia (AML) was diagnosed in 32 cases; acute lymphoblastic leukemia (ALL) was diagnosed in 18 (14 B lineage and 4 T line age). Four cases showed mixed lineage leukemia, and undifferentiated acute leukemia was diagnosed in one case. The mean/range of SPF for these groups were 3.76/0.33-6.91, 6.25/0.15-21.4, 2.89/0.35-10.64, 2.60/0.72-6.94 and 7.34, respectively. Aneuploidy was detected in two cases of B-lineage ALL and tetraploidy in a case of AML-M7, while all others were diploid p53. Was detected in 6 of 55 cases (10.90%). Follow-up was available for 24 patients. Five patients relapsed, and four had B-cell type ALL and were diploid and expressed no p53 gene. SPF% did not show any correlation with outcome. These data suggest that within acute leukemia subtypes, there is a wide variation in SPF. SPF does not seem to correlate with outcome. Immunophenotyping is essential to determine the lineage in myeloperoxidase-negative cases. It is perhaps the only way to diagnose mixed lineage leukemia and aberrant expression of markers presently. The p53 gene was detected less frequently. However, more studies are required from different centers with longer follow-up to evaluate prognostic significance.

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

    International Nuclear Information System (INIS)

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

    1984-01-01

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

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

    International Nuclear Information System (INIS)

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

    1994-01-01

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

  4. 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.

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

    DEFF Research Database (Denmark)

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

    2014-01-01

    experienced seven OUH patients, initially presenting negative CDC T-cell crossmatches, who developed weakly positive CDC T-cell crossmatch shortly prior to transplantation coinciding with a significant DSA reduction. The complication was not observed in AUH patients who received apheresis. This led us...

  6. [Flow cytometric test using eosin-5'-maleimide (EMA) labelling of red blood for diagnosis of hereditary spherocytosis].

    Science.gov (United States)

    Wang, Jiying; Zheng, Bin; Zhao, Yuping; Chen, Xuejing; Liu, Yan; Bo, Lijin; Zheng, Yizhou; Zhang, Fengkui; Ru, Kun; Wang, Huijun

    2015-07-01

    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. EMA flow cytometry test, NaCl-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. 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 that of acidified glycerol lysis test was 1.000 and 0.318, respectively. Although the sensitivity of NaCl-osmotic fragility test and acidified glycerol lysis test was a little higher than that of EMA binding test, the specificity of the former two methods was poor, they couldn't clearly distinguish whether spherocytosis is hereditary spherocytosis. The experiment results showed that EMA was sensitive to the temperature and should not be stored in a small aliquots at -80 ℃ over a period of 6 months. The stability of the HS sample was better, 6 days storage at 4 ℃ and 3 days storage at room temperature had no influence on the results. EMA binding test by flow cytometry showed good sensitivity and specificity for HS diagnosis. EMA reagent should be stored at-80 ℃ and the HS samples should be tested within 6 days storage at 4 ℃ and 3 days at room temperature.

  7. Cr(VI) induces DNA damage, cell cycle arrest and polyploidization: a flow cytometric and comet assay study in Pisum sativum.

    Science.gov (United States)

    Rodriguez, Eleazar; Azevedo, Raquel; Fernandes, Pedro; Santos, Conceição

    2011-07-18

    Chromium(VI) is recognized as the most toxic valency of Cr, but its genotoxicity and cytostaticity in plants is still poorly studied. In order to analyze Cr(VI) cyto- and gentotoxicity, Pisum sativum L. plants were grown in soil and watered with solutions with different concentrations of Cr up to 2000 mg/L. After 28 days of exposure, leaves showed no significant variations in either cell cycle dynamics or ploidy level. As for DNA damage, flow cytometric (FCM) histograms showed significant differences in full peak coefficient of variation (FPCV) values, suggesting clastogenicity. This is paralleled by the Comet assay results, showing an increase in DNA damage for 1000 and 2000 mg/L. In roots, exposure to 2000 mg/L resulted in cell cycle arrest at the G(2)/M checkpoint. It was also verified that under the same conditions 40% of the individuals analyzed suffered polyploidization having both 2C and 4C levels. DNA damage analysis by the Comet assay and FCM revealed dose-dependent increases in DNA damage and FPCV. Through this, we have unequivocally demonstrated for the first time in plants that Cr exposure can result in DNA damage, cell cycle arrest, and polyploidization. Moreover, we critically compare the validity of the Comet assay and FCM in evaluating cytogenetic toxicity tests in plants and demonstrate that the data provided by both techniques complement each other and present high correlation levels. In conclusion, the data presented provides new insight on Cr effects in plants in general and supports the use of the parameters tested in this study as reliable endpoints for this metal toxicity in plants. © 2011 American Chemical Society

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

    Science.gov (United States)

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

    2018-01-01

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

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

    Science.gov (United States)

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

    2015-08-01

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

  10. Multiparameter flow cytometric remission is the most relevant prognostic factor for multiple myeloma patients who undergo autologous stem cell transplantation

    Science.gov (United States)

    Paiva, Bruno; Vidriales, Maria-Belén; Cerveró, Jorge; Mateo, Gema; Pérez, Jose J.; Montalbán, Maria A.; Sureda, Anna; Montejano, Laura; Gutiérrez, Norma C.; de Coca, Alfonso García; de las Heras, Natalia; Mateos, Maria V.; López-Berges, Maria C.; García-Boyero, Raimundo; Galende, Josefina; Hernández, Jose; Palomera, Luis; Carrera, Dolores; Martínez, Rafael; de la Rubia, Javier; Martín, Alejandro; Bladé, Joan; Lahuerta, Juan J.; Orfao, Alberto

    2008-01-01

    Minimal residual disease (MRD) assessment is standard in many hematologic malignancies but is considered investigational in multiple myeloma (MM). We report a prospective analysis of the prognostic importance of MRD detection by multiparameter flow cytometry (MFC) in 295 newly diagnosed MM patients uniformly treated in the GEM2000 protocol VBMCP/VBAD induction plus autologous stem cell transplantation [ASCT]). MRD status by MFC was determined at day 100 after ASCT. Progression-free survival (PFS; median 71 vs 37 months, P < .001) and overall survival (OS; median not reached vs 89 months, P = .002) were longer in patients who were MRD negative versus MRD positive at day 100 after ASCT. Similar prognostic differentiation was seen in 147 patients who achieved immunofixation-negative complete response after ASCT. Moreover, MRD− immunofixation-negative (IFx−) patients and MRD− IFx+ patients had significantly longer PFS than MRD+ IFx− patients. Multivariate analysis identified MRD status by MFC at day 100 after ASCT as the most important independent prognostic factor for PFS (HR = 3.64, P = .002) and OS (HR = 2.02, P = .02). Our findings demonstrate the clinical importance of MRD evaluation by MFC, and illustrate the need for further refinement of MM re-sponse criteria. This trial is registered at http://clinicaltrials.gov under identifier NCT00560053. PMID:18669875

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

    Science.gov (United States)

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

    2010-05-01

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

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

    Science.gov (United States)

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

    2018-02-01

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

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

    Science.gov (United States)

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

    2017-11-01

    Within recent years, there has been growing interest in the prediction of bull fertility through in vitro assessment of semen quality. A model for fertility prediction based on early evaluation of semen quality parameters, to exclude sires with potentially low fertility from breeding programs, would therefore be useful. The aim of the present study was to identify the most suitable parameters that would provide reliable prediction of fertility. Frozen semen from 18 Italian Holstein-Friesian proven bulls was analyzed using computer-assisted semen analysis (CASA) (motility and kinetic parameters) and flow cytometry (FCM) (viability, acrosomal integrity, mitochondrial function, lipid peroxidation, plasma membrane stability and DNA integrity). Bulls were divided into two groups (low and high fertility) based on the estimated relative conception rate (ERCR). Significant differences were found between fertility groups for total motility, active cells, straightness, linearity, viability and percentage of DNA fragmented sperm. Correlations were observed between ERCR and some kinetic parameters, and membrane instability and some DNA integrity indicators. In order to define a model with high relation between semen quality parameters and ERCR, backward stepwise multiple regression analysis was applied. Thus, we obtained a prediction model that explained almost half (R 2=0.47, P<0.05) of the variation in the conception rate and included nine variables: five kinetic parameters measured by CASA (total motility, active cells, beat cross frequency, curvilinear velocity and amplitude of lateral head displacement) and four parameters related to DNA integrity evaluated by FCM (degree of chromatin structure abnormality Alpha-T, extent of chromatin structure abnormality (Alpha-T standard deviation), percentage of DNA fragmented sperm and percentage of sperm with high green fluorescence representative of immature cells). A significant relationship (R 2=0.84, P<0.05) was observed between

  14. Flow cytometric sex sorting affects CD4 membrane distribution and binding of exogenous DNA on bovine sperm cells.

    Science.gov (United States)

    Domingues, William Borges; da Silveira, Tony Leandro Rezende; Komninou, Eliza Rossi; Monte, Leonardo Garcia; Remião, Mariana Härter; Dellagostin, Odir Antônio; Corcini, Carine Dahl; Varela Junior, Antônio Sergio; Seixas, Fabiana Kömmling; Collares, Tiago; Campos, Vinicius Farias

    2017-08-01

    Bovine sex-sorted sperm have been commercialized and successfully used for the production of transgenic embryos of the desired sex through the sperm-mediated gene transfer (SMGT) technique. However, sex-sorted sperm show a reduced ability to internalize exogenous DNA. The interaction between sperm cells and the exogenous DNA has been reported in other species to be a CD4-like molecule-dependent process. The flow cytometry-based sex-sorting process subjects the spermatozoa to different stresses causing changes in the cell membrane. The aim of this study was to elucidate the relationship between the redistribution of CD4-like molecules and binding of exogenous DNA to sex-sorted bovine sperm. In the first set of experiments, the membrane phospholipid disorder and the redistribution of the CD4 were evaluated. The second set of experiments was conducted to investigate the effect of CD4 redistribution on the mechanism of binding of exogenous DNA to sperm cells and the efficiency of lipofection in sex-sorted bovine sperm. Sex-sorting procedure increased the membrane phospholipid disorder and induced the redistribution of CD4-like molecules. Both X-sorted and Y-sorted sperm had decreased DNA bound to membrane in comparison with the unsorted sperm; however, the binding of the exogenous DNA was significantly increased with the addition of liposomes. Moreover, we demonstrated that the number of sperm-bound exogenous DNA was decreased when these cells were preincubated with anti-bovine CD4 monoclonal antibody, supporting our hypothesis that CD4-like molecules indeed play a crucial role in the process of exogenous DNA/bovine sperm cells interaction.

  15. Flow cytometric analysis reveals the high levels of platelet activation parameters in circulation of multiple sclerosis patients.

    Science.gov (United States)

    Morel, Agnieszka; Rywaniak, Joanna; Bijak, Michał; Miller, Elżbieta; Niwald, Marta; Saluk, Joanna

    2017-06-01

    The epidemiological studies confirm an increased risk of cardiovascular disease in multiple sclerosis, especially prothrombotic events directly associated with abnormal platelet activity. The aim of our study was to investigate the level of blood platelet activation in the circulation of patients with chronic phase of multiple sclerosis (SP MS) and their reactivity in response to typical platelets' physiological agonists. We examined 85 SP MS patients diagnosed according to the revised McDonald's criteria and 50 healthy volunteers as a control group. The platelet activation and reactivity were assessed using flow cytometry analysis of the following: P-selectin expression (CD62P), activation of GP IIb/IIIa complex (PAC-1 binding), and formation of platelet microparticles (PMPs) and platelet aggregates (PA) in agonist-stimulated (ADP, collagen) and unstimulated whole blood samples. Furthermore, we measured the level of soluble P-selectin (sP-selectin) in plasma using ELISA method, to evaluate the in vivo level of platelet activation, both in healthy and SP MS subjects. We found a statistically significant increase in P-selectin expression, GP IIb/IIIa activation, and formation of PMPs and PA, as well as in unstimulated and agonist-stimulated (ADP, collagen) platelets in whole blood samples from patients with SP MS in comparison to the control group. We also determined the higher sP-selectin level in plasma of SP MS subjects than in the control group. Based on the obtained results, we might conclude that during the course of SP MS platelets are chronically activated and display hyperreactivity to physiological agonists, such as ADP or collagen.

  16. 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). Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Use of internal control T-cell populations in the flow cytometric evaluation for T-cell neoplasms.

    Science.gov (United States)

    Hunt, Alicia M; Shallenberger, Wendy; Ten Eyck, Stephen P; Craig, Fiona E

    2016-09-01

    Flow cytometry is an important tool for identification of neoplastic T-cells, but immunophenotypic abnormalities are often subtle and must be distinguished from nonneoplastic subsets. Use of internal control (IC) T-cells in the evaluation for T-cell neoplasms was explored, both as a quality measure and as a reference for evaluating abnormal antigen expression. All peripheral blood specimens (3-month period), or those containing abnormal T-cells (29-month period), stained with CD45 V500, CD2 V450, CD3 PE-Cy7, CD7 PE, CD4 Per-CP-Cy5.5, CD8 APC-H7, CD56 APC, CD16&57 FITC, were evaluated. IC T-cells were identified (DIVA, BD Biosciences) and median fluorescence intensity (MFI) recorded. Selected files were merged and reference templates generated (Infinicyt, Cytognos). IC T-cells were present in all specimens, including those with abnormal T-cells, but subsets were less well-represented. IC T-cell CD3 MFI differed between instruments (p = 0.0007) and subsets (p < 0.001), but not specimen categories, and served as a longitudinal process control. Merged files highlighted small unusual IC-T subsets: CD2+(dim) (0.25% total), CD2- (0.03% total). An IC reference template highlighted neoplastic T-cells, but was limited by staining variability (IC CD3 MFI reference samples different from test (p = 0.003)). IC T-cells present in the majority of specimens can serve as positive and longitudinal process controls. Use of IC T-cells as an internal reference is limited by variable representation of subsets. Analysis of merged IC T-cells from previously analyzed patient samples can alert the interpreter to less-well-recognized non-neoplastic subsets. However, application of a merged file IC reference template was limited by staining variability. © 2016 Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.

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

    Directory of Open Access Journals (Sweden)

    Zhou F

    2018-03-01

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

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

    Science.gov (United States)

    Dasgupta, Alakananda; Mahapatra, Manoranjan; Saxena, Renu

    2013-04-01

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

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

    International Nuclear Information System (INIS)

    Pinkel, D.

    1983-01-01

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

  1. Cytometric approaches to biological dosimetry

    International Nuclear Information System (INIS)

    Burger, G.

    1983-01-01

    Automatic cytometric techniques for detecting chromosomal aberrations are being tested but will not be used in routine examinations for some time to come. Automatic micronuclei counts are more promising but not sufficiently sensitive in the low dose range ( [de

  2. Emergency heart retransplantation with a positive donor crossmatch.

    Science.gov (United States)

    Ippoliti, G; Martinelli, L; Minzioni, G; Goggi, C; Graffigna, A; Rinaldi, M; Campana, C; Ascari, E; Vigano, M

    1989-01-01

    We report a case of one patient who underwent emergency retransplantation with a highly positive donor crossmatch. Standard immunosuppression was integrated by the addition of plasma exchange during extracorporeal circulation, polyclonal IgG, and cyclophosphamide for the first 30 days. After transplantation the clinical outcome was normal; immunosuppression induced a complete disappearance of the donor-specific antibody. In spite of the heavy immunosuppression, we did not observe any infectious complications. We suggest that a greater immunosuppression established soon after the transplant and adjusted on the basis of immunological monitoring may allow a heart transplant with a positive crossmatch.

  3. Deep sequencing and flow cytometric characterization of expanded effector memory CD8+CD57+ T cells frequently reveals T-cell receptor Vβ oligoclonality and CDR3 homology in acquired aplastic anemia.

    Science.gov (United States)

    Giudice, Valentina; Feng, Xingmin; Lin, Zenghua; Hu, Wei; Zhang, Fanmao; Qiao, Wangmin; Ibanez, Maria Del Pilar Fernandez; Rios, Olga; Young, Neal S

    2018-05-01

    Oligoclonal expansion of CD8 + CD28 - lymphocytes has been considered indirect evidence for a pathogenic immune response in acquired aplastic anemia. A subset of CD8 + CD28 - cells with CD57 expression, termed effector memory cells, is expanded in several immune-mediated diseases and may have a role in immune surveillance. We hypothesized that effector memory CD8 + CD28 - CD57 + cells may drive aberrant oligoclonal expansion in aplastic anemia. We found CD8 + CD57 + cells frequently expanded in the blood of aplastic anemia patients, with oligoclonal characteristics by flow cytometric Vβ usage analysis: skewing in 1-5 Vβ families and frequencies of immunodominant clones ranging from 1.98% to 66.5%. Oligoclonal characteristics were also observed in total CD8 + cells from aplastic anemia patients with CD8 + CD57 + cell expansion by T-cell receptor deep sequencing, as well as the presence of 1-3 immunodominant clones. Oligoclonality was confirmed by T-cell receptor repertoire deep sequencing of enriched CD8 + CD57 + cells, which also showed decreased diversity compared to total CD4 + and CD8 + cell pools. From analysis of complementarity-determining region 3 sequences in the CD8 + cell pool, a total of 29 sequences were shared between patients and controls, but these sequences were highly expressed in aplastic anemia subjects and also present in their immunodominant clones. In summary, expansion of effector memory CD8 + T cells is frequent in aplastic anemia and mirrors Vβ oligoclonal expansion. Flow cytometric Vβ usage analysis combined with deep sequencing technologies allows high resolution characterization of the T-cell receptor repertoire, and might represent a useful tool in the diagnosis and periodic evaluation of aplastic anemia patients. (Registered at clinicaltrials.gov identifiers: 00001620, 01623167, 00001397, 00071045, 00081523, 00961064 ). Copyright © 2018 Ferrata Storti Foundation.

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

    DEFF Research Database (Denmark)

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

    2016-01-01

    OBJECTIVES: To understand the worldwide scope of RBC crossmatching and issuing practices and measure efficiency using a novel quality indicator, the crossmatch/issue (C/I) ratio. METHODS: An electronic survey was disseminated to hospital transfusion services collecting details about RBC...... crossmatching and issuing practices. Respondents were asked to enumerate the number of RBCs crossmatched and issued at their institutions during the 2014 calendar year to calculate the C/I ratio. RESULTS: Fifty-two survey responses were received, mostly from North American transfusion services (28/52, 54......%). The electronic crossmatch was the most common technique (n = 29), and most respondents performed the crossmatch at the time that an order for RBCs was received in the transfusion service (even if an order to issue the RBCs was not received). Data to calculate the C/I ratio were supplied by 22 respondents...

  5. Towards a Next-Generation Catalogue Cross-Match Service

    Science.gov (United States)

    Pineau, F.; Boch, T.; Derriere, S.; Arches Consortium

    2015-09-01

    We have been developing in the past several catalogue cross-match tools. On one hand the CDS XMatch service (Pineau et al. 2011), able to perform basic but very efficient cross-matches, scalable to the largest catalogues on a single regular server. On the other hand, as part of the European project ARCHES1, we have been developing a generic and flexible tool which performs potentially complex multi-catalogue cross-matches and which computes probabilities of association based on a novel statistical framework. Although the two approaches have been managed so far as different tracks, the need for next generation cross-match services dealing with both efficiency and complexity is becoming pressing with forthcoming projects which will produce huge high quality catalogues. We are addressing this challenge which is both theoretical and technical. In ARCHES we generalize to N catalogues the candidate selection criteria - based on the chi-square distribution - described in Pineau et al. (2011). We formulate and test a number of Bayesian hypothesis which necessarily increases dramatically with the number of catalogues. To assign a probability to each hypotheses, we rely on estimated priors which account for local densities of sources. We validated our developments by comparing the theoretical curves we derived with the results of Monte-Carlo simulations. The current prototype is able to take into account heterogeneous positional errors, object extension and proper motion. The technical complexity is managed by OO programming design patterns and SQL-like functionalities. Large tasks are split into smaller independent pieces for scalability. Performances are achieved resorting to multi-threading, sequential reads and several tree data-structures. In addition to kd-trees, we account for heterogeneous positional errors and object's extension using M-trees. Proper-motions are supported using a modified M-tree we developed, inspired from Time Parametrized R-trees (TPR

  6. Perioperative Desensitization Improves Outcomes Among Crossmatch Positive Recipients of Deceased Donor Renal Transplants.

    Science.gov (United States)

    Sharma, Amit; King, Anne; Kumar, Dhiren; Behnke, Martha; McDougan, Felecia; Kimball, Pamela M

    2016-06-01

    Graft failure due to chronic rejection is greater among renal transplant patients with donor-specific antibody (DSA) than among DSA-free patients. For patients dependent on deceased donor transplantation, preoperative desensitization to eliminate DSAs may be impractical. We speculated that perioperative desensitization might eliminate preexisting DSAs and prevent de novo DSAs and improve graft outcomes. We report that brief perioperative desensitization using either intravenous immunoglobulin (IVIG) or plasmapheresis/IVIG (PP/IVIG) treatment improves clinical outcomes among patients with positive crossmatches. Immediately following deceased donor transplantation, 235 renal recipients were assigned points for PRA and flow crossmatches (FCXM): delayed graft function (DGF) ≤ 1 point received standard therapy; 2 points received high-dose IVIG; and ≥3 points received PP/IVIG. The DSAs were serially monitored by single antigen bead luminex for 1 year. Five-year clinical outcomes were determined from the chart review. All desensitized patients had preoperatively positive FCXM with DSA. Rejection was more common (P desensitized than nonsensitized groups. However, overall graft survivals were similar between the groups (P = not significant) and superior to historic untreated patients (P 90% in all desensitizated patients with DSA elimination as well as PP/IVIG patients with residual DSA. In contrast, IVIG patients with persistent DSA had poorer graft survival (45%, P desensitization improved overall graft survival of sensitized patients compared to historic untreated patients. Plasmapheresis/IVIG had greater impact on DSA eradication and graft survival than IVIG alone. © 2016, NATCO.

  7. A radiolabeled antiglobulin test for crossmatching platelet transfusions

    International Nuclear Information System (INIS)

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

    1983-01-01

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

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

    DEFF Research Database (Denmark)

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

    2012-01-01

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

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

    DEFF Research Database (Denmark)

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

    2006-01-01

    High hyperdiploid acute lymphoblastic leukemia in children is related to a good outcome. Because these patients may be stratified to a low-intensity treatment, we have investigated the sensitivity of flow cytometry (FCM), G-band karyotyping (GBK), and high-resolution comparative genomic hybridiza......High hyperdiploid acute lymphoblastic leukemia in children is related to a good outcome. Because these patients may be stratified to a low-intensity treatment, we have investigated the sensitivity of flow cytometry (FCM), G-band karyotyping (GBK), and high-resolution comparative genomic...

  10. An improved flow cytometric method using FACS lysing solution for measurement of ZAP-70 expression in B-cell chronic lymphocytic leukemia

    NARCIS (Netherlands)

    Bekkema, Roelof; Tadema, Afke; Daenen, Simon M. G. J.; Kluin-Nelemans, Hanneke C.; Mulder, Andre B.

    Background: B-cell expression of ZAP-70, normally expressed in T and NK cells, correlates with poor prognosis in B-CLL. Poor discrimination between ZAP-70 positive and negative cells hampers routine application of flow cytometry. We examined the usefulness of FACS Lysing Solution. Methods: ZAP-70

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

    Science.gov (United States)

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

    2018-04-01

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

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

    Directory of Open Access Journals (Sweden)

    Ian McGowan

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    DEFF Research Database (Denmark)

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

    2013-01-01

    Characterization of circulating microparticles (MPs) is usually performed by flow cytometry. Annexin V, a protein that Ca(2+)-dependently binds to phosphatidylserine, has been used to define entire microparticle (MP) populations, but not all MPs bind AnxV. Recent reports have correlated Anx...... for comprehensive assessment of circulating MPs directly from platelet-poor plasma with characterization of AnxV-binding and of cellular origin of MPs....

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

    Directory of Open Access Journals (Sweden)

    Yingzi Ming

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

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

    Science.gov (United States)

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

    2016-02-01

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

  17. Flow Cytometric 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. PMID:26111048

  18. 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.

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

    Science.gov (United States)

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

    2016-09-01

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

  20. Flow cytometric detection of growth factor receptors in autografts and analysis of growth factor concentrations in autologous stem cell transplantation: possible significance for platelet recovery

    DEFF Research Database (Denmark)

    Schiødt, I; Jensen, Charlotte Harken; Kjaersgaard, E

    2000-01-01

    In order to improve prediction of hematopoietic recovery, we conducted a pilot study, analyzing the significance of growth factor receptor expression in autografts as well as endogenous growth factor levels in blood before, during and after stem cell transplantation. Three early acting (stem cell......-CSF receptor positive, CD34+ progenitor cells were measured by flow cytometry in the leukapheresis product used for transplantation in a subgroup of 15 patients (NHL, n = 8, MM, n = 7). Three factors were identified as having a significant impact on platelet recovery. First, the level of Tpo in blood...... at the time of the nadir (day +7). Second, the percentage of re-infused thrombopoietin receptor positive progenitors and finally, the percentage of Flt3 receptor positive progenitors. On the other hand, none of the analyzed factors significantly predicted myeloid or erythroid recovery. These findings need...

  1. Flow cytometric minimal residual disease assessment of peripheral blood in acute lymphoblastic leukaemia patients has potential for early detection of relapsed extramedullary disease.

    Science.gov (United States)

    Keegan, Alissa; Charest, Karry; Schmidt, Ryan; Briggs, Debra; Deangelo, Daniel J; Li, Betty; Morgan, Elizabeth A; Pozdnyakova, Olga

    2018-03-27

    To evaluate peripheral blood (PB) for minimal residual disease (MRD) assessment in adults with acute lymphoblastic leukaemia (ALL). We analysed 76 matched bone marrow (BM) aspirate and PB specimens independently for the presence of ALL MRD by six-colour flow cytometry (FC). The overall rate of BM MRD-positivity was 24% (18/76) and PB was also MRD-positive in 22% (4/18) of BM-positive cases. We identified two cases with evidence of leukaemic cells in PB at the time of the extramedullary relapse that were interpreted as MRD-negative in BM. The use of PB MRD as a non-invasive method for monitoring of systemic relapse may have added clinical and diagnostic value in patients with high risk of extramedullary disease. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

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

    International Nuclear Information System (INIS)

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

    1992-09-01

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

  3. Flow cytometric estimation on cytotoxic activity of leaf extracts from seashore plants in subtropical Japan: isolation, quantification and cytotoxic action of (-)-deoxypodophyllotoxin.

    Science.gov (United States)

    Masuda, Toshiya; Oyama, Yasuo; Yonemori, Shigetomo; Takeda, Yoshio; Yamazaki, Yuko; Mizuguchi, Shinichi; Nakata, Mami; Tanaka, Tomochika; Chikahisa, Lumi; Inaba, Yuzuru; Okada, Yoshihiko

    2002-06-01

    The cytotoxic activity of methanol extracts of leaves collected from 39 seashore plants in Iriomote Island, subtropical Japan was examined on human leukaemia cells (K562 cells) using a flow cytometer with two fluorescent probes, ethidium bromide and annexin V-FITC. Five extracts (10 microg/mL) from Hernandia nymphaeaefolia, Cerbera manghas, Pongamia pinnata, Morus australis var. glabra and Thespesia populnea greatly inhibited the growth of K562 cells. When the concentration was decreased to 1 microg/mL, only one extract from H. nymphaeaefolia still inhibited the cell growth. A cytotoxic compound was isolated from the leaves by bioassay-guided fractionation and was identified as (-)-deoxypodophyllotoxin (DPT). The fresh leaves of H. nymphaeaefolia contained a remarkably high amount of DPT (0.21 +/- 0.07% of fresh leaf weight), being clarified by a quantitative HPLC analysis. DPT at 70-80 pM started to inhibit the growth of K562 cells in an all-or-none fashion and at 100 pM or more it produced complete inhibition in all cases. Therefore, the slope of the dose-response curve was very steep. DPT at 100 pM or more decreased the cell viability to 50%-60% and increased the number of cells undergoing apoptosis (annexin V-positive cells). The results indicate that DPT contributes to the cytotoxic action of the extract from the leaves of H. nymphaeaefolia on K562 cells. Copyright 2002 John Wiley & Sons, Ltd.

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

    Science.gov (United States)

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

    2018-03-13

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

  5. Flow cytometric characterization of culture expanded multipotent mesenchymal stromal cells (MSCs) from horse adipose tissue: towards the definition of minimal stemness criteria.

    Science.gov (United States)

    Pascucci, L; Curina, G; Mercati, F; Marini, C; Dall'Aglio, C; Paternesi, B; Ceccarelli, P

    2011-12-15

    In the last decades, multipotent mesenchymal progenitor cells have been isolated from many adult tissues of different species. The International Society for Cellular Therapy (ISCT) has recently established that multipotent mesenchymal stromal cells (MSCs) is the currently recommended designation. In this study, we used flow cytometry to evaluate the expression of several molecules related to stemness (CD90, CD44, CD73 and STRO-1) in undifferentiated, early-passaged MSCs isolated from adipose tissue of four donor horses (AdMSCs). The four populations unanimously expressed high levels of CD90 and CD44. On the contrary, they were unexpectedly negative to CD73. A small percentage of the cells, finally, showed the expression of STRO-1. This last result might be due to the existence of a small subpopulation of STRO-1+ cells or to a poor cross-reactivity of the antibody. A remarkable donor-to-donor consistency and reproducibility of these findings was demonstrated. The data presented herein support the idea that equine AdMSCs may be easily isolated and selected by adherence to tissue culture plastic and exhibit a surface profile characterized by some peculiar differences in comparison to those described in other species. Continued characterization of these cells will help to clarify several aspects of their biology and may ultimately enable the isolation of specific, purified subpopulations. Copyright © 2011 Elsevier B.V. All rights reserved.

  6. Multi-color CD34⁺ progenitor-focused flow cytometric assay in evaluation of myelodysplastic syndromes in patients with post cancer therapy cytopenia.

    Science.gov (United States)

    Tang, Guilin; Jorgensen, L Jeffrey; Zhou, Yi; Hu, Ying; Kersh, Marian; Garcia-Manero, Guillermo; Medeiros, L Jeffrey; Wang, Sa A

    2012-08-01

    Bone marrow assessment for myelodysplastic syndrome (MDS) in a patient who develops cytopenia(s) following cancer therapy is challenging. With recent advances in multi-color flow cytometry immunophenotypic analysis, a CD34(+) progenitor-focused 7-color assay was developed and tested in this clinical setting. This assay was first performed in 73 MDS patients and 53 non-MDS patients (developmental set). A number of immunophenotypic changes were differentially observed in these two groups. Based on the sensitivity, specificity and reproducibility, a core panel of markers was selected for final assessment that included increased total CD34(+) myeloblasts; decreased stage I hematogones; altered CD45/side scatter; altered expression of CD13, CD33, CD34, CD38, CD117, and CD123; aberrant expression of lymphoid or mature myelomonocytic antigens on CD34(+) myeloblasts; and several marked alterations in maturing myelomonocytic cells. The data were translated into a simplified scoring system which was then used in 120 patients with cytopenia(s) secondary to cancer therapy over a 2-year period (validation set). With a median follow-up of 11 months, this assay demonstrated 89% sensitivity, 94% specificity, and 92% accuracy in establishing or excluding a diagnosis of MDS. Copyright © 2012 Elsevier Ltd. All rights reserved.

  7. Cell kinetics of hypoxic cells in a murine tumour in vivo: flow cytometric determination of the radiation-induced blockage of cell cycle progression

    International Nuclear Information System (INIS)

    Rutgers, D.H.; Niessen, D.P.P.; Linden, P.M. van der

    1987-01-01

    Cells from the small cell population of viable cells in the large necrotic centre of murine M8013 tumours were investigated with respect to their cell kinetics. Flow cytometry (FCM) of this part of subcutaneously transplanted tumours revealed the presence of tumour cells with G1,S and G2 + M phase DNA-contents. These severely hypoxic cells could have stopped cell cycle progression due to the nutritional deprivation, irrespective of their position within the cell cycle. Labelling methods, used to disclose the cell kinetics of this cell population, are hampered by the absence of a transport system in these large necrotic areas. Therefore FCM was used to monitor radiation induced changes in the cell cycle distribution. From this investigation it was concluded that hypoxic cells in the necrotic centre of the M8013 tumour progress through the cell cycle. As well as a cell population with a cell cycle time (Tsub(c)) of approximately 84 hr, a subpopulation with a Tsub(c) of approximately 21 hr occurred. (author)

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

    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

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

    International Nuclear Information System (INIS)

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

    1984-01-01

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

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

    LENUS (Irish Health Repository)

    Al-Sibae, Mohamad R

    2012-02-01

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

  11. Protein a Immunoadsorption May Hamper the Decision to Transplant Due to Interference With CDC Crossmatch Results

    DEFF Research Database (Denmark)

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

    2017-01-01

    using a highly sensitive time-resolved fluorescent assay. In conclusion, the results emphasize the importance of carefully considering CDC crossmatch results subsequent to IA, before a planned transplantation is either postponed or cancelled. J. Clin. Apheresis 32:163-169, 2017. © 2016 Wiley Periodicals...

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

    Directory of Open Access Journals (Sweden)

    Mehmet Özen

    2018-02-01

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

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

    Science.gov (United States)

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

    2017-09-01

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

  14. Audit on the Efficient Use of Cross-Matched Blood in Elective Total Hip and Total Knee Replacement

    Science.gov (United States)

    Rogers, BA; Johnstone, DJ

    2006-01-01

    INTRODUCTION This prospective audit studies the use of cross-matched blood in 301 patients over a 1-year period undergoing total knee (TKR) and total hip replacement (THR) surgery in an orthopaedic unit. PATIENTS AND METHODS Analysis over the first 6 months revealed a high level of unnecessary cross-matched blood. The following interventions were introduced: (i) to cease routine cross-matching for THR; (ii) all patients to have a check full blood count on day 2 after surgery; and (iii) Hb < 8 g/dl to be considered as the trigger for transfusion in patients over 65 years and free from significant co-morbidity. These changes are in accordance with published national guidelines [Anon. Guidelines for the clinical use of red cell transfusions. Br J Haematol 2001; 113: 24–31]. RESULTS In the next 6 months, the number of units cross-matched but not transfused fell by 96% for THR, and the cross-match transfusion (C:T) ratio reduced from 3.21 to 1.62. Reductions were also observed for the TKR cohort. These results provide evidence of a substantial risk and cost benefit in the use of this limited resource. A telephone survey of 44 hospitals revealed that 20 hospitals routinely cross-matched blood for THR and 11 do so for TKR. CONCLUSIONS Changes can be made to the Maximum Surgical Blood Ordering Schedules (MSBOS) in other orthopaedic units according to national guidelines. PMID:16551419

  15. Assignment of C1q-binding HLA antibodies as unacceptable HLA antigens avoids positive CDC-crossmatches prior to transplantation of deceased donor organs.

    Science.gov (United States)

    Juhl, David; Marget, Matthias; Hallensleben, Michael; Görg, Siegfried; Ziemann, Malte

    2017-03-01

    Soon, a virtual crossmatch shall replace the complement-dependent cytotoxicity (CDC) allocation crossmatch in the Eurotransplant region. To prevent positive CDC-crossmatches in the recipient centre, careful definition of unacceptable antigens is necessary. For highly sensitized patients, this is difficult by CDC alone. Assignment of all antibodies detected by sensitive assays, however, could prevent organ allocation. To assess the usefulness of the Luminex C1q-assay to prevent positive CDC-crossmatches, all CDC-crossmatches performed prior to deceased kidney transplantation in a 16-month-period were reviewed. Sera causing positive crossmatches were investigated by the C1q-assay. 31 out of 1432 crossmatches (2.2%) were positive. Sera involved in 26 positive crossmatches were available. C1q-binding donor-specific antibodies were detected in 19 sera (73.1%). The other sera were from recipients without any HLA antibodies detectable by CDC or common solid phase assays. Three patients had known Non-HLA antibodies causing positive CDC-results. Four crossmatches were only weak positive. Therefore, avoidance of donors with HLA antigens against whom C1q-binding antibodies were detected would have prevented all positive crossmatches due to HLA antibodies. Provided that all HLA specificities against which antibodies are detected by the Luminex C1q-assay are considered as unacceptable antigens, CDC-crossmatches prior to transplantation might safely be omitted in many patients. They should be maintained in highly immunized patients, however, for whom assignment of all C1q-positive antibodies as unacceptable antigens could lead to a significant delay or even prevention of transplantation. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. 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.

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

    International Nuclear Information System (INIS)

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

    1990-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1978-08-01

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

  19. 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.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2014-12-01

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

  2. Cross-Matching Source Observations from the Palomar Transient Factory (PTF)

    Science.gov (United States)

    Laher, Russ; Grillmair, C.; Surace, J.; Monkewitz, S.; Jackson, E.

    2009-01-01

    Over the four-year lifetime of the PTF project, approximately 40 billion instances of astronomical-source observations will be extracted from the image data. The instances will correspond to the same astronomical objects being observed at roughly 25-50 different times, and so a very large catalog containing important object-variability information will be the chief PTF product. Organizing astronomical-source catalogs is conventionally done by dividing the catalog into declination zones and sorting by right ascension within each zone (e.g., the USNOA star catalog), in order to facilitate catalog searches. This method was reincarnated as the "zones" algorithm in a SQL-Server database implementation (Szalay et al., MSR-TR-2004-32), with corrections given by Gray et al. (MSR-TR-2006-52). The primary advantage of this implementation is that all of the work is done entirely on the database server and client/server communication is eliminated. We implemented the methods outlined in Gray et al. for a PostgreSQL database. We programmed the methods as database functions in PL/pgSQL procedural language. The cross-matching is currently based on source positions, but we intend to extend it to use both positions and positional uncertainties to form a chi-square statistic for optimal thresholding. The database design includes three main tables, plus a handful of internal tables. The Sources table stores the SExtractor source extractions taken at various times; the MergedSources table stores statistics about the astronomical objects, which are the result of cross-matching records in the Sources table; and the Merges table, which associates cross-matched primary keys in the Sources table with primary keys in the MergedSoures table. Besides judicious database indexing, we have also internally partitioned the Sources table by declination zone, in order to speed up the population of Sources records and make the database more manageable. The catalog will be accessible to the public

  3. 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

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

    International Nuclear Information System (INIS)

    Tokita, N.; Raju, M.R.

    1982-01-01

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

  5. 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)

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

    Science.gov (United States)

    Peperzak, Louis; Brussaard, Corina P D

    2011-06-01

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

  7. 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 virus-mediated processes for better understanding of the microbial food web and the biogeochemistry. 1. Suttle, C. A., Nature, 2005, 437, 356– 361. 2. Danovaro, R. et al., Freshwater Biol., 2008, 53, 1186–1213. 3. Suttle, C. A., Nature, 2007, 5... of the microbial food web, with abundance in marine waters ranging from 10 6 ml –1 in the deep sea to 10 8 ml –1 in coastal waters and 10 9 g –1 of dry weight in the marine sediments 1,2 , which is usually 15-fold greater than bacterial and archael...

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

    African Journals Online (AJOL)

    Jocky

    2012-02-23

    Feb 23, 2012 ... species. A systematic comparison of nuclear lysis buffers has been ... All experiments were carried out with 3 replicates (n = 3) per treatment. ... Na2EDTA, 0.5 mM spermine.4HCl, 80 mM KCl, 20 mM NaCl, 0.1%. (v/v) Triton ...

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

    Czech Academy of Sciences Publication Activity Database

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

    2003-01-01

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

  10. Comparison Between Conventional and Automated Techniques for Blood Grouping and Crossmatching: Experience from a Tertiary Care Centre.

    Science.gov (United States)

    Bhagwat, Swarupa Nikhil; Sharma, Jayashree H; Jose, Julie; Modi, Charusmita J

    2015-01-01

    The routine immunohematological tests can be performed by automated as well as manual techniques. These techniques have advantages and disadvantages inherent to them. The present study aims to compare the results of manual and automated techniques for blood grouping and crossmatching so as to validate the automated system effectively. A total of 1000 samples were subjected to blood grouping by the conventional tube technique (CTT) and the automated microplate LYRA system on Techno TwinStation. A total of 269 samples (multitransfused patients and multigravida females) were compared for 927 crossmatches by the CTT in indirect antiglobulin phase against the column agglutination technique (CAT) performed on Techno TwinStation. For blood grouping, the study showed a concordance in results for 942/1000 samples (94.2%), discordance for 4/1000 (0.4%) samples and uninterpretable result for 54/1000 samples (5.4%). On resolution, the uninterpretable results reduced to 49/1000 samples (4.9%) with 951/1000 samples (95.1%) showing concordant results. For crossmatching, the automated CAT showed concordant results in 887/927 (95.6%) and discordant results in 3/927 (0.32%) crossmatches as compared to the CTT. Total 37/927 (3.9%) crossmatches were not interpretable by the automated technique. The automated system shows a high concordance of results with CTT and hence can be brought into routine use. However, the high proportion of uninterpretable results emphasizes on the fact that proper training and standardization are needed prior to its use.

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

    International Nuclear Information System (INIS)

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

    1984-01-01

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

  12. Multiparameter cytometric analysis of complex cellular response

    Czech Academy of Sciences Publication Activity Database

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

    2018-01-01

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

  13. Transplantation with positive complement-dependent microcytotoxicity crossmatch in contemporary kidney transplantation: Practice patterns and associated outcomes

    Directory of Open Access Journals (Sweden)

    Ralph J Graff

    2012-01-01

    Full Text Available We analyzed clinical factors and graft survival associated with complement-dependent microcytotoxicity (CDC crossmatch (XM positive (+ kidney transplants in 1995 to 2009 United Network of Sharing (UNOS registry data. CDCXM negative (- transplants were selected from centers and years in which at least one CDCXM+ transplant was performed at a given center in a given year. CDCXM+ and CDCXM- results were compared with bivariate and multivariate survival analysis. Our observations are as follows: (1 The risk of graft loss with CDCXM+ vs. CDCXM- results was markedly lower than the risk observed historically, e.g., living donor (LD-CDCXM+ absolute all-cause graft survival reductions were 0.7% at 24 hours (P=0.007, 2.9% at one year (P <0.0001, 3.7% at five years (P<0.0001; deceased donor (DD-CDCXM+ absolute graft survival reductions were 0.7% at 24 hours (P=0.02, 3.5% at one year (P <0.0001, 2.7% at five years (P=0.0009. On covariate adjustment, the only significant association of CDCXM+ vs. CDCXM- results was with one-year graft loss risk: LD aHR 1.44 (95% CI 1.05-1.96, DD aHR 1.33 (CI 1.10-1.61. (2 CDCXM+ transplantation was more commonly performed among groups disadvantaged with respect to transplant access, including sensitized, previously transplanted women and black recipients. (3 In CDCXM+ recipients, there was a high percentage of flow cytometry (FC XM- and autoXM+ results. After removing these groups, outcomes with CDCXM+ results were relatively good. (4 CDCXM+/FCXM+ vs. CDCXM-/FCXM- graft loss risk was observed only in LD recipients transplanted at centers performing fewer than 10 such transplants during the study period: 11.0% reduction (P<0.0001 and aHR of 2.86 (CI 1.18-6.94 at one year; 14.7% reduction (P<0.0001 and aHR of 1.77 (CI 0.88-3.58 at five years. Although using CDCXM+ as a contraindication to transplantation has been associated with virtual elimination of hyperacute rejection, the negative effect of a CDCXM+ in contemporary

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

    Science.gov (United States)

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

    2018-04-01

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

  15. Multivariate analysis of flow cytometric data using decision trees

    Directory of Open Access Journals (Sweden)

    Svenja eSimon

    2012-04-01

    Full Text Available Characterization of the response of the host immune system is important in understanding the bidirectional interactions between the host and microbial pathogens. For research on the host site, flow cytometry has become one of the major tools in immunology. Advances in technology and reagents allow now the simultaneous assessment of multiple markers on a single cell level generating multidimensional data sets that require multivariate statistical analysis. We explored the explanatory power of the supervised machine learning method called 'induction of decision trees' in flow cytometric data. In order to examine whether the production of a certain cytokine is depended on other cytokines, datasets from intracellular staining for six cytokines with complex patterns of co-expression were analyzed by induction of decision trees. After weighting the data according to their class probabilities, we created a total of 13,392 different decision trees for each given cytokine with different parameter settings. For a more realistic estimation of the decision trees's quality, we used stratified 5-fold cross-validation and chose the 'best' tree according to a combination of different quality criteria. While some of the decision trees reflected previously known co-expression patterns, we found that the expression of some cytokines was not only dependent on the co-expression of others per se, but was also dependent on the intensity of expression. Thus, for the first time we successfully used induction of decision trees for the analysis of high dimensional flow cytometric data and demonstrated the feasibility of this method to reveal structural patterns in such data sets.

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

    Directory of Open Access Journals (Sweden)

    Pathak Sangeeta

    2011-01-01

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

  17. Long-term outcomes of kidney transplantation across a positive complement-dependent cytotoxicity crossmatch.

    Science.gov (United States)

    Riella, Leonardo V; Safa, Kassem; Yagan, Jude; Lee, Belinda; Azzi, Jamil; Najafian, Nader; Abdi, Reza; Milford, Edgar; Mah, Helen; Gabardi, Steven; Malek, Sayeed; Tullius, Stefan G; Magee, Colm; Chandraker, Anil

    2014-06-27

    More than 30% of potential kidney transplant recipients have pre-existing anti-human leukocyte antigen antibodies. This subgroup has significantly lower transplant rates and increased mortality. Desensitization has become an important tool to overcome this immunological barrier. However, limited data is available regarding long-term outcomes, in particular for the highest risk group with a positive complement-dependent cytotoxicity crossmatch (CDC XM) before desensitization. Between 2002 and 2010, 39 patients underwent living-kidney transplantation across a positive CDC XM against their donors at our center. The desensitization protocol involved pretransplant immunosuppression, plasmapheresis, and low-dose intravenous immunoglobulin±rituximab. Measured outcomes included patient survival, graft survival, renal function, rates of rejection, infection, and malignancy. The mean and median follow-up was 5.2 years. Patient survival was 95% at 1 year, 95% at 3 years, and 86% at 5 years. Death-censored graft survival was 94% at 1 year, 88% at 3 years, and 84% at 5 years. Uncensored graft survival was 87% at 1 year, 79% at 3 years, and 72% at 5 years. Twenty-four subjects (61%) developed acute antibody-mediated rejection of the allograft and one patient lost her graft because of hyperacute rejection. Infectious complications included pneumonia (17%), BK nephropathy (10%), and CMV disease (5%). Skin cancer was the most prevalent malignancy in 10% of patients. There were no cases of lymphoproliferative disorder. Mean serum creatinine was 1.7±1 mg/dL in functioning grafts at 5 years after transplantation. Despite high rates of early rejection, desensitization in living-kidney transplantation results in acceptable 5-year patient and graft survival rates.

  18. SkyQuery - A Prototype Distributed Query and Cross-Matching Web Service for the Virtual Observatory

    Science.gov (United States)

    Thakar, A. R.; Budavari, T.; Malik, T.; Szalay, A. S.; Fekete, G.; Nieto-Santisteban, M.; Haridas, V.; Gray, J.

    2002-12-01

    We have developed a prototype distributed query and cross-matching service for the VO community, called SkyQuery, which is implemented with hierarchichal Web Services. SkyQuery enables astronomers to run combined queries on existing distributed heterogeneous astronomy archives. SkyQuery provides a simple, user-friendly interface to run distributed queries over the federation of registered astronomical archives in the VO. The SkyQuery client connects to the portal Web Service, which farms the query out to the individual archives, which are also Web Services called SkyNodes. The cross-matching algorithm is run recursively on each SkyNode. Each archive is a relational DBMS with a HTM index for fast spatial lookups. The results of the distributed query are returned as an XML DataSet that is automatically rendered by the client. SkyQuery also returns the image cutout corresponding to the query result. SkyQuery finds not only matches between the various catalogs, but also dropouts - objects that exist in some of the catalogs but not in others. This is often as important as finding matches. We demonstrate the utility of SkyQuery with a brown-dwarf search between SDSS and 2MASS, and a search for radio-quiet quasars in SDSS, 2MASS and FIRST. The importance of a service like SkyQuery for the worldwide astronomical community cannot be overstated: data on the same objects in various archives is mapped in different wavelength ranges and looks very different due to different errors, instrument sensitivities and other peculiarities of each archive. Our cross-matching algorithm preforms a fuzzy spatial join across multiple catalogs. This type of cross-matching is currently often done by eye, one object at a time. A static cross-identification table for a set of archives would become obsolete by the time it was built - the exponential growth of astronomical data means that a dynamic cross-identification mechanism like SkyQuery is the only viable option. SkyQuery was funded by a

  19. Successful kidney transplantation across a positive complement-dependent cytotoxicity crossmatch by using C1q assay-directed, bortezomib-assisted desensitization

    Science.gov (United States)

    Lee, Juhan; Park, Borae G.; Jeong, Hyang Sook; Park, Youn Hee; Kim, Sinyoung; Kim, Beom Seok; Kim, Hye Jin; Huh, Kyu Ha; Jeong, Hyeon Joo; Kim, Yu Seun

    2017-01-01

    Abstract Rationale: Human leukocyte antigen (HLA) is the major immunologic barrier in kidney transplantation (KT). Various desensitization protocols to overcome the HLA barrier have increased the opportunity for transplantation in sensitized patients. In addition, technological advances in solid-phase assays have permitted more comprehensive assessment of donor-specific antibodies. Although various desensitization therapies and immunologic techniques have been developed, the final transplantation decision is still based on the classic complement-dependent cytotoxicity (CDC) crossmatch (XM) technique. Some patients who fail to achieve negative XM have lost their transplant opportunities, even after receiving sufficient desensitization therapies. Patient concerns: A 57-year-old male with end-stage renal disease secondary to chronic glomerulonephritis was scheduled to have a second transplant from his son, but CDC XM was positive. Diagnoses: Initial CDC XM (Initial T-AHG 1:32) and flow-cytometry XM were positive. Anti-HLA-B59 donor specific antibody was detected by Luminex single antigen assay. Interventions: Herein, we report a successful case of KT across a positive CDC XM (T-AHG 1:8 at the time of transplantation) by using C1q assay-directed, bortezomib-assisted desensitization. After confirming a negative conversion in the C1q donor-specific antibody, we decided to perform KT accepting a positive AHG-CDC XM of 1:8 at the time of transplantation. Outcomes: The posttransplant course was uneventful and a protocol biopsy at 3 months showed no evidence of rejection. The patient had excellent graft function at 12 months posttransplant. Lessons: The results of XM test and solid-phase assay should be interpreted in the context of the individual patient. PMID:28953652

  20. Successful kidney transplantation across a positive complement-dependent cytotoxicity crossmatch by using C1q assay-directed, bortezomib-assisted desensitization: A case report.

    Science.gov (United States)

    Lee, Juhan; Park, Borae G; Jeong, Hyang Sook; Park, Youn Hee; Kim, Sinyoung; Kim, Beom Seok; Kim, Hye Jin; Huh, Kyu Ha; Jeong, Hyeon Joo; Kim, Yu Seun

    2017-09-01

    Human leukocyte antigen (HLA) is the major immunologic barrier in kidney transplantation (KT). Various desensitization protocols to overcome the HLA barrier have increased the opportunity for transplantation in sensitized patients. In addition, technological advances in solid-phase assays have permitted more comprehensive assessment of donor-specific antibodies. Although various desensitization therapies and immunologic techniques have been developed, the final transplantation decision is still based on the classic complement-dependent cytotoxicity (CDC) crossmatch (XM) technique. Some patients who fail to achieve negative XM have lost their transplant opportunities, even after receiving sufficient desensitization therapies. A 57-year-old male with end-stage renal disease secondary to chronic glomerulonephritis was scheduled to have a second transplant from his son, but CDC XM was positive. Initial CDC XM (Initial T-AHG 1:32) and flow-cytometry XM were positive. Anti-HLA-B59 donor specific antibody was detected by Luminex single antigen assay. Herein, we report a successful case of KT across a positive CDC XM (T-AHG 1:8 at the time of transplantation) by using C1q assay-directed, bortezomib-assisted desensitization. After confirming a negative conversion in the C1q donor-specific antibody, we decided to perform KT accepting a positive AHG-CDC XM of 1:8 at the time of transplantation. The posttransplant course was uneventful and a protocol biopsy at 3 months showed no evidence of rejection. The patient had excellent graft function at 12 months posttransplant. The results of XM test and solid-phase assay should be interpreted in the context of the individual patient.

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

    Directory of Open Access Journals (Sweden)

    Lucky Onotai

    2013-01-01

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

  2. ASVCP quality assurance guidelines: control of preanalytical and analytical factors for hematology for mammalian and nonmammalian species, hemostasis, and crossmatching in veterinary laboratories.

    Science.gov (United States)

    Vap, Linda M; Harr, Kendal E; Arnold, Jill E; Freeman, Kathleen P; Getzy, Karen; Lester, Sally; Friedrichs, Kristen R

    2012-03-01

    In December 2009, the American Society for Veterinary Clinical Pathology (ASVCP) Quality Assurance and Laboratory Standards committee published the updated and peer-reviewed ASVCP Quality Assurance Guidelines on the Society's website. These guidelines are intended for use by veterinary diagnostic laboratories and veterinary research laboratories that are not covered by the US Food and Drug Administration Good Laboratory Practice standards (Code of Federal Regulations Title 21, Chapter 58). The guidelines have been divided into 3 reports: (1) general analytical factors for veterinary laboratory performance and comparisons; (2) hematology, hemostasis, and crossmatching; and (3) clinical chemistry, cytology, and urinalysis. This particular report is one of 3 reports and provides recommendations for control of preanalytical and analytical factors related to hematology for mammalian and nonmammalian species, hemostasis testing, and crossmatching and is adapted from sections 1.1 and 2.3 (mammalian hematology), 1.2 and 2.4 (nonmammalian hematology), 1.5 and 2.7 (hemostasis testing), and 1.6 and 2.8 (crossmatching) of the complete guidelines. These guidelines are not intended to be all-inclusive; rather, they provide minimal guidelines for quality assurance and quality control for veterinary laboratory testing and a basis for laboratories to assess their current practices, determine areas for improvement, and guide continuing professional development and education efforts. © 2012 American Society for Veterinary Clinical Pathology.

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

    Science.gov (United States)

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

    2018-02-20

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

  4. Flow cytometry detection of planktonic cells with polycyclic aromatic hydrocarbons sorbed to cell surfaces

    KAUST Repository

    Cerezo, Maria I.; Linden, Matthew; Agusti, Susana

    2017-01-01

    Polycyclic aromatic hydrocarbons are very important components of oil pollution. These pollutants tend to sorb to cell surfaces, exerting toxic effects on organisms. Our study developed a flow cytometric method for the detection of PAHs sorbed

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

    International Nuclear Information System (INIS)

    Gledhill, B.L.

    1983-01-01

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

  6. 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.

  7. 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

  8. 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

  9. Flow cytometry and integrated imaging

    Directory of Open Access Journals (Sweden)

    V. Kachel

    2000-06-01

    Full Text Available It is a serious problem to relate the results of a flow cytometric analysis of a marine sample to different species. Images of particles selectively triggered by the flow cytometric analysis and picked out from the flowing stream give a valuable additional information on the analyzed organisms. The technical principles and problems of triggered imaging in flow are discussed, as well as the positioning of the particles in the plane of focus, freezing the motion of the quickly moving objects and what kinds of light sources are suitable for pulsed illumination. The images have to be stored either by film or electronically. The features of camera targets and the memory requirements for storing the image data and the conditions for the triggering device are shown. A brief explanation of the features of three realized flow cytometric imaging (FCI systems is given: the Macro Flow Planktometer built within the EUROMAR MAROPT project, the Imaging Module of the European Plankton Analysis System, supported by the MAST II EurOPA project and the most recently developed FLUVO VI universal flow cytometer including HBO 100- and laser excitation for fluorescence and scatter, Coulter sizing as well as bright field and and phase contrast FCI.

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

    International Nuclear Information System (INIS)

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

    2011-01-01

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

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

    OpenAIRE

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

    2014-01-01

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

  12. 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

    on their surface. Polyclonal antibodies against defined peptides in the Env- and Gag-regions of the HERVs were raised in rabbits and used in antibody-dependent cell-mediated cytotoxicity (ADCC) -assays. Rituximab® (Roche), a chimeric monoclonal antibody against CD20 expressed primarily on B cells, was used...

  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

    human leukemia cell line, stained as a methanol-fixed nuclear suspension. The BrUrd-induced fluorescence signals were highest with the antibody ABDM (Partec), moderate but reproducible with B-44 (Becton Dickinson), variable or low with BR-3 and IU-4 (Caltag), and not detectable with Bu20a (DAKO...... the variation of RNA synthesis during the cell cycle. The BrUrd incorporation was high in the S and G2 phase, variable in G1, and negligible in mitosis. Similar results were obtained using other cell types....

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

    DEFF Research Database (Denmark)

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

    1983-01-01

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

  15. 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...

  16. Multiplex ready flow cytometric immunoassay for total insulin like growth factor 1 in serum of cattle

    NARCIS (Netherlands)

    Bremer, M.G.E.G.; Smits, N.G.E.; Haasnoot, W.; Nielen, M.W.F.

    2010-01-01

    The European Union has banned the use of recombinant bovine somatotropins (rbST, growth hormones) to increase milk yield in dairy cattle. As direct detection of rbST in serum is problematic, methods based on the detection of changes in multiple rbST-dependent biomarkers have high potential for

  17. Multiplex flow cytometric immunoassay for serum biomarker profiling of recombinant bovine somatotropin

    NARCIS (Netherlands)

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

    2013-01-01

    Recombinant bovine somatotropin (rbST) is licensed for enhancing milk production in dairy cows in some countries, for instance the United States, but is banned in Europe. Serum biomarker profiling can be an adequate approach to discriminate between treated and untreated groups. In this study a

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

    International Nuclear Information System (INIS)

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

    1986-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Saumyadipta Pyne

    Full Text Available 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/.

  20. Flow-cytometric identification of vinegars using a multi-parameter analysis optical detection module

    Science.gov (United States)

    Verschooten, T.; Ottevaere, H.; Vervaeke, M.; Van Erps, J.; Callewaert, M.; De Malsche, W.; Thienpont, H.

    2015-09-01

    We show a proof-of-concept demonstration of a multi-parameter analysis low-cost optical detection system for the flowcytometric identification of vinegars. This multi-parameter analysis system can simultaneously measure laser induced fluorescence, absorption and scattering excited by two time-multiplexed lasers of different wavelengths. To our knowledge no other polymer optofluidic chip based system offers more simultaneous measurements. The design of the optofluidic channels is aimed at countering the effects that viscous fingering, air bubbles, and emulsion samples can have on the correct operation of such a detection system. Unpredictable variations in viscosity and refractive index of the channel content can be turned into a source of information. The sample is excited by two laser diodes that are driven by custom made low-cost laser drivers. The optofluidic chip is built to be robust and easy to handle and is reproducible using hot embossing. We show a custom optomechanical holder for the optofluidic chip that ensures correct alignment and automatic connection to the external fluidic system. We show an experiment in which 92 samples of vinegar are measured. We are able to identify 9 different kinds of vinegar with an accuracy of 94%. Thus we show an alternative approach to the classic optical spectroscopy solution at a lowered. Furthermore, we have shown the possibility of predicting the viscosity and turbidity of vinegars with a goodness-of-fit R2 over 0.947.

  1. Flow cytometric viability assessment and transmission electron microscopic morphological study of Bacteria in Glycerol

    NARCIS (Netherlands)

    Saegeman, V.S.M.; Vos, de R.; Tebaldi, N.D.; Wolf, van der J.M.; Bergervoet, J.H.W.; Verhaegen, J.; Lismont, D.; Verduyckt, B.; Ectors, N.L.

    2007-01-01

    Human cadaveric skin allografts are used in the treatment of burns and can be preserved in glycerol at high concentrations. Previously, glycerol has been attributed some antimicrobial effect. In an experimental set-up, we aimed at investigating this effect of prolonged incubation of bacteria in 85%

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

    International Nuclear Information System (INIS)

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

    1992-10-01

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

  3. Quantification of silver nanoparticle toxicity to algae in soil via photosynthetic and flow-cytometric analyses

    OpenAIRE

    Nam, Sun-Hwa; Il Kwak, Jin; An, Youn-Joo

    2018-01-01

    Soil algae, which have received attention for their use in a novel bioassay to evaluate soil toxicity, expand the range of terrestrial test species. However, there is no information regarding the toxicity of nanomaterials to soil algae. Thus, we evaluated the effects of silver nanoparticles (0–50 mg AgNPs/kg dry weight soil) on the soil alga Chlamydomonas reinhardtii after six days, and assessed changes in biomass, photosynthetic activity, cellular morphology, membrane permeability, esterase ...

  4. 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.

  5. Image Cytometric Analysis of Algal Spores for Evaluation of Antifouling Activities of Biocidal Agents.

    Science.gov (United States)

    Il Koo, Bon; Lee, Yun-Soo; Seo, Mintae; Seok Choi, Hyung; Leng Seah, Geok; Nam, Taegu; Nam, Yoon Sung

    2017-07-31

    Chemical biocides have been widely used as marine antifouling agents, but their environmental toxicity impose regulatory restriction on their use. Although various surrogate antifouling biocides have been introduced, their comparative effectiveness has not been well investigated partly due to the difficulty of quantitative evaluation of their antifouling activity. Here we report an image cytometric method to quantitatively analyze the antifouling activities of seven commercial biocides using Ulva prolifera as a target organism, which is known to be a dominant marine species causing soft fouling. The number of spores settled on a substrate is determined through image analysis using the intrinsic fluorescence of chlorophylls in the spores. Pre-determined sets of size and shape of spores allow for the precise determination of the number of settled spores. The effects of biocide concentration and combination of different biocides on the spore settlement are examined. No significant morphological changes of Ulva spores are observed, but the amount of adhesive pad materials is appreciably decreased in the presence of biocides. It is revealed that the growth rate of Ulva is not directly correlated with the antifouling activities against the settlement of Ulva spores. This work suggests that image cytometric analysis is a very convenient, fast-processable method to directly analyze the antifouling effects of biocides and coating materials.

  6. Preoperative selective desensitization of live donor liver transplant recipients considering the degree of T lymphocyte cross-match titer, model for end-stage liver disease score, and graft liver volume.

    Science.gov (United States)

    Hong, Geun; Yi, Nam-Joon; Suh, Suk-won; Yoo, Tae; Kim, Hyeyoung; Park, Min-Su; Choi, YoungRok; Lee, Kyungbun; Lee, Kwang-Woong; Park, Myoung Hee; Suh, Kyung-Suk

    2014-05-01

    Several studies have suggested that a positive lymphocyte cross-matching (XM) is associated with low graft survival rates and a high prevalence of acute rejection after adult living donor liver transplantations (ALDLTs) using a small-for-size graft. However, there is still no consensus on preoperative desensitization. We adopted the desensitization protocol from ABO-incompatible LDLT. We performed desensitization for the selected patients according to the degree of T lymphocyte cross-match titer, model for end-stage liver disease (MELD) score, and graft liver volume. We retrospectively evaluated 230 consecutive ALDLT recipients for 5 yr. Eleven recipients (4.8%) showed a positive XM. Among them, five patients with the high titer (> 1:16) by antihuman globulin-augmented method (T-AHG) and one with a low titer but a high MELD score of 36 were selected for desensitization: rituximab injection and plasmapheresis before the transplantation. There were no major side effects of desensitization. Four of the patients showed successful depletion of the T-AHG titer. There was no mortality and hyperacute rejection in lymphocyte XM-positive patients, showing no significant difference in survival outcome between two groups (P=1.000). In conclusion, this desensitization protocol for the selected recipients considering the degree of T lymphocyte cross-match titer, MELD score, and graft liver volume is feasible and safe.

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

    International Nuclear Information System (INIS)

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

    2011-01-01

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

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

    Science.gov (United States)

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

    2018-04-01

    Coming high-cadence wide-field optical telescopes will image hundreds of thousands of sources per minute. Besides inspecting the near real-time data streams for transient and variability events, the accumulated data archive is a wealthy laboratory for making complementary scientific discoveries. The goal of this work is to optimise column-oriented database techniques to enable the construction of a full-source and light-curve database for large-scale surveys, that is accessible by the astronomical community. We adopted LOFAR's Transients Pipeline as the baseline and modified it to enable the processing of optical images that have much higher source densities. The pipeline adds new source lists to the archive database, while cross-matching them with the known cataloguedsources in order to build a full light-curve archive. We investigated several techniques of indexing and partitioning the largest tables, allowing for faster positional source look-ups in the cross matching algorithms. We monitored all query run times in long-term pipeline runs where we processed a subset of IPHAS data that have image source density peaks over 170,000 per field of view (500,000 deg-2). Our analysis demonstrates that horizontal table partitions of declination widths of one-degree control the query run times. Usage of an index strategy where the partitions are densely sorted according to source declination yields another improvement. Most queries run in sublinear time and a few (< 20%) run in linear time, because of dependencies on input source-list and result-set size. We observed that for this logical database partitioning schema the limiting cadence the pipeline achieved with processing IPHAS data is 25 s.

  9. 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.

  10. Measurement of Soluble Biomarkers by Flow Cytometry

    OpenAIRE

    Antal-Szalm?s, P?ter; Nagy, B?la; Debreceni, Ildik? Beke; Kappelmayer, J?nos

    2013-01-01

    Microparticle based flow cytometric assays for determination of the level of soluble biomarkers are widely used in several research applications and in some diagnostic setups. The major advantages of these multiplex systems are that they can measure a large number of analytes (up to 500) at the same time reducing assay time, costs and sample volume. Most of these assays are based on antigen-antibody interactions and work as traditional immunoassays, but nucleic acid alterations ? by using spe...

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

    International Nuclear Information System (INIS)

    Yishai, Yitzhak; Fixler, Dror; Cohen-Kashi, Meir; Zurgil, Naomi; Deutsch, Mordechai

    2003-01-01

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

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

    Science.gov (United States)

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

    2017-10-01

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

  13. Clinical cytometry and progress in HLA antibody detection.

    Science.gov (United States)

    Bray, Robert A; Tarsitani, Christine; Gebel, Howard M; Lee, Jar-How

    2011-01-01

    For most solid organ and selected stem cell transplants, antibodies against mismatched HLA antigens can lead to early and late graft failure. In recognition of the clinical significance of these antibodies, HLA antibody identification is one of the most critical functions of histocompatibility laboratories. Early methods employed cumbersome and insensitive complement-dependent cytotoxicity assays with a visual read-out. A little over 20 years ago flow cytometry entered the realm of antibody detection with the introduction of the flow cytometric crossmatch. Cytometry's increased sensitivity and objectivity quickly earned it popularity as a preferred crossmatch method especially for sensitized recipients. Although a sensitive method, the flow crossmatch was criticized as being "too sensitive" as false positive reactions were a know drawback. In part, the shortcomings of the flow crossmatch were due to the lack of corresponding sensitive and specific HLA antibody screening assays. However, in the mid 1990s, solid phase assays, capable of utilizing standard flow cytometers, were developed. These assays used microparticles coated with purified HLA molecules. Hence, the era of solid-phase, microparticle technology for HLA antibody detection was born permitting the sensitive and specific detection of HLA antibody. It was now possible to provide better correlation between HLA antibody detection and the flow cytometric crossmatch. This flow-based technology was soon followed by adaptation to the Luminex platform permitting a mutltiplexed approach for the identification and characterization of HLA antibodies. It is hoped that these technologies will ultimately lead to the identification of parameters that best correlate with and/or predict transplant outcomes. Copyright © 2011 Elsevier Inc. All rights reserved.

  14. 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

  15. 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...

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

    Czech Academy of Sciences Publication Activity Database

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

    2016-01-01

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

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

    Czech Academy of Sciences Publication Activity Database

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

    2014-01-01

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

  18. Bizarre (pseudomalignant) granulation-tissue reactions following ionizing-radiation exposure. A microscopic, immunohistochemical, and flow-cytometric study

    International Nuclear Information System (INIS)

    Weidner, N.; Askin, F.B.; Berthrong, M.; Hopkins, M.B.; Kute, T.E.; McGuirt, F.W.

    1987-01-01

    Two patients developed extremely bizarre (pseudomalignant) granulation-tissue reactions in the larynx and facial sinuses, following radiation therapy for carcinoma. Containing pleomorphic spindle cells and numerous (sometimes atypical) mitotic figures, both tumefactive lesions simulated high grade malignancies. While the pleomorphic cells contained vimentin immunoreactivity, they were nonreactive for low or high molecular weight keratin. Flowcytometric study of paraffin-embedded tissues revealed DNA indexes of 0.75 and 1.0. Neither recurred locally nor spread distantly after therapy. Their granulation-tissue growth pattern, and the presence of stromal and endothelial cells showing similar degrees of cytologic atypia were central to their recognition as benign. These findings show that severely atypical, sometimes aneuploid, granulation-tissue reactions can occur following radiation exposure. Care should be taken not to misinterpret these lesions as malignant

  19. L-Carnitine in rooster semen cryopreservation: Flow cytometric, biochemical and motion findings for frozen-thawed sperm.

    Science.gov (United States)

    Fattah, A; Sharafi, M; Masoudi, R; Shahverdi, A; Esmaeili, V; Najafi, A

    2017-02-01

    Rooster semen cryopreservation is not efficient for artificial insemination in breeder flocks. L-Carnitine (LC) has been evaluated for effectiveness in cryopreservation media on the characteristics of rooster sperm after freeze-thawing. Motility characteristics, membrane functionality, abnormal morphology, apoptotic like changes, mitochondria activity and lipid peroxidation of rooster sperms were assessed after freeze-thawing with different concentrations of LC in Beltsville medium. Semen samples were collected from 12 roosters, twice a week, and diluted in the extenders that contained different concentrations of LC. Supplementation of Beltsevile with 1 and 2 mM LC was found to result in higher total motility (68.2± 1.7% and 69.1± 1.7%, respectively), progressive motility (28.4± 1.6%, 29.8± 1.6%), membrane functionality (76.2± 1.9% and 75.9± 1.9%), viability (58.2 ± 1.1%, 59.1 ± 1.1%) and lower significant of lipid peroxidation (2.53 ± 0.08 nmol/ml, 2.49 ± 0.08 nmol/ml) compared to control group containing no LC. Lower motility, progressive motility, and viability were observed in frozen-thawed sperm in extender containing 8 mM LC (35.8± 1.7%, 9.6± 1.2% and 27.1 ± 1.2%, respectively) compared to control. Morphology and mitochondrial activity were not affected by different concentrations of LC. Our results showed that supplementation of Beltsville extender with 1 and 2 mM LC significantly improved the quality of rooster sperm quality after freeze-thawing. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Fertility and flow cytometric evaluations of frozen-thawed rooster semen in cryopreservation medium containing low-density lipoprotein.

    Science.gov (United States)

    Shahverdi, A; Sharafi, M; Gourabi, H; Yekta, A Amiri; Esmaeili, V; Sharbatoghli, M; Janzamin, E; Hajnasrollahi, M; Mostafayi, F

    2015-01-01

    Frozen-thawed rooster semen is not reliable for use in artificial insemination in commercial stocks. Low-density lipoprotein (LDL) has been assessed for effectiveness as a cryoprotectant in the extender to improve the quality of frozen-thawed rooster semen. Although LDL has been evaluated in a few studies in other species for semen cryopreservation, so far no study has been conducted to examine this cryoprotectant for cryopreservation of fowl semen. Thus, this study aims to analyze the effects of different concentrations of LDL (0%, 2%, 4%, 6%, and 8%) in a Beltsville extender for cryopreservation of rooster spermatozoa. In experiment 1, motion parameters, membrane integrity, acrosome integrity, apoptosis status, and mitochondria activity were assessed after freeze-thawing. The highest quality frozen-thawed semen was selected to be used for evaluation of the fertility rate in experiment 2. Semen was collected from six roosters, twice weekly, then extended in a Beltsville extender that contained different concentrations of LDL as follows: 0% (control), 1% (Beltsville plus 1% LDL [BLDL1]), 2% (BLDL2), 4% (BLDL4), 6% (BLDL6), and 8% (BLDL8). Supplementation of the Beltsville extender with 4% LDL produced the most significant percentage of motility (43.1 ± 1.3), membrane integrity (59.4 ± 2.1),mitochondria activity (49.1 ± 1.19), and viable spermatozoa (45 ± 2.28) compared with the control treatment with the results of 22.7 ± 1.3 (motility), 38.4 ± 2.1 (membrane integrity), 40.25 ± 1.19 (mitochondrial activity), and 37.8 ± 2.28 (viability). In experiment 2, a significantly higher percentage of fertility rate was observed for frozen-thawed semen in the extender supplemented with 4% LDL (49.5 ± 1.6) compared with the control (29.2 ± 2.9). Progressive motility and acrosome integrity were not affected by LDL levels in the extenders. The results revealed that supplementation of the Beltsville extender with 4% LDL resulted in higher quality of frozen-thawed rooster sperm.

  1. 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.

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

    KAUST Repository

    Van Der Merwe, Riaan; Hammes, Frederik A.; Lattemann, Sabine; Amy, Gary L.

    2014-01-01

    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

  3. 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

    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...

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

    NARCIS (Netherlands)

    Vrieling, EG; vandePoll, WH; Vriezekolk, G; Gieskes, WWC

    The ichthyotoxic dinoflagellates Gyrodinium aureolum and Gymnodinium nagasakiense were cultured under different environmental conditions to test possible variability in immunochemical labelling intensity of cell-surface antigens using species-specific monoclonal antibodies. Variation of antigen

  5. Flow cytometric purification of Colletotrichum higginsianum biotrophic hyphae from Arabidopsis leaves for stage-specific transcriptome analysis.

    Science.gov (United States)

    Takahara, Hiroyuki; Dolf, Andreas; Endl, Elmar; O'Connell, Richard

    2009-08-01

    Generation of stage-specific cDNA libraries is a powerful approach to identify pathogen genes that are differentially expressed during plant infection. Biotrophic pathogens develop specialized infection structures inside living plant cells, but sampling the transcriptome of these structures is problematic due to the low ratio of fungal to plant RNA, and the lack of efficient methods to isolate them from infected plants. Here we established a method, based on fluorescence-activated cell sorting (FACS), to purify the intracellular biotrophic hyphae of Colletotrichum higginsianum from homogenates of infected Arabidopsis leaves. Specific selection of viable hyphae using a fluorescent vital marker provided intact RNA for cDNA library construction. Pilot-scale sequencing showed that the library was enriched with plant-induced and pathogenicity-related fungal genes, including some encoding small, soluble secreted proteins that represent candidate fungal effectors. The high purity of the hyphae (94%) prevented contamination of the library by sequences derived from host cells or other fungal cell types. RT-PCR confirmed that genes identified in the FACS-purified hyphae were also expressed in planta. The method has wide applicability for isolating the infection structures of other plant pathogens, and will facilitate cell-specific transcriptome analysis via deep sequencing and microarray hybridization, as well as proteomic analyses.

  6. A modified method of flow cytometric seed screen simplifies the quantification of progeny classes with different ploidy levels

    Czech Academy of Sciences Publication Activity Database

    Krahulcová, Anna; Suda, Jan

    2006-01-01

    Roč. 50, č. 3 (2006), s. 457-460 ISSN 0006-3134 R&D Projects: GA AV ČR IAA6005203 Institutional research plan: CEZ:AV0Z60050516 Keywords : facultative apomixis * reproduction routes * polyhaploids Subject RIV: EF - Botanics Impact factor: 1.198, year: 2006

  7. Naturalized plants have smaller genomes than their non-invading relatives: a flow cytometric analysis of the Czech alien flora

    Czech Academy of Sciences Publication Activity Database

    Kubešová, M.; Moravcová, Lenka; Suda, Jan; Jarošík, V.; Pyšek, Petr

    2010-01-01

    Roč. 82, č. 1 (2010), s. 81-96 ISSN 0032-7786 R&D Projects: GA ČR GA206/09/0563; GA ČR GD206/08/H049; GA MŠk LC06073 Institutional research plan: CEZ:AV0Z60050516 Keywords : cytometry * ploidy * genome size Subject RIV: EF - Botanics Impact factor: 2.792, year: 2010

  8. Genome size variation in the pine fusiform rust pathogen Cronartium quercuum f.sp. fusiforme as determined by flow cytometry

    Science.gov (United States)

    Claire L Anderson; Thomas L Kubisiak; C Dana Nelson; Jason A Smith; John M Davis

    2010-01-01

    The genome size of the pine fusiform rust pathogen Cronartium quercuum f.sp. fusiforme (Cqf) was determined by flow cytometric analysis of propidium iodide-stained, intact haploid pycniospores with haploid spores of two genetically well characterized fungal species, Sclerotinia sclerotiorum and Puccinia graminis f.sp. tritici, as size standards. The Cqf haploid genome...

  9. Flow cytogenetic studies in chromosomes and whole cells for the detection of clastogenic effects

    International Nuclear Information System (INIS)

    Otto, F.J.; Oldiges, H.

    1980-01-01

    Flow cytometric measurements of the chromosomal DNA content have been used to develop a screening method for the detection of chemically- or physically-induced cytogenetic damage. The reproducibility of this flow cytogenetic assay was shown in a series of subcultures of a Chinese hamster cell clone. The accuracy and sensitivity was tested in cultures treated with chemical mutagens and x-rays. The clastogenic effectiveness was quantified and the dose-effect relationship was established by the increase of the coefficient of variation of the peak of the largest chromosome type in the flow histograms. Since structural chromosome aberrations cause an unequal division of the DNA at mitosis, it is expected that clastogenic effects can be detected also in whole cells of growing populations as an increased dispersion of the cellular DNA content. In order to test this feature, high resolution flow cytometric measurements were performed in x-irradiated hamster cells in vitro and mouse bone marrow cells in vivo

  10. 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.

  11. Morphologic, cytometric and functional characterization of the common octopus (Octopus vulgaris) hemocytes.

    Science.gov (United States)

    Castellanos-Martínez, S; Prado-Alvarez, M; Lobo-da-Cunha, A; Azevedo, C; Gestal, C

    2014-05-01

    The hemocytes of Octopus vulgaris were morphologically and functionally characterized. Light and electron microscopy (TEM and SEM), and flow cytometry analyses revealed the existence of two hemocyte populations. Large granulocytes showed U-shaped nucleus, a mean of 11.6 μm±1.2 in diameter with basophilic granules, polysaccharide and lysosomic deposits in the cytoplasm. Small granulocytes measured a mean of 8.1 μm±0.7 in diameter, and have a round nucleus occupying almost the entire cell and few or not granules in the cytoplasm. Flow cytometry analysis showed that large granulocytes are the principal cells that develop phagocytosis of latex beads (rising up to 56%) and ROS after zymosan stimulation. Zymosan induced the highest production of both ROS and NO. This study is the first tread towards understanding the O. vulgaris immune system by applying new tools to provide a most comprehensive morpho-functional study of their hemocytes. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. Standardization and validation of a cytometric bead assay to assess antibodies to multiple Plasmodium falciparum recombinant antigens

    Directory of Open Access Journals (Sweden)

    Ondigo Bartholomew N

    2012-12-01

    Full Text Available Abstract Background Multiplex cytometric bead assay (CBA have a number of advantages over ELISA for antibody testing, but little information is available on standardization and validation of antibody CBA to multiple Plasmodium falciparum antigens. The present study was set to determine optimal parameters for multiplex testing of antibodies to P. falciparum antigens, and to compare results of multiplex CBA to ELISA. Methods Antibodies to ten recombinant P. falciparum antigens were measured by CBA and ELISA in samples from 30 individuals from a malaria endemic area of Kenya and compared to known positive and negative control plasma samples. Optimal antigen amounts, monoplex vs multiplex testing, plasma dilution, optimal buffer, number of beads required were assessed for CBA testing, and results from CBA vs. ELISA testing were compared. Results Optimal amounts for CBA antibody testing differed according to antigen. Results for monoplex CBA testing correlated strongly with multiplex testing for all antigens (r = 0.88-0.99, P values from Conclusion With optimization, CBA may be the preferred method of testing for antibodies to P. falciparum antigens, as CBA can test for antibodies to multiple recombinant antigens from a single plasma sample and produces a greater range of values in positive samples and lower background readings for blank samples than ELISA.

  13. SPICE: exploration and analysis of post-cytometric complex multivariate datasets.

    Science.gov (United States)

    Roederer, Mario; Nozzi, Joshua L; Nason, Martha C

    2011-02-01

    Polychromatic flow cytometry results in complex, multivariate datasets. To date, tools for the aggregate analysis of these datasets across multiple specimens grouped by different categorical variables, such as demographic information, have not been optimized. Often, the exploration of such datasets is accomplished by visualization of patterns with pie charts or bar charts, without easy access to statistical comparisons of measurements that comprise multiple components. Here we report on algorithms and a graphical interface we developed for these purposes. In particular, we discuss thresholding necessary for accurate representation of data in pie charts, the implications for display and comparison of normalized versus unnormalized data, and the effects of averaging when samples with significant background noise are present. Finally, we define a statistic for the nonparametric comparison of complex distributions to test for difference between groups of samples based on multi-component measurements. While originally developed to support the analysis of T cell functional profiles, these techniques are amenable to a broad range of datatypes. Published 2011 Wiley-Liss, Inc.

  14. Standardization and validation of a cytometric bead assay to assess antibodies to multiple Plasmodium falciparum recombinant antigens.

    Science.gov (United States)

    Ondigo, Bartholomew N; Park, Gregory S; Gose, Severin O; Ho, Benjamin M; Ochola, Lyticia A; Ayodo, George O; Ofulla, Ayub V; John, Chandy C

    2012-12-21

    Multiplex cytometric bead assay (CBA) have a number of advantages over ELISA for antibody testing, but little information is available on standardization and validation of antibody CBA to multiple Plasmodium falciparum antigens. The present study was set to determine optimal parameters for multiplex testing of antibodies to P. falciparum antigens, and to compare results of multiplex CBA to ELISA. Antibodies to ten recombinant P. falciparum antigens were measured by CBA and ELISA in samples from 30 individuals from a malaria endemic area of Kenya and compared to known positive and negative control plasma samples. Optimal antigen amounts, monoplex vs multiplex testing, plasma dilution, optimal buffer, number of beads required were assessed for CBA testing, and results from CBA vs. ELISA testing were compared. Optimal amounts for CBA antibody testing differed according to antigen. Results for monoplex CBA testing correlated strongly with multiplex testing for all antigens (r = 0.88-0.99, P values from <0.0001 - 0.004), and antibodies to variants of the same antigen were accurately distinguished within a multiplex reaction. Plasma dilutions of 1:100 or 1:200 were optimal for all antigens for CBA testing. Plasma diluted in a buffer containing 0.05% sodium azide, 0.5% polyvinylalcohol, and 0.8% polyvinylpyrrolidone had the lowest background activity. CBA median fluorescence intensity (MFI) values with 1,000 antigen-conjugated beads/well did not differ significantly from MFI with 5,000 beads/well. CBA and ELISA results correlated well for all antigens except apical membrane antigen-1 (AMA-1). CBA testing produced a greater range of values in samples from malaria endemic areas and less background reactivity for blank samples than ELISA. With optimization, CBA may be the preferred method of testing for antibodies to P. falciparum antigens, as CBA can test for antibodies to multiple recombinant antigens from a single plasma sample and produces a greater range of values in

  15. Characterization of glycosylphosphatidylinositol biosynthesis defects by clinical features, flow cytometry, and automated image analysis

    DEFF Research Database (Denmark)

    Knaus, Alexej; Pantel, Jean Tori; Pendziwiat, Manuela

    2018-01-01

    , the increasing number of individuals with a GPIBD shows that hyperphosphatasia is a variable feature that is not ideal for a clinical classification. METHODS: We studied the discriminatory power of multiple GPI-linked substrates that were assessed by flow cytometry in blood cells and fibroblasts of 39 and 14...... those with PIGA mutations. Although the impairment of GPI-linked substrates is supposed to play the key role in the pathophysiology of GPIBDs, we could not observe gene-specific profiles for flow cytometric markers or a correlation between their cell surface levels and the severity of the phenotype...

  16. Detection of bacteriophage-infected cells of Lactococcus lactis using flow cytometry

    DEFF Research Database (Denmark)

    Michelsen, Ole; Cuesta-Dominguez, Álvaro; Albrektsen, Bjarne

    2007-01-01

    Bacteriophage infection in dairy fermentation constitutes a serious problem worldwide. We have studied bacteriophage infection in Lactococcus lactis by using the flow cytometer. The first effect of the infection of the bacterium is a change from cells in chains toward single cells. We interpret...... describe a new method for detection of phage infection in Lactococcus lactis dairy cultures. The method is based on flow cytometric detection of cells with low-density cell walls. The method allows fast and early detection of phage-infected bacteria, independently of which phage has infected the culture...

  17. Flow cytometric evaluation of the effects of 3-bromopyruvate (3BP) and dichloracetate (DCA) on THP-1 cells: a multiparameter analysis

    NARCIS (Netherlands)

    Verhoeven, H.A.; Griensven, van L.J.L.D.

    2012-01-01

    Two human leukemia cells K562 and THP-1, the breast cancer lines MCF-7 and ZR-75-1, and the melanoma line MDA-MB-435S were compared by flowcytometry for their behaviour at increasing levels of 3BP. K562 and THP-1 responded to 3BP by membrane depolarization and increased ROS; MCF-7 and ZR-75-1 showed

  18. Flow cytometric analysis of lymphocytes in aplastic anemia among atomic bomb survivors. With special reference to immunological mechanisms and bolus methylprednisolone therapy

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-11-01

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

  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

    distribution could be demonstrated following the treatment. The results indicate that the oestradiol-induced cell kill could be explained to some extent by the induction of polyploid cells, which eventually die. Since the cell cycle changes monitored by FCM in the receptor-positive breast carcinoma appeared...

  20. Detection of HIV-RNA-positive monocytes in peripheral blood of HIV-positive patients by simultaneous flow cytometric analysis of intracellular HIV RNA and cellular immunophenotype.

    Science.gov (United States)

    Patterson, B K; Mosiman, V L; Cantarero, L; Furtado, M; Bhattacharya, M; Goolsby, C

    1998-04-01

    Determinations of plasma HIV viral RNA copy numbers help to define the kinetics of HIV-1 infection in vivo and to monitor antiretroviral therapy. However, questions remain regarding the identity of various infected cell types contributing to this free virus pool and to the in vivo lifecycle of HIV during disease progression. Characterization of a novel fluorescence in situ hybridization (FISH) assay employing a pool of labeled oligonucleotide probes directed against HIV RNA was done followed by coupling of the FISH assay with simultaneous surface immunophenotyping to address these questions. In vitro characterizations of this assay using tumor necrosis factor-alpha stimulated and unstimulated ACH-2 cells demonstrated the ability to detect < 5% HIV RNA positive cells with a sensitivity of < 30 RNA copies per cell. Peripheral blood mononuclear cells from 39 HIV-seropositive patients on no, single, combination, or triple drug therapy and 8 HIV-seronegative patients were examined. The majority of HIV-positive patients (24/39) harbored monocytes positive for HIV RNA and a significantly higher fraction of patients with high plasma viral load carried positive monocytes (13/16) than did patients in the low plasma viral load group (11/23). These results demonstrate the effectiveness of a novel FISH assay for identifying and monitoring HIV-infected cell populations in the peripheral blood of HIV-positive patients. In addition, monocytes are a major source of cellular HIV virus in the peripheral blood of HIV patients, even with progression of disease.

  1. CHANGES OF BUOYANT DENSITY DURING THE S-PHASE OF THE CELL-CYCLE - DIRECT EVIDENCE DEMONSTRATED IN ACUTE MYELOID-LEUKEMIA BY FLOW-CYTOMETRIC

    NARCIS (Netherlands)

    DAENEN, S; HUIGES, W; MODDERMAN, E; HALIE, MR

    Studies with synchronized or exponentially growing bacteria and mammalian cell lines are not able to demonstrate small changes in buoyant density during the cell cycle. Flowcytometric analysis of density separated acute myeloid leukemia cells, a system not dependent on time-related variables, shows

  2. The value of flow cytometric analysis of platelet glycoprotein expression of CD34+ cells measured under conditions that prevent P-selectin-mediated binding of platelets

    NARCIS (Netherlands)

    Dercksen, M. W.; Weimar, I. S.; Richel, D. J.; Breton-Gorius, J.; Vainchenker, W.; Slaper-Cortenbach, C. M.; Pinedo, H. M.; von dem Borne, A. E.; Gerritsen, W. R.; van der Schoot, C. E.

    1995-01-01

    In the present study, we show by adhesion assays and ultrastructural studies that platelets can bind to CD34+ cells from human blood and bone marrow and that this interaction interferes with the accurate detection of endogenously expressed platelet glycoproteins (GPs). The interaction between these

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

    NARCIS (Netherlands)

    E.H.A. Dekking (E. H A); V.H.J. van der Velden (Vincent); A. Varro (Andras); H. Wai; S. Böttcher (Stephan); M. Kneba (Michael); E. Sonneveld (Edwin); A. Koning; N. Boeckx; N. Van Poecke; P. Lucio (Paulo); A. Mendonça; L. Sedek (Lukasz); T. Szczepanski (Tomasz); T. Kalina (Tomas); V. Kanderová (V.); P.G. Hoogeveen (Patricia); J. Flores-Montero (Juan); C. Chillón (Carmen); A. Orfao (Alberto); J.M.M. Almeida (Julia); P.A.S. Evans; C. Cullen; A.L. Noordijk; P.M. Vermeulen (P.); M.T. de Man (M.); E.P. Dixon (Eric); W.M. Comans-Bitter; J.J.M. van Dongen (Jacques)

    2012-01-01

    textabstractThe 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

  4. Effect of lecithin nanoliposome or soybean lecithin supplemented by pomegranate extract on post-thaw flow cytometric, microscopic and oxidative parameters in ram semen.

    Science.gov (United States)

    Mehdipour, Mahdieh; Daghigh Kia, Hossein; Nazari, Maryam; Najafi, Abouzar

    2017-10-01

    This investigation was carried out to study the effect of soybean lecithin 1.5% (wt/vol) (0, 2.5, 5 and 7.5 mg l -1 pomegranate extract (PE)) or PE-loaded lecithin nanoliposome (0, 2.5, 5 and 7.5 mg l -1 ) to Tris-based extender. Sperm motility (CASA), viability, membrane integrity (HOS test), abnormalities, mitochondrial activity, apoptosis status, lipid peroxidation, total antioxidant capacity (TAC)) and antioxidant activities (GPX, SOD) were investigated following freeze-thawing. No significant differences were detected in motility parameters, viability, membrane integrity, and mitochondria activity after thawing sperm between soybean lecithin and lecithin nanoliposomes. It was shown that PE5 significantly improved sperm total and progressive motility, membrane integrity, viability, mitochondria activity, TAC and reduced lipid peroxidation (malondialdehyde concentration). Moreover, the percentage of apoptotic sperm in PE5 extenders was significantly the lowest among other treatments. Sperm abnormalities, SOD and GPX were not affected by the antioxidant supplements. For apoptotic status, no differences were observed between soybean lecithin and lecithin nanoliposome. We showed that lecithin nanoliposome extender can be a beneficial alternative extender to protect ram sperm during cryopreservation without any adverse effects. It was also observed that regarding pomegranate concentration, PE5 can improve the quality of ram semen after thawing. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Fluorescence-intensity multiplexing: simultaneous seven-marker, two-color immunophenotyping using flow cytometry.

    Science.gov (United States)

    Bradford, Jolene A; Buller, Gayle; Suter, Michael; Ignatius, Michael; Beechem, Joseph M

    2004-10-01

    Conventional immuno-based multiparameter flow cytometric analysis has been limited by the requirement of a dedicated detection channel for each antibody-fluorophore set. To address the need to resolve multiple biological targets simultaneously, flow cytometers with as many as 10-15 detection channels have been developed. In this study, a new Zenon immunolabeling technology is developed that allows for multiple antigen detection per detection channel using a single fluorophore, through a unique method of fluorescence-intensity multiplexing. By varying the Zenon labeling reagent-to-antibody molar ratio, the fluorescence intensity of the antibody-labeled cellular targets can be used as a unique identifier. Although demonstrated in the present study with lymphocyte immunophenotyping, this approach is broadly applicable for any immuno-based multiplexed flow cytomety assay. Lymphocyte immunophenotyping of 38 clinical blood specimens using CD3, CD4, CD8, CD16, CD56, CD19, and CD20 antibodies was performed using conventional flow cytometric analysis and fluorescence-intensity multiplexing analysis. Conventional analysis measures a single antibody-fluorophore per photomultiplier tube (PMT). Fluorescence-intensity multiplex analysis simultaneously measures seven markers with two PMTs, using Zenon labeling reagent-antibody complexes in a single tube: CD19, CD4, CD8, and CD16 antibodies labeled with Zenon Alexa Fluor 488 Mouse IgG(1) labeling reagent and CD56, CD3, and CD20 antibodies labeled with Zenon R-Phycoerythrin (R-PE) Mouse IgG(1) or IgG(2b) labeling reagents. The lymphocyte immunophenotyping results from fluorescence-intensity multiplexing using Zenon labeling reagents in a single tube were comparable to results from conventional flow cytometric analysis. Simultaneous evaluation of multiple antigens using a single fluorophore can be performed using antibodies labeled with varying ratios of a Zenon labeling reagent. Labeling two sets of antibodies with different Zenon

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

    KAUST Repository

    Garcí a, Francisca C.; Alonso-Sá ez, Laura; Moran, Xose Anxelu G.; Ló pez-Urrutia, Á ngel

    2015-01-01

    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

  7. Standardization of 8-color flow cytometry across different flow cytometer instruments: A feasibility study in clinical laboratories in Switzerland.

    Science.gov (United States)

    Glier, Hana; Heijnen, Ingmar; Hauwel, Mathieu; Dirks, Jan; Quarroz, Stéphane; Lehmann, Thomas; Rovo, Alicia; Arn, Kornelius; Matthes, Thomas; Hogan, Cassandra; Keller, Peter; Dudkiewicz, Ewa; Stüssi, Georg; Fernandez, Paula

    2017-07-29

    The EuroFlow Consortium developed a fully standardized flow cytometric approach from instrument settings, through antibody panel, reagents and sample preparation protocols, to data acquisition and analysis. The Swiss Cytometry Society (SCS) promoted a study to evaluate the feasibility of using such standardized measurements of 8-color data across two different flow cytometry platforms - Becton Dickinson (BD) FACSCanto II and Beckman Coulter (BC) Navios, aiming at increasing reproducibility and inter-laboratory comparability of immunophenotypic data in clinical laboratories in Switzerland. The study was performed in two phases, i.e. a learning phase (round 1) and an analytical phase (rounds 2 and 3) consisting of a total of three rounds. Overall, 10 laboratories using BD FACSCanto II (n=6) or BC Navios (n=4) flow cytometers participated. Each laboratory measured peripheral blood samples from healthy donors stained with a uniform antibody panel of reagents - EuroFlow Lymphoid Screening Tube (LST) - applying the EuroFlow standardized protocols for instrument setup and sample preparation (www.EuroFlow.org). All data files were analyzed centrally and median fluorescence intensity (MedFI) values for individual markers on defined lymphocyte subsets were recorded; variability from reference MedFI values was assessed using performance scores. Data troubleshooting and discussion of the results with the participants followed after each round at SCS meetings. The results of the learning phase demonstrated that standardized instrument setup and data acquisition are feasible in routine clinical laboratories without previous experience with EuroFlow. During the analytical phase, highly comparable data were obtained at the different laboratories using either BD FACSCanto II or BC Navios. The coefficient of variation of MedFI for 7 of 11 markers performed repeatedly below 30%. In the last study round, 89% of participants scored over 90% MedFI values within the acceptance criteria

  8. Reticulocyte maturity index by flow cytometry: its applicability in radioinduced bone marrow aplasia

    International Nuclear Information System (INIS)

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

    1995-01-01

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

  9. Use of flow cytometry for the possible identification of radio-induced changes in DNA of animal cells

    International Nuclear Information System (INIS)

    Spano, M.; Leonardi, M.; Cordelli, E.

    1991-01-01

    Since DNA is the main cellular target of ionizing irradiation, methods fit for analyzing DNA alterations should be able to discern irradiated versus control cells. Flow cytometry allows the rapid measurement of DNA content of single chromosomes or cell nuclei at very high resolution on a statistically significant sample. Alterations of chromatine structure can also be analyzed by flow cytometry. Briefly, evaluation of in situ DNA resistance to denaturation can be evaluated by flow cytometric analysis of different staining pattern of single versus double strange regions of DNA. In the present work both approaches were used with the aim to recognize cells derived from an irradiated sample of breast chicken. Although flow cytometry has been demonstrated to be a useful tool to detect DNA alterations and has been widely used to detect damages on DNA induced by several physical and chemical agents, it was unable to detect clastogenic effects induced by electrons on DNA of chicken breast cells. Heavily irradiated nuclei, even if challenged by denaturating treatments that partially collapse chromatine organization, do not present differences from non irradiated samples after flow cytometric DNA content measurement. (16 refs)

  10. Detection and quantification of live, apoptotic, and necrotic human peripheral lymphocytes by single-laser flow cytometry.

    Science.gov (United States)

    Liegler, T J; Hyun, W; Yen, T S; Stites, D P

    1995-05-01

    Regulation of peripheral lymphocyte number involves a poorly understood balance between cell renewal and loss. Disrupting this balance leads to a large number of disease states. Methods which allow qualitative and quantitative measurements of cell viability are increasingly valuable to studies directed at revealing the mechanisms underlying apoptotic and necrotic cell death. Here, we have characterized a method using single-laser flow cytometry that differentiates and quantifies the relative number of live, apoptotic, and late-stage apoptotic and necrotic peripheral lymphocytes. Following in vitro gamma irradiation and staining with acridine orange in combination with ethidium bromide, three distinct populations were seen by bivariate analysis of green versus red fluorescence. The identity of each distinct fluorescent population (whether live, apoptotic, or necrotic) was determined by sorting and examination of cellular morphology by electron microscopy. This flow cytometric method is directly compared with the techniques of trypan blue exclusion and DNA fragmentation to quantify cell death following exposure to various doses of in vitro gamma irradiation and postirradiation incubation times. We extend our findings to illustrate the utility of this method beyond analyzing radiation-induced apoptotic peripheral blood mononuclear cells (PBMC); similar fluorescent patterns are shown for radiation- and corticosteroid-treated murine thymocytes, activated human PBMC, and PBMC from human immunodeficiency virus-infected individuals. Our results demonstrate that dual-parameter flow cytometric analysis of acridine orange-ethidium bromide-stained lymphocytes is overall a superior method with increased sensitivity, greater accuracy, and decreased subjectivity in comparison with the other methods tested. By using standard laser and filter settings commonly available to flow cytometric laboratories, this method allows rapid measurement of a large number of cells from a

  11. Construction of BAC Libraries from Flow-Sorted Chromosomes.

    Science.gov (United States)

    Šafář, Jan; Šimková, Hana; Doležel, Jaroslav

    2016-01-01

    Cloned DNA libraries in bacterial artificial chromosome (BAC) are the most widely used form of large-insert DNA libraries. BAC libraries are typically represented by ordered clones derived from genomic DNA of a particular organism. In the case of large eukaryotic genomes, whole-genome libraries consist of a hundred thousand to a million clones, which make their handling and screening a daunting task. The labor and cost of working with whole-genome libraries can be greatly reduced by constructing a library derived from a smaller part of the genome. Here we describe construction of BAC libraries from mitotic chromosomes purified by flow cytometric sorting. Chromosome-specific BAC libraries facilitate positional gene cloning, physical mapping, and sequencing in complex plant genomes.

  12. Rapid assay for cell age response to radiation by electronic volume flow cell sorting

    International Nuclear Information System (INIS)

    Freyer, J.P.; Wilder, M.E.; Raju, M.R.

    1987-01-01

    A new technique is described for measuring cell survival as a function of cell cycle position using flow cytometric cell sorting on the basis of electronic volume signals. Sorting of cells into different cell age compartments is demonstrated for three different cell lines commonly used in radiobiological research. Using flow cytometric DNA content analysis and [ 3 H]thymidine autoradiography of the sorted cell populations, it is demonstrated that resolution of the age compartment separation is as good as or better than that reported for other cell synchronizing techniques. Variation in cell survival as a function of position in the cell cycle after a single dose of radiation as measured by volume cell sorting is similar to that determined by other cell synchrony techniques. Advantages of this method include: (1) no treatment of the cells is required, thus, this method is noncytotoxic; (2) no cell cycle progression is needed to obtain different cell age compartments; (3) the cell population can be held in complete growth medium at any desired temperature during sorting; (4) a complete radiation age - response assay can be plated in 2 h. Applications of this method are discussed, along with some technical limitations. (author)

  13. Rotating flow

    CERN Document Server

    Childs, Peter R N

    2010-01-01

    Rotating flow is critically important across a wide range of scientific, engineering and product applications, providing design and modeling capability for diverse products such as jet engines, pumps and vacuum cleaners, as well as geophysical flows. Developed over the course of 20 years' research into rotating fluids and associated heat transfer at the University of Sussex Thermo-Fluid Mechanics Research Centre (TFMRC), Rotating Flow is an indispensable reference and resource for all those working within the gas turbine and rotating machinery industries. Traditional fluid and flow dynamics titles offer the essential background but generally include very sparse coverage of rotating flows-which is where this book comes in. Beginning with an accessible introduction to rotating flow, recognized expert Peter Childs takes you through fundamental equations, vorticity and vortices, rotating disc flow, flow around rotating cylinders and flow in rotating cavities, with an introduction to atmospheric and oceanic circul...

  14. Flow visualization

    CERN Document Server

    Merzkirch, Wolfgang

    1974-01-01

    Flow Visualization describes the most widely used methods for visualizing flows. Flow visualization evaluates certain properties of a flow field directly accessible to visual perception. Organized into five chapters, this book first presents the methods that create a visible flow pattern that could be investigated by visual inspection, such as simple dye and density-sensitive visualization methods. It then deals with the application of electron beams and streaming birefringence. Optical methods for compressible flows, hydraulic analogy, and high-speed photography are discussed in other cha

  15. Flow regimes

    International Nuclear Information System (INIS)

    Kh'yuitt, G.

    1980-01-01

    An introduction into the problem of two-phase flows is presented. Flow regimes arizing in two-phase flows are described, and classification of these regimes is given. Structures of vertical and horizontal two-phase flows and a method of their identification using regime maps are considered. The limits of this method application are discussed. The flooding phenomena and phenomena of direction change (flow reversal) of the flow and interrelation of these phenomena as well as transitions from slug regime to churn one and from churn one to annular one in vertical flows are described. Problems of phase transitions and equilibrium are discussed. Flow regimes in tubes where evaporating liquid is running, are described [ru

  16. Analysis of the Budding Yeast Cell Cycle by Flow Cytometry.

    Science.gov (United States)

    Rosebrock, Adam P

    2017-01-03

    DNA synthesis is one of the landmark events in the cell cycle: G 1 cells have one copy of the genome, S phase cells are actively engaged in DNA synthesis, and G 2 cells have twice as much nuclear DNA as G 1 cells. Cellular DNA content can be measured by staining with a fluorescent dye followed by a flow-cytometric readout. This method provides a quantitative measurement of cell cycle position on a cell-by-cell basis at high speed. Using flow cytometry, tens of thousands of single-cell measurements can be generated in a few seconds. This protocol details staining of cells of the budding yeast Saccharomyces cerevisiae for flow cytometry using Sytox Green dye in a method that can be scaled widely-from one sample to many thousands and operating on inputs ranging from 1 million to more than 100 million cells. Flow cytometry is preferred over light microscopy or Coulter analyses for the analysis of the cell cycle as DNA content and cell cycle position are being directly measured. © 2017 Cold Spring Harbor Laboratory Press.

  17. Flow cytometry detection of planktonic cells with polycyclic aromatic hydrocarbons sorbed to cell surfaces

    KAUST Repository

    Cerezo, Maria I.

    2017-02-17

    Polycyclic aromatic hydrocarbons are very important components of oil pollution. These pollutants tend to sorb to cell surfaces, exerting toxic effects on organisms. Our study developed a flow cytometric method for the detection of PAHs sorbed to phytoplankton by exploiting their spectral characteristics. We discriminated between cells with PAHs from cells free of PAHs. Clear discrimination was observed with flow cytometer provided with 375 or 405nm lasers in addition to the standard 488nm laser necessary to identify phytoplankton. Using this method, we measured the relationship between the percentages of phytoplankton organisms with PAHs, with the decrease in the growth rate. Moreover, the development of this method could be extended to facilitate the study of PAHs impact on cell cultures from a large variety of organisms.

  18. Improved and Reproducible Flow Cytometry Methodology for Nuclei Isolation from Single Root Meristem

    Directory of Open Access Journals (Sweden)

    Thaís Cristina Ribeiro Silva

    2010-01-01

    Full Text Available Root meristems have increasingly been target of cell cycle studies by flow cytometric DNA content quantification. Moreover, roots can be an alternative source of nuclear suspension when leaves become unfeasible and for chromosome analysis and sorting. In the present paper, a protocol for intact nuclei isolation from a single root meristem was developed. This proceeding was based on excision of the meristematic region using a prototypical slide, followed by short enzymatic digestion and mechanical isolation of nuclei during homogenization with a hand mixer. Such parameters were optimized for reaching better results. Satisfactory nuclei amounts were extracted and analyzed by flow cytometry, producing histograms with reduced background noise and CVs between 3.2 and 4.1%. This improved and reproducible technique was shown to be rapid, inexpensive, and simple for nuclear extraction from a single root tip, and can be adapted for other plants and purposes.

  19. Lipid nanoparticles assessment by flow cytometry.

    Science.gov (United States)

    Bryła, Anna; Juzwa, Wojciech; Weiss, Marek; Lewandowicz, Grażyna

    2017-03-30

    Liposomes are promising carriers for drugs and bioactive compounds. Size and structure are their crucial parameters. Thus, it is essential to assess individual vesicles as prepared. Currently available techniques fail to measure liposome's size and structure simultaneously, with a high throughput. To solve this problem, we have developed a novel, flow cytometric method quantifying liposomes. Firstly, the following fluorescent staining combinations were tested: DiD/TO, Rh123/DiD, Syto9/DiD. Further, chosen fluorochromes were used to compare three populations of vesicles: raw (R), obtained by thin film hydration and extruded ones (populations E10 and E21). Dynamic light scattering (DLS) was used for determination of average diameter and size distribution of nanocarriers. Structural differences between the raw and the extruded liposomes, as well as additional information concerning vesicles size were acquired employing atomic force microscopy (AFM). DLS analysis indicated that, three distinct populations of vesicles were obtained. Liposomes were characterized by mean diameter of 323nm, 220nm and 170nm for population R, E10 and E21 respectively. All the populations were stable and revealed zeta potential of -29mV. AFM confirmed that raw and extruded liposomes were differed in structure. DiD/TO was the optimal fluorochrome combination that enabled to resolve distinctly the sub-populations of liposomes. Results obtained by flow cytometry were in a good agreement with those from DLS and AFM. It was proved that, flow cytometry, when proper fluorescent dyes are used, is an adequate method for liposomes assessment. The proposed method enables fast and reliable analysis of liposomes in their native environment. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Flow cytometry with gold nanoparticles and their clusters as scattering contrast agents: FDTD simulation of light-cell interaction.

    Science.gov (United States)

    Tanev, Stoyan; Sun, Wenbo; Pond, James; Tuchin, Valery V; Zharov, Vladimir P

    2009-09-01

    The formulation of the finite-difference time-domain (FDTD) approach is presented in the framework of its potential applications to in-vivo flow cytometry based on light scattering. The consideration is focused on comparison of light scattering by a single biological cell alone in controlled refractive-index matching conditions and by cells labeled by gold nanoparticles. The optical schematics including phase contrast (OPCM) microscopy as a prospective modality for in-vivo flow cytometry is also analyzed. The validation of the FDTD approach for the simulation of flow cytometry may open up a new avenue in the development of advanced cytometric techniques based on scattering effects from nanoscale targets. 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

  1. Flow visualization

    International Nuclear Information System (INIS)

    Weinstein, L.M.

    1991-01-01

    Flow visualization techniques are reviewed, with particular attention given to those applicable to liquid helium flows. Three techniques capable of obtaining qualitative and quantitative measurements of complex 3D flow fields are discussed including focusing schlieren, particle image volocimetry, and holocinematography (HCV). It is concluded that the HCV appears to be uniquely capable of obtaining full time-varying, 3D velocity field data, but is limited to the low speeds typical of liquid helium facilities. 8 refs

  2. Flow regimes

    International Nuclear Information System (INIS)

    Liles, D.R.

    1982-01-01

    Internal boundaries in multiphase flow greatly complicate fluid-dynamic and heat-transfer descriptions. Different flow regimes or topological configurations can have radically dissimilar interfacial and wall mass, momentum, and energy exchanges. To model the flow dynamics properly requires estimates of these rates. In this paper the common flow regimes for gas-liquid systems are defined and the techniques used to estimate the extent of a particular regime are described. Also, the current computer-code procedures are delineated and introduce a potentially better method is introduced

  3. Absolute counting of neutrophils in whole blood using flow cytometry.

    Science.gov (United States)

    Brunck, Marion E G; Andersen, Stacey B; Timmins, Nicholas E; Osborne, Geoffrey W; Nielsen, Lars K

    2014-12-01

    Absolute neutrophil count (ANC) is used clinically to monitor physiological dysfunctions such as myelosuppression or infection. In the research laboratory, ANC is a valuable measure to monitor the evolution of a wide range of disease states in disease models. Flow cytometry (FCM) is a fast, widely used approach to confidently identify thousands of cells within minutes. FCM can be optimised for absolute counting using spiked-in beads or by measuring the sample volume analysed. Here we combine the 1A8 antibody, specific for the mouse granulocyte protein Ly6G, with flow cytometric counting in straightforward FCM assays for mouse ANC, easily implementable in the research laboratory. Volumetric and Trucount™ bead assays were optimized for mouse neutrophils, and ANC values obtained with these protocols were compared to ANC measured by a dual-platform assay using the Orphee Mythic 18 veterinary haematology analyser. The single platform assays were more precise with decreased intra-assay variability compared with ANC obtained using the dual protocol. Defining ANC based on Ly6G expression produces a 15% higher estimate than the dual protocol. Allowing for this difference in ANC definition, the flow cytometry counting assays using Ly6G can be used reliably in the research laboratory to quantify mouse ANC from a small volume of blood. We demonstrate the utility of the volumetric protocol in a time-course study of chemotherapy induced neutropenia using four drug regimens. © 2014 International Society for Advancement of Cytometry.

  4. Ploidy level variability in South American fescues (festuca L., poaceae): use of flow cytometry in up to 5 1/2-year-old caryopses and herbarium specimens.

    Science.gov (United States)

    Smarda, P; Stancík, D

    2006-01-01

    Ploidy levels and chromosome numbers for 24 species of Festuca L. from 29 sites in Bolivia, Colombia, Ecuador and Venezuela are given. The ploidy level of 22 species is reported for the first time. A higher proportion of tetraploids in northern South America and the high frequency of polyploids in the whole continent are documented. In combination with chromosome counts, ploidy level was determined using flow cytometry in 4- to 5 1/2 -year-old herbarium specimens and mature caryopses. Flow cytometric determination from seeds was more reliable than determination from herbarium specimens. In herbarium specimens, the youngest, fresh green leaves, still hidden in sheaths, seem to be most suitable for cytometric determination. In old, brownish leaves, or poorly preserved herbarium specimens, the degradation of DNA signal in flow histograms was documented. DNA content measured in seeds was always higher than that measured in herbarium specimens, which may be caused by the presence of different cytosolic compounds. Differences of about 15% in relative DNA content of F. sodiroana and F. vaginalis was found in simultaneous measurements in seeds.

  5. Measurement of separase proteolytic activity in single living cells by a fluorogenic flow cytometry assay.

    Directory of Open Access Journals (Sweden)

    Wiltrud Haaß

    Full Text Available ESPL1/Separase, an endopeptidase, is required for centrosome duplication and separation of sister-chromatides in anaphase of mitosis. Overexpression and deregulated proteolytic activity of Separase as frequently observed in human cancers is associated with the occurrence of supernumerary centrosomes, chromosomal missegregation and aneuploidy. Recently, we have hypothesized that increased Separase proteolytic activity in a small subpopulation of tumor cells may serve as driver of tumor heterogeneity and clonal evolution in chronic myeloid leukemia (CML. Currently, there is no quantitative assay to measure Separase activity levels in single cells. Therefore, we have designed a flow cytometry-based assay that utilizes a Cy5- and rhodamine 110 (Rh110-biconjugated Rad21 cleavage site peptide ([Cy5-D-R-E-I-M-R]2-Rh110 as smart probe and intracellular substrate for detection of Separase enzyme activity in living cells. As measured by Cy5 fluorescence the cellular uptake of the fluorogenic peptide was fast and reached saturation after 210 min of incubation in human histiocytic lymphoma U937 cells. Separase activity was recorded as the intensity of Rh110 fluorescence released after intracellular peptide cleavage providing a linear signal gain within a 90-180 min time slot. Compared to conventional cell extract-based methods the flow cytometric assay delivers equivalent results but is more reliable, bypasses the problem of vague loading controls and unspecific proteolysis associated with whole cell extracts. Especially suited for the investigaton of blood- and bone marrow-derived hematopoietic cells the flow cytometric Separase assay allows generation of Separase activity profiles that tell about the number of Separase positive cells within a sample i.e. cells that currently progress through mitosis and about the range of intercellular variation in Separase activity levels within a cell population. The assay was used to quantify Separase proteolytic

  6. Functional characterization of neotropical snakes peripheral blood leukocytes subsets: Linking flow cytometry cell features, microscopy images and serum corticosterone levels.

    Science.gov (United States)

    de Carvalho, Marcelo Pires Nogueira; Queiroz-Hazarbassanov, Nicolle Gilda Teixeira; de Oliveira Massoco, Cristina; Sant'Anna, Sávio Stefanini; Lourenço, Mariana Mathias; Levin, Gabriel; Sogayar, Mari Cleide; Grego, Kathleen Fernandes; Catão-Dias, José Luiz

    2017-09-01

    Reptiles are the unique ectothermic amniotes, providing the key link between ectothermic anamniotes fish and amphibians, and endothermic birds and mammals; becoming an important group to study with the aim of providing significant knowledge into the evolutionary history of vertebrate immunity. Classification systems for reptiles' leukocytes have been described by their appearance rather than function, being still inconsistent. With the advent of modern techniques and the establishment of analytical protocols for snakes' blood by flow cytometry, we bring a qualitative and quantitative assessment of innate activities presented by snakes' peripheral blood leukocytes, thereby linking flow cytometric features with fluorescent and light microscopy images. Moreover, since corticosterone is an important immunomodulator in reptiles, hormone levels of all blood samples were measured. We provide novel and additional information which should contribute to better understanding of the development of the immune system of reptiles and vertebrates. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Sex-sorting sperm using flow cytometry/cell sorting.

    Science.gov (United States)

    Garner, Duane L; Evans, K Michael; Seidel, George E

    2013-01-01

    The sex of mammalian offspring can be predetermined by flow sorting relatively pure living populations of X- and Y-chromosome-bearing sperm. This method is based on precise staining of the DNA of sperm with the nucleic acid-specific fluorophore, Hoechst 33342, to differentiate between the subpopulations of X- and Y-sperm. The fluorescently stained sperm are then sex-sorted using a specialized high speed sorter, MoFlo(®) SX XDP, and collected into biologically supportive media prior to reconcentration and cryopreservation in numbers adequate for use with artificial insemination for some species or for in vitro fertilization. Sperm sorting can provide subpopulations of X- or Y-bearing bovine sperm at rates in the 8,000 sperm/s range while maintaining; a purity of 90% such that it has been applied to cattle on a commercial basis. The sex of offspring has been predetermined in a wide variety of mammalian species including cattle, swine, horses, sheep, goats, dogs, cats, deer, elk, dolphins, water buffalo as well as in humans using flow cytometric sorting of X- and Y-sperm.

  8. Detection of mycoplasmas in goat milk by flow cytometry.

    Science.gov (United States)

    Assunção, Patricia; Davey, Hazel M; Rosales, Ruben S; Antunes, Nuno T; de la Fe, Christian; Ramirez, Ana S; de Galarreta, Carlos M Ruiz; Poveda, Jose B

    2007-12-01

    The detection of mycoplasma in milk can be performed by either culture techniques or polymerase chain reaction (PCR) based methods. Although PCR can reduce the average diagnostic time to 5 h in comparison with the several days for the isolation of the agent, there is still a need to develop methods, which could give earlier results. For this purpose, we tested the ability of flow cytometry (FC) to detect mycoplasmas in milk samples. Milk samples inoculated with four different mycoplasmas, Mycoplasma agalactiae, Mycoplasma putrefaciens, Mycoplasma capricolum subsp. Capricolum, or Mycoplasma mycoides subsp. mycoides large-colony type, known to cause contagious agalactia in goats, were stained with the DNA stain SYBR Green I and analyzed by FC. Three goat milk samples, from which mycoplasmas have been isolated in broth medium were also analyzed. All mycoplasmas were easily distinguished from debris of milk samples, but it was not possible to distinguish between the different mycoplasma species. In our conditions, the detection limit of the technique was of the order of 10(3)-10(4) cells ml(-1). Furthermore, mycoplasmas were also distinguished from Staphylococcus aureus. FC together with SYBR Green I was able to distinguish between mycoplasma cells and debris present in milk samples and gave results in 20-30 min. This is an important first step in developing a robust, routine flow cytometric method for the detection of mycoplasmas in milk samples. (c) 2007 International Society for Analytical Cytology

  9. Biology and flow cytometry of proangiogenic hematopoietic progenitors cells.

    Science.gov (United States)

    Rose, Jonathan A; Erzurum, Serpil; Asosingh, Kewal

    2015-01-01

    During development, hematopoiesis and neovascularization are closely linked to each other via a common bipotent stem cell called the hemangioblast that gives rise to both hematopoietic cells and endothelial cells. In postnatal life, this functional connection between the vasculature and hematopoiesis is maintained by a subset of hematopoietic progenitor cells endowed with the capacity to differentiate into potent proangiogenic cells. These proangiogenic hematopoietic progenitors comprise a specific subset of bone marrow (BM)-derived cells that homes to sites of neovascularization and possess potent paracrine angiogenic activity. There is emerging evidence that this subpopulation of hematopoietic progenitors plays a critical role in vascular health and disease. Their angiogenic activity is distinct from putative "endothelial progenitor cells" that become structural cells of the endothelium by differentiation into endothelial cells. Proangiogenic hematopoietic progenitor cell research requires multidisciplinary expertise in flow cytometry, hematology, and vascular biology. This review provides a comprehensive overview of proangiogenic hematopoietic progenitor cell biology and flow cytometric methods to detect these cells in the peripheral blood circulation and BM. © 2014 International Society for Advancement of Cytometry.

  10. Monitoring of Legionella pneumophila viability after chlorine dioxide treatment using flow cytometry.

    Science.gov (United States)

    Mustapha, Pascale; Epalle, Thibaut; Allegra, Séverine; Girardot, Françoise; Garraud, Olivier; Riffard, Serge

    2015-04-01

    The viability of three Legionella pneumophila strains was monitored after chlorine dioxide (ClO2) treatment using a flow cytometric assay. Suspensions of L. pneumophila cells were submitted to increasing concentrations of ClO2. Culturable cells were still detected when using 4 mg/L, but could no longer be detected after exposure to 6 mg/L of ClO2, although viable but not culturable (VBNC) cells were found after exposure to 4-5 mg/L of ClO2. When testing whether these VBNC were infective, two of the strains were resuscitated after co-culture with Acanthamoeba polyphaga, but neither of them could infect macrophage-like cells. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  11. The use of flow cytometry to examine calcium signalling by TRPV1 in mixed cell populations.

    Science.gov (United States)

    Assas, Bakri M; Abdulaal, Wesam H; Wakid, Majed H; Zakai, Haytham A; Miyan, J; Pennock, J L

    2017-06-15

    Flow cytometric analysis of calcium mobilisation has been in use for many years in the study of specific receptor engagement or isolated cell:cell communication. However, calcium mobilisation/signaling is key to many cell functions including apoptosis, mobility and immune responses. Here we combine multiplex surface staining of whole spleen with Indo-1 AM to visualise calcium mobilisation and examine calcium signaling in a mixed immune cell culture over time. We demonstrate responses to a TRPV1 agonist in distinct cell subtypes without the need for cell separation. Multi parameter staining alongside Indo-1 AM to demonstrate calcium mobilization allows the study of real time calcium signaling in a complex environment. Copyright © 2017. Published by Elsevier Inc.

  12. Flow chemistry vs. flow analysis.

    Science.gov (United States)

    Trojanowicz, Marek

    2016-01-01

    The flow mode of conducting chemical syntheses facilitates chemical processes through the use of on-line analytical monitoring of occurring reactions, the application of solid-supported reagents to minimize downstream processing and computerized control systems to perform multi-step sequences. They are exactly the same attributes as those of flow analysis, which has solid place in modern analytical chemistry in several last decades. The following review paper, based on 131 references to original papers as well as pre-selected reviews, presents basic aspects, selected instrumental achievements and developmental directions of a rapidly growing field of continuous flow chemical synthesis. Interestingly, many of them might be potentially employed in the development of new methods in flow analysis too. In this paper, examples of application of flow analytical measurements for on-line monitoring of flow syntheses have been indicated and perspectives for a wider application of real-time analytical measurements have been discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Vortical flows

    International Nuclear Information System (INIS)

    Wu, Jie-Zhi; Ma, Hui-Yang; Zhou, Ming-De

    2015-01-01

    This book is a comprehensive and intensive book for graduate students in fluid dynamics as well as scientists, engineers and applied mathematicians. Offering a systematic introduction to the physical theory of vortical flows at graduate level, it considers the theory of vortical flows as a branch of fluid dynamics focusing on shearing process in fluid motion, measured by vorticity. It studies vortical flows according to their natural evolution stages,from being generated to dissipated. As preparation, the first three chapters of the book provide background knowledge for entering vortical flows. The rest of the book deals with vortices and vortical flows, following their natural evolution stages. Of various vortices the primary form is layer-like vortices or shear layers, and secondary but stronger form is axial vortices mainly formed by the rolling up of shear layers. Problems are given at the end of each chapter and Appendix, some for helping understanding the basic theories, and some involving specific applications; but the emphasis of both is always on physical thinking.

  14. Vortical flows

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Jie-Zhi [Peking Univ., Beijing (China). College of Engineering; Ma, Hui-Yang [Univ. of Chinese Academy of Sciences, Beijing (China). Dept. of Physics; Zhou, Ming-De [Arizona Univ., Tucson, AZ (United States). Dept. of Aerospace and Mechanical Engineering

    2015-11-01

    This book is a comprehensive and intensive book for graduate students in fluid dynamics as well as scientists, engineers and applied mathematicians. Offering a systematic introduction to the physical theory of vortical flows at graduate level, it considers the theory of vortical flows as a branch of fluid dynamics focusing on shearing process in fluid motion, measured by vorticity. It studies vortical flows according to their natural evolution stages,from being generated to dissipated. As preparation, the first three chapters of the book provide background knowledge for entering vortical flows. The rest of the book deals with vortices and vortical flows, following their natural evolution stages. Of various vortices the primary form is layer-like vortices or shear layers, and secondary but stronger form is axial vortices mainly formed by the rolling up of shear layers. Problems are given at the end of each chapter and Appendix, some for helping understanding the basic theories, and some involving specific applications; but the emphasis of both is always on physical thinking.

  15. Using flow cytometry for counting natural planktonic bacteria and understanding the structure of planktonic bacterial communities

    Directory of Open Access Journals (Sweden)

    Josep M. Gasol

    2000-06-01

    Full Text Available Flow cytometry is rapidly becoming a routine methodology in aquatic microbial ecology. The combination of simple to use bench-top flow cytometers and highly fluorescent nucleic acid stains allows fast and easy determination of microbe abundance in the plankton of lakes and oceans. The different dyes and protocols used to stain and count planktonic bacteria as well as the equipment in use are reviewed, with special attention to some of the problems encountered in daily routine practice such as fixation, staining and absolute counting. One of the main advantages of flow cytometry over epifluorescence microscopy is the ability to obtain cell-specific measurements in large numbers of cells with limited effort. We discuss how this characteristic has been used for differentiating photosynthetic from non-photosynthetic prokaryotes, for measuring bacterial cell size and nucleic acid content, and for estimating the relative activity and physiological state of each cell. We also describe how some of the flow cytometrically obtained data can be used to characterize the role of microbes on carbon cycling in the aquatic environment and we prospect the likely avenues of progress in the study of planktonic prokaryotes through the use of flow cytometry.

  16. Granular flow

    DEFF Research Database (Denmark)

    Mitarai, Namiko; Nakanishi, Hiizu

    2012-01-01

    Granular material is a collection of macroscopic particles that are visible with naked eyes. The non-equilibrium nature of the granular materials makes their rheology quite different from that of molecular systems. In this minireview, we present the unique features of granular materials focusing...... on the shear flow of dry granular materials and granule-liquid mixture....

  17. 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

    fraction of polyploid cells. The results suggest that estradiol induces a dose-dependent cell killing effect in the T61 human breast carcinoma. The correlation between the treatment-induced growth delay and the effect on the cell cycle distribution indicates that the changes in the cell cycle...

  18. Plant cytokinin analogues with inhibitory activity on cyclin-dependent kinases exert their antiproliferative effect through induction of apoptosis initiated by the mitochondrial pathway: Determination by a multiparametric flow cytometric analysis

    Czech Academy of Sciences Publication Activity Database

    Vermeulen, K.; Strnad, Miroslav; Havlíček, Libor; Onckelen, H.; Lenjou, M.; Nijs, G.; Bockstaele, D. R.; Berneman, Z. N.

    2002-01-01

    Roč. 30, č. 10 (2002), s. 1107-1114 ISSN 0301-472X R&D Projects: GA MŠk OC 844.10 Institutional research plan: CEZ:AV0Z5038910 Keywords : PLASMA MEMBRANE * PURINE ANALOGS * CELLS Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.366, year: 2002

  19. Flow Game

    DEFF Research Database (Denmark)

    Olsen, Jesper Lind

    2003-01-01

    Flow Game er et dialogspil, der kan bruges som ledelsesværktøj, ledertræning, samtaletræning, coachingtræning og ideudvikling m.m. Gennem dilemmakort provokeres en dialog og teori-U inspireret afklaring- og udviklingsproces, hvor der enten arbejdes på en gruppes eller et individs vision/innovatio......Flow Game er et dialogspil, der kan bruges som ledelsesværktøj, ledertræning, samtaletræning, coachingtræning og ideudvikling m.m. Gennem dilemmakort provokeres en dialog og teori-U inspireret afklaring- og udviklingsproces, hvor der enten arbejdes på en gruppes eller et individs vision...

  20. Media Flow

    DEFF Research Database (Denmark)

    Kabel, Lars

    2016-01-01

    News and other kinds of journalistic stories, 16-17 hours a day, all year round, on all platforms, also the moderated social media. The key research thesis behind this article is that the continuous and speedy stream of news stories and media content now is becoming the centre of the production...... processes and the value creation in converged multimedia newsrooms. The article identify new methods and discuss editorial challenges in handling media flow....

  1. Real-time cytometric assay of nitric oxide and superoxide interaction in peripheral blood monocytes: A no-wash, no-lyse kinetic method.

    Science.gov (United States)

    Balaguer, Susana; Diaz, Laura; Gomes, Angela; Herrera, Guadalupe; O'Connor, José-Enrique; Urios, Amparo; Felipo, Vicente; Montoliu, Carmina

    2017-05-01

    Nitric oxide (NO) and its related reactive nitrogen species (RNS) and reactive oxygen species (ROS) are crucial in monocyte responses against pathogens and also in inflammatory conditions. Central to both processes is the generation of the strong oxidant peroxynitrite (ONOO) by a fast reaction between NO and superoxide anion. ONOO is a biochemical junction for ROS- and RNS cytotoxicity and causes protein nitrosylation. Circulating by-products of protein nitrosylation are early biomarkers of inflammation-based conditions, including minimal hepatic encephalopathy in cirrhotic patients (Montoliu et al., Am J Gastroenterol 2011; 106:1629-1637). In this context, we have designed a novel no-wash, no-lyse real-time flow cytometry assay to detect and follow-up the NO- and superoxide-driven generation of ONOO in peripheral blood monocytes. Whole blood samples were stained with CD45 and CD14 antibodies plus one of a series of fluorescent probes sensitive to RNS, ROS, or glutathione, namely 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate, dihydrorhodamine 123, MitoSOX Red, dihydroethidium, and 5-chloromethylfluorescein diacetate. Samples were exposed sequentially to a NO donor and three different superoxide donors, and analyzed in real time by kinetic flow cytometry. Relevant kinetic descriptors, such as the rate of fluorescence change, were calculated from the kinetic plot. The generation of ONOO, which consumes both NO and superoxide, led to a decrease in the intensity of the cellular fluorescence of the probes sensitive to these molecules. This is a fast and simple assay that may be used to monitor the intracellular generation of ONOO in physiological, pathological, and pharmacological contexts. © 2015 International Clinical Cytometry Society. © 2015 International Clinical Cytometry Society.

  2. Automated Blood Sample Preparation Unit (ABSPU) for Portable Microfluidic Flow Cytometry.

    Science.gov (United States)

    Chaturvedi, Akhil; Gorthi, Sai Siva

    2017-02-01

    Portable microfluidic diagnostic devices, including flow cytometers, are being developed for point-of-care settings, especially in conjunction with inexpensive imaging devices such as mobile phone cameras. However, two pervasive drawbacks of these have been the lack of automated sample preparation processes and cells settling out of sample suspensions, leading to inaccurate results. We report an automated blood sample preparation unit (ABSPU) to prevent blood samples from settling in a reservoir during loading of samples in flow cytometers. This apparatus automates the preanalytical steps of dilution and staining of blood cells prior to microfluidic loading. It employs an assembly with a miniature vibration motor to drive turbulence in a sample reservoir. To validate performance of this system, we present experimental evidence demonstrating prevention of blood cell settling, cell integrity, and staining of cells prior to flow cytometric analysis. This setup is further integrated with a microfluidic imaging flow cytometer to investigate cell count variability. With no need for prior sample preparation, a drop of whole blood can be directly introduced to the setup without premixing with buffers manually. Our results show that integration of this assembly with microfluidic analysis provides a competent automation tool for low-cost point-of-care blood-based diagnostics.

  3. Induction of chromosome damage by ultraviolet light and caffeine: correlation of cytogenetic evaluation and flow karyotype

    International Nuclear Information System (INIS)

    Cremer, C.; Cremer, T.; Gray, J.W.

    1982-01-01

    Asynchronously growing cells of a M3-1 Chinese hamster line were ultraviolet (UV) irradiated (lambda . 254 nm) with UV fluences up to 7.5 J/m(2). After irradiation cells were incubated with or without 2 mM caffeine for 20 hr, then mitotic cells were selected by mechanical shaking. Their chromosomes were isolated, stained with Hoechst 33258 and chromomycin A3, and measured flow cytometrically. While the fluorescence distributions of chromosomes (flow karyo-types) from cells treated with UV alone or with caffeine alone were very similar to those of untreated controls, the flow karyo-types of UV + caffeine-treated cells showed a debris continuum that increased with increasing UV fluence suggesting an increased number of chromosome fragments. Visual evaluation of metaphase plates revealed that the percentage of cells with chromosome damage also increased steadily with increasing UV fluence. A high degree of correlation was observed between the relative magnitude of the debris level from flow karyotypes and the percentage of cells with chromosome damage and with generalized chromosomes shattering, respectively, as determined from metaphase spreads

  4. Flow Control

    Science.gov (United States)

    2013-04-08

    can be written as â fj (t) =WO tanh( WIx (t)+bI)+bO, (38) where WI , WO are the input and output matrices, respectively, and bI and bO are the input...applications, present on envisioned airborne optical platforms . One of the problems is that all adaptive optical systems rely on mechanically moving some...of successfully controlling the optical aberration due to the flow over the aperture of airborne optical platforms . As outlined above, systems

  5. Distinct neutrophil subpopulations phenotype by flow cytometry in myelodysplastic syndromes.

    Science.gov (United States)

    Vikentiou, Myrofora; Psarra, Katerina; Kapsimali, Violetta; Liapis, Konstantinos; Michael, Michalis; Tsionos, Konstantinos; Lianidou, Evi; Papasteriades, Chryssa

    2009-03-01

    The cardinal feature of myelodysplastic syndromes (MDS) is dysplasia involving one or more myeloid cell lineages. In the present study, we used 4-color flow cytometric analysis to investigate dysgranulopoiesis in bone marrow specimens from 65 patients with MDS. The antigen expression patterns of total neutrophil granulocytes (TNG) and of the two distinct neutrophil granulocytic subpopulations (NGSs), NGS-1 (dimmer CD45 expression) and NGS-2 (stronger CD45 expression) identified on the side scatter (SS) vs. CD45-intensity plot, were studied. The neutrophil granulocytes from patients with MDS showed characteristic antigen expression aberrancies which were more pronounced in NGS-2 subpopulation. Studying separately the NGS-2 subpopulation with the CD16/MPO/LF combination, the low CD16(+)/MPO(+) and low CD16(+)/LF(+) percentages seemed to discriminate between lower-risk and higher-risk patients with MDS in most occasions. Furthermore, a detailed assessment of the NGS-1 and NGS-2 immunophenotypic patterns revealed early dysplastic changes, not otherwise observed by standard TNG analysis, especially in cases of lower-risk MDS.

  6. Early stage detection of chemotherapeutic effect on 203 GL glioma in mice as studied by P-31 NMR and flow cytometry

    Energy Technology Data Exchange (ETDEWEB)

    Itoh, Masamitsu; Yoshikawa, Koki; Nishikawa, Junichi; Iio, Masahiro; Shibui, Soichiro; Nomura, Kazuhiro; Saito, Hazime; Kodama, Masahiko

    1988-08-01

    The effect of chemotherapy against glioma in mouse was evaluated by /sup 31/P NMR spectroscopy and flow cytometry. We found that administration of ACNU or tegafur at a dose less than LD/sub 50/ resulted in the partial suppression of the ratio of inorganic phosphate (Pi)/phosphocreatine (PCr) and phosphomonoester (PME)/creatine phosphate (PCr) after 24 or 48 hr, although these ratios are usually increased together with growth of tumors. Flow cytometric analysis of glioma in vivo showed an accumulation in cells containing tetraploid DNA by G/sub 2/M block 24 - 48 hr after treatment. However, the change occurred at a period slightly later than that of the Pi/PCr ratio. In contrast, histological change was noted at eight days after administration. Hence, it is concluded that in vivo /sup 31/P NMR spectroscopy can detect a change in metabolic pathways in tumors as early as 24 - 48 hr after the administration of chemotherapeutic agents.

  7. Astrophysical Flows

    Science.gov (United States)

    Pringle, James E.; King, Andrew

    2003-07-01

    Almost all conventional matter in the Universe is fluid, and fluid dynamics plays a crucial role in astrophysics. This new graduate textbook provides a basic understanding of the fluid dynamical processes relevant to astrophysics. The mathematics used to describe these processes is simplified to bring out the underlying physics. The authors cover many topics, including wave propagation, shocks, spherical flows, stellar oscillations, the instabilities caused by effects such as magnetic fields, thermal driving, gravity, shear flows, and the basic concepts of compressible fluid dynamics and magnetohydrodynamics. The authors are Directors of the UK Astrophysical Fluids Facility (UKAFF) at the University of Leicester, and editors of the Cambridge Astrophysics Series. This book has been developed from a course in astrophysical fluid dynamics taught at the University of Cambridge. It is suitable for graduate students in astrophysics, physics and applied mathematics, and requires only a basic familiarity with fluid dynamics.• Provides coverage of the fundamental fluid dynamical processes an astrophysical theorist needs to know • Introduces new mathematical theory and techniques in a straightforward manner • Includes end-of-chapter problems to illustrate the course and introduce additional ideas

  8. Molecular Characterization of Gastric Epithelial Cells Using Flow Cytometry

    Directory of Open Access Journals (Sweden)

    Kevin A. Bockerstett

    2018-04-01

    Full Text Available The ability to analyze individual epithelial cells in the gastric mucosa would provide important insight into gastric disease, including chronic gastritis and progression to gastric cancer. However, the successful isolation of viable gastric epithelial cells (parietal cells, neck cells, chief cells, and foveolar cells from gastric glands has been limited due to difficulties in tissue processing. Furthermore, analysis and interpretation of gastric epithelial cell flow cytometry data has been difficult due to the varying sizes and light scatter properties of the different epithelial cells, high levels of autofluorescence, and poor cell viability. These studies were designed to develop a reliable method for isolating viable single cells from the corpus of stomachs and to optimize analyses examining epithelial cells from healthy and diseased stomach tissue by flow cytometry. We performed a two stage enzymatic digestion in which collagenase released individual gastric glands from the stromal tissue of the corpus, followed by a Dispase II digestion that dispersed these glands into greater than 1 × 106 viable single cells per gastric corpus. Single cell suspensions were comprised of all major cell lineages found in the normal gastric glands. A method describing light scatter, size exclusion, doublet discrimination, viability staining, and fluorescently-conjugated antibodies and lectins was used to analyze individual epithelial cells and immune cells. This technique was capable of identifying parietal cells and revealed that gastric epithelial cells in the chronically inflamed mucosa significantly upregulated major histocompatibility complexes (MHC I and II but not CD80 or CD86, which are costimulatory molecules involved in T cell activation. These studies describe a method for isolating viable single cells and a detailed description of flow cytometric analysis of cells from healthy and diseased stomachs. These studies begin to identify effects of

  9. Multiplex flow cytometry barcoding and antibody arrays identify surface antigen profiles of primary and metastatic colon cancer cell lines.

    Directory of Open Access Journals (Sweden)

    Kumar Sukhdeo

    Full Text Available Colon cancer is a deadly disease affecting millions of people worldwide. Current treatment challenges include management of disease burden as well as improvements in detection and targeting of tumor cells. To identify disease state-specific surface antigen signatures, we combined fluorescent cell barcoding with high-throughput flow cytometric profiling of primary and metastatic colon cancer lines (SW480, SW620, and HCT116. Our multiplexed technique offers improvements over conventional methods by permitting the simultaneous and rapid screening of cancer cells with reduced effort and cost. The method uses a protein-level analysis with commercially available antibodies on live cells with intact epitopes to detect potential tumor-specific targets that can be further investigated for their clinical utility. Multiplexed antibody arrays can easily be applied to other tumor types or pathologies for discovery-based approaches to target identification.

  10. Cytometric evaluation of intracellular IFN-γ and IL-4 levels in thyroid follicular cells from patients with autoimmune thyroid diseases

    Directory of Open Access Journals (Sweden)

    Bossowski Artur

    2011-09-01

    Full Text Available Abstract Background In recent few years is underlined that altered balance of pro- and anti-inflammatory cytokines play an important role in the pathogenesis of AITD. The aim of this study was to estimate intracellular INF-γ and IL-4 levels in thyroid-infiltrating lymphocytes and thyrocytes isolated from thyroid tissues in 54 adolescent patients aged 8-21 years, with Graves' disease (GD; n = 18, Hashimoto's thyroiditis (HT; n = 18 and non-toxic multinodular goiter (NTMG; n = 18. Methods Fresh thyroid tissues were taken on culture medium RPMI -1640, it was mechanically prepared. In next step were added cell activators -12- myristate 13- the acetate (PMA and Ionomycin as well as the inhibitor of transportation of proteins - Breferdin A. They were cultured 24 hours in 50 ml flasks at 37°C in a 5-95% CO2-air water-saturated atmosphere. After that, thyrocytes were identified by mouse mAb directed against human TPO epitope 64 conjugated with rabbit anti-mouse antibodies IgG (Fab'2 labeled by FITC. After incubation at room temperature to each of samples added reagent A fixative the cellular membrane. In next step into the cell suspensions were added reagent B to permeabilization of cellular membrane and specific anti-IL-4-PE or anti-IFN-γ-PE mAbs. Identification of intracellular cytokines in T lymphocytes was performed in the same procedure with application of anti-CD4-PerCP and anti-CD8-PerCP mAbs specific for T lymphocytes. The cells were analyzed in a flow cytometry (Coulter EPICS XL. Results In examined group of patients with GD we observed statistically significant higher mean percentage of cells with phenotype CD4+IL-4 (p Conclusions We conclude that human thyrocytes in autoimmune thyroid disorders could be a source of cytokine production and that their activation influences local interaction with T lymphocytes inflowing to the thyroid gland.

  11. Quality assessment program for EuroFlow protocols: summary results of four-year (2010-2013) quality assurance rounds.

    Science.gov (United States)

    Kalina, Tomas; Flores-Montero, Juan; Lecrevisse, Quentin; Pedreira, Carlos E; van der Velden, Vincent H J; Novakova, Michaela; Mejstrikova, Ester; Hrusak, Ondrej; Böttcher, Sebastian; Karsch, Dennis; Sędek, Łukasz; Trinquand, Amelie; Boeckx, Nancy; Caetano, Joana; Asnafi, Vahid; Lucio, Paulo; Lima, Margarida; Helena Santos, Ana; Bonaccorso, Paola; van der Sluijs-Gelling, Alita J; Langerak, Anton W; Martin-Ayuso, Marta; Szczepański, Tomasz; van Dongen, Jacques J M; Orfao, Alberto

    2015-02-01

    Flow cytometric immunophenotyping has become essential for accurate diagnosis, classification, and disease monitoring in hemato-oncology. The EuroFlow Consortium has established a fully standardized "all-in-one" pipeline consisting of standardized instrument settings, reagent panels, and sample preparation protocols and software for data analysis and disease classification. For its reproducible implementation, parallel development of a quality assurance (QA) program was required. Here, we report on the results of four consecutive annual rounds of the novel external QA EuroFlow program. The novel QA scheme aimed at monitoring the whole flow cytometric analysis process (cytometer setting, sample preparation, acquisition and analysis) by reading the median fluorescence intensities (MedFI) of defined lymphocytes' subsets. Each QA participant applied the predefined reagents' panel on blood cells of local healthy donors. A uniform gating strategy was applied to define lymphocyte subsets and to read MedFI values per marker. The MedFI values were compared with reference data and deviations from reference values were quantified using performance score metrics. In four annual QA rounds, we analyzed 123 blood samples from local healthy donors on 14 different instruments in 11 laboratories from nine European countries. The immunophenotype of defined cellular subsets appeared sufficiently standardized to permit unified (software) data analysis. The coefficient of variation of MedFI for 7 of 11 markers performed repeatedly below 30%, average MedFI in each QA round ranged from 86 to 125% from overall median. Calculation of performance scores was instrumental to pinpoint standardization failures and their causes. Overall, the new EuroFlow QA system for the first time allowed to quantify the technical variation that is introduced in the measurement of fluorescence intensities in a multicentric setting over an extended period of time. EuroFlow QA is a proficiency test specific for

  12. Quantitative analysis of gold and carbon nanoparticles in mammalian cells by flow cytometry light scattering

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Gang [Nanjing University, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences (China); Liu, Naicheng; Wang, Zhenheng [Nanjing University, Department of Orthopedics, Jinling Hospital, School of Medicine (China); Shi, Tongguo; Gan, Jingjing; Wang, Zhenzhen; Zhang, Junfeng, E-mail: jfzhang@nju.edu.cn [Nanjing University, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences (China)

    2017-02-15

    Nanoparticle-based applications for diagnostics and therapeutics have been extensively studied. These applications require a profound understanding of the fate of nanoparticles (NPs) in cellular environments. However, until now, few analytical methods are available and most of them rely on fluorescent properties or special elements of NPs; therefore, for NPs without observable optical properties or special elements, the existing methods are hardly applicable. In this study, we introduce a flow cytometry light scattering (FCLS)-based approach that quantifies in situ NPs accurately in mammalian cells. Continuous cells of heterogeneous human epithelial colorectal adenocarcinoma (Caco-2 cells), mouse peritoneal macrophages (MPM), and human adenocarcinomic alveolar basal epithelia (A549 cells) were cultured with NPs with certain concentrations and size. The intensity of the flow cytometric side scattered light, which indicates the quantity of NPs in the cells, was analyzed. The result shows an accurate size- and dose-dependent uptake of Au NPs (5, 30, 250 nm) in Caco-2 cells. The size- and dose- dependence of Au NPs (5, 30, 250 nm) and carbon NPs (50, 500 nm) in cells was validated by transmission electron microscope (TEM). This paper demonstrates the great potential of flow cytometry light scattering in the quantitative study of the size and dose effect on in situ metallic or non-metallic NPs in mammalian cells.

  13. Quantitative analysis of gold and carbon nanoparticles in mammalian cells by flow cytometry light scattering

    Science.gov (United States)

    Zhou, Gang; Liu, Naicheng; Wang, Zhenheng; Shi, Tongguo; Gan, Jingjing; Wang, Zhenzhen; Zhang, Junfeng

    2017-02-01

    Nanoparticle-based applications for diagnostics and therapeutics have been extensively studied. These applications require a profound understanding of the fate of nanoparticles (NPs) in cellular environments. However, until now, few analytical methods are available and most of them rely on fluorescent properties or special elements of NPs; therefore, for NPs without observable optical properties or special elements, the existing methods are hardly applicable. In this study, we introduce a flow cytometry light scattering (FCLS)-based approach that quantifies in situ NPs accurately in mammalian cells. Continuous cells of heterogeneous human epithelial colorectal adenocarcinoma (Caco-2 cells), mouse peritoneal macrophages (MPM), and human adenocarcinomic alveolar basal epithelia (A549 cells) were cultured with NPs with certain concentrations and size. The intensity of the flow cytometric side scattered light, which indicates the quantity of NPs in the cells, was analyzed. The result shows an accurate size- and dose-dependent uptake of Au NPs (5, 30, 250 nm) in Caco-2 cells. The size- and dose- dependence of Au NPs (5, 30, 250 nm) and carbon NPs (50, 500 nm) in cells was validated by transmission electron microscope (TEM). This paper demonstrates the great potential of flow cytometry light scattering in the quantitative study of the size and dose effect on in situ metallic or non-metallic NPs in mammalian cells.

  14. Laboratory Tests of Multiplex Detection of PCR Amplicons Using the Luminex 100 Flow Analyzer

    Energy Technology Data Exchange (ETDEWEB)

    Venkateswaran, K.S.; Nasarabadi, S.; Langlois, R.G.

    2000-05-05

    Lawrence Livermore National Laboratory (LLNL) demonstrated the power of flow cytometry in detecting the biological agents simulants at JFT III. LLNL pioneered in the development of advanced nucleic acid analyzer (ANM) for portable real time identification. Recent advances in flow cytometry provide a means for multiplexed nucleic acid detection and immunoassay of pathogenic microorganisms. We are presently developing multiplexed immunoassays for the simultaneous detection of different simulants. Our goal is to build an integrated instrument for both nucleic acid analysis and immuno detection. In this study we evaluated the Luminex LX 100 for concurrent identification of more than one PCR amplified product. ANAA has real-time Taqman fluorescent detection capability for rapid identification of field samples. However, its multiplexing ability is limited by the combination of available fluorescent labels. Hence integration of ANAA with flow cytometry can give the rapidity of ANAA amplification and the multiplex capability of flow cytometry. Multiplexed flow cytometric analysis is made possible using a set of fluorescent latex microsphere that are individually identified by their red and infrared fluorescence. A green fluorochrome is used as the assay signal. Methods were developed for the identification of specific nucleic acid sequences from Bacillus globigii (Bg), Bacillus thuringensis (Bt) and Erwinia herbicola (Eh). Detection sensitivity using different reporter fluorochromes was tested with the LX 100, and also different assay formats were evaluated for their suitability for rapid testing. A blind laboratory trial was carried out December 22-27, 1999 to evaluate bead assays for multiplex identification of Bg and Bt PCR products. This report summarizes the assay development, fluorochrome comparisons, and the results of the blind trial conducted at LLNL for the laboratory evaluation of the LX 100 flow analyzer.

  15. Ultrasonic flow meter

    NARCIS (Netherlands)

    Lötters, Joost Conrad; Snijders, G.J.; Volker, A.W.F.

    2014-01-01

    The invention relates to an ultrasonic flow meter comprising a flow tube for the fluid whose flow rate is to be determined. The flow meter comprises a transmitting element for emitting ultrasonic waves, which is provided on the outer jacket of the flow tube. A receiving element, which is provided on

  16. Advances in Automated Plankton Imaging: Enhanced Throughput, Automated Staining, and Extended Deployment Modes for Imaging FlowCytobot

    Science.gov (United States)

    Sosik, H. M.; Olson, R. J.; Brownlee, E.; Brosnahan, M.; Crockford, E. T.; Peacock, E.; Shalapyonok, A.

    2016-12-01

    Imaging FlowCytobot (IFCB) was developed to fill a need for automated identification and monitoring of nano- and microplankton, especially phytoplankton in the size range 10 200 micrometer, which are important in coastal blooms (including harmful algal blooms). IFCB uses a combination of flow cytometric and video technology to capture high resolution (1 micrometer) images of suspended particles. This proven, now commercially available, submersible instrument technology has been deployed in fixed time series locations for extended periods (months to years) and in shipboard laboratories where underway water is automatically analyzed during surveys. Building from these successes, we have now constructed and evaluated three new prototype IFCB designs that extend measurement and deployment capabilities. To improve cell counting statistics without degrading image quality, a high throughput version (IFCB-HT) incorporates in-flow acoustic focusing to non-disruptively pre-concentrate cells before the measurement area of the flow cell. To extend imaging to all heterotrophic cells (even those that do not exhibit chlorophyll fluorescence), Staining IFCB (IFCB-S) incorporates automated addition of a live-cell fluorescent stain (fluorescein diacetate) to samples before analysis. A horizontally-oriented IFCB-AV design addresses the need for spatial surveying from surface autonomous vehicles, including design features that reliably eliminate air bubbles and mitigate wave motion impacts. Laboratory evaluation and test deployments in waters near Woods Hole show the efficacy of each of these enhanced IFCB designs.

  17. Flow of Aqueous Humor

    Science.gov (United States)

    ... Home Flow of Aqueous Humor Flow of Aqueous Humor Most, but not all, forms of glaucoma are ... remains normal when some of the fluid (aqueous humor) produced by the eye's ciliary body flows out ...

  18. Comparative analysis of minimal residual disease detection using four-color flow cytometry, consensus IgH-PCR, and quantitative IgH PCR in CLL after allogeneic and autologous stem cell transplantation.

    Science.gov (United States)

    Böttcher, S; Ritgen, M; Pott, C; Brüggemann, M; Raff, T; Stilgenbauer, S; Döhner, H; Dreger, P; Kneba, M

    2004-10-01

    The clinically most suitable method for minimal residual disease (MRD) detection in chronic lymphocytic leukemia is still controversial. We prospectively compared MRD assessment in 158 blood samples of 74 patients with CLL after stem cell transplantation (SCT) using four-color flow cytometry (MRD flow) in parallel with consensus IgH-PCR and ASO IgH real-time PCR (ASO IgH RQ-PCR). In 25 out of 106 samples (23.6%) with a polyclonal consensus IgH-PCR pattern, MRD flow still detected CLL cells, proving higher sensitivity of flow cytometry over PCR-genescanning with consensus IgH-primers. Of 92 samples, 14 (15.2%) analyzed in parallel by MRD flow and by ASO IgH RQ-PCR were negative by our flow cytometric assay but positive by PCR, thus demonstrating superior sensitivity of RQ-PCR with ASO primers. Quantitative MRD levels measured by both methods correlated well (r=0.93). MRD detection by flow and ASO IgH RQ-PCR were equally suitable to monitor MRD kinetics after allogeneic SCT, but the PCR method detected impending relapses after autologous SCT earlier. An analysis of factors that influence sensitivity and specificity of flow cytometry for MRD detection allowed to devise further improvements of this technique.

  19. Interlaboratory assessment of mitotic index by flow cytometry confirms superior reproducibility relative to microscopic scoring.

    Science.gov (United States)

    Roberts, D J; Spellman, R A; Sanok, K; Chen, H; Chan, M; Yurt, P; Thakur, A K; DeVito, G L; Murli, H; Stankowski, L F

    2012-05-01

    A flow cytometric procedure for determining mitotic index (MI) as part of the metaphase chromosome aberrations assay, developed and utilized routinely at Pfizer as part of their standard assay design, has been adopted successfully by Covance laboratories. This method, using antibodies against phosphorylated histone tails (H3PS10) and nucleic acid stain, has been evaluated by the two independent test sites and compared to manual scoring. Primary human lymphocytes were treated with cyclophosphamide, mitomycin C, benzo(a)pyrene, and etoposide at concentrations inducing dose-dependent cytotoxicity. Deming regression analysis indicates that the results generated via flow cytometry (FCM) were more consistent between sites than those generated via microscopy. Further analysis using the Bland-Altman modification of the Tukey mean difference method supports this finding, as the standard deviations (SDs) of differences in MI generated by FCM were less than half of those generated manually. Decreases in scoring variability owing to the objective nature of FCM, and the greater number of cells analyzed, make FCM a superior method for MI determination. In addition, the FCM method has proven to be transferable and easily integrated into standard genetic toxicology laboratory operations. Copyright © 2012 Wiley Periodicals, Inc.

  20. Flow Cytometry Enables Multiplexed Measurements of Genetically Encoded Intramolecular FRET Sensors Suitable for Screening.

    Science.gov (United States)

    Doucette, Jaimee; Zhao, Ziyan; Geyer, Rory J; Barra, Melanie M; Balunas, Marcy J; Zweifach, Adam

    2016-07-01

    Genetically encoded sensors based on intramolecular FRET between CFP and YFP are used extensively in cell biology research. Flow cytometry has been shown to offer a means to measure CFP-YFP FRET; we suspected it would provide a unique way to conduct multiplexed measurements from cells expressing different FRET sensors, which is difficult to do with microscopy, and that this could be used for screening. We confirmed that flow cytometry accurately measures FRET signals using cells transiently transfected with an ERK activity reporter, comparing responses measured with imaging and cytometry. We created polyclonal long-term transfectant lines, each expressing a different intramolecular FRET sensor, and devised a way to bar-code four distinct populations of cells. We demonstrated the feasibility of multiplexed measurements and determined that robust multiplexed measurements can be conducted in plate format. To validate the suitability of the method for screening, we measured responses from a plate of bacterial extracts that in unrelated experiments we had determined contained the protein kinase C (PKC)-activating compound teleocidin A-1. The multiplexed assay correctly identifying the teleocidin A-1-containing well. We propose that multiplexed cytometric FRET measurements will be useful for analyzing cellular function and for screening compound collections. © 2016 Society for Laboratory Automation and Screening.

  1. Panel development for multicolor flow-cytometry testing of proliferation and immunophenotype in hMSCs.

    Science.gov (United States)

    Bradford, Jolene A; Clarke, Scott T

    2011-01-01

    Adult human mesenchymal stem cells (hMSC) are rare fibroblast-like cells capable of differentiation into a variety of cell tissues which include bone, cartilage, muscle, ligament, tendon, and adipose. Normal adult bone marrow and adipose tissue are the most common sources of these cells. The International Society for Cellular Therapy (ISCT) has proposed a set of standards to define hMSC for laboratory investigations and preclinical studies: adherence to plastic in standard culture conditions; in vitro differentiation into osteoblasts, adipocytes, and chondroblasts; and specific surface antigen expression. Direct measurement of proliferation combined with simultaneous detection of the ISCT-consensus immunophenotypic profile provides data that is used to determine the differentiation status and health of the cells. Flow cytometry provides a powerful technology that is routinely used to simultaneously and rapidly measure multiple parameters in a single sample. This chapter describes a flow cytometric panel for the simultaneous detection of immunophenotypic profile, proliferative capacity, and DNA content measurement in hMSC. Because a relatively small number of cells are needed with this approach, measurements can be made with minimal impact on expansion potential. The ability to assess antigen expression and proliferative status enables the investigator to make informed decisions on expansion and harvesting.

  2. A Flow Cytometry Protocol to Estimate DNA Content in the Yellowtail Tetra Astyanax altiparanae

    Directory of Open Access Journals (Sweden)

    Pedro L. P. Xavier

    2017-09-01

    Full Text Available The production of triploid yellowtail tetra Astyanax altiparanae is a key factor to obtain permanently sterile individuals by chromosome set manipulation. Flow cytometric analysis is the main tool for confirmation of the resultant triploids individuals, but very few protocols are specific for A. altiparanae species. The current study has developed a protocol to estimate DNA content in this species. Furthermore, a protocol for long-term storage of dorsal fins used for flow cytometry analysis was established. The combination of five solutions with three detergents (Nonidet P-40 Substitute, Tween 20, and Triton X-100 at 0.1, 0.2, and 0.4% concentration was evaluated. Using the best solution from this first experiment, the addition of trypsin (0.125, 0.25, and 0.5% and sucrose (74 mM and the effects of increased concentrations of the detergents at 0.6 and 1.2% concentration were also evaluated. After adjustment of the protocol for flow cytometry, preservation of somatic tissue or isolated nuclei was also evaluated by freezing (at −20°C and fixation in saturated NaCl solution, acetic methanol (1:3, ethanol, and formalin at 10% for 30 or 60 days of storage at 25°C. Flow cytometry analysis in yellowtail tetra species was optimized using the following conditions: lysis solution: 9.53 mM MgCl2.7H20; 47.67 mM KCl; 15 mM Tris; 74 mM sucrose, 0.6% Triton X-100, pH 8.0; staining solution: Dulbecco's PBS with DAPI 1 μg mL−1; preservation procedure: somatic cells (dorsal fin samples frozen at −20°C. Using this protocol, samples may be stored up to 60 days with good accuracy for flow cytometry analysis.

  3. Analysis of UV-excited fluorochromes by flow cytometry using near-ultraviolet laser diodes.

    Science.gov (United States)

    Telford, William G

    2004-09-01

    Violet laser diodes have become common and reliable laser sources for benchtop flow cytometers. While these lasers are very useful for a variety of violet and some ultraviolet-excited fluorochromes (e.g., DAPI), they do not efficiently excite most UV-stimulated probes. In this study, the next generation of InGaN near-UV laser diodes (NUVLDs) emitting in the 370-375-nm range have been evaluated as laser sources for cuvette-based flow cytometers. Several NUVLDs, ranging in wavelength from 370 to 374 nm and in power level from 1.5 to 10 mW, were mounted on a BD Biosciences LSR II and evaluated for their ability to excite cells labeled with the UV DNA binding dye DAPI, several UV phenotyping fluorochromes (including Alexa Fluor 350, Marina Blue, and quantum dots), and the fluorescent calcium chelator indo-1. NUVLDs at the 8-10-mW power range gave detection sensitivity levels comparable to more powerful solid-state and ion laser sources, using low-fluorescence microsphere beads as measurement standards. NUVLDs at all tested power levels allowed extremely high-resolution DAPI cell cycle analysis, and sources in the 8-10-mW power range excited Alexa Fluor 350, Marina Blue, and a variety of quantum dots at virtually the same signal-to-noise ratios as more powerful UV sources. These evaluations indicate that near-UV laser diodes installed on a cuvette-based flow cytometer performed nearly as well as more powerful solid-state UV lasers on the same instrumentation, and comparably to more powerful ion lasers on a jet-in-air system, and. Despite their limited power, integration of these small and inexpensive lasers into benchtop flow cytometers should allow the use of flow cytometric applications requiring UV excitation on a wide variety of instruments. Copyright 2004 Wiley-Liss, Inc.

  4. What Is Fanconi Anemia?

    Science.gov (United States)

    ... people who don't have FA. Cytometric Flow Analysis Cytometric flow analysis, or CFA, is done in a lab. This ... to know about resources that can give you emotional support and helpful information about FA and its ...

  5. Measurement of Lipid Accumulation in Chlorella vulgaris via Flow Cytometry and Liquid-State ¹H NMR Spectroscopy for Development of an NMR-Traceable Flow Cytometry Protocol

    Science.gov (United States)

    Bono Jr., Michael S.; Garcia, Ravi D.; Sri-Jayantha, Dylan V.; Ahner, Beth A.; Kirby, Brian J.

    2015-01-01

    In this study, we cultured Chlorella vulgaris cells with a range of lipid contents, induced via nitrogen starvation, and characterized them via flow cytometry, with BODIPY 505/515 as a fluorescent lipid label, and liquid-state 1H NMR spectroscopy. In doing so, we demonstrate the utility of calibrating flow cytometric measurements of algal lipid content using triacylglyceride (TAG, also known as triacylglycerol or triglyceride) content per cell as measured via quantitative 1H NMR. Ensemble-averaged fluorescence of BODIPY-labeled cells was highly correlated with average TAG content per cell measured by bulk NMR, with a linear regression yielding a linear fit with r 2 = 0.9974. This correlation compares favorably to previous calibrations of flow cytometry protocols to lipid content measured via extraction, and calibration by NMR avoids the time and complexity that is generally required for lipid quantitation via extraction. Flow cytometry calibrated to a direct measurement of TAG content can be used to investigate the distribution of lipid contents for cells within a culture. Our flow cytometry measurements showed that Chlorella vulgaris cells subjected to nitrogen limitation exhibited higher mean lipid content but a wider distribution of lipid content that overlapped the relatively narrow distribution of lipid content for replete cells, suggesting that nitrogen limitation induces lipid accumulation in only a subset of cells. Calibration of flow cytometry protocols using direct in situ measurement of TAG content via NMR will facilitate rapid development of more precise flow cytometry protocols, enabling investigation of algal lipid accumulation for development of more productive algal biofuel feedstocks and cultivation protocols. PMID:26267664

  6. Measurement of lipid accumulation in Chlorella vulgaris via flow cytometry and liquid-state ¹H NMR spectroscopy for development of an NMR-traceable flow cytometry protocol.

    Directory of Open Access Journals (Sweden)

    Michael S Bono

    Full Text Available In this study, we cultured Chlorella vulgaris cells with a range of lipid contents, induced via nitrogen starvation, and characterized them via flow cytometry, with BODIPY 505/515 as a fluorescent lipid label, and liquid-state 1H NMR spectroscopy. In doing so, we demonstrate the utility of calibrating flow cytometric measurements of algal lipid content using triacylglyceride (TAG, also known as triacylglycerol or triglyceride content per cell as measured via quantitative 1H NMR. Ensemble-averaged fluorescence of BODIPY-labeled cells was highly correlated with average TAG content per cell measured by bulk NMR, with a linear regression yielding a linear fit with r2 = 0.9974. This correlation compares favorably to previous calibrations of flow cytometry protocols to lipid content measured via extraction, and calibration by NMR avoids the time and complexity that is generally required for lipid quantitation via extraction. Flow cytometry calibrated to a direct measurement of TAG content can be used to investigate the distribution of lipid contents for cells within a culture. Our flow cytometry measurements showed that Chlorella vulgaris cells subjected to nitrogen limitation exhibited higher mean lipid content but a wider distribution of lipid content that overlapped the relatively narrow distribution of lipid content for replete cells, suggesting that nitrogen limitation induces lipid accumulation in only a subset of cells. Calibration of flow cytometry protocols using direct in situ measurement of TAG content via NMR will facilitate rapid development of more precise flow cytometry protocols, enabling investigation of algal lipid accumulation for development of more productive algal biofuel feedstocks and cultivation protocols.

  7. Measurement of lipid accumulation in Chlorella vulgaris via flow cytometry and liquid-state ¹H NMR spectroscopy for development of an NMR-traceable flow cytometry protocol.

    Science.gov (United States)

    Bono, Michael S; Garcia, Ravi D; Sri-Jayantha, Dylan V; Ahner, Beth A; Kirby, Brian J

    2015-01-01

    In this study, we cultured Chlorella vulgaris cells with a range of lipid contents, induced via nitrogen starvation, and characterized them via flow cytometry, with BODIPY 505/515 as a fluorescent lipid label, and liquid-state 1H NMR spectroscopy. In doing so, we demonstrate the utility of calibrating flow cytometric measurements of algal lipid content using triacylglyceride (TAG, also known as triacylglycerol or triglyceride) content per cell as measured via quantitative 1H NMR. Ensemble-averaged fluorescence of BODIPY-labeled cells was highly correlated with average TAG content per cell measured by bulk NMR, with a linear regression yielding a linear fit with r2 = 0.9974. This correlation compares favorably to previous calibrations of flow cytometry protocols to lipid content measured via extraction, and calibration by NMR avoids the time and complexity that is generally required for lipid quantitation via extraction. Flow cytometry calibrated to a direct measurement of TAG content can be used to investigate the distribution of lipid contents for cells within a culture. Our flow cytometry measurements showed that Chlorella vulgaris cells subjected to nitrogen limitation exhibited higher mean lipid content but a wider distribution of lipid content that overlapped the relatively narrow distribution of lipid content for replete cells, suggesting that nitrogen limitation induces lipid accumulation in only a subset of cells. Calibration of flow cytometry protocols using direct in situ measurement of TAG content via NMR will facilitate rapid development of more precise flow cytometry protocols, enabling investigation of algal lipid accumulation for development of more productive algal biofuel feedstocks and cultivation protocols.

  8. Intelligent Flow Control Valve

    Science.gov (United States)

    Kelley, Anthony R (Inventor)

    2015-01-01

    The present invention is an intelligent flow control valve which may be inserted into the flow coming out of a pipe and activated to provide a method to stop, measure, and meter flow coming from the open or possibly broken pipe. The intelligent flow control valve may be used to stop the flow while repairs are made. Once repairs have been made, the valve may be removed or used as a control valve to meter the amount of flow from inside the pipe. With the addition of instrumentation, the valve may also be used as a variable area flow meter and flow controller programmed based upon flowing conditions. With robotic additions, the valve may be configured to crawl into a desired pipe location, anchor itself, and activate flow control or metering remotely.

  9. Flow cytometry-assisted rapid isolation of recombinant Plasmodium berghei parasites exemplified by functional analysis of aquaglyceroporin

    Science.gov (United States)

    Kenthirapalan, Sanketha; Waters, Andrew P.; Matuschewski, Kai; Kooij, Taco W.A.

    2012-01-01

    The most critical bottleneck in the generation of recombinant Plasmodium berghei parasites is the mandatory in vivo cloning step following successful genetic manipulation. This study describes a new technique for rapid selection of recombinant P. berghei parasites. The method is based on flow cytometry to isolate isogenic parasite lines and represents a major advance for the field, in that it will speed the generation of recombinant parasites as well as cut down on animal use significantly. High expression of GFP during blood infection, a prerequisite for robust separation of transgenic lines by flow cytometry, was achieved. Isogenic recombinant parasite populations were isolated even in the presence of a 100-fold excess of wild-type (WT) parasites. Aquaglyceroporin (AQP) loss-of-function mutants and parasites expressing a tagged AQP were generated to validate this approach. aqp− parasites grow normally within the WT phenotypic range during blood infection of NMRI mice. Similarly, colonization of the insect vector and establishment of an infection after mosquito transmission were unaffected, indicating that AQP is dispensable for life cycle progression in vivo under physiological conditions, refuting its use as a suitable drug target. Tagged AQP localized to perinuclear structures and not the parasite plasma membrane. We suggest that flow-cytometric isolation of isogenic parasites overcomes the major roadblock towards a genome-scale repository of mutant and transgenic malaria parasite lines. PMID:23137753

  10. Introduction to compressible fluid flow

    CERN Document Server

    Oosthuizen, Patrick H

    2013-01-01

    IntroductionThe Equations of Steady One-Dimensional Compressible FlowSome Fundamental Aspects of Compressible FlowOne-Dimensional Isentropic FlowNormal Shock WavesOblique Shock WavesExpansion Waves - Prandtl-Meyer FlowVariable Area FlowsAdiabatic Flow with FrictionFlow with Heat TransferLinearized Analysis of Two-Dimensional Compressible FlowsHypersonic and High-Temperature FlowsHigh-Temperature Gas EffectsLow-Density FlowsBibliographyAppendices

  11. Practical flow cytometry

    National Research Council Canada - National Science Library

    Shapiro, Howard M

    2003-01-01

    ... ... Conflict: Resolution ... 1.3 Problem Number One: Finding The Cell(s) ... Flow Cytometry: Quick on the Trigger ... The Main Event ... The Pulse Quickens, the Plot Thickens ... 1.4 Flow Cytometry: ...

  12. Review of zonal flows

    International Nuclear Information System (INIS)

    Diamond, P.H.; Itoh, S.-I.; Itoh, K.; Hahm, T.S.

    2004-10-01

    A comprehensive review of zonal flow phenomena in plasmas is presented. While the emphasis is on zonal flows in laboratory plasmas, zonal flows in nature are discussed as well. The review presents the status of theory, numerical simulation and experiments relevant to zonal flows. The emphasis is on developing an integrated understanding of the dynamics of drift wave - zonal flow turbulence by combining detailed studies of the generation of zonal flows by drift waves, the back-interaction of zonal flows on the drift waves, and the various feedback loops by which the system regulates and organizes itself. The implications of zonal flow phenomena for confinement in, and the phenomena of fusion devices are discussed. Special attention is given to the comparison of experiment with theory and to identifying direction for progress in future research. (author)

  13. Load flow optimization and optimal power flow

    CERN Document Server

    Das, J C

    2017-01-01

    This book discusses the major aspects of load flow, optimization, optimal load flow, and culminates in modern heuristic optimization techniques and evolutionary programming. In the deregulated environment, the economic provision of electrical power to consumers requires knowledge of maintaining a certain power quality and load flow. Many case studies and practical examples are included to emphasize real-world applications. The problems at the end of each chapter can be solved by hand calculations without having to use computer software. The appendices are devoted to calculations of line and cable constants, and solutions to the problems are included throughout the book.

  14. Fluid flow control system

    International Nuclear Information System (INIS)

    Rion, Jacky.

    1982-01-01

    Fluid flow control system featuring a series of grids placed perpendicular to the fluid flow direction, characterized by the fact that it is formed of a stack of identical and continuous grids, each of which consists of identical meshes forming a flat lattice. The said meshes are offset from one grid to the next. This system applies in particular to flow control of the coolant flowing at the foot of an assembly of a liquid metal cooled nuclear reactor [fr

  15. Hyperspectral imaging flow cytometer

    Science.gov (United States)

    Sinclair, Michael B.; Jones, Howland D. T.

    2017-10-25

    A hyperspectral imaging flow cytometer can acquire high-resolution hyperspectral images of particles, such as biological cells, flowing through a microfluidic system. The hyperspectral imaging flow cytometer can provide detailed spatial maps of multiple emitting species, cell morphology information, and state of health. An optimized system can image about 20 cells per second. The hyperspectral imaging flow cytometer enables many thousands of cells to be characterized in a single session.

  16. Flowing holographic anyonic superfluid

    Science.gov (United States)

    Jokela, Niko; Lifschytz, Gilad; Lippert, Matthew

    2014-10-01

    We investigate the flow of a strongly coupled anyonic superfluid based on the holographic D3-D7' probe brane model. By analyzing the spectrum of fluctuations, we find the critical superfluid velocity, as a function of the temperature, at which the flow stops being dissipationless when flowing past a barrier. We find that at a larger velocity the flow becomes unstable even in the absence of a barrier.

  17. Thermal flow micro sensors

    NARCIS (Netherlands)

    Elwenspoek, Michael Curt

    1999-01-01

    A review is given on sensors fabricated by silicon micromachining technology using the thermal domain for the measurement of fluid flow. Attention is paid especially to performance and geometry of the sensors. Three basic types of thermal flow sensors are discussed: anemometers, calorimetric flow

  18. OpenFlow cookbook

    CERN Document Server

    Smiler S, Kingston

    2015-01-01

    This book is intended for network protocol developers, SDN controller application developers, and academics who would like to understand and develop their own OpenFlow switch or OpenFlow controller in any programming language. With basic understanding of OpenFlow and its components, you will be able to follow the recipes in this book.

  19. STOCHASTIC FLOWS OF MAPPINGS

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    In this paper, the stochastic flow of mappings generated by a Feller convolution semigroup on a compact metric space is studied. This kind of flow is the generalization of superprocesses of stochastic flows and stochastic diffeomorphism induced by the strong solutions of stochastic differential equations.

  20. Distributed Power Flow Controller

    NARCIS (Netherlands)

    Yuan, Z.

    2010-01-01

    In modern power systems, there is a great demand to control the power flow actively. Power flow controlling devices (PFCDs) are required for such purpose, because the power flow over the lines is the nature result of the impedance of each line. Due to the control capabilities of different types of

  1. Flow lab.: flow visualization and simulation

    International Nuclear Information System (INIS)

    Park, Chung Kyun; Cho, Won Jin; Hahn, Pil Soo

    2005-01-01

    The experimental setups for flow visualization and processes identification in laboratory scale (so called Flow Lab.) has developed to get ideas and answer fundamental questions of flow and migration in geologic media. The setup was made of a granite block of 50x50cm scale and a transparent acrylate plate. The tracers used in this experiments were tritiated water, anions, and sorbing cations as well as an organic dye, eosine, to visualize migration paths. The migration plumes were taken with a digital camera as a function of time and stored as digital images. A migration model was also developed to describe and identify the transport processes. Computer simulation was carried out not only for the hydraulic behavior such as distributions of pressure and flow vectors in the fracture but also for the migration plume and the elution curves

  2. Is there elliptic flow without transverse flow?

    International Nuclear Information System (INIS)

    Huovinen, Pasi; Kolb, Peter F.; Heinz, Ulrich

    2001-01-01

    Azimuthal anisotropy of final particle distributions was originally introduced as a signature of transverse collective flow. We show that finite anisotropy in momentum space can result solely from the shape of the particle emitting source. However, by comparing the differential anisotropy to recent data from STAR collaboration we can exclude such a scenario, but instead show that the data favour strong flow as resulting from a hydrodynamical evolution

  3. Signal flow analysis

    CERN Document Server

    Abrahams, J R; Hiller, N

    1965-01-01

    Signal Flow Analysis provides information pertinent to the fundamental aspects of signal flow analysis. This book discusses the basic theory of signal flow graphs and shows their relation to the usual algebraic equations.Organized into seven chapters, this book begins with an overview of properties of a flow graph. This text then demonstrates how flow graphs can be applied to a wide range of electrical circuits that do not involve amplification. Other chapters deal with the parameters as well as circuit applications of transistors. This book discusses as well the variety of circuits using ther

  4. Separation of flow

    CERN Document Server

    Chang, Paul K

    2014-01-01

    Interdisciplinary and Advanced Topics in Science and Engineering, Volume 3: Separation of Flow presents the problem of the separation of fluid flow. This book provides information covering the fields of basic physical processes, analyses, and experiments concerning flow separation.Organized into 12 chapters, this volume begins with an overview of the flow separation on the body surface as discusses in various classical examples. This text then examines the analytical and experimental results of the laminar boundary layer of steady, two-dimensional flows in the subsonic speed range. Other chapt

  5. Physics of zonal flows

    International Nuclear Information System (INIS)

    Itoh, K.; Fujisawa, A.; Itoh, S.-I.; Yagi, M.; Nagashima, Y.; Diamond, P.H.; Tynan, G.R.; Hahm, T.S.

    2006-01-01

    Zonal flows, which means azimuthally symmetric band-like shear flows, are ubiquitous phenomena in nature and the laboratory. It is now widely recognized that zonal flows are a key constituent in virtually all cases and regimes of drift wave turbulence, indeed, so much so that this classic problem is now frequently referred to as ''drift wave-zonal flow turbulence.'' In this review, new viewpoints and unifying concepts are presented, which facilitate understanding of zonal flow physics, via theory, computation and their confrontation with the results of laboratory experiment. Special emphasis is placed on identifying avenues for further progress. (author)

  6. Physics of zonal flows

    International Nuclear Information System (INIS)

    Itoh, K.; Itoh, S.-I.; Diamond, P.H.; Hahm, T.S.; Fujisawa, A.; Tynan, G.R.; Yagi, M.; Nagashima, Y.

    2006-01-01

    Zonal flows, which means azimuthally symmetric band-like shear flows, are ubiquitous phenomena in nature and the laboratory. It is now widely recognized that zonal flows are a key constituent in virtually all cases and regimes of drift wave turbulence, indeed, so much so that this classic problem is now frequently referred to as 'drift wave-zonal flow turbulence'. In this review, new viewpoints and unifying concepts are presented, which facilitate understanding of zonal flow physics, via theory, computation and their confrontation with the results of laboratory experiment. Special emphasis is placed on identifying avenues for further progress

  7. CANDU channel flow verification

    International Nuclear Information System (INIS)

    Mazalu, N.; Negut, Gh.

    1997-01-01

    The purpose of this evaluation was to obtain accurate information on each channel flow that enables us to assess precisely the level of reactor thermal power and, for reasons of safety, to establish which channel is boiling. In order to assess the channel flow parameters, computer simulations were done with the NUCIRC code and the results were checked by measurements. The complete channel flow measurements were made in the zero power cold condition. In hot conditions there were made flow measurements using the Shut Down System 1 (SDS 1) flow devices from 0.1 % F.P. up to 100 % F.P. The NUCIRC prediction for CANDU channel flows and the measurements by Ultrasonic Flow Meter at zero power cold conditions and SDS 1 flow channel measurements at different reactor power levels showed an acceptable agreement. The 100 % F.P. average errors for channel flow of R, shows that suitable NUCIRC flow assessment can be made. So, it can be done a fair prediction of the reactor power distribution. NUCIRC can predict accurately the onset of boiling and helps to warn at the possible power instabilities at high powers or it can detect the flow blockages. The thermal hydraulic analyst has in NUCIRC a suitable tool to do accurate predictions for the thermal hydraulic parameters for different steady state power levels which subsequently leads to an optimal CANDU reactor operation. (authors)

  8. Genome-size Variation in Switchgrass (Panicum virgatum: Flow Cytometry and Cytology Reveal Rampant Aneuploidy

    Directory of Open Access Journals (Sweden)

    Denise E. Costich

    2010-11-01

    Full Text Available Switchgrass ( L., a native perennial dominant of the prairies of North America, has been targeted as a model herbaceous species for biofeedstock development. A flow-cytometric survey of a core set of 11 primarily upland polyploid switchgrass accessions indicated that there was considerable variation in genome size within each accession, particularly at the octoploid (2 = 8 = 72 chromosome ploidy level. Highly variable chromosome counts in mitotic cell preparations indicated that aneuploidy was more common in octoploids (86.3% than tetraploids (23.2%. Furthermore, the incidence of hyper- versus hypoaneuploidy is equivalent in tetraploids. This is clearly not the case in octoploids, where close to 90% of the aneuploid counts are lower than the euploid number. Cytogenetic investigation using fluorescent in situ hybridization (FISH revealed an unexpected degree of variation in chromosome structure underlying the apparent genomic instability of this species. These results indicate that rapid advances in the breeding of polyploid biofuel feedstocks, based on the molecular-genetic dissection of biomass characteristics and yield, will be predicated on the continual improvement of our understanding of the cytogenetics of these species.

  9. Minimal disease detection of B-cell lymphoproliferative disorders by flow cytometry: multidimensional cluster analysis.

    Science.gov (United States)

    Duque, Ricardo E

    2012-04-01

    Flow cytometric analysis of cell suspensions involves the sequential 'registration' of intrinsic and extrinsic parameters of thousands of cells in list mode files. Thus, it is almost irresistible to describe phenomena in numerical terms or by 'ratios' that have the appearance of 'accuracy' due to the presence of numbers obtained from thousands of cells. The concepts involved in the detection and characterization of B cell lymphoproliferative processes are revisited in this paper by identifying parameters that, when analyzed appropriately, are both necessary and sufficient. The neoplastic process (cluster) can be visualized easily because the parameters that distinguish it form a cluster in multidimensional space that is unique and distinguishable from neighboring clusters that are not of diagnostic interest but serve to provide a background. For B cell neoplasia it is operationally necessary to identify the multidimensional space occupied by a cluster whose kappa:lambda ratio is 100:0 or 0:100. Thus, the concept of kappa:lambda ratio is without meaning and would not detect B cell neoplasia in an unacceptably high number of cases.

  10. The mean Evershed flow

    Science.gov (United States)

    Hu, W.-R.

    1984-09-01

    The paper gives a theoretical analysis of the overall characteristics of the Evershed flow (one of the main features of sunspots), with particular attention given to its outward flow from the umbra in the photosphere, reaching a maximum somewhere in the penumbra, and decreasing rapidly further out, and its inward flow of a comparable magnitude in chromosphere. Because the inertial force of the flow is small, the relevant dynamic process can be divided into a base state and a perturbation. The base-state solution yields the equilibrium relations between the pressure gradient, the Lorentz force, and gravity, and the flow law. The perturbation describes the force driving the Evershed flow. Since the pressure gradient in the base state is already in equilibrium with the Lorentz force and the gravity, the driving force of the mean Evershed flow is small.

  11. Kidney transplantation after desensitization in sensitized patients: a Korean National Audit.

    Science.gov (United States)

    Huh, Kyu Ha; Kim, Beom Seok; Yang, Jaeseok; Ahn, Jeongmyung; Kim, Myung-Gyu; Park, Jae Berm; Kim, Jong Man; Chung, Byung-Ha; Kim, Joong Kyung; Kong, Jin Min

    2012-10-01

    The number of end-stage renal disease (ESRD) patients with preformed antibodies waiting for a kidney transplant has been increasing lately. We conducted a nationwide study on the outcomes of kidney transplantation after desensitization in Korea. Six transplant centers have run desensitization programs. The patients who underwent living donor kidney transplantation after desensitization from 2002 to 2010 were retrospectively analyzed. A total of 86 cases were enrolled. Thirty-five of these were cases of re-transplantation (40.7 %). Indications of desensitization were positive complement-dependent cytotoxicity (CDC) cross-match responses (CDC(+), 36.0 %), positive flow-cytometric cross-match responses (FCX(+), 54.7 %), and positive donor-specific antibodies (DSA(+), 8.1 %). The desensitization protocols used pre-transplant plasmapheresis (95.3 %), intravenous immunoglobulin (62.8 %), and rituximab (67.4 %). Acute rejection occurred in 18 patients (20.9 %), graft failure occurred in 4 patients, and the 3-year graft survival rate was 93.8 %. The presence of DSA increased the acute rejection rate (P = 0.015) and decreased the 1-year post-transplant estimated glomerular filtration rate (P = 0.006). Although rejection-free survival rates did not differ significantly between the CDC(+) and FCX(+) groups, the 1-year estimated glomerular filtration rate was lower in the CDC(+) group (P = 0.010). Infectious and significant bleeding complications occurred in 15.5 % and 4.7 % of cases, respectively. Kidney transplantation after desensitization had good graft outcomes and tolerable complications in Korea, and therefore, this therapy can be recommended for sensitized ESRD patients.

  12. Contribution of flow cytometry to the diagnosis of gastric lymphomas in endoscopic biopsy specimens.

    Science.gov (United States)

    Almasri, N M; Zaer, F S; Iturraspe, J A; Braylan, R C

    1997-07-01

    Gastric lymphomas seem to have unique clinical, pathologic, and immunophenotypic features that set them apart from nodal lymphomas. Microscopic examination of endoscopic biopsy specimens is the most frequent procedure used to diagnose gastric tumors, but it is very difficult, and sometimes impossible, to recognize lymphomas in endoscopic samples by histologic or even immunohistologic methods. Because most gastric lymphomas are of B-cell origin, we used flow cytometry to assess B-cell clonality in gastric biopsy specimens containing dense lymphocytic infiltrates thought to represent lymphoma. We prepared viable cell suspensions from unfixed specimens obtained from 29 consecutive patients who had a previous microscopic diagnosis of suspicious gastric lymphoid infiltrates. We performed immunophenotypic studies with multicolor flow cytometry, and we assessed clonality by examination of immunoglobulin (Ig) light-chain expression analyzed exclusively on B cells identified by anti-CD20 or CD19 antibodies. The mean number of cells recovered was 1.04 x 10(6), from an average of 5.5 gastric biopsy fragments per patient. In 26 of the 29 patients, the number of cells was adequate for analysis. We detected B-cell monoclonality in 16 cases, including 5 in which the percentage of clonal B cells was less than 5%. Of the 16 cases, only 8 could be diagnosed as lymphomas on morphologic grounds alone; the remaining 8 patients had either suspicious lymphoid infiltrates or chronic gastritis. The three cases with an insufficient number of cells were considered non-neoplastic either on histologic grounds alone or in conjunction with Southern analysis of Ig genes. We conclude that flow cytometric immunophenotypic analysis of freshly prepared cell suspensions obtained from endoscopic biopsy specimens can be used to evaluate gastric lymphocytic infiltrates. Specifically, the analysis of surface Ig light-chain expression on B cells distinguishes between monoclonal (lymphoma) and polyclonal

  13. FLOCK cluster analysis of mast cell event clustering by high-sensitivity flow cytometry predicts systemic mastocytosis.

    Science.gov (United States)

    Dorfman, David M; LaPlante, Charlotte D; Pozdnyakova, Olga; Li, Betty

    2015-11-01

    In our high-sensitivity flow cytometric approach for systemic mastocytosis (SM), we identified mast cell event clustering as a new diagnostic criterion for the disease. To objectively characterize mast cell gated event distributions, we performed cluster analysis using FLOCK, a computational approach to identify cell subsets in multidimensional flow cytometry data in an unbiased, automated fashion. FLOCK identified discrete mast cell populations in most cases of SM (56/75 [75%]) but only a minority of non-SM cases (17/124 [14%]). FLOCK-identified mast cell populations accounted for 2.46% of total cells on average in SM cases and 0.09% of total cells on average in non-SM cases (P < .0001) and were predictive of SM, with a sensitivity of 75%, a specificity of 86%, a positive predictive value of 76%, and a negative predictive value of 85%. FLOCK analysis provides useful diagnostic information for evaluating patients with suspected SM, and may be useful for the analysis of other hematopoietic neoplasms. Copyright© by the American Society for Clinical Pathology.

  14. Make peak flow a habit

    Science.gov (United States)

    Asthma - make peak flow a habit; Reactive airway disease - peak flow; Bronchial asthma - peak flow ... 2014:chap 55. National Asthma Education and Prevention Program website. How to use a peak flow meter. ...

  15. Concentric Split Flow Filter

    Science.gov (United States)

    Stapleton, Thomas J. (Inventor)

    2015-01-01

    A concentric split flow filter may be configured to remove odor and/or bacteria from pumped air used to collect urine and fecal waste products. For instance, filter may be designed to effectively fill the volume that was previously considered wasted surrounding the transport tube of a waste management system. The concentric split flow filter may be configured to split the air flow, with substantially half of the air flow to be treated traveling through a first bed of filter media and substantially the other half of the air flow to be treated traveling through the second bed of filter media. This split flow design reduces the air velocity by 50%. In this way, the pressure drop of filter may be reduced by as much as a factor of 4 as compare to the conventional design.

  16. Polyoxometalate flow battery

    Science.gov (United States)

    Anderson, Travis M.; Pratt, Harry D.

    2016-03-15

    Flow batteries including an electrolyte of a polyoxometalate material are disclosed herein. In a general embodiment, the flow battery includes an electrochemical cell including an anode portion, a cathode portion and a separator disposed between the anode portion and the cathode portion. Each of the anode portion and the cathode portion comprises a polyoxometalate material. The flow battery further includes an anode electrode disposed in the anode portion and a cathode electrode disposed in the cathode portion.

  17. Defining Quantum Control Flow

    OpenAIRE

    Ying, Mingsheng; Yu, Nengkun; Feng, Yuan

    2012-01-01

    A remarkable difference between quantum and classical programs is that the control flow of the former can be either classical or quantum. One of the key issues in the theory of quantum programming languages is defining and understanding quantum control flow. A functional language with quantum control flow was defined by Altenkirch and Grattage [\\textit{Proc. LICS'05}, pp. 249-258]. This paper extends their work, and we introduce a general quantum control structure by defining three new quantu...

  18. Microparticle Flow Sensor

    Science.gov (United States)

    Morrison, Dennis R.

    2005-01-01

    The microparticle flow sensor (MFS) is a system for identifying and counting microscopic particles entrained in a flowing liquid. The MFS includes a transparent, optoelectronically instrumented laminar-flow chamber (see figure) and a computer for processing instrument-readout data. The MFS could be used to count microparticles (including micro-organisms) in diverse applications -- for example, production of microcapsules, treatment of wastewater, pumping of industrial chemicals, and identification of ownership of liquid products.

  19. Flow patterns in vertical two-phase flow

    International Nuclear Information System (INIS)

    McQuillan, K.W.; Whalley, P.B.

    1985-01-01

    This paper is concerned with the flow patterns which occur in upwards gas-liquid two-phase flow in vertical tubes. The basic flow patterns are described and the use of flow patter maps is discussed. The transition between plug flow and churn flow is modelled under the assumption that flooding of the falling liquid film limits the stability of plug flow. The resulting equation is combined with other flow pattern transition equations to produce theoretical flow pattern maps, which are then tested against experimental flow pattern data. Encouraging agreement is obtained

  20. Forecasting freight flows

    DEFF Research Database (Denmark)

    Lyk-Jensen, Stéphanie

    2011-01-01

    Trade patterns and transport markets are changing as a result of the growth and globalization of international trade, and forecasting future freight flow has to rely on trade forecasts. Forecasting freight flows is critical for matching infrastructure supply to demand and for assessing investment...... constitute a valuable input to freight models for forecasting future capacity problems.......Trade patterns and transport markets are changing as a result of the growth and globalization of international trade, and forecasting future freight flow has to rely on trade forecasts. Forecasting freight flows is critical for matching infrastructure supply to demand and for assessing investment...

  1. A neural flow estimator

    DEFF Research Database (Denmark)

    Jørgensen, Ivan Harald Holger; Bogason, Gudmundur; Bruun, Erik

    1995-01-01

    This paper proposes a new way to estimate the flow in a micromechanical flow channel. A neural network is used to estimate the delay of random temperature fluctuations induced in a fluid. The design and implementation of a hardware efficient neural flow estimator is described. The system...... is implemented using switched-current technique and is capable of estimating flow in the μl/s range. The neural estimator is built around a multiplierless neural network, containing 96 synaptic weights which are updated using the LMS1-algorithm. An experimental chip has been designed that operates at 5 V...

  2. Flow chemistry is starting to flow

    NARCIS (Netherlands)

    Duisterwinkel, A.E.

    2012-01-01

    One good thing about this symposium on flow chemistry is that at least half of the papers was on actual applications: summarized one member of the audience of the IPIT symposium in Rotterdam, 25 May 2012. This remark can be viewed as a compliment to the organizer, TNO, a Dutch contract research

  3. Flow Around Steep Topography

    Science.gov (United States)

    2015-09-30

    Flow around steep topography T. M. Shaun Johnston Scripps Institution of Oceanography University of California, San Diego 9500 Gilman Drive, M...tall, steep, submarine topography and islands. During the Flow Encountering Abrupt Topography (FLEAT) DRI, investigators will determine: • Whether...estimates from making accurate statistical/deterministic predictions at ᝺ km resolution around submarine topography and islands? How can we

  4. Biomimetic Flow Sensors

    NARCIS (Netherlands)

    Casas, J.; Liu, Chang; Krijnen, Gijsbertus J.M.

    2012-01-01

    Biomimetic flow sensors are biologically inspired devices that measure the speed and direction of fluids. This survey starts by describing the role and functioning of airflow-sensing hairs in arthropods and in fishes, carries on with the biomimetic MEMS implementations, both for air and water flow

  5. Pressure Driven Poiseuille Flow

    DEFF Research Database (Denmark)

    Stotz, Ingo Leonardo; Iaffaldano, Giampiero; Davies, D. Rhodri

    2018-01-01

    The Pacific plate is thought to be driven mainly by slab pull, associated with subduction along the Aleutians–Japan, Marianas–Izu–Bonin and Tonga–Kermadec trenches. This implies that viscous flow within the sub–Pacific asthenosphere is mainly generated by overlying plate motion (i.e. Couette flow...

  6. Flow cytometry protocols

    National Research Council Canada - National Science Library

    Jaroszeski, Mark J; Heller, Richard

    1998-01-01

    ... are individually analyzed, and it is typical for flow cytometers to quantitatively process thousands of individual particles in a matter of seconds. This a powerful analytic feat particularly if one relates it to the time required to examine several thousand individual cells using a microscope. This leaves little doubt regarding why the field of flow cytometry has...

  7. Airport Network Flow Simulator

    Science.gov (United States)

    1978-10-01

    The Airport Network Flow Simulator is a FORTRAN IV simulation of the flow of air traffic in the nation's 600 commercial airports. It calculates for any group of selected airports: (a) the landing and take-off (Type A) delays; and (b) the gate departu...

  8. Flow visualization using bubbles

    International Nuclear Information System (INIS)

    Henry, J.P.

    1974-01-01

    Soap bubbles were used for visualizing flows. The tests effected allowed some characteristics of flows around models in blow tunnels to be precised at mean velocities V 0 5 . The velocity of a bubble is measured by chronophotography, the bulk envelope of the trajectories is also registered [fr

  9. AUTO-EXPANSIVE FLOW

    Science.gov (United States)

    Physics suggests that the interplay of momentum, continuity, and geometry in outward radial flow must produce density and concomitant pressure reductions. In other words, this flow is intrinsically auto-expansive. It has been proposed that this process is the key to understanding...

  10. Elementary chaotic snap flows

    International Nuclear Information System (INIS)

    Munmuangsaen, Buncha; Srisuchinwong, Banlue

    2011-01-01

    Highlights: → Five new elementary chaotic snap flows and a generalization of an existing chaotic snap flow have been presented. → Three of all are conservative systems whilst three others are dissipative systems. → Four cases need only a single control parameter and a single nonlinearity. → A cubic case in a jerk representation requires only two terms and a single nonlinearity. - Abstract: Hyperjerk systems with 4th-order derivative of the form x .... =f(x ... ,x .. ,x . ,x) have been referred to as snap systems. Five new elementary chaotic snap flows and a generalization of an existing flow are presented through an extensive numerical search. Four of these flows demonstrate elegant simplicity of a single control parameter based on a single nonlinearity of a quadratic, a piecewise-linear or an exponential type. Two others demonstrate elegant simplicity of all unity-in-magnitude parameters based on either a single cubic nonlinearity or three cubic nonlinearities. The chaotic snap flow with a single cubic nonlinearity requires only two terms and can be transformed to its equivalent dynamical form of only five terms which have a single nonlinearity. An advantage is that such a chaotic flow offers only five terms even though the (four) dimension is high. Three of the chaotic snap flows are characterized as conservative systems whilst three others are dissipative systems. Basic dynamical properties are described.

  11. Elbow mass flow meter

    Science.gov (United States)

    McFarland, A.R.; Rodgers, J.C.; Ortiz, C.A.; Nelson, D.C.

    1994-08-16

    The present invention includes a combination of an elbow pressure drop generator and a shunt-type mass flow sensor for providing an output which gives the mass flow rate of a gas that is nearly independent of the density of the gas. For air, the output is also approximately independent of humidity. 3 figs.

  12. Optimised Renormalisation Group Flows

    CERN Document Server

    Litim, Daniel F

    2001-01-01

    Exact renormalisation group (ERG) flows interpolate between a microscopic or classical theory and the corresponding macroscopic or quantum effective theory. For most problems of physical interest, the efficiency of the ERG is constrained due to unavoidable approximations. Approximate solutions of ERG flows depend spuriously on the regularisation scheme which is determined by a regulator function. This is similar to the spurious dependence on the ultraviolet regularisation known from perturbative QCD. Providing a good control over approximated ERG flows is at the root for reliable physical predictions. We explain why the convergence of approximate solutions towards the physical theory is optimised by appropriate choices of the regulator. We study specific optimised regulators for bosonic and fermionic fields and compare the optimised ERG flows with generic ones. This is done up to second order in the derivative expansion at both vanishing and non-vanishing temperature. An optimised flow for a ``proper-time ren...

  13. Pengalaman Flow dalam Belajar

    Directory of Open Access Journals (Sweden)

    Lucky Purwantini

    2017-08-01

    Full Text Available Flow is a condition when individual merges within his/her activity. When a person in flow state, he/she can develop his/her abilities and more success in learning. The purpose of the study is to understand flow experience in learning among undergraduate student. The study used case study qualitative approach. Informant of this research was an undergraduate student which had flow experience. Data was collected by an interview. According to the result, the subject did not experience flow in the learning process, as likes he was in meditation. It happened because when he learned something, he felt be pressed by tasks. It’s important for individual to relax when they are learning.

  14. Multiparametric flow cytometry in the diagnosis and characterization of low-grade pulmonary mucosa-associated lymphoid tissue lymphomas.

    Science.gov (United States)

    Zaer, F S; Braylan, R C; Zander, D S; Iturraspe, J A; Almasri, N M

    1998-06-01

    Primary mucosa associated lymphoid tissue (MALT) lymphomas are rare neoplasms that seem to have a better prognosis than nodal lymphomas. Morphologic diagnosis of these lesions may be difficult because of features that overlap with those of benign lymphoid infiltrates. In this study, we assessed the contribution of multi-parametric flow cytometry in demonstrating clonality and further characterizing pulmonary MALT lymphomas. Based on a clinical or pathologic suspicion of MALT-lymphoma, 3 transbronchial biopsies, 4 fine needle aspirates, 1 core needle biopsy, and 13 wedge excisions of lung were submitted fresh (unfixed) to our laboratory for evaluation. Among the 13 cases diagnosed as MALT lymphomas, B-cell monoclonality was established by identifying expression of a single immunoglobulin light chain on CD20 or CD19-positive cells in 12 cases. One case lacked expression of both light chains on B-cells. Of 11 lymphoma cases in which CD5 and CD10 surface antigens were assessed, no cases expressed CD10, and 1 case demonstrated weak CD5 expression. Nine of 10 cases studied were diploid and 1 case was hyperdiploid. All of the lymphomas displayed low (< or = 3%) S-phase fractions consistent with low grade processes. In 10 patients with short follow-up, none died of their disease and the majority had no evidence of lymphoma dissemination. In seven of the remaining eight cases, B-cells were polyclonal consistent with reactive processes. In one morphologically reactive case, flow cytometric analysis was unsuccessful because of poor cell viability. The pulmonary MALT lymphomas in this study represent a group of B-cell tumors with distinctive morphologic, immunophenotypic, and cell kinetic characteristics. Multi-parametric flow cytometry is useful for confirming B-cell monoclonality and illustrating an antigenic profile compatible with this diagnosis. Flow cytometry can be particularly helpful when working with small biopsies and cytologic samples with limited diagnostic

  15. Flow in bedrock canyons.

    Science.gov (United States)

    Venditti, Jeremy G; Rennie, Colin D; Bomhof, James; Bradley, Ryan W; Little, Malcolm; Church, Michael

    2014-09-25

    Bedrock erosion in rivers sets the pace of landscape evolution, influences the evolution of orogens and determines the size, shape and relief of mountains. A variety of models link fluid flow and sediment transport processes to bedrock incision in canyons. The model components that represent sediment transport processes are increasingly well developed. In contrast, the model components being used to represent fluid flow are largely untested because there are no observations of the flow structure in bedrock canyons. Here we present a 524-kilometre, continuous centreline, acoustic Doppler current profiler survey of the Fraser Canyon in western Canada, which includes 42 individual bedrock canyons. Our observations of three-dimensional flow structure reveal that, as water enters the canyons, a high-velocity core follows the bed surface, causing a velocity inversion (high velocities near the bed and low velocities at the surface). The plunging water then upwells along the canyon walls, resulting in counter-rotating, along-stream coherent flow structures that diverge near the bed. The resulting flow structure promotes deep scour in the bedrock channel floor and undercutting of the canyon walls. This provides a mechanism for channel widening and ensures that the base of the walls is swept clear of the debris that is often deposited there, keeping the walls nearly vertical. These observations reveal that the flow structure in bedrock canyons is more complex than assumed in the models presently used. Fluid flow models that capture the essence of the three-dimensional flow field, using simple phenomenological rules that are computationally tractable, are required to capture the dynamic coupling between flow, bedrock erosion and solid-Earth dynamics.

  16. Allogeneic Mesenchymal Stem Cell Treatment Induces Specific Alloantibodies in Horses

    Directory of Open Access Journals (Sweden)

    Sean D. Owens

    2016-01-01

    Full Text Available Background. It is unknown whether horses that receive allogeneic mesenchymal stem cells (MSCs injections develop specific humoral immune response. Our goal was to develop and validate a flow cytometric MSC crossmatch procedure and to determine if horses that received allogeneic MSCs in a clinical setting developed measurable antibodies following MSC administration. Methods. Serum was collected from a total of 19 horses enrolled in 3 different research projects. Horses in the 3 studies all received unmatched allogeneic MSCs. Bone marrow (BM or adipose tissue derived MSCs (ad-MSCs were administered via intravenous, intra-arterial, intratendon, or intraocular routes. Anti-MSCs and anti-bovine serum albumin antibodies were detected via flow cytometry and ELISA, respectively. Results. Overall, anti-MSC antibodies were detected in 37% of the horses. The majority of horses (89% were positive for anti-bovine serum albumin (BSA antibodies prior to and after MSC injection. Finally, there was no correlation between the amount of anti-BSA antibody and the development of anti-MSC antibodies. Conclusion. Anti allo-MSC antibody development was common; however, the significance of these antibodies is unknown. There was no correlation between either the presence or absence of antibodies and the percent antibody binding to MSCs and any adverse reaction to a MSC injection.

  17. Gas Flow Detection System

    Science.gov (United States)

    Moss, Thomas; Ihlefeld, Curtis; Slack, Barry

    2010-01-01

    This system provides a portable means to detect gas flow through a thin-walled tube without breaking into the tubing system. The flow detection system was specifically designed to detect flow through two parallel branches of a manifold with only one inlet and outlet, and is a means for verifying a space shuttle program requirement that saves time and reduces the risk of flight hardware damage compared to the current means of requirement verification. The prototype Purge Vent and Drain Window Cavity Conditioning System (PVD WCCS) Flow Detection System consists of a heater and a temperature-sensing thermistor attached to a piece of Velcro to be attached to each branch of a WCCS manifold for the duration of the requirement verification test. The heaters and thermistors are connected to a shielded cable and then to an electronics enclosure, which contains the power supplies, relays, and circuit board to provide power, signal conditioning, and control. The electronics enclosure is then connected to a commercial data acquisition box to provide analog to digital conversion as well as digital control. This data acquisition box is then connected to a commercial laptop running a custom application created using National Instruments LabVIEW. The operation of the PVD WCCS Flow Detection System consists of first attaching a heater/thermistor assembly to each of the two branches of one manifold while there is no flow through the manifold. Next, the software application running on the laptop is used to turn on the heaters and to monitor the manifold branch temperatures. When the system has reached thermal equilibrium, the software application s graphical user interface (GUI) will indicate that the branch temperatures are stable. The operator can then physically open the flow control valve to initiate the test flow of gaseous nitrogen (GN2) through the manifold. Next, the software user interface will be monitored for stable temperature indications when the system is again at

  18. Epigenetic targeting in acute myeloid leukemia: use of flow cytometry in monitoring therapeutic effects.

    Science.gov (United States)

    Ryningen, Anita; Bruserud, Øystein

    2007-12-01

    Flow cytometric techniques have emerged as a powerful tool in hematology allowing fast, sensitive and reproducible multi-parametric analyses at the single cell level of heterogeneous samples. Small subsets of cells can be studied with high degree of accuracy, and a broad and constantly increasing specter of antibodies is available. Flow cytometry has therefore become the method of choice for evaluation of therapeutic effects at single cell level. These methodological approaches can easily be used to study hematological malignancies, and the future use of this strategy in other malignancies will depend on the development of laboratory techniques to prepare suspensions of viable cells also from tumor biopsies. The selection of biological parameters for evaluation of treatment effects should probably be based on (i) molecular markers involved in cancer-associated genetic abnormalities; (ii) other molecular markers showing altered expression in the malignant cells and thought to be involved in leukemogenesis or having a prognostic impact; (ii) functional assays known to reflect biological characteristics that are important in carcinogenesis (e.g. cell cycle distribution, functional evaluation of apoptosis regulation). These molecules will in addition often represent the therapeutic targets when new anticancer drugs are developed. In this review we use treatment of acute myeloid leukemia with histone deacetylase inhibitors as an example. Based on the criteria mentioned above we suggest that the monitoring of therapeutic effects on the cancer cells in these patients should include differentiation status, histone acetylation, cell cycle distribution, pro- and anti-apoptotic signaling balance and intracellular levels of various transcription factors.

  19. Pulsatile pipe flow transition: Flow waveform effects

    Science.gov (United States)

    Brindise, Melissa C.; Vlachos, Pavlos P.

    2018-01-01

    Although transition is known to exist in various hemodynamic environments, the mechanisms that govern this flow regime and their subsequent effects on biological parameters are not well understood. Previous studies have investigated transition in pulsatile pipe flow using non-physiological sinusoidal waveforms at various Womersley numbers but have produced conflicting results, and multiple input waveform shapes have yet to be explored. In this work, we investigate the effect of the input pulsatile waveform shape on the mechanisms that drive the onset and development of transition using particle image velocimetry, three pulsatile waveforms, and six mean Reynolds numbers. The turbulent kinetic energy budget including dissipation rate, production, and pressure diffusion was computed. The results show that the waveform with a longer deceleration phase duration induced the earliest onset of transition, while the waveform with a longer acceleration period delayed the onset of transition. In accord with the findings of prior studies, for all test cases, turbulence was observed to be produced at the wall and either dissipated or redistributed into the core flow by pressure waves, depending on the mean Reynolds number. Turbulent production increased with increasing temporal velocity gradients until an asymptotic limit was reached. The turbulence dissipation rate was shown to be independent of mean Reynolds number, but a relationship between the temporal gradients of the input velocity waveform and the rate of turbulence dissipation was found. In general, these results demonstrated that the shape of the input pulsatile waveform directly affected the onset and development of transition.

  20. Flow Cytometry Pulse Width Data Enables Rapid and Sensitive Estimation of Biomass Dry Weight in the Microalgae Chlamydomonas reinhardtii and Chlorella vulgaris

    Science.gov (United States)

    Chioccioli, Maurizio; Hankamer, Ben; Ross, Ian L.

    2014-01-01

    Dry weight biomass is an important parameter in algaculture. Direct measurement requires weighing milligram quantities of dried biomass, which is problematic for small volume systems containing few cells, such as laboratory studies and high throughput assays in microwell plates. In these cases indirect methods must be used, inducing measurement artefacts which vary in severity with the cell type and conditions employed. Here, we utilise flow cytometry pulse width data for the estimation of cell density and biomass, using Chlorella vulgaris and Chlamydomonas reinhardtii as model algae and compare it to optical density methods. Measurement of cell concentration by flow cytometry was shown to be more sensitive than optical density at 750 nm (OD750) for monitoring culture growth. However, neither cell concentration nor optical density correlates well to biomass when growth conditions vary. Compared to the growth of C. vulgaris in TAP (tris-acetate-phosphate) medium, cells grown in TAP + glucose displayed a slowed cell division rate and a 2-fold increased dry biomass accumulation compared to growth without glucose. This was accompanied by increased cellular volume. Laser scattering characteristics during flow cytometry were used to estimate cell diameters and it was shown that an empirical but nonlinear relationship could be shown between flow cytometric pulse width and dry weight biomass per cell. This relationship could be linearised by the use of hypertonic conditions (1 M NaCl) to dehydrate the cells, as shown by density gradient centrifugation. Flow cytometry for biomass estimation is easy to perform, sensitive and offers more comprehensive information than optical density measurements. In addition, periodic flow cytometry measurements can be used to calibrate OD750 measurements for both convenience and accuracy. This approach is particularly useful for small samples and where cellular characteristics, especially cell size, are expected to vary during growth. PMID

  1. Report of the results of the International Clinical Cytometry Society and American Society for Clinical Pathology workload survey of clinical flow cytometry laboratories.

    Science.gov (United States)

    Wolniak, Kristy; Goolsby, Charles; Choi, Sarah; Ali, Asma; Serdy, Nina; Stetler-Stevenson, Maryalice

    2017-11-01

    Thorough review of current workload, staffing, and testing practices in clinical laboratories allows for optimization of laboratory efficiency and quality. This information is largely missing with regard to clinical flow cytometry laboratories. The purpose of this survey is to provide comprehensive, current, and accurate data on testing practices and laboratory staffing in clinical laboratories performing flow cytometric studies. Survey data was collected from flow cytometry laboratories through the ASCP website. Data was collected on the workload during a 1-year time period of full-time and part-time technical and professional (M.D./D.O./Ph.D. or equivalent) flow cytometry employees. Workload was examined as number of specimens and tubes per full time equivalent (FTE) technical and professional staff. Test complexity, test result interpretation, and reporting practices were also evaluated. There were 205 respondent laboratories affiliated predominantly with academic and health system institutions. Overall, 1,132 FTE employees were reported with 29% professional FTE employees and 71% technical. Fifty-one percent of the testing performed was considered high complexity and 49% was low complexity. The average number of tubes per FTE technologist was 1,194 per year and the average number of specimens per FTE professional was 1,659 per year. The flow cytometry reports were predominantly written by pathologists (57%) and were typically written as a separate report (58%). This survey evaluates the overall status of the current practice of clinical flow cytometry and provides a comprehensive dataset as a framework to help laboratory departments, directors, and managers make appropriate, cost-effective staffing decisions. © 2016 International Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.

  2. Flow in rod bundles

    International Nuclear Information System (INIS)

    Hazi, G.; Mayer, G.

    2005-01-01

    For power upgrading VVER-440 reactors we need to know exactly how the temperature measured by the thermocouples is related to the average outlet temperature of the fuel assemblies. Accordingly, detailed knowledge on mixing process in the rod bundles and in the fuel assembly head have great importance. Here we study the hydrodynamics of rod bundles based on the results of direct numerical and large eddy simulation of flows in subchannels. It is shown that secondary flow and flow pulsation phenomena can be observed using both methodologies. Some consequences of these observations are briefly discussed. (author)

  3. Magnetic vortex filament flows

    International Nuclear Information System (INIS)

    Barros, Manuel; Cabrerizo, Jose L.; Fernandez, Manuel; Romero, Alfonso

    2007-01-01

    We exhibit a variational approach to study the magnetic flow associated with a Killing magnetic field in dimension 3. In this context, the solutions of the Lorentz force equation are viewed as Kirchhoff elastic rods and conversely. This provides an amazing connection between two apparently unrelated physical models and, in particular, it ties the classical elastic theory with the Hall effect. Then, these magnetic flows can be regarded as vortex filament flows within the localized induction approximation. The Hasimoto transformation can be used to see the magnetic trajectories as solutions of the cubic nonlinear Schroedinger equation showing the solitonic nature of those

  4. Initiation of slug flow

    Energy Technology Data Exchange (ETDEWEB)

    Hanratty, T.J.; Woods, B.D. [Univ. of Illinois, Urbana, IL (United States)

    1995-12-31

    The initiation of slug flow in a horizontal pipe can be predicted either by considering the stability of a slug or by considering the stability of a stratified flow. Measurements of the shedding rate of slugs are used to define necessary conditions for the existence of a slug. Recent results show that slugs develop from an unstable stratified flow through the evolution of small wavelength waves into large wavelength waves that have the possibility of growing to form a slug. The mechanism appears to be quite different for fluids with viscosities close to water than for fluids with large viscosities (20 centipoise).

  5. Refrigeration. Two-Phase Flow. Flow Regimes and Pressure Drop

    DEFF Research Database (Denmark)

    Knudsen, Hans-Jørgen Høgaard

    2002-01-01

    The note gives the basic definitions used in two-phase flow. Flow regimes and flow regimes map are introduced. The different contributions to the pressure drop are stated together with an imperical correlation from the litterature.......The note gives the basic definitions used in two-phase flow. Flow regimes and flow regimes map are introduced. The different contributions to the pressure drop are stated together with an imperical correlation from the litterature....

  6. Use of flow cytometry for the adhesion analysis of Streptococcus pyogenes mutant strains to epithelial cells: investigation of the possible role of surface pullulanase and cysteine protease, and the transcriptional regulator Rgg

    Directory of Open Access Journals (Sweden)

    Finne Jukka

    2006-02-01

    Full Text Available Abstract Background Flow cytometry based adherence assay is a potentially powerful but little used method in the study of bacterial binding to host structures. We have previously characterized a glycoprotein-binding activity in Streptococcus pyogenes called 'strepadhesin' binding to thyroglobulin, submaxillar mucin, fetuin and asialofetuin. We have identified surface-associated pullulanase (PulA and cysteine protease (SpeB as carriers of strepadhesin activity. In the present paper, we investigated the use of flow cytometry as a method to study the binding of Rgg, SpeB and PulA knock-out strains to cultured human epithelial cells. Results Streptococcal mutants were readily labelled with CFDA-SE and their binding to epithelial cells could be effectively studied by flow cytometry. A strain deficient in Rgg expression showed increased binding to the analyzed epithelial cell lines of various origin. Inactivation of SpeB had no effect on the adhesion, while PulA knock-out strains displayed decreased binding to the cell lines. Conclusion These results suggest that the flow cytometric assay is a valuable tool in the analysis of S. pyogenes adherence to host cells. It appears to be an efficient and sensitive tool for the characterization of interactions between the bacteria and the host at the molecular level. The results also suggest a role for Rgg regulated surface molecules, like PulA, in the adhesion of S. pyogenes to host cells.

  7. Flow Cytometry Section

    Data.gov (United States)

    Federal Laboratory Consortium — The primary goal of the Flow Cytometry Section is to provide the services of state-of-the-art multi-parameter cellular analysis and cell sorting for researchers and...

  8. The disappearance of flow

    International Nuclear Information System (INIS)

    Soff, S.; Hartnack, C.; Stoecker, H.; Greiner, W.

    1995-01-01

    We investigate the disappearance of collective flow in the reaction plane in heavy-ion collisions within a microscopic model (QMD). A systematic study of the impact parameter dependence is performed for the system Ca+Ca. The balance energy strongly increases with impact parameter. Momentum dependent interactions reduce the balance energies for intermediate impact parameters b∼4.5 fm. Dynamical negative flow is not visible in the laboratory frame but does exist in the contact frame for the heavy system Au+Au. For semi-peripheral collisions of Ca+Ca with b∼6.5 fm a new two-component flow is discussed. Azimuthal distributions exhibit strong collectiv flow signals, even at the balance energy. (orig.)

  9. Border information flow architecture

    Science.gov (United States)

    2006-04-01

    This brochure describes the Border Information Flow Architecture (BIFA). The Transportation Border Working Group, a bi-national group that works to enhance coordination and planning between the United States and Canada, identified collaboration on th...

  10. Upscaling of Forchheimer flows

    KAUST Repository

    Aulisa, Eugenio; Bloshanskaya, Lidia I.; Efendiev, Yalchin R.; Ibragimov, Akif I.

    2014-01-01

    analytical results (Aulisa et al., 2009) [1] and formulate the resulting system in terms of a degenerate nonlinear flow equation for the pressure with the nonlinearity depending on the pressure gradient. The coarse scale parameters for the steady state

  11. Flow in data racks

    Directory of Open Access Journals (Sweden)

    Manoch Lukáš

    2014-03-01

    Full Text Available This paper deals with the flow in data racks. The aim of this work is to find a new arrangement of elements regulating the flow in the data rack so that the aerodynamic losses and the recirculation zones were minimized. The main reason for solving this problem is to reduce the costs of data racks cooling. Another problem to be solved is a reverse flow in the servers, thus not cooled, occuring due to the underpressure in the recirculation zones. In order to solve the problem, the experimental and numerical model of 27U data rack fitted with 10 pieces of server models with a total input of 10 kW was created. Different configurations of layout of elements affecting the flow in the inlet area of the data rack were compared. Depending on the results achieved, design solutions for the improvement of existing solutions were adopted and verified by numerical simulations.

  12. Complex Flow Workshop Report

    Energy Technology Data Exchange (ETDEWEB)

    none,

    2012-05-01

    This report documents findings from a workshop on the impacts of complex wind flows in and out of wind turbine environments, the research needs, and the challenges of meteorological and engineering modeling at regional, wind plant, and wind turbine scales.

  13. Water Flow Experiments

    Indian Academy of Sciences (India)

    year undergraduate student at Ashoka University,. Sonipat, Haryana. This article studies how the height of water varies with time when water ... Experiment using a one-bottle system with a small bore tube at- tached to .... restricting free flow.

  14. Flow energy conversion system

    International Nuclear Information System (INIS)

    Sargsyan, R.A.

    2011-01-01

    A cost-effective hydropower system called here Flow Energy Converter was developed, patented, manufactured and tested for water pumping, electricity generation and other purposes especially useful for the rural communities. The system consists of water-driven turbine with plane-surface blades, power transmission means and pump and/or generator. Working sample of the Flow Energy Converter was designed and manufactured at the Institute of Radio Physics and Electronics

  15. Stability of parallel flows

    CERN Document Server

    Betchov, R

    2012-01-01

    Stability of Parallel Flows provides information pertinent to hydrodynamical stability. This book explores the stability problems that occur in various fields, including electronics, mechanics, oceanography, administration, economics, as well as naval and aeronautical engineering. Organized into two parts encompassing 10 chapters, this book starts with an overview of the general equations of a two-dimensional incompressible flow. This text then explores the stability of a laminar boundary layer and presents the equation of the inviscid approximation. Other chapters present the general equation

  16. Stability of radial swirl flows

    International Nuclear Information System (INIS)

    Dou, H S; Khoo, B C

    2012-01-01

    The energy gradient theory is used to examine the stability of radial swirl flows. It is found that the flow of free vortex is always stable, while the introduction of a radial flow will induce the flow to be unstable. It is also shown that the pure radial flow is stable. Thus, there is a flow angle between the pure circumferential flow and the pure radial flow at which the flow is most unstable. It is demonstrated that the magnitude of this flow angle is related to the Re number based on the radial flow rate, and it is near the pure circumferential flow. The result obtained in this study is useful for the design of vaneless diffusers of centrifugal compressors and pumps as well as other industrial devices.

  17. Hydrodynamical description of collective flow

    OpenAIRE

    Huovinen, Pasi

    2003-01-01

    I review how hydrodynamical flow is related to the observed flow in ultrarelativistic heavy ion collisions and how initial conditions, equation of state and freeze-out temperature affect flow in hydrodynamical models.

  18. Phenolic composition, antioxidant capacity of Salvia verticcilata and effect on multidrug resistant bacteria by flow-cytometry.

    Science.gov (United States)

    Tekeli, Yener; Karpuz, Esra; Danahaliloglu, Hatice; Bucak, Serbay; Guzel, Yelda; Erdmann, Helmuth

    2014-01-01

    Antioxidants are of great importance for preventing oxidative stress that may cause several degenerative diseases. Studies have indicated phytochemicals have high free-radical scavenging activity, which helps to reduce the risk of chronic diseases. The aim of the present study is the determination of antioxidant properties, polyphenolic content and multidrug resistant bacteria of Salvia verticcilata L. Methanol was used as the extraction solvent. The total phenolic content was calculated using Folin-Ciocalteau method and phenolic composition was determined by HPLC. The radical scavenging activity of plant was evaluated in vitro based on the reduction of the stable DPPH free radical. The reducing capacity was identified by using the FRAP method. The ability of Salvia verticcilata L. to increase the permeability of multidrug resistant bacterial cells was conducted by flow cytometric assay on Listeria innocua and E-coli. The amount of total phenolics was found to be 347.5 mg GA/g extract. The IC50 value and FRAP assay are 0.61, and 0.944 respectively, Free radical scavenging effect and FRAP values are less than synthetic antioxidant compounds (BHA and BHT). Eight phenolic compounds were found in Salvia verticcilata L. Intense concentration of S. verticcilata L. has destroyed 97 % of living cells for Listeria innocua and 94.86% for E-coli. This study shows that methanolic extracts of Salvia verticcilata L. is a potential source of natural antioxidants and antimicrobial agent and can form the basis for pharmacological studies.

  19. Fertility and flow cytometry study of frozen-thawed sperm in cryopreservation medium supplemented with soybean lecithin.

    Science.gov (United States)

    Masoudi, R; Sharafi, M; Zareh Shahneh, A; Towhidi, A; Kohram, H; Esmaeili, V; Shahverdi, A; Davachi, N Dadashpour

    2016-08-01

    Semen cryopreservation can provide genetic resources for a large number of females from a small number of superior males. Optimization of cryopreservation media to achieve the highest quality of post-thaw semen is crucial. Soybean lecithin has evaluated as a plant-based cryoprotectant for substitution of egg yolk in ram semen extender. Flow cytometric and fertility assessments were applied following cryopreservation procedure in two experimental groups (SL group: extender containing 1% w/v soybean lecithin and EY group: extender containing 20% v/v egg yolk). The higher percentage of live sperm and the lower percentage of dead sperm were obtained in SL (47.66 ± 1.38, 52.33 ± 1.69, respectively) extender compared to EY (41.16 ± 1.38, 58.83 ± 1.69). For motion characteristics, plasma membrane integrity, acrosome integrity and mitochondria activity, no significant difference was observed between SL and EY extenders. In artificial insemination experiment, there was no significant difference in pregnancy rate, lambing rate and twining rate between SL and EY extenders. It can be concluded that SL extender can be an efficient alternative extender to preserve ram sperm during cryopreservation procedure without adverse effects. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. 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.

  1. Flow Cytometry Detection of Bacterial Cell Entrapment within the Chitosan Hydrogel and Antibacterial Property of Extracted Chitosan

    Directory of Open Access Journals (Sweden)

    Nafise Sadat Majidi

    2016-09-01

    Full Text Available Background:   Chitosan is unbranched polysaccharide composed of D-glucosamine and N-acetyl-D-glucosamine. Chitosan, derived from shrimp shell, has broad antimicrobial properties against Gram-negative, Gram-positive bacteria and fungi. Methods:  Chitosan was extracted from shrimp shell and studied for cell entrapment and anti-bacterial properties. The hydrogel chitosan was used as the beads for cell entrapment and chitosan beads were designed to deliver cells and nutrients. These data confirmed with flow cytometric analyses.                 Results:   Experimental results exhibited that internal diffusion through the chitosan matrix was the main mechanism for whole gelation by TPP (Tri-polyphosphate. The minimum inhibitory concentration (MIC for chitosan against Staphylococcus aureus and Escherichia coli was 16 and 32 μg/ml respectively. Conclusion:  Despite the antimicrobial properties of chitosan, trapped bacteria in the gel network were alive and were chelated indicating that their access to the outside was limited.

  2. Effects of in vitro storage time and semen-extender on membrane quality of boar sperm assessed by flow cytometry.

    Science.gov (United States)

    Waterhouse, K E; De Angelis, P M; Haugan, T; Paulenz, H; Hofmo, P O; Farstad, W

    2004-12-01

    The Norwegian AI company Norsvin has used the short-term semen-extender BTS to extend and store boar semen since the late 1980s. Fertility results have been consistent when extended semen has been used for AI within 3 days after collection, however, from a production and economic point of view it is preferable that semen stored for up to 5 days can be used. The aim of this study was to compare membrane quality of sperm stored in BTS for 3 days with sperm stored in the long-term semen-extenders Androstar, Mulberry III and X-cell for 5 days. Using a split-sample design, plasma membrane- and acrosome-integrity were assessed flow cytometrically by use of Yo-Pro-1 and PNA-FITC, and fluidity and phospholipid asymmetry of the membrane were assessed by use of MC540 and Annexin V-FITC. Due to observed sperm fragmentation in Androstar after Day 1, the data for Androstar were excluded from the analyses. After 5 days of storage, the membrane quality of X-cell-stored sperm was not statistically different from that of sperm stored in BTS for 3 days, while membrane quality of sperm stored in Mulberry III was statistically better on Day 5 compared to BTS on Day 3. In conclusion, Mulberry III and X-cell preserve sperm quality, as well as that of BTS on Day 3, for up to 5 days after collection.

  3. Oscillatory flow chemical reactors

    Directory of Open Access Journals (Sweden)

    Slavnić Danijela S.

    2014-01-01

    Full Text Available Global market competition, increase in energy and other production costs, demands for high quality products and reduction of waste are forcing pharmaceutical, fine chemicals and biochemical industries, to search for radical solutions. One of the most effective ways to improve the overall production (cost reduction and better control of reactions is a transition from batch to continuous processes. However, the reactions of interests for the mentioned industry sectors are often slow, thus continuous tubular reactors would be impractically long for flow regimes which provide sufficient heat and mass transfer and narrow residence time distribution. The oscillatory flow reactors (OFR are newer type of tube reactors which can offer solution by providing continuous operation with approximately plug flow pattern, low shear stress rates and enhanced mass and heat transfer. These benefits are the result of very good mixing in OFR achieved by vortex generation. OFR consists of cylindrical tube containing equally spaced orifice baffles. Fluid oscillations are superimposed on a net (laminar flow. Eddies are generated when oscillating fluid collides with baffles and passes through orifices. Generation and propagation of vortices create uniform mixing in each reactor cavity (between baffles, providing an overall flow pattern which is close to plug flow. Oscillations can be created by direct action of a piston or a diaphragm on fluid (or alternatively on baffles. This article provides an overview of oscillatory flow reactor technology, its operating principles and basic design and scale - up characteristics. Further, the article reviews the key research findings in heat and mass transfer, shear stress, residence time distribution in OFR, presenting their advantages over the conventional reactors. Finally, relevant process intensification examples from pharmaceutical, polymer and biofuels industries are presented.

  4. Tolerance Induction of Temperature and Starvation with Tricalcium Phosphate on Preservation and Sporulation in Bacillus amyloliquefaciens Detected by Flow Cytometry.

    Science.gov (United States)

    Shahrokh Esfahani, Samaneh; Emtiazi, Giti; Shafiei, Rasoul; Ghorbani, Najmeh; Zarkesh Esfahani, Seyed Hamid

    2016-09-01

    The Bacillus species have many applications in the preparation of various enzymes, probiotic, biofertilizer, and biomarkers for which the survival of resting cells and spore formation under different conditions are important. In this study, water and saline along with different mineral substances such as calcium carbonate, calcium phosphate, and silica were used for the detection of survival and preservation of Bacillus amyloliquefaciens. The results showed intensive death of resting cells at 8 °C, but significant survival at 28 °C after one month. However, preservation by minerals significantly decreased the rate of death and induced sporulation at both the temperatures. The resting cells were maintained at room temperature (about 60 % of the initial population survived after a month) in the presence of tricalcium phosphate. The results showed that temperature has more effect on sporulation compare with starvation. The sporulation in normal saline at 28 °C was 70 times more than that at 8 °C; meanwhile, addition of tricalcium phosphate increases sporulation by 90 times. Also, the FTIR data showed the interaction of tricalcium phosphate with spores and resting cells. The discrimination of sporulation from non-sporulation state was performed by nucleic acid staining with thiazole orange and detected by flow cytometry. The flow cytometric studies confirmed that the rates of sporulation in pure water were significantly more at 28 °C. This is the first report on the detection of bacterial spore with thiazole orange by flow cytometry and also on the interaction of tricalcium phosphate with spores by FTIR analyses.

  5. A complementary role of multiparameter flow cytometry and high-throughput sequencing for minimal residual disease detection in chronic lymphocytic leukemia: an European Research Initiative on CLL study.

    LENUS (Irish Health Repository)

    Rawstron, A C

    2016-04-01

    In chronic lymphocytic leukemia (CLL) the level of minimal residual disease (MRD) after therapy is an independent predictor of outcome. Given the increasing number of new agents being explored for CLL therapy, using MRD as a surrogate could greatly reduce the time necessary to assess their efficacy. In this European Research Initiative on CLL (ERIC) project we have identified and validated a flow-cytometric approach to reliably quantitate CLL cells to the level of 0.0010% (10(-5)). The assay comprises a core panel of six markers (i.e. CD19, CD20, CD5, CD43, CD79b and CD81) with a component specification independent of instrument and reagents, which can be locally re-validated using normal peripheral blood. This method is directly comparable to previous ERIC-designed assays and also provides a backbone for investigation of new markers. A parallel analysis of high-throughput sequencing using the ClonoSEQ assay showed good concordance with flow cytometry results at the 0.010% (10(-4)) level, the MRD threshold defined in the 2008 International Workshop on CLL guidelines, but it also provides good linearity to a detection limit of 1 in a million (10(-6)). The combination of both technologies would permit a highly sensitive approach to MRD detection while providing a reproducible and broadly accessible method to quantify residual disease and optimize treatment in CLL.

  6. Monitoring human neutrophil granule secretion by flow cytometry: secretion and membrane potential changes assessed by light scatter and a fluorescent probe of membrane potential

    International Nuclear Information System (INIS)

    Fletcher, M.P.; Seligmann, B.E.

    1985-01-01

    Purified human peripheral blood polymorphonuclear neutrophils (PMN) were incubated at 37 degrees C with the fluorescent membrane potential sensitive cyanine dye di-O-C(5)(3) and exposed to a number of stimulatory agents (N-formylmethionylleucylphenylalanine (FMLP), cytochalasin B (cyto B) + FMLP, phorbol myristate acetate (PMA). Flow cytometry was utilized to measure changes in forward light scatter (FS), orthogonal light scatter (90 degrees-SC), and fluorescence intensity of individual cells over time. A saturating (10(-6) M) dose of FMLP lead to a significant increase in the cells' FS without a change in 90 degrees-SC as well as a heterogeneous loss of di-O-C(5)(3) fluorescence. PMA (100 ng/ml) also caused an increase in FS but a uniform loss of dye fluorescence by all cells (apparent depolarization). Cyto B + FMLP produced an increase in FS, a marked loss of 90 degrees-SC, and a uniform loss of fluorescence. Secretion experiments under identical incubation conditions indicated a significantly positive relationship between loss of enzyme markers or cell granularity and orthogonal light scatter (r . 0.959, 0.998, and 0.989 for loss of 90 degrees-SC vs lysozyme, beta-glucuronidase, and granularity index, respectively). Flow cytometric light scatter measurements may yield important information on the extent of prior cell degranulation or activation

  7. Tomographic multiphase flow measurement

    International Nuclear Information System (INIS)

    Sætre, C.; Johansen, G.A.; Tjugum, S.A.

    2012-01-01

    Measurement of multiphase flow of gas, oil and water is not at all trivial and in spite of considerable achievements over the past two decades, important challenges remain (). These are related to reducing measurement uncertainties arising from variations in the flow regime, improving long term stability and developing new means for calibration, adjustment and verification of the multiphase flow meters. This work focuses on the first two issues using multi gamma beam (MGB) measurements for identification of the type of flow regime. Further gamma ray tomographic measurements are used for reference of the gas/liquid distribution. For the MGB method one Am-241 source with principal emission at 59.5 keV is used because this relatively low energy enables efficient collimation and thereby shaping of the beams, as well as compact detectors. One detector is placed diametrically opposite the source whereas the second is positioned to the side so that this beam is close to the pipe wall. The principle is then straight forward to compare the measured intensities of these detectors and through that identify the flow pattern, i.e. the instantaneous cross-sectional gas-liquid distribution. The measurement setup also includes Compton scattering measurements, which can provide information about the changes in the water salinity for flow segments with high water liquid ratio and low gas fractions. By measuring the transmitted intensity in short time slots (<100ms), rapid regime variations are revealed. From this we can select the time sections suitable for salinity measurements. Since the salinity variations change at the time scale of hours, a running average can be performed to increase the accuracy of the measurements. Recent results of this work will be presented here. - Highlights: ► Multiphase flow gas-fraction and flow regime measurements by multi gamma ray beams. ► High-speed gamma ray tomograph as reference for the flow pattern and gas fraction. ► Dual modality

  8. Tomographic multiphase flow measurement

    Energy Technology Data Exchange (ETDEWEB)

    Saetre, C., E-mail: camilla@ift.uib.no [Department of Physics and Technology, University of Bergen (Norway); Michelsen Centre for Industrial Measurement Science and Technology (Norway); Johansen, G.A. [Department of Physics and Technology, University of Bergen (Norway); Michelsen Centre for Industrial Measurement Science and Technology (Norway); Tjugum, S.A. [Michelsen Centre for Industrial Measurement Science and Technology (Norway); Roxar Flow Measurement, Bergen (Norway)

    2012-07-15

    Measurement of multiphase flow of gas, oil and water is not at all trivial and in spite of considerable achievements over the past two decades, important challenges remain (). These are related to reducing measurement uncertainties arising from variations in the flow regime, improving long term stability and developing new means for calibration, adjustment and verification of the multiphase flow meters. This work focuses on the first two issues using multi gamma beam (MGB) measurements for identification of the type of flow regime. Further gamma ray tomographic measurements are used for reference of the gas/liquid distribution. For the MGB method one Am-241 source with principal emission at 59.5 keV is used because this relatively low energy enables efficient collimation and thereby shaping of the beams, as well as compact detectors. One detector is placed diametrically opposite the source whereas the second is positioned to the side so that this beam is close to the pipe wall. The principle is then straight forward to compare the measured intensities of these detectors and through that identify the flow pattern, i.e. the instantaneous cross-sectional gas-liquid distribution. The measurement setup also includes Compton scattering measurements, which can provide information about the changes in the water salinity for flow segments with high water liquid ratio and low gas fractions. By measuring the transmitted intensity in short time slots (<100ms), rapid regime variations are revealed. From this we can select the time sections suitable for salinity measurements. Since the salinity variations change at the time scale of hours, a running average can be performed to increase the accuracy of the measurements. Recent results of this work will be presented here. - Highlights: Black-Right-Pointing-Pointer Multiphase flow gas-fraction and flow regime measurements by multi gamma ray beams. Black-Right-Pointing-Pointer High-speed gamma ray tomograph as reference for the flow

  9. Radiotracer techniques for measuring fluid flow and calibrating flow meters

    International Nuclear Information System (INIS)

    Cooper, E.L.

    1987-08-01

    Radiotracer techniques can be used to measure accurately both gas and liquid flow rates under operating conditions in a wide range of flow systems. They are ideally suited for calibrating flow meters as well as for measuring unmetered flows in industrial plants. Applications of these techniques range from measuring the flows of fuels and process fluids for energy and mass balance studies to measuring the flows of liquid and airborne effluents for pollution control. This report describes the various radiotracer techniques which can be used to measure fluid flows. The range of application and inherent accuracy of each technique is discussed

  10. Rapid flow imaging method

    International Nuclear Information System (INIS)

    Pelc, N.J.; Spritzer, C.E.; Lee, J.N.

    1988-01-01

    A rapid, phase-contrast, MR imaging method of imaging flow has been implemented. The method, called VIGRE (velocity imaging with gradient recalled echoes), consists of two interleaved, narrow flip angle, gradient-recalled acquisitions. One is flow compensated while the second has a specified flow encoding (both peak velocity and direction) that causes signals to contain additional phase in proportion to velocity in the specified direction. Complex image data from the first acquisition are used as a phase reference for the second, yielding immunity from phase accumulation due to causes other than motion. Images with pixel values equal to MΔΘ where M is the magnitude of the flow compensated image and ΔΘ is the phase difference at the pixel, are produced. The magnitude weighting provides additional vessel contrast, suppresses background noise, maintains the flow direction information, and still allows quantitative data to be retrieved. The method has been validated with phantoms and is undergoing initial clinical evaluation. Early results are extremely encouraging

  11. Upscaling of Forchheimer flows

    KAUST Repository

    Aulisa, Eugenio

    2014-08-01

    In this work we propose upscaling method for nonlinear Forchheimer flow in heterogeneous porous media. The generalized Forchheimer law is considered for incompressible and slightly-compressible single-phase flows. We use recently developed analytical results (Aulisa et al., 2009) [1] and formulate the resulting system in terms of a degenerate nonlinear flow equation for the pressure with the nonlinearity depending on the pressure gradient. The coarse scale parameters for the steady state problem are determined so that the volumetric average of velocity of the flow in the domain on fine scale and on coarse scale are close. A flow-based coarsening approach is used, where the equivalent permeability tensor is first evaluated following streamline methods for linear cases, and modified in order to take into account the nonlinear effects. Compared to previous works (Garibotti and Peszynska, 2009) [2], (Durlofsky and Karimi-Fard) [3], this approach can be combined with rigorous mathematical upscaling theory for monotone operators, (Efendiev et al., 2004) [4], using our recent theoretical results (Aulisa et al., 2009) [1]. The developed upscaling algorithm for nonlinear steady state problems is effectively used for variety of heterogeneities in the domain of computation. Direct numerical computations for average velocity and productivity index justify the usage of the coarse scale parameters obtained for the special steady state case in the fully transient problem. For nonlinear case analytical upscaling formulas in stratified domain are obtained. Numerical results were compared to these analytical formulas and proved to be highly accurate. © 2014.

  12. Transient flow combustion

    Science.gov (United States)

    Tacina, R. R.

    1984-01-01

    Non-steady combustion problems can result from engine sources such as accelerations, decelerations, nozzle adjustments, augmentor ignition, and air perturbations into and out of the compressor. Also non-steady combustion can be generated internally from combustion instability or self-induced oscillations. A premixed-prevaporized combustor would be particularly sensitive to flow transients because of its susceptability to flashback-autoignition and blowout. An experimental program, the Transient Flow Combustion Study is in progress to study the effects of air and fuel flow transients on a premixed-prevaporized combustor. Preliminary tests performed at an inlet air temperature of 600 K, a reference velocity of 30 m/s, and a pressure of 700 kPa. The airflow was reduced to 1/3 of its original value in a 40 ms ramp before flashback occurred. Ramping the airflow up has shown that blowout is more sensitive than flashback to flow transients. Blowout occurred with a 25 percent increase in airflow (at a constant fuel-air ratio) in a 20 ms ramp. Combustion resonance was found at some conditions and may be important in determining the effects of flow transients.

  13. Turbine flow meter response in two-phase flows

    International Nuclear Information System (INIS)

    Shim, W.J.; Dougherty, T.J.; Cheh, H.Y.

    1996-01-01

    The purpose of this paper is to suggest a simple method of calibrating turbine flow meters to measure the flow rates of each phase in a two-phase flow. The response of two 50.8 mm (2 inch) turbine flow meters to air-water, two-phase mixtures flowing vertically in a 57 mm I.D. (2.25 inch) polycarbonate tube has been investigated for both upflow and downflow. The flow meters were connected in series with an intervening valve to provide an adjustable pressure difference between them. Void fractions were measured by two gamma densitometers, one upstream of the flow meters and the other downstream. The output signal of the turbine flow meters was found to depend only on the actual volumetric flow rate of the gas, F G , and liquid, F L , at the location of the flow meter

  14. Particles in flows

    CERN Document Server

    Galdi, Giovanni; Nečasová, Šárka

    2017-01-01

    This book aims to face particles in flows from many different, but essentially interconnected sides and points of view. Thus the selection of authors and topics represented in the chapters, ranges from deep mathematical analysis of the associated models, through the techniques of their numerical solution, towards real applications and physical implications. The scope and structure of the book as well as the selection of authors was motivated by the very successful summer course and workshop "Particles in Flows'' that was held in Prague in the August of 2014. This meeting revealed the need for a book dealing with this specific and challenging multidisciplinary subject, i.e. particles in industrial, environmental and biomedical flows and the combination of fluid mechanics, solid body mechanics with various aspects of specific applications.

  15. Two-phase flow

    International Nuclear Information System (INIS)

    Olive, J.

    1990-01-01

    The design, operation and safety of nuclear components requires increasingly accurate knowledge of two-phase flows. This knowledge is also necessary for some studies related to electricity applications. The author presents some concrete examples showing the range of problems and the complexity of the phenomena involved in these types of flows. Then, the basic principles of their numerical modelling are explained, as well as the new tendency to use increasingly local and refined models. The newest computer codes developed at EDF are briefly presented. Experimental studies dealing with twophase flow are also referred to, and their connections to numerical modelling are explained. Emphasis is placed on the major efforts devoted to the development of new test rigs and instrumentation [fr

  16. Robust Optical Flow Estimation

    Directory of Open Access Journals (Sweden)

    Javier Sánchez Pérez

    2013-10-01

    Full Text Available n this work, we describe an implementation of the variational method proposed by Brox etal. in 2004, which yields accurate optical flows with low running times. It has several benefitswith respect to the method of Horn and Schunck: it is more robust to the presence of outliers,produces piecewise-smooth flow fields and can cope with constant brightness changes. Thismethod relies on the brightness and gradient constancy assumptions, using the information ofthe image intensities and the image gradients to find correspondences. It also generalizes theuse of continuous L1 functionals, which help mitigate the effect of outliers and create a TotalVariation (TV regularization. Additionally, it introduces a simple temporal regularizationscheme that enforces a continuous temporal coherence of the flow fields.

  17. Designing reliability information flows

    International Nuclear Information System (INIS)

    Petkova, Valia T.; Lu Yuan; Ion, Roxana A.; Sander, Peter C.

    2005-01-01

    It is well-known [Reliab. Eng. Syst. Saf. 75 (2002) 295] that in modern development processes it is essential to have an information flow structure that facilitates fast feedback from product users (customers) to departments at the front end, in particular development and production. As information is only relevant if it is used when taking decisions, this paper presents a guideline for building field feedback information flows that facilitate the decision taking during the product creation and realisation process. The guideline takes into consideration that the type of decisions depends on the span-of-control, therefore following Parsons [Structure and Process in Modern Societies (1990)] the span-of-control is subdivided into the following three levels: strategic, tactic, and executive. The guideline is illustrated with a case in which it is used for analysing the quality of existing field feedback flows

  18. Hypogenetic chaotic jerk flows

    International Nuclear Information System (INIS)

    Li, Chunbiao; Sprott, Julien Clinton; Xing, Hongyan

    2016-01-01

    Removing the amplitude or polarity information in the feedback loop of a jerk structure shows that special nonlinearities with partial information in the variable can also lead to chaos. Some striking properties are found for this kind of hypogenetic chaotic jerk flow, including multistability of symmetric coexisting attractors from an asymmetric structure, hidden attractors with respect to equilibria but with global attraction, easy amplitude control, and phase reversal which is convenient for chaos applications. - Highlights: • Hypogenetic chaotic jerk flows with incomplete feedback of amplitude or polarity are obtained. • Multistability of symmetric coexisting attractors from an asymmetric structure is found. • Some jerk systems have hidden attractors with respect to equilibria but have global attraction. • These chaotic jerk flows have the properties of amplitude control and phase reversal.

  19. Choked flow through cracks

    International Nuclear Information System (INIS)

    Feburie, V.; Giot, M.; Granger, S.; Seynhaeve, J.M.

    1992-06-01

    The leaks through steam-generator cracks are the subject of a research carried out in cooperation between EDF and UCL. A software called ECREVISSE to predict the mass flow rate has been developed and has been successfully validated. The purpose of the paper is to present the mathematical model used in ECREVISSE as well as some comparison between the results and the presently available data. The model takes into account the persistence of some metastable liquid in the crack and the special flow pattern which appears in such particular geometry. Although the model involves the use of several correlations (friction, heat transfer), no adjustment of parameters against the data has been needed, neither in the single-phase part of the flow, or in the two-phase part. (authors). 8 figs., 1 tab., 20 refs

  20. Probable C4d-negative accelerated acute antibody-mediated rejection due to non-HLA antibodies.

    Science.gov (United States)

    Niikura, Takahito; Yamamoto, Izumi; Nakada, Yasuyuki; Kamejima, Sahoko; Katsumata, Haruki; Yamakawa, Takafumi; Furuya, Maiko; Mafune, Aki; Kobayashi, Akimitsu; Tanno, Yudo; Miki, Jun; Yamada, Hiroki; Ohkido, Ichiro; Tsuboi, Nobuo; Yamamoto, Hiroyasu; Yokoo, Takashi

    2015-07-01

    We report a case of probable C4d-negative accelerated acute antibody-mediated rejection due to non-HLA antibodies. A 44 year-old male was admitted to our hospital for a kidney transplant. The donor, his wife, was an ABO minor mismatch (blood type O to A) and had Gitelman syndrome. Graft function was delayed; his serum creatinine level was 10.1 mg/dL at 3 days after transplantation. Open biopsy was performed immediately; no venous thrombosis was observed during surgery. Histology revealed moderate peritubular capillaritis and mild glomerulitis without C4d immunoreactivity. Flow cytometric crossmatching was positive, but no panel-reactive antibodies against HLA or donor-specific antibodies (DSAbs) to major histocompatibility complex class I-related chain A (MICA) were detected. Taken together, we diagnosed him with probable C4d-negative accelerated antibody-mediated rejection due to non-HLA, non-MICA antibodies, the patient was treated with steroid pulse therapy (methylprednisolone 500 mg/day for 3 days), plasma exchange, intravenous immunoglobulin (40 g/body), and rituximab (200 mg/body) were performed. Biopsy at 58 days after transplantation, at which time S-Cr levels were 1.56 mg/dL, found no evidence of rejection. This case, presented with a review of relevant literature, demonstrates that probable C4d-negative accelerated acute AMR can result from non-HLA antibodies. © 2015 Asian Pacific Society of Nephrology.

  1. HLA antibody-incompatible kidney transplantation between jehovah's witnesses--a case report.

    Science.gov (United States)

    Greenberg, A; Macphee, I; Popoola, J; Sage, D; Iqbal, R; Fossati, N; Heap, S; Morsy, M; Kessaris, N

    2013-06-01

    Desensitization before HLA antibody-incompatible (HLAi) transplantation involves nonspecific apheresis of HLA antibodies. Clotting factors and albumin are also removed and have to be replaced. This makes transplantation difficult because it increases the risk of bleeding. Such risk is further compounded when certain blood products are refused on religious grounds. We present a case of successful HLAi transplantation in a Jehovah's Witness across a positive-flow cytometric HLA crossmatch from a live donor who was also a Jehovah's Witness. This was achieved by giving rituximab 1 month before transplantation and starting prednisolone, tacrolimus, and mycophenolate mofetil 10 days before surgery. In preparation, the patient also underwent 4 sessions of double-filtration plasma exchange each followed by low-dose intravenous immunoglobulin. The night before transplantation, the fibrinogen was low, requiring 2 pools of cryoprecipitate. The organ was retrieved through laparoscopic hand-assisted retroperitoneoscopic nephrectomy and transplanted into the recipient with no complications. In addition, the patient received basiliximab during surgery. Sixteen months after transplantation the serum creatinine was 70 μmol/L (0.79 mg/dL) and there were no rejection episodes. To our knowledge this is the world's first live-related kidney transplant across the HLAi barrier between 2 Jehovah's Witnesses. This case may allow further HLAi transplants to be carried out in Jehovah's Witnesses in the future around the world. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. Evaluation of flow hood measurements for residential register flows; TOPICAL

    International Nuclear Information System (INIS)

    Walker, I.S.; Wray, C.P.; Dickerhoff, D.J.; Sherman, M.H.

    2001-01-01

    Flow measurement at residential registers using flow hoods is becoming more common. These measurements are used to determine if the HVAC system is providing adequate comfort, appropriate flow over heat exchangers and in estimates of system energy losses. These HVAC system performance metrics are determined by using register measurements to find out if individual rooms are getting the correct airflow, and in estimates of total air handler flow and duct air leakage. The work discussed in this paper shows that commercially available flow hoods are poor at measuring flows in residential systems. There is also evidence in this and other studies that flow hoods can have significant errors even when used on the non-residential systems they were originally developed for. The measurement uncertainties arise from poor calibrations and the sensitivity of exiting flow hoods to non-uniformity of flows entering the device. The errors are usually large-on the order of 20% of measured flow, which is unacceptably high for most applications. Active flow hoods that have flow measurement devices that are insensitive to the entering airflow pattern were found to be clearly superior to commercially available flow hoods. In addition, it is clear that current calibration procedures for flow hoods may not take into account any field application problems and a new flow hood measurement standard should be developed to address this issue

  3. Flow Rate Measurement in Multiphase Flow Rig: Radiotracer and Conventional

    International Nuclear Information System (INIS)

    Nazrul Hizam Yusoff; Noraishah Othman; Nurliyana Abdullah; Amirul Syafiq Mohd Yunos; Rasif Mohd Zain; Roslan Yahya

    2015-01-01

    Applications of radiotracer technology are prevalent throughout oil refineries worldwide, and this industry is one of the main users and beneficiaries of the technology. Radioactive tracers have been used to a great extent in many applications i.e. flow rate measurement, RTD, plant integrity evaluation and enhancing oil production in oil fields. Chemical and petrochemical plants are generally continuously operating and technically complex where the radiotracer techniques are very competitive and largely applied for troubleshooting inspection and process analysis. Flow rate measurement is a typical application of radiotracers. For flow measurements, tracer data are important, rather than the RTD models. Research is going on in refining the existing methods for single phase flow measurement, and in developing new methods for multiphase flow without sampling. The tracer techniques for single phase flow measurements are recognized as ISO standards. This paper presents technical aspect of laboratory experiments, which have been carried out using Molybdenum-99 - Mo99 (radiotracer) to study and determine the flow rate of liquid in multiphase flow rig. The multiphase flow rig consists of 58.7 m long and 20 cm diameter pipeline that can accommodate about 0.296 m 3 of liquid. Tap water was used as liquid flow in pipeline and conventional flow meters were also installed at the flow rig. The flow rate results; radiotracer and conventional flow meter were compared. The total count method was applied for radiotracer technique and showed the comparable results with conventional flow meter. (author)

  4. Three-Dimensional Flows

    CERN Document Server

    Araujo, Vitor; Viana, Marcelo

    2010-01-01

    In this book, the authors present the elements of a general theory for flows on three-dimensional compact boundaryless manifolds, encompassing flows with equilibria accumulated by regular orbits. The book aims to provide a global perspective of this theory and make it easier for the reader to digest the growing literature on this subject. This is not the first book on the subject of dynamical systems, but there are distinct aspects which together make this book unique. Firstly, this book treats mostly continuous time dynamical systems, instead of its discrete counterpart, exhaustively treated

  5. Magnetohydrodynamic flow phenomena

    International Nuclear Information System (INIS)

    Gerbeth, G.; Mutschke, G.; Eckert, S.

    1995-01-01

    The MHD group of the Institute of Safety Research performs basic studies on fluid dynamics and heat/mass transfer in fluids, particularly for electrically conducting fluids (liquid metals) exposed to external magnetic fields (Magnetohydrodynamics - MHD). Such a contactless influence on transport phenomena is of principal importance for a variety of applied problems including safety and design aspects in liquid metal cooled fusion reactors, fast reactors, and chemical systems. Any electrically conducting flow can be influenced without any contact by means of an external electromagnetic field. This, of course, can change the known hydromechanically flow patterns considerably. In the following two examples of such magnetic field influence are presented. (orig.)

  6. Mechanics of fluid flow

    CERN Document Server

    Basniev, Kaplan S; Chilingar, George V 0

    2012-01-01

    The mechanics of fluid flow is a fundamental engineering discipline explaining both natural phenomena and human-induced processes, and a thorough understanding of it is central to the operations of the oil and gas industry.  This book, written by some of the world's best-known and respected petroleum engineers, covers the concepts, theories, and applications of the mechanics of fluid flow for the veteran engineer working in the field and the student, alike.  It is a must-have for any engineer working in the oil and gas industry.

  7. Flow cytometry for intracellular SPION quantification: specificity and sensitivity in comparison with spectroscopic methods

    Directory of Open Access Journals (Sweden)

    Friedrich RP

    2015-06-01

    Full Text Available Ralf P Friedrich,1 Christina Janko,1 Marina Poettler,1 Philipp Tripal,1 Jan Zaloga,1 Iwona Cicha,1 Stephan Dürr,1,2 Johannes Nowak,3 Stefan Odenbach,3 Ioana Slabu,4 Maik Liebl,4 Lutz Trahms,4 Marcus Stapf,5 Ingrid Hilger,5 Stefan Lyer,1 Christoph Alexiou1 1Department of Otorhinolaryngology, Head and Neck Surgery, Section of Experimental Oncology and Nanomedicine, University hospital Erlangen, 2Department of Otorhinolaryngology, Head and Neck Surgery, Section of Phoniatrics and Pediatric Audiology, University hospital Erlangen, Erlangen, 3Technische Universität Dresden, Chair of Magnetofluiddynamics, Measuring and Automation Technology, Dresden, 4Physikalisch-Technische Bundesanstalt Berlin, Berlin, 5Department of Radiology, Division of Diagnostic and Interventional Radiology, Experimental Radiology, University hospital Jena, Jena, Germany Abstract: Due to their special physicochemical properties, iron nanoparticles offer new promising possibilities for biomedical applications. For bench to bedside translation of superparamagnetic iron oxide nanoparticles (SPIONs, safety issues have to be comprehensively clarified. To understand concentration-dependent nanoparticle-mediated toxicity, the exact quantification of intracellular SPIONs by reliable methods is of great importance. In the present study, we compared three different SPION quantification methods (ultraviolet spectrophotometry, magnetic particle spectroscopy, atomic adsorption spectroscopy and discussed the shortcomings and advantages of each method. Moreover, we used those results to evaluate the possibility to use flow cytometric technique to determine the cellular SPION content. For this purpose, we correlated the side scatter data received from flow cytometry with the actual cellular SPION amount. We showed that flow cytometry provides a rapid and reliable method to assess the cellular SPION content. Our data also demonstrate that internalization of iron oxide nanoparticles in human

  8. Transient two-phase flow

    International Nuclear Information System (INIS)

    Hsu, Y.Y.

    1974-01-01

    The following papers related to two-phase flow are summarized: current assumptions made in two-phase flow modeling; two-phase unsteady blowdown from pipes, flow pattern in Laval nozzle and two-phase flow dynamics; dependence of radial heat and momentum diffusion; transient behavior of the liquid film around the expanding gas slug in a vertical tube; flooding phenomena in BWR fuel bundles; and transient effects in bubble two-phase flow. (U.S.)

  9. Using Crossflow for Flow Measurements and Flow Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Gurevich, A.; Chudnovsky, L.; Lopeza, A. [Advanced Measurement and Analysis Group Inc., Ontario (Canada); Park, M. H. [Sungjin Nuclear Engineering Co., Ltd., Gyeongju (Korea, Republic of)

    2016-10-15

    Ultrasonic Cross Correlation Flow Measurements are based on a flow measurement method that is based on measuring the transport time of turbulent structures. The cross correlation flow meter CROSSFLOW is designed and manufactured by Advanced Measurement and Analysis Group Inc. (AMAG), and is used around the world for various flow measurements. Particularly, CROSSFLOW has been used for boiler feedwater flow measurements, including Measurement Uncertainty Recovery (MUR) reactor power uprate in 14 nuclear reactors in the United States and in Europe. More than 100 CROSSFLOW transducers are currently installed in CANDU reactors around the world, including Wolsung NPP in Korea, for flow verification in ShutDown System (SDS) channels. Other CROSSFLOW applications include reactor coolant gross flow measurements, reactor channel flow measurements in all channels in CANDU reactors, boiler blowdown flow measurement, and service water flow measurement. Cross correlation flow measurement is a robust ultrasonic flow measurement tool used in nuclear power plants around the world for various applications. Mathematical modeling of the CROSSFLOW agrees well with laboratory test results and can be used as a tool in determining the effect of flow conditions on CROSSFLOW output and on designing and optimizing laboratory testing, in order to ensure traceability of field flow measurements to laboratory testing within desirable uncertainty.

  10. ECAL Energy Flow Calibration

    CERN Multimedia

    CERN. Geneva

    2015-01-01

    My talk will be covering my work as a whole over the course of the semester. The focus will be on using energy flow calibration in ECAL to check the precision of the corrections made by the light monitoring system used to account for transparency loss within ECAL crystals due to radiation damage over time.

  11. Is flow verification necessary

    International Nuclear Information System (INIS)

    Beetle, T.M.

    1986-01-01

    Safeguards test statistics are used in an attempt to detect diversion of special nuclear material. Under assumptions concerning possible manipulation (falsification) of safeguards accounting data, the effects on the statistics due to diversion and data manipulation are described algebraically. A comprehensive set of statistics that is capable of detecting any diversion of material is defined in terms of the algebraic properties of the effects. When the assumptions exclude collusion between persons in two material balance areas, then three sets of accounting statistics are shown to be comprehensive. Two of the sets contain widely known accountancy statistics. One of them does not require physical flow verification - comparisons of operator and inspector data for receipts and shipments. The third set contains a single statistic which does not require physical flow verification. In addition to not requiring technically difficult and expensive flow verification, this single statistic has several advantages over other comprehensive sets of statistics. This algebraic approach as an alternative to flow verification for safeguards accountancy is discussed in this paper

  12. Flow of nuclear matter

    International Nuclear Information System (INIS)

    Ritter, H.G.; Doss, K.G.R.; Gustafsson, H.A.

    1985-08-01

    The systems Nb + Nb and Au + Au have been measured at different energies at the Bevalac with the Plastic Ball spectrometer. Distributions of the flow angles as a function of charged particle multiplicity are presented. Also shown is a transverse momentum analysis for 400 MeV per nucleon Nb + Nb. 25 refs., 5 figs., 1 tab

  13. Heat flow method

    International Nuclear Information System (INIS)

    Chen Yunmei

    1994-01-01

    In this paper we study the heat flow of harmonic maps between two compact Riemannian manifolds. The global existence of the regular solution and the weak solution, as well as the blow up of the weak solution are discussed. (author). 14 refs

  14. Lateral flow assays

    NARCIS (Netherlands)

    Posthuma-Trumpie, G.A.; Amerongen, van A.

    2012-01-01

    A simple version of immunochemical-based methods is the Lateral Flow Assay (LFA). It is a dry chemistry technique (reagents are included); the fluid from the sample runs through a porous membrane (often nitrocellulose) by capillary force. Typically the membrane is cut as a strip of 0.5*5 cm. In most

  15. Delta Flow Modulator

    NARCIS (Netherlands)

    Stamhuis, Eize; Lengkeek, W

    2015-01-01

    A support structure (2) is installed in or near a water (50). The support structure is holding a deltalike-wing (3) under an angle of incidence relative to an incoming flow (54), caused by at least a prevailing current in the water, thus generating a vortex (77). The action of the vortex is

  16. Flow cytometry apparatus

    Science.gov (United States)

    Pinkel, D.

    1987-11-30

    An obstruction across the flow chamber creates a one-dimensional convergence of a sheath fluid. A passageway in the obstruction directs flat cells near to the area of one-dimensional convergence in the sheath fluid to provide proper orientation of flat cells at fast rates. 6 figs.

  17. Diffusion or bulk flow

    DEFF Research Database (Denmark)

    Schulz, Alexander

    2015-01-01

    is currently matter of discussion, called passive symplasmic loading. Based on the limited material available, this review compares the different loading modes and suggests that diffusion is the driving force in apoplasmic loaders, while bulk flow plays an increasing role in plants having a continuous...

  18. Proportionate Flow Shop Games

    NARCIS (Netherlands)

    Estevez Fernandez, M.A.; Mosquera, M.A.; Borm, P.E.M.; Hamers, H.J.M.

    2006-01-01

    In a proportionate flow shop problem several jobs have to be processed through a fixed sequence of machines and the processing time of each job is equal on all machines.By identifying jobs with agents, whose costs linearly depend on the completion time of their jobs, and assuming an initial

  19. Probabilistic Load Flow

    DEFF Research Database (Denmark)

    Chen, Peiyuan; Chen, Zhe; Bak-Jensen, Birgitte

    2008-01-01

    This paper reviews the development of the probabilistic load flow (PLF) techniques. Applications of the PLF techniques in different areas of power system steady-state analysis are also discussed. The purpose of the review is to identify different available PLF techniques and their corresponding...

  20. Techniques of Flow Visualization

    Science.gov (United States)

    1987-12-01

    are sensitive. Within the bandwidth of their sensitivity, up to ten color hues may be discriminated by the eye. The visible edge between two colors...can be performed with conven- tional photography or cinematography . Video recording is of advantage for the further pro- cessing of the flow pictures

  1. Erosion in extruder flow

    Science.gov (United States)

    Kaufman, Miron; Fodor, Petru S.

    A detailed analysis of the fluid flow in Tadmor's unwound channel model of the single screw extruder is performed by combining numerical and analytical methods. Using the analytical solution for the longitudinal velocity field (in the limit of zero Reynolds number) allows us to devote all the computational resources solely for a detailed numerical solution of the transversal velocity field. This high resolution 3D model of the fluid flow in a single-screw extruder allows us to identify the position and extent of Moffatt eddies that impede mixing. We further consider the erosion of particles (e.g. carbon-black agglomerates) advected by the polymeric flow. We assume a particle to be made of primary fragments bound together. In the erosion process a primary fragment breaks out of a given particle. Particles are advected by the laminar flow and they disperse because of the shear stresses imparted by the fluid. The time evolution of the numbers of particles of different sizes is described by the Bateman coupled differential equations used to model radioactivity. Using the particle size distribution we compute an entropic fragmentation index which varies from 0 for a monodisperse system to 1 for an extreme poly-disperse system.

  2. Upscaling of reactive flows

    NARCIS (Netherlands)

    Kumar, K.

    2012-01-01

    The thesis deals with the upscaling of reactive flows in complex geometry. The reactions which may include deposition or dissolution take place at a part of the boundary and depending on the size of the reaction domain, the changes in the pore structure that are due to the deposition process may or

  3. Flow analysis of HANARO flow simulated test facility

    International Nuclear Information System (INIS)

    Park, Yong-Chul; Cho, Yeong-Garp; Wu, Jong-Sub; Jun, Byung-Jin

    2002-01-01

    The HANARO, a multi-purpose research reactor of 30 MWth open-tank-in-pool type, has been under normal operation since its initial critical in February, 1995. Many experiments should be safely performed to activate the utilization of the NANARO. A flow simulated test facility is being developed for the endurance test of reactivity control units for extended life times and the verification of structural integrity of those experimental facilities prior to loading in the HANARO. This test facility is composed of three major parts; a half-core structure assembly, flow circulation system and support system. The half-core structure assembly is composed of plenum, grid plate, core channel with flow tubes, chimney and dummy pool. The flow channels are to be filled with flow orifices to simulate core channels. This test facility must simulate similar flow characteristics to the HANARO. This paper, therefore, describes an analytical analysis to study the flow behavior of the test facility. The computational flow analysis has been performed for the verification of flow structure and similarity of this test facility assuming that flow rates and pressure differences of the core channel are constant. The shapes of flow orifices were determined by the trial and error method based on the design requirements of core channel. The computer analysis program with standard k - ε turbulence model was applied to three-dimensional analysis. The results of flow simulation showed a similar flow characteristic with that of the HANARO and satisfied the design requirements of this test facility. The shape of flow orifices used in this numerical simulation can be adapted for manufacturing requirements. The flow rate and the pressure difference through core channel proved by this simulation can be used as the design requirements of the flow system. The analysis results will be verified with the results of the flow test after construction of the flow system. (author)

  4. Measurement of circulating cell-derived microparticles by flow cytometry: sources of variability within the assay.

    Science.gov (United States)

    Ayers, Lisa; Kohler, Malcolm; Harrison, Paul; Sargent, Ian; Dragovic, Rebecca; Schaap, Marianne; Nieuwland, Rienk; Brooks, Susan A; Ferry, Berne

    2011-04-01

    Circulating cell-derived microparticles (MPs) have been implicated in several disease processes and elevated levels are found in many pathological conditions. The detection and accurate measurement of MPs, although attracting widespread interest, is hampered by a lack of standardisation. The aim of this study was to establish a reliable flow cytometric assay to measure distinct subtypes of MPs in disease and to identify any significant causes of variability in MP quantification. Circulating MPs within plasma were identified by their phenotype (platelet, endothelial, leukocyte and annexin-V positivity (AnnV+). The influence of key variables (i.e. time between venepuncture and centrifugation, washing steps, the number of centrifugation steps, freezing/long-term storage and temperature of thawing) on MP measurement were investigated. Increasing time between venepuncture and centrifugation leads to increased MP levels. Washing samples results in decreased AnnV+MPs (P=0.002) and platelet-derived MPs (PMPs) (P=0.002). Double centrifugation of MPs prior to freezing decreases numbers of AnnV+MPs (P=0.0004) and PMPs (P=0.0004). A single freeze thaw cycle of samples led to an increase in AnnV+MPs (P=0.0020) and PMPs (P=0.0039). Long-term storage of MP samples at -80° resulted in decreased MP levels. This study found that minor protocol changes significantly affected MP levels. This is one of the first studies attempting to standardise a method for obtaining and measuring circulating MPs. Standardisation will be essential for successful development of MP technologies, allowing direct comparison of results between studies and leading to a greater understanding of MPs in disease. Crown Copyright © 2010. Published by Elsevier Ltd. All rights reserved.

  5. Visualization study of flow in axial flow inducer.

    Science.gov (United States)

    Lakshminarayana, B.

    1972-01-01

    A visualization study of the flow through a three ft dia model of a four bladed inducer, which is operated in air at a flow coefficient of 0.065, is reported in this paper. The flow near the blade surfaces, inside the rotating passages, downstream and upstream of the inducer is visualized by means of smoke, tufts, ammonia filament, and lampblack techniques. Flow is found to be highly three dimensional, with appreciable radial velocity throughout the entire passage. The secondary flows observed near the hub and annulus walls agree with qualitative predictions obtained from the inviscid secondary flow theory.

  6. Critical flow rate in a single phase flow. Blocking concept

    International Nuclear Information System (INIS)

    Giot, Michel

    1978-01-01

    After referring to the phenomena accompanying the appearance of a critical flow rate in a nozzle and presenting equations governing single phase flows, the critical condition is defined. Several particular cases are then examined; the horizontal and vertical isentropic flow, Fanno's flow and Raleigh's and the isothermal flow. The entropy deviation is calculated on either side of a normal impact. To conclude, the link existing between the concepts of critical flow and the propagation rate of small perturbations is demonstrated. To do so, the method of perturbations, that of Prandtl and that of characteristic directions are applied in turn [fr

  7. Using Crossmatch tests for serological compatibility assessment intra- and interspecific at dogs and cats

    Directory of Open Access Journals (Sweden)

    Sergiu Adrian Muntean

    2016-11-01

    Conclusions: The intraspecific evaluations revealed a high level of blood compatibility in the case of dogs unsensitivized through previous blood transfusions, yet without excluding the possibility of some atypical sensitivization for clinical interest. Having all the interspecific tests exclusively highly positive, we can not sustain a probable xenotransfusion.

  8. AstroDAbis: Annotations and Cross-Matches for Remote Catalogues

    Science.gov (United States)

    Gray, N.; Mann, R. G.; Morris, D.; Holliman, M.; Noddle, K.

    2012-09-01

    Astronomers are good at sharing data, but poorer at sharing knowledge. Almost all astronomical data ends up in open archives, and access to these is being simplified by the development of the global Virtual Observatory (VO). This is a great advance, but the fundamental problem remains that these archives contain only basic observational data, whereas all the astrophysical interpretation of that data — which source is a quasar, which a low-mass star, and which an image artefact — is contained in journal papers, with very little linkage back from the literature to the original data archives. It is therefore currently impossible for an astronomer to pose a query like “give me all sources in this data archive that have been identified as quasars” and this limits the effective exploitation of these archives, as the user of an archive has no direct means of taking advantage of the knowledge derived by its previous users. The AstroDAbis service aims to address this, in a prototype service enabling astronomers to record annotations and cross-identifications in the AstroDAbis service, annotating objects in other catalogues. We have deployed two interfaces to the annotations, namely one astronomy-specific one using the TAP protocol (Dowler et al. 2011), and a second exploiting generic Linked Open Data (LOD) and RDF techniques.

  9. Improving Face Verification in Photo Albums by Combining Facial Recognition and Metadata With Cross-Matching

    Science.gov (United States)

    2017-12-01

    with our method. The third chapter presents the description and implementation of our approach. We provide a definition of the dataset, the...a means of classification using the shape and the texture. 16 Figure 7. 3D Alignment Pipeline. Adapted from [20]. In 2014, Facebook announced...Stating that face recognition consists of four main stages, detect ⟹ align ⟹ represent ⟹ classify, the Facebook team’s intent is to revisit the

  10. Evaluation of CD307a expression patterns during normal B-cell maturation and in B-cell malignancies by flow cytometry.

    Science.gov (United States)

    Auat, Mariangeles; Cardoso, Chandra Chiappin; Santos-Pirath, Iris Mattos; Rudolf-Oliveira, Renata Cristina Messores; Matiollo, Camila; Lange, Bárbara Gil; da Silva, Jessica Pires; Dametto, Gisele Cristina; Pirolli, Mayara Marin; Colombo, Maria Daniela Holthausen Perico; Santos-Silva, Maria Claudia

    2018-02-24

    Flow cytometric immunophenotyping is deemed a fundamental tool for the diagnosis of B-cell neoplasms. Currently, the investigation of novel immunophenotypic markers has gained importance, as they can assist in the precise subclassification of B-cell malignancies by flow cytometry. Therefore, the purpose of the present study was to evaluate the expression of CD307a during normal B-cell maturation and in B-cell malignancies as well as to investigate its potential role in the differential diagnosis of these entities. CD307a expression was assessed by flow cytometry in normal precursor and mature B cells and in 115 samples collected from patients diagnosed with precursor and mature B-cell neoplasms. CD307a expression was compared between neoplastic and normal B cells. B-acute lymphoblastic leukemia cases exhibited minimal expression of CD307a, displaying a similar expression pattern to that of normal B-cell precursors. Mantle cell lymphoma (MCL) cases showed the lowest levels of CD307a among mature B-cell neoplasms. CD307a expression was statistically lower in MCL cases than in chronic B lymphocytic leukemia (CLL) and marginal zone lymphoma (MZL) cases. No statistical differences were observed between CD307a expression in neoplastic and normal plasma cells. These results indicate that the assessment of CD307a expression by flow cytometry could be helpful to distinguish CLL from MCL, and the latter from MZL. Although these results are not entirely conclusive, they provide a basis for further studies in a larger cohort of patients. © 2018 International Clinical Cytometry Society. © 2018 International Clinical Cytometry Society.

  11. Structural power flow measurement

    Energy Technology Data Exchange (ETDEWEB)

    Falter, K.J.; Keltie, R.F.

    1988-12-01

    Previous investigations of structural power flow through beam-like structures resulted in some unexplained anomalies in the calculated data. In order to develop structural power flow measurement as a viable technique for machine tool design, the causes of these anomalies needed to be found. Once found, techniques for eliminating the errors could be developed. Error sources were found in the experimental apparatus itself as well as in the instrumentation. Although flexural waves are the carriers of power in the experimental apparatus, at some frequencies longitudinal waves were excited which were picked up by the accelerometers and altered power measurements. Errors were found in the phase and gain response of the sensors and amplifiers used for measurement. A transfer function correction technique was employed to compensate for these instrumentation errors.

  12. Slow viscous flow

    CERN Document Server

    Langlois, William E

    2014-01-01

    Leonardo wrote, 'Mechanics is the paradise of the mathematical sciences, because by means of it one comes to the fruits of mathematics' ; replace 'Mechanics' by 'Fluid mechanics' and here we are." -    from the Preface to the Second Edition Although the exponential growth of computer power has advanced the importance of simulations and visualization tools for elaborating new models, designs and technologies, the discipline of fluid mechanics is still large, and turbulence in flows remains a challenging problem in classical physics. Like its predecessor, the revised and expanded Second Edition of this book addresses the basic principles of fluid mechanics and solves fluid flow problems where viscous effects are the dominant physical phenomena. Much progress has occurred in the nearly half a century that has passed since the edition of 1964. As predicted, aspects of hydrodynamics once considered offbeat have risen to importance. For example, the authors have worked on problems where variations in viscosity a...

  13. Fracture flow code

    International Nuclear Information System (INIS)

    Dershowitz, W; Herbert, A.; Long, J.

    1989-03-01

    The hydrology of the SCV site will be modelled utilizing discrete fracture flow models. These models are complex, and can not be fully cerified by comparison to analytical solutions. The best approach for verification of these codes is therefore cross-verification between different codes. This is complicated by the variation in assumptions and solution techniques utilized in different codes. Cross-verification procedures are defined which allow comparison of the codes developed by Harwell Laboratory, Lawrence Berkeley Laboratory, and Golder Associates Inc. Six cross-verification datasets are defined for deterministic and stochastic verification of geometric and flow features of the codes. Additional datasets for verification of transport features will be documented in a future report. (13 figs., 7 tabs., 10 refs.) (authors)

  14. The Flow of Energy

    Science.gov (United States)

    Znidarsic, F.; Robertson, G. A.

    In this paper, the flow of energy in materials is presented as mechanical waves with a distinct velocity or speed of transition. This speed of transition came about through the observations of cold fusion experiments, i.e., Low Energy Nuclear Reactions (LENR) and superconductor gravity experiments, both assumed speculative by mainstream science. In consideration of superconductor junctions, the LENR experiments have a similar speed of transition, which seems to imply that the reactions in the LENR experiment are discrete quantized reactions (energy - burst vs. continuous). Here an attempt is made to quantify this new condition as it applies to electrons; toward the progression of quantized energy flows (discrete energy burst) as a new source of clean energy and force mechanisms (i.e, propulsion).

  15. Hawaii Lava Flows

    Science.gov (United States)

    2001-01-01

    This sequence of ASTER nighttime thermal images shows the Pu'u O'o lava flows entering the sea at Kamokuna on the southeast side of the Island of Hawaii. Each image covers an area of 9 x 12 km. The acquisition dates are April 4 2000, May 13 2000, May 22 2000 (upper row) and June 30 2000, August 1 2000 and January 1 2001 (lower row). Thermal band 14 has been color coded from black (coldest) through blue, red, yellow and white (hottest). The first 5 images show a time sequence of a single eruptive phase; the last image shows flows from a later eruptive phase. The images are located at 19.3 degrees north latitude, 155 degrees west longitude. The U.S. science team is located at NASA's Jet Propulsion Laboratory, Pasadena, Calif. The Terra mission is part of NASA's Science Mission Directorate.

  16. Optimal Power Flow Pursuit

    Energy Technology Data Exchange (ETDEWEB)

    Dall' Anese, Emiliano; Simonetto, Andrea

    2018-03-01

    This paper considers distribution networks featuring inverter-interfaced distributed energy resources, and develops distributed feedback controllers that continuously drive the inverter output powers to solutions of AC optimal power flow (OPF) problems. Particularly, the controllers update the power setpoints based on voltage measurements as well as given (time-varying) OPF targets, and entail elementary operations implementable onto low-cost microcontrollers that accompany power-electronics interfaces of gateways and inverters. The design of the control framework is based on suitable linear approximations of the AC power-flow equations as well as Lagrangian regularization methods. Convergence and OPF-target tracking capabilities of the controllers are analytically established. Overall, the proposed method allows to bypass traditional hierarchical setups where feedback control and optimization operate at distinct time scales, and to enable real-time optimization of distribution systems.

  17. Flow Control Technology

    Science.gov (United States)

    2010-07-01

    known as Darrieus turbines or, after the German inventors of these devices, Voith-Schneider propellers. Their main advantage is the ability to produce... turbines (VAWT), named for the typical orientation of the main shaft. While their efficiency is similar to that of the more common horizontal axis wind ...Oscillating Systems’, Cambridge University Press, 2002 [11] G. M. Darrieus , ’ Turbine having its rotating shaft transverse to the flow of the current

  18. Flow Injection Analysis

    DEFF Research Database (Denmark)

    Hansen, Elo Harald

    1998-01-01

    Learning objectives:* To provide an introduction to automated assays* To describe the basic principles of FIA * To demonstrate the capabilities of FIA in relation to batch assays and conventional continuous flow systems* To show that FIA allows one to augment existing analytical techniques* To sh...... how FIA offers novel analytical procedures which are not feasible by conventional means* To hightlight the potentials of FIA in selected practical assays...

  19. Invariant submanifold flows

    Energy Technology Data Exchange (ETDEWEB)

    Olver, Peter J [School of Mathematics, University of Minnesota, Minneapolis, MN 55455 (United States)], E-mail: olver@math.umn.edu

    2008-08-29

    Given a Lie group acting on a manifold, our aim is to analyze the evolution of differential invariants under invariant submanifold flows. The constructions are based on the equivariant method of moving frames and the induced invariant variational bicomplex. Applications to integrable soliton dynamics, and to the evolution of differential invariant signatures, used in equivalence problems and object recognition and symmetry detection in images, are discussed.

  20. Secondary Flow in Cascades.

    Science.gov (United States)

    1984-06-01

    Int. .7. Neat adMs rnfr 9 the auction Vo1. iS. p. 1.157, 12 1.7r aeTase WOW ugte 0In the comuted 2. Briley# V.R., umenrical Method for AW16 wereo at...S. ft, C.R., -A General Theory of Three saemnit u thecsg the rotation Dimensional Flow in subsonic -a super- injoes Wer sonic Turhasachines of Axial